M"bod' fot E>1lma"'1 RJU of Ch<m""llnjY'Y H\lllU. ll><l I'oo·bumao B;oa ll><l &0<"""""Edll«l ~. V. B. VO"t. G C. Bu'l«. D. G H<J<I ,TId D B. P,,1llu1lC 1~5SCOPE

4 Approaches to Measuring ChemicalInjury in Non·human Biota andEcosystems*

4.1 II'.'TRODUCTION

Con"d.nng lhallhoro are about 0,4 million id.nlifiod plam .pock. and $Om. 1.2million known animal SPOC'OS, and lhal thOllS<lnds of man-made chomi""l. arebolng relea«d or aro hl:.ly 10 bo rol.a«d to lh••n\'ironm.nt, lho imeractionsbolween .pec;., and of speci., and .nvirommntal chemicah aro ofl.n v.ryoompl.x. This 8'ves ecotoxicolo8'''' a las\: of .normou. magnitudo andimti<acy Only caroful «l«tion of eh.micah, 5peci<S and ..SIS will allow thoocotoxicologi'IS 10 focus on conditions in whieh eb.mical, or. lil:.ly 10 cau«..riou. ecologICal damas.,

In ecotoxieololY lb. accoptabl. rish vary groatly, Domostie annuals andendang.r<d 'pee,•• are con$lderod al lho lnd,vi<!uall.v.l. The deJl."« of damag.a=plabl. for ,low breeding "'arm bloodod ammals 1$ much lo,,'or than forInsec'" wllb blgh r<prodll<"tton pot.utial. For slo'" brt<dlng speeio~. lbo offOC1.S onsmall groups or mmor populalion, aro lbo major concom.: for speeio. Wilh bighr<product'''. pol.ntial only .tTeets on communitios arO likoly to bo 'ignlficanl.The .".nt of l!to 10.. is also ;mporlam: for e.ampk, monality of a fow fish atth. point of discharge of a cb.m;cal moy bo accoptabl•. Wh.f.as widosproadmOrlality in 0 r;v.r i. not.

Undor nalural conditions, an;mal and plant populalion, ftuctuat., $Om.limosdra'lically. bUI wilhin a Hmitod range. An array of llme-dependent chanse,repre..n" a nalural phenomonon in all OCM)'5I.m$. Ecolosical damaso, as th.l.m! is u«d in lhi' r.porl, mean. adv..... cbans•• in ecosystem, frequenll)'reflecled in re<lll<"tions in 'ize of population. 10 level. bolow Ibeir normallo"'erlimil'. In ",me cas« il is moro con,~nienlIO ,ubSlanuale ¢coloSical damase byob..rving raleS of ocosystem proce,«s. ,uch as flu.eo; of en.rgy and ofel;«mialnutrient>, ralber lhan by moniloring l!to SilO of populatIons.

• PT<pored by D. B. P..hll (0..;"""0), D Colamon, H. C",,,,!. W. H",t,t«, T. T. Koal"...."V. Uado. L M_lOr•. P Sb«..... R, K T",t... 00<1 M Wol<licbut.

e,

86 M"~adJfor £Slimming RISk ofCh",,,cal injury

4.2 STEPWIS[ APPROACH TO lISTING

The tool' available for appropnate as<eS.menl alld prediclion of ad'..... effect'in ecosystems are limned m number and adequacy. In te'ting for effects ofenvironmental chem;cals, a slepwise approach may provide a conceplualframework for a p.-acllcal organizalion of 'esting. In consecullve ,Iep', thecomple,ity of tem generally i""rease, "'ith the I..'el of ecoloiical organizationbeing 'Iudied. The stepwise procedure mlgh, have the following .Iructure andindude the follo"'ing types of tem.

Ut'RII encompa'ses. generally. shon·term tem a' well as some gro"1h andreprodu<:tive sludies on microorganisms and microin"erleb.-ales.

wRI 1 mdudes a limited search for sensitive speeies and poSSibly forecolyslem properties and or pr~..s, such a. pholo,yn,hesIS, IIkolj' to beaffected by exposure to chemical,. AI this le"'l. biOChemical mechani'm. ofaction are al.., .tudied.

Le..cl3 incorporales tem for evidence of cumulalive effecl'.i1l'RI 4 Includes multi,peeies and miCrOCOlm t",1S examining effecIs Ihal

depend on interaclions among population•. Multi,peei., systems ;n"ol,..interaclion. of population•. such as predalion and competition, as well ase<:o$j'$lem pr~ ..., like producli"lty and nutrient cycllng,

The final level (/.,..15) would encilmpasslarg.·scalc field and ecosystem testlng'il e,amine changes in .Iructure and funetion;ns of Ihe ec<><Y'lem a$ a "'hole,

A promlSms approach \0 the inteiralion of ecotox,,:;olo~aldata is tbe use ofmalhcmatical model. to deSCribe and "mulate ecological reaclion. 10 chemicals,

Praclical conslde.-atlon. generally diclate thai all potentiall}' to,ic chemical.he tested at the lo"'e.t I;er. Addilional testing a hisher 1.,...1, i' ad"isable if keyeffecn are observed. The Intent of the slep,,'i.. approach i.to mmim",e the u.. ofanilable fund' and labour while providing a logical ..t of te.ting procedure., Inthis repon. tesl;ng method' Will he re"iewed wnhin Ihis conceptual frame"ork.

4.J SISGl.£ SPECIES TESTS

4.J.1 Rulc T",t.

,""vel I tesling i' repre..nted by the OrSanisation for Ecilnomic Cooperalion andDe"elopmont minimum premarke, Ie... (OECD. 1979, 198(1) or the ba..·..t oft..l. ofl!lt CommiSSion of the European Communilies (CEC, 1979). The OECDpr~market testmi ,ystem comprises the 96·hour alialgrowth leSl, lhe 24-hourimmobilization 1~'I. acut~ and l~ay oral 10~icilY t..ts, reproduction test onDi>ph~io 'pp, and the 96-hour aculewxicily (LC,.) lest for fish. In addilion. t..l.on ra" (acu'e (LO'Q) and 14· 10 28-da}' repeated do.. oral to~icit}' 1"'IS],mutallenicit}' tests and tem for various physicochemical properlle. are ..'allable.While Ihese lesl' are generally aCute 'oxiclly 'est,. 'he algal and Di>phma t..t$include "".. ral cell divisions and a nearl}' cilmplele life cycle, respeelively

JhlJSllrillQ CMmiral In}II')' in N""·h"",,,n B'ota and ua'yJlems 87

Primary' prodonfl. ,n parlicular planklonic algae. are lhe basi. offood c~msin aquatic ecosystems, Alpe can be cultured quickly m lal"i" quanlilies in pureculture; olon«of un,form I<"neliccomposilion can be ea.il)· rna in lained and algalas....y mel hod. are relali~ely ine~pensi.'e. In a5$OSsmg lhe ,mpaOI of. ohemical on.n aqualic syStem. alpl as....y< can pro.'ide informal ion .boUI the .".ilability ofchemic.l> for uptake and lheu sumulatory or mrub,lo')" elfeel'.

In order 10 ...... Ihe hazard ofchemical. to lhe .lruclure .nd funclion of lhoe.uIOIrophic componenl of aquauc ec05)'1tem<, Ihe elfect. of chemic.l. can hemeasured in bolh monocu!ture .nd m,xed populalions, Field .nd conlrolledecosyslem .ludie. c.n .150 be carried oUI.

In alp! balch assays. lhere i•• llme-<lependent rel.lion.hip bel .....n uposure.nd elfect. lhe effect con.iSling mlllnly ofred....uon of Iro",h ,n lerm. of bolhmaXImum y,eld .nd IrO""lh rale_ SIl'>Ce m.ny individual orpni.ms are ,nvol"ed,th,s assay m.y be considered as a populalion a...y. So.'eral general ion. aree~posed for a shon pc:nod and the r.le ofcell di~",on lS ob:served, Algal balcha...y. h••-e been "'idely used in eutrophic.tion stud,« and ,he da\ll ob\ll'ne<!ha,c lxen u..rul for lhe O"erall comprehen":on of phyloplankton reaction 10cen.in chem"'.I5. The melhod conlinues 10 be employ'ed for ,den'ific.uon ofa"'ilable gro""lh limning nUlrienls.nd for "".lu.tion of trophic condilion' inorder to eSUma'e lhe perm'"'ble load of nulnenlS (sec Calaman .' 01.. lh,svolume),

Algal ....y••re increasin&!)' used for a.5eSSilllloxic elfee" of ehemic.l. inaqu.;u'" ~"ronm~t-S bec.use s....h ....ys can gener.te qu.n\ll.lI~e data onfICH,.. lhale.n inftucnce pnmal'} prodllClion. 8ecau.. they.re .imple, lle~ible.nd rehable, alpl batch 1..1$ .re ..'ell.adapted for Standard,zed assays_ Howe~e'.

lhere are inherent pnfall. in ,nterpretation. if il i••,sumed thai the reoullS of ineilto euhure sludies can explain the dynamics of phenom~. tak,ng place ,n!ta,ural aquatIC ec05yStemS,

Short·term toxlCny l..ls ,n ba'ch culture< can be carried ou, by measunnl "cupl.ke during. ). 10 4-hour incubat,on ,mmedia'ely fono..',ng addit,on of thechoem>cal to be 'ested, Advantages of 'ueh leSts are redllClion ,n lime .pent ...nsili";ly and e••y t..hnl of "ola"le. unslable or reacli'e chem,cal.,

Unlike ba'ch CUllu.... conllnuou, cullures provide a con"an, en~"onm~lonce lhey re.ch .te.dy..lale conc!luons. Continuou. cuhureo eliminale 'hevanat,on wllh ume found in balch culmres.•nd belle' represent lhe rclauonsh,pbet"-..,n • constant envlronmen, and lhe elfecls ofchemical' on growth rale o"era lonl period ofume, So far. the u.. ofC<lnhnuou,eul,urcs m lo~",i'y studies hasbeen limiled. although It ha. be<-n ",dely applied in nulnen' uplake sludies.

The OECD Daphnkl .pp. 15' C<lmpri... t..'o pam: delerminatlon of24·OOurEC,o (acute immob,liutlon). and a 14-day reprod ....tlon 'eSl. which gives Le,."alue. al inlervals up 10 14 days, lnformalion i. oblained on t,m~ required forlhe emergence of first brood. as "ell as on number of eus and live and dead)'ounl-

Cooctlllflilioo-mpomecuo"t$(1lI be prepartrl.lbe llope Oflhe ume-mpoo\CCUfW can Ii"" mf(lf1!QlllOfl OIIlhe ralt and. <kgrtt of .boorptlOfl. <kIO~ocallOfl

aDd tbmlR.llOfIoflbc ehtm>o:al."The rallO ofthe l~y' to 2.........., Le•• >-al....pro,-.x. .... md'Cl1or of tlu"onlC dl"ccu (DECO. (983)

T1lt <:o<><X'1ltrallOll~ R'1alJ<>rUlnp for aqua", orpnu.ms upowd 10 •

chemJaI IS .......11). cunllmeu. TIle '"""""'"~ II "pre_tal by thopoIDl 011 the (:()<>(l<1llrallOll ..... bdo1o" ..loch ,he UISI"'I~, udI·""' canDtII 1DtUlIR' lbe respomc Tbn poID' io ,0meUIIItS mUR'd to III tho"no-df """,,",,tnuoa or moR' .ppoopo ..td) .lIO-Obotr.'O(\~tc"I'f;OQClI'IlU'a.

bon.. TIle mo:haIlt1.kIl (:()<>(l<1ltnl1lOlr., LC... io • .aDdard _urc attou:tl) of• man...,! and. «:c'lIn alonl ,he OOftClI<I,tnI1>011-responst CIU'~ btyond tho .......obott'\'O(\~ poIlil TIle IqIOIItII het.."UII lS ....rded III tho ratllt of s..bCtlW~Ira of ,be: ebtlmal. Dda~'O(\ dfeeu of dItm>calo 011 aq 11C

orpn m1lSl ..... be tat... 11110 aocounl. for ¢nmpk. r.......n.alll 0fPII......apoocd to tad"",um .tId _ orpnoo;hJorult compolltlds lhe.. II a IaltllC)"p<1'JoO<I btfo.. dtalh OCCU"- 10 ordt1" '0 dttta dday"Cd~. l1 IS u.wful 100011'''''''' ,he ox"""'.. ror an a1tndtd period he}""" lha'lIOnllIUy u.wd '" astandard ,hon·ltmt l>loIy.

4.3.2 AoIdi'ioooal Sped" 'N (cosy,l.. PrOOOHO T"IS

If there i. C<IfIa,n ellhe, .boUI tCOlo.lCOlopcll dala or aboul phY.ICochtmM:;lIproptrl it,ofehem,eal. (f0' talmplt, imile." ng pe'tn"al fo' b!QKC:um ula noo 0'

,e,istance to b,odti,adallon) or 'flhe "olum<: of the chemical i. la,.. alld l1S uSCpauem .ualltm ucapt' to Ihe tn"lronment. funhcr tClllnl (le"e1 2) 's n:cessa'Y.The type of ')'ilems 10 be Imcd will dtptnd on Ihe sensili";I)' found In lhe firstround of t<'<IS and on e~poturc Inlly,.. ""hich "ould p<lint 10 seclion, of Ihet<»<y,t<m mosl hxel~' 10 be expos.ed 10 the ohemlCal.

R<,<p<lllst'S of lerrcstnal plantS to chemicals WIll vary ...idely deptndini on,pecie.. dones, ceOtl'pes and eullJvars: 1,","lh pattern' and ck"clopmcntal.tap; en>i,onmcnw rcJlll'k'l; pl.nt "lour; slid .ptCIC'i C<>mptllllOn (_Kozkw,.x,. 'hi' "olumc)

Mttbod' c"",,,",", should fCOOII\lZt Wt ehel11JCl.ls '"'l.lItfIlt&111 JD811t1U plantsal the ...o. orpnllm••~ commun'l) aDd ecosystem It\~l Beau," oflal;t oflUl..blc ODOtroI plots la ,be field II " of'en d<-<uablc 10 Ii'" o«cea etrccu ofch"'''Clls ID ODO"oll«I-tft>'fO<\D>C'O' chambers (pb)lot'01l~) la lhe field.expomrc chambers CI.lI ohea be IISCd hanl&J'COU'ly '0 crcalt IUltablc eon,roIpIotI. Such chambers arc ad>_blc for ·"ad.) crop pb.nu aDd other......uplants """''''"''' 10~ IllDdra. bop and. shn&\>.dom.Nltal ClOIIIIDUII1.....

AmoII& tho ....... u.wful _mrocnu '" plan" arc dwtJ" '" pplCllu aDdDtn of plooIOl)"DthtRs. H...."''U, al1tfa.lJ()ft of rain oL plooI<»)nlhtsd II _ah>llY" llOOd ll'eUun of",u'uallwma" cbatlF bcc:a1lst crowth potenltll1 iolIOlIun>caI 11) tho -.onal ~11tnI of pbotOl)lI.~ ~ntl1Oll1'" at p/lo<o-

Measurin!! Chemical Injury in i'>'on_n""",n Biola and Eco,pum. 89

.}'nthate "'ithin the plan, and the rela'ion of pho'o,ynthesi, '0 re,piration(Kramer and KOllo"..ki. 1979),

One of the mos' u~ful md"es of the impac, of chemical' is a change in 'hegrow'h of eco.}'..em components. Thi' ,nformatIon can be oblained bycompanng 'M growth of plan,s .n Ihe presence and ab~nce of the chemical, Itshould be recoJllized. howe"or. 'ha' net changes .n biomas, are affected byweIght losses due 10 respira'ion. insecl injury. disease,. ncnnal ,hedding anddea'h of plan' pa"s and hat\'e,'ing,

Effon.should be made to tran'fer llro"'th and y,.ld data '0 a form usable foreconomic a'....'ment or ecoloillcal evaluation (Heck fI al" 1979), For mostplants 'he grea'es' interes' i' in abo"e-llround pam. How",'",. for rOOl crops lheemphasis often i. on root srowth (Kramer and Kozlo""ki. lQ79).

For 'mall planl' (annual plant•. crop plant•. old field,. grassland,. lundra.bogs. and shrub-.dominated communitIes). ~quenhal ha",e,'s .hould be takenon sample plants (or plants pam) and dry_welghl mcrement determined,

Insects, both as regard. lhe number of species and 'he number of indi~idual,.

repre~nt a major componen' of ,he fauna, Insects are adap,ed 10 specialecolollical condlt,ons by lheir morphology. phy..iolollical funclion, and be­ha,'iour. They react in a ~ery ~n"",'e way to specIfic chanlles in the en"ironmentat 1M le~el of indiVIdual.. populalion, alKl commun,l,.,. In conneclion wilhpesticide production. method' have been developed for testing chemical. bolh inthe IaboralOT)' and under field condilions,

The most "'idely used lest invol,'mga terreslrial ...nebrale is lhe ral LD••. Thedala ba~ obtamed from 'a' LD,o "alues ha. been used 10 predlCllethali'y inolher .pecies. For example. lD,. 'alues for tM ral and ''''0 avian 'pecles(starhng and red-,,'inged blackbird) were compared by Schafer (1972). For 61d,fferem pesticides and olher chem"al. bolh a~ian ,peci", ",ere, on the a"'rage,more sen'it"'e than 1M rat. lhe "arlinll beinll roullhly fi"e limes and lhe red­winged blackbird len lime' more sensili~e. Only a 'mall percentage ofdifference' of LD," nlu",. hO"",er, faU outside two orders of magnitude. Moredelails on the scope and limitalion' of ,hI' ty-po of e~!rapolalionare conSIderedelsewhere (Peakall and Tucker. thiS "olume),

The compaflson of oral LD,o and 5-.day dietary LC,. "alues (Kenaga. 1974)fOT fi"e compound. are poorly oorrelaled because oflhe e!feclS lhal can be causedby compound. 'hal are ..adil)' absorbed and rapIdly metabolized. compared 10those lhal ar. poorly absorbed and persistent. Somewha' beller correlation.were found u'ing a larll"r number of compound. {Healh n ~I.. 1972).

Desplle 1M "'eaknesse' of the lD,o Or lC,o tests (see Dobson. lhis ~olume).

the acule ~thalily teslS g"'e a reasonable firsl approxlmalion of the number ofdeaths 'hal can be e~pecled at a given .~posure 10 a Chemical. i,e.. in ,pill.ilUalions or ifdehberalely used, for example. as a peSliClde. The predicli~e nlueofacute l~lha\il}' t"'ls for effects on populalion, or ecosy".m. i, likely 10 be verylow. Un~.. I.D,o or LC•• values correlate well wilh life..:ycle tests or some more

9() M~lhod< for EJ'imali"{J RiJk of Ch~miral Injury

limiled ltsli lhal mclude reproduction, lhen ilCIlle lelhalily i~ unlikely 10 prediclchronic effeclS. Allhough a large body ofdata does nN e.ist al present. it seem,unlikely lhal any .Irong correlation ""ill be found.

If there IS ,uffic,ent e>'idenee for ecological ConCern from dala on ..miliveSpec1<> or processes and if Ihe chem"'al IS permlent. Ihereby maIntaIningappreciable concenlrations in the en,-ironment. or i. exp«led to be a,"ilableIhrough conlinuou, rele..." teStS for chron'" lo"city' are recommended

4.3.3 Tesl! for Reprodoethe C.p.dty.1>d Oll>er Long_lumT••

Complex protocols ex"l for Ie", for reproducti"e capacily ofbolh mammals andbirds (level 3 lesnng), The difficulty IS In knowmg when to use Ihese comple,h'''s. E"en ",'ilh Ih... lests...vcral en"ironmental problems of Ihe pa'i wouldha"e mi'sed using commonly a"allable laDora lory ammal•. for e.ample. the.ffect off'CBs on mInk reprod""lion (Ihi, SP«les,S much more sensitive than Iheral, Aulerich and Ringer. 1977) and on fi,h-eating bird, (beha'-ioural respon'"Ihar Would not.how up In caplivily. Fo'" al" 1978) or DDE.,nduced egg,hellthinning (most commonly used lesl 'p«les are 001 sen."i.., although someord.... of bird' are highly sen.ilive. PeakaH. 1975). Early-life 51age lesl. miihlreplace fulilife""y'cle studies, The easiOS1 early-life-cycle 'est thai i. available forterreslnal ,..rtebrales ,nvolves injeclion of chemIcal' inlo lhe aVIan egg. ThISprocedure mlgh, be worthwhIle for reaSCInably '<able compoundsJudged by thephy.icochemical data. e'p«ially if Ihere is any likelihood of bioaccumulalion,However, the predlcl,,'e .. alue of Ihi' lesl need' 10 be evaluated,

A variely of behavioural effeclS has been osed to tesl Ihe loxicity ofchemicals,Warner ~I at (1966) emphasized Ihal such effeelS Integral< va"ou, biochemIcaland physiological changes and are ",n.itive indicators of sublethal to,icilY,There i•. ho"'e>er, conSIderable difficulty m evaluatmg Ihe significance ofrndr>ced behavioural abnormalilie' as regard, the .o,,"'ival of individuals andpopulalion, in Iheir nalural habilats.

For Ihe ma"ne ~n,'ironmenl.crileria ha"e been de"eloped by lhe JOInt Groopof Experl$ on the Sciemific A$pects of Manne Pollution (GESAMP) for theseleclion ofenvironmental variables for inclusion in waler pollolion moniloringprOjrammes. A, a first Slep in ",leclion of "anables. Ihese cri,."a mIght beapplied for predICtive purpose, to nomerou, technique, no"" a"ailable formeasuring effects of~hemical,on aquatic animal•. Thecriletla Include ecologicalsignIficance. relevance to other effects.•pecificity, reveroibilil}', range of taxaaffected. quamitali"e aspectS (does the effeel bear a qoanlitalive or prediclablerelallonship 10 Ihe cause?). sen,ilivily, scope. response rale, .ignal noi'" ratio.preci,ion, cosl and range of applicalion (see Waldichok. this ,-olomo)_

Using a ,ystem of rankIng hiological variables lhal can be measored for anestimate of pollulion effeclS, GESAMP (l9g0) amved al a series of meaSUre--

Mtiif>jring CMmieallnjlU}I in N",..human BiOla IJIU1 Uo'YJIMU 91

mcnlS OuI of J.6 conSldertd. whIch