application of atomic energie in agriculture annual report …
TRANSCRIPT
ASSOCIATION EURATOM - ITAL
STICHTTNG fTAL
APPLICATION OFATOMIC ENERGIE IN AGRICULTURE
Annual Report 1980
1981
C O N T E N T S
page
ASSOCIATION EURATOM - ITAL 1
STICHTING ITAL 1
STAFF OF THE ASSOCIATION EURATOM-ITAL 2
INTRODUCTION * • ^
ACTIVITIES OF GENERAL PUBLIC INTEREST 6
SUMMARY OF THE CONTAMINATION RESEARCH GROUP ~ 12
Project 83 • • •<• • I**
Project 8*» , ... ......r.. 2A
Project 85 31
Project 86 .„.„ ...—.. 35
Project 87 0̂Project 88 ~ MProject 89 „. „ 50
Project 90 . .. « 5&
Project 91 61
SUMMARY OF THE PLANT NUTRITION - SOILS RESEARCH GROUP 69
Project 93 70SUMMARY OF THE PROPERTIES OF CROP PLANTS RESEARCH GROUP A 75
Project 95 76
Project 99 80
Project 120 83
SUMMARY OF THE PROPERTIES OF CROP PLANTS RESEARCH GROUP £, Gene
mutations - Chromosome manipulation „ 85
Project 103 87
Proiect 10^ .. . 90
Project 118 „ „ 93
Project 119 „ 97
Project 136 1..... , 102
SUMMARY OF THE CROP PROTECTION RESEARCH GROUP „ 104
Project 106 „..„ , 106
Project 107 111Project 134 ....'. „ 112Project 135 „„. „ 115
SUMMARY OF THE PRODUCT TREATMENT RESEARCH GROUP 119
Project 108 121
Project 1Q9 124
Project 111 126
Protect 112 . 134
Project 123 , 136
SUMMARY OF THE EXTERNAL SERVICES GROUP 141
Project 105 , , , „., 142
Project 113 143
Project 116 145
" " * " 122 -•••"-- m
PUBLICATIONS , , 152PUBLICATIONS IN PRESS „„..„.. t 156
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A S S O C I A T I O N E U R A T O M - I T A L
B O A R D O F G O V E R N O R S A N D A D V I S O R S
MANAGEMENT COMMITTEE: •
P. BOURDEAU C h a i r m a n
G. WANS INK S e c r e t a r y
R. CRAPS
H . N . HASSELO
F. VAN HOECK
P. VAN DER SCHAMS
H . F . LINSKENS
S T I C H T I N G I T A L
BOARD OF GOVERNORS:
J.B. RITZEMA VAN IKEMA ChairmanG. WANS INK SecretaryT.J. BARENDREGTH.F. LINSKENSH.N. HASSELOP. VAN DER SCHANS
.X
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S T A F F O F T H E A S S O C I A T I O N
E U R A T O M - I T A L
CONTAMINATION
W.R.R. ten Broeke, K.H. Chadwick, 6.HI. Desmet, W.G. Oirkse, J,H. van Ginkel,B. Heije, R.M.J. Pennders, K.J. Puite, J. Sinnaeve, E.T.A, Zwemmer.
PLANT NUTRITION - SOILS
D.W. Bannink, F. van Dorp, H, Kleinhout, W.A. van Lienden, J.A. van Veen.
PROPERTIES OF CROP PLANTS
A. Nutrient uptake and (re)distribution
J.H. Becking, H. Breteler, S.C. van de Geijn, Miss Ch.H. Hänisch ten Cate,M.J.H. Jansen, G.L. Jupijn, D.K. Ketel, P.J.J. Pikaar, H.W. Roelofsen,A. de Ruyter, T.W. Strubbe.
' JB. Gene mutations - Chromosome manipulation
J. Blaas, Miss G.S. Bokelmann, G.M.M. Bredemeijer, C. Broertjes, Mrs. L.M.W.; Dellaert, P. Dijkhuis. K. Sree Ramulu, S. Roest, P.A.Th.J. Werry.
CROP PROTECTION - GENETIC CONTROL
VI.J. van den Brink, A.M. Feldmann, C. van Heemert, A.S. Robinson, G.C.Schelling.
PRODUCT TREATMENT - DISINFECTION - Dl S INFESTATION
W.W.A. Bergers, A.F. Groneman, A. den Hartog, H.G. Heins, J.G. van Kooij,: D.ls. Langerak, H.P. Leenhouts, H.B. Leveling, D. Rintjema, H. Stegeman,: J.W.G.M. Wilmer.
; EXTERNAL SERVICES
; P.H. Dignum, H. Dirkse, J.C. Huisman, W.F. Oosterheert, W.F. Pieters,; J.F. Stoutjesdijk, J.G. de Swart.
í SERVICES AND ADMINISTRATION[ ' ' "••••""••
; G.W.Th. Albers, J.C. Baars, H. Beijer, C. Bongaards, C M . Brouwer, Mrs.: H,P. Brouwer-Engberink, K.H. van Del len, C.H. Deijs, S. van Dijk, E.J.H.f Driever, M. Drost, V.R. den Dunnen, J.F. Eikelenstam, W. Elands, H.G. Elbers,I J.M. van Eidik, N. Griffioen, Mrs. G. van Harsselaar-Heyink, Mrs. M.J.I 't Hart-Versteeg, J.F. Heufkens, G. van den Heuvel, Mrs. H. Jacobs-vanI Brake), H. Jager, C.H.J. Keijman, D.J. van Kleef, F. de Koning, A. Kooyman,I Mrs. C. Mons-Enklaar, C. van de Peppel, J.J. Ronk, P. van Roon, T.G. Rozijn,I J.W. Ruisch, P. Schiphuis, J. van Schothorst, J. Schouten, H.J. Sonies,s; J.J. Tempe1, W.Th. Vérmaten, A.P. de Vries, B. Wenting, J.H. de Winkel,I A.W.H.C.F.M. Wolfs.
I DIRECTION TEAM
I. A. Ringoet, M.J. Frissel, B. de Groot, N. van der Klugt, P.H. van Nierop.
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GUESTWORKERS
R. Merckx (Belgium)
Y. Sagawa (USA)Ta Toa (Vietnam)A. Essaka (Ghana)Mrs. A. Cebulska-Was ilewska (Poland)J.K. Martin (Australia)
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INTRODUCTION
The activity of the Institute in 1980 was characterized by the transitionfrom the old task and programme of the "Institute for Atomic Sciences inAgriculture" to its new task as the Institute of the ITAL Foundation orITAL.The present report has been worked out along the lines of the former pro-gramme; this is a logical approach as the main efforts in 1980 were stillconcentrated on collecting the final results for projects which had to becompleted.The Governing Board of ITAL met five times. The Management Committee of theAssociation EURATOM-ITAL, which manages the contract-programmes with theCommission of the European Community, met twice. Important topics fordiscussion in Board and Committee were:
- the new task of the Institute and its future long term programme.- the organization of the Institute in relation to its new task, as well as
personnel, material and budgetary consequences of the planned set-up.- the progress of research and the programme proposals for 1981; the related
personnel situation and budgetary matters.- the participation of the Association in research programmes of the
Commission of the European Community and of other internationalorganizations.
In the course of 1980 a special task group of selected members of thepersonnel, reinforced with representatives of the Directions for Personneland Organization and Efficiency of the Ministry of Agriculture and Fisheriesworked out the final proposal for the new organization of the Institute, aswell as for a strategy for the transition from the old to the new programme.This proposal was approved by the Board of Governors and has now been sub-mitted for approval to the Ministry.
. The Institute's personnel council met monthly during 1980. The group functionsV as an advisory body to the management on matters of personnel, administration,- budget and research. Important topics of discussion were, of course the newŕ programme, the reorganization and its consequences. Special attention was', paid to the progress made by the above mentioned task-group.
- The work in the different research groups and the various aspects of thenew programme have been discussed in seminars at the Institute, as well asin working groups with participants from Universities and other Institutes.
! Scientists of the Institute also participated in symposia, workshops,. congresses elsewhere; discussions during the meetings were mainly focussed
on topics related to their discipline and of interest to the new programme(see travel reports).
j In 1980 scientists from western and eastern European countries and fromí developing countries visited the Institute for short or longer periods.
I Five s c i e n t i s t s , L.M.W. Oel laert, W.W.A. Bergers, F.H.G. Smeulders, J.W.G.M.1 Wilmer and G. Z u r l i n i , presented t h e i r Doctoral Theses on subjects studied] at the Institute.
\ As stated previously much of the scientific programme was carried out in| cooperation with scientific Organizations and Institutes in the Netherlands,I in otl.er countries of the European Community and elsewhere in the world.i Soťiite examples of this collaboration are:
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the application of nuclear energy in agriculture in working groups of theEuropean Society of Nuclear methods in Agriculture (ESNA);
cooperative studies on pollution, radioactive and other, mainly withInstitutes in the Netherlands, Belgium, France and Germany;
analysis of radiation effects on biological material (e.g. in the Europeanworking group for Microdosimetry);
standardization of absorbed dose and dose distribution measurements; 1980was the last year of collaboration within the European Late Effects ProjectGroup, EULEP;
studies on food irradiation, on radiation effects on living material with-in the frame of the Cultural Agreement of the Netherlands with severaleastern European Countries;
the project on food irradiation technology (IFFIT) with the InternationalAtomic Energy Agency (IAEA) at Vienna and the Food and AgriculturalOrganization (FAO) at Rome;
programmes of the joint (AEA/FAO Oivision on e.g. mutation breeding ofvegetatively propagated crops, radiation dosimetric aspects, fertilizerefficiency, food irradiation, genetic control of insect pest*;;
' contacts with the World Health Organization (Geneva), the RockefellerFoundation, Ross Institute, London and specialized Institutions in Pakistan,India, in relation to the working out of a programme on a genetic methodfor control of a malaria mosquito.
In 1980 we lost P. Poelstra, who was involved with the activities of theInstitute almost since the start. We really miss the personal attention ofa good friend as well as the knowledge and know-how of an excellentspecialist in the field of his discipline.
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A C T I V I T I E S OF GENERAL PUBLIC INTEREST
1. Cadmium Pollution
One of the heavy metals which, according to r isk evaluations, is extreme-ly dangerous is cadmium. This element accumulates qu i te well in s o i l s .Cadmium contamination may resul t from the appl icat ion of contaminatedsewage sludge and from the appl icat ion of phosphate f e r t i l i z e r s wi th are la t ive ly high cadmium content,
I t appeared that in a few speci f ic situations accumulation of cadmiumwould occur, even when cadmium levels in the supplied material are low,In the majority of soi ls the leaching of cadmium to the subsoil preventsa severe accumulation. Differences between so i ls are high. The use ofaverage data to calculate permissible levels or permissible supplies istherefore not permitted. I t would lead to too severe rest r ic t ions in onecase, and to unsuff ic ient res t r i c t ions in others.
2. Mutation breeding of Chrysanthemum
Ornamentals, such as bulbs, potplants, cut flowers and shrubs, are aneconomically very important group of hor t i cu l tu ra l plants in the Nether-lands. The production value in 1980, for instance, amounted to as much asOfl . 3,3 x 109 representing 37»7% of the total production value of h o r t i -cultural and agr icul tura l crops. Cut flowers especial ly contribute to thislarge production value, to the extent of D f l . 2,1 x 1CP with a few cropsplaying a dominant role. Within the ornamentals the chrysanthemum isextremely important with a to ta l sale of D f l . 187,6 x 106 (10Š of theto ta l : Table 1).
Table 1 - Annual turnover of the most important cut flower crops at thefour largest auctions in the Netherlands (1980).(Data taken from Vakbl. Bloem., 9 (1981): kG-kJ)
Roses D f l . 339 x 106
Al l Year Round chrysanthemum (spray types) - 187,6 x 106
Carnation - H l x 106
Freesia - 107 x 106
Gerbera - 104, *• x 106
'. Tulip 96,8 x 106
• Total at four auctions - 1878 x 106
J Total at a l l auctions - 2058 x 106
j I t is not surprising that breeders are very interested in the improvementi of such crops, in view of their economic interest,, One of the methods7 used is cross-breeding by which genotypes-are produced with importantf characteristics such as high yield and quality under poor l ight conditions\ and/or at lower temperatures (energy.1). As soon as outstanding cult ivars: are obtained mutation breeding is usually applied to induce the\ necessary flower colour "sports", without altering the combination of| favourable characteristics of the cultivar in question.
í One of the most successful and recent examples concerns the cultivar) Horim. This seedling cultivar, obtained in 1972, is an excellent grower\ and producer of cut flowers under our winter conditions but was not very| popular because of i ts unfavourable flower colour (unattractive white).
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Despite its excellent properties i t was about to disappear when a very palepink spontaneous mutant was found in 1975 which revived the interest in thecul ti var. This pinkish "sport" was the start of a very large mutationbreeding programme. Within a few years hundreds of flower colour mutantswere produced of which several were put on the market (Plate 1).
Plate 1 - Four radi ation-induced flower colour mutants of the chrysanthemumcultivar Horim. Upper le f t : Milonka (white); upper right: Mikrop(pink); lower left: Mi lava (yellow); lower right: Middelry (darkyellow). The flower colour of the original cultivar was very palepink.
The mutants were re-irradiated, not only producing other and improved flowercolours, but in addition mutant genotypes with a better quality cut flowers(sturdier and longer stems, larger inflorescences).
These mutants of cv. Horim and later of cv. Miros proved to be very popular(Fig. 1): within a few years they have superceeded the so-called spider-
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types, the previous leading group. In addition, several radiation-inducedmutants of other cult ivars (such as of cv. VJestland) play an importantrole.
7 0 r up to 1975 no detailsare available on thecontribution perc pgroup of cultivars
\m>idtr lyptf
•72 "73 75 •76 •77 •78 •79Fig, 1 Relative contribution to the total production of cut flowers from a few groups of all-year-round(AYR) chrysanthemum spray-type cultivars. Data taken from the Annual Reports of the CCWS-Veiling atHonselersdijk. Since 55-60% of the AYR chrysanthemum cut flowers produced in the Netherlands are soldnt this auction, the '.'gurcs are representative for total production in the Netherlands.
(Broertjes, C., Euphytica, 29 (1980): 525-530),.
3. Radiation Protection
Research has been carried out to investigate whether a synergistic in ter -action occurred when ionizing radiation and a chemical mutagen were givenin a combined treatment. Analysis of data in the l i te ra tu re had indicateda synergistic interaction for the case of cel l k i l l i n g at relat ively highradiation doses. The research in 1980 indicated that synergism also arosein the induction of somatic mutations in Tradescantia when EHS (ethylmethanesulfonate) was combined wi th rather low doses of X-rays.
A theoretical model for the ef fect of radiation on biological ce l ls ha~-been further developed. The model indicates that a good correlationexists between di f ferent endpoints such as cel l ki11 ing, mutations,chromosome aberrations and malignancy. The l inear-quadratic dose re la t ion-ship derived for these effects is based on the proposal that the DNAdouble strandbreak is the crucial radiation induced lesion which Causesradiation effects in man. The implications of the model including thosefor Radiological Protection are discussed in a book by Chadwick andLeenhouts en t i t l ed The Molecular Theory of Radiation Biology.
Research using African v io le t [Saintpaulia ionantha) has shown that instationary epidermis cel ls the repair of radiation damage can bestimulated by pretreatment of the cel ls with a small dose of radiationsome 8 - 2 4 hours before the main radiation dose. This repair is notcompletely error- f ree as the number of mutations induced by the radiationdoes not decrease in the same proportion as cel l survival inc eases.
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k. Courses
a *In 1980 ITAL decided to terminate its course activities. In 1958 theinstitute organized the f irst general radionuclide courses. Since thattime these have been held annually.
Since 1967 a liquid scintillation course was also organized, as thistechnique was extremely suitable for the quantitative determination ofsoft e-emitters such as 3H and ^ C , which are used on a large scale inbiological research. After the introduction of the Nuclear Energy Lawthe need arose to also organize a health physics course and since 1971*ITAL has given seven of these courses as a completion to the generalcourse.
Data concerning the numbers of participants and their origin have beenassembled In table 2.
I t appears from the table that 73% of the participants of the generalcourse originated'from agricultural institutions. The liquid scintillation-and the healt physics courses draw more participants from otherorganizations: for example about 30% of the participants came from medicalinstitutes. >
As well as these courses five special courses have also been organizedfor the benefit of a school of analysts.
b. JFFJT
One of the main tasks of IFFIT (the International Facility for FoodIrradiation Technology), an international project jointly established atthe Pilot Plant for Food Irradiation, Wageningen by the IAEA, the FAO andthe Ministry of Agriculture and Fisheries of the Netherlands, is to offertraining in the f ie ld of food irradiation for scientists from developingcountries. To achieve this aim, IFFIT organizes training courses andaccepts fellowship-holders for longer-term applied research training.
The second IFFIT Inter-Regional Training Course on Food Irradiation washeld from 1 September to 3 October 1980 at the Foundation ITAL and thePilot Plant for Food Irradiation. The five-week course consisted oflectures for 5*t hours, laboratory exercises for 50 hours, numerousdiscussions, country reports and f ield trips. Emphasis was placed onunderstanding the principles of food irradiation as a method for foodpreservation and of minimizing post-harvest losses. Present technologicaland economic feasibility as well as wholesomeness and legislative aspectswere pointed out. Practical training in food irradiation technology andtechniques, including1 dosimetry and operation of a pilot food irradiatorwas provided.
Ten full-time participants of the training course came from the followingcountries: Afghanistan, Egypt, Ghana, Indonesia, Kenya, Nigeria, ThePhilippines, Poland, Thai land and Zambia. Part-time participation wasprovided also for the IFFIT fellows from Chile and Ghana, who worked atthat time in IFFIT under longer-term applied research training. Thecourse programme was presented by eight invited foreign experts, fivespecialists from the Netherlands, fifteen staff members of ITAL and thePilot Plant for Food Irradiation, and the Project Director.
Table 2 ITAl-Rad i onuci i de courses
Nature of Course
General Radio-nuclide course(3-4 weeks)
LiquidScintillationCourse(2 weeks)
Health PhysicsCourse(2 weeks)
Total
Special courseof one week fora school ofanalysts
Internationalcourses of5-8 weeks
Number of
courses
28
23
7
58
5
5
participants
469
354
117
940
135
68
Percentage of the total number of participants, originatingfrom different institutions
ITAL
%
20
16
12
17
1
AgriculturalUniversi ty
31
14
11
22
Agriculturalinstitutes
atWage-ningen
%
12
6
3
9
1
outsideWage-n i ngen
%
10
8
7
9
Otherinstitutes
ml see l -1aneous
%
16
28
32
23
medical
%
11
29
34
21
98
oI
x
řIr.
řr
The trainees' reports as well as formal and informal discussions formedvery important parts of this training course. During these talks i tbecame clear that there are many common problems in the developingcountries regarding the application of food irradiation, which call forcooperative efforts between the participants, their institutions andIFFIT. The success of this second IFFIT course showed again that theIFFIT project, through training and assistance, can significantly furtherthe use of food irradiation technology and make developing countriesaware of the possibilities of this technique to lessen their energyproblems, and to bring about improvements in the supply of foods of desir-able shelf-l ife, hygienic quality and biological safety.
The third IFFIT training course on food irradiation wi l l be held from 12October to 20 November 1981 and a three-week Special Course on PublicHealth Aspects, Proper Application and Control of Food Irradiation isplanned for January 1982.In 1980, seven scientists: Mrs. I . Kramomtong (Thailand), Or. M. El-DinEl Fouly (Egypt), Mrs. V. Appiah (Ghana), Mr. G.T. Odamtten (Ghana),Mrs. W.T. Rubio-CabeUo (Chile), Dr. B.M. Shirzad (Afghanistan) and Mr.M.G.O. Nyambati (Kenya) have obtained applied research training at IFFITunder 6 - 15 month fellowship programmes. For this training the scientificguidance and laboratory faci l i t ies were provided by ITAL. The investigatedtopics included the sheIf-life extension of fermented cocoa-beans, maize,limes and grapes, and enhancing the juice yield of limes and grapes.Effect of irradiation on pathogenic microorganisms on chicken carcasses,and radiation decontamination of Chilean fish meal was also studied byone of the fellows.
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SUMMARY OF THE 1980 ANNUAL REPORT OF THE CONTAMINATION RESEARCH GROUP
INVOLVING PROJECTS 83, 84, 85, 86, 87, 88, 89, 90 and 91.
The study of various aspects of the effects of radiation on living cellsmaking use of plant cells and related material " • •, - ,
Haplopappuo graoilia call cultures we developed for cellular radiobiologicalstudies and cell survival and mutatloi were studied. A very pronounced re-pair of potentially ietna1 damage was found. In Saintpaulia icriontha the re-generation of epidermal cells was used to study a radiation stimulated repairof potentially lethal damage for X rays and neutrons. Analysis of literaturedata demonstrated a close correlation between survival, mutations and chro-mosomal aberrations.In Oenathera organenaie, mutation studies at the S-locus suggest that lowdoses or radiation induce heritable changes. . r
Dosimetry intercomparisons studies were made to enable coordination of re-search on late effects of radiation, especially on partial body irradiationof rats. Lyoluminescence studies demonstrated that mannose and glutamlne canbe used as suitable dosimeters for intermediate doses. The clear perspexdosimeter was investigated for its dependence of oxygen diffusion, temperatureand humidity during storage.
Behaviour of radiocontaminants in soils and vegetations of specific sites
A scheme to examine the possibilities for decontamination of agriculturalareas after severe nuclear accidents has been prepared. The study takesinto account both the short term pathway (grass -cow - milk - man) and thelong term pathway (soil - grass - cattle - milk/meat - man). Numerousmeasures are discussed with special attention being paid to their practic-ability. , _•;-
The possibility to fix 90Sr in soil with alginates was studied. The pers-pectives áre disappointing. ; V ; .
The migration of 90Sr, 1 3 7Cs, 2 3 8Pu and 2 3 9» 2 4 0Pu in soils was studied bylaboratory column elements and for 90Sr and 137Cs since 1957 by samplingpastures. Models to describe the migration were refined and tested. Resultsareisatisfactory. "
Soils and sediments of the river Rhine delta were analysed to determine apossible contamination of Pu and Am. Some slight enrichment could be ob-served; a soil of the forelands of the Rhine had a 239»2l>,0Pu content of500 mBq.kg"1 versus 300 mBq in a soil which was never flooded with Rhinew a t e r . . '"•. • ,;. • > - ' . ' , - - - " ' ' , - • • "• ' • • '' ''• '.',-
Tetren (tetraethylenepentamine) appeared to be a chemical ligand whichconsiderably decreases the availability of transition metals in claýcontaining soils. The roots cultivated in a 10~6 molar Cu solution contained20 times as much Cu as the roots of plants which were grown in a 10"6 molarCu-tetren solution, the Cu-tetren complex once taken up by the roots, isvery mobile within the plants. A drawback of using such synthetic ligandsi s that they do not react spec i f i cally wi th rad i oact i ve trans i t i on meta1 s i
Transfer coefficients and (re)di stri but ion of some radiocontaminants(corrosion products and transi tion metals) in plants have been determinedon nutrient solutions and two soils. The transfer coefficients are apparent-1 y f unct 1 onél 1 y dependent on the staiSl é í sotope concent rat i on and the pi ant
.|; age, but independent on the water uptake at the g iven concent rat ions. The
ii I
t
mobility of the rad iocontaminants is different, being for example very lowfor 65Zn, but fast for 60Co. The transfer of 65Zn from soils contaminatedin their top layer is rare due to limited depth migration.
In 1974 an unexpected phenomenon was observed in our laboratories. It seemedthat two isotopes of Cs, at very tow concentrations, behaved differently. A Hsamples of this experiment were stored and they were recounted during thiscontract period; the aberrant behaviour seemed consistent. All experimentsthereafter did not show, however, any different behaviour of the two Csisotopes. . .... .-• , ';_ _ :, •• -, • : ,',,'. , .. ': * -.
The possibility that Cd, originating from phosphate fertilizers, accumulatesin the soil,was studied. The accumulation depends mainly on the distributionof Cd over the soil solid phase and soil solution phase; this distributiondepends strongly on the pH. With a precipitation surplus of 1000 cm (re-presenting 25 to 50 years) only a small percentage of the Cd supplied, willaccumulate in the top cm of a soil with a pH of 4,5• at a pH of J tííispercentage increases to 50. This means that general recommendations forpermissible Cd supplies to soils and/or Cd levels in soils cannot be basedon average values of Cd In soils and crops. The use of average values willinduce high risks in particular areas, and at the same time induceexcessive restrictions in other areas. . ,
L
PROJECT No. 83 ANNUAL REPORT 1980
K.H. Chadwick, H.P. Leenhouts, P.A.Th.J. Werry, K. Sree Ramulu
A STUDY OF DOSE EFFECT RESPONSE AND THE INTER-RELATIONSHIP BETWEEN CELLSURVIVAL, MUTATION AND CHROMOSOMAL ABERRATION IN PLANT CELLS
\. Haplopappua gygoilia (Nutt.) Gray cell cultures
A relating efficiency of 80% was obtained for single cells of Hezplo-pappus gvaoilis by using a replating technique in which small aggre-gates of cells were replated on top of a feeder layer. The smallaggregates were grown from single cells plated at high density in softagar on top of a feeder layer. This technique was used for survivalstudies,A different technique was used for mutation studies when much largernumbers of cells needed to be treated. In this case 2 ml of cellsuspension culture in stationary phase was plated on top of B-5 agarcontaining the selective agent cycloheximide.
B. Survivalstud|es
a) .Acute_
Measurements of cell survival have been made using exponentiallygrowing cells and stationary phase cells using X-rays and fissionneutrons.
- Fission neutrons are more effective than X-rays and an RBE valueof 3 to 5 is found, in accordance with data reported for mammaliance11 s.
- Cells in stationary phase are more sensitive to both X-rays andI fission neutrons than cells in exponential growth.
• - The plating efficiency (PE) of cells irradiated with doses of lessthan k Gy (400 rad) of X-rays exceeded that of the control cells.
- This "stimulated" PE was ascribed to an increase in cell elongation ,; possibly caused by radiation induced endogenous cytokinine'} production. The increased cell growth improved the cells chance to: form small aggregates in the first phase of the plating process.
- The "stimulated" PE was found to be dose rate dependent for X-rays,• extending to a higher dose at lower dose rate. Acute neutrons did
i- 'I not give an observable "stimulation" but it was found for low dose- ,I • rate neutrons. \
i i b) De_layjet[ j>ljatjji£ ̂nd_ decrease^ dosej-ate :
' f The repair of both potentially lethal damage (PLD) and sub-lethal I• damage (SLD) was observed in the in vitro cultivated cells. |
' \! - Repair of PLD after X-irradiation is completed after a delay of\ plating of 12 hours under normal conditions; at low temperatureI the repair process is retarded.I - Repair of .PLD after irradiation with fission neutrons follows a
. I b i-phasic pattern with respect to the reaction rate of the repairÍ f process and with respect to the sensitivity to low temperature.
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- The stimulation effect at low dose of X-rays is not manifest whenthe cells - after the irradiation - are kept in condition thatallows full repair of PUD.
Mutation studies in the Haptopappus cells have been initiated makinguse of the induction of resistance to the antibiotic cycloheximide,
a) Jheorea i£aj_ bas i£ £
Cycloheximide is a potent inhibitor of protein synthesis: it pre-vents elongation of the peptide chain "growing" at the ribosomeby competitively binding to the ribosome - based on a change in theONA coding for the ribosomal structure - prevents such binding andrenders the cell resistant to a concentration of cycloheximide,that would normally kill the cell by protein deprivation.
b) De$ejsffeet £eja_t i£n£hj_p__a£te_r_i£radj_a_ti£n_wj_th_ J<-jraxs_arjd_fj_sjij£nneutToňs ~"""The cell killing effect and the mutation induction effect are bothcorrelated to the applied dose in a strictly similar way. Fissionneutrons are more effective in cell killing and mutation inductionthan X-rays, however, the net yield of mutants at the optimum dosefor mutation induction is similar for both types of radiation. Theoptimum dose for net mutation induction is 10 Gy (1000 rad) for X-rays (3.1 Gy-min"1 (310 raďmin" 1)) and 5 Gy (500 rad) for fissionneutrons (1 Gymin" 1 (100 rad.min"1)).
c ) X n e. EfÍFSt-PĹ É?LaX.eŔ E'jL'J/íä LoHowijT£ ,X-_ray_sWhen the plating of the cells is delayed such that PLD repair canbe completed, it appears that concomitantly with PLD repair, themutation frequency decreases. However, since PL0 repair is morepronounced than the decrease in mutation frequency, the net in-crease of mutants as a result of delayed plating is observed.
2. Saintpaulia
A technique was developed to quantify the effect of radiation on thestationary (Go) epidermal cells at the base of several leaves. Using thistechnique the effect of radiation on the regeneration of these cells wasfound to be similar to cell survival. A dose rate effect was measured andin a fractionation experiment the repair of sub-lethal damage was foundto be efficient, a first order process with time having a half-life of 2hours.Very little repair of potentially lethal damage was detected unless theleaves were pretreated with a small radiation dose 2k hours prior to achallenging dose. In this case the stimulated repair process enabled thecells to repair a considerable amount of potentially lethal damage. Thedevelopment of the repair process was time dependent and was optimal at12 - 2k hours after the pretreatment dose. The repair process was de-pendent on the size of the pretreatment dose and could be induced by bothX-rays and fission neutrons. Analysis indicated that the repair processwas induced in a single-hit process and had a neutron RBE of unity. Itwas shown that with delayed cultivation of the leaves after a single ir-radiation the repair process can develop and eventually repair somepotentially lethal damage induced by the irradiation. This process is moreefficient at low doses of X-rays and leads to a change in the shape of theX-ray dose response curve for regeneration which develops a flat shoulderat low doses. Very little effect on the fission neutron response curve is
•' mi •
- 16 -
seen and this stimulated repair process combined with delayed platingresults in an increase in the low dose RBE for neutrons from 10 forimmediate cultivation to 35 for delayed cultivation. This increase in RBEis solely due in this case to a decrease in the effectiveness of the X-rays,The induction by a low pretreatment dose of a "protective" effect in theepidermal cells correlates very closely with the effect found at theplantlet level in Saintpaulia previously. The plantlets are apparentlyderived from single regenerating epidermal cells and the results indicatethat the effect found at the plantlet (organism) level can be ascribed toan effect occurring at the cellular level; it is not due to a cellcompetition or tissue organizational effect. The facts that plantlets arederived from single epidermal cells and that they can be grown to fullplants and scored for mutations made it possible to study the effect ofthe simulated repair process on the frequency of mutations. The resultsshowed that the pretreated leaves gave a lower mutation frequency thanthe untreated leaves at each dose indicating that the repair ofpotentially lethal damage led to a reduction in the mutation frequency.However, the reduction factor for the mutation frequency was smaller thanthe increase in cell survival (regeneration) induced by the pretreatmentand this indicates that whilst part of the repair of potentially lethaldamage might be error-free, part of the repair is not error-free andstill leads to mutations.
3. Analysis
In the model developed over the past eight years it is assumed thatradiation induced DNA double strand breaks (dsb) are the crucial lesionsresponsible for biological effects such as cell reproductive death,chromosome aberrations and mutations. This assumption implies that inexperiments where different biological end-points are determined fromthe same population of treated cells direct correlations between thedifferent end-points should exist. The model predicts quantitativecorrelations between the end-points as follows:
if the number of double strand breaks (N) induced by a dose (D) ofradiation is given by
N = ctD + BD 2 (1)
and p is the probability that a dsb leads to cell killing;q is the probability that a dsb leads to a specific mutation;c is the probability that a dsb leads to a scorabie chromosomal
aberration;then cell survival S = expl-pN] (2)
mutation frequency M = 1 - exp [-qN] s qN (3)and aberration yield Y = cN (i»)N, the number of crucial lesions, is common to the equations 2, 3and k and elimination of N between these equations leads to thecorrelative equations:
In S =-•
In S -ť(5)
(6)
(7)
(8)
í
\
•\í.
- 17 -
Figuře 3-1 illustrates the various straight-line correlations betweenthe different end-points which are predicted by equations 5» 6» 7 and8 for data taken from a small sample of suitable experiments publishedin the 1iterature.
These correlations also mean that comparable effects of dose-rate,fractionation, radiation quality and sensitizers and protectors willbe found for these different end-points. At low doses of radiation,where the linear (a) term in dose dominates the dose responses, thelimiting relative biological effectiveness (RBEO) will be the samefor different end-points measured in the same cell population. ThisRBEO is relevant for the quality factor used in radiologicalprotection.
The model has also been developed to provide a qualitative analysisof the synergistic interaction between ionizing radiation and otherDMA damaging agents. The analysis of data on the interaction of UVand X-rays, and an alkylating nitrosourea compound (BCNU) and X-rayshas shown that the experimental results are compatible with thesynergistic interaction model. The quantitative analysis predictsthat the interaction between a DNA damaging agsnt and radiation leadsto an increase in the linear component of the radiation responsewhich is significant at low doses.
Genetic effects of the very low doses of X-rays and fast neutrons
on the S-locus of Oenotheva organensis Munz.
The analyses on the genetic effects after treatment of pollen mothercells with low doses (0,025 - 0,2 Gy (2,5 - 20 rad) of fast neutronsand X-rays in clone 111-4/55 of Oenotheva organensis Munz. werefurther continued. The results showed that fast neutrons and X-raysproduced significantly higher frequencies of seeds and seedlingsthan those obtained spontaneously (table 1). The genetic analysis of79 progeny plants revealed that the low doses of fast neutrons andX-rays induced both permanent mutations (pollen-part) and reversiblemutations at the S locus, while spontaneously only reversiblemutations occurred (inspite of screening approx. 25 million pollengrains), the frequency being significantly lower than that inducedby fast neutrons or X-rays. Among the 50 progeny plants derived fromthe treated series, so far analysed, kl showed reversible mutationsand 3 permanent mutations. Thus, the present data suggest that thelow doses of radiations induce heritable changes.
The implications of these results for Radiological Protectionare:
1. the repair of sublethal damage is a different process thanthe repair of potentially lethal damage;
2. one common type of lesion seems to be responsible for differentbiological end-points;
3. in general a comparable effect of dose rate, fractionation,radiation quality, sensitizers and protectors can be anticipatedfor different biological end-points;
k. the limiting RBE at low doses will be the same for differentend points in the same cell population;
- 1 8 -
5. in testing cells radiation can stimulate a repair process whichis not entirely error-free;
6. the stimulated repair can affect the low dose RBE value;
7. synergism between a chemical mutagen and radiation can lead toan increase in the linear dose component which is effective atlow doses;
8. very low doses of X-rays and neutrons can induce permanentinheritable mutations,
Publications
CHADWICK, K.H, and H.P, LEENHOUTS. The correlation between mutationfrequency and cell survival following different mutageni c treatments.Theor. and Appl. Genetics J»7 (1976): 5-8.CHADWICK, K.H., H.P. LEENHOUTS, I. SZUMIEL and A.H.W. NIAS. An analysisof the interaction of a platinum complex and radiation with CHO cellsusing the molecular theory of cell survival. Int. J. Rad I at. Biol. .30.(1976): 511-524.LEENHOUTS, H.P. and K.H. CHADWICK. An analysis of rad i at í on-induceddamage in the spider mite. Biological and environmental effects of low-level radiation. IAEA-SM-202/202 (1976): 53"65.LEENHOUTS, H.P. and K.H. CHADWICK. Stopping power and the radiobiologicaleffect of electrons, gamma rays and ions. Fifth Symp. on Microdosimetry,
j- Verbania Pallanza, Sept. 22-26 (1975), EUR 5452 d-e-f (1976): 289-310.
\ SPANJERS W.A., F.M. ENGELS, P.A.Th.J. WERRY, K.H. CHADWICK and H.P.LEENHGUTS. New evidence on chromosome joining after radiation. Fifth Symp.
i- on Microdosimetry, Verbania Pallanza, Sept. 22-26, 1975 EUR 5452 d-e-fi (1976): 545-556.ŕ CHADWICK, K.H. and H.P. LEENHOUTS. The rejoining of DNA double strandbreaksIr and a mode) for the formation of chromosomal rearrangements. Int. J. Rad i at.J Biol. 21 (1978): 517-529.; CHADWICK, K.H. and H.P. LEENHOUTS. The implications for radiologicalJ protection on the synergistic interaction between radiation and other DNAÍ damaging agents. 6th Symposium on Microdosimetry (1978): 1123-1136, EUR 6064.
j LEENHOUTS, H.P. and K.H. CHADWICK. An analysis of radiation induced malignancy| *jased on somatic mutation. Int. J. Radiat. Biol. JJ3. (1978): 357*370.
s LEENHOUTS, H.P. and K.H. CHADWICK. An analysis of synergistic sensitizationI Brit. J. Cancer 27 Suppl. Ill (1978): 198-201.| LEENHOUTS, H.P., K.H. CHADWICK and M.J. SIJSMA. The influence of radiationI stimulated repair processes on the shape of dose-effect curves. 6thI Symposium on Microdosimetry (1978): 1011-1022, EUR 6064.
[ LEENHOUTS, H.P. and K.H. CHADWICK. The crucial role of DNA double-strandI breaks in cellular radiobiological effects. Advances in Radiation Biologyí 7 (1978) - Academic Press Inc. (1978): 55-101.
| LEENHOUTS, H.P., K.H. CHADWICK and D.F. DEEN. An analysis of the inter-| action between two nitrosourea compounds and X-radiation in rat brain
tumour cells. Int. J. Radiat. Biol. ̂ 7 (1980): 169-181.
"' rn • • ľ" r r n ~ " ri
i
- 19 -
CHAOWICK, K.H. and H.P. LEENHOUTS. A molecular model for the synergisticinteraction between cytotoxic chemicals and radiation. In; RadiationBiology and Chemistry; Research Developments (eds. H.E, Edwards et at.)(Elsevier, Amsterdam) pp. 333-331* (1979).LEENHOUTS, H.P., M.J. SIJSMA, M. UTWINISZYN, C. BROERTJES and K.H. CHADWICK.Radiation stimulated repair in Saintpaulia cells in vivo. In: RadiationBiology and Chemistry: Research Developments (eds. H.E. Edwards et al.)(Elsevier, Amsterdam) pp. 227-236 (1979).LEENHOUTS, H.P. and K.H, CHADWICK. The need for an integral approach tothe protection of man against ionizing radiation and other mutagens.Topical Seminar in the practical implications of the ICRP recommendations(1977). Vienna, IAEA (1979) pp. Wl5kM
LEENHOUTS, H.P. Beschouwingen over analyses van de doodsoorzaak vanradiologische werkers te Hanford, Nederlandse Vereniging voor Stralings-hygiěne, NVS Nieuws, 5. (1) (1980): 5-13.
CHADWICK, K.H. and H.P. LEENHOUTS. Dose effect relationship for malignancyin cells with different genetic characteristics. Late Biological Effectsof Ionizing Radiations (1978), pp. 327-329 (IAEA, Vienna;.
LEENHOUTS, H.P. and K.H. CHADWICK. The interation of chemical mutagensand radiation in the induction of malignancy. Late Biological Effectsof Ionizing Radiations (1978), pp. 409-^22 (IAEA, Vienna).
ENGELS, F.M., F.M. VAN DER LAAN, H.P. LEENHOUTS and K.H. CHADWICK.The regeneration of epidermal cells of Saintpaulia leaves as a new plant-tissue system for cellular radiation biology. Int. J. Radiat. Biol. 3̂5(1980): 309-321.
LEENHOUTS, H.P. and K.H. CHADWICK. An analytical approach to the Inductionof Trans Iocat ions in the Spermatogonia of the Mouse (submitted to MutationRes.) (1980).
CHADWICK, K.H. and H.P. LEENHOUTS. Interplay between radiation and biologicalparameters in the induction of malignancy in animals. Seventh Symposium onMicrodosimetry. Oxford (1980) (in press).
WERRY, P.A.Th.J., F.M. ENGELS, A.W. SPANJERS. Rad i at i on-induced telomere-involved chromosome rearrangements in Haplopappus graailis. ITAL, externalreport 57 (1977).
WERRY, P.A.Th.J., K. STOFFELSEN, F.M. ENGELS, F. VAN DER LAAN, A.W. SPANJERS.The relative arrangements of chromosomes in mitotic interphase andmetaphase in Haplopappus graailis. Chromosoma £2 (1977): 93-101.
WERRY, P.A.Th.J., K.M. STOFFELSEN. Factors affecting the colony formingability of free cells of Haplopappus graailis (Nutt.) Gray. Acta Horticultura78 (1977): 3 M 4 8
WERRY, P.A.Th.J., K.M. STOFFELSEN. Conditions for a high plating efficiencyof free cell suspensions of Haplopappus gvaoilis (Nutt.) Gray. Theor. Appl.Genet. ̂ (1978): 161-167.
WERRY, P.A.Th.J., K.M. STOFFELSEN. The effect of ionizing radiation onsurvival of free plant cells cultivated in suspension cultures. Int.J.Radiat. Biol._35 (1979): 293-298.
WERRY, P.A.Th.J., K.M. STOFFELSEN. Theoretical and practical aspects ofradiation induced mutagenesis in plant cells In: Plant Cell Cultures:Results and Perspectives. F. Sala, B. Parisi, R. Cella and 0. Ciferi eds.Elsevier, Amsterdam (I980): 115-120.
- 20 -
WERRY, P.A.Th.J. Radiation induced mutagenesis in in vitro cultivated plantcells. Radiat. Environ. Biophys- YT. (1980): 292.
SREE RAMULU, K,, HANNELORE SCHIBILLA and P. DUKHUIS, Self-incompatibilitysystem of Oenothera organensis for the detection of genetic effects at lowradiation doses. NIEHS/EPA conf. on Pollen Systems to detect biologicalactivity of environmental pollutants, Knoxville, TN, May 5-8 (1980).Environmental Health Perspectives (suppl.) (in press).LecturesCHADWICK, K.H, An analysis of the sensitizing action of cytotoxic chemicalson the cellular radiation effect. Univ. of Lexington. (1978).
CHAOWICK, K.H. An analysis of the sensitizing action of cytotoxic chemicalson the c.llular radiation effect. Penn. State University, University Park.(1978).CHADWICK, K.H. An analysis of the sensitizing action of cytotoxic chemicalson the cellular radiation effect. Yale University, New Haven. (1978).
CHADWICK, K.H. An analysis of the sensitizing action of cytotoxic chemicalson the cellular radiation effect. York University, Toronto. (1978).CHADWICK, K.H. An analysis of the sensitizing action of cytotocic chemicalson the cellular radiation effect. Univ. of Pennsylvania, Philadelphia. (1978).
LEENHOUTS, H.P. and K.H. CHADWICK., A molecular model for the synergisticinteraction of ionizing radiation with chemical mutagens and its implicationsfor radiation therapy and radiological protection. ľ»th Meeting of EuropeanSociety of Radiation Biology. Jill ich. (1978).LEENHOUTS, H.P., M.M. LITWINISZYM and M.J. SIJSMA. A radiation stimulatedrepair process in Saintpaulia cells in vivo for X-rays and fast neutrons.141h Meeting of European Society of Radiation Biology. Jill ich. (1978).
LEENHOUTS, H.P. A molecular model for the synergistic interaction of chemicalmutagens and radiation. Inst. of Nuclear Research. Warsaw. (1978).
LEENHOUTS, H.P. A molecular model for the synergistic interaction of chemicalmutagens and radiation. Inst. of Nuclear Physics, Krakow. (1978).LEENHOUTS, H.P. Radiation stimulated repair in Saintpaulia epidermal cellsin vivo. inst. of Nuclear Physics. Krakow. (1978).
LEENHOUTS, H.P. Biophysical aspects of dose effect relationships in radio-biology. Autumn School, Polish Ass. Radiat. Research. Kowary. (1978).
LEENHOUTS, H.P., M.J. SIJSMA, M. LITWINISZYN, C. BROERTJES, K.H. CHADWICK.\ Radiation stimulated repair in Saintpaulia cells in vivo. NederlandseÍ Society for Radiobiology. 20th Jan. 1979.
[ CHADWICK, K.H. and H.P. LEENHOUTS. The synergistic interaction betweenI ionizing radiation and other mutagenic agents. Nederlandse Society for\ Radiobiology, 20th Jan. 1979.I CHADWICK, K.K. and H.K LEENHOUTS. Synergism between radiation and chemicalI mutagens and the implications for chemical carcinogenesis presented at IAEA[• Advisory Group Meeting on Comparative Health impacts of Nuclear and Alter-i native Energy Sources. Trieste, May 1979.
I LEENHOUTS, H.P. The effectiveness of X-rays and fast neutrons for thei stimulation of a potentially lethal damage repair in Saintpaulia epidermisI cells. Microdosimetry Contractors Meeting, EEC. April Dundee, Scotland.
- 21 -
CHADWICK, K.H. and H.P. LEENHOUTS. Sensitizers and fractionated radiationregimes. Meeting of French, Dutch and Belgian Radiobiological Societies.Brussels, October 1979-
LEENHOUTS, H.P. and K.H. CHADWICK. The importance of physical and chemicalfactors on the effectiveness of different types of ionizing radiation.Meeting of French, Dutch and Belgian Radiogiological Societies. Brussels,October 1979-
CHADWICK, K.H. and H.P. LEENHOUTS. The correlation between differentradiation induced biological effects. lnst» Nuclear Physics. Krakow,June 1980,
CHADWICK, K.H. and H.P. LEENHOUTS. The correlation between different radiationinduced biological effects. 15th Meeting European Soc. Rad i at. BiologyRotterdam. Aug. 1980.
LEENHOUTS, H.P. and K.H. CHADWICK. An analytical approach to radiation inducedtranslocations in mouse spermatogonia. 15th Meeting European Soc. Radiat.Biology. Rotterdam, Aug. 1980.
STOFFELSEN, K.M. en P,.A.Th.J. WERRY. Hoge plating efficiency bij lageplating density van celsuspensie cultures van Haplopappus gvacilis.Voordracht Werkgemeenschap plantecel- en Weekselkweek. Wageningen (1977) -
WERRY, P.A.Th.J. and K.M. STOFFELSEN. Factors affecting the colony formingability of free cells of Haplopappus graailis (Nutt.) Gray. InternationalSymposium Tissue Culture for Horticultural purposes. Gent. September 1977.
WERRY, P.A.Th.J. and K.M. STOFFELSEN. Increased colony forming ability offree plant cells following low dose irradiation. 13th Annual meeting E.S.R.B.Luik, October 1977.
STOFFELSEN, K.M. en P.A.Th.J. WERRY. Suspensie cultures van Haplopappusgraoilis voor radibbiologisch onderzoek. 11e Weefselkweekdag, Wageningen.Febr. 1978.
WERRY, P.A.Th.J- and K.M. STOFFELSEN. Effect of ionizmg radiation onHaplopappus graoilis cells grown in vitro. Poster IVth IAPTC ConferenceCalgary, Canada, 12-25 August 1978.
WERRY, P.A.Th.J. and K.M. STOFFELSEN. Effect of dose rate and delayedplating on the response of free plant cells to ionizing radiation.XlVth Annual Meeting of the European Society of Radiation Biology. Julich,October 9-12, 1978.
WERRY, P.A.Th.J. and K.M. STOFFELSEN. Theoretical and practical aspects ofradiation induced mutagenesis in plant cells. Workshop on Plant Cell Cultures:Results and Perspectives. Pavia, 1-3 August 1979.
WERRY, P.A.Th.J. Kwantitatieve aspecten van mutatie-inductie in plantecel-suspensies door ioniserende straling. 13e Weefselkweekdag, Leiden-Wageningen,23 April 1980.
WERRY, P.A.Th.J. Radiation induced mutagenesis in iri vitro cultivated plantcells. XVth Annual Meeting of the European Society for Radiation Biology.Erasmus University, Rotterdam. (1980).
SREE RAMULU, K. The self-incompatibility system of Oenothera organensis forthe detection of genetic effects at low radiation doses, NIEHS/EPA conf. onpollen systems, Knoxville, U.S.A.
Table 1 - Frequencies of capsules and seeds after crosses between unirradiated S3S4 plants, used as femaleparents and irradiated and control S3S4 plants in Oenothera ovganenais Munz.
Irradiationtreatments
ControlFast neutronsGy rad
0,025 2,5
0,05 50,1 10X-raysGy rad
0,025 2,50,05 5
0,1 10
0,2 20
Flowerspol 1inated
5380
1081
1056
889
787869824768
CapsulesWith seeds
No.
54
513626
23
36
29
31
No. per 100pol Iinations
1,00
4,72
3,41
2,92
2,92
4,14
3,52
4,04
With viable seed
No.
23
171511
1No. per 100pol 1ination
0,43
1,571,421,24
SeedsNo.
147
60
37
33
32474460
Frequency
Perpo11 i na t i on
0,(ň7
0,055
0,035
0,037
0,041
0,054
0,053
0,065
Permi 11 ionpollengrains*
13,978,959,50
9,0912,0511,9214,53
Viable seedsNo.
1
44
171511
Frequency
Perpollination
0,008
0,016
0,014
0,012
Permi 11 ionpollengrains
2,02
3,96
3,63
3,17
rhe mean no. of pollen grains per pollination was 4483 ± 121
N>
I
23 -
1
0.1
oJ| a o i
0.00
vv*
r -
Chinese
g ,
D • •• •
\\
1 r-
hamster cells
X-rayí
x-Z*̂ «
\
\
1
• -
1 '
1 2 3 4 5 6chromosomal aberrations/ceU
S•e(U
3Q1
001
y •DO- C0-6CI-rays O«Gv/min
v - 7.6MrV(WUtions
o - 14.7M>V nvutroni
c)Jo.i i
dictntrics/celi
1.0
0.1
o Chinese homster cells
• Human diploid cells
b)2 4 6 8
mutations/survivorK)
G04 0.08 0.12 0.16pink mutations/hair
Fig. 3-1 - The correlations between different biological end points aspredicted by equations 5, 6, 7 and 8.
a) - £ . Y , b) in S - - -J -M, e) -i -Y2, d) M, = -1 -M2.
PROJECT No. 8i»
K.J. Puite and K.H. Chadwick
ANNUAL REPORT 1980
DEVELOPMENT AND IMPROVEMENT IN ACCURACY AND REPRODUCIB ILITY OF DOS I METRYSYSTEMS FOR X-RAY INTERCOMPARISON, NEUTRON AND HIGH LEVEL GAMMA IRRADIATION
1, Dosimetry intercomparison for evaluation of late effects of ionizingradiation
A prerequisite for coordination of research programmes to study thelate somatic effects of ionizing radiation in mammalian organisms isthe standardization of experimental methods, such as the dosimetry.Investigations in radiation biology and radiation therapy havedemonstrated that differences of 10 per cent in absorbed dose willproduce clearly observable variations in biologic response. In general,cell survival analyses do not allow the prediction of variations inabsorbed dose determinations smaller than 5 per cent. It has beensuggested therefore that an accuracy of 5 to 6 per cent and a precisionof 2 to 3 per cent is required for the determination of absorbed dosein biologic applications.
For the institutes cooperating within the European Late Effects ProjectGroup (EULEP) intercompariscns of doses and dose distributions havebeen performed in 1971, 1973, 1976 and 1930. These studies haveresulted in improvements with regard to the accuracy and precision ofthe X-ray dosimetry at these EULEP laboratories. The third and fourthintercompari son was considered to be essential for a periodical check onthe dosimetry procedures and for the benefit of new groups joiningEULEP in the intervening years. The intercompari sons were carried outusing mailed thermoluminescent (TL) dosemeters.
The fourth dose intercomparison was organized in close cooperationwith the National Institute of Public Health at Bilthoven. The dosemeterswere evaluated both at this institute and at ITAL. Future work on behalfof the EULEP programme will be carried out by the Institute or PublicHealth.
Late effects caused by the irradiation of specific organs, e.g. lungand liver, are also studied in the EULEP research programme. For thesestudies col limated X-ray beams have to be employed, since it isnecessary to restrict the dose to other essential organs in the animal.In connection with these programmes it was decided to initiate anintercomparison of the X-ray dose and dose distribution for partialbody irradiation in 1978. The exposures at the participating institutehad to be performed under conditions similar to those actually employedfor rat partial body irradiations. The dose in the irradiated organs aswell as the scattered doses in the phantoms have been measured andcompared with the dose values from a standardization laboratory. Onthe basis of these partial body X-ray dosimetry studies (Fig. 1) anumber of conclusions can be drawn:(1) Dual capsules with LI F and CaF2'.Mn TL powder appear to be well
suited for this type of dosimetry with X-ray beams having an HVLabove 1,3 mm Cu. However, one should be aware of the disturbancecaused by the capsules in the lung and liver equivalent material.
- 25 -
(2) At an FSD of 50 cm large dose gradients are present in case ofunilateral irradiations and minimal backscatter conditions: forexample 23% for an HVL of 1,5 mm Cu in the lung substitutematerial and 51$ in the liver substitute material. Full back-scatter geometry reduces ihe dose gradients in the lung to 18%,but hardly influences the gradient in the liver. One shouldrealize that the dose variation over the lung and liver (3 cmdepth) will be equal to 12% due to the inverse square law alone.
(3) The scattered doses in the centra] part of the phantom are of theorder of 1 to 5% of the dose in the primary beam, depending onthe scatter material (lung or liver material) and the presenceof backscatter material. The scattered dose in the distal partof the phantom amounts to 0,1 and 0,5% of the dose in the direct-ly irradiated part,
(h) The main objective of this study was to obtain information on theaccuracy of X-ray dosimetry and the adequacy of exposure arrange-ments for partial-body irradiations of rats. The doses deliveredby the participants in the two sessions showed good reproducibi1 i ty;however, large discrepancies were observed (up to 36% from thestandard value). In a number of cases the sources of error wererather elementary, and it is to be expected that the results ofthis intercomparison will inspire the participants to performbetter and more careful measurements. Four of the eight institutesgenerally perform partial body irradiations of rats by unilateralexposure. The large dose gradients (up to 68%) are unacceptablefor radiobiological studies. Consequently for these institutesthe introduction of a bilateral exposure arrangement is stronglyrecommended,
2. Lyoluminescence dosimetry
- The lyoluminescence (LL) dosimetry technique is based on the pro-duction by radiation of free radicals in organic, closely tissue-equivalent, materials. Upon dissolution of these materials inwater light emission can be recorded due to excited states of re-action products. This light signal is a measure of the absorbed dosein the material. Due to the low material costs, simple equipment,extended dose range and tissue equivalency, use of the LL techniquein the field of radiobiology, radiotherapy, fast neutron dosimetryand high dose level dosimetry offers distinct advantages over systemsin current use.
- A luminescence measuring system has been developed at ITAL which hasa closed water circuit under gas pressure to carefully control theexperimental conditions (Fig. 2). The monosaccharide mannose andthe ami no acid glutaniine have been investigated as potential LLdosemeters.
- Mannose can be used in the dose range of 0,1 Gy up to 500 Gy(10 rad - 50 krad) in photon and fast neutron beams. The variationof the dose values above 3 Gy (300 rad) is 3 to k%. The relativeeffectiveness of neutrons ranges from Oťik (fission neutrons) to
í 0.71* (15 HeV neutrons).
- 26 -
- Glutamine can in principle be used in the dose range of 0,01 to100 kGy (1 - 10000 krad). The glutamine system at ITAL has beentested for doses of 0,1 to 25 kGy (10 - 2500 krad). The variationin the dose determination is less than $%. Therefore this dosi-metry system seems to be very valuable for high-dose standardizationand intercomparison for industrial radiation processing. The IAEA(International Atomic Energy Agency) has initiated research forthe development of a dosemeter suitable for high dose dosimetry.During a dose intercomparison (dose range 0,01 to 3 kGy (1 - 300krad) organized by the IAEA in 1980 the use of mailed LL dose-meters has also been tested. The results showed that the largestdeviation of the dose values obtained with the ITAL glutaminedosemeters from the nominal doses was 3% for doses above 0,1 kGy(10 krad). In 1981 the lyoluminescence system will be tested ina dose intercomparison under radiation processing plant conditionsusing a dose range of 0,01 to 100 kGy (1 - 10000 krad).
- After 1930 no lyoluminescence dosimetry research will be performedat ITAL. The state of art of LL dosimetry will be described in ajoint review article together with Dr. K.V. Ettinger of theUniversity of Aberdeen. Although no LL dosimetry readers areavailable commercially it is possible to use some existing com-mercial apparatus after simple modifications e.g. the LKB Lumino-meter 1250 and the Packard Pico-Lite Luminometer.
3. Clear Perspex Dosimetry
The influence of two environmental factors, temperature and humidity,on the stability, reproducibi)i ty and accuracy of the clear perspexdosemeter system have been studied. The results can be summarizedbriefly:
- oxygen diffusion which causes a fading of the radiation inducedabsorbance A (optical density, OD) at all wavelengths istemperature dependent. It proceeds more rapidly at higher temperatures.The speed of oxygen diffusion is also dependent on the batch ofperspex;
- when the diffusion of oxygen is prevented by treatment in vacuumthe conversion of the induced free radical species Ri to thesecondary free radical species R2 can be studied. Rj and R2 havedifferent optical absorption spectra and the R1 to R2 conversionalters the optical density spectrum, in general low wavelengthsincrease and higher wavelengths decrease. It has been found thatthe R\ to R2 conversion is also temperature dependent and proceedsmore rapidly at higher temperatures. At kS °C under vacuum thecross-over wavelength caused by the spectral change is not constantbut is dose dependent, under these conditions of storage it isnot possible to select one wavelength for stable OD measurementand accurate dose estimate;
- on storage in air at different temperatures a reasonably stablecross-over wavelength has been found for at least 2^ h afterradiation at 35 °C. Dose estimates at this wavelength can be madewith 3 - 5 ? reproducibi1 i ty at 99% confidence limits and an ab-solute accuracy of less than 5% should be achievable even whenirradiation takes place at k$ °C. This is not true for 305 nmmeasurements made Ik h after radiation, the OD at this wavelengthat 2k h is very temperature dependent and errors of 10% caneasily be made;
X"••«"? • ' .
i-M'-síaHiaWMfUWiWíWjSMiM^-iiiKifti.jVuBŕJiĽkS^ií.Jiŕ.--.-'-.1-----'-.
• A - •
- 27 -
- irradiation at kS °C for 16 h to a total dose of 35 kGy (3,5 Mrad)indicated no oxygen diffusion induced fading during irradiationand the OD was the same as 35 kGy (3,5 Mrad) given at 20 °C in3 h when measured at 31*1 nm. This indicates that the OD-doserelationship remains constant for irradiation temperatures upto kS °C and irradiation times of less than 20 h when measure-ments are made at 314 nm;
- the newer batch 5 of the HX dosimetry perspex is thicker (1,75 mm),has a slower O7 diffusion induced fading and demonstrates no buildup of OD at 305 nm after a short irradiation at room temperature.Irradiation at 45 °C for 10 h or 20 °C for 3 h to the same dosegave the same OD at 314 nm but a different OD at 305 nm. This meansthat although the OD at 305 nm is more stable after irradiationprolonged irradiation at higher temperatures can lead to errorsif dose estimates are made at 305 nm. At 314 nm the OD remainedconstant for 9 days at 45 °C under vacuum. The new batch 5 HXperspex is a more stable dosemeter than the previous batches;
- perspex takes up water to 2% in weight, the increase is initiallyquite rapid even at room temperature but proceeds more rapidly athigher temperatures. At 20 °C saturation takes several days, at80 °C saturation takes 48 h. The water molecules are absorbed asquickly from water vapour or water;
- comparison of the dose response of samples saturated at differentlevels of relative humidity at 20 °C has demonstrated very littledifferences from 0% RH to 100% RH water vapour although thehigher RH samples tended to give a slightly higher OD reading.Samples saturated in water at 80 °C for 48 h prior to irradiationshowed a radically different radiation response above 10 kGy (1 Mrad)the dose curve saturating rapdily;
- measurements with samples saturated with water at 20 °C, at 80 °Cfor 48 h or treated at 80 °C for 48 h in air have revealed thatit is neither the water saturation nor the 80 °C treatment butthe combination of water and 80 °C that leads to the alterationin the dose response relationship and that this treatment mainlyreduces the unstable free radical component of the induced opticaldensity. Since pre-saturation of perspex with water at 80 °C isUnlikely to occur in practice it can be concluded that humiditydifferences should not affect the response of the clear perspexdosemeter in practical applications.
Since 1977 the institute has cooperated in the IAEA High-DoseStandardization and Intercomparison Programme by irradiating severaldosemeter packages to known doses in intercomparison trials andproviding consultant expert advice on programme development.
- 28 -
Pub!icat ions
ad 1.PUITE, K.J. A thermoluminescence system for the intercomparison of absorbeddose and radiation quality of X-rays with an HVL of 0,1 to 3,0 mm Cu. Phys.Med. Biol. 21., (1976): 216-225.PUITE, K.J., D.L.J.M. CREBOLDER and J. VAN DE BERG. Thermoluminescentmeasurement of X-ray dose and radiation quality after exposure in beams withan HVL of 0,1 to 3 mm Cu. Proc. VI M e Congrěs International de la SociétéFrancaise de Radioprotection, Saclay, 23-26 March, 1976.
PUITE, K.J. Perspectives for using lyoluminescence in photon and fast neutronfields. Proc. Fourth Int. Congress of the Int. Radiation ProtectionAssociation. Vol. 4, pp. 1273-1275, 1977-
PUITE, K.J. and D.L.J.M. CREBOLDER. Experience with X-ray dose intercomparisonprojects; Measurement of beam quality and absorbed dose using a two-phosphorsystem; Energy independent systems based on luoluminescence intercomparisonProcedures in the Dosimetry of Photon Radiation, Technicap Report Series No.182, IAEA, Vienna, pp 71-76, 123-135, 147-151, 1978.
ZOETELIEF, J., J.J. BROERSE and K.J. PUITE. The need for repeated inter-comparisons and standardization of X-ray dosimetry for the coordination oflate effects research in Europe. Proc. Int. Symp. on National and Inter-,national Standardization of Radiation Dosimetry, pp. 305-317, Vienna, 1978.
BROERSE, J.J., J. ZOETELIEF and K.J. PUITE. Dosimetry intercomparisons forevaluation of late effects of ionizing radiation. Acta Radiologica Oncology17. (1978): 225-23i».PUIYE, K.J., M.S. AGRAWAL, J.J. BROERSE and J. ZOETELIEF. Dosimetry inter-comparison for partial body X-irradiation of rats. Phys. Med. Biol. 2S_ (1980):13-24.
ad 2.
PUITE, K.J. and D.L.J.M. CREBOLDER. Lyoluminescence dosimetry in photon andfast neutron beams. Phys. Med. Biel. 22 (1977): 1136-1145.
PUITE, K.J., D. RI NTJEMA and D.L.J.M. CREBOLDER. High dose level dosimetryusing the lyoluminescence technique. In: Proc. Int. Symp. of Food Preservationby Irradiation, IAEA, Vienna, vol. 2, pp. 335-343 (1978).
PUITE, K.J. and J. ZOETELIEF. The application of lyoluminescence in fastneutron fields. Proc. 3rd Symp. on Neutron Dosimetry in Biology andMedicine. Eds. G. Burger and H. Ebert, EUR 5848, Brussels, pp. 727-735 (1978).PUITE, K.J. A lyoluminescence dosimetry system useful for high-dose inter-comparison studies. Nucl. Inst. Meth., J_75 (1980): 122-125.PUITE, K.J. and K.V. ETTINGER, Lyoluminescence dosimetry. State of the art.Int. J. applied Rad!at. Isotopes, (in press).
ad 3.CHADWICK, K.H., D.E. EHLERMANN and W.L. McLAUGHLIN. Manual of Food IrradiationDosimetry (IAEA, Vienna), Techn. Rep. Ser. No. 178 (1977).
CHADWICK, K.H. Solid state dosimetry at high doses. Ionizing Radiation Metro-logy 1977, ed. E. Casnati (Editrice Compositori Bologna) (1977): 195-211.
Í J ^ňi£j-> _ j . t„„.W £ ifÄsÍ.>«Uftí*ili 6&S*
CHADWICK, K.H. Precision and Accuracy in Radiation Processing and Sterilization. Ionizing Radiation Metrology 1977, ed. E. Casnatie (Editrice Compo-sitori Bologna( (1977): k ^
CHADWICK, K.H. Some observations on the results of the preliminary inter-comparison held in 1977* IAEA Advisory Group on High Dose Standardizationand Intercomparison, Vienna, Techn. Tep. Series, (1980).
CHADWICK, K.H,, D. RINTJEMA and W.R.R. TEN BROEKE. The accuracy of thecalibration curve of the clear perspex dosemeter. Food Preservation byIrradiation, Vol. II (IAEA, Vienna) IAEA, SM-221 (1978): ^
CHADWICK, K.H. Radiation measurements and quality control. Proc. 2nd Int.Cong, on Radiation Processing, Miami. Rad i at. Phys. Chem. Jjt (1979): 203.
Lectures
PUITE, K.J. A lyoluminescence dosimetry system useful for high-dose inter-comparison studies. Sixth Int. Conf. of Solid State Dos imetry, Toulouse \-kApril, 1980.
PUITE, K.J. Final improvements of the LL dosimetry system using glutamine.Advisory Group Meeting on Dosimetry for High Doses employed in IndustrialRadiation Processing, IAEA, Vienna, 17-21 November, 1980.
CHADWICK, K.H. Humidity effects in the clear perspex dosemeter. AdvisoryGroup Meeting on Dosimetry for High Doses employed in Industrial RadiationProcessing, IAEA, Vienna, 17-21 November, (1980).
5,
- 30 -
Pwcent deviation with respect to standard dose
30
20
10
0
-10
-20
10
0
-10
InstituteA B C D E F G H
Before consultation
P Lung exposure
H Liver exposure
J. J „t After consultation
Figure 1 - Diagram showing the results of the EULEP partial bodyX-ray close intercomparison before and after consultationwith the participating institutes.
glass cup-sample
injection needlé j ^ s h u t t e rPM
j H.T. j H DC ampl j H integrator I
Figure 2 - Lyoluminescence apparatus
\
s.I\
- 31 -
PROJECT No. 85 ANNUAL REPORT 198O
F. van Dorp, M.J.Frissel, P. Poelstra, A. Ringoet
SCHEME FOR DECONTAMINATION OF AGRICULTURAL AREAS AFTER SEVERE NUCLEARACCIDENTS
In 1980 this project was completed by preparing the final report "Agri-cultural measures to reduce radiation doses to man caused by severe nuclearaccide ts", which will, be published early 1980. This report contains allthe information collected in the period 1976-1980. It is divided into threeparts. The first part describes models to calculate radiation doses to manfrom deposited radionuclldes via agricultural pathways. The second partdescribes possible measures to reduce the radiation dose to man, theirlimitations and their practicability. The last part describes fields forfuture research. A summary of this report is included in the following pages.
Experimental work in 1980 has been carried out in cooperation with Dr. C.van der Borght (CEN/SCK, Mol, Belgium) concerning temporary fixation of 9 0Srin soil with alginates. Alginates in combination with Sr ions form in-soluble compounds, therefore it was expected that alginates could immobilize9 0Sr in soil. Experiments with undisturbed soil columns showed, however,that the alginates are decomposed by microbial activity in soil. Thereforethe perspectives for the application of alginates are disappointing. Theresults of this experiment are also presented in the following pages.
F. van Dorp, R. Eleveld, M.J. FrisselAGRICULTURAL MEASURES TO REDUCE RADIATION DOSES TO MAN CAUSED BYSEVERE NUCLEAR ACCIDENTSAgricultural land and products may become contaminated after a severenuclear accident. If radiation doses to man caused by the ingestionof contaminated agricultural products from such areas will be un-acceptably high, measures to reduce this rad "• at i on dose will have tobe taken. Radiation doses to man can be estimated by using models ;which describe quantiatively the transfer of radionuclides through :the biosphere. The following processes and pathways are describedin this study:
- accidental releases into atmospheric environments and subsequentnearby deposition (= not world wide fallout) (atmosphericdispersion and deposition models are not included in this study) '
- contamination of crops by direct deposition and the subsequent ;f short term pathway (e.g. grass-cow-milk-man)I - contamination of soil and the subsequent long term pathway (e.g.\ soil-crop^mant soil-grass-cattle-milk/meat-man). \
j Depending on the degree of contamination (to be predicted by atmos- jI pheric dispersion models, or to be determined by direct measurements), jI and depending on the estimated radiation doses to man (to be calculated jÉ by using the models described in this study) measures can be advised.s The most important measures are the following.I Short term measures:I - feeding oattle with uncontaminated food if possibleť - production and storage of milk powder when the contaminationí consists of 1 3 1 I ;
- 32 -
- administration of ber.tonite to cattle to reduce the uptake ofcesium from feeds
- administration of calcium to cattle to reduce the uptake ofstrontium from feeds
- decontamination of milk by removal of strontium and cesium onionexchange columns.
Long term measures:- removal of contaminated crops if presents- removal of the contaminated top layer of the soil, applicable
only on stable and smooth soilss- application of lime or gypsum to reduce the uptake of Sr by
crops from soils- producing crops with a low transfer of radionuclides from soil to
edible productss- reduction of the radionualide content in the final consumable
products by processing treatments.Special attention has been paid to the practicability of the measures.A coit-analysis, however, was not carried out.
Some of the fields for future research are:- the improvement of transfer modelss
• - the determination of realistic parameters to be used in these modelss- the study of more efficient methods to measure contamination levels
- in the fields- the study of the effects of application of some complexing agents
to soils or cattle to reduce the transfer of radionuclides s'-• - the 3tudy of product treatments which reduce the radionuclide content
of the final product.
': 0. van der Borght, M.J. Frissel, P. Poelstra, D. Bannink
i TEMPORARY FIXATION OF 90Sr IN SOIL VilTH ALGINATES
( Alginates react easily with Sr ions forming insoluble compounds.: Therefore it was expected that alginates could temporarily immobilize; 90Sr in soils. Alginates are extracted from seaweed and consist mainly} of polyguluronic acid and polymannurcnic acid. The Na-form is soluble. in water. A k% solution in water is somewhat viscous, but still easy to: handle, so from a technical point of view the method seemed promising.
•: An experiment has been carried out on four soil columns. Two soil. columns were treated with alginates, two were used as controls. The; soil was a sandy soil with a low organic matter content, so aI soil in which 90Sr migration is rather fast compared to other soils.
v The experiment was carried out in the ITAL soil column installationI (Bannink et al., 1977); the main characteristics of this installationi are that the soils are not saturated with water and that artificial\ rain is supplied as droplets by a rain simulator. The length of the) columns was 100 cm, the diameter 12 cm and the soils were undisturbed.
f At the beginning of the experiment the soils were brought to water| equilibrium (i.e. efflux equals influx). Thereafter the soil surface| was contaminated with [85Sr]Cl2 by applying a carrierfree solution| of 10 ml (0,1 ml cm"2), After 2^ h 113 ml of a k% alginate solution
(1 ml cm"2) was supplied. Again after 2k h, leaching started byapplying 1 cm of artificial rain (composition rain 6,9 x 10"5 N CaCl2,
. 2,3 x 10"5 N NaCI, 2,3 x 10"5 N KC1, acidified with H2SOi, to pH 4,5).
- 33 -
For half a year the columns were scanned every 14 days with a soilcolumn scanner. The results of the scannings after 16 and 184 days areshown in fig. 1. After 16 days there is a slight indication that the8r'Sr in the columns with alginates moved a little faster than in thecolumns without alginates. Because the experiments were carried outin natural, undisturbed, soils, such slight differences can also becaused by natural differences between the soil in the different columns.After 168 days a real difference became clear: in the columns withalginates the migration was delayed by a factor of about 1/3, comparedto the controls; increased tailing counteracted this effect.
Additional observations were: measurement of the redox potentialwithin the column 10 cm below the soil surface and measurement ofthe pH of the effluent. These observations showed that in the columnswithout alginates no significant changes of redox potential or pHoccurred, indicating no important microbiological activity. In thecolumns with alginates the redox potential dropped from about 730 mVto about 500 mV, while the pH of the effluent increased from 4,6 to 6 - 7-This indicates significant microbiological activity. It must thereforebe assumed that alginates were decomposed. This would explain the smalldifferences between the two sets of soil columns. The tailing whichwas observed in the columns with alginates may be ascribed to small quantitiesof complexing agents which were formed during the decomposition of thealginates.
So far, the perspectives for the application of alginates for thetemporary fixation of 90Sr on soils seem disappointing. Of courseit could be attempted to sterilize the soils, but in practice sucha sterilization is not possible.
Publi cat ions
BANNINK, D.W., P. POELSTRA, M.J. FRISSEL. An installation for leaching andaccumulation studies on undisturbed soil columns. Neth. J. of Agric.Science 25 (1977): 126-133-
DORP, F. VAN, R. ELEVELD and M.J. FRISSEL. A new approach for a soil-planttransfer calculation. Proceedings of the International Symposium on Bio-logical Implications of Radionuclides Released from Nuclear Industries, inVienna 26th - 30th March 1979, Vol. II, pp. 399-406, IAEA, Vienna 1979.
DORP, F. VAN, R. ELEVELD and M.J. FRISSEL. Agricultural measures to reduceradiation doses to man caused by severe nuclear accidents (X + 114 p, 7 figs,13 tables, 93 refs., in English), submitted for publicationto the Commissionof the European Communities, Brussels.
Lectures
DORP, F. VAN, R. ELEVELD and M.J. FRISSEL. A new approach for soil-planttransfer calculation. At the International Symposium on Biological Impli-cations of Radionuclides Released from Nuclear Industries, in Vienna,26th - 30th March 1979, organized by the IAEA.
85Sr (orbitrary units)
10
5
4/ N1 1I 1' \1 1• 1
"J \1 »1 3 t
• IÍY•/I \•/I \
'/Z \2// A- / \/ \
ff
• w
with alginates
without alginates
8
NN
• 1 1 1 1 "**"*• T " 1
5 10depth (cm )
Fig. I. The migration of 85Sr in soil columns. Curves 1-4 positionof 85Sr after 16 days of leaching, curves 5-8 positionafter 134 days of leaching. The total amounts of 85Srdiffer slightly from column to column. Amounts of 85Sr arecorrected for decay, therefore they are expressed asarbitrary units.
- 35 -
PROJECT No. 86 ANNUAL REPORT 1980
M.J. Frissel, P. Poelstra, N. van der Klugt, F. van Dorp
MIGRATION OF 9 0Sr, 137Cs AND Pu IN SOILS. VERIFICATION OF A COMPUTER MODELON THE BEHAVIOUR OF THESE RADIOCONTAMINANTS IN SOILS OF WESTERN EUROPE
This project was based on and is partly a continuation of an earlierproject which started in 1957- From that time on four pastures weresurveyed for their 90Sr content. Four layers at depths of 0-58 5-10»10-15 and 15-20 cm were analysed. From these field data and fromlaboratory column experiments a model was derived to predict thebehaviour of 90Sr in soil. Gradually the number of sampling sitesincreased and also attempts were made to survey l 3 7Cs.
At this stage of the investigations the 1976-1980 project started.Aims were:1. Verification of the 90Sr model.2. Development of a simple method to determine 1 3 7Cs. Development
and test of a model for the behaviour of 137Cs in soil3. Development of methods to determine Z38Pu and 239,2>+0pu jn s o n .
Development and test of a model for the behaviour of Pu in soil.
In general it can be stated that all these aims have been achieved.
Further a method to determine 2 4 1Am (at fallout levels) is also inthe process of development.
During the course of the project 25 different sampling sites wereused, but only a limited number of sites could be continuouslyused during the whole period from 1957 until the present.The results from 14 sites, together with simulation results havebeen described in the report "Modeling of the transport andaccumulation of the radionuclides of Sr, Cs and Pu in soil. Experimentalverification by Frissel, Jakubiek, Van der Klugt, Pennders, Poelstraand Zwemmer, which will appear early 1981. Table 1 summarizes thedata for Pu and Am.Three representative graphs of the concentration of 9 0Sr, 137Cs and239,240pu j n the course of time are shown in the Figures 1, 2 and 3respectively. These figures show also the simulated results, i.e.the curves which were calculated using the models. There are, however,considerable differences in the degree of sophistication of the modelsfor Sr, Cs and Pu. The model for Sr is a simulation model. Inprinciple all parameters can be obtained from independent measurements.In practice it appears that some adaptation is necessary.The models for Cs and Pu are pseudo residence time models. Thereason why these simpler models were used was the difficulty inpredicting irreversible adsorption/desorption phenomena. Relevantdata for the model have therefore to be determined from the siteunder study. In fact the model is a kind of extrapolation model.
A literature study showed that observed data for 9 0Sr and othertheories confirmed the model developed. For 1 3 7Cs there are lessdata available, but there is no reason to assume that the model is notgenerally applicable. For 239,2foPu the n u m | j e r of observations in soilis very limited, only part of the existing data support the residencetime model. A study of Pu chemistry shows that so many complicationsmay occur that it must be assumed that in numerous situations the modelwill not be applicable.
Table 1 - Concentration of Pu and Am in the upper layer of three soils. Mean values mBq/kg (pCi/kg)
LocationNud i deDepthcm
0 - 5
5 - 1 0
10 - 15
15 - 20
20 - 25
25 - 30
30 - 35
35 - 40
40 - 50
50 - 60
60 - 70
70 - 80
Groot
239,2't0pu
mBq/kg
655400
110
26
26
11
26
18
15
74
4
pCi/kg
17,7
10,8
2,9
0,7
0,7
0,3
0,7
0,5
0,50,2
0,1
0,1
Ammers2 1 t lAm
mBq/kg
340
190
100
70
pCi/kg
9,1
5,1
2,72,0
Hooglanderveer
238pu
mBq/kg
22
11
4
pCi/kg
0,6
0,3
i
239,2l*0pu
mBq/kg
240
126
11
77
74
77
4
pCi/kg
6,4
3,4
0,3
0,2
0,2
0,2
0,1
0,2
0,2
0,1
Schoonebeek
238pu
mBq/kg
730
11
4
74
7
pCi/kg
0,2
0,8
0,3
0,1
0,2
0,1
0,2
239,2»»0pu
mBq/kg
800
560
130
1522
7<4
pCi/kg
21,6
15,1
3,50,4
0,6
0,2
<0,1 I
- 37 -
Pub!icat ions
BANNINK, D.W., P. POELSTRA and M.J. FRISSEL. An installation for leachingand accumulation studies on undisturbed soil columns, Netherlands J. ofAgricultural Science, 25 (1977): 126-133-
FKISStL, M.J. and ľ. POELSTRA. Radioacticve slof Ten in "Handboek voor Milieu-beheer", IV Bodembescherming. 1978.
FRISSEL, M,J. and F. VAN DORP. Terrestrial pathway models in the Netherlands.IAEA, Neuherberg, 1978.DORP, F. VAN, R. ELEVELD and M.J. FRISSEL. A new approach for Soil-Planttransfer calculations. In "Biological implications of radionuclides re-leased from nuclear industries" Vienna, IAEA. (1979)-
FRISSEL, M.J. and A.T. JAKUBICK. Transport and Accumulation of radionuclidesin soil. In "Radiookologie Berichtsband der Fachtagung Radiookologie desDeutschen Atomforum e.V., Bonn". Vulkán Verlag, Essen, p. 131-ľ»6 (1979)•
FRISSEL, M.J., P. POELSTRA and K. SMIERZCHALSKA. The verification of modelsfor the migration of 1 3 t t. 1 3 7Cs in soil. In "lie Symposium international deRadioecologie", CEA, Cadarache, France, p o 711-732 (1979).
FRISSEL, M.J.,, A.T. JAKUBICK, N. VAN DER KLUGT, R. PENNDERS, P. POELSTRA,E. ZWEMMER. Modeling of the transport and accumulation of Sr, Cs and Pu. -Experimental Verification. EEG report (In preparation).
"SrmCi/hm1
40
30
20
10
x 0 - 5cm
o 5.10cm
• • 10-15cm
a 15- 20cm
1965 1970 1975
Fig. 1. 90Sr activity concentration in a mucky peat soil near GrootAmmers.Dots refer to observations, curves to calculations based on asimulation model. The upper curve refers to layer 0-5 cm, thesecond one to layer 5-10 cm, etc.
mCi 1S7Cs per km8 —
70-
20
10-
Fig. 2.137Cs activity concentration ina mucky peat soil near GrootAmmers.Dots refer to observations,curves refer to calculations;pseudo residence time 3 a cm"1.
1960 1970 1980
- 39 -
2.0-
1.5.
1.0-
0.5
calculation: observation:curvei.lav* 0 - 5cm •
• 2 - 5.10cm •. 3' . 10.15cm •- 4. « 15-20cm *
1955 1960 1965 1970 1975 1980
Fig. 3- 239,2U0pu ac t iv i ty concentration in a podsol soi l fromHooglanderveen.Dots refer to observations. Curves refer to calculations;pseudo residence time 2,5 a cm"1.
- hO -
PROJECT No. 87 ANNUAL REPORT 1980
P. Poelstra, N. van der Klugt, M.J. Frissel
A SURVEY OF THE CONTAMINATION OF PLUTONIUM, AMERICIUM AND RADIOACTIVE METALSIN SOILS OF THE RHINE RIVER DELTA
The first years of the contract period were utilized to develop reliablemethods to determine ct-emitters at fallout and waste out.levels.Two problems had to be solved: 1. The chemical separation of Pu andAm from the sediment samples and 2. The development of suitable countingtechniques. The determination of Pu has been reliable since 1979, the Amdetermination is in development. In the last years attention was mainlyfocussed on the determination of Pu and to a lesser degree of Am insediment samples of the Rhine river. Samples were taken from a clay-typesoil from the forelands of the river Rhine near Valburg. This area isfrom time to time flooded by water of the Rhine. During such floodsconsiderable deposition of clay and other particles occurs. Because itmust be assumed that almost all Pu and Am is adsorbed on clay and otherparticles, the Pu content of the soil will reflect the presence of Puand Am in the river. At Valburg five sites were sampled. Differences inPu content may be caused by analytical difficulties and/or actualdifferences.
At the moment the standard deviation of the 239>21*0Pu data when determinedfrom one (homogenized) soil sample and with the same chemicals and 2 3 6Pustandard is 45 mBq/kg (1,2 pCi/kg) for levels of 0,5 Bq/kg (15 pCi/kg).(236pu recovery varies, depending on soil type, between 30 and 80%). Thereported differences must therefore be attributed to actual differencesof Pu levels.
The second sampling location was in the Biesbosch, an area consisting ofreed banks, old and recent river deposites, creeks and streams. Foursites were sampled, some consisted of clay/sand mixtures, otherscontained almost only organic matter. In another research programme wehad found remarkable accumulation of heavy metals at some of thesesampling sites. Therefore we expected similar results for Pu and Am.
A third sampling site was.'-tjaken outside the delta, namely a sandy soilnear Amersfoort. This area -has never been flooded and is used as areference. Results for all three locations are shown in the tables 1 - 3 .From the tables it appears that the 239,2»»0pu c o ntent of the uppersoil layer from Valburg has a somewhat higher Pu content than thosefrom Amersfoort (500 mBq/kg (13,4 pCi/kg) versus 300 mBq/kg (8,06 pCi/kg),mean values for the 0-5 cm layer). Pu content decreases with depth inboth soils, but the difference between the soi 1 remains (for example inthe 20-25 cm layer 70 versus 11 mBq/kg (1,9 versus 0,3 pCi/kg)). A ;slight enrichment of Pu in the foreland soils of the Rhine might there-fore be present. The enrichment of Pu and Am is certainly less than jthat of heavy metals such as Hg.
The values for the Biesbosch are again slightly higher than for Valburg.Pu is also observed at greater depth (6JO mBq/kg (16,4 pCi/kg) for theupper 40 cm) and considerable enrichment seems to occur. It can, however,not be concluded that considerable accumulation occurred. The density
- 41 -
of the sediment from the Biesbosch is only one quarter of that of thesoil from Amersfoort. Therefore the enrichment expressed per unitsurface area is only 1,2. Because of the great heterogeneity of theBiesbosch sediments this value has an indicative meaning only.
Further research on possible accumulation of transuranium elementsin the Rhine and Scheldt deltas will be carried out in cooperationwith the Delta Institute at Yerseke.
Table 1 - Pu and Am contents of soils from Val burg
RadionucIide:
Sampling site:
Sampling date:
Depthcm
0 - 5
5 - 1 0
10 - 15
15 - 20
20 -25
25 - 30
238pu
1
1/78
m
44
30
2
4/78
Bq-kg-
26
26
18
7
3
4/78
l
26
30
263018
18
7715
7
1
1/78
600
560630560
239,
2
4/78
mBq
415
545
125
70
240pu
3
4/78
• kg"1
445
415
5*55451451257870
?836
5
10/78
420
74
33
30
2^Am
4
4/80
mBq*
120
70
38
21
5
10/78
kg"1
160
240
- 42 -
Table 2 - Pu content of sediments from the Biesbosch
Radionuclide:
Sampling site:
SampI ing date;
Depthcm
0 - 5
0 - 2 0
0 - 1 0
10-20
20 - 30
30 - 40
40 - 50
50 - 60
60 - 70
70 - 80
238pu
2
2/78
3
2/78
4
3/78
mBq-kg"1
56
59
52
53
48
63
63
56
>56
33
22
11
7
11
7
239,2<»0pu
1
4/7C
2
2/78
3
2/78
4
3/78
mBq-kg""1
850
775
805
850
560
555
550
600
475
465
545
470
460
>460
700
610
220
81
48
52
- 43 -
Table 3 - Pu and Am content of a soi l from Amersfoort
Radionuclide:
Sampling site:
Sampling date:
Depthcm
0 - 5
5 - 1 0
10 - 15
15 - 20
20 - 25
25 - 30
30 - 35
35 - 40
40 - 50
50 - 60
7 0 - 8 0
80 - 100
Z38pu
1
5/77
2
11/79
mBq-kg"1
22
22
22
26
11
11
74
< 4
7
15
*
239,2i»0pu
1
5/77
2
11/79
mBq- kg-*
310
365285
335330
330
155
150
1511
44
74
< 4
255
237314
92
66
7
11
7
74
774
2"lAm
2
11/79
mBq•kg"1
96
141
70
15
Publications
FRISSEL, M.J . , F. VAN DORP, P. POELSTRA. Use of residence time models inecological studies of transuranics. Proceedings IAEA-CEA meeting "Behaviourof transuranics in the aquatic environment and sediment-water exchanges",ISPRA, (1980).
PROJECT No, 88 ANNUAL REPORT 1980
J. Sinnaeve, F. Smeulders, M.J, Frissel, A, Ringoet, P, Poelstra
IMMOBILIZATION AND REDUCTION OF THE AVAILABILITY OF RAO IOCONTAMINANTS INSOILS
The aim of the project was to investigate to what extent syntheticligands could be used to reduce the availability of rad i©contaminantsfor plant uptake and the percolation to groundwater. A polyarn!ne-typeligand, tetraethylene-pentamine (tetren or T), was used. It formsstable complexes with some transition metals, to which some radio-contaminants belong. The complex obtains an extra-stabilization whenadsorbed on alumino-si1icates. The basic assumptions underlying theextra-stabilization effect were described in extenao in the 1976annual report. Throughout the study, a pure clay mineral, illite,characteristic clay mineral of most West-European soils, a sandy-loam soil from Horst, The Netherlands and a clay soil from Winsum, TheNetherlands, have been used.
A. Soil physico-chemical aspects
Data of adsorption experiments are given as isotherms and as thenaturel logarithms of the stoichiometric equilibrium constant asa function of the adsorbed fraction. Figure 1 illustrates theeffect of complex formation with tetren on the ion exchange selectivityof Zn, Mi, and Cd in the Winsum soil. These results are similar tothose found for Cu (partly depicted (Fig. 1)). In the absence oftetren, a high selectivity is observed at low coverage: theselectivities decrease in the order Cu > Zn > Hi > Cd. The tetren-complex selectivity data roughly correspond to a two-order-of-magnitude increase of the stability constant.Figure 2 illustrates the results obtained with the Horst soil. Incontrast to what is observed for the Winsum soil, complex formationwith tetren results in a shift of the metal (-complex) into theliquid phase, the effect becoming more pronounced with increasingcoverage. This indicates that the stability of the tetren complexesis higher than the stability of the metal-humus complexes. Evident-ly, if the metal complex is less retained by the humic fraction andif only minor amounts of clay are available, the equilibriumconcentration levels of the complex in the liquid phase are boundto increase.
B. Stoichiometric aspects of the uptake of Cu++ and Cu-tetren byplants
Maize plants {Zea mays L. cv. spec.) were grown for three weeks ona complete Hoagland-Arnon I (HA-1) nutrient solution. After pre-conditioning during 2k hours on a two times refreshed nutrientsolution without microelements, the plants were transferred to a10 times diluted HA-1 solution without microelements but containing10"6 mole/1 Cu(N03)2 labelled with
6l*Cu or 10 - s mole/1 Cu-tetrenlabelled with 6l*Cu and ^C-marked tetren. The plants were harvestedafter an uptake period of 26 hours (of which 13 hours light). Theresults are summarized in table 1.
Complex formation leads to a marked decrease of the Cu-content ofthe roots (195 ymole/kg versus 3,6 mmole/kg, the reverse beingobserved for the stem (39 versus 3.4 ymole/kg) and for the leaves(9 versus 0,4 vimole/kg).
Table 1 - Stoichiometcy, of the uptake of Cu and tetren suppliedas Cu T 10"e mole/1 and comparison with the uptake ofCu supplied as Cu(N0j)2 10~
G mole/1 by 3 weeks old maizeplants after a 26 hours absorption period. Mean values(and standard deviation) for 3 replicates in nmole-g"1
per 26 h.
HA-1 1/10 solutionwithout microelements
Plant organ
RootsStemLeaves
Cu
195 +39 +9 +
Cu-tetren
2323
10"6
tetren
167378
+ 15+ 4+ 2
mole/1
tetren/Cu
0,86 +0,94 +0,87 +
0,0,0,
131135
Cu(NO3)210"6 mole/1
Cu
3645 + 6413,43 + 0,(0,43 + 0,1
The ratio tetren/Cu is only slightly different from unity and thisprovides evidence that Cu enters the root system as a Cu T complexand because of the higher stem and leaves contents, it is alsotranslocated as such. In vivo measurements of the 6t|Cu translocationwith semi-conductor radiation detectors placed along the stem re-vealed that translocation of the Cu-tetren was five tines higher.This was also evidenced by the distributions of Cu++, Cu T++ andTH3+ (protonated tetren) in a papyrus stem (Cyperus papyrus L. cv.spec), to which labelled solutions were supplied for 330 minutes.Copper supplied as Cu(N0.3)2 concentrated in the lower part of thestem whereas chelation of the Cu ion resulted in a lower con-centration at the base of the stem and a distribution over its length(figure 3).The mobility of the trivalent TH5+ is between that of Cu++ and thatof Cu
C. Growth experiments using a natural clay soil
The final growth experiments, using the heavy clay soil (Winsum)were carried out in the Experimental Soil Plant Atmosphere System(ESPAS) at ITAL. Four treatments were considered: 1) the controlsoil, b) a Cu contaminated soil (9,5 meq/kg or 300 mg/kg), c) a Cu Ttreated soil (9,5 meq/kg Cu with a molar ratio tetren/Cu equal to 1,5)and d) a tetren treated soil (7,1 mmole/kg, i.e an equal amount oftetren as in treatment c). Maize seeds were germinated directly insoil columns and after 20, 34, 44 and 55 days respectively, threeplants were harvested. The yields of root and shoot are depictedin figure 4. It is readily seen that chelation of Cu neutralizesmetal toxicity. Comparison of the yield data of control and tetrentreated soil shows a lower relative growth of the latter up to 35days and a higher relative growth in the following period. Thiswas due to a delayed senescence of the oldest leaves of the plantsgrowing on the tetren treated soil. The application of tetren ona copper contaminated soil resulted in a drastic decrease in the
kecopper concentration of the root (175 versus *»30 mg/kg) and anormal concentration in the shoot (10 versus 21 mg/kg). Unchelatedtetren decreased the Cu concentration of the roots; Zn and Mnconcentrations increased but the iron concentration was hardlymodified. This demonstrates the very beneficial effect of tetren ~at toxic concentrations of transition metals (e.g. Cu, Co, Zn)whereas no deficiency symptoms occur at trace levels.
D. Biodegradation of chelated and unchelated tetren in soils
In the clay soil (Winsum), the total amount of [ll|C]02 recoveredafter 1 year, suggested an overall degradation of about 20% ofthe chelated tetren. The decomposition of unchelated tetren wasmuch faster and reached about 50& after 1 year. The extra-stabilization effect of the chelated form may account for this.In both cases, increasing humidity and higher temperatureaccelerated the breakdown. The presence of a root microfloraalso enhanced the degradation. In soil columns with a denseroot system, the [ll*C]02 recovery was 6,2 and 5>2% for Cu T andT respectively, whereas without cropping it was 1,2 and 1,5%. Inthe sandy loam soil, the rate of degradation was much faster.After 1 year, the amount of [1'*C]02 recovered indicated a totaldegradation of about 70% per year.
E. In situ experiment
A field experiment on a Zn-contaminated soil (about 100 mg/kg Znin the top layer) is in progress. The first results indicate afavourable effect of a tetren treatment on plant growth and thetreatment eliminates Zn toxicity symptoms. The preliminary resultsconfirm the conclusions drawn from the above reported laboratoryexperiments.
F. General conclusion
In the light of the reported results, some important conclusionsmay be drawn.
1) Due to the low chemical concentrations of rad iocontami nantseven after accidental contamination and due to the presencein the soil of carrier ions, it is impossible to immobilizethe radiocontaminant in a selective way.
2) In cases of local contamination, complexing agents of thepolyamine type (e.g. tetren plus clay mineral) may be used asa strong "temporary sink" for stabilizing the situation inorder to prevent further spreading before adequate measures(e.g. removal of the top soil layer) can be taken.
3) Complexing agents, although not suited to reduce completelythe availability of radiocontaminants for uptake by plants,may be used to reduce the soil-plant transfer withoutprovoking nutrient deficiencies.
k) In case of contamination of agricultural land, the appropriatemeasures to be taken must be evaluated in the light of thelocal circumstances. The use of complexing agents cancontribute to limit the collective dose commitment by ingestion.
Publications
SMEULDERS, F.G.H. In situ immobilization of transition metal ions in soils,w.th tetreethylenepentamine. Doctoral thesis, K.U.L., Leuven, Belgium (1980)SMEULDERS, F.G.H., J. SINNAEVE and A. CREMERS. In situ immobilization ofheavy metals ,n so.Is. Proc. 8th Int. Coll. on Plant Analysis andFert.hzer Problems, Auckland, New Zealand, 1978 (Ed. by DSIR, New Zealand).
Meo<te(jieq/g)
50
40
30
20
10
ZnT
lnKc
5
4
3
Zn
-1
10 20 30 40 50 60 70 80Meods(jjeq/g)
Fig. 1 - /\. Ion exchange isotherms for the Cd T-Ca, Zn T-Ca, Ni T-Ca andCa-Ca, Zn-Ca, Ni-Ca pairs of the Winsum heavy clay soil.
JJ. Selectivity coefficients for the exchanges Cd-Ca, Zn-Ca andNi-Ca in the absence and presence of tetren as a function of themetal ion surface composition.
In K.
15-1
10-
5- -150
- 4 - 3 2 - 1 0logSMe
CuT
ia 15Mead, (jieq/g)
Fig. 2 - £. Ion exchange isotherms for the Cu T-Ca, Zn T-Ca and Cu-Ca, Zn-Capairs of the Horst sandy loam soil.
j>. Selectivity coefficients for the exchanges Cu-Ca and Zn-Ca in theabsence and presence of tetren as a function of the metal ionsurface composition.
TRANSPORT PERIÓDE ShSOnň
rfwcíc
Fig. 3 - Distr ibution of 6**Cu or 1 4 C - t e t r e n in a papyrus stem a f t e r 330minutes of absorption. The solutions were:A: Cu-Tetren 10" 5 mole/l, Ca (1(03)2 5.10"1* mole/1
10" 5 l / 1 C ( N 0 ) 5-10"1* mole/15-10"1* mole/1
A: Cu-Tetren 1 0 5 mole/l, Ca (1(03)2B: Cu(N03)2 10" 5 mole/1, Ca(N0 3 ) 2
C: tetren H§+ 10" s mole/l, Ca(N0 3 ) 2
Control Cu 300ppm CuT 300 optn Control Tetren
yield (9)14,
121
4l
2!
20 34 44 55
, -
20 34 44 55II IL
20 34 44 55 20 34 44 55
I
shoo*
age
root
4|
Fig. k - Yield of maize plants (g) as a function of time (days) grown on aclay soil at a Cu(-tetren) level of 300 mg/kg. The "control tetren"treatment was amended with the same amount of tetren ad the Cu Ttreatment.
- 50 -
PROJECT No. 89 ANNUAL REPORT 1980
G. Desmet, J, Sinnaeve, S.C. van de Geijn, A. Ringoet
ROOT AND FOLIAR UPTAKE OF RADIOACTIVE CONTAMINANTS BY CROPS; THEIR TRANSPORTAND REDISTRIBUTION IN DIFFERENT PLANT ORGANS (e.g. EDIBLE PARTS, REPRODUCTIVEORGANS)
The aim of this project has been the determination of transfercoefficients and (redistribution of some radiocontaminants in plants.The subject has been assessed in two different ways. Firstly ex-periments have been performed with plants growing on nutrient solutionsto deal with problems of growth mechanisms which often provoke con-flicting interpretations of transfer functions, and secondly ex-periments have been performed in soils in order to obtain insight intothe order of magnitude of the transfer coefficients.
The nutrient solution experiments utilized both spinach (Spinaoeaoleraoea L. cv. Verbeterd Breedblad) and tomato plants {LyooperaioonescuLentvm Mill. cv. Moneymaker). The data on spinach relating ageand transfer are presented in table 1. The 5*»Mn and 6 0Co transfershave been studied at one fixed stable isotope concentration and thee5Zn transfer at three stable isotope concentrations. Large differencesamong the three rad ioisotopes' transfers were observed with a cleardependence on the age of the plants.Moreover in the 65Zn experiments a dependence on the stable isotopeconcentration was established. In tomato plants the roots and evenmore the fruits were the most important sites of accumulation of 65Zn(table 2).For all plant organs, the amount of 65Zn accumulated variedconsiderably with the degree of maturation at harvest time. All thesedata are firm evidence of the existence of predictable transfer functionsinstead of randomly occurring transfer factors.
A comparison between the amount of Zn "supplied" with the water takenup and the actual Zn consumption of a spinach plant rules out theopportunity to uti 1 i ze water-uptake data for transfer calculations ofradiocontaminants in forecasting models (table 3).
The mobility in spinach leaves of 6 5Zn and 60Co has also been studiedby investigating the redistribution of 65Zn after a short period ofcontamination, and by measuring the time necessary for 65Zn and 60Coto exchange with stable Zn and Co in mature non-growing leaves. Thelatter method will be published in detail elsewhere. Both approachesshowed a strong binding of zinc, with almost no redistribution andwith very slow isotopic exchange rates. The 60Co in contrast showeda very rapid exchange rate. In order to study the distribution of6SZn within tomato plants a natural sandy-loam soil column wascontaminated over its complete length at different times after theappearance of the first flowers. The 65Zn content of the lower partof the stem was higher than of the upper part and this differencesharply increased with a shortening of the contamination period (afactor k for a kO days contamination period compared to a factor 15for an 18 days contamination period). However, the S 5Zn distribution,being mainly in the seeds and the peel was hardly influenced by timeof the contamination.
- 51 -
Prior to the determination of crfinsfer coefficients of 6 5Zn froma well defined clay soil to the spinach, several introductory studiesof some important parameters have been done. These included the growthrate of spinach at various soil humidities, the rate of extraction ofstable Zn and the total availability of the native Zn and of theartificially supplied 6 5Zn. In this soil about hQ% of the 6 5Znappeared to be tightly adsorbed, the other 60& being extractable bythe commonly used soil extractant DTPA.The transfer of 6 5Zn after both a top and a bottom layer contaminationof the clay soil has been investigated in pot experiments. The toplayer contamination caused rather small contaminations of the leaves(table k) because of the very small depth migration of the 6 5Zn (alsoverified by soil column scanning experiments) and the fact that theroots were rapidly penetrating the soil. This penetration was verifiedin the bottom layer contamination experiment.Comparative transfer studies of different radionuclides were performedwith several plant species growing on a sandy-loam soil. The plantsused were tomato, maize (Zea rmiyt? L. cv. s p e c ) , bean (Phaseolusvulgca-us L. cv. Witte Krombek), barley (Hordeum vulgare L. cv. Piccolo),lupine (Lupinus lutens L. cv. Barpine), wheat {Triticum sativwn L. cv.Tundra), clover (Tin.folium r<epens L. cv. Retor), spinach and ryegrass{Lolium perenne L. cv. Pelo). The tomato plants were grown in auniformly labelled soil whereas for the other cultures only the upper7 cm of the soil were contaminated. The results are presented in table5. The transfer coefficients of the divalent cations with the exceptionof iron are rather high, being on average higher than of the monovalentcation Cs. Calculations of transfer coefficients are hazardous hithertoin top layer contamination since no certainty exists about the radio-contaminants' concentration at the sites where it is picked up by theindividual roots. They depend moreover on soil water content. Furtherresearch is therefor urgently needed to assess these problems.
Table 1 - The dependence of the transfer coefficient (TC) upon age orconcentration. TC = cpm per kg fresh weight/cpm per 1 nutrientsolution
1
\
\
\
í
i;
)
\
r
ii
Stable Znconcentrationmole/1 soln.1,7«.1O"7
4,40.10"6
2,64.10"5
Stable Mnconcentration1.16.10"5
Stable Coconcentration1.10"8
Age ofplantsdays
610131722610131722
610131722
9111<t1821
18132127
Transfer coefficients
cotyledons
108465649472333322923
6,78,8108,810
1319213457
M! 2,3! 2,3
2.61 2.1
first leaves
1761648058544042342922
1010119,29,2
1217182424
5,13,41,61,4
second leaves
238167118127
55362422
8,68,76,56,9
613141615
5,21,51,2
- 53 -
Table 2 - 6r'Zn content in plant organs at different ages
Age orplant
Old
Med i um
Young
Base
Top
part oforgan
GSZn content,
leaves
uCi/g
0
1
2
,59,13
,23
MBq/kg
22
42
83
expressed in the
petioles
pCi/g
1,55
2,58
2,93
MBq/kg
5795103
dry
wCi
0,
1,
matter
stem
/g
43
36
MBq/kg
16
50
Table 3 - Comparison between the amount of zinc present in the dailywater consumption by a spinach plant at different zincconcentrations and the total amount of zinc accumulated inone day. The mean and standard deviation are presented,n = 4 with 9 plants on a holder at 5 days, and 3 per harvestat 17 days. - means shortage; + means surplus.
Parameter
Age of plant: 5 days
Zinc in daily waterconsumption, nmole
Zinc accumulated inone day, nmolc
Difference, nmole
%
Age of plant: 17 days
Zinc in da ily waterconsumption, nmole
Zinc accumulated inone day, nmole
Difference, nmole
%
1,76 x
0,
4,
-98
1,
57-55,
-97
Concentration
10"7mole/l
07+ 0
15+ 1
08+ 1
67+ 0
+36
3 +36
,00
,98
,98
,14
,4,4
4,40 x
1,
9,
-7,-31
54,
147-92,
-63
of Zn
10~6mole/l
77+ 0
15+ 3
38+ 3
6 + 3
+67
4 +67
,02
,44
,4
,5,6
2,64 x
10,
20,
-9,-48,
372
350
+22
+6
10~5mole/l
4+ 0,48
2+ 8,6
8+ 8,6
5
+ 31,4
+253
+255
- 5*1 -
Table 4 - Depth migration of 6 5Zn after a top layer contaminationand transfer coefficients (counts in 5 min per g dryweight of plant / counts in 5 min per g dry soil toplayer) in spinach.
Soilwatercontent
15
25
Depth migrationlayerscm
0 -
2 -
4 -6 -
0 -
2 -
4 -6 -
2
468
2
468
Countsper gtotal
237406285514
21510
289210
50
in 5 mindry soilextract
15850407345
12350
19913732
Plant part
cotyledons
first leaves
second leaves
cotyledons
first leaves
second 1 eaves
Transfercoefficient
0,50 + 0,18
0,60 + 0,04
0,57 + 0,16
0,11 + 0,02
0,15 + 0,11
0,14 + 0,09
Table 5 - Mean transfer coefficients of the shoot (activity concentrationof dry matter divided by activity concentration of dry soil) andstandard deviations (four replicates) for different radionuclidesand different plant species.
Plantspecies
tomato*
bean
barley
maizelupine
wheat
clover
spinach
ryegrass
60Co
0,03+0,02
3,29+1,10
0,64+0,08
0,17+0,09
6,66+3,130,41+0,11
2,76+1,682,97+0,950,64+0,44
Transfer65 Z n
2,46+1
0,52+0
0,30+0
0,41+0
0,42+0
0,28+0
0,53+02,00+0
0,38+0
,15,24
,18
,09,34,04
,1*,72,08
0
coefficient of
59 F e
-
,06**
0,09+0,03
0
00
0
0
0
,03**
,10+0,
,06+0
,09+0
,04+0
,07**
0802
02
,01
2
1
5*Mn
,60.11,33+0
0,38+0
0
10
02
0
,70+0
,15+1
,56+0
,76+0,70+0,44+0
the shoot
,04,68,18,41,21
,13,20
,56,02
9°Sr-
1,47+0
0,38+0
0,30+0
1,32+0
0,27+0
0,94+0
0,64+0
0,42+0
,28
.1*,ľ»,93,07,17,23
,13
13<*Cs
2,22+0,24
0,05+0,03
0,11+0,08
0,05+0,02
0,15+0,14
0,11+0,04
0,14+0,06
0,04+0,01
0,07+0,01
homogeneous contamination of the soil
mean value of two replicates
- 55 -
Publications
BALEN, E. VAN, S.C. VAN DE GE1JN and G.M. DESMET. Autoradiographic evidencefor the incorporation of cadmium into calcium oxalate crystals. Z. Pflanzen-physiol. 97 (1980): 123-133.
DESMET, G.M. and W.G. DIRKSE. Growth and zinc accumulation in spinach. Phy-siol. Plant. &Q (1980): 31*»-318.
GINKEL, J.H. VAN and J. SINNAEVE, Determination of total nitrogen in plantmaterial with Nessler's reagent by continuous flow analysis. Analyst, 105(1980): 1199-1203.
JUPUN, G.L. and G.M. DESMET. Description of the use of an EPROM for theautomatic baseline control of a single-beam spectrophotometer. J. Phys. E.Sci. Instrum., 12 (1979): 29^-297.
MIEDEMA, P, and J. SINNAEVE. Photosynthesis and resporation of maize seed-lings at suboptimal temperatures. Exp. Bot., _3]_ (1980): 813-819.
SINNAEVE, J., F. VAN DORP. Mixtures of synthetic ion exchangers as anartificial soil for plant nutrition studies. Proc. 8th Int. Coll. on PlantAnalysis and Fertilizer problems, Auckland, New Zealand, 1978.
SINNAEVE, J. A growth chamber for the integrated study of the plant-soil-atmosphere interactions. Application to the influence of soil temperatureon the mineral composition and development of plants. Proc. 8th Int. Coll.on Plant Analysis and Fertilizer Problems, Auckland, New Zealand, 1978.
SINNAEVE, J. Technical description of a growth chamber for the study ofsoil-plant-atmosphere interactions. J. Exp. Bot. (In press).
Lectures
SINNAEVE, J. Description d'une approche expérimentale pour 1'etude desinteractions sol-plante-atmosphere„ Unite de Physiologie végétale, Louvain-la-Neuve, Belgium.
SINNAEVE, J. An original growth-chamber for the study of plant-soil-atmosphere interactions. Division of Plant Industry (CSIRO) and ControlledEnvironment Research Laboratory (CSIRO), Canberra, Australia.
SINNAEVE, J. A growth chamber for the integrated study of the plant-soil-atmosphere interactions: influence of soil temperature on plant uptake.Division of Soils (CSIRO) and Waste Institute (University of Adelaide), GlenOsmond, Austral i a.
- 56 -
PROJECT No. 90 ANNUAL REPORT 1980
J. Sinnaeve, S.C. van de Geijn, M.J. Frissel, N. van der Klugt
BEHAVIOUR OF RAD I ONUCU DES IN BIOLOGICAL AND NON-BIOLOGICAL PROCESSES ATVERY LOW CONCENTRATIONS
The aim of the project was to investigate an observed aberrant be-haviour of two isotopes of the same element at very low concentrations.These results were reported in 197^. The data discussed here wereobtained under experimental conditions identical throughout the study.However, new counting equipment, i.e. a germanium-!ithium drifteddetector with a resolution of 2,1 keV at the 1,31 MeV photopeak of60Co, has been used. For all counting results, the peak areas of theidentified photopeaks (corresponding with the yphotons with 605 and796 keV for l3l*Cs and 662 keV for l37Cs) were computed by Gaussianfitting and base-line correction using a modified "GASPAN" fittingprocedure. After correction for radioactive decay, the Z-value ofthe sample, i.e. the ratio of the activity concentrations of 13ltCsand 137Cs in the plant material divided by that ratio in thereference sample, mostly the nutrient solution, was computed.
A. Biological aspects
Plant uptake experiments were carried out with intact tomato plants{.Lyaopersicon exculentum Mill,, cv. Marette VF). During the ex-perimental period, the nutrient solution contained stable caesium(mostly between 0,3 and 3 mg per liter), and 13l*Cs and 137Cs at theindicated activity ratio. The results of the key experiment (re-ported in 197*0 clearly indicated aberrant Z-values for the rootsamples at different activity ratios. After four years of storageof the samples, they were recounted with the new counting equipment.An extract of the results is given in table 1. The second countingconfirmed the anomalies reported previously and this led to severalexperiments in order to answer specific questions.
1. Is an absorption step involved which is linked to metabolism?Absorption experiments were carried out with nutrient solutionslabelled with 1,9 kBq/1 (0,05 pCi/1) 131|Cs and 90 kBq/1 (2,5 pCi/1)137Cs and kept at 22, 12 and 2 °C respectively during the ex-perimental period. To investigate whether a time dependentphenomenon was involved, two plants of each treatment were harvestedafter 150, 300, 450 and 600 minutes. The results are given intable 2. As no influence of time was observed, the mean values ofthe eight replicates per treatment are given. Taking into accountthe rather large standard deviations (can be up to 8%), it can beconcluded that none of the Z-values is significantly differentfrom 1.
2. Are different physico-chemical forms of the isotopes involved?Plants were grown on a mixture of a weak acid and a weak base ionexchanger saturated with the nutrient elements. 11, 7, 5, 3 and1 day before harvest of the plants (age 30 days), 20 ml of asolution containing 2k mg stable caesium per liter,1,9 kBq (0,05pCi) 13l»Cs and 900 kBq (25 yCi) 1 3 7Cs, were applied on top of thesubstrate. The Z-values were computed using as a reference sample
• i - "
- 57 -
Table 1 - Mean Z-values and their standard deviations (five replicates)for root samples using different labelling procedures
Countingdate
Dec. 1973
Dec. 1977
Dec. 1977
m.Csphoto-peak
keV
796
796
605
j
Mean Z-values + standard deviation1 3 l*Cs/ 1 3 7Cs activity concentrations in kBq/1 (uCi/l)
1,85/92,5(0,05/2,5)
2,11 + 0,05
1,92 + 0,09
1,73 + 0,11
18,5/185(0,5/5,0)
1,00 + 0,01
1,02 + 0,03
1,02 + 0,03
185/18,5(5,0/0,5)
0,78 + 0,02
0,77 i 0,05
0,78 + 0,06
92,5/1,85(2,5/0,05)
0,35 + 0,04
0,36 +0,10
0,36 + 0,10
Table 2 - Mean Z-values and their standard deviations for roots (R),stems (S) and leaves (L.) using different photopeaks of 13l|Cs
Temperature
22 °C
12 °C
2 °C
Plantorgan
R
S
L
R
S
L
R
S
13'*Cs photopeaks used
605 keV
1,00 _+ 0,02
0,99 + 0,01
1,00 + 0,01
0,98 + 0,03
0,99 + 0,03
1,02 + 0,01
1,00 + 0,03
1,00 + 0,02
796 keV
1,02 + 0,0í*
1,00 + 0,01
1,00 + 0,02
1,00 + 0,03
1,01 + 0,03
1,10 + 0,05
1,01 + 0,02
1,00 + 0,06
605 + 796 keV
1,01 + 0,02
0,99 + 0,01
1,00 + 0,01
0,99 + 0,03
1,00 + 0,03
1,05 + 0,02
1,02 + 0,02
1,01 + 0,04
the equilibrium solution of the substrate and the substrate itself.Given the extreme activity ratio used, no significantly deviatingZ-values were found.
3. Are biophysical exchange phenomena involved?Exchange chromatography using biological exchange columns (cutpapyrus stem - Cypemto papyrus L. spec, -with a cation exchangecapacity of about 8,10"J eq/m and a length of about 90 cm) wascarried out to check whether different isotopic behaviour at thesolution-membrane interphase might occur. Untreated, protonated andpotassium charged "exchange columns" were used. In total, somefifteen experiments with different acitivty ratios were carried outbut no aberrant behaviour could be observed.
- 5 8 -
B, Hon-bioloqical aspects
1 . Exchange chromatographyTo test whether aberrant behaviour at the solution-root interphasemight occur, exchange chromatography experiments with glass pearls,having a low cation exchange capacity, were carried out. Prior toexchange with a solution containing 1,9 kBq/1 (0,05 pCi/1) a31*Csand 900 kBq/1 (25 yCi/1) l^Cs - with and without 25 mq/1 stablecaesium -, the exchange sites were brought in the H -form. TheZ-values of all percolated fractions were normal
2. Counting equipmentThe response of the counting equipment towards various m C s / 1 3 7 C sactivity and concentration ratios in the absence of any physical,chemical or biological effects has been tested. The ratio of theactivity concentrations A of both isotopes varied in geometricsequence ranging from 13"^»5 to 132»5 (1,6 x 10"3 to 610) byexponential increments of 0,5. The logarithmic base 13 being equalto the ratio of the decay constants, the activity series can besuperposed to a mass concentration ratio ranging from 13~1»5 to133»5 (0,02 to 7900). The deviations of the measured activityratios from the prepared ones, expressed in percentage of thetheoretical values, are represented in figure 1. A systematiceffect exists which reduces the 137Cs/13l*Cs ratio when the activityratio is greater than 50.In collaboration with Drs. Myttenaere and Ronneau (U.C.L., Louvainla Neuve, Belgium) and Or. Guillot (CEC, Brussels, Belgium) theinfluence of counting rate on the ratio of the activityconcentrations of m C s and 1 3 7Cs was checked. The results givenin figure 2 illustrate a slight systematic shift over the entirerange of counting rates of the samples. These deviations cannotexplain the reported anomalies.
C. Conclusion
In the light of the reported data, it may be concluded that systematicdifferences in biological and non-biological processes of isotopes andstable elements at extreme concentration ratios are absent. Theanomalies observed before must therefore be attributed to an artefact- most probably a difference in physico-chemical form - and thisconclusion stresses the necessity of careful use and interpretationof experiments on and with radiotracers.
- 59 -
A 1 3 7 Cs/A 1 3 4 C•/•deviation -zs -10 -15 -1.0 -0.5 130 .0.5 *i.o .15 .2.0 .25
Measured Activity ratio
Cs by605keVp134Cs by 796 .,
-15 -1.0 -0.5 13O .0.5 .1.0 «1S
Figuře * - Deviations of act iv i ty ratios.
2.0 »ao .35| 1 3 7 C s l / l 1 3 4 C s j
- 60 -
Ratio
0,00.400
390
380
370
360
0.00.350
340
330
320
310
0.003000 2 * 6 8 10 cm
distance from the head of th» detector
Fig. 2 - m C s / 1 3 7 C s ra t io of a sample (based on the 795 keV photopeakof 13l*Cs) as a function of counting rate (decreasing with in-creas i ng dis tance).
- 61 -
PROJECT No. 91
P. Poelstra, M.J. Frissel, N. van der Klugt
ANNUAL REPORT 1980
THE CONTAMINATION OF VEGETATION, SURFACE WATER AND DEEP GROUNDWATER(DRINKING WATER) BY HEAVY METALS UNINTENTIONALLY RELEASED INTO SOILS
During the contract period two topics were studied: 1. Behaviour ofCd in soil and 2. Retention capacity of soils for contaminantspresent in percolation water from a refuse dump.1. Behaviour of Cd in soil
Within the study the following elements can be considerd: a. Cd ad-sorption and desorption measurements on soils. Data obtained wereused to predict accumulation in topsoils and leaching to subsoils,b. Determination of Cd leaching in soil columns. These data were usedto verify the predictions stated under a. c. Literature review on Cduptake studies by crops. The review provided data to predict the up-take of Cd by the crop from data measured under a. d. Further pre-dictions based on assumed Cd input data and calculations of Cdleaching to subsoil and Cd removal by the crop.
So U s
Five soils were used. Two of these soils have a higher than normalCd concentration, two of the other soils were specially selected tostudy the fate of Cd from phosphate fertilizer dressings.
j>andy_ jL°l'_B£ajisdTwej£: Slightly acid sandy soil used since 1895 forclumping sewage water with an average of 900 mm annually. Exchangecapacity top layer 31,6 m per 100 g, at a depth of 100 cm approx.2 me per 100 g. Total Cd content: top layer 3,5 mg Cd kg"1, decreasingto 0,75 mg Cd kg"1 at a depth of 40 cm, pH 6,5 at the top and 5,4 at adepth of 1 meter.
£ e£ t_ s£'I Schoonebeek: N.E. part of the Netherlands. Peaty soil usedas permanent pasture, organic matter content for layer 0-100 cm 95$,pH (H2O) 5,1. Total Cd concentration 1,4 mg-kg"1 in the upper 10 cm,decreasing to 0,2 mg-kg"1 at 20 cm depth.
Cl.a.y_so_i_!_ \/a_j_bu_rg_: Soil from the foreland of the Rhine river, used aspermanent pasture. Layer 0-30 cm: Fraction < 2 ym 23%, organic matter6?,, traces CaC03m, Layer 30-100 cm: Fraction < 2 pm 20%, organicmatter U , traces CaC03. pH (H2O) of both layers 7,4. Total Cd con-centration at soil surface 6,5 mg-kg"1 decreasing to 1 mg-kg"1 at adepth of 35 cm, and decreasing further to 0,5 mg-kg"1 at a depth of100 cm.
SanjJy. ̂p_N_D£e_t_ij]chem: Old arable land. pH (in artificial rain water)Z,Q. Organic matter "Ô-5 cm 6,3%, 5-10 cm 4,4%, at lower depths 4%.Exchangeable cations in 0-5 cm layer: K 0,4, Na 0,1, Ca 5,4 and Mg1,1 me per 100 g. Extractable Cd 0-20 cm 0,2 mg per kg soil.
Loss_s£iJ_ H_aasdaJ_. Arable land. pH (in artificial rain water) 5,1.Organic matter 0-5 cm 3,8%, 5-10 cm 1,9%. Exchangeable cations in0-5 cm layer: K 0,2, Na 0,1, Ca 5,1 and Mg 0,2 me per 100 g.Extractable Cd varies between 0,45 and 0,87 mg/kg for the upper 20 cm.
- 62 -
Adsorption was measured in 0,01 mole*!"1 CaCl2 or in a 1,7x10"^N mixture of chlorides of Ca, Na and K (ratio 3 : 1 : 1 ) ' The pHwas varied by HC1 or NťtyOH; pH and Cd in solution were measuredafter 24 h equilibration. Redox potential was varied by addingsmall amounts of glucose and keeping the sample vial airtight.Bacterial activity induced low oxygen concentration and low redoxpotential; equilibration time varied from 15 min to 1 week. Forhigh Cd concentrations stable Cd was determined by atomic absorp-tion, for low Cd concentrations 1 1 5 m C d was used as a tracer. Someresults are shown in table 1. It appears that adsorption is not afunction of the redox potential, but is strongly dependent on the pH.Adsorption curves were linear to 0,03 mmole I"1 for pH values up to6,5 and to 0,01 mmole I"1 for a pH up to 7. The influence of the pHon the sandy soil from Ooetinchem and loss soil from Haasdal isshown in fig. 1. The influence of the pH seems even more importantthan the soil type. The Kn (distribution ratio of Cd between soiland solution in g«cm"3) varies from 25 g-cm"3 at pH k,l\ to valuesbetween 600 and 700 g«cm~3 at pH values above 6,2. These differencesmean that the leaching properties must also vary by a factor of 20to 30, depending on pH. The differences in Cd uptake by the cropare probably somewhat less pronounced, however, enormous differencesin Cd uptake (for equal Cd levels in the soil) must be expected.This means that general recommendations for permissible Cd levelsin soil cannot be based on average values. The use of average valueswill induce high risks in particular areas, and at the same timeinduce excessive restrictions in other areas.
Transport was observed in undisturbed soil columns, length 120 cm,diameter 12 cm. The soil was non-watersaturated. Rain was appliedby a rain simulator. 1 1 5 mCd transport was measured by scanning (nondestructively) or by slicing the column and analysing each slide.There were aerobic and anaerobic sections in the column. Some resultsare shown in the figures 2 and 3. The Kn values which were used forthe calculations are indicated; in table 1 they are compared withthe KQ values obtained from adsorption/desorption measurements. Theagreement is good, except for anaerobic conditions. More data arepresented by Poelstra et al. (1979). From the data it can be con-cluded that there exists a good agreement between actual leachingand calculated leaching (based on adsorption/desorption measurements)Only the behaviour under anaerobic conditions is unpredictable. Withvery low redox potentials transport may cease completely.In a special experiment the transport of Cd at levels which mightresult from Cd containing phosphate fertilizers was investigated.A phosphate fertilization during 25 years, with an intensity of100 kg ha"1 y"1 of ? vnth a fertilizer containing 60 mg Cd per kg P,produces in the top layer of a soil (thickenss 1 cm) 1,25 mg Cd perkg of soil. Rainfall during 25 years is (in the Netherlands) about1800 cm of which approximately one third evaporates without causingany leaching. Another third passes the rooting layer and causesleaching. The last third penetrates into the soil but evaporates viasoil pores or is taken up by the crop and transpired. The effectiveprecipitation, i.e. the fraction which contributes to leachingranges, in a 25 years period, therefore from 600. to 1200 cm.
- 63 -
It was impossible to do a leaching experiment with this amountwithin the period which was available for experimentation. Theleaching experiment was therefore performed with 100 and 200 cmrespectively. The artificial rain contained an amount of Cd whichwould induce the previously mentioned 1 mg/kg Cd level. The Cdwas labelled with l l 5 mCd to facilitate the measurement of thedistribution within the soil after leaching. Soils: loess soilfrom Haasdal and sandy soil from Dcetinchem. Calculated andmeasured results of the Cd accumulation in the upper soil layerare shown in table 2. The agreement between calculations andobserved values is rather poor. This is probably caused by samplingerrors. The experiments were carried out with undisturbed soil mono-liths and it is difficult to separate exactly a one cm slice fromthe soil surface. The concentration in the 0 to 0,5 cm layer isabout three times as high as in the 0,5 - 1,0 cm layer. Smallsampling errors induce therefore large errors in the values ofthe Cd content. (This difficulty had been previously realized, buttime did not permit a longer lasting period with its more accuratemeasurements). Despite the poor agreement it is assumed that theKQ values as shown in fig. 1 can be used to predict the fate of Cdwhich is, together with the fertilizer, sdded to a soil.Table 3 shows the expected accumulation in the top cm of a soilfor 1000 ml of effective precipitation. (This represents thus aperiod of 25 to 50 years).It appears that the accumulation is considerably counteracted byleaching. Even with a very high KQ value of 800 (maximumaccumulation) only 50% of the added amount is still present in theupper cm. Table 3 shows once again that the use of mean values forCd accumulation is not applicable.
_íi£Sl£!i_žÍyY showed that Cd is taken up by crops rather well.When a dry matter production of 1 kg per 500 kg transpired water isassumed it appears that between 20 and 100% of the Cd present in thesoil solution which is taken up by the crop, is transferred to the
, crop. This fraction is called the selectivity coefficient. Of coursenot all Cd is removed together with the crop. Yet is can be concludedthat the Cd removal via the crop and by leaching is considerable.There are many situations in which a moderate supply of Cd will be
• compensated for by a removal of Cd. An example of such a calculationis shown in Fig. k. The Cd supply is set at 0,2 kg-ha" 1^" 1, the
: amount of drainage water is set at 20 ml-cm" 2^" 1 and the trans-piration by the crop at 20 ml-cm" 2^" 1.For more details see Fig. k and Poelstra et al. (1979). Fig. k shows
• that for Kn values as they were found in the sandy soil fromj Braunschweig the Cd level reaches an equilibrium at about 10 kg CdI per ha (layer 0-30 cm). For Kn values as measured for the clay soil[ from Valburg the equilibrium level is not reached even after a period| of a few hundred years. (Although it is easy to extend the calculationsj to longer periods, this is not done; the model is not sophisticated| enough for such calculations).[•
f-
2. Retention capacity of soils for contaminants present inpercolation water of a refuse dump
The study was carried out in cooperation with the Stichting Ver-wijdering Afvalstoffen at Amersfoort, The retention was studiedby supplying the percolation water from a refuse dump to soilcolumns in the laboratory.The percolation water was obtained from the refuse dump at Ambt-Delden. During sampling, transport and addition to the soilprecautions were taken to prevent oxidation effects within thepercolation water. For the filtration experiments undisturbedsoil columns were used. Soil types were: sandy soil, peaty soil,sea clay soil and river clay soil. The following observationsw?r e made in the percolation water from the refuse dump, in thesoils (as function of depth) and in the effluent from the columns:pH, T0C (Total organic carbon content), chloride, bicarbonate,nitrogen (Kjeldahl), phosphate, sulfates, calcium, chromium,potassium, sodium, magnesium, manganese, nickel, iron and zinc.From the soils the redox potential was also measured. The redoxpotential is an important variable. In a soil treated withperculation water from a refuse dump it is much lower than in anormal soil. This induces adsorption and desorption reactions,which mainly for heavy metals differ from those of a normal soil.
In general, adsorption and desorption are in agreement withexpectations based on redox-pH diagrams, physico-chemical adsorptionrules, etc. Purification of the influent in the sandy soil was low,this must be attributed to a low exchange capacity and a low pH.The redox potential decreases, but not sufficiently so that sul-fates are not reduced to sulfides and heavy metals are precipitatedas sulfides. Purification is highest in the peat soils,attributable to physical adsorption, but does not reach 100 percent. Both clay soils retain zinc well but only 50% of the nickeland chromium is retained. The redox potential is sufficiently lowso that numerous heavy metals precipitate as sulfides and manganeseoxides are reduced to soluble products. Thus manganese is leached.Iron is adsorbed in all four soil, probably as an organic-ironcomplex. Adsorption of organic matter is not very pronounced inany of the four soils.
Details and a discussion of the accuracy and reproducibi1ity of thestudy, possibilities for extrapolation, etc. are discussed in thereport "Onderzoek naar de f iltereigenschappen van de bodem voorpercolatiewater afkomstig van vuiIstortplaatsen" by Frissel et al.(1980).
ŕ 1
I \í - s
- 65 -
Table 1 - Some Kn values for Cd
Soil
Val burg, clay
Schoonebeek,
Braunschweig
Val burg, clay
Schoonebeek,
Braunschweig
Braunschweig,
soil
peat soil
0-20 cm
30-40 cm
0- 5 cm
0- 5 cm
0- 5 cm
0- 5 cm
soi 1
peat soil
anaerobic
hg-cnT3
670230
3318
5.6
4,951
1100
710
320
3343
pH
7.5.6.6,
5.4,6,
8,
41
5
50
8
75
EH
mV
143206
206
206
Highestads./des
mmole
0,
0,
o,0,
0,
o,0,
o,
cone. i n, series
r1
01
01
03
03
2
2
02
001
Remarks
ads./des.
ads. /des.
ads./des.
ads./des.
ads./des.
ads./des.
ads./des.
ads./des.
column
column
column
column
Table 2 - Accumulation of Cd in top cm of soil as percentage of the totalamount supplied. Continuous supply. Calculated results based onKQ values of fig. 3, experimental results see text. Cd levelless than 1 mg per kg of soil.
Loess soil , Haasdal
rain supplied
102,5 ml
calc.
64,2
exp.
42,2
200
calc.
45,4
ml
exp.
71,3
Sandy soil, Doetinchem
rain supplied
102,5 ml
calc.
89,7
exp.
84,9
200 ml
calc.
81,4
exp.
67,6
- 66 -
Table 3 - Accumulation of Cd as percentage of the total amount supplied.Continuous supply. Calculated values for an effective amountof precipitation of 1000 cm (25 to 50 years) and a soil densityof 1,2 g dry matter per cm3 of bulk soil.
g«cm~3
25
50
100
200
1*00
800
Accumulated fractiont
1,9
M8,2
16,5
31,5
51,7
PublicationsBANNINK, D.W., P. P0F.LSTRA and M.J. FRISSEL. An installation forleaching and accumulation studies on undisturbed soil columns.Netherlands J. of Agricultural Science, 25 (1977): 126-133.
POELSTRA, P., M.J. FRISSEL and N. EL-BASSAM. Transport andaccumulation of Cd-ions. Z. Pflanz. u. Bodenk. V}2 (1979):8it8-86<t.
FR.ISSEL, M.J., P. POELSTRA, D.W. BANNINK en R.M.J. PENNDERS.Onderzoek naar de f i 1tereigenschappen van de bodem voor percolatie-water afkomstig van vui lstort.plaatsen. ITAL, Wageningen (1980).
I
- 67 -
1000
500
100
50
10
o
a sandy soil• lotu soil
7 pH
Fig. 1 - The Kp value of Cd, in g-cm"3, for a loess soil (Haasdal)and a sandy soil (Doetinchem). Cd level 1 mg Cd per kg soil.Natural pH in simulated rainwater (1,7 x 10"4 N mixture ofNaCI, KC1 and CaCl2) of the loess soil is 5,1, of the sandysoil 6,0 respectively.
mg Cd hg~' »•!2OQ 400 600
dmut
5
10
19
70
^ ^
DtttSO* OamnfW
ŕ
clay sail Vaiourg
r caiľirtr « r«»«-Bt«ní- ,"?a ——
Fig. 2 - Calculated and observed distribution of Cd in undisturbedsoil columns. The time elapsed since the beginning of theapplication of Cd is indicated near the explanation of thecurves. Cd concentration of the influent: 5 mg-l"*. Furtherdetails: Peat soil: influent rate approx. 1 cm-d"1, KQ = 320;Clay soil: influent rate approx. 0,7 cm-d"1, KQ = 710; Sewagefield: influent rate approx. 2 cm ď"1 during the first \k& d,thereafter approx. O,** cm^d"1, Kp = 33.
•~7 J
- 68 -
depthcm
10
15
20-
mg Cd kg'1 soil100 200 300
concentration l1SmCd
r J
Sewagelield BraunschweigI anaerobic conditions
calculation observation275 d
Sewogelield Braunschweiganaerobic conditions
calculation observation163 d209 d
Fig. 3 - Calculated and observed distirbution of stable and radio-active Cd in undisturbed soil columns. Cd concentration ofinfluent 5 mg*!"1, influent rate approx. 2 cm.d"1, KQ = k"}.
kqCd/ha
10
30
20
10
K, =1000
/ ^ - K„'3J
f ^ 1 . 1
-
KQ31Q00
/ ^
• -
i
yr
• / ^
100 200 100 200 100 200 year
Fig. k - Accumulation of Cd on arable land. Kn values are indicated.From lefthand side to righthand side: selectivity coefficientfor Cd uptake: 10%, 30% and 100%.(With a layer thickness of30 cm and a density of ̂ ,k, 5 ppm Cd eqals about 20 kg Cdper ha).
- 69 -
SUMMARY OF THE 1 9 8 0 ANNUAL REPORT OF THE P L A N T N U T R I T I O N - S O I L S
RESEARCH GROUP INVOLVING PROJECT 9 3 .
Nitrogen cycles
The first phase in the development of computer simulation models of theterrestrial nitrogen cycle was ended with the organization of a small inter-national meeting of scientists who have modelled different aspects of thenitrogen cycle. Besides discussing on the several models, the participantsdiscussed the present state of knowledge on classification and on the soilphysical, plant physiological and microbiological aspects of the soil N-cycle in relation to modelling.
The work on the nitrogen cycle in a short rotation poplar forest was finished.Based on the experimental results presented in the annual report of 1979 anitrogen balance was made. At the present production level, the system needsan annual nitrogen input of approx. 120 kg^ha"1. Extrapolation to higherproduction levels, indicated that in spite of higher N-fertMizer inputs,the net energy production might increase considerably.
The effect of the matric potential of soil moisture on the microbial activitywas simulated by growing microorganisms isolated from soil on top of a semi-permeable membrane that was in contact with a solution of PEG 20,000 in amedium suitable to the particular microorganism. Microbial activity wasdetermined from measurements of the (^-production. The five microorganismsthat were used, reacted very differently to osmotic stress, with fungi show-ing the smallest effect, i , "
• r . . ' ' v 1 - . '... . •' •- ' - , . • " ' ,
- 70 -
PROJECT No. 93
J.A. van Veen, M.J. Frissel, H, Breteler
ANNUAL REPORT 1980
EXPERIMENTAL ANALYSIS AND COMPUTER SIMULATION OF NITROGEN CYCLING IN ATMO-SPHERE-SOIL-PLANT INTERRELATION
1. Modelling terrestrial N-cycle
The first phase in the development of computer simulation models on theterrestrial N-cycle was completed with the organization of a small inter-national meeting of scientists who have modelled different aspects of thenitrogen cycle. It was held from 28 January to 1 February 1980 and wassupported by the International Union of Biological Sciences (IUBS), Paris,the Scientific Committee on Problems of Environment of the InternationalCouncil of Scientific Unions (SCOPE) and the Dutch Ministry of Agriculture.
The first two days were spent in open sessions with all participantslistening to lectures on the models (working papers had been circulatedpreviously) and discussing the papers in depth. Separate panels then dis-cussed the Classification, soil physics, soil microbiology and plantphysiology of the various models. They reviewed the "state of art" andidentified the factors limiting progress..
The main conclusions on the present "state of art" were with respect to:
- soil physical aspects: the physics of water movement through weaklystructured, non-swelling soils are sufficiently wel1 understood tojustify the use of mechanistic type models. This is also valid withrespect to transport of solutes such as NO3. However, the descriptionof those processes in well-structured soils containing cracks and largepores, where preferentially leaching occurs, is much more difficult,although a limited number of conceptual models exists. Those descriptionsare, however, not coherent with descriptions of other processes of theN-cycle. Similar problems occur when describing the gas diffusion pro-cesses related to denitrification and ammonia volatilization.
- plant physiological aspects: the understanding of transport processesin soils and plants is much greater than our understanding of rootdevelopment, root function and nitrogen nutrition. It is recognizedthat major limitations to progress are the lack of technology forstudying roots in the soil and the problems of val i dat ing complex models.
- soil microbiological processes: data on control mechanisms areparticularly lacking. Progress is limited by the lack of re 1 i able methodsfor measurement of growth and activity of the microbial biomass and forfractionating soil organic matter to quantify the several components,that are utilized by microorganisms as substrate. A serious restrictionis the lack of data on the combined effect of environmental factors,such as temperature, pH, moisture, on the soil microbtal activity.
In the second phase of this study the results and experiences, that wereobtained during the development and use of the models, will be used tostudy the behaviour of N in the soil-plant interphase.
2. Nitrogen cycling in a short-rotation poplar forest
This work was completed with the development of a nitrogen and energybalance, which was based on the results presented in the annual report of1979. The balance is schematically presented in Fig. 1.
- 71 -
Based on this balance the system needs an annual inorganic N input of122 kg ha"1 under the present conditions.
Table 1 shows that the present annual production of ľ» tons dry matterper hectare had a net energy output of 5k *»00 MJ-ha"1. An increase ofthe dry matter production to a theoretical maximum of approximately25 ton-ha'i-y1, which seems possible when planting the trees at higherdensity, would raise the net energy output to 96 700 MJ-ha-^y"1, inspite of higher fertilizer applications. Although this study showed thatat the present dry matter production level short-rotation forestry is anenergy-producing system both the dry matter production and the energyprices have to increase considerably to make the system economicallyfeasible (see Annual reports of 1979 and 1980).
Table 1 - Energy balance of a short rotation forest system
Production level (dry matter)
INPUTS
Fuel, electricity
Fertilizers, chemicals
Depreciation
Other costs (including irrigation)
Transport
Total
OUTPUTCombustion
MET OUTPUT
ľ» 000 kg/ha/y
MJ
3 0006 000
6 000
2 000
7 0002t» 000
78 toO
5k 'tOO
25 000 kg/ha/y
MJ
5 000
13 3008 000
k 000
12 500
43 300
1̂ 0 000
96 700
3. Moisture stress on microbial activity
The osmotic effect of soil moisture on the activity of four soil micro-organisms, two bacteria, a fungus and a yeast, was simulated by growingthe organisms on top of a membrane, that is in contact with media ofvarious osmotic potentials. Osmotic stresses were effected by additionof polyethylene glycol, 20000 (PEG 20000) to the medium. The activity ofthe microorganisms was determined by the measurement of the CO2 productionduring 150 hours incubation period at room temperature. Fig. 2 shows atypical relation between osmotic tension and.CO2 production.Table 2 gives the effect of osmotic stress on the activity of the micro-organisms related to the activity of the organisms when no PEG 20000 isadded to the medium. Obviously, soil microorganisms react very different-ly on osmotic stresses with the fungus showing the smallest effect. Thisproject will be continued by incubation at different temperatures andosmotic potentials to determine the combined effect of temperature andmoisture.Analysis of the cell content of the microorganisms grown under thevarious conditions will also be carried out, with special reference toK, Na, Mg and Ca, which are known to play an important role in theosmotic regulations of microbial cells.
••"' :•<":[ r
- 72 -
Table 2 - Effect of osmotic stress on the activity of soil microorganismsafter incubation during 100 h at room temperature related tothe activity without osmotic stress
Osmotic StresskPa
0
300
700
2000
Bacterium 1
1,00
1,00
0,52
0,17
Bacterium 2
1,00
0,<t1
0,20
0,15
Fungus
1,00
1,000,8/*
0,75
Yeast
1,00
0,56
0,21
0,11
Pub!ications
FRISSEL, M.J., J.A. VAN VEEN and G.J. KOLENBRANDER. The use of submodels inthe simulation of nitrogen transformations in soils. In; A. Banin & U.Kafkafi (eds.) Agrochemicals in soils, p. 253-265 (1980). Pergamon Press,Oxford,
FRISSEL, M.J. and J.A. VAN VEEN. Soil-nitrogen transformations in relationto leaching. In: Soil nitrogen as fertilizer or pollutant. Proč. IAEA/FA07GSF Coord, meeting, Piracicaba, Brasil, p. 61-75 (1980).
VAN VEEN, J.A., W.B. McGILL, H.W. HUNT, M.J. FRISSEL and C.V. COLE.Simulation models of the terrestrial nitrogen cycle. In: Clar, F.E. andRosswall, T. (eds.) Nitrogen cycling in terrestrial ecosystems. Ecol. Bull.(Stockholm) 33, 25-48 (1981).
FRISSEL, M.J. and J.A. VAN VEEN. Some aspects of irrigation relevant to theterrestrial nitrogen cycle. In: Clar, F.E. and Rosswall, T. (eds.)Nitrogen cycling in terrestrial ecosystems. Ecol. Bull. (Stockholm) 33.603-6T* (1981). —
FRISSEL, M.J. van J.A. VAN VEEN. Validation of a simulation model fornitrogen immobi1ization and mineralization. In: fskander, I. (ed.)Simulating nutrient transformations and transport during land treatment ofwastewater. Chapter 13, Wiley £ Sons Publ. (In press).
VAN VEEN, J.A. and E.A. PAUL. Organic carbon dynamics in grassland soils.I. Background information and computer simulation. Can. J. Soil Sci.(accepted for publication).
FRISSEL, K.J. 6 J.A. VAN VEEN (eds.).: Simulation of nitrogen behaviour insoil-plant systems. 277 pp. Pudoc, Wageningen (1981).
VAN VEEN, J.A. and M.J. FRISSEL. Review of simulation models for nitrogenbehaviour in soil in relation to plant uptake and emission. IIASA, Vienna,Austria, (accepted for publication) (1981).
FRISSEL, M.J. The definition of residence times in ecological systems.Ecol. Bui K (Stockholm) 2i, 117-121 (1981). .
Lectures
VAN VEEN, J.A. Simulation model of the behaviour of N in soil. Internationalmeeting on N-modelling in soil-plant systems. January 28 - February 2.
VAN VEEN, J.Ai Computer simulatie model len in de bodembiologie. Contactcie.Bodembiologie, April, Wageningen.
- 73 -
VAN VEEN, J.A. Review on nitrogen models in soil-plant systems. IAEA/FA0/GSF-seminar on nitrogen residues, 25th - 29th August, Vienna, Austria.
VAN VEEN, J.A. Modelling nutrient cycling in agro ecosystems. Intern. Symp.on nutrient cycling in agro ecosystems, 22nd - 26th September. Univ. ofGeorgia, Athens, CA, USA.
VAN VEEN, J.A. Modelling terrestrial nitrogen cycle. Univ. Claude Bernard -Lyon, France. November 1980.
FRISSEL, M.J. Modelling the nitrogen cycle in agro ecosystems with emphasison disturbance of the environment. I(ASA, Vienna, Austria, 2nd - 6th June.
FRISSEL, M.J. Modelling the nutrient cycle, ESNA, Debrecen, Hungary,25th - 29th August,
FRISSEL, M.J. The use of models for improvement of fertilizer recommendations,Collegios Postgraduadas, Chapingo, Mexico, 22nd - 26th September.
FRISSEL, M.J. Survey of existing nitrogen models. Uppsala, Sweden, 21st -2<tth October.
C 18
Fig. 1 - Schematic presentation of the nitrogen cycle in a short rotationpoplar forest. The figures indicated with asterix are amounts ofN (kg.ha--} after a growing period of 5 years. Other figures(except for the soil-N content) refer to annual N fluxes in kg.ha"1
Ji.
60
~ 50
sQ.
LJ 30
-§ 20
10
Effect of PEC 20,000 on theactivity of a yeast.
fov. PEG
10 V . PEC
a
50 100 150
hours
Fig. 2 - Effect of PEG 20 000 on the activity of a yeast.
- 75 -
SUMMARY OF THE 1980 ANNUAL REPORT OF THE PROPERTIES OF CROP PLANTS
RESEARCH GROUP A, INVOLVING PROJECTS 95, 99 and 120.
Nutrient uptake and (re)distribution
Nitrogen metabolism, and the complexatipn of copper and cadmium in therhizosphere and during xyletn transport were the main topics investigated in1 9 8 0 . ••. . ; -• . ••';• •• -.•.-. - . . . • • ' . . • ' ; - • _ • . ;• ••• • '
The fixation of atmospheric nitrogen by Azolla species and the subsequentuse of Azolla as an organic nitrogen manure is a common agricultural practicein a number of tropical countries. Research was carried out on the optimalcombination of clones and environmental conditions for the cultivation ofthis inexpensive source of nitrogen for crop production. The temperatureoptimum for nitrogenase activity was about 30 °C for A. pimata and con-siderably lower for A. filiauloidea. In view of the new research programmepreliminary experiments were performed on the colonization of Rhisobia inthe rhizosphere of a legume and a non-legume.
During initial nitrate nutrition, nitrate utilization by dwarf bean plantsis localized mainly in the root system. Two key-steps in the uti1izationpathway, namely the uptake and reduction of NOJ, were affected by the carbo-hydrate status and the hormonal balance of the roots. Uptake and reduction,however, were not similarly affected by for example plant growth regulatorsor exogenous sugars, indicating separate control mechanisms for uptake andreduction.
Binding of cadmium in the rhizosphere of tomato plants was confirmed bydigestion of the nutrient solution after removal of the roots. This procedureconverted all the bound Cd to the ionic form (Cd2+). Binding of Cd2+ was notobserved in the rhizosphere of barley plants, the upward transport of copperin cut papyrus stems was enhanced by carboxylates and ami no acids. A modelstudy was made on the effect of complexing substances on the partition ofcopper over the liquid phase and the adsorption sites of a synthetic ionexchange resin. Research on the occurrence and biological significance ofion complexation by plant-borne compounds and its consequences for plant-growth and nutrition will be continued. ' • ! •
- 76 -
PROJECT No. 95
D.H. Ketel, S.C. van de Geijn
ANNUAL REPORT 1980
THE MINERAL NUTRITION OF AGRICULTURAL CROPS IN RELATION TO THE COMPOSITIONOF THE NUTRITIONAL MATRIX WITH EMPHASIS ON THE NITRATE AND PHOSPHATE AB-SORPTION. RELATED MODEL STUDIES
1. Cation transport in plantsa •Our previous work on cut stems of Cyperua papyrus L, spec, as a modelsystem to study xylem transport was continued. The effect of variouscarboxylates (citrate, ma late) and amino acids (cysteine, methionine,glycerine) and of EDTA on the mobility of copper in the xylem wasexamined. The cut end of stems was placed into 64Cu-label led CuSOj,solutions (lO'^mole-dm"3, pH 6,5), without or with carboxylates or aminoacids at molar ratios of 0,5, 1 and 10, relative to Cu. The mobility ofCu in the xylem increased with all organic additions of citrate, with acitrate: Cu ratio of 10, being the most effective. Malate and glycinestimulated mobility much less, though still significantly as compared tothe treatment without organic additions. A quantitative expression of theresults is difficult because the organic Cu-complexes turned out to besubject to continuous association and dissociation, resulting in anappreciable loss of copper to the exchange sites on the walls of xylemvessels. In case of Cu-EDTA, no dissociation occurred and the entirecomplex moved along with the transpiration stream at a speed of 10 to 15m«h~l. The Cu-carboxylate complexes migrated at a speed of 5 m-h"1
(citrate) down to 0,1 m-h"1 (malate). Copper bound at the vessel wallswas only partly removed after flushing the xylem with carboxylates oramino acids. In this respect too, citrate was the most and malate theleast effective compound.
Our results show that organic ligands have an important effect!on the up-ward transport of copper and indicate the possible significance ofsimilar complexation processes at or near the root surface for themineral nutrition of plants. Part of this work will be continued in theframe-work of a new project on the impact of rhizosphere processes onplant growth.
Pa£tit^on_of_cogger_over_carboxyl_atesi_amj_ng_ac_iids_and_iion_excnange
i i I I ISolutions containing Cu2+, carboxylates, amino acids and EDTA, asdescribed above (see a.) were prepared to study the kinetics ofequilibration between the solution and a synthetic ion exchange resin ofthe carboxylate type (Dowex CCR-2). At equilibrium (after 1-2 d) thesolutions were devoid of Cu, except in the presence of EDTA. The variouscomplexing agents hardly affected the time required to reach equilibrium,probably because of the dominating effect of "particle diffusion" at thebead size used (0,5 mm). Citrate tended to shorten the equilibrationperiod. The latter effect of citrate could play a role in absorption oradsorption processes in the rhizosphere. Its biological significance,however, can only be assessed after further experimentation.
I
- 77 -
c, Plgsmo^sis^of_root_endoderma^_cgiis
Optimal conditions for the observation of band plasmolysis in endodermalcells of maize were established. During this type of plasmolysis, theplasmalemma remains attached to the Casparian strip, resulting in a lens-shaped band of cytoplasm in the plasmo)ysed cell. We examined whether theeffect of polyvalent cations on the plasmolytic pattern is related to theuptake and transfer of ions from the rhizosphere into the stele of rootcells. Along the rood, the occurrence of band plasmolysis coincided withthe primary development of the endodermis. Research on the relationbetween plasmolytic pattern and ion uptake will be continued in 1981.
Characterization of cadmium uptake and compiexation in the root medium oftomato plants and barley
In the 1979 annual report, we described a rapid complexation of Cd2+ inthe presence of tomato roots. Similar experiments were done with roots ofintact barley plants {Hordem vulgare L. cv. Piccolo). Complexation wasestimated as the difference between total Cd (115mCd) and ionic Cd (Cd2+
electrode). The difference between both measurements never exceeded 7%and turned out to be not significant. For tomatoes, in contrast, asignificant difference ranging between 10 and 30% was found. To avoidproblems in the exact calculation of initial Cd concentrations, thedepletion of Cd and Cd2+ in the medium was computed as percentage of theinitial concentration for each method (Fig. 1). In an experiment withrepetitive provision of Cd to barley plants, most of the given cadmiumdisappeared from the medium within 20 min. The curves of figure 1 clearlydemonstrate the suitability of the Cd 2 + electrode to monitor rapid changesin Cd concentration of a nutrient solution.
Experiments with intact tomato plant roots were repeated and the re-maining nutrient solutions were digested with HNO3. Cadmium was measuredin the digests with a callibrated electrode, and the amount of acid-liberated Cd 2 + was compared with the amount of bound Cd, calculated asdescribed above. All of the Cd bound in a Cd-cysteine mixture was re-covered by the chosen method of digestion and the method also turned outto be adequate to free bound cadmium from nutrient media, in which theplants had been grown (Table 1). In line with other data for barley, noacid-liberated Cd was measured in barley media.
Our data thus supply further evidence for the rapid binding of Cd 2 + inthe presence of roots of tomato, but not of barley plants. Work on ioncomplexation in the rhizosphere will be continued in the frame-work ofthe new research programme.
- 73 -
Table 1 - Total, ionic and bound cadmium in a stepwise Cd 2 + (labelled with115mCd) addition experiment with tomato plants. Total Cd wasestimated by measuring radioactivity and correction for specificactivity, ionic cadmium by a Cd2+electrode, and bound Cd as thedifference between the data obtained by tehse methods. At the endof the experiment Cd2+ was measured in the solution before andafter digestion with HNO3, Each time step represents 12 min andthe amount of Cd is given in ug. The experimental volume was 0,5dm3.
1
2
34
56
total
Cd2+Cd*+
supplied
257
6051356
2777
5712
11760
22467
Decrease perelectrode
before digestionafter digestion
111
286
701
1573
2352
5740
10763
stepnamed
1,65 x 10"*»3,67 x 10-1*
51
70
131610
3546781894
Boundper step
60
216
570
9631998
5062
8869
mol e-dm1"3 (a) ,mole»dm"3 (b)
Cdtotal
60
276846
1809
3807
8869
a/b = 0
totaltotal
l.r
Cd boundCd
0,
0,
0,
0,
0,
0,
present
29
37
4344
40
43
Publi cat ions
VAN BALEN, E., S.C. VAN DE GEIJN and G.M. OESMET. Autoradiographic evidencefor the incorporation of cadmium into calcium oxalate crystals. Z. Pflanzen-physiol. 97 (1980): 123-133.
VAN DE GEIJN, S.C. and F. SMEULDERS. Calcium translocation towards leavesand meristems in tomato and maize plants: a mechanistic approach. PlantPhysiol. 65S (1980): 60.
VAN DE GEIJN, S.C. and F. SMEULDERS. Diurnal changes in the flux of calciumtowards meristems and transpiring leaves in tomato and maize plants. Planta(In press).
Lectures
KETEL, D.H. Beschouwingen over Cd-opname door intacte worte1systeme,n met hetoog op een door Cd geTnduceerde exudatie van Cd-complexerende organische ver-bindingen. Colloquium Landbouwhogeschool, Vakgroep Plantenfysiologie, Wage-ningen, februári I98O.
KETEL, D.H. Introductie van het HPLC systeem ten behoeve van snelle en accu-rate scheidingen. ITAL, november I980.
Travel Report
VAN DORP, F., D.H. KETEL, J.K. MARTIN and R. MERCKX.
- 79 -
60 minute
Fig. 1 - Time course of total Cd ( x — x , 1 1 5 mCd assay) and ionic Cd2+
(electrode assay) after three successive Cd*+ additions (arrows)to barley plants. Initial Cd2+ concentration 17s5 vitnol •dm"
3„
vil-
- 80 -
PROJECT No. 99
H. Breteler, Ch.H. HSnisch ten Cate
ANNUAL REPORT 1980
NITROGEN NUTRITION OF PLANTS IN RELATION TO ENERGY BALANCE AND ACCUMULATIONOF AHI NO ACIDS AND PROTEINS
Research on nitrate uptake and nitrate reduction by dwarf bean was continuedwith alterations being focused mainly on the role of sugars and plant growthregulators. More information about the regulation of initial nitrateutilization by dwarf bean will be published in subsequent years, and someof our work will be continued in the frame-work of a project on ionicbalances in the rhizosphere.
Sugar status and nitrate utilizationNitrate uptake and in vivo nitrate reductase activity (NRA) in roots ofľhaseolus vulgcwis L. cv, Witte Krombek were measured in nitrogen-depletedplants of varying sugar status. Variation in sugar status was achieved atthe start of nitrate nutrition by excision, ringing, darkness or administr-ation of sugars to the root medium.
The shape of the apparent induction pattern of nitrate uptake was not in-fluenced by the sugar status of the absorbing tissue. When measured after 6h of nitrate nutrition (0,1 mmole-dm" 3), steady state nitrate uptake androot NRA were in the order intact > dark > ringed > excised. Exogenoussucrose restored NRA in excised roots to the level of intact plants. Thenitrate uptake rate of excised roots, however, was not fully restored bysucrose (0,03 - 300 mmole-dm"3).
When plants were decapitated after an 18 h NO3 pretreatment, the net uptakerate declined gradually to become negative after three hours. This declinewas slowed down by exogenous fructose, whilst glucose rapidly (sometimeswithin 5 min) stimulated NO3 uptake. The NRA of excised roots was alsohigher in the presence of glucose than in the presence of fructose after 6h of nitrate nutrition presumably due to a difference in NO3 uptake. Thesugar-stimulation of oxygen consumption as well as the release of [ll|C]02from freshly absorbed (U-llfC) sugar was the same for glucose and fructose.Therefore, we propose a glucose-specific effect on NO3 uptake that is dueto the presence of glucose rather than to its utilization in root respiration.A differential glucose-fructose effect on nitrate reductase activity in-dependent of the effect on NO3 uptake was not indicated.
A constant level of NRA occurred in roots of NO3 induced plants. Removal ofnutrient nitrate from these plants caused an exponential NRA decay with anapproximate half-life of 12 h in intact plants and 5,5 h in excised roots.The latter value was also found in roots that were excised in the presenceof nitrate, indicating that the sugar status primarily determines theapparent rate of nitrate reductase decay in excised roots.
Plant growth regulators and nitrate utilization
We examined the effect of pretreatments (18 h at 5 nmole-dm"3) with abscisicacid (ABA), the ethylene-releasing substance ethephon, gibberellic acid(GA3), indoleacetic acid (IAA), kinetin (Ki) and zeatin on nitrate uptakeand in vivo NRA in roots of nitrogen-depleted Phaseolus vulgaris L. Nitrateuptake showed an apparent induction pattern with a steady state after 6 h,in all treatments. Initial nitrate uptake, in proportion to steady stateuptake, was somewhat slower after pretreatment with ABA, IAA or ethephon.
[£&»flh!í£SE£iŤEI
- 81 -
The absolute rate of nitrate uptake after 6 h was unaffected or at most 30%lower after treatments with plant growth regulators (PGR's) at 5 ymole-dm"3.However, Ki at 25 umole-dnf3 inhibited NO3 uptake by 80%.
The cytokinins, Ki and zeatin, and GA3, induced substantial NRA in roots in theabsence of nitrate. Ethephon enhanced NRA only during nitrate nutrition.Kinet in-induced NRA (Ki-NRA) was maximal after a Ki pretreatment concentrat-ion of 1 pmole-dm"3, and showed a lag phase of 6 to 8 h. Ki-NRA was additiveto N03-lnduced NRA (NO3-NRA) for at least 24 h, independent of the inductionsequence. After full Induction, Ki-NRA was approximately 20fc of NO3-NRA.ABA counteracted the development of KI-NRA, but not of NO3-NRA. Cyclo-heximide and tungstate were equally effective to suppress nitrate reductaseinduction by Ki or NO3.
Our data are consistent with the operation of two independent enzymefractions (Ki-NRA and NO3-NRA) with identical properties but with separatecontrol mechanisms. The absence of major effects of PGR's on the time-course and rate of nitrate uptake suggests that exogenous regulators andpossibly endogenous phytohormones are of minor importance for initialnitrate uptake. The differential effect of some regulators on nitrate up-take and root NRA furthermore indicates that the processes of uptake andreduction of NO3 are not obligatory or exclusively coupled to each other.
Publications
BRETELER, H. and Ch.H. HANISCH TEN CATE. Fate of nitrate during initialnitrate utilization by nitrogen-depleted dwarf bean. Physiol. Plant. Jt8(1980): 292-296.
BRETELER, H. and Ch.H. HANISCH TEN CATE. Effect of glucose on nitrate up-take by bean roots not linked to respiration. Plant Physiol. 65S (1980): 57.
HANISCH TEN CATE, Ch.H. and H. BRETELER. Role of sugars in nitrateutilization by roots of dwarf bean. Physiol. Plant, (in press).
HANISCH TEN CATE, Ch.H. and H. BRETELER. Nitrate utilization and phyto-hormones in roots of dwarf bean. Fed. Eur. Soc. Plant Physiol. (FESPP) 1_(1980): 395-396.
BRETELER, H. and Ch.H. HANISCH TEN CATE. Effect of nitrogen history onnitrate utilization by dwarf bean. Fed. Eur. Soc. Plant Physiol, (FESPP) 2(1980) 246-247.
HANISCH TEN CATE, Ch.H. and H. BRETELER. Nitrate utilization by dwarf bean.Kurzfassungen Botaniker Tagung Bochum (1980): 168.
Lectures
BRETELER, H. Stikstofomzettingen in gewassen. PAGV Leiystad, februári 1930.
BRETELER, H. Stikstofopname en de ionenbalans van hogere planten. CABO,Wageningen, maart 1980.
BRETELER, H. Ionic balance in relation to nitrogen conversion. University ofLeeds, September 1980.
BRETELER, H. Nitrate uptake and nitrate assimilation in plants - Currentaspects. MacauIay Institute, Aberdeen, September I98O.
BRETELER, H. Effect van phytohormonen op de nitraatbenutting door stamsla-bonen. B ION Biolog ische Stikstofkringloop, Leiden, november 1980.
- 82 -
Travel Reports
BRETELER, H. and Ch.H, HÄNISCH TEN CATE. Federation of European Societiesof Plant Physiology (FESPP), Santiago de Compostela (Spain), 27th July -1st August 1980. Travel Report 268.
BRETELER, H. A visit to three laboratories in the U.K. working on ionicbalances In higher plants. September 1980. Travel Report 271.
- 83 -
PROJECT No. 120 ANNUAL REPORT 1980
J.H. Becking
SYMBIOTIC NITROGEN FIXATION, INCLUDING SELECTION OF RHIZOBIUM STRAINS
1. Experiments on nitrogen fixation by Azolla spp.
Experiments on the physiology and ecology of Azolla were continued. Inparticular the influence of external conditions on nitrogen fixationwas studied, because the various Azolla species (clones) showed dif-ferences in nitrogenase activity under different conditions. In 1979 ex-periments were conducted with various media (nutrient levels) and withvarious pH's, in the experiments discussed below special attention waspaid to the effect of temperature.Nitrogenase activity of the various Azolla species was studied under thesame light conditions (15 000 lux) at different temperatures. The N2(C2H2) activity of A. pimata reaches a maximum at about 30 °C, but evenat 40 °C, there is still an appreciable N2 (C2H2)-ase activity. In con-trast optimal nitrogen fixation in A. filiouloides is shifted to thelower temperatures, because at 10 and 20 °C the specific N2 (C2H2)~aseactivity is higher, whereas at kQ °C the activity is iower than that ofA. pinnata. At k2 °C, the N2~ase activity of 4. pinnata fronds grown at29 °C is decreased, although sometimes there is a small recovery ofactivity in the fifth and sixth hour of incubation. At k5 - ^6 °C, thereis no measurable N2 (C2H2)-ase activity in both species.
The molecular basis was investigated for the different behaviour patternsof Azolla species with regard to formazan production (reducing power)from tetrazolium salts. With refined microscopic methods for the ab-sorption of chlorophyll and phycocyanin in individual cells and epi-fluorescence of these pigments a qualitative estimate was made of thepigment composition of the various cell-types of the symbiotic Anabaenaazollae extracted from the leaf chambers of the leaves. It was observedthat the relative pigment composition was different in the variousspecies, but also varied with the age of the leave, i.e. the age of thecyanobacteriurn clump. For this reason the pigment distribution wasstudied in individual leaves and concomittantly cell counts were made ofheterocyst frequency and N2 (C2H2) activity was measured. It was foundthat in Azolla pinnata fluorescence yield of chlorophyll £ was low inthe heterocysts and that the vegetative cells showed distinct gradientsin phycocyanin concentration. This pigment distribution is consistentwith the view that only the heterocysts are able to fix molecularnitrogen. In the other Ázolla species a significant amount of phyco-cyanin was found in the heterocysts, suggesting that they have lower N2-ase activity. The latter has been confirmed by N2~ase determinations. Itis evident that Azolla pinnata, which in SE Asia is used in agriculturefor soil fertilization, had by its "domestication" and selection a highN2~ase activity. This opens the possibility for further selection, cellmanipulation and gene mutation to produce more effective Azolla cloneshighly suitable for agricultural purposes.
2. Colonization of Rhizobium bacteria in the rhizosfeer
In view of the new project preliminary experiments were carried out onthe colonization of Rhizobia in the rhizosphere of a legume and a non-legume. Difficulties were experienced to obtain sufficiently thin
preparations to study the colonization under the microscope. It was,however, evident that the colonization was rather selective, probablybecause of root exudates from the host plant. Further experiments arein progress on the patterns of root colonization (also on ElectronMicroscopic (E.M.) level) and the probable role of root exudates bymeans of High Pressure Liquid Chromatography,
vsi. '
- 85 -
SUMMARY OF THE 1980 ANNUAL REPORT OF THE PROPERTIES OF CROP PLANTS
RESEARCH GROUP B GENE MUTATIONS - CHROMOSOME MANIPULATI ON, INVOLVING
PROJECTS 103. 10*», 118, 119 and 136. ^ ľ
Evaluation of the BARN"reactorThe experiments concerning the effect of fast neutrons, as compared withX-rays, upon mutation frequency and mutation spectrum \n Arabidopaia tha-liana (L) Heynh. and Hordeum vulgare I. in relation to evaluation of theBARN-reactor were completed and the results were published in severalarticles as well as in a thesis (Dellaert, L.M.W., 1980. X-ray and fastneutron-induced mutations in Arabidopaia thaliam, and the effect of di-thiothreitol upon the mutant spectrum).
Homozygosing of translocations induced by X-ray and fast neutron treatmentsin Arabidopaia thalima (I) Heynh. was continued to estimate the backgrounddamage Induced by the two types of radiation. Of the 19 translocations, 10were induced by fast neutrons and 9 by X-rays. The homozygotes occurred in18 transiocation lines. However, in 3 translocation lines no normals occur-red after inbreeding. But, in two of the three translocation lines, normalswere recovered- after outcrossing. The translocation Which was not homozygousviable was induced by X-rays and the three trans I ocations without normals byfast neutrons. The present sample is still insufficient to arrive at aconclusion on the extent of background damage induced by the two types ofradiation.
In barley 27 translocätions in heterozygous condition have been detected,19 being induced by fast neutrons and 8 by X-rays. The frequency of trans-locations was 3,35%. Inbreeding, backcrossing and outcrossing of trans-location heterozygptes were carried out to identify homozygote viabilitydifferences. • .; - => "' '• .'". / • .-. . .- .-'. -. .'•;
Methodological aspects of mutation breeding ;In vitro propagation techniques, through the formation of adventitious shoots,have been developed in several floricultural crops 1 ike Chryaanthemum morirfolium, Begonia x hiSmalis, Dianthus aaxyaphyilua.,pUnhprbia puloherrrimaand Foraythia x intermedia, the bulbous crop msaariarmeniaaum and the :-agricultural crop Solanum tuberoaum. After irradiation of explants and invitro propagation through adventitious shoots, either normal (non-mutated)plantiets or almost exclusively sol id (non-chimeric) mutants have beenproduced in Chrysanthemum, Begonia, Dianthua and Solomon. In Muacari it isbeing- investigated whether after irradiation and microprppagation solidmutants can be produced. Since many plant species can be propagated throughadventitious shoots, such in vitro adventitious bud techniques have a greatpotential for mutation breeding. .
Use of Tradeaaantia as Indicator of mutagenic agents in the neighbourhoodof power station's " ~™ . /; • -". - ••• .- - ^~' T~-'^
P ink and colourless mutati ons in Tradeaaantia s tamen ha i rs were used todemonstrate a synergistic interaction between EMS (ethylmethanesulpnonate)and, X- rays. the 1owest s i gn i f}cant effect, of X-ŕaýs was;found; at"a dpser a t e o f 3 m r a d / h . .•"•;'"•'.•;•.-.. '•-."','. •;' r'-::: ,-. ''-'•" ' v ;"•"-'• •.'••."'_" -.,'.. •.' .V-...''.:",-'>-'v.:--'- .' ~
,1- M l
- 86 -
Somatic cell genetics in plants
Haplopappm qvaeilia
- A synchronization procedure for cell suspensions was further developed.
- A practical and efficient method for mutation induction by radiation wasdeveloped and used to produce mutants with respect to N-metabolism. Somecell lines were selected having different tolerance for Zn**.
- Cytotogicat analysis of genetic stability of calU cultivated in severalmedia was continued.
- The flow cytometer (FACS IV) was installed and put into operation. Aprocedure for the production of protoplasts, suited for flow cytometry,was developed.
- Methods for extraction and electrophoresis of excretion products of cellsuspensions were further developed.
Solcmum tuberoaum
- The isolation and regeneration of protoplasts has proceeded to the stagethat cell wall formation, cell divisions and callus formation are observed.The regeneration of shoots (plants) is currently being studied.
- Cell suspension cultures were set up and optimal growth conditions arebeing defined.
- 87 -
PROJECT No. 103
L.M.W. Detlaert
ANNUAL REPORT 1980
THE EFFECT OF FAST NEUTRONS, AS COMPARED WITH X-RAYS UPON MUTATION SPECTRUMAND MUTATION FREQUENCY IN ARABIDOPSIS THALIANA (L.) HEYNH. AND HORDEUMVULGARE L. IN RELATION TO EVALUATION OF THE BARN-REACTOR
The work on this project has been completed and reported in a thesis.
The genetic effects of X-ray and fast neutron seed-irradiation on kvdbi-dop8ia thaliana (L.) Heynh., and the influence of a pre-i rradi ation treat-ment with the radio-protector dithiothreitol (DTT), are the main subjectsof this thesis.
Chapters I and 11 deal with the effects of radiation - with or without apre-lrradiation treatment - on M^-plant development, ovule sterility,embryonic lethality and frequency of chlorophyll deficient embryos in theM^-siliques. These investigations revealed that;
- with increasing radiation doses, no further increase in chlorophyll mutantfrequency is found at relatively high doses, for Ardbidopsis progenies ofthe "main" inflorescence;
- this observed "saturation" in chlorophyll mutant frequency is due partlyto scoring of progenies from initial cells - forming the sub-epidermalcell layer - which have heterogeneous radiation-sensitivities;
- this heterogeneity, which was observed after some of the X-ray and fastneutron treatments, is most likely caused by replacement of the originalinitial cells by less sensitive cells;
- fast neutron-irradiation of pre-soaked (3 hours) Arabidopsis seeds isapproximately seven times more effective than X-irradiation with respectto the induction of Mi-ovule sterility end M2~embryonic lethality;
- dithiothreitol (DTT) provides considerable protection against X- and fastneutron irradiation, using Mi-ovule sterility, and, to a lesser extent,the number of embryonic lethals and chlorophyll mutants as parameters.This, as compared to irradiation without DTT, leads to more mutants at agiven level of Mi-ovule sterility.
To compare the genetic effects of different mutagens, a standardized mutagendose with reference to a specific=nenetic effect is necessary. The applieddoses of X-rays and fast neutrons as reported in chapter II - the X-raydoses were seven times the applied fast neutron doses - induced similarlevels of ovule sterility and embryonic lethality in Arabidopais. Therefore,these doses were applied in the analysis of the X-ray and fast neutron in-duced mutant spectra. In chapter III the results of a study on the effectsof (1) radiation type, (2) radiation dose and (3) a DTT pre-irradiationtreatment, on the spectra of X-ray- and fast neutron-induced morphologicalmutants are reported. These investigations demonstrated that:
- the X-ray- and fast neutron-induced spectra of different types of viablemutants vary significantly,
- throughout the studied dose range, some mutant types occur significantlymore frequently with fast neutrons than with X-rays, i.e. mutants withloosely packed leaves with long petioles and the eaeriferum mutants;
- mutants with closely packed leaves with short petioles were more frequent-ly induced with X-rays. Other mutants, e.g. the vital chlorophyll
.,• - v í - -
- 88 -
deficient mutants, were induced with equal frequencies by X-rays and fastneutrons in the studied range of radiation doses;
- certain mutant types are relatively less frequent after irradiation inthe presence of DTT. Since DTT preferentially protects against radiation-induced single and double strand-breaks, this observation suggests thatthese mutants more often than other mutants, originate from strand-breakdamage. It is striking that these mutant types were more frequently in-duced with fast neutrons than with X-rays,
In order to compare different mutagenic treatments, the mutation frequencyshould be expressed per cell. For the transformation of the mutant frequencyscores, expressed as the number of mutant plants among the total number ofM2~plants (or embryos in the case of MUller's embryo test), a mutant fre-quency of twenty per cent in the offspring of a heterozygous parent and anequal viability of mutant and non-mutant plants have been assumed for alltreatments in chapters I, II and III.
In chapter IV, the segregation frequency of mutants induced by various X-ray and fast neutron doses, with and without DTT pre-treatment, is studied.From this investigation the following conclusions can be drawn:
- the average mutant segregation frequencies of X-ray and fast neutron-in-duced mutants are equal;
- a OTT pre-irradiation treatment does not influence the segregation fre-quency of rad i at ion-induced mutants;
- there is no consistent change in the mutant segregation frequency with in-creasing radiation dose;
- progenies from single flowers in the top of the Mj-main inflorescenceoriginate, in general, from genetically homogeneous tissue;
- the average mutant segregation frequency is 21,5 per cent. Since no dif-ference in viability of mutant and non-mutant plants is observed, themutant deficit is due to reduced transmission of the mutant gene through(probably the male) gametophytes;
- the observations lead to the conclusion that the mutant segregation fre-quency in the offspring of a heterozygous parent can be estimated fromthe (^-segregation frequencies, provided that progenies from a singleflower from the top of the Mj-main inflorescence are tested. The obtainedvalue can then be used directly for the calculation of the mutant fre-quency per cell.
Avabždopsis mutants with a reduced or absent wax coating, i.e. eeeriferummutants, are relatively more frequently induced with fast neutrons thanwith X-rays (chapters III and V ) . The phenotyp i c analyses-by macroscopic andscanning-electron microscopic techniques, reported in chapter V - of the waxcoating on stems and siliques of the wild type and of 30 fast neutron-, 11X-ray-, and 10 EMS-induced and of 1 spontaneous mutant, indicate that:
- in eoeviferum mutants with visually reduced wax coating, the quantity of ]f rodlet-shaped wax estrusions is less than in the wild type; I
| - in eaeriferm mutants, with visually absent wax coating, the wax extrusionsI are affected in specific ways. Compared to the wild type, the rodlet-
shaped crystals are absent or rare; the plate-like wax structures are ab-sent or reduced and often there is, in contrast to the wild type, a thickorganized wax layer present on the siliques and stems.
- 89 -
Genetic analysis of the eoeriferum mutants, reported in chapter V, revealedthat the mutants were conditioned by many different loci (minimum 14,maximum M O . The ultrastructural research of the eoevifervm mutants withabsent wax coating demonstrated that, at the level of resolution of thescanning-electron microscope, we can distinguish between differentialeffects of different loci. However, the number of mutants per locus wasstill too low to pronounce upon X-ray or fast neutron specificity forindividual loci.
In chapter VI, various methods to select lines, mutated for a specificcharacteristic and with an otherwise undisturbed genetic background, arediscussed. A mutation breeding programme is proposed. It is based on thedetermination of Mi-heterozygous plants by means of single seed progenytesting. Subsequently the desired mutant line is selected in the progeny ofthese Mj-plants of which M2 spare seed is available. As compared to M2"bulktesting, the mutation frequency can be increased considerably with theproposed method. Besides, the number of independent mutants is highercompared to Mj-progeny testing (without spare seed) and M2~bulk testing.The mutant frequency and the number of spare seed determine to what extent.
Publications
DELLAERT, L.M.W. Comparison of selection methods for specified mutants inself-fertilizing crops: theoretical approach. In: "Seed protein improvementin cereals and grain legumes". Vol J_: 57-7^ International Atomic EnergyAgency, Vienna 0979).
DELLAERT, L.M.W. Ecepiferm mutant•> in Arabidopsis thaliana (L.) Heynh.:I. Induction by X-rays and fast neutrons. Arabidopsis Information Service.16: 1-9 (1979).
DELLAERT, L.M.W. X-ray- and fast neutron-induced mutations in Arabidopsisthaliana, and the effect of dithiothreitol upon the mutant spectrum. ThesisAgricultural University of Wageningen, February 1980, 169 pp.
DELLAERT, L.M.W. Dose response curves after irradiation of Arabidopsis seeds:A possible explanation for the "saturation" in mutant frequency at highradiation doses. Environm. and Exp. Botany £0_: 309-323 (1980).
DELLAERT, L.M.W. The effect of dithiothreitol on radiation-induced geneticdamage in Arabidopsis thaliana (L.) Heynh. Mutation Research 71: 109-126(1980). —
DELLAERT, L.M.W. Segregation frequencies of rad iation-induced viable mutantsin Arabidopsis thaliana (L.) Heynh. Theoretical and Applied Genetics 57:137-H3 (1980). ~~
DELLAERT, L.M.W. Comparison of X-ray- and fast neutron-induced mutant spectra.Experiments i»» Arabidopsis thaliana (L.) Heynh. Environm. and Exp. Botany(In press).
DELLAERT, L.M.W., J.Y.P. van Es and M. Koornneef. Eceviferwn mutants inArabidopaia thaliana (L.) Heyn.: II. Phenotypic and genetic analysis.Arabidopsis Information Service 16: 10-26 (1979).
- 90 -
PROJECT No. 104 ANNUAL REPORT 1980
C. Broertjes, S. Roest, G.S. Bokelmann
METHODOLOGICAL ASPECTS OF MUTATION BREEDING
In view of the termination of this project, this annual report will be thefinal one, summarizing the results of 1980 and preceding years.
The so-called "adventitious bud technique" has demonstrated to be of greatpotential value for mutation breeding of vegetatively propagated floricul-tural crops. This in vivo method is based on the phenomenon that (the apexof) an adventitious bud, produced at the base of the petiole of a freshlydetached leaf, ultimately originates from a single (epidermal) cell. Henceafter irradiation of leaves and the subsequent in vivo formation ofadventitious shoots, either normal (non-mutated) plants or almost exclusive-ly solid (non-chimeric) mutants are obtained. The significance of in vivoadventitious bud techniques, also from a commercial point of view, has beendemonstrated in ornamentals such as Aohimenea, Begonia, KalanahoS, Saint-paulia and Streptoaarpua. Commercial mutants were put on the market within arelatively short time after irradiation and adventitious shoot formation ondetached leaves. Since a large and rapidly increasing number of plant speciescan be propagated in vitro, it seemed worthwhile to develop and evaluate invitro propagation techniques, preferably through the formation of adventitiousshoots, in crops which cannot or are not rapidly propagated through ad-ventitious shoots in vivo.
1. Development of micropropagation techniques
During the past seven years micropropagation techniques, through ad-ventitious shoot formation on different types of explants and subsequentroot formation of excised shoots, have been developed in the followingcrops: Chrysanthemum morifolium Ram., Solanum tuberosum L., Begonia xhiSmdlis, Dianthus oaryophyllus L., Musaari armeniaaum (2n and An) andM. botryotdes cv. Album, Euphorbia puloherrima and Forsythia x intermedia.
After irradiation and the in vitro formation of adventitious shoots, solid(non-chimeric) mutants have been produced in Chrysanthemum morifolium Ram,Solanum tuberosum L. and Begonia x hiSmalis. Further spontaneous chromo-some doubling of diploid interspecific potato hybrids has been achievedthrough adventitious shoot formation on in vitro cultivated leaf explants.
Using similar techniques it is still under investigation whether solidmutants can be produced in Dianthus aaryophyllus L. (carried out in co-operation with the Institute of Horticultural Plant Breeding at Wageningen)and in Mueaari.
In Euphorbia pulaherrima and Forsythia x intermedia supplementary researchwill be essential for the realization of a rapid and large-scale micro-propagation, which is essential for the application of in vitro ad-ventitious bud techniques in mutation breeding programmes.
In conclusion, in vitro propagation techniques through the formation ofadventitious shoots, have been developed within a relatively short timefor several crops. It was further demonstrated that, after irradiation ofexplants and using in vitro adventitious bud techniques, solid mutants canbe produced in several vegetatively propagated crops, which is of con-siderable interest for mutation breeding, since many plant species can bepropagated through the in vitro formation of adventitious shoots.
2, The comparison of mutant frequency and spectrum in diploid and autotetra-ploid forms of a few vegetatively propagated ornamentals
During the time that several gesneriads were used to investigate thesignificance of the in vivo adventitious bud technique for mutationbreeding, autotetraploids of a few Aehimenes, Saintpaulia and Strepto-earpua cultivars were produced by colchicine treatment of detached leaves.They were used to compare the radiosensitivity of the autotetraploidswith that of their respective diploid forms. Especially with Aehimenesrather high mutant frequencies were found at tetraploid level as comparedto the very low mutant frequencies at diploid level. Large-scale ex-periments with a few Aohimenes cultivars confirmed the prel iminary results.They were explained on the basis of a low heterozygosity of the cultivarsused and the subsequent low mutant frequency at diploid level. On tetra-ploid level the explantation is less obvious, one of the speculativesolutions being the dominant expression of chromosome aberrations whichare not lethal on tetraploid level. This phenomenon was studied as wellby large-scale irradiation of several diploid cultivars of Saintpauliaand of their respective colchicine-induced autotetraploid forms. The re-sults were much less convincing, as compared to Aohimenes, since onlyslightly higher mutant frequencies were found in the tetraploids as com-pared to the (already rather high mutant frequencies of the) diploids.The mutant spectrum at tetraploid level seemed to be different as com-pared to the one at diploid level, in the sense that higher frequenciesof rather drastic mutant forms (dwarfs, for instance) were found.
Publications
BROERTJES, C. Mutation breeding of autotetraploid Aahimenes cultivars.Euphytica 25: 297-304 (1976).
BROERTJES, C. The significance of in vitro adventitious bud techniques formutation breeding of vegetatively propagated crops. In Proc. 4th Coord.Meeting of the IAEA Panel on "Improvement of vegetatively propagated cropsand tree crops through induced mutations". India, Coimbatore, 1980.(In press).
BROERTJES, C. and A.M. VAN HARTEN. Application of mutation breeding methodsin the improvement of vegetatively propagated crops. An interpretiveliterature review. Elsevier Scientific Publ. Co., Amsterdam 316 p (1978).
BROERTJES, C. and A. KEEN. Adventitious buds: do they develop from one cell?Euphytica 29 (1): 73-87 (1980),
BROERTJES, C., S. ROEST and G.S. BOKELMANN. Mutation breeding of Chrysanthe-mum morifolium Ram. using in vivo and in vitro adventitious bud techniques.Euphytica 25: 11-19 (1976).
HARTEN, A.M. VAN, H. BOUTER and C. BROERTJES. In vitro adventitious budtechniques for vegetative propagation and mutation breeding of potato(SoLanim tuberosum L.). II. Significance for mutation breeding. Euphytica.30 (In press). 1981.
HARTEN, A.M. VAN, H. BOUTER en C. BROERTJES. Toepassing van een in vitroadventiefspruit-methode bij de mutat i everede1 ing van aardappel. 2. Mutatie-frequentie en chimaerie. Landbouwk. Tijdschr. (In press). I98I.
HERMSEN, J.G.Th., M.S. RAMANNA, S. ROEST and G.S. BOKELMANN. Chromosomedoubling through adventitious shoot formation on in vitro cultivated leaf ex-plants from diploid interspecific potato hybrids. EuphyticaJ£ (in press). 1981,
ROEST, S. In vitro adventitious bud techniques and mutation breeding. InProc. EUCARPIA Meeting (Section Vegetables) on "In vitro culture applied tovegetable breeding". Versailles, 1980, pp. 186-191 (1980).
#(i^j^3s^
- 92 -
ROEST, S., M.A.E. VAN BERKEL, G.S. BOKELMANN and C. BROERTJES. The use of anin vitro adventitious bud technique for mutation breeding of Begonia xhi'ômalia. Euphytica 30 (In press). 1981.
ROEST, S. and G.S. BOKELMANN. Vegetative propagation of Chrysanthemum mori-J'olium Ram. in vitro. Scientia Horticulture y. 317-330 (1975).
ROEST, S. en G.S. BOKELMANN. Vermeerdering van Begonia in kweekbuizen. Vak-bl. Bloem. 35 (5): 116-117 (1980).
ROEST, S. en G.S. BOKELMANN. Mutatieveredeling Begonia. Vakbl. Bloem. 3>5 (6):56-57 (1980).
ROEST, S. and G.S. BOKELMANN. In vitro adventitious bud techniques for ve-getative propagation and mutation breeding of potato (Solatium tuberoaum L.).I, Vegetative propagation in vitvo through adventitious shoot formation.Potato Res. 23,: 167-181 (1980).
ROEST, S. en G.S. BOKELMANN. Forsythia vegetatief vermeerderen in kweekbui-zen. Vakbl. Bloem. 35 (45): 3^-35 (1980).
ROEST, S. en G.S. BOKELMANN. Vegetatieve vermeerdering van Poinsettia inkweekbuizen. Vakbl. Bloem. 35 (47): 36-37 (1980).
ROEST, S. and G.S. BOKELMANN. Vegetative propagation of carnation in vitrothrough multiple shoot development. Scientia Hort. J M 4 ) : 357"366 (1981).
ROEST, S. en G.S. BOKELMANN. Toepassing van een in vitro advent iefsprui ti-me t hode bij de mutatieveredeling van aardappel. 1. Vegetatieve vermeerderingvia adventieve scheutvorming in vitro. Landbouwk. Tijdschr. (In press). 1981.
ROEST, S. en G.S. BOKELMANN. Vegetatieve vermeerdering van Musaari in kweek-buizen. Vakbl. Bloem. 36. (5): 134-135 (1981).
Lecture
ROEST, S. In vitro adventitious bud techniques and mutation breeding. EUCAR-PIA Meeting (Section Vegetables) on "In vitro culture applied to vegetablebreeding11, Versailles (1980).
- 93 -
PROJECT No. 118
K, Sree Ramulu
ANNUAL REPORT 1980
COMPARISON OF FAST NEUTRONS ANO X-RAYS IN RESPECT TO GENETIC EFFECTSACCOMPANYING INDUCED CHROMOSOME ABERRATIONS, IN RELATION TO THE EVALUATIONOF THE BARN-REACTOR
The analyses of the M2 and M3 plants derived from X-ray and fast neutrontreatments were further continued to isolate translocation heterozygotes,to study homozygote viability differences and to estimate the amount ofbackground damage induced by X-rays and fast neutrons in Arabidopsisbhaliam (L,) Heynh. cv. Landsberg erecta (2n = 10) and Hovdeum vulgare I.cv. Arami r (2n = \k).
1. Arabidopais thaliana (L.) Heynh.
Of the 147 semi-sterile M2 lines isolated after seed treatments withoptimum doses of X-rays and fast neutrons, 42 have been so faranalysed in meiosis. Nineteen lines contained translocations inheterozygous condition, 10 from fast neutron treatments and 9 fromX-ray treatments, whereas the other 23 lines had consistently 5 pairsof chromosomes (Table 1) and hence probably had no translocations.All the translocation lines contained 1 quadrivalent (ring and chainquadrivalents of zig-zag-or non-zig-zag-type) and 3 bivalents.
The translocation hterozygotes showed semi-sterility in pollen andovules. Although the degree of semi-sterility differed to some extentbetween the translocation lines, it was more or less similar betweenpollen and ovule sterility.
Table 1 - Frequency of translocation heterozygotes among the M2 semi-sterile lines derived from X-ray and fast neutron treatmentsin Avabidopsis thaliana (L.) Heynh.
Treatments
X-rays 140 Gy*
233 Gy
326 Gy
Fast neutrons 20 Gy
33 Gy
47 Gv
Total
No. of M2semi-sterile1 ines analysed
8
6
7
7
7
7
42
No. of 1 i nes withheterozygoustranslocations
3
3
3
2
44
19
No. of lineswith 5||
5
3
4
5
3
3
23
: 1 Gy = 100 rad
b. Viabi2jit^_of_traj2siocation>ihomozygotes
The data on the segregation of M3~progenies, so far analysed after in-breeding of the M2-translocatlon heterozygotes, and the segregation ofprogenies after selfing of translocation heterozygotes obtained frombackcrossing to partent genotype (a genie male sterile line) or but-crossing to another eultivar (cv. Estland) are shown in tables Z-**.The comparison of the viability of the homozygotes derived after in-breeding, backcrossing and outcrossing permits an estimate to be madeof the extent of background damage and the breakpoint damage inducedby X-rays and fast neutrons. As can be seen from the data in table 2,homozygotes occurred in all the translocation lines except T13 (in-duced by X-rays). The homozygotes were also recovered among the pro-genies derived after backcrossing or outcrossing (Tables 3 and k).They were, normal in morphology, growth and fertility.
Normals were recovered in all the translocation lines except T2, T4and T6 (Table 2) indicating that lethal recessives might have beeninduced in the normal chromosomes. However, normals in Tk and T6 wererecovered among the progenies obtained from outcrossing to anothereultivar, cv. Estland (Table k) which differs in genetic background.The trans locations T2, T** and T6 were induced by fast neutrons. Thepresent sample is still insufficient to arrive at a conclusion onhomozygote viability differences and on the extent of backgrounddamage induced by X-rays and fast neutrons.
Table 2 - Segregation of M3~prpgenies after inbreeding of the M2 trans-location heterozygotes in Arábidopsis thaliana (L.) Heynh.
Trans lo-cations
T1
T2
T3
ik
T5
T6
T7
T8
T9
T10
T11
T12
T13
T1í»
Total
No. of progenyplants analysed
20
20
U a
35
13
32
37b
28
33
27
56b
37
*»3
33
<t58
Frequency of plants segregating insemi-ste"ri le's(heterozygotes)
10
15
29
22
7
.1622
17
21
16
32
13
23
15
258
Fertileshomozygotes
3
5
k
13
3
16
2
5
5
7
3
9
0
5
80
normals
70
80
3
0
1»
6
7
7
15
20
13
a: includes 3 male steriles.b: nine fertiles in T7 and T» fertiles in T11 are yet to be analysed.
- 95 -
Table 3 - Segregation of progenies after selfing of translocationheterozygotes obtained from backcrossing of translocationheterozygotes in Arabidopsis thaliana (L.) Heynh,
Trans lo-cations
T2
T4
T7T8
T9T10
Total
No. ofprogenyplants
analysed
23
4228
3642
34
205
Frequency of plants
Semi-ster i les(heterozygotes)
10
914
15
1713
78
segregat
Fertileshomozygbt'es
511
88
11
10
55
normals
0
96
68
534
ng in
male ster i les
8
130
76
6
40
Table 4 - Segregation of F2-progenies after selfing of translocationheterozygotes obtained from crossing of translocationheterozygotes with cv. Estland in Arabidopsis thaliana (L.)Heynh.
Trans lo-cations
T2
T3T4
T5T6
T7T8
T9T10
Til
Total
No. of progenyplants
analysed
4741
52
44
5416
433941
40
417
Frequency of plants segregating inSemi-steriles(heterozygotes)
30
23
25
24
32
924
21
21
22
231
Fertileshomozygotes
16
10
4
76
31
781
63
normals
0
814
10
11
4
811
6
11
83
undecidec
1
0
9
3
50
10
0
6
6
40
2. Hordeum vulgare (L.)
The analyses on fertility of (^-plants derived from X-ray and fast neutrontreatments were further continued. Among the 1117 plants analysed untilthe present 234 (2U) were semi-steriles. Of the 169 semi-sterile plantsexamined in meiosis, 27 had translocations in heterozygous condition, 19being induced by fast neutrons and 8 by X-rays, and 23 were characterized
- 96 -
by various meiotic abnormalities. The frequency of translocation hetero-zygotes in the M2 was 3.35%. About 16% of the semi-steri les containedtranslocations. All contained 1 quadrivalent and 5 bivalents, except twolines which had 1 hexavalent and k bivalents.
The data on chromosome association in meiosis of the twelve translocationheterozygotes isolated this year showed that the proportion of cells withalternate and adjacent orientation of the multivalents varied among thedifferent translocation lines. The degree of semi-sterility differedbetween lines or between pollen and ovule sterility.
Inbreeding, backcrossing and outcrossing of translocation heterozygoteswere carried out to assess the background damage and breakpoint damageby studying homozygote viability differences.
Publi cat ions
SREE RAMULU, K. Failure of EMS to induce S-locus mutations in Niaotianaalata Link and Otto, Environ. Expt. Bot. 20 (1980) ľ»9~155.
CAPPADOCIA, M. and K. SREE RAMULU. Plant regeneration from in vitrocultures of anthers and stem internodes in an interspecific hybrid,Lycopersicon esaulentum L. x L. peruviamm Mill, and cytogenetic analysisof the regenerated plants. Plant Science Lett. 20 (1980) 157-166.
BREDEMEIJER, G.M.M., K. SREE RAMULU and P. DIJKHUIS. The influence ofgamma-i rrad i at i on of Nicotiana alata styles on peroxidase isoenzymes andpollen tube growth. Incompatibility Newslett. 13 (in press).
ANCORA, G. and K. SREE RAMULU, Plant regeneration from in vitro culturesof stem internodes in triploid of Lycopersiaum peruvianum and cytogeneticanalysis of regenerated plants. Plant Science Lett. (In press).
Lectures
SREE RAMULU, K. Application of Flow Cytometry in sorting and analysis ofplant cells and chromosomes - an introduction. Lawrence LivermoreLaboratory, Univ. California, Livermore, U.S.A., May I98O.
Travel Reports
SREE RAMULU, K. Travel Report No. 265.A. NIEHS/EPA conference on pollen systems to detect biological activity
of environmental pollutants. University of Tennessee, Knoxville, Tenn.,May 5-8, 1980.
B. Visits to Laboratories in U.S.A. in relation to research on geneticmanipulation. May 2, May 12-24, i960.
f í
[ • I\ • 'A
97 -
PROJECT No. 119 ANNUAL REPORT 1980
P.A.Th.J. Werry, G.M.M. Bredemeijer, S. Roest, K. Sree Rámu 1 u, Ch.H. Hänischten Cate, K.J. Puite, L.J.W.J. Gilissen (gastmedewerker)
SOMATIC CELL GENETICS IN PLANTS
This is the final report of this project which was set up as a preparatorystudy for the development of the new research area; advanced new geneticmethods for plant breeding.
Haplopappu8 graoilia was used as model plant for preparatory studies withrespect to synchronisation (For chromosome isolation), mutation induction(for genetic markers), cytodifferentiation (for genetic analysis), flowcytometry (for measurement and physical characterization of cells and cellconstituents) and biochemical aspects of in vitro cultivation of planteel Is.
Potato was used for studies aimed at the definition of optimum conditionsfor in vitro cultivation of potato cells, protoplast isolation and re-generation of plants from isolated protoplasts.
1. Hgpíopgppus gradlia
a. Synchronization
Based on our previous observation that a relatively short period ofreduced temperature (10 °C) results (after the restoration of thenormal temperature (28 °C)) in a small "wave" of synchronous mitoses,it was proposed that low temperature shocks at regular time intervalswould produce a highly synchronous culture of H. graoilis cells.However, problems with contamination in a newly purchased incubatorprevented the experiment being carried out. These problems weresolved recently and new experiments to develop optimal conditions forthe synchronisation of plant cells are again in progress.
The further development of an efficient and convenient procedure forthe induction of mutants (genetic markers) in in vitro cultivatedplant cells has resulted in the development of a standard procedure.
- A cell culture in stationary phase of growth is irradiated with X-rays up to a dose of 30 Gy.
- Following irradiation the culture is further cultivated instationary phase conditions for 2k h, thus allowing full repair ofpotentially lethal damage.
- Following the delay the cells are plated on top of the selectivemedium at a density of 107 cells-ml"1 (2-107 eells/petridish) aodincubated until the frowing colonies can be clearly distinguishedfrom the mass of dead cells.
The procedure as described yields an estimated cell survival of 1%and a net mutation frequency of 5*10~6. It is calculated that thecontribution of preexisting spontaneous mutants in the number ofmutants found after irradiation is negligible.Two lines of cycioheximide-resistant cells have so far beenestablished.Experiments are currently in progress to study optimal conditions forthe induction of chlorate resistance.
'•^íi .
- 98 -
Considerable progress has already been achieved in the developmentof Zn + + tolerant lines.Zinc is a metal, the ions of which catalyze a number of enzymic re-actions in the nucleus related to nucleic acid metabolism. Highconcentrations of zinc ions are toxic and probably mutagenic. Thetoxicity of zinc is used as a tool to identify mutant cells, whichare Zn-resistant. The aim of this research is to obtain marker celllines, which can be used in genetic manipulation.
The Zn-toxicity appeared to be maximal at a concentration of 7xlO"3 Mand higher. This toxic concentration was independent of the salts inwhich Zn + + was added to the growth medium. An increase of Zn SQ^,ZnCl2 or Zn(NO3/2 in liquid or in solid media reduced cell growthsimilarly.
This concentration of 7x10"3 M Zn** WČSS used as a selection barrier.Cell suspensions were plated on solid growth medium containing thisconcentration of Zn^-ions. Zn-resistant cells formed colonies, whichwere isolated, multiplied and resuspended in liquid medium containinga sublethal concentration of Zn^-ions (= 2,1x10"3 M). In this way,156 colonies were isolated. The 19 best growing colonies were used forfurther selection experiments. This reselection, based on growth inliquid medium containing 2,lx10~3 M Zn + + and on colony formation solidmedium containing 7*10"3 M Zn"1"*", resulted in seven cell lines, ofwhich the stability of the Zn-resistance has now to be investigated.
During the course of the experiments it appeared that due to theaddition of Zn-salts to the growth medium a precipitate of zincphosphate was formed. So, addition of high amounts of Zn caused aconsiderable decrease of the phosphate concentration in the medium.However, simulation experiments, in which liquid growth medium wasused containing as much phosphate as remained in the medium afteraddition of the Zn-salt, showed a reduced growth of the cell suspension,but this reduced growth was still considerably higher in comparisonto the growth in the same phosphate concentration in the presence ofzinc. In conclusion the reduced phosphate concentration in the mediumhas much less influence on growth of the cell suspension of H. graai-lis than has the high Zn-ion concentration.
c <
In order to have adequate knowledge of the factors influencing cyto-logical stability, further experiments were carried out on the in-duction and analysis of callus from stem on leaf expiants, its qualityand quantity during induction and maintenance, and on differentiationfrom callus. In all, six modified media of White have been studied.All these media differ in the quantitative ratio of auxin and cyto-kinin. Both callus production and quality varied on different media.Of the six media, the one containing IAA 1 mg/1 (Wh-1) proved to be agood general medium for callus induction and differentiation: abundantgreen-white call i were produced and roots formed consistently on thesurface of the call i. The other media that contained the combinationof both auxin (IAA or NAA or 2,4-0) and kinetin in different ratiosgave in general, poor growth of call i although marked differences inquantity and quality were observed depending on auxin-cytokinincombination and their ratio. The analyses on the cytological stabilityof the call i induced by various media are now in progress.
- 99 -
EÍ2w_SY.Í9G!S£r.Y.Prerequisites for the selection of cells to be used for genetic mani-pulation studies include a characterization of the individual cellsand the sorting of the cells. Flow cytometry techniques permit theanalysis of the cell cycle (ONA histogram) and the sorting of arelatively pure sample of single compartment cells, e.g. G2/M cells,in a non-destructive way. Fortunately, the FACS IV (fluorescenceactivated cell sorter) could be installed this year and thisinstrument will be used for these mentioned studies.
Protoplasts from Haplopappua graailia cells have been prepared using0,4 M sorb i toI, 5% driselase (Kyowa Hakko, Japan) and 0,5% pectyolaseY-23 (Seishin, Japan). After 3 h of gentle shaking and after washingwith 0,4 M saccharose, 2-5x105 protoplasts per m) were obtained. About75% of the protoplasts were viable as measured by fluorescein di-acetate (FDA). The fluorescence of the FDA-treated protoplasts,together with the scatter signal from the protoplasts, have also beenmeasured on the FACS.Preliminary experiments with protoplasts incubated in a rich proto-plast medium (V-KM medium with 0,35 M glucose) show some budding, butalso cell divisions. After a few weeks colony formation was observed.
e-In order to characterize mutant cell lines of H. gracilis by means ofelectrophoretic separation of proteins and isoenzymes it was necessaryto adapt methods used in previous studies on Nicotiana alata.It appeared that the methods used for extraction and electrophoresisof peroxidase isoenzymes from styles of ff. aZatacould also be appliedto the cells of a H. gracilis suspension culture. For proteins, anumber of extraction procedures were compared on the basis of electro-phoretic patterns obtained by electrofocusir.g. A simple extractionwith buffers gave rise to protein patterns with intense backgroundstaining and tailing of several lanes. It appeared necessary to re-move pigments and low molecular weight substances before extractionwith buffers.
2. Potato (Solanum tuherosum)a •
For genetic manipulation (chromosome transplantation and gene trans-formation) it is of essential importance that plantléts can be re-generated from manipulated protoplasts. The phenomenon of plantlet re-generation from protoplasts has so far been realized in about 40 plantspecies (30 of them belonging to the Solanaaeae) and was studied inpotato (Solanum tuberosum L.). By enzymatic treatment protoplasts wereisolated from leaflet-blades of the tetraploid cvs "Bintje" and"Russet Burbank" at densities of 106-107 per g leaf tissue; theseprotoplasts proved to be non-viable a few hours after isolation.However, following a period of practical training (Miss Bokelmann) inprotoplast handling in the Federal Republic of Germany (Max-Planck-
[ Institut fiir Zuchtungsforschung, Koln) an improvement of the viabilityi of isolated protoplasts was obtained. This improvement occasionallyí resulted in cell wall formation, cell division and callus formationI from plated protoplasts of two German dihaploid potato clones (HH 258I and 260). Attempts to obtain the first regenerated plantlets fromi ' these call i are now being carried out.
- 100 -
b. Cell susgension_cuHure
For the use of the genetic manipulation high quantities of uniformprotoplasts are needed. Those protoplasts could be obtained directlyfrom leaves or via the culture of cells in suspension by dissolvingthe eel 1 wall.
In this research the culture of potatoes in cell suspensions has beenstarted. Two diploid lines were obtained from the Max-Planck Institute(Cologne) e.g. HH258 and 260, suitable for regeneration from leafprotoplasts or in growing in cell suspensions.
The growth of cells in cell suspension was set up. The sterile hand-ling of the cell cultures is learned. A method to measure the growthwithout harvesting the samples is established. The effect of thecomposition of different media is now tested. Cytokinins in ratherhigh concentrations (10~5 M) are needed for cell division and sur-pressing the formation of root-like structures.The culture of the tetraploid Bintje has been started. Callus was in-duced on leaf explants; the growth rate of the callus is low, and hasstill to be improved. Also the growth rate of the Bintje cells insuspension needs improving.
Publications
BREDEMEIJER, G.M.M. and J. BLAAS. S-specific proteins in styles of Nicotianaalata. Incompatibi1ity Newsl. 12: ki-kk (1980).
BREDEMEIJER, G.M.M. and J. BLAAS. Do S allele-specific peroxidase isoenzymesexist in self-incompatibie Nicotiana alata. Theor. Appl. Genet. 57: 119-123(1980).
BREDEMEIJER, G.M.M. and J. BLAAS. S-specific proteins in styles of self-incompatible Nicotiana. alata. Thero. Appl. Genet. (In press).
BREDEMEIJER, G.M.M., K. SREE RAMULU and P. DIJKHUIS. The influence of gamma-irradiation of Nicotiana alata styles on peroxidase isoenzymes and pollentube growth. Incompatibility Newsl. 13 (In press).
GILISSEN, L.J.W. (1980). Apfelschorf. Naturwissensch. Rundschau _33 (7) 292-293.GILISSEN, L.J.W. (1980), Appelschurft. Vakbl. Biol. Jj», voorplaat.
WERRY, P.A.Th.J. Radiation induced mutagenesis in in vitro cultivated plantcells. Rad. Environ. Biophys. Jľ7» 29 2> 19Ô0.WERRY, P.A.Th.J. Fysiologische principes van plantecel en weefselkweek in:Weefselcultures in de Farmacie (ed.: H. Huizing) Groningen, Univers iteit,1980.
WERRY. P.A.Th.J. and K.M. STOFFELSEN. Theoretical and practical aspects ofradiation induced mutagenesis in plant cells. In: Plant eel) cultures;Results and Perspectives (eds. F. Sala, B. Parisi, R. Cella and 0. Ciferi).Elsevier, Amsterdam 1980 pp. 115-120.
Lectures
BREDEMEIJER, G.M.M. S-specifieke eiwitten in Nicotiana alata stijlen. BiONWerkgemeenschap GEFYBO. Oudleusen 1980.
GILISSEN, L.J.W. (1980). De zintuigfunctie van de bloemstijl. Bion-werkge-meenschap GEFYBO, Oudleusen.
,A
- 101 -
GILISSEN, L.J.W. (1980). Pollination and the role of the style as a sense-organ. 5th International Palynological Conference, Cambridge.
VAN DEN ENDE, G., GILISSEN, L.J.W. (I98O) The phyllosphere of apple.Poster session International Phyllosphere Symposium, Aberdeen (Scotland).
WERRY, P.A.Th.J. Moleculair-biologische methoden voor de genetische recon-structie van pIanten met behu)p van fusie- en mutatietechnieken.13e Weefselkweekdag, Ned. Weefselkweekclub. Leiden-Wageningen, 1980.
WERRY, P.A.Th.J. Kwantitatieve aspecten van mutatieinductie in plantecel-suspensies door ioniserende straling. 13e Weefselkweekdag, Hed. Weefsel-kweekclub, Leiden-Wageningen, 1980.
WERRY, P.A.Th.J. Radiation induced mutagenesis in in vitro cultivated plantcelh- XVth Annual meeting of the European Society for Radiation Biology.Rotterdam, 1980.
WERRY. P.A.Th.J. Plantenveredeling met behulp van geTsoleerde cellen*Ned, Ver. voor Landbouwjournalistiek, Wageningen, 1980.
- 102 -
PROJECT No. 136
H.P. Leenhouts
ANNUAL REPORT 1980
USE OF TRADESCANTIA AS INDICATOR OF MUTAGENIC AGENTS IN THE NEIGHBOORHOUDOF POWER STATIONS
Tradesaantia, clone KU 9, has been exposed to acute and chronic ionizingradiation and a chemical mutagen, ethylmethanesulphonate (EMS). Theinduction of pink mutations in cells forming the stamen hairs has been usedas end-point. The acute dose response relationship for X-rays was found tobe comparable to data for other clones published elsewhere. EMS was foundto induce mutations when cuttings were exposed in a solution of EMS + phos-phate buffer for 2 - 8 hours. It was found that the combination of EMS +X-rays induced more mutations than was expected from the sum of theindividual treatments (Fig. 1). This synergistic effect nas important con-sequences for radiological protection and also for the risk assessment forexposures to complex mixtures pf chemical mutagens.
It was found that the synergistic interaction between EMS and X-rays dis-appeared when an interval of 24 or *»8 hours was allowed between the treat-ments. As the cells in the stamen hairs divide each 2k hours, it is notclear whether the cell division or the repair of the damage induced by theEMS is responsible for the disappearance of the synergism.
Complete plants were exposed to different low dose rates of gamma radiationfor 1 month. The mutation frequency increased during the first 20 days andreached a plateau value, which was maintained for the period of exposure.On removal of the source the mutation frequency gradually decreased. Thevalue of the plateau mutation frequency was found to be linearly related tothe dose rate and a minimum detactable effect was found at a dose rate of30 nGy/h (3 mrad/h).
Apparatus has been constructed which permits the exposure of Tradescantiato gaseous pollutants (e.g. S02, ozone, etc.) under controlled conditionsof temperature, light and humidity.
Publicat ions
LEENHOUTS, H.P., K.H. CHADWICK, A. CEBULSKA-WASILEWSKA. 1980. An analyticalapproach to the comparison of chemical and radiation hazards to man.Radiation Protection: A systematic approach to safety. Pergamon Press(Oxford) Vol. 11, 1155-1158.
- 103 -
10
1.15
C
'ä
Tradescantia KU 9
EMS ond X - rayscombined
12 14 16day after treatment
18
Fig. 1 - The number of mutation events in Tvad&aeantia stamen hairs inducedby 0,6 Gy (60 rad) X-rays, an exposure of 8 hours to EMS solution,and a combination of both where the radiation dose was givenimmediately following the EMS treatment. The dotted line indicatesthe sum of the effects of the X-rays and of the EMS treatment.
SUMMARY OF THE 1980 ANNUAL REPORT OF THE CROP PROTECTION RESEARCH GROUP
INVOLVING PROJECTS 106, 107, 13^ and 135.
A total of seventy chromosomal rearrangements were induced and studied inthe onion fly Hylemya antiqua (106) and several were chosen for inbreedingto produce homozygotes. In four of the lines homozygous individuals wereclearly identified. It can therefore be concluded that the absence of geneticmarkers in the onion fly is not a serious obstacle to the development of suchlines.Results from population studies revealed critical relationships betweenlarval behaviour and larval density. Further, large differences were re-vealed between the laboratorymaintained strains ana newly colonized'wiIdstrains. Such data reveal the importance of "quality" for the applicationof genetic techniques involving the release of laboratory reared insects,
Cytological studies on sex-ratio showed how Variable this character was andthe póssibilities for its use in genetic control. However, the mechanism ofthe sex-ratio distortion was not sensitive to manipulation and distortionremained a somewhat random process.
This report represents the completion of the project. During tb,e course ofthe project the scientific results have been reported in 52 publications.
In I960 further efforts on the induction of temperature sensitive lethalsin the two spotted spider mite Tetranychus upticae (Koch) yielded 10 lineshamozygous for these mutations (107). 11 has been'proven that the temperaturerange between the restrictive and permissive level should be higher than 7 °Cin order to obtain temperature sensitive lethals in a homozygous condition.
Initial work was begun on the biology arid g'&netics of the malaria mosquito,Anopheles stephenai (131*). Population biological work revealed 1) the in-sensitivity of a long established laboratory population to increases inadult density and 2) the same population was characterized by only producinga single batch of eggs.
A new larval rearing system has been designed and preliminary resultsindicate its high potential for the. rearing of single-families.
A start has been made on the cytology of this insect and poiytene pre-parations have so far been very successful. Meiotic and mitotic divisionsin the testes of older larvae or young pupae are also very suitable and thethree pairs of chromosomes can be discriminated.
A first experiment has been carried out for the induction and isolation ofchromosomal fans locations, a total *of hi families were isolated but rathersurprisingly as yet no chromosomal rearrangements have been cytologicallyidentified.
In relation to the development of a genetic sex ing method for Ceratitisaapitata male-1 inked trané locations have been isolated (135). As geneticmarkers.are available in this species these have been utilized. It wasrelatively easy to isolate such translocations but rather disturbinglygenetic recombination was observed in mates of this: species. This makes thei solat i on of a genet i c sexIng method : ráthér, móre dlfficu It. Howeve r, • trans-location lines were isolated which showed extremely tight 1inkage betweenthe marker gene and the male sex. "', • y ^Population studies with a white-eye mutant revealed severe fítriěšs ré-J
- 105 -
auctions in males of this genotype. Nevertheless in random, mating populationsthe mutant aUele remained in the population albeit at a very low frequency.
Electrophoretic studies for alcohol dehydrogenase (AOH) polymorphism re-vealed no variability in the majority of stocks but in a wild strains from ,Tenerife variation was identified and characterized.
Using Drosaphila a genetic sexing mechanism was developed utilizing an-ADHnull mutant and a trans location. The ease with which this system was'pro-duced indicates its applicability for Ceratitia oapitata.
I
- 106 -
PROJECT No. 106
C. van Heemert, A.S. Robinson
ANNUAL REPORT 1980
GENETIC CONTROL OF THE ONION FLY (HYLEMXA ANTIQUA ME I GEN) USING STRUCTURALCHROMOSOME REARRANGEMENTS
1. Trans locations induced by fast neutrons and X-rays, cytogenetic analysisand breakpoint distribution in Hylemya antiqua Meigen
The project on "genetic control of the onion fly" has come to an end andthe results of 8 years research on the biology, genetics and cytogeneticshave been published in $2 pub1 i cat ions and can be summarized as follows:
Over seventy chromosomal rearrangements have been isolated in the onion-fly with X-rays and fast neutrons on the basis of reduced fertilitymeasurements and cytological analysis. No difference in efficiency betweenthe two radiation types could be observed. However, with fast neutronsmany multiple trans locations were induced including a quadruple ( 2 x 4chromosomes) translocation. One male-linked and one X-linked translocationwere isolated. Most rearrangements were asymmetrical reciprocal translo-cations and two pericentric inversions were established. Both meiotic andsomatic pairing configurations were utilized.An interesting phenomenon was the viability of "duplication" larvaecarrying a large duplication and a small deficiency.Relatively more breakpoints were observed in chromosomes 2 and 6. Itcould be concluded from sibcrosses of (mainly) asymmetrical trans locationsthat homozygotes can be produced without the use of morphological markers.The cytological analysis of adults following the assessment of reducedfertility is essential for the isolation of translocation homozygousindividuals. From four trans locations viable homozygous adults wereobserved.
2. Re-irradiation of a male-linked translocation T61 (Y-2)
In order to develop a genetic sexing method based on dieldrin resistanceit is necessary to link the dieldrin resistant allele with the maledetermining chromosome. A fertile Y-linked translocation was irradiatedwith 2 Gy fast neutrons and new complexes were induced involving the"lengthened" Y chromosome. Table 1 gives a list of these translocationstogether with the chromosomes involved and the sterility of malescarrying the complex. Ten translocations involved two autosomes and theY chromosome and one included 3 autosomes and the Y chromosome. Thereappeared to be no relationship between the number of autosomes involvedand the sterility of the males.
Translocations 3*»7» W* and 165 were tested for linkage of the dieldrinresistant locus to the Y chromosome using the technique described in theAnnual Report 1979. The preliminary results indicated the absence oflinkage although there were difficulties in the expression of thisresistance gene. If indeed linkage was absent then the gene forresistance is located on chromosome k and unfortunately no complex in-volving chromosome k was isolated.
t,
I-
M - C1T.T.-
- 107 -
Table 1 - Cytology and sterility of complex Y-linked trans locations inDelia antiqua
Trans locationnumber
TT
TTT
T
T
TT
T
TT
T
108
165
3*7W 7 TWk7k619
698
710
887903,919*
936
Chromosomesi n vo1ved
Y-2-51
Y-2-61
Y-2-51
Y-2-3I-6'
Y-2-31
Y-2-65
Y-2-6
Y-2-5?
Y-2-31
Y-2-31
Y-2-3s
Steri
30,
38.
50,
34,63,
17.
31,
35,
26
%lity
-
72 +
52 +
57 +
32 +
'5 +
56 +
90 +
93 +
27 +
+_
1
1
1
2
41
1
1
1
S.E.
,80
,55
.85
,86
,51
,36
.84
,62
No. malesiSSt€G
For steri1 i ty-
12
18
13
14
10
9
6
19
13
3. Population Biology
During 1980 a thesis entitled "Population Biological Aspects of GeneticControl of the Onion Fly, Delia (= Hylemya) antiqua" was presented at theUniversity of Utrecht by 6. Zurlini.
During the study, the paramount role which "quality" plays in all aspectsof genetic control became apparent. Quality is the final manifestation ofmany components of the biology of the species at an individual andpopulation level.
Several of these components were studied as they represent what could beassessed as important parameters under field conditions in the light ofthe available understanding of Delia antiqua ecology. For these reasonsthe majority of the work involved the larval stage. The choice of thisstage proved to be valid as it was shown that this stage determines to anoverriding extent many adult fitness components. It emphasized the im-portance which must be placed on this stage in the mass rearing proceduresof flies for release. The "internal quality" of the strain, i.e. itslaboratory fitness, is also of course, to a great extent determined bythe larval stage, therefore efficiency in mass rearing i.e. optimal eggproduction, survival, mating success etc., requires an appreciation of theimportance of the larval rearing. In order to assess quality, comparisonswith a newly colonized wild strain are essential; as these comparisonswere being made, interesting and important differences in larval behaviourpattern became obvious. The most relevant of these was the reduction inlarval dispersal activity of the laboratory strain. This observation wasinterpreted with reference to the routine laboratory rearing where larvaldispersal is selected against as dispersing larvae inevitably perish.Rearing under these conditions for many generations would rapidly changethis larval behaviour pattern producing a much more sedentary strain.
- 108 -
Just how quickly this adaptation can occur was demonstrated in the secondpaper of the thesis.
Clearly the physical environment in which strains are reared has im-portant consequences for many population parameters. Early in the studythe question arose as to the relevance of the constant temperature towhich the flies were subjected in the laboratory, when it is clear thatin the field a completely different temperature regime is present, in-volving changes between day and night temperatures of up to 10 °C. Ex-periments on this aspect using the wild and laboratory strains showedthat an alternating temperature regime has many advantages in comparisonwith constant temperature. It can in fact be suggested that the adoptionof alternating temperature regime should be seriously considered for massrearing facilities.
One of the immediate changes faced by a recently colonized strain whichis to be used in the future for mass rearing is an immediate order ofmagnitude increase in adult density. Although a wild strain was notavailable to assess this particular aspect, information was obtainedusing a laboratory strain. This data could be used to identify for thisstrain an optima) density range for mass rearing.
It is considered that the results obtained from the laboratory studiespresented in this thesis can be used with confidence in highlighting somecritical aspects of relevance to the field ecology of the species. Theimportance of quality studies for genetic control using translocationscannot be overemphasized; the results presented in this thesis illustratethis point very succinctly; however, one has always to bear in mind thedanger of overanalysis.
. Translocations and sex ratio distortion in the onion fly, Hylemya antiqua(Mei gen), and their relevance to genetic controT(Ph.D. thesis L. Vosselman)
Experiments on sex determination on the onion fly were carried out withthe aim of establishing if sex ratio distortion, as observed in certaincrosses, could be used for genetic control. Two types of males, XYj andXXY2, were found and the aberrant sex ratios appeared only to occur inprogenies of XXY? males. Sex-ratio distortion is attributed to numericalvariation of Y2 between primordial germ cells. Some males were XXY2Y2 andshowed a highly distorted sex ratio. Unfortunately effective selection ofthese males was not possible. Other subjects treated in chapters 1 and 2of the thesis are gynandromorphism, a polymorphism with respect to thelength of the X-chromosome and X-polysomy. The meiotic segregation of aV\-\ inked translocatlon T61 is reported in the third chapter. The highpercentage of alternate segregation observed in T61-heterozygous maleswas in good agreement with the high fertility of this translocation (95%).Due to an early separation (or asynapsis) of the centromeric regions ofthe acrocentrlc X and Y2, chain quadrivalents with X and Y2 in terminalposition predominated in diakinesis/prometaphase I stages.
Double-trans location heterozygous T 1 V T 6 1 males were produced by inter-crossing Ti4-homozygous females and T61-heterozygous males. These doubleheterozygotes showed several segregation types but four predominated.The total frequency of duplication/deficiency gametes was 60-65%.Application possibilities for these heterozygotes in genetic control ofthe onion fly were discussed. The meiotic segregation of five differentautosomal reciprocal translocations are reported in chapter 5.For translocation Tl*» a pure breeding stock of homozygotes could be ob-
- 109 -
tained using egg-hatch percentages and cytology for the recognition ofthe karyotypes. The result of cage experiments indicated that nosubstantial differences in fitness between TT and ++ exist.
In field cage.experiments a high diapause sensitivity was obtained inboth the TT and ++ stock.
Publications
HEEMERT, C. VAN and I. WITTEVEEN-PILLEN. 1980. Location of the alcoholdehydrogenase gene in the onion fly Hylemya antiqua (Me i gen) using atranslocation tester set. J. of Hered. 71: 36I»~365-
HEEMERT, C. VAN and L. VOSSELMAN. 1980. A male-linked translocation withhigh fertility in the onion fly Hylemya antiqua (Meigen), Genetica 51, 2i
m-m.HEEMERT, C. VAN and A.S, ROBINSON. Cytology and breakpoint distribution ofradiation induced interchanges in Delia antiqua, Genetica. (in press).ROBINSON, A.S. 1980. Effect of sex ratio at three densities on reproductionin laboratory colonies of Delia (= Hylemya) antiqua. Z. ang. Ent. 9.0: 82-89.
ROBINSON, A.S., M. HERFST and L. VOSSELMAN. 1980. Genetic control of Hyle-mya antiqua. Sensitivity to diapause interfering with genetic control.Bull. ent. Res. 70: 103-111.
ROBINSON, A.S. and C. VAN HEEMERT. 1980. Genetic control of the onion fly,Delia antiqua with chromosomal rearrangements. In "Integrated Control ofInsect Pests in the Netherlands". Pudoc, Wageningen, pp. 39-102.
ROBINSON, A.S. Genetic control of Hylemya antiqua. IV. Irradiation offertilized females to produce translocations. Int. J. Appl. Rad. andIsotopes, (in press).
ROBINSON, A.S. and C. VAN HEEMERT. Translocations in the onion fly, Deliaantiqua induced by fast neutrons and X-rays. Theoret. Appl. Genet, (in press).
ROBINSON, A.S. and C. VAN HEEMERT. Complex Y-linked translocations in Deliaantiqua produced by irradiation of a fertile Y-linked translocation.Heredity. (In press).
ROBINSON, A.S. and G. ZURLINI. Mating success of differently sized onionflies Delia (= Hylemya) antiqua. Ent. Exp. ec Appl. (In press).
VOSSELMAN, L. and C. VAN HEEMERT. 1980. Meiotic disjunction and embryoniclethality in sex-linked double translocation heterozygous males of theonion fly Hylemya antiqua (Meigen). T.A.G. 58: 161-167.
ZURLINI, G. and A.S. ROBINSON. Preimaginal environments determining fitnesscomponents of Delia (= Hylemya) antiqua. Oecologia. (in press).
ZURLINI, G. and A.S. R08INS0N. The effect of crowding on adult populationsof Delia (» Hylemya) antiqua. Res. Popn. Ecol. (in press).
Lectures
HEEMERT, C. VAN. Genetische bestrijding. Seminar + course, University Utrecht.22 November 1980.
HEEMERT, C. VAN. Effect of radiation on insects and genetic control of post-harvest insect pests. September 1980. 2 seminars + IFFIT course.
.HEEMERT, C. VAN. Genetische bestrijding van insektenplagen. Introduction forbiology students of the Agricultural University. 23rd May 1980.
- 110 -
HEEMERT, C. VAN, Genetic systems in the onion fly and Drosophila.(Robinson and van Heemert) Japan. Int. Entom. Congress, August 1980.
ROBINSON, A.S. and C. VAN HEEMERT. Male-linked translocation complexes inthe onion fly, Delia antiqua. ESNA. September 1980.
ROBINSON, A.S. Genetics and the control of insect pests. Colloquium Univ.Utrecht, November 1980.
^^^
- I l l -
PROJECT No. 107 ANNUAL REPORT 1980
A.M. Feldmann
GENETIC CONTROL OF THE TWO SPOTTED SPI DERM ITE TETRANJCRUS URTICAE (KOCH)
The induction of temperature sensitive lethal s
It was reported in the previous annual report that a difference of temperat-ure of 7 degrees Celsius between the permissive (28 °C) and restrictivetemperature (35 °C) was too small to obtain the temperature sensitive (T.S.)mutations in a homozygous condition. During 1980, a new experiment had beenperformed, having the permissive temperature at 22 °C and the restrictivetemperature at 35 °C. The experimental conditions for the application of themutagens ethylmethanesulphonate (E.M.S.) were not changed.In this experiment 144 males were treated with E.M.S. of which 70 could besuccessfully mated individually to 3 vergin females for 3 hours immediatelyafter the treatment. They were mated again in the same way in the period of24 to 27 hours after the treatment. 2302 ?\-virgin females were tested fortemperature sensitivity; 124 of these females gave indications on thepresence of a temperature sensitive lethal in a heterozygous condition. Out-crossing of F2~<í<í (produced by the Fj-females presumably heterozygous for atemperature sensitive mutation) with wildtype females resulted in a numberof lines which gave rise following inbreeding and repeated testing at therestrictive temperature to 10 families each being homozygous for a temperat-ure sensitive mutation. These lines will be further tested on homogeneityand full expression of the mutation.
The experiment showed that it is possible to induce and isolate temperaturesensitive mutations in T. urtiaae and that a temperature difference of 7degrees Celsius is too small for obtaining these mutations in a homozygouscondition.
Publications
FELDMANN, A.M. Life table and male mating competitiveness of wild type andof a structural chromosomal mutation strain of Tetranychus urticae, inrelation to genetic pest control. Entomol. exp. 6 appl. 29 (1981) pp. 134-146.
FELDMANN, A.M. and M.W. SABELIS. Karyotype displacement in a laboratorypopulation of the two spotted spider mite Tetranyohus urtiaae Koch. Experi-ments and computer simulations. Genetika, April 1981, 18 pp.
FELDMANN, A.M. Genetic control of the greenhouse spider mite Tetranydhu8upticae. In; Integrated Control of insect pests in the Netherlands. PudocWageningen, 1980, pp. 137-140.
- 112 -
PROJECT No, 13^
A.M. Feldmann, C, van Heemert, A.S. Robinson
GENETICS OF THE MALARIA MOSQUITO, ANOPHELES STEPHENSI
ANNUAL REPORT 1930
1. Population Biology
In the middle of this year a laboratory colony of A. stephenai was intro-duced from the laboratory of Medical Parasitology of the University ofNijmegen. Initial studies were started to obtain insight in the biologyand genetics of this mosquito species. A literature review was made byPUDOC for basic information on previous work on this species.
Since little information on the influence of the adult density on severalparameters is known an experiment on this subject was carried out byM. Theslngh (student).
Using adult densities varying from SO to 500 individuals/cage (23 cm x24 cm x 25 cm) the following parameters were measured: adult survival,mating success, eggs/female and egg hatchability. Rather surprisingly noeffect of density could be demonstrated for any of these parameters. Itwas concluded that adult density effects would only become apparent at amuch higher level and further, the strain which was used was already pre-adapted to extremely high adult densities.
Using the same strain a life table was constructed by R. van Soest(student). He concluded that this particular strain was characterized byonly one oviposition despite the frequent availability of a blond source.This was probably due to the fact that for many generations in Nijmegenonly the first ovipostion was used for propagation of the colony.Repeated selection for this behaviour pattern resulted in the productionof a strain which only oviposited once.
2, Larval Rearing Technique
In order to arrive at a more efficient and reliable rearing method forsingle families of this mosquito, a necessity for genetic work a newlarval rearing system was developed and tested. The new "open" systeminvolved the placing of rearing boxes in a circulating water system. Toallow water to pass through the boxes, fine gauze inserts were made onthe sides of the boxes. The circulated water originated from anaquarium,
Abdullah Essaka used various larval densities with two replicates to assessthe efficiency of this "open" method in comparison with a "closed" systemi.e. the size of box in which the water remained for the whole of thelarval rearing period. The results can be seen in table 1.
From these initial experiments it can be concluded that at larval densi-ties up to 100 variation between replicate is much less in the opensystem. Further at larval densities in excess of 100 survival was poor inboth systems. However, it is considered that modifications in the opensystem will lead to appreciable increase in larval survival even at thehigher densities.
- 113 -
Table 1 - Larval survival in an open and closed larval rearing systemfor Anopheles stephenai
No. larvae
25
50
75
100
150
200
No. (%) pupaeOpen
19 (76)18 (72)
38 (76)k\ (82)
62 (83)57 (76)
86 (86)86 (86)
18 (12)*»7 (3D
18 ( 9)7 ( 3)
Closed
25 (100)19 ( 76)
38 ( 76)*9 ( 88)
W ( 59)55 ( 73)
73 ( 73)8<t ( 8<t)
17 ( 11)T» ( 9)
8 ( 3)12 ( 6)
% variationOpen
<t
6
7
0
19
6,5
Closed
2h
22
lit
11
2
3,5
3- Cytology
Cytology of the mosquitoes was started by Dr. Toan Ta, a guestworker fromthe Department of Genetics, Hanoi University, Vietnam. A technique to pre-pare polytene chromosomes was developed by using half gravid females(16,5 h after bloodfeeding at 27 °C). In this stage good quality pre-parations were obtained. Meiotic and mitotic divisions in testes of olderlarvae or young pupae were suitable and three pairs of chromosomes can bediscriminated.Chromosomes in larval brains and eggs were less assessible.
k. Genetic Studies
For the initiation of genetic studies four different strains were obtainedfrom the PMRC laboratory in Lahore, Pakistan, i.e.:- a laboratory strain, originally from Karachi;- a white eye strain (X-linked);- a striped larvae strain;- a wild strain collected in the field in August 1980.
These four populations will be continuously reared in our laboratory inlarge numbers. Bloodfeeding of the females was done with anaesthetizedwhite mice on top of the cage.
A selection was made in the striped larvae stock for full expression ofthe dorsal white stripes in all a 1 domina) segments. A cross between thewhite eye, non-striped stock and the red, full-striped stock was made inorder to assess firstly if striped is dominant, recessive or codominantand secondly to establish.if linkage or independent inheritance exists.
For the induction of structural chromosome aberrations, h day old maleswere Irradiated with 35 Gy (239,7 rad/min) and subsequently mated tovirgin females. Five egg collections (average E.H. = 31,6%; control E.H.= 89,5%) and 313 females (control) were separated individually in smallcages after mating with Fj males in mass cages. Approximately 50? (15*0
of the females oviposited. Egg hatch analysis was done similarly as inthe onion fly; empty, light brown (non-fertilized or early embryonicdeath) and dark brown (late embryonic death). In table 2 the results areshown. 43 F2 families showed semi-sterility ( 29& - 73%) but no structuralchromosome mutations have been as yet observed cytologically.
Table 2 - Results of first irradiation experiment in Anopheles stephenai.(35 Gy on 4 day old males)
Egg batchof
P females
First
Second
Third
Fourth
Fifth
Total
F] number
6
85
127
84
6732
395
9
90
135
90
8550
450
Number ofseparated
Fj females
66
87
54
67
39
313
Number of¥] females
ovipositing
4546
21
24
18
154
Number ofFi females
showing ster i 1 i ty
12
9
6
9
7
43
Percentage
26,7
19,6
28,6
37,5
38,9
29,0
Lectures
FELOMANN, A.M. Research on genetic control of insect pests in the Netherlands.9th May 1980. London School of Hygiene and Tropical Medicine, London.
HEEMERT, C. VAN. Genetische bestrijding van de malariamuskiet. Capita selectavan Tropische vectorziekten en de moge1 i jkheden en bezwaren van vectorbe-strijding. 24 October 1980. Dept. of Entomology, Agricultural University,Wagentngen.
ROBINSON, A.S. The role of genetics in vector control. Dutch Society forParasitology, October 1980.
ROBINSON, A.S. Population replacement as a means for malaria control.Malaria Colloquium ITAL, February 1980.
FELDMANN, A.M. A new approach in malaria control. The genetic method.WHO Meeting of Malariologists, 27 May, 1980, Geneva, Switzerland.
- 115 -
PROJECT No. 135
A.S. Robinson, C. van Heemert
GENETIC SEX ING IN THE MEDFLY, CERATITIS CAPITATA
ANNUAL REPORT 1980
1. Male-linked translocations
In order to develop the genetic sexing male-linked translocations arenecessary. There are come indications in the literature that geneticrecombination occurs in males of this species so that light linkage ofa resistance gene for use in genetic sexing would be difficult. In orderto resolve this problem males carrying a mutant eye colour (see 2) wereirradiated and mated to wild-type females. Females were mated to mutantfemales and the F2 adults checked for linkage between sex and eye colour.Four families were identified in which there was absolute linkage of thesex with eye colour. However, in two other families there was evidencethat recombination was occurring leading to loss of linkage.
From these results it is suggested that by selecting male linked trans-locations in which the autosomal break point is close to the mutant geneextremely tight linkage can be obtained. Nevertheless it does support theprevious data that indeed crossing-over does occur in males carrying suchtranslocations.
2. Viability of a white-eye mutant
Following inbreeding of irradiated lines a mutant-eye phenotype was ob-served in males and females. It was given the symbol bl (blonde eye).As no mating took place between the mutant individuals, the females wereoutcrossed to normal males; following inbreeding of this progeny a truebreeding line was established, the mutant allele was recessive and auto-somal. In a series of tests the fitness of the mutant line was assessed.The results can be summarized as follows:
- the mutant males showed severely reduced competitiveness (Table 1) andlarval survival was reduced under competitive situations (Table 2 ) .
Table 1. Mating competitiveness of blond eye males (bl/bl) in Ceratitisaapitata
A
óbl/bl
5
5
dul t ratios
6
+/+
5
5
9+/+
5
9bl/bl
5
n
10
10
%femalesmated
70,8
61*. 6
No. females mated with
+/-KS
31
20
bl/bld
0
0
+/+ and bl/bltíd
3
0
- in a population cage experiment where wild-type and mutant individualswere present the frequency of the bl allele declined to a very low levelbut it nevertheless persisted in the population. This indicates thatthe hybrid shows some superiority for this trait over both the wildtype and mutant.
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Table 2. Effect of larval density on larval survival for three genotypesof Ceratitis oapitata
La rva 1genotype
+/+
bl/+
bl/bl
+/+and
bl/b?
No.Larvae
200300400
200300400
200300400
2003001)00
n
333
333
333
333
% Pupation +_ S.E.
89,3 + 1,728,2 + 2,979,3 + 0.7
85,0 + 1,97^,8 + 3,581,5 + 2,4
74,0 + 5,579,1 + 2.773,6 + 0,08
83,6 + 3,865,3 + 1,384,6 + 1,3
Hypoth. tested d.f.
Density x Surv. 2Genotype x Surv. 3Interaction _6
Density x Surv. x 17Genotype indepencence
243,74'
3. Electrophoresis
Alcohol-dehydrogenase polymorphism was analysed eiectrophoretically withthe isoelectric focusing method. Strach gel appeared to be not suitable.In the laboratory-material from Vienna (Seibersdorf, IAEA) no poly-morphism was observed. In contrast to this, pupal material from Tenerifecontained new isozymes which were not previously observed. Further in-breeding of this material was carried out and a third phenotype wasvisualized. In figure 1 the three different isozyme patterns WW (fromVienna), WT (hybrid) and TT (homozygous phenotype from Tenerife) areshown. Further selection towards a pure TT stock has not yet been success-ful.Adult TT phenotypes could be established in each generation of the in-breeding experiment but the TT phenotypes probably show reduced fitness.
4. Supporting research using Drosophila melanoaastep
The system that w i l l be developed for the Medfly depends on the inductionand isolation of an alcohol dehydrogenase null a l i e l e (n ) . In Drosophilasuch a l le les ex is t . I t was therefore decided to see i f the theoreticalsystem proposed for Medfly could be achieved in Drosophila*
By incorporating ethanol (4% v/v) in the larval rearing medium of aspecially constructed D. melanogastev strain i t was indeed possible toproduce exclusively male adults, the female larvae died (Table 3 ) .
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Table 3 - Effect of ethanol on the survival of male and female larvae ofDrosophila differing in their ADH activity. (Fn is heterozygousfor a nul1 allele)
Larvalgenotype
nn Fn
nn Fn
Larvalmed i um
0 % ethanol
ethanol
No. larvaetrans-ferred
200
300
ReplicafcesMean no. (&'emerged adults
9 dMean survival
% + S.E.
82,6(W,60(OS)
115,1(100%)
83,3+ 1,2
39,4 + 3,2
The male-determining chromosome was linked with the positive (F) ADHallele by a translocetion, the females were hotnozygous for the nuil alleleand hence sensitive to ethanol.
The success of <•> system indicates its feasibility as a genetic sex ingmethod for the Medfly
5. Pentenol, Pentynol and Allylaicohoi as toxicants for adult Drosophila
For the development of a male-female separation system the use ofsecondary alcohols have been suggested. Since the flies which are homo-zygous for two ADH positive alleles (F or S) are sensitive for low con-centrations of pentenol etc, and ADH negative flies are not, it was in-tended to develop a technique by which ADH heterozygous flies (FN or SN)could be positively selected from either FF, SS or FS positive flies Thealternative procedure, in which electrophoresis is involved requiresconsiderable work, although it is a safe method.
Several experiments with pentenol and pentynol were carried out on FF, SS,FS, FN, SN and NN Drosophila adults of more than b days old. Treatmentsin which the time was changed were more reliable than when the con-centrations were changed and the time fixed. SS and SN phenotypes were ingeneral more sensitive as compared to FF and FN respectively. NN was re-latively less sensitive at the levels at which the other types were deadafter 24 hours. Allyl-alcohol, in contrast to the other two compounds issoluble in water and therefore more useful. The results in most ex-periments were rather variable.
Using 8% allyl alcohol on adult DrosopkLlas preliminary results indicatethat after approximately 5 minutes FF, SS and FS (all homozygous positive)were killed and the FN and SN after 7 minutes. The NN types survive after7 minutes treatment.
The application of this approach to Medfly needs further investigationin order to assess after which period, at a certain concentration, ADH-positive adults will die. Using this Drosophila information it is hopedthat following an EMS treatment, new ADH negative mutants can be inducedand isolated.
Publications
HEEMERT, C. VAN and W.J. VAN DEN BRINK. 1980. Alcohol dehydrogenase poly-morphism in the mediterranean fruit fly Ceratitis aapitata. Ent. exp. appl.28, 2: 229-231.
- 118 -
ROBINSON, A.S. and C. VAN HEEMERT. Genetic sexing in Drosophila using theADH locus and a Y-linked translocation. Theoret. Appl. Genet. (In press).
ROBINSON, A.S. and G.C. SCHELLING. Reduced male mating competitiveness ina "blond eye" mutant strain of the Mediterranean fruit fly, Ceratitisaapitata. Environ. ' ntomol, ,(ln press).
Lectures
HEEMERT, C. VAN. Genetic sexing and alcohol dehydrogenase in insect control.(C. van Heemert and A.S. Robinson) Japan, August 1980. Int. Entom. Congress.
ROBINSON, A.S. and C, VAN HEEMERT. Genetic sexing in Dvoaophila. ESNA,September 1980.
ROBINSON, A.S. and C. VAN HEEMERT. Progress in genetic sexing of the Medfly.IAEA. Experts Meeting, December 1980.
Fig. 1 - ADH polymorphism in Ceratitie aapitataW - Vienna alleleT - Tenerife allele
- 119 -
SUMMARY OF THE 1980 ANNUAL REPORT OF THE PRODUCT TREATMENT RESEARCH
GROUP INVOLVING PROJECTS 108, 109, 1 1 1 , 112 and 123.
General
In 1980 the Joint Expert Committee on the Wholesomeness of Irradiated Food(JEFCI), which is supported by the Food and Agriculture Organization (FAO),the World Health Organization (WHO) and the International Atomic EnergyAgency (IAEA), gave the following clearance. No toxicologlcal hazard iscaused by irradiating, for conservation, any food up to a dose of 10 kGy(1 Mrad), and hence foods treated in this way no longer need to be testedfor toxicity. At present, for approximately 95 per cent of food items to betreated by irradiation, the dose needed is much lower than 10 kGy.
The JEFCI founded in 1961, recommended already in 1976 strawberries, papayaand chicken, and provisional acceptance of irradiated rice, onions and cod-redfish mixture treated at levels of irradiation below 10 kGy. Based on itsrecommendations 22 countries have already given clearance for unconditionalor provisional irradiation conservation for foods such as deep-frozen mealsin hospitals, cod and haddock fillets, chicken, spices, strawberries, mush-rooms, onions and potatoes.
In 1979 the Codex Alimentarius Commission adopted a Recommended InternationalGeneral Standard for Irradiated Foods. The above conclusion that toxicplogicaltesting is no longer necessary for foods conserved by a dose of not morethan 10 kGy is expected to facilitate the wider use of food irradiation. TheCommission is requested by the Codex Committee on Food Additives to initiatethe procedure for the amendment of the Recommended Codex Standard. It willbe considered by the ľith Session of the Codex Alimentarius Commission inJune 1981.
The 2nd IFFIT Inter-Regional Training Course on Food Irradiation was held atthe Foundation ITAL and the Pilot Plant for Food Irradiation from 1 Septem-ber to 3 October. Lectures were given and practical exercises conducted bydifferent specialists from ITAL and other Dutch Institutions and some foreignexperts.
Fellows from Ghana, Chile, Afghanistan and Kenya received applied trainingin food irradiation for a period of six or twelve months within the framework of IFFIT at ITAL.
Mutagenicity
The contents of phenol i c cinnamic acids and coumarins as well as of the glyco-alkaloids of gamma irradiated potato tubers has been studied in detail.Gamma irradiation till 3 kGy had no effect on the glycoalkaloids contentsin two potato tuber varieties during a 4 months storage period. The phenoliccompounds behave differently and show a considerable change during storagein potatoes Irradiated at the highest dose level, 3 kGy. A time dependentchange of phenolic extracts was observed. This change of phenolic compoundscould be partly described to the p-blycos ide of scopoletin (coumarin, 7-hy-droxy-6-methoxy) and was accompanied by a general decrease of chlorogenicacid, the main hydroxy-cinnamic acid of potatoes.
The major conclusions of the investigation to the mutagenicity of gamma-irradiated solutions of nucleic acid constituents were:
1. Irradiated solutions of 2-deoxy-D-ribose, the sugar moiety in DNA, areclearly mutagenic in Salmonella typhimurium and induce sister-chromatid
- 120 -
exchanges in CHO cells. The mutagenicity of these solutions is not due tohydrogen peroxide or malonaldehyde.
2, Irradiated solutions of nucleic acid bases do not show mutagenic activityin the Salmonella test.
3, Irradiated solutions of nucleosides are mutagenic in Salmonella typhi-muHm. The mutagenicity is due to the radiolytic products of the sugarmoiety.
4, Irradiated solutions of the nucleotide, thymldine-S'-monophosphate, arenot mutagenic in Salmonella typhimupinm.
Combined EffectsStudies on the radiosensttization of microorganisms by selective inorganicradicals showed that Brjj was intrinsically a more effective radiosensitizingagent than oxygen, ,The effect of radiation on some pathogenic organisms In chicken carcasseswas studied. The D-values were 0,33 and 0,54 kGy for EnterobaoteHaoeae and0,3*1 and 0,55 kGy for Eeohevichia eoli in fresh and frozen chicken, respect-ively. The 0-value of S. niloese on artificially contaminated carcasses at4 °c varied between 0,55 and 0,68 kGy, while at -18 °C it was about 1,25 kGy.Decay in potatoes is mainly caused by the fungi Phytophthora infestans,Fuaarivm aotani and Phoma eadgua. An irradiation dose of 0,24 kGy inactivatedcompletely P. infeatana. Wuaavixm and Phoma wrre, however, very resistant toheat and irradiation and only a combination of 45 °C and 1,6 kGy and 50 °Cand 1,6 kGy can inactivate both fungi.Experiments carried out with apples showed that a combined treatment has apositive effect on the control of fungi but a small negative effect on theorganoteptic properties of the fruits. The optimum combination was 45 °C and1.2 kGy.Maize grains, coca beans, animal feed and cotton seeds were inoculated withAapepgiltuB flaous Link NRRL 5906 and subsequently heated under high (> 85%R.H.) and low (< 45% R.H.) humidity conditions and then irradiated at 0, 3,5and 4,0 kGy followed by storage at 28 °C with an R.H. of 75 and 90% respect-ively.A heat treatment at 60 °C with 85% R.H. prior to a gamma irradiation with4,0 kGy inactivated A. flovua in maize grains, animal feed and cotton seeds.For cocoa beans a temperature of 80 °C (85% R.H.) was necessary. Thelongest storage times were obtained at an R.H. of 75%.The formation of aflatoxin Bj in the products were prevented by the abovementioned combined treatment. THe total amount of aflatoxin Bj formed wasdirectly related to the spore density.The combination of mild heat in the form of hot water dipping at 40, 45 and50% and tow dose irradiation at 0,25 to 1,5 kGy were applied to auberginesfor the control of BotryHe ainerea aná to increase the shelf life.Doses of 1,0 and f,5 kGy decreased the resistance of the fruits to Botrytísattack. The combined treatment delayed senescence but discoloured the calyxof the aubergines even at a dose of 0,25 kGy. V ? ť :
- 121 -
PROJECT No, 108 ANNUAL REPORT 1980
H. Stegeman, H.B. Leveling
STUDIES CONCERNING COMPLEMENTARY EFFECTS OF IRRADIATION AND OTHER PRE-SERVATION TECHNIQUES ON MICROORGANISMS FOR IMPROVEMENT OF FOOD HYGIENE
1. Radlosensitization by selective inorganic radicals
In cooperation with Dr. J. Schubert, Professor of toxicological chemistryat the University of Maryland, USA we have examined bacterial survivalfollowing attack on crucial ami no acids by radiolytically generatedradical an ions, X|> where X = Br~orCNS~. The radical anions aregenerated by OH radicals;
X" + OH -»• -X + OH"
Depending on pH radical anions react selectively or specifically withcysteine, tryptophan, tyrosine and histidine. No measurable reactiontakes place with other amino acids as components of proteins. Consequent-ly, when one or more of the amino acids crucial for bacterial survival isattacked by a radical anion, a high degree of radiosensitization isreal i zed.
Irradiation of Streptocoaaus faecalis in a neutral, N20/Br" system leadsto practically complete killing with extraordinary low doses of ir-radiation, namely a Djo of 13 Gy compared to ^70 Gy in N2, 250 Gy in N2Oand 16O Gy in O2. However, irradiation and chemical investigations de-monstrated that the lethal action in neutral, N20/Br~ is due mainly tobromine, Br2, rather than Br^ or the radical anion, Br2. For example,addition of unirradiated bacteria to a previously irradiated neutralsolution of N2O/Br" reduces survival. The medium effects are eliminatedby radiation chemical and/or chemical reactions which destroy bromine,such as occur in basic solutions, or in N2/Br~ or 02/Br" systems becauseof back reactions of eaq with Br2 in the former and with H2O2 and 0$ inthe latter. Neither are medium effects produced in N2O/Br" systems atpH's > 9. However, in N2/Br~ the Dig = 82 Gy compared to 160 Gy in O2which indicates that BrJ is intrinsically a more effective radiosensitiz-ing agent than oxygen.
The crucial amino acid for the survival of S. faecalis appears to betyrosine. It is postulated that the radiosensitizing action of radicalanions involves the inhibition of DNA repair of strand breaks by deprivingthe cells of energy.
2. Elimination of pathogenic organisms from chicken carcasses by irradiationrganirholt(in cooperation with Spelderholt Institute for Poultry Research)
í (I. Kramomtong).
{ Commercially scalded, plucked and eviscerated broilers, aerob i call yI packed into polythylene bags were radiation treated at +4 °C (fresh{ chicken) and -18 °C (frozen chicken), respectively, and viable counts ofí Enteróbacteriaoeae, Escheriohia coli, Streptococcus faecalis, Staphylo-| aocaue aureus and Salmonella were enumerated on various media before andI ' after irradiation of chicken. Gamma radiation doses applied were 0,5,
1,0 and 1,5 kGy.
- 122 -
D-values were 0,33 and 0,54 kGy for the Enterobacteviaceae and 0,34 and0,55 kGy for Escheriekia coli in fresh and frozen chicken, respectively.There was a tendency that pre-incubation on non-selective media beforepouring the plates with selective media was more effective in enumerationof stressed cells of these microorganisms. D-values of Streptococcusfaecalis were 0,69 kGy (fresh chicken) and 1,22 kGy (frozen chicken).Regarding D-values, Staphyloooccus aueeus showed the same radiationsensitivity as Enterobaateriaceae, Three samples from 20 untreatedchicken proved to be Salmonella positive. No Salmonella were found in anyof the irradiated chicken.
Radiation survival oftSalmoneÍÍa.«iÍ2egg_on_artificial>]i^>iicontam|natedčhTčkěňrTfioh!ě~Ž7~Ě!:FOuTý7The effect of irradiation on the number of Salmonella niloeae inartificially contaminated chicken carcasses was investigated. The rangeof irradiation dose used was between 0,17 and 3,40 kGy (17 - 340 krad).The irradiation was performed at +4 and -18 °C, respectively.Irradiation considerably decreased the number of Salmonella from thechicken especially when the irradiation was done at 4 °C. The D-value at4 °C ranged from 0,55 to 0,68, while at -18 °C it was about 1,25 kGy. Forthe reduction of 0,7 log-cycles, a dose of at least 4,8 kGy and 8,76 kGyare needed for the refrigerated and deep frozen carcasses, respectively.
This project has been completed. Results of further studies will be reportedin project 111.
PublicationsSTEGEMAN, H. De bacteriespore., In: Sporevormende bacterien in voedingsmidde-len (H. Stegeman, H.M.C. Put and I.P.M. Langeveld, red.) p 6-25 (1980) PudocWageningen.
SCHUBERT, J., STEGEMAN, H. and SWILDENS, M. Radiosensitization of micro-organisms by radical anions. I. Medium Effects. Int. J. Radiat. Biol. (Inpress).
SCHUBERT, J., H. STEGEMAN and A.F. GRONEMAN. Radiosensitization of micro-organisms by radical anions. 2. Stveptoaoaous faecalis. Int. J. Radiat. Biol.(In press).SCHUBERT, J.. and H. STEGEMAN. Sensitization of microorganisms and enzymes byrad iat ion-induced selective inorganic radical anions. In: Combination Pro-cesses in Food Irradiation. Proceedings of a symposium. Colombo, 24-28 No-vember 1980. IAEA, Vienna. (In press).
Technical and preliminary research reports
STEGEMAN, H., J.G. VAN K00IJ and H.B. LEVELING. The suitability of irradiatedcocoa beans for processing. Techn. and Prelim. Res. Report No. 90. 1980.
KRAM0MT0NG, I. and M.Z. EL-FOULY. Elimination of pathogenic organisms fromchicken carcasses by irradiation. 1. Effect of radiation on some pathogenicorganisms in chicken carcasses. IFFIT Report No. 1. February 1980.
EL-FOULY, M.Z. and I. KRAM0MT0NG. Elimination of pathogenic organisms fromchicken carcasses by irradiation. 2. Radiation survival of S. niloese onartificially contaminated chickens. IFFIT Report No. 2. February 1980.
Lectures
STEGEMAN, H. Basic effects of radiation on microorganisms. 2nd IFFIT Inter-Regional Training Course on Food Irradiation. 1980.
v V-1É5(íSSIiSBíS5K3:>s.'X.'uJi«r.
- 123 -
STEGEMAN, H. Microbiological aspects of radiation preservation of foods2nd IFFIT Inter-Regional Training Course on Food Irradiation. 1980.
1ií>
i
í,vf
i;f,
ř"> •
' * " ' " • ! ,
r 12** -
ANNUAL REPORT 1980PROJECT No. 109
J.W.G.M. Wilmer, H.B. Leveling
RAD I AT I ON-INDUCED CHANGES IN NUCLEIC ACID CONSTITUENTS AND THEIR BIOLOGICALACTIVITY
The work on this project has been completed and reported in a thesis.
Deoxyribonucleic acid (DNA) is the most critical target for the lethal,mutagenic and carcinogenic effects of ionizing radiation on living organisms.Rad i at i on-induced damage to DNA includes single-strand and double-strandbreaks, modification and liberation of nucleic acid bases, alterations inthe sugar moiety, and release of inorganic phosphate. Although many radio-lytic products of DNA constituents have now been identified, it is largelyunknown which changes in DNA are lethal, mutagenic or carcinogenic.The aim of this study was to investigate the mutagenicity of gamma-irradiatedsolutions of .nucleic acid constituents. For this purpose, two short-termmutagenicity test systems were used: the Salmonella/mammalian mlcrosome test(Ames test) and the in vivo Sister-chromatid exchange test with Chinesehamster ovary cells.The major conclusions of the investigations are:
1. Irradiated solutions of 2-deoxy-D-ribose, the sugar moiety in DNA, areclearly mutagenic in Salmonella typhimwrim and induce sister-chromatidexchanges in CHO cells. The mutagenicity of these solutions is not due tohydrogen peroxide or malonaldehyde.
2. Irradiated solutions of nucleic acid bases do not show mutagenic activityin the Salmonella test.
3 Irradiated solutions of nucleosides are mutagenic in Salmonella typhi-muŕium. The mutagenicity is due to the radiolytic products of the sugarmoiety.
k. Irradiated solutions of the nucleotide, thymidine-5'-monophosphate, arenot mutagenic in Salmonella typhimurium.
In addition to these conclusions, the following remarks can be made.(1) C,'-TMP does not probably represent the other nucleotides. From the•iterature it is known, for example, that irradiated 3'-monophosphate-nucleo-tides and 3',5'-diphosphate-nucleotides generally eliminate more readily thephosphate groups than irradiated 5'-nucleotides. Subsequently, mutagenicradiolytic products might arise from the sugar moiety of the resultingnucleosides. (2) Upon irradiation of DNA an identical situation may arise.Very recent investigations of Schulte-Frohlinde (Proceedings of the SixthInternational Congress of Radiation Research, Tokyo, 1979) show that inirradiated solutions of DNA products are formed - initially by breakage ofphosphate ester bonds or by a C-C bond rupture in the sugar skeleton - whichare identical to the radlolytic products of 2-deoxy-D-ribose.It is difficult to extrapolate the presented results and considerations toirradiated foods. In principal, it seems possible that radiolytic sugar pro-ducts of DNA are formed in irradiated foods. It can be assumed that, at thedoses applied in food irradiation processes e.g. to inhibit sprouting of
' potatoes or onions, the concentrations of these products will be very lowand/or that they react with other components in foods, in such a way that
- 125 -
they become harmless. The results of the investigations on the wholesomenessof irradiated foods, which never showed any deletrious effect, seem tojustify this assumption.
Publications
WILMER, J.W.G.M. Mutagenicity of gamma irradiated nucleic acid constituents.Thesis University of Nijmegen, September 1980, 93 pp.
WILMER, J.W.G.M., H. LEVELING and J. SCHUBERT. Mutagenicity of irradiatedsolutions of 2-deoxy-D-rlbose. Mutation Research 78 (1980) 85-90.
WILMER, J.W.G.M., J. SCHUBERT and H. LEVELING. Mutagenicity of irradiatedoxygenated and deoxygenated solutions of 2-deoxy-D-ribose and D-r i bose inSalmonella typhimurim. Mutation Research.(in press).
WILMER, J.W.G.M. and J. SCHUBERT. Mutagenicity of irradiated solutions ofnucleic acid bases and nucieosides in Salmonella typkimurium. MutationResearch. (In press).
WILMER, J.W.G.M. and A.T. NATARAJAN, Induction of sister-chromatid exchangesand chromosome aberrations by irradiated nucleic acid constituents in CHOcells. Mutation Research 88 (1981) 99-107.
- 126 -
PROJECT No. 111
D. Is. Langerak
APPLICATION ASPECTS OF FOOD IRRADIATION TO IMPROVE FOOD HYGIENE
ANNUAL REPORT 1980
Combined heat and irradiation treatment to control mould contamination infruit and vegetables " — — — — — — — — — — — — _ ^ _
Experiments to study the effect of a combination of heat and irradiationfor controlling mould infection have been continued using potatoes, applesand tropical products (IFFIT programme).
Potatoes (Solanm tuberosum L. cv. Bintje)
Decay in potatoes is mainly caused by the fungi Phytophthora infestana,Fuaarim aolani aoerulewn and Phoma exigua.The aim of the research was to inactivate these fungi by the combined treat-ment. The experiments were carried out in collaboration with the ResearchInstitute for Plant Protection (IPO).The potatotubers were artificially infected with the fungi in the top, basaland lateral sides with a 101* spore-suspension per spot.
Preliminary experiments showed that P. infeatans was rather resistant toheat but very sensitive to irradiation. For that reason the experimentswere carried out with spores in a more resistant stage by using a longerincubation time.
After an incubation of 24, 48 and 72 hours at 15 °C, the infected potatoeswere dipped in water at 20, 40, 45 and 50 °C for 5 min and subsequentlyirradiated with 0, 0,08 and 0,24 kGy and stored at 15 °C at R.H. of 90%.
With an increasing incubation time P. infestcms appeared to become moreresistant to irradiation; even a combination of 45 °C with a dose of 0,08kGy was unsuffictent to inactivate the spores. A dose of 0,24 kGy, however,inactivated the fungus, even without a heat treatment.
Fuaarium_solaniLAfter infection the potatoes were incubated for 48 hours at 15 °C and sub-sequently dipped into water at temperatures of 20, 40 and 45 °C for 5 minand then irradiated with 0,2 - 1,6 kGy.
Fig. 1 shows that in comparison with P. infestane, Fusarium is clearly moreresistant to irradiation; even a dose of 1,6 kGy is unsufficient to in-activate this fungus. Decay due to Fusapium, however, can be prevented bya combination of heat (45 °C) and irradiation (1,6 kGy).
Phgma exigua.
Similar experiments were carried out with Phoma. Fig. 2 shows that Phoma israther resistant to heat and irradiation; even a heat treatment of 50 °Cor an Irradiation dose of 1,6 kGy is not capable of completely inactivatingthis fungus. An irradiation treatment enhances even the percentage of decayprobably because some phytoalexines such as rishitine, are degraded, whichsubstance is known as a fungistatic compound in potatoes.
A combination of heat and irradiation, however, reduces considerably thepercentage of decay. The best results were obtained with the combination of50 °C and 1,6 kGy.
- 127 -
The combination of mild heat in the form of hot water dipping at 20, 40, 45and 50 °C and low dose irradiation at 0,4 to 1,6 kGy were applied to thevarieties Cox Orange Pippin and Laxton Superbe for the control of Botrytisainerea infection and for the ma intai nance of the quality.
The experiments were carried out at the end of the storage period. Duringstorage at 15 °C the apples were evaluated on decay, colour (reflectometer),firmness (Instron Shear Press) and sensor i c properties (panel tests).
The results on decay are presented in figures 3 and 4.
Heat treatment of 45 °C and higher reduces the percentage of rot in apples.A dipping temperature of 50 °C, however, causes peel damaging.
The largest reduction in delay is obtained by the combination of 45 °C and1,2 kGy but 0,8 kGy also gives some delay.
Colour measurements carried out with a reflectometer indicated that a heattreatment delays the colour changing during storage. This phenomenon isintensified by irradiation. Estimation of the colour by means of a colourcard shows, however, the opposite; an explanation of this difference cannotbe given.
Instron Shear Press measurements show that the firmness of the apples de-creases a little with an increasing irradiation dose. The effect of a heattreatment on the firmness is not clear.
Sensoric tests showed that only a combined treatment of 45 °C and 1,6 kGyhas a small unfavourable effect on the consistency and taste of the apples.
In conclusion a combined treatment has a positive effect on the control offungi but a small negative effect on organoleptic properties of fruit atthe end of the storage period (end of climacterium).
This research will be continued with fruits at the beginning of the storageperiod (in pre-cIImacterium).
Tl2DÍSii_BJI2^y£í5 (V. Appiah, G.T. Odamtten)
_i/ľ__wtro_s<tudj£s_on J^e jefj[.ect_of_ £omb_inatjp£ treatment £f_h£a_t and_ j_r^jraitatTon £n_sjjores_ ofJispepgillus^fl^us^ i'UK-N^Rr J9O6_
Studies have been carried out using Aspergillus flavus NRRL 5906 todemonstrate the fungicldal and synergistic effect of the combination treat-ment of heat (40 - 60 °C) and gamma irradiation (0,25 - 4,0 kGy) on thegrowth of the spores employing two treatment methods. The state of thespores (dry or moist) determined the heat and dose requirements for completeinactivation.
Heating for at least 5 min at 59 °C was sufficient to inactivate the sporesof A. ftaous treated in the moistened state and raised on maize meal agar.Generally 5 min heating at 53 °C in combination with 0,75 kGy of gammairradiation was synergistic in its effect resulting in inactivation ofspores treated in the moistened state. Ue obtained similar D|o values of0,19 and 0,18 kGy after 5 and 10 min heating respectively at 53 °C.
A higher killing effect was recorded when dry spores were heat-treated(60 °C for 30 min) under humid conditions (< 85£ R.H.) prior to irradiationthan when low humidity (< 45% R.H.) was applied prior to irradiation with3,5 or 4,0 kGy.
- 128 -
on
In the absence of a readily-built chamber with facilities for moist heattreatment, the possibility of using a climate room, an incubator and an ovenwas investigated. An incubator and an oven with facilities for moist heatwere found to be the most suitable for the heat treatment of grains andcocoa respectively.
Contra), ofjnoujds ca.usi.ng <LeJte£Í£rj»tjoni of Ji»*«L9i£a!.n.S. IP^ôomoTnä*tToň T«aTme"nT:~a_p7eTliňi£a7y""mô*d¥l—s"tuďy~w7th "kevPKSOS"Preliminary investigations using maize grains artificially inoculated withA. f lama Link. NRRL 5906 have shown that a simple heating apparatus pro-viding either low (< 45% R.H.) or high (> 85% R.H.) humidity during the heattreatment can be employed to treat grains and obtain good control of mouldgrowth when the grains were first heated at 60 °C for 30 min under highhumidity conditions prior to irradiation with 4,0 kGy of gamma radiation.It was expedient to irradiate the grains within 30 min after the heat treat-ment In order to maintain the lethal sensitization effect of the moist heattreatment (> 85% R.H.) on the spores of A. flows prior to irradiation.
Evidence is presented that the fungistatic effect of the combination treat-ment was strongly dependent on the storage humidity in prolonged storageconditions since greater reduction of infection could be obtained at thesame dose and heat treatment humidity conditions if grains were kept at 75%R.H. as compared by 90% R.H.
The £,ffe£t_of_ ,the wmbj/ratton ^treatment, o_f_heat a_nd £am_ma_ J.r£ad_ia_t_ioji onj 5 O T O ~ p £ y T l č a 7 a n d c a n o n i c a l J P > X P £ e 7 t T e š f Z I O i 5 X ia n < f d T ] l j t £ " ™ ~ " " " -Consumer acceptance test was performed with a popular Ghanaian meal, kenkey,prepared from the control and combined treated samples in the conventionalway. The product was presented for the first time to Dutch adult panelists.
In the organoleptic testing Kramers1s Quick Rank Test was employed. Thetwenty (20) adult panelists could not detect any significant difference(P • 0,05) in colour, flavour and taste between the control and the combinedtreated maize grains. The results give indications of the market acceptanceof the heat treated and irradiated maize.
The starch viscosity measurement of samples showed a degradation withtreated samples as compared with the controls but this was not directlyrelated to the dose and heat treatment. The combined treated grains yieldedmore reducing sugars than the control samples. This perhaps renders thecombined treated samples more digestible.
£rodjicj|jon_of_ äflätox'J! &L^Í ApEPSgilUu^ £l3PiL8J-Ĺnk. in_siib£eJľ3£cuj.tu.re jäfter̂ £oj<[bTrô»tTon treatment, £f_hea_t and_ Ijamma TnFaillätjonThe production of aflatoxin Bi by a toxigenic strain of Aspergillus flavusLink in static liquid culture, after the combination treatment of heat(60 °C for 30 min under high humidity > 85* R.H. or low humidity < kS% R.H.)and irradiation (0, 3,5 and 4,0 kGy) has been investigated. The attenuationof aflatoxin Bi formation was related to the ambient humidity during theheating of the spores. Aflatoxin B) formation was curtailed when the sporeswere heat-treated at 60 °C for 30 min under high humidity (> 85% R.H.)conditions prior to irradiation with 4,0 kGy of gamma irradiation. Theparallel treatment of spores of A. flaous under low humidity (< 45% R.H.)conditions resulted in the formation of a copious amount of aflatoxin B].
- 129 -
The total amount of aflatoxin Bj formed was directly related to the sporedensity used in the inoculation of the flask and less so with dose.High spore concentration (105 - 106 c.f.u.) resulted in preponderance ofwhite vegetative growth and little or no aflatoxin B] was formed.Reduction of spore concentration resulted in higher yields of aflatoxin Bjand the cultures were green to dark green.
Preliminary studUg £n_the_ef_fect £f_tjiejc£mlbijia_ti£n_t£e«ttnejit_of_ J2eat_and_g a m m a T r T a d i j [ t T o n ^ O i l O s S r l L ' Z i s Z s i P ' i ' J E y - ? — L ' ^ E ÍE i t É ~"The combination treatment of heat, (60 °C applied for 30 min under highhumidity conditions (> 851 R.H.) and irradiation 3,5 - 4,0 kGy could reducethe mould count of artificially infected animal feed from about 106 downto 10 colony forming units g"1 (c.f.u. g"1) in six weeks storage at 75fc R.H.At 80% R.H,, the reduction was from 106 to 102 c.f.u, g"1; irradiation alonewas also equally effective. At a storage humidity of 90% there was a delayIn onset of infection for 2 weeks, after which there was an outburst ofprolific growth and the animal feed was completely overgrown by A. flavusafter 6 weeks storage. Higher amounts of about 126 yg kg"1 of aflatoxin Bjwas formed in the combined treated samples under low humidity conditions andkept at 90% R.H. than in the controls.
Heat treatment alone did not significantly reduce the microbial load of thefeed samples but was effective together with gamma irradiation in eliminatingthe Enterobaoteriaaeae contamination in the feed samples tested.
Results for Cotton seed were similar to that obtained for animal feed andthe treatment did not adversely affect the fat composition of the cottonseed after 6 weeks storage. Only short chain fatty acids, acetic, propionicand butyric acids could be detected in trace amounts in all the samples butthis was not due to the combination treatment.
Similar to the animal feed, the enteropathogenic organisms, using thecriterlum of Enterobaoteriaaeae counts were drastically reduced as a resultof the combination treatment of heat and gamma irradiation employed inthese investigations.
a £Ojnbjjrration £fheat jradjatjhoncemtToT mou l£" i.n~djFi ®c'Zc£c£aZ'55.a£?_Dried cocoa beans were inoculated with Aspergillus flavus Link NRRL 5906,treated with a combination of heat applied at 80 °C low humidity (< kQ%)and high {> 85%) humidity for 30 minutes and radiation at 3,5 and k,Q kGyfollowed by storage in polypropylene bags at 28 °C, 80% relative humidityfor four months. Treatment combination of 80 °C high humidity and 4,0 kGyreduced the population of the colony forming units of A. flavus to less than10 per gram of cocoa. The storage relative humidity of 80% prevented mouldingfor the period of four months and there was no significant change in thenumber of colony forming units of A. ftavus.
The £valua£l£n_of £Otne_jjhy£Í£aJ_ ,g.ua_lj_ty_ £a£ametersMcrf £oaoa after £omb_j,nedTnaaTniinT £ f h e a t ][n<í jJl líT "~ "" "~ _ — —The organolepttc evaluation of the quality of cocoa after combined treatmentof heat and radiation was performed by a laboratory panel. No statisticaldifferences were found between the odour (flavour), colour and taste of thesamples. The determination of odour of the alcoholic extract using absorb-ance in a spectrophotometer also did not detect any differences due to thecombined treatment. The combined treatment of the beans did not cause anychange In the quality of cocoa butter prepared from them.
- 130 -
effect of mij[d hea_t .treatment; a_nd_ j.rradj at k m £n_a^bergj JNM j.nf,e£ted_
The combination of mild heat in the form of hon water dipping at kQt ^5 and: 50 °C and low dose irradiation at 0,5 to 1,5 kGy were applied to aubergines
for the control of Botvytia ainevea and to increase the shelf life.
Doses of 1,0 and 1,5 kGy decreased the resistance of the fruits to Botrytisattack and decreased shelf life but doses of 0,5 and 0,75 kGy in combinationwith 45 °C dipping decreased less the natural resistance to the disease.The combination treatment delayed senescence and decreased loss of weightduring storage but the caJyx of the aubergines is too sensitive to ir-radiation even low dose of 0,25 kGy resulted in the browning of the calyxand so although the fruits were firmer they were not acceptable to theconsumer.
Publications
UVNGERAK, D.is. Effect van de combinatie verpakken en bestraling op de kwa-liteit van gesneden groenten. Voedlngsmiddelentechnologie Jj$ (1980) nr. 23.13-18.
LANGERAK, D.ls. Verpakking in relatle tot ioniserende stralen. Syllabus"Verpakking van voedingsmiddelen" Stichting Post-Academisch Onderwijs -L.H. (in press).
Technical and preliminary research reports
APPIAH, V., G.T. ODAMTTEN and D.ls. LANGERAK. In vitro studies of the com-bination of heat and radiation on conidia of Aspergillua flaous Link.IFFIT Report No. 3, 1980.
APPIAH, V., G.T. ODAMTTEN and D.ls. LANGERAK. The technology of heat treat-ment of cocoa beans. IFFIT Report No. k, 1980.
• APPIAH, V., G.T. ODAMTTEN and D.ls. LANGERAK. Studies on the possibility• of using a combination of moist heat and radiation to control mould i ness: in dried cocoa beans. I. A preliminary study. IFFIT Report No. 5, 1980,,
APPIAH, V., G.T. ODAMTTEN and D.ls. LANGERAK. Studies on the possibilityof using a combination of heat and radiation to control mould i ness in driedcocoa beams. II. IFFIT Report No. 6, 1980.
' APPIAH, V., G.T. ODAMTTEN and D.ls. LANGERAK. The evaluation of some physical, quality parameters after combined treatment of heat and radiation. IFFIT
Report No. 7, 1980.
i í APPIAH, V., G.T. ODAMTTEN and D.ls. LANGERAK. The effect of mild heat treat-| i ment and irradiation on aubergines infected with Botrytia cinerea. IFFIT{, : Report No. 8, 1980.
; ODAMTTEN, G.T. and D.ls. LANGERAK. Moisture sorption of two maize varieties| kept under different relative humidities. IFFIT Report No. 9, 1980.
! ODAMTTEN, G.T., V. APPIAH and D.ls. LANGERAK. Studies on the TechnologicalI' Feasibility of the application of dry or moist heat to grains and grainf products prior to gamma irradiation. IFFIT Report No. 10, 1980.
| ODAMTTEN, G.T., V. APPIAH and D.ls. LANGERAK. In vitro studies on the effects of the combination treatment of heat and irradiation on spores of AspergillusI flams Link NRRL 5906. IFFIT Report No. 11, 198O.
I. I ' ODAMTTEN, G.T., V. APPIAH and D.|s. LANGERAK. Control of moulds causingj I deterioration of maize grains in storage by combination treatment: A pre-
I liminary model study with Aspergillua flavus Link NRRL 5906. IFFIT ReportI i No. 12, 1980.
- 131 -
ODAMTTEN, G.T., V, APPIAH and D.ls. LANGERAK. Production of Aflatoxin Bjduring storage of maize grains subjected to the combination treatment ofheat and gamma irradiation. IFFIT Report No. 13, 1980.
ODAMTTEN, G.T., V. APPIAH and D.ls. LANGERAK. The effect of the combinationtreatment of heat and gamma irradiation on some physical and chemical pro-perties of maize grains; Sensory evaluation and quality of product. IFFITReport No. H», 1980.
ODAMTTEN, G.T., V, APPIAH and D.ls,. LANGERAK. Production of Aflatoxin Bi byAupergillua flavus Link in submerged, static culture after combination treat-ment of heat and gamma irradiation. IFFIT Report No. 55, 1980.
ODAMTTEN, G.T., V. APPIAH and D.ls, LANGERAK, Preliminary studies on theeffect of the combination treatment of heat and gamma irradiation on thekeeping quality of animal feed and cotton seeds. IFFIT Report No. 16, 1980.
HEINS, H.G. and D.ls. LANGERAK. Quality evaluation of heat treated and/orirradiated papayas and strawberries. An air-borne trial shipment from SouthAfrica. Preliminary Research Report No. 91, December 1980.
Lectures
LANGERAK, D.ls. Voedselbestraling. Rijks Hogere Tu inbouwschool, Utrecht.April 198O, Wageningen,
LANGERAK, D.ls. Het bestralen van voedsel. Lerarenopleiding. Tu inbouwschoolMechelen, April 1980. Wageningen.
LANGERAK, D.ls. Het conserveren van voedsel d.m.v. straling. Hogere Tuin-bouwschool Utrecht, May 1980, Utrecht.
LANGERAK, D.ls. Verpakking in relatie tot ioniserende stralen. PAO-cursus"Verpakkings- en voedingsmiddelen". May-June, Wageningen.
LANGERAK, D.ls. Foodpreservation by means of irradiation. InternationalSummer Course on Industrialization, NUFFIC, June, 1980. Wageningen.
LANGERAK, D.ls. Preservation of fruits and vegetables: Introduction. IFFITTraining course on Food Irradiation, September 1980, Wageningen.
LANGERAK, D.ls. Preservation of vegetables by ionizing radiation. IFFITTraining course on Food Irradiation. September 1980, Wageningen.
LANGERAK, D.ls. Preservation of fruits by ionizing radiation. IFFIT Trainingourse on Food Irradiation. September I98Ó, Wageningen.
LANGERAK, D.ls. Irradiation and packaging in relation to food preservation.International Packaging Congress, Paris, 18-19 November I98O.
- 132 -
-.1
decay (%)
100
80
60
W
20
no dipping
C3
5' 208C
'í
S1 W»C S' W'C 5' SO'C
0 0,20,81,6 0 0.2 0,81,6 0 0,2 0.61,6 0 0.2 031.6 0 0,2 0,81,6
dose (kGy)
Fig. I - Effect of the combination of heat and irradiation on the percentagedecay in potatoes, artificially infected with Fusarivm solemicoeruleum, after a storage of 28 days at 15 °C.
decay (%)
innIVV
80
60
40
20
0
;
•
•
nc dipping
;•'•;
ŕ * 4
%
'Ú
';/t >
5• 20°C
•;.'•
.•[.•
•;•'•
/ /
' ' '
i
P
I
5 °c 5
Š;1
4S
;!•;
Vy
°C 5' 5 0 ^
0 0.2 0.81,6 0.2 0.8\6 0 0,2 0,81,6 0 0,2 0.81,6 0 0,2 0.81,6
dose (kGy)
Fig. 2 - Effect of the combination of heat and irradiation on the percentagedecay in potatoes, artificially infected with Phoma exiguaafter a storage of 28 days at 15 °C.
- 133 -
decay (%)
-
-
-
no dipping
V.
'.'v
• : • :
* /*é
14
'S
*
/é
J/ 1
51 20°
• • ; •
c
-
hi
5*4(
''•\.v'
;".'•_'.
',y
• • ' .
°c s•45°C 51 50°C
yt/í
*,•
y. 771 .
100
eo
60
to
20
O 0A0JS1.2 O 0.4 0,81,2 O 0.40.81,2 O 0,4 0^1,2 O 0,40,81,2
dose (kGy)
Fig. 3 - Effect of the combination of heat and irradiation on the percentagedecay in Cox Orange Pippin, artificially infected with Botrytiscinevea, after a storage of 9 days at 15 ° C
decoy (%)
100 L.
80
60
40
20
no dipping 5' 20°C
YKVJ
I4
5'40oC 5"45°C 5* 50»C
0 0,4 0̂ 1,2 0 0,40.81,2 0 0.4 Or 1.2 0 0.4 0.61,2 0 0,40,81,2
dose (kGy)
Fig. k - Effect of the combination of heat and irradiation on the percentagedecay in Laxton Superbe, artificially infected with Botrytisainerea, after a storage of 9 days at 15 ° C
PROJECT No. 112
W.W,A. Bergers, J,G. van Kooij
ANNUAL REPORT 1980
STUDIES ON CHANGES IN PHENOLIC AND ALKALOIDAL COMPOUNDS OF IRRADIATEDPOTATOES DURING POSTHARVEST STORAGE
The work on this project has been completed and reported in a thesis.
Irradiation is a recent preservation method. With the aid of ionizingradiation microorganisms in food can be killed or specific physiologicalprocesses in vegetable products can be influenced.
In order to study the effects of metabolic radiation stress on quantitativechemical changes in vegetable products, specific target compounds were in-vestigated in stored irradiated potatoes. These target compounds, i.e. gly-coalkaioids and phenolic compounds were chosen with a view to food toxico-logy and food sensor i c quality.
Much attention has been spent on quantitative analyses of the pre-selectedtarget compounds in the potato samples. Enzymic changes of the polyphenoliccompounds were kept to a minimum by the direct freezing of potato slices inliquid nitrogen, freeze-drying and extraction by boiling in 80% ethanol.
A quantitative assay for solanidine potato glycoalkaioids was developedfrom a pre-existing method with minor changes, by which the rapidity of theassay is improved, without affecting its sensitivity. As a specificapplication, this method was used for the analysis of solanidine glyco-alkaioids in industrial potato protein. Because of objections to the colourassays for glycoalkaioids, concerning their specificity (Fitzpatrick 6Osman, \31k; Coxon et al., 1979), these quantitative reactions were in-vestigated.
Quantitative analyses of phenolics and coumarins were done, starting fromthe alcoholic extracts. Qualitative data were obtained by analysis of UVspectra and fluorescence of diluted extracts and TLC chromatography oncellulose plates. For quantitative analyses a method was developed by HPLCchromatography of alcoholic extracts. Evidence with respect to identificationof scopolin and scopoletin in the alcoholic extracts was obtained by compar-ison of extracts with and without enzymic hydrolysis with (3-glycosidase.Scopoletin was also directly identified by UV, IR and Mass Spectra.
Results of glycoalkaioids, analysed over several seasons, show no significantchanges with regard to irradiation dose or storage time. On the other hand achange in phenolic compounds and coumarins was observed.A 10 to 30 fo,ld accumulation of scopolin was found in irradiated (3 kGy)potatoes of the Eba variety after approx. one month's storage at 10 °C, 30%RH and also a decrease in the chlorogenic acid content. For irradiated(3 kGy) potatoes of the Bintje variety, the increase in fluorescent compoundswas smaller. Several unidentified phenolic compounds increase in 3 kGy ir-radiated Eba potatoes, which were detected by UV absorbance at 310 nm.Chemical analyses of samples of irradiated Eba potatoes indicate a dosethreshold for accumulation of fluorescent compounds. Below 0,5 kGy no in-crease is observed.
By using the high fluorescence of the accumulated coumarins it was possibleto detect accumulation of fluorescent compounds by simply examining potato
- 135 -
halves under long wave UV light. The results agree with the chemical ana-lyses of extracts of irradiated potatoes of the Eba and Bintje varieties.In this way potatoes of several varieties irradiated with a dose of 3 kGyand stored for at least 2 weeks at 10 °C could be examined. The results in-dicate pronounced differences between varieties. Further, a threshold forthe accumulation of fluorescent compounds in irradiated Eba potatoes couldbe determined. In Eba potatoes irradiated above 0,5 kGy accumulation offluorescent compounds could be seen. By fluorescence microscopy of sectionsof tubers it was observed that specific cells accumulate fluorescent couma-rins. Examination of the fluorescent cells after plasmolysis, indicates avacuolar origin of these compounds. Similar results have been reported forfungal infected potatoes (Clarke, 1973; Clarke s Baines, 1976). The increasein scopoletin can be explained by the increase in phenyl ammonia lyase,which has been shown to increase in irradiated citrus fruits (Riov et al.,1972) and Irradiated potatoes (Pendharkar & Nair, 1975).
In view of conflicting earlier reports of increases in mutagenic compoundsIn alcoholic extracts of irradiated potatoes, glycoalkaloids and phenoliccompounds as well as the alcoholic extracts of irradiated and controlpotatoes were examined using a bacterial mutagenici ty test system. No In-creased mutagenici ty of extracts or reference compounds were found. Theseresults agree with the negative findings of Levinsky & Wilson (1975) formutagenic evaluation of extracts of irradiated potatoes and mutagenicitystudies on irradiated potatoes and chlorogenic acid by Hossain et al. (1976).
Rad iat ion-induced increase in coumarins is to be expected primarily invegetable products having a natural coumarin content. Chemical changes as aresult of radiolytic processes in a theoretical foodstuff can be estimated.Diehl & Schertz (1975) calculated 55 mg/kg radiolytic decomposition productsfor a 5 kGy dose. In comparison, in this study an increase in scopolin con-tent from 2 to 60 mg/kg was found in 3 kGy irradiated Eba potatoes after amonth's storage at 10 °C caused by metabolic stress.
Publications
BERGERS, W.W.A. Glycoalkaloids and phenolic compounds in gamma irradiatedpotatoes. A food irradiation study on rad i ati on-induced stress in vegetableproducts. Thesis Agricultural University of Wageningen, June 1980, 1»9 pp.
BERGERS, W.W.A. A rapid quantitative assay for solanidine glycoalkaloids inpotatoes and industrial potato protein. Potato Res. 23: 105-110 (1980).
BERGERS, W.W.A. On the colour reactions of potato glycoalkaloids in strongacids in the presence of (para)formaldehyde. Food Chemistry 6: 123-131(1980-81).
BERGERS, W.W.A. Investigation of the contents of phenolic and alkaloidalcompounds of gamma irradiated potatoes during storage. Food Chemistry (Inpress).
BERGERS, W.W.A. and G.M. ALINK. Toxic effects of the glycoalkaloids solanineand tomati ne on cultures neonatal rat heart cells. Toxicology Letters 6: 29-32 (1980).
: V „Wý&XfíUH • lij, j I Vtai **
PROJECT No. 123
A . F . Groneman
- 136 -
ANNUAL REPORT 1980
DISINFECTION AND PHYSICAL-CHEMICAL CHANGES IN SEWAGE SLUDGE AND AGRICULTURALWASTES
In many situations, the options for disposal of sludges and agriculturalwastes are strictly limited. Incineration is usually not an attractive dis-posal method for wastes owing to its high cost. Dumpint at sea is notregarded as acceptable.
Often the only realistic alternative is recycling to land. Agriculturalutilization is socially beneficial. However, most communities become notonly more aware of the advantages of recycling sludges but also of thepotential problems.
The waste products must be tailored according to the needs and requirementsof the farming community and much care must be exercised. The use of ionizingradiation has interesting potentials, as a clean technology for upgradingdifferent kinds of wastes, killing pathogenic microorganisms and easing theseparation between solids and water.
There were no opportunities to investigate new aspects of the inactivationof microorganisms by ionizing radiation in 1980. Therefore research wasconcentrated on aspects of the effects of ionizing radiation on physical-chemical properties, that are associated with improvements in dewateringproperties by radiation.
Dewatering of sludge prior to its use is a costly part of the waste watertreatment.
Many experiments were concerned with the ramification of earlier reportedfindings. Those indicated that it is the radioiytically generated oxidizingOH radical, which is responsible for the improvement of the dewateringproperties.The effects of high temperature on the OH radical reactions were in-vestigated because it is known that radiation combined with heat hasdesirable synergistic sterilization properties.
Typical results of series of experiments are summarized in Table 1 andindicate the existence of a strong synergistic effect between radiationand heat on dewatering properties of various sludges. The specificresistance to filtration is selected as parameter to reflect changes in thedewatering properties of sludges. A decrease in the specific resistance tofiltration indicates an improvement of dewatering properties.
The data are expressed in percents compared to the untreated control. Theyindicate that the thermal treatment deteriorates the dewatering propertiesin all sludges. The two anaerobically digested sludges of Renkum and Zeistrespond to the irradiation treatment, showing improvements in filter-ability of k3% and h0% respectively.
Results also indicate that the conditioning effect of radiation increaseswhen the temperature of the heat treatment increases, showing indeed thjtthere is also a synergistic effect between radiation and heat in the'con-ditioning of sewage sludges.
- 137 -
Table 1 - Effects of radiation and heat treatment on the normalizedspecific resistance to filtration of different types of sludges
Type of Sludge
Zeist
Anaerobi call ydigested sludge3,6% Susp. Solids
Renkum
Anaerob i call ydigested sludge2,0% Susp, Solids
Bennekom
Raw Sludge3,1% Susp. Solids
DosedaGy(kratf)
0300
0300
0300
Temperature tjf
20 °C
%
10060
10051
10089
50 °C
%
13558
13646
131134
neat treatment
70 °C
%
215134
262113
216175
95 °C
%
306150
816273
357205
The values of the specific resistances to filtration are normalized inpercents.with respect to the value of the control samples. The absolutevalues of the controls were 10,0, 8,5 and 13,9 in kg"1 for the sludges ofZeist, Renkum and Bennekom, respectively.
Interesting is also to notice that the raw sludge of Bennekom, that doesnot respond to radiation treatments at ambient temperature, does respondsignificantly at temperatures of 70 °C and higher. Obviously the highertemperatures make the compounds of raw sludge more susceptible to effectsof radiation.
The synergistic effect of heat and radiation appears to be more effectivethan an increase of the yield of generated OH radicals by adding nitrousoxide during irradiation. N2O is known to convert the hydrated electron tohydroxyl radicals and is a means to create an approximately uni-radicalsystem in pure water.
Experimental evidence was collected indicating that the increase of theOH radical yield by N2O, even when irradiation was performed in a 100% N2Oatmosphere and pressurized in reaction vessels up to overpressures ofapproximately 500 mbar resulted only in slight increases of irradiationeffects on the dewatering properties.
Effects of radiation combined with heat on chemical properties of the liquidphase of sludges were also investigated. Experimental data on the totalorganic carbon (TOC) concentrations are summarized in Table 2 as illustration.They show that heat treatments of 70 °C and higher dissolve to a largerdegree organic compounds from the solid phase of the sludge than occurswith an irradiation treatment of 3 kGy (300 krad). Synergistic effectsbetween radiation and heat do occur, but in a smaller extent and only inanaerobicall y digested sludge from Zeist and raw sludge from Bennekom.
Experiments were set up and analyses were performed in order to collectmore experimental information on the composition of the dissolved organiccompounds and their molecular weight.
' '-••' 'Í - '
- 138 -
Table 2 - Effects of radiation and heat treatment on the solubi1izationof organic matter in the liquid phase of various types of sludges,
Type of Sludge
Zeist
Anaerob icallydigested sludge3,6?; Susp. Sol ids
Renkum
Anaerob icallydigested sludge2,0?, Susp. Solids
Bennekom
Raw sludge3,U Susp. Solids
DosedaGy(krad)
0300
0300
0300
Temperature of
20 °C
%
100256
10025*t
100367
50 °C
%
\(>k330
H»2361
207611
heat treatment
70 °C
%
**90
*»9361Í*
522737
95 °C
%
809978
878957
8291197
The values of the Total Organic Carbon (TOC) concentration are normalizedin percents with respect to the concentration in the untreated controlsamples. The TOC concentrations in the filtrate of the sludges of Zeist,flenkum and Bennekom were 125, 88 and 150 mg/kg respectively.
Results indicated that the irradiation and heat treatment does not onlyincrease the total organic carbon (TOC) concentration in the liquid phaseof the sludge system but also increases the concentration of total nitrogen.
Attempts to determine the molecular weight of those compounds were done byestimating changes in the osmotic pressure of the liquid phase with anadvanced freezing-point osmometer. The data obtained indicated that thedissolved organic compounds are non-ionic and have very large molecularmasses. The dissolved organic particles excerted negligible effects on theconcentration of the liquid phase. The osmolality generally fell into therange of 30 to 110 mOsm/kg (mol/m 3-kg).The ammonium carbonate of the liquid phase contributes for the largest partin the osmotic pressure of the liquid phase of the sludge system.
The determination of the reactivity of OH radicals with solutes in nontransparant sludge and with components of the solid phase of different kindsof sludges was performed using the radioactively labelled benzoate instationary-state radiolysis experiments that were developed and describedlast year. Data of series of systematic investigations all indicate that thereactivity of OH radicals with the solid phase is much greater than thereactivity of the OH radicals with solutes of the liquid phase.
The obtained experimental evidence clearly shows, that published reactionschemes, which use the theoretical equation of Smoluchowsky and whichassume that the rate of reaction of OH radicals is determined by particlesize, lead to a misleading rationale and to wrong conclusions. These werethat sludge particles in the solid phase cannot compete with solutes in theliquid phase for the reactive OH radicals.
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The investigations concerning the kinetics of radiolytically generatedradicals was extended to the hydrated electrons because of the unexplainedsmall effects of radiation treatments in the presence of N2O. At firstnew analytical procedures had to be developed. Preliminary experiments in-dicated that the employ of chloroacetic acid gives information on the re-activity of the hydrated electron. Reactions of the undissociated fractionof the acid becomes competitive as the concentration of chloroacetic acidincreases according reaction:
e qC1CH2 C I" + CH2C00H
This reaction has a relatively high reaction rate constant of 1,1 x 109
mol'M-s" 1, The produced Cl'ion could be quantitatively measured with ahomogene-solid-state Cl electrode.This method is in development for the determination of the reactivity of thehydrated electron with non-transparant systems. The results obtained sofaragree reasonably well with data obtained by pulsed radiolysis experimentsin transparant solutions, The reaction rates of the hydrated electron canbe measured directly by a pulsed radiolysis technique since the hydratedelectron has a known spectrum and a high extinction coefficient. Figure 1shows a typical osci1logram, from where information can be obtainedconcerning growth, decay, yield and reaction order of hydrated electrons.
Publications
GRONEMAN, A.F. Radiation applications concerning waste treatment. In: Pro-ceedings of the seminar "Joniserande straining - ti1 lsmpningar inom lantbrukoch industri. Swedish University of Agricultural Sciences, Allmänt 30.Uppsala 1980, p. 51.
GRONEMAN, A.F. Irradiation of organic wastes for the purpose of animal foodEditor of Proceedings on International Workshop of ESNA Working-Group on"Waste Irradiation", Hanover, FRG, 1980.
GRONEMAN, A.F. Radiation induced chemical changes in and disinfection oforganic wastes suitable for supplemental feed. In: Proceedings of an Inter-national Workshop of ESNA Working-Group on Waste Irradiation. Hanover, FRG,1980.
Lectures
GRONEMAN, A.F. The use of scavengers in sludge irradiation research.Presented at the Xlth annual ESNA meeting at Debrecen, Hungary, August 1980.
GRONEMAN, A.F. Ongoing international research, development and demonstrationefforts concerning the treatment of liquid and solid wastes by ionizingradiation for use in agriculture. Presented at the Xlth Annual ESNA meetingat Debrecen, Hungary, August 1980.
GRONEMAN, A.F. The waste irradiation programme in the Netherlands. COOCWaterkwaliteit, June 1980.
GRONEMAN, A.F. The use of ionizing radiation in the valorization of sludgeof slaughterhouses as animal feed. Contactcommissie: Verwerking bijproduktenin Veevoeder.
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Fig. 1 - Ose 11 logram showing the production and decay of eag in the aqueousde-aerated 3. x diluted liquid phase of anaerob icalíy digestedsludge, buffered at pH 8,7 after a pulse of 0,3 usec and 2,2 daGy.Wave length 650 nm; abscissa: 1 jisec per large division.Ord i nate 16,6% change in transmission per large division.t£ • 1 psec.
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SUMMARY OF THE 1980 ANNUAL REPORT OF THE EXTERNAL SERVICES GROUP IN-
VOLVING PROJECTS 105, 113. 116 and 122.
Nuclear Methodology •'..:..'...'.- - v-\ ''•'^ ',.•>';;:,. -.../;.:--;_.• •• •. •• .
For the promotion and application of nuclear methods four courses have beenorganized. Cooperation with other institutes has been continued. In ,particular good results were achieved with measurements of adsorptionphenomena of human plasma albumin (HPA) on glass surfaces with the help of*2SI labelled HPA. , •-,.
M u t a t i o n B r e e d i n g '. ' •>••'•..- • . ..'[._ '•[. '••••' -•'••:•„:•::'.', - ^ ,• ; . -;-•-
The interest in the application of mutation breeding insLthods continues to belarge, especially among breeders of ornamental crops. This is illustrated bythe increase in number of cooperative projects between the Association and(private) breeders (no less than 25 projects were started in 1980). Severalmutants (13) of chrysanthemum were put on the market.
Application of Food Irradiation ,
The increasing quantities of food compounds and items irradiated by thePilot Plant for Food Irradiation show that this technology has proved itsworth. However, the general introduction and consumption of irradiated foodis seriously hampered by the consumers attitude. This problem can only betackled by instituting an expert public education about the process on an(inter)natlonal scale, which will result in a better understanding andconsequently in a higher degree of acceptability of this technology.
IfFIT • -,/>'-. : , - , • •"-. .:. i ._.-_
During the first full year of IFFIT, the International Facility for FoodIrradiation Technology, a second inter-regional training course has beenorganized and 7 long-term fellows have been trained at ITAL.
E S N A . y '••".•• • •'•:-;-•..-"- • •" ;•,•••":•• .•.-;• ' •
The annual ESNA meeting 1980 at Debrecen, Hungary, has been organized andthe annual report for.1979 has been prepared, v :
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PROJECT No. 105
C. Broertjes
ANNUAL REPORT 1980
COORDINATION OF MUTATION BREEDING, PREDOMINANTLY IN VEGETATIVELY PROPAGATEDCROPS
The significantly increased interest in the application of mutation breedingmethods in (ornamental) crop plants, as mentioned in the Annual Report 1979,has continued into 1980. In total 25 new projects were started; this re-presents almost the same number as in 1979 (27) (the average in the fore-going years was 15).
The following crops were used: Abies, Brodiaea, Calathea, carnation, chry-sant (3), Crocus, Croton, Cymbidium, Dahlia, Eohevevia, Gentium, Haemanthus,KalanohoS (2), Lilium (2), PotentilZa, RhipsalidopaiB, roses, Saintpauliaand tulip (3).
The total number of projects, begun since 1959 has exceeded 300, of whichappro*. 50% were completed or replaced by others.
Several mutant cultivars were put on the market by cooperating breeders,namely 13 flower colour mutants of the chrysant cultivars Snapper andM i ros and one Alstroemeria mutant.
Publications
BROERTJES, C , P. KOENE and J.W.H. VAN VEEN, 1980. A mutant of a mutant ofa mutant of a ... : Irradiation of progressive radiation-induced mutantsin a mutation-breeding programme with Chrysanthemum morifolium Ram.Euphytlca, 29 (3): 525-530.
PROJECT No, 113 ANNUAL REPORT 1980
H.G. Heins
COMMERCIALIZATION OF FOOD IRRADIATION FOR PRESERVATION PURPOSES
Quantities of commercially irradiated food
The commercially applied irradiation of foods is steadily increasing, thoughmainly in quantity. During the year of 1980 the following quantities of foodcompounds and items were treated by the Pilot Plant for Food Irradiation:
- dyes (i»2,700 kg)- spices and condiments (19,700 kg)- frozen frog legs (133,800 kg)- frozen shrimps (193i700 kg)- defatted cocoa-powder and -extractions (219,300 kg)- potatoes (6000 kg).
Potatoes
In close cooperation with scientists of the South African Atomic Board, anexperiment was carried out to assess the influence of growing circumstanceson a possible after-cooking browning of irradiated tubers. Potatoes wereboth grown in South Africa and the Netherlands from seed potatoes of thesame Dutch origin. Samples from both lots were irradiated and stored in thiscountry, and cooked during the storage period. It appeared that the SouthAfrican grown tubers were less sensitive to after-cooking browning than theDutch ones, due to differences in climate and in condition of the soil.
The traditional sale of irradiated potatoes to ITAL personnel was continued;6000 kg were sold, of which 25% to other buyers. This increasing percentageover the years indicates that consumers prefer irradiated potatoes, once theyhave experienced the practical advantages of this food irradiation method.This finding underlines the standpoint that the decision as whether an ir-radiated food item should be traded, has also to be supported by the ex-perience of early consumers and not decided exclusively by marketingcompanies as happened with irradiated potatoes. Their experts fear consumerdisapproval because of a possible aftercooking browning. This fear is con-tradictory to our experiences.
Status of commercialization
With regard to the above paragraph it must be emphasized that the last partof the distribution chain for the introduction of irradiated food is themost important one: the consumer.
It has been our experience that most consumers are willing to buy irradiatedfood as soon as their benefits (i.e. economy, quality) are apparent andinformation about the irradiation process has been supplied. It can betherefore concluded that a wide-spread consumption of irradiated foods mayonly be expected after a professionally carried out (inter)national publiceducation. It must also be stressed that it is high time this initiativewas taken as two basic conditions have been fulfilled, namely:1) the FAO and the WHO have recognized irradiated food (up to and including
an absorbed dose level of 10 kGy) as unconditionally safe for humanconsumption, and
2) Food irradiation has been developed to such a technological degree, thatpractical application on a commercial scale is feasible now, and in manycases even preferable from various points of view.
This project is to be terminated as per 31 December 1980. Further activitieswithin this frame will be accounted under project number 111, entitled"Application aspects of food irradiation to improve food hygiene".
LecturesHEINS, H.G. Food irradiation in practice, present status in the Netherlands.Paper presented during a visit of NUFFiC members,
HEINS, H.G. Sprout inhibition of root crops by irradiation. Lecture during2nd IFF IT course.
HEINS, H.G. Food irradiation in practice, market testing in the Netherlands.Lecture during 2nd IFF IT course.
HEINS, H.G. Inter-country shipment studies of irradiated food. Lecture during2nd IFFIT course.
HEINS, H.G, Dutch experiences with inter-country trial shipments of ir-radiated food. Paper presented during the 11th ESNA meeting at Debrecen,Hungary.
Travel report
HEINS, H.G. Visit to Hungary, 25 August - 3 September 1980. AssociationEuratom-ITAL, travel report Nr. 274, November 1980.
Technical and preliminary research report
HEINS, H.G. and D.ls. LANGERAK. Quality evaluation of heat treated and/orirradiated papayas and strawberries. An air-borne trial shipment from SouthAfrica. Association Euratom-ITAL, preliminary technical report nr. 91,December 1980.
ľ
PROJECT No. 116 ANNUAL REPORT 1980
JUF. Stoutjesdijk, J.G. de Swart, W.F. Oosterheert, A.F. Groneman,M,J. Frissel
DEVELOPMENT, IMPROVEMENT AND PROMOTION OF APPLICATION OF NUCLEAR AND RELATEDPHYSICAL AND CHEMICAL METHODS IN BIOLOGY AND AGRICULTURE
1, CoursesDuring this period four courses have been organizeda. General Radionuclide Course - \k participants (3)b. Liquid Scintillation Course - 13 participants (2)c. Health Physics Course - 9 participants (3)d. Short Radionuclide Course - 31 participants.
For courses a, b and c the number of ITAL participants is given betweenbrackets.
In course c an examination was held under the auspices of the Ministry ofHealth and Environmental Protection and the Ministry of Social Affairs;only one candidate was unsuccessful.
2. Cooperation with other institutes
.1 Cooperation with Dr. W. Norde and Mrs. P. Bosman of the Department ofPhysical and Colloid Chemistry of the Agricultural University at Wage-n i ngen.
The influence has been studied of physical and chemical properties on theadsorption of human plasma albumin (HPA) on hydrophilic and hydrophobiaglass with the help of 12SI labelled HPA. Adsorption isotherms up to 300mg/kg of HPA were found to be in good agreement with previous researchand with literature data.
Maximum adsorption was found to occur after "}Q-kO min on both surfacesfor a HPA concentration of 100 mg/kg.On hydrophobic glass the adsorption was about 2,5 times higher than onhydrophilic glass.
.2 Analyses for the Nuclear Reactor Plant at Dodewaard: 228 samples of thecooling water were analysed for 3H by means of liquid scintillationcounting.
.3 Cooperation with Ir. J. van Bruchem and T.v.d.Lende of the Department ofAnimal Physiology of the Agricultural University of Wagen i ngen concerningthe retention time of food in the abomasum of sheep.
Sheep were fitted with an abomasal infusion tube and with re-entrantcannulae in the distal duodenum. In order to measure abomasal outflow ofdigesta and the mean retention time of digesta in the abomasum, 1,65 MBq(50 nCi) 57Co, chelated to EDTA, were injected into the abomasum. Theradioactivity of digesta leaving the abomasum was observed with two G.M.counters along the cannula, with i» mm thick lead shielding between thedetectors and the body of the sheep.
Soya-protein, introduced directly into the abomasum of the sheep has aninfluence on abomasal emptying and the retention time of the digesta inthe proximal duodenum.
Adaptation to the changed circumstances in the abomasum appears mainlyin the flow of digesta through the proximal duodenum. As a consequence
of this the retention time of the digests in the abomasum will dropafter an initial increase until it equals the value which it had beforethe extra soysrprotein-offer into the abomasum. The volume of the con-tents of the abomasum is increased because of the extra soya-protein-offer.
Z.k Cooperation with Drs, H.L. Elenbaas and Dr. W.G. de Ruig of the StateInstitute for Quality Control of Agricultural Products (RIKILT) atWageningen,
Twelve samples of milk were analysed for 90Sr and 1 3 7Cs in behalf ofthe National Measuring Programme of the Coordinating Committee for theMonitoring of Radioactive and Xenobiotic Substances (CCRX).
.5 Cooperation with hospitals at Ede (Julianaziekenhuis) and Bennekom(Protestants Christelijk Streekziekenhuis) involving the performance ofradioimmuno-assays in the radiochemical laboratory of ITAL.
.6 Cooperation with Comprimo B.V. at Zaandam concerning the use of radiationtreatments to ease the dewatering of liquid organic sludges of a foodfactory. A research programme was performed to investigate to whatextent ionizing treatments can effectively improve the dewateringcharacteristics of food processing wastes produced in the manufacturing ,of chicory. _ i
.7 Cooperation with Prof.Dr. W. Kuhn of the Institute for Applied Physicsin Hanover (FRG).
Ten samples of thyroids of cows were collected and freezedried to in-vestigate the 1 2 9I content of cow thyroids. Five samples were taken fromcows in the neighbourhood of nuclear reactors and five samples fromelsewhere.The results are not yet known.
3- Internal cooperation
.1 Determination of the liquid waste load of the Institute.
The liquid waste load has been determined during the year in order toarrive at the correct assessment of pollution tax to be charged to theInstitute for 1980 (A.F. Groneman).
*». Work in behalf of ESNA and other organizations
.1 The proceedings of a workshop sponsored by the "Deutsche Forschungs-gemeinschaft" on "Irradiation of organic wastes for the purpose ofanimal food" held In Hanover (FRG) was edited and published by A.F.Groneman.
.2 The organization of the eleventh annual meeting of ESNA, that hasconvened in Debrecen, Hungary, was coordinated by P.H. van Mierop,secretary, M.J. Frissel and A.F. Groneman, chairmen of several workinggroups.
.3 The annual report 1979 of ESNA has been prepared by P.H. van Nierop andJ.F. Stoutjesdijk.
.4 One issue of the "Incompatibility Newsletter" has been published(G.M.M. Bredemeijer).
5. Committee Work
.1 Work in behalf of the Coordinating Committee for the Monitoring ofRadioactivity and Xenobiotic Substances (CCRX).Stoutjesdijk, as the technical secretary for radioactivity, has preparedthe annual report for 1980 for radioactive substances. In the samecapacity he sat on a subcommittee of the Interdepartmental Committee forNuclear Energy (ICK), which is in charee of the preparation of alarmschemes for nuclear reactors.
.2 Van der Klugt, Stoutjesdijk and De Swart have participated in a workinggroup of the "Landbouw Advies Commissie voor Mi I ieukritische Stoffen"(LAC), for the replacement of monitors for the measurement of the radio-active contamination of milk,
,3 Van der Klugt and De Swart, the latter as secretary, have contributed tothe advisory work of the Committee for Automatization of LaboratoryApparatus (COALA) at Wageningen.
Publi cations
DEN HARTOG, A. en A.F. GRONEMAN. 1980. Ontwateringseigenschappen van afval-produkten van Douwe Egberts. Techn. en Preliminary Res. Rep. No. 89.
•í"
PROJECT No. 122 ANNUAL REPORT 1980
W.F. Oosterheert, J. Farkas, D.Is. Langerak, H.G. Heins, H. Stegeman,H.B. Leveling
INTERNATIONAL FACILITY FOR FOOD IRRADIATION TECHNOLOGY (IFFIT)
IFFIT is an international project initiated by the Dutch Government to speedup progress in the practical introduction of the process of food irradiation.The IFFIT Agreement came into force late 1978 between FAO, IAEA and theMinistry of Agriculture and Fisheries of the Netherlands.
IFFIT is hosted by the Pilot Plant for Food Irradiation, Uageningen and theFoundation ITAL, which make available to the project their facilities. BothFAO and IAEA make a cash-contribution of $ 25 000 per year whereby the IAEAalso provides a project director. Financial contribution is given to IFFIT'straining programme by the International Technical Assistance Department ofthe Ministry of Foreign Affairs of the Netherlands,
The purposes of IFFIT are:
- to offer training and to assist in international coordination of develop-ment and research in the fields of the technology, economics and com-mercialization of food irradiation;
- to assist national and international agencies in their assessment of thefeasibility of applying radiation preservation techniques to foods;
- to develop and facilitate the dissemination of information on the abovetopics without proprietory restrictions.
Special attention is paid in the IFFIT programme to train scientists fromdeveloping countries in various aspects of technology and practical.ap-plications of food irradiation.
The longer-term IFFIT training involves technology-oriented research workon subjects of practical importance to developing countries. Thesefeasibility studies are defined in close cooperation with the visitingscientists prior to start of their work at IFFIT.
In 1980 the following scientists obtained applied research training at ITALunder 6 - 1 5 months fellowship programmes:
Mrs. Indhira Kramomtong (Thailand) and Or. M. El-Din El-Fouly (Egypt)studied the elimination of pathogenic microorganisms from eviscerated andrefrigerated or frozen chicken by radiation treatment; (see project No. 108).
Mrs. Victoria Appiah (Ghana) and Mr. G.T. Odamtten (Ghana) investigated thecombined effects of heat and irradiation with the aim to prevent spoilageby moulds of maize, fermented cocoa-beans, poultry-feed and aubergines;(see project No. Ill).
Mrs. W.T. Rubio-Cabello (Chile) investigated the effects of water-activity,radiation and heat treatment on the survival and recovery of Enterobacte-viaoeae in Chilean fish meal; (see project No. 108).
Dr. B.M. Shi rzad (Afghanistan) studied the combined effects of irradiation,mild heat and/or SO2-treatment on the shelf-life of table grapes and thepossibilities to enhance juice-yield of grapes by application of irradiationand/or pectolytic enzymes; (see project No. 111).
Mr. M.6.0. Nyambati (Kenya) investigated the shelf-life extension of limefruits by combination of irradiation with mild heat or SO2-treatment, andthe possibilities to enhance juice-yield of limes by application of ir-radiation and/or pectolytic enzymes, (zee project No. 111).
IFFIT organizes from time to time regional or inter-regional trainingcourses for two to six weeks duration. From 1 September to 3 October 1980the 2nd IFFIT Inter-Regional Training Course on Food Irradiation was heldfor 10 participants from 10 developing countries. The 23 lectures of thetraining course came partly from ITAL partly from other Dutch and foreigninstitutions. This five-weeks course consisted of lectures for 5h hours,laboratory exercises for 50 hours, as well as discussions, country-reportsand field trips. Emphasis was placed on understanding the principles offood Irradiation as a method for food preservation and of minimizing post-harvest losses. Present technological and economic feasibility as well aswholesomeness and legislation aspects were pointed out. Practical trainingin food irradiation technology and techniques, including dosimetry andoperation of a pilot food i rrad i ator was provided. A Training Manual wasprepared for the participants and the course papers as well as many addition-al publications and source materials were given to them.
Negotiations were initiated during the course of 1980 with severalinstitutions in various countries, which are seeking assistance from IFFITin their technological and/or economic feasibility studies, or willing tocontribute to IFFIT's training activities as associated regional centers.Hopefully several such relations will soon reach the form of a formalagreement between IFFIT and the participating institutions.
In order to satisfy the request of IFFIT fellows and institutions from thedeveloping countries literature data and reprints on up-to-date progress infood irradiation were collected and systematized by the Project's office.Outlines as well as an action plan for en international cooperation on foodirradiation development were prepared.
The Project Director served as Scientific Secretary at the IAEA/FA0 Inter-national Symposium on Combination Processes in Food Irradiation, held inColombo, Sri Lanka, 24-28 November 1980.
Publications
iFFIT Second Training Course on Food Irradiation. Course-book, IFFIT,Wageningen, 1980.
IFFIT Manual of Food Irradiation. Part I. Fundamentals, IFFIT, Wageningen,1980.
PARKAS, J. Programme of the International Facility for Food IrradiationTechnology (IFFIT). Food Irradiation Newsletter J», No. 2 (1980) 22-24.
IFF IT-reports:
KRAM0MT0NG, I. and M.Z. EL-FOULY. Elimination of pathogenic organisms fromchicken carcasses by irradiation. I. Effect of irradiation on some patho-genic organisms in chicken carcasses. IFFIT Report No. 1, Wageningen, 1980.
EL-FOULY, M.Z. and I. KRAM0MT0NG. Elimination of pathogenic organisms fromchicken carcasses by irradiation. II. Radiation survival of Salmonellaniloeee on artificially contaminated chickens. IFFIT Report No. 2, Wageningen,1980.
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APPIAH, V., G.T, ODAMTTEN and D.ls. LANGERAK. In vitro studies of thecombination of heat and radiation on conidia of Aspergillus flavus Link,IFFIT Report No. 3, Wagenlngen, 1980.
APPIAH, V., G.T. ODAMTTEN and 0.1s. LANGERAK. The technology of heat treat-ment of cocoa beans. IFFIT Report No. k, Wageningen, 1980.
APPIAH, V., G.T. ODAMTTEN and D.ls. LANGERAK. Studies on the possibilitiesof using a combination of moist heat and radiation to control mould i nessin dried cocoa beans. 1. A preliminary study. IFFIT Report No. 5, Wageningen,1980.
APPIAH, V., G.T. ODAMTTEN and D.ls. LANGERAK. Studies on the possibility ofusing a combination of moist heat and radiation to mould i ness in driedcocoa beans. II. IFFIT Report No. 6, Wageningen, 1980.
APPIAH, V., G.T. ODAMTTEN and D.ls. LANGERAK. The evaluation of some physicalquality parameters of cocoa after combined treatment of heat and radiation.IFFIT Report No. 7, Wageningen, 1980.
APPIAH, V., G.T. ODAMTTEN and D.ls. LANGERAK. The effect of mild heat treat-ment and irradiation on aubergines infected with Botrytia ainerea. IFFITReport No. 8, Wageningen, 1980.
ODAMTTEN, G.T. and D.ls. LANGERAK. Moisture sorption of two maize varietieskept under different ambient relative humidities. IFFIT Report No. 9, Wage-ningen, 1980.
ODAMTTEN, G.T., V. APPIAH and D.ls. LANGERAK. Short communication. Studieson the technological feasibility of the application of dry or moist heat tograins and grain products prior to gamma irradiation. IFFIT Report No. 10,Wageningen, 1980.
ODAMTTEN, G.T., V. APPIAH and D.ls. LANGERAK. In vitro studies on the effectof the combination treatment of heat and irradiation on spores of Aspergillusflavue Link NRRL 5906. IFFIT Report No. 11, Wageningen, 1980.
ODAMTTEN, G.T., V. APPIAH and D.ls. LANGERAK. Control of moulds causingdeterioration of maize grains in storage by combination treatment: A pre-liminary model study with Aspergillus flavus Link NRRL 5906. IFFIT ReportNo. 12, Wageningen, 1980.
ODAMTTEN, G.T., V. APPIAH and D.ls. LANGERAK. Production of aflatoxin B]during storage of maize grains subjected to the combination treatment ofheat and gamma irradiation. IFFIT Report No. 13, Wageningen, 1980.
ODAMTTEN, G.T., V. APPIAH and D.ls. LANGERAK. The effect of the combinationtreatment of heat and gamma irradiation on some physical and chemicalproperties of maize grains. Sensory evaluation and quality of product.IFFIT Report No. 1*», Wageningen, 1980.
ODAMTTEN, G.T., V. APPIAH and D.ls. LANGERAK. Production of aflatoxin Q] byAapergillua flavus Link in submerged static culture after combination treat-ment of heat and gamma irradiation. IFFIT Report No. 15, Wageningen. 1980.
ODAMTTEN, G.T., V. APPIAH and D.ls. LANGERAK. Preliminary studies on theeffect of the combination treatment of heat and gamma irradiation on thekeeping quality of animal feed and cotton seeds. IFFIT Report No. 16,Wageningen, 198O.
RUB 10 CABELLO, W.T., H. STEGEMAN and H.B. LEVELING. Combined effects of wateractivity, radiation, heating and storage on the survival and regrowth ofEriberobaeteriaceae in Chilean fish meal. IFFIT Report No. 17, Wageningen,1980.
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LecturesFARKA'j, J. Economic feasibility of food irradiation with special referenceto the International Facility for Food Irradiation Technology (IFFIT).NUFlf|C Summer Course on Industrialization, Wageningen, 12 June 19itO.
FARKAS, J. Principles of food irradiation. 2nd IFFIT inter-Regional TrainingCourse on Food Irradiation, Wageningen, September 1980. '
FARKAS, J. Irradiation of meat and meat products. 2nd IFFIT inter-RegionalTraining Course on Food Irradiation, Wageningen, September 1980.
FARKAS, J. Objectives and activities of the International Facility for FoodIrradiation Technology. IAEA/FAO International Symposium on CombinationProcesses In Food Irradiation, Colombo, November 1980.
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P U B L I C A T I O N S
VAN BALEN, E., S.C, VAN DE GEIJN and G.M. DESMET. Autoradiographic evidencefor the incorporation of cadmium into calcium oxalate crystals. Z. Pflanzen-physiol, 97 (1980): 123-133-
BERGERS, W.W.A. Glycoalkaloids and phenolic compounds in gamma irradiatedpotatoes. A food irradiation study on rad i at ion-induced stress in vegetableproducts. Thesis Agricultural University of Wageningen, June 1980, kS pp.
BERGERS, W.W.A. A rapid quantitative assay for solanjdine glycoalkaloids inpotatoes and industrial potato protein'. Potato Res. 23. (1980): 105-110.
BERGERS, W.W.A, On the colour reactions of potato glycoalkaloids in strongacids in the presence of (para)formaldehyde. Food Chem. 6 (1980-81): U 3 -131.BERGERS, W.W.A. and G.M. ALINK. Toxic effects of the glycoalkaloids solanineand tomatine on cultures neonatal rat heart cells. Toxicology Letters 6_(1980): 29-32.
BREDEMEIJER, G.M.M. and J. BLAAS. S-specific proteins in styles of Niootiaria.alata. Incomp. Newsl. VZ (1980): k)-kk.
BREDEMEIJER, G.M.M. and J. BLAAS. Do S allele-specific peroxidase isoenzymesexist in sel f-incompatible Nicotiana alata. Theor. Appl. Genet. 57 (1980):119-123.BRETELER, H. and Ch.H. HÄNISCH TEN CATE. Effect of nitrogen history onnitrate utilization by dwarf bean. Fed. Eur. Soc. Plant Physiol. (FESPP)(1980): l 6 f
BRETELER, H. and Ch.H. HÄNISCH TEN CATE. Fate of nitrate during initialnitrate utilization by nitrogen-depleted dwarf bean. Physiol. Plant. jt8_(1980): 292-296.
BRETELER, H. and Ch.H. HÄNISCH TEN CATE. Effect of glucose on nitrate up-take by bean roots not linked to respiration. Plant Physiol. 65S, (1980): 57-
BROERTJES, C. and A. KEEN. Adventitious buds: do they develop from one cell?Euphytica 29 (1980): 73-87.
BROERTJES, C , P. KOENE and J.W.H. VAN VEEN. A mutant of a mutant of amutant of a ......: Irradiation of progressive rad i at i on-induced mutants ina mutation-breeding programme with Chrysanthemum morifolium Ram. Euphytica29. (1980): 525-530.
CAPPADOCIA, M. and K. SREE RAMULU. Plant regeneration from in vitrocultures of anthers and stem i ntemodes in an interspecific hybrid,Lyoopeveioon esculentum L. x L. peruviamon Mill, and cytogenetic analysisof the regenerated plants. Plant Science Lett. £0 (1980): 157-166.
DELLAERT, L.M.W. X-ray- and fast neutron-induced mutations inArabidopsisthaliana, and the effect of dithiothreitoi upon.the mutant spectrum. ThesisAgricultural University of Wageningen,' February 1980, 169 pp.
DELLAERT, L.M.W. Dose response curves after irradiation of Avabidopsia seeds:A possible explanation for the "saturation" in mutant frequency at highradiation doses. Environ. Exp. Bot. 20 (1980): 309-323.
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DELLAERT, L.M.W. The effect of dithiothreitol on rad i at i on-induced geneticdamage in Arabidopais thalicma (L.) Heynh. Mut. Res. 2 ! (1980): 109-126.
DELLAERT, L.M.W. Segregation frequencies of radiation-induced viable mutantsin Arabidopsis thaliana (L,) Heynh. Theor. Appl. Genet. 57 (1980): 137-ľ»3.
DESMET, G,M. and W.G. OIRKSE. Growth and zinc accumulation in spinach.Physiol. Plant. 50 (1980): 3H-318.
ENGELS, F.M., F.M. VAN DER LAAN, H.P. LEENHOUTS and K.H, CHADWICK.JThe re-generation of epidermal eel Is of Saintpaulia leaves as a new plant-tissuesystem for cellular radiation biology. Int. J. Rad. Biol. 38_ (1980): 309-321.
FARKAS, ,J. Programme of the International Facility for Food IrradiationTechnology (IFFIT). Food Irradiation Newsletter j» (1980): 22-24.
FELDMANN, A.M. Genetic control of the greenhouse spider mite Tetranyohusuvtioae. In; Integrated Control of insect pests in the Netherlands. PudocWagenlngen (1980): 137-140.
FRISSEL, M.J., J.A. VAN VEEN and G.J. KOLENBRANDER. The use of submodels inthe simulation of nitrogen transformations in soils. In: A. Banin & U.Kafkafi (eds.) Agrochemicals in soils. Pergamon Press, Oxford (1980): 253-265.FRISSEL, M.J. and J.A. VAN VEEN. Soil-nitrogen transformations in relationto leaching. In: Soil nitrogen as fertilizer or pollutant. Proč. IAEA/FA0/GSH Coord, meeting, Pi racicaba, Brasil (1980): 61-75.
FRISSEL, M.J., F. VAN DORP, P. PQELSTRA. Use of residence time models inecological studies of transuränics. Proceedings IAEA/CEA meeting "Behaviourof transuranics in the aquatic environment and sediment-water exchange",ISPRA, (1980).
FRISSEL, M.J., P. POELSTRA, D.W. BANNINK en R.M.J. PENNDERS. Onderzoek naarde fiItereigenschappen van de bodem voor percolatiewater afkomstig van vuil-stortplaatsen. ITAL, Wageningen (1980).
VAN DE GEIJN, S.C. and F. SMEULDERS. Calcium translocation towards leavesand merIsterns in tomato and maize plants: a mechanistic approach. PlantPhysioi. 65S (1980): 60.
VAN GINKEL, J.H. and J. SINNAEVE. Determination of total nitrogen in plantmaterial with Nessler1s reagent by continuous flow analysis. Analyst 105(1980): 1199-1203.
HÄNISCH TEN CATE, Ch.H. and H. BRETELER. Nitrate utilization by dwarf bean.Kurzfassungen Botaniker Tagung Bochum (198O): 168.
HÄNISCH TEN CATE, Ch.H. and H. BRETELER. Nitrage uti1izatlon and phyto-hormones in roots of dwarf bean. Fed. Eur. Soc. Plant Physiol. (FESPP) 2(1980): 395-396-HEEMERT, C. VAN and W.J. VAN DEN BRINK. Alcohol dehydrogenase polymorphismin the mediterranean fruit fly Ceratitia capitata. Ent. Exp. Appl. 28 (1980)2 2 9 - 2 3 1 . '' •• -'. .'• ".;;.;• :-;., • ; • , - •
;' • • " "
HEEHERT, C. VAN and I. WlTTEVEEN-PILLEN. Locationof the alcohol dehydro-genase gene in the onion fly Hylemya antique. (Heigén) using a translocationtester set. J. of Hered. 2 1 (1980): 36*»-365.HEEMERT, C. VAN and L. VOSSELMAN. A male-linked translocation with highfertility in the onion fly Bylemya antiqua (Meigen). Theor. Appl. Genet.51 (1980): 111-114.
LANGERAK, D.Is. Effect van de corabinatie verpakken en bestraling op de kwa-liteit van gesneden groenten. Voedingsmiddelentechn. J_3 (1980); 13-18.
LEENHOUTS, H.P,, K.H. CHADWICK, A. CEBULSKA-WASILEWSKA. An analyticalapproach to the comparison of chemical and radiation hazards to man.Radiation Protection: A systematic approach to safety. Pergamen Press(Oxford) U_ (1980); 1155-1158.
LEENHOUTS, H.P,, K.H. CHADWICK and D.F. DEEN. An analysis of the interactionbetween two nitrosourea compounds and X-rad i at i on in rat brain tumour cells.Int.J. Rad. Bioi. 37 (1980): 169-181.
LEENHOUTS, H.P. Beschouwingen over analyses van de doodsoorzaak van radio-log ische werkers te Hanford. Nederlandse Vereniging voor Stralingshygiene,NVS Nieuws, 5 (1980): 5-13.
Ml EDEMA, P. and J. SINNAEVE. Photosynthesis and respiration of maize seed-lings at suboptimal temperatures. Exp. Bot., 3J. (1980): 813-819.
PUITE, K.J. A lyoluminescence dosimetry system useful for high-dose inter-comparison studies. Nucl. Inst. Meth., YJ1 (1980): 122-125.
PUITE, K.J., M.S. AGRAWAL, J.J. BROERSE and J. ZOETELIEF. Dosimetry inter-comparison for partial body X-i rradiation of rats. Phys. Med. Biol. T5_(1980): 13-24.
ROBINSON, A.S. Effect of sex ratio at three densities on reproduction inlaboratory colonies of Delia (= Hylemyä) antiqua. Z. ang. Ent. 90 (1980):82-89.
ROBINSON, A.S., M. HERFST and L. VOSSELMAN. Genetic control of Hylemyäantiqua. Sensitivity to diapause interfering with genetic control. Bull. ent.Res. 22. (1980): 103-111.
ROBINSON, A.S. and C. VAN HEEMERT. Genetic control of the onion fly, Deliaantiqua with chromosomal rearrangements. In "Integrated Control of InsectPests in the Netherlands". Pudoc, Wageningen (1980): 99-102.
ROEST, S. In vitro adventitious bud techniques and mutation breeding. InProc. EUCARPIA Meeting (Section Vegetables) on "In vitro culture applied tovegetable breeding". Versailles (1980): 186-191.
ROEST, S. en G.S. BOKELMANN. Vermeerdering van Begonia in kweekbuizen. Vak-bl. Bloem. 35 (1980): 116-117;
ROEST, S. en G.S. BOKELMANN. Mutatieveredeling Begonia. Vakbl. Bloem. 35(1980): 56-57. . """
ROEST, S. and G.S. BOKELMANN. In vitro adventitious bud techniques forvegetative propagation and mutation breeding of potato (Solanim UiberpawnL.). I. Vegetative propagation in vitro through adventitious shoot formation.Potato Res. 23 (1980): 167-181.
ROEST, S. en G.S. BOKELMANN. Eoraythia vegetatief vermeerderen in kweek-buizen. Vakbl. Bloem. .35 (1980): 34-35.
ROEST, S. en G.S. BOKELMANN. Vegetati eve vermeerdering van Poinaettia inkweekbuizen. Vakbl. Bloem. 35. (1980): 36-37.SMEULDERS, F.G.H. Insitu immobilization of transition metal ions in soilswith tetraethylenepentamine. Doctoral thesis, K.U.L., Leuven, Belgium (1980).
SREE RAMULU, K. Failure of EMS to induce S-locus mutations in Niaotianaalata Link and Otto. Environ. Exp. Bot. 20 (1980): 149-155.
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STEGEMAN, H. De bacteriespore. In: Sporevormende bacterien in voedingsmidde-len (H. Stegeman, H.M.C. Put and L.P.N. Langevled, red). Pudoc, Wageningen(1980): 6-25.VOSSELMAN, L. and C. VAN HEEMERT, Meiotic disjunction and embryonic lethal-ity in sex-linked double translocation heterozygous males of the onion flyHylemya antique. (Meigen). Theor. Appi .Genet. 58 (1980): 161-167.
WERRY, P.A.Th.J. Radiation induced mutagenesis in in vitro cultivated plantcells. Rad. Environ. Blóphys. V7_ (1980): 292.WERRY, P.A.Th.J. Fysiologische princ i pes van plantecel en weefselkweek in:WeefseUultures in de Farmácie (ed,: H. Huizing) Groningen, Universlteit,(1980).
WERRY, P.A.Th.J. and K.M. STOFFELSEN, Theoretical and practical aspects ofradiation induced mutagenesis in plant cells. In; Plant cell cultures;Results and Perspectives (eds. F. Sala, B. Parisi, R. Cella and 0. Ciferi).Elsevier, Amsterdam (1980): 115-120.
WILMER, J.W.G.M. Mutagenicity of gamma irradiated nucleic acid constituents.Thesis University of Nijmegen, September 1980, 93 pp.WILMER, J.W.G.M., H. LEVELING and J. SCHUBERT. Mutagenicity of irradiatedsolutions of 2-deoxy-D-ribose. Mut. Res. 78 (1980): 85-90.
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IN P R E S S
ANCORA. G. and K. SREE RAMULU. Plant regeneration from in vitro cultures ofstem i n te modes in triploid of Lyoopevsiausm peruvianum and cytogeneticanalysis of regenerated plants. Plant Science Lett.
BERGERS, W.W.A. Investigation of the contents of phenolic and alkaloidalcompounds of gamma irradiated potatoes during storage. Food Chemistry.
BREDEMEIJER, G.M.M. and J. BLAAS. S-specific proteins in styles of self-incompatible Niaotiana alata. Theor. Appl. Genet.
BREOEMEIJER, G.M.M., K. SREE RAMULU and P, DIJKHUIS. The influence of gamma-irradiation of Niootiana alata styles on peroxidase isoenzymes and pollentube growth, Incomp. Newsl.
BROERTJES, C. The significance of in vitro adventitious bud techniques formutation breeding of vegetatively propagated crops. In Proc. *tth Coord.Meeting of the IAEA Panel on "Improvement of vegetatively propageted cropsand tree crops through induced mutations". India, Coimbatore, 1980.
CHADUICK, K.H. and H.P. LEENHOUTS. Interplay between radiation and biologicalparameters in the induction of malignancy in animals. Seventh Symposium onMicrodosimetry. Oxford (1980).
DELLAERT, L.M.W. Comparison of X-ray- and fast neutron-induced mutant spectra.Experiments in Arabidopeis thaliana (L.) Heynh. Environ. Exp. Bot.
DORP, F. VAN, R. ELEV/ELD and M.J. FRISSEL. Agricultural measures to reduceradiation doses to man caused by severe nuclear accidents (X + 114 p, 7 figs,13 tables, 93 refs, in English), submitted for publication to the Commissionof the European Communities, Brussels.
FELOMANN, A.M. Life table and male mating competitiveness of wild type andof a structural chromosomal mutation strain of Tetranyahus uptiaae, inrelation to genetic pest control. Entomol. exp. & appJ.
FELOMANN, A.M. and M.W. SABELIS. Karyotype displacement in a laboratorypopulation of the two spotted spider mite Tetvanyohua urticae Koch.Experiments and computer simulations. Genetika.
FRISSEL, M.J. and J.A. VAN VEEN. Some aspects of irrigation relevant to theterrestrial nitrogen cycle. In: Clar, F.E. and RosswaU, T. (eds.) Nitrogencycling in terrestrial ecosystems. EcoI. Bull. (Stockholm).
FRISSEL, M.J. and J.A. van VEEN. Val idát ion of a simulation model fornitrogen immobilization and mineralization. In: Iskander, I. (ed.)Simulating nutrient transformations and transport during land treatment ofwastewater. Chapter 13, Wiley & Sons Publ.
FRISSEL, M.J. The definition of residence times in ecological systems.Ecol. Bull. (Stockholm).
FRISSEL, M.J. and J.A. VAN VEEN (Eds.).: Simulation of nitrogen behaviour insoi I-piant systems. Pudoc, Wageningen.
VAN DE GEIJN, S.C. and F. SMEULDERS. Diurnal changes in the flux of calciumtowards meristems and transpiring leaves in tomato and maize plants. Planta.
HÄNISCH TEN CATE, Ch.H. and H. BRETELER. Role of sugars in nitrate' utilization by roots of dwarf bean. Physiol. Plant.
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HARTEN, A.M. VAN, H, BOUTER and C. BROERTJES. In vitro adventitious budtechniques for vegetative propagation and mutation breeding of potato(Volanum tuberoaum L.). II. Significance for mutation breeding. Euphytica.
HARTEN, A.M. VAN, H. BOUTER en C. BROERTJES. Toepassing van een in vitroadventiefspruit-methode bij de mutatieveredeling van aardappel. 2. Mutatie-frequentie en chimaerie. Landbouwk. Tijdschr.
HEEMERT, C. VAN and A.S. ROBINSON. Cytology and breakpoint distribution ofradiation induced interchanges in Delia antique/.. Genet i ca.
HERMSEN, J.G.Th., M.S. RAMANNA, S. ROEST and G.S. BOKELMANN. Chromosomedoubling through adventitious shoot formation on in vitro cultivated leafexplants from diploid interspecific potato hybrids. Euphytica.
LANGERAK, D.Is. Verpakking in relatie tot ioniserende stralen. Syllabus"Verpakking van voedingsmiddelen" Stichting Post-Academisch Onderwijs - L.H.
PUITE, K.J. and K.V. ETTINGER. Lyoluminescence dosimetry. State of the art.Int.J.Appl.Rad. and Isotopes.
ROBINSON, A.S. end C. VAN HEEMERT. Genetic sexing in Drosophila using theADH locus and a Y-linked trans location. Theor. Appl. Genet.
ROBINSON, A.S. and G.C. SCHELLING. Reduced male mating competitiveness in a"blond eye" mutant strain of the Mediterranean fruit fly, Ceratitis capitata.Entomol.
ROBINSON, A.S. Genetic control of Hylemya antiqua. IV. Irradiation offertilized females to produce trans locations. Int.J.Appl.Rad. and Isotopes.
ROBINSON, A.S. and C. VAN HEEMERT. Trans locations in the onion fly, Deliaantiqua induced by fast neutrons and X-rays. Theor. Appl. Genet.
ROBINSON, A.S. and C. VAN HEEMERT. Complex Y-linked trans locations in Deliaantiqua produced by irradiation of a fertile Y-linked trans location.Heredity.
ROBINSON, A.S. and G. ZURLINI. Mating success of differently sized onionflies Delia (- Hylemya) antiqua. Ent. Exp. et Appl.
ROEST, S. and G.S. BOKELMANN. Vegetative propagation of carnation in vitrothrough multiple shoot development. Scientia Hort.
ROEST, S. en G.S. BOKELMANN. Toepassing van een in vitro adventiefspruit-methode bij de mutatieveredeling van aardappel. 1. Vegetatieve vermeerderingvia advent!eve scheutvorming in vitro. Landbouwk. Tijdschr.
ROEST, S. en G.S. BOKELMANN. Vegetatieve vermeerdering van Musoari in kweek-buizen. Vakbl. Bloem.
ROEST, S., M.A.E. VAN BERKEL, G.S. BOKELMANN and C. BROERTJES. The use of anin vitro adventitious bud technique for mutation breeding of Begonia xhiSmalia. Euphytica 30.
SCHUBERT, J., H4 .STEGEMAN and M. SWILDENS. Radiosensitization of micro-organisms by radical anions. I. Medium Effects. Int.J.Rad.Biol.
SCHUBERT, J., H. STEGEMAN and A.F, GRONEMAN. Radiosensitization of micro-organisms by radical anions. 2. Streptococcus faecalis. Int.J.Rad. Biol.
SCHUBERT, J. and H. STEGEMAN. Sensítízatíon of microorganisms and enzymes byrad i at ion-induced selective inorganic radical anions. In: Combination Pro-cesses in Food Irradiation. Proceedings of a symposium. Colombo, 24-28 No-vember 1980. IAEA, Vienna.
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SINNAEVE, J. Technical description of a growth chamber for the study of soil-plant-atmosphere interactions. J. Exp. Bot.SREE RAMULU, K., HANNELORE SCHIBILLA and P. DUKHUIS. Self-incompatibilitysystem of Oenothera organaneie for the detection of genetic effects at lowradiation doses. NIEHS/EPA conf, on Pollen Systems to detect biologicalactivity of environmental pollutants, Knoxvilte, TN, Nay 5-8 (1980).Environmental Health Perspectives (suppl.).
VAN VEEN, J.A., W.B. McGILL, H.W. HUNT. M.J. FRISSEL and C.V. COLE, Simulationmodels of the terrestrial nitrogen cycle. In: Clar, F.E. and Rosswall, T.(eds.) Nitrogen cycling in terrestrial ecosystems. Ecol. Bull. (Stockholm).VAN VEEN, J.A. and M.J. FRISSEL. Review of simulation models for nitrogenbehaviour in soil in relation to plant uptake and emission, NASA, Vienna,Austria, (accepted for publication).VAN VEEN, J.A. and E.A. PAUL. Organic carbon dynamics in grassland soils.I. Background information and computer simulation. Can. J. Soil Sci.(accepted for publication).WILMER, J.W.G.M., J. SCHUBERT and H. LEVELING, Mutagenlcity of irradiatedoxygenated and deoxygenated solutions of 2-deoxy-D-ribose and D-ri bose inSalmonella typkimupium. Mut. Res.WILMER, J.W.G.M. and J. SCHUBERT. Mutagenicity of irradiated solutions ofnucleic acid bases and nucleosides in Salmonella typhimurinm. Mut. Res.WILMER, J.W.G.M. and A.T. NATARAJAN. Induction of sister-chromatic exchangesand chromosome aberrations by irradiated nucleic acid constituents in CHOcells. Mut. Res. 'ZURLINI, G. and A.S. ROBINSON. Preimaginal environments determining fitnesscomponents of Delia (• Hylemya) antiqua. OecoIogia.ZURLINI, G. and A.S. ROBINSON. The effect of crowding on adult populationsof Delia (• Hylemya) antiqua. Res. Popn. Ecol. ]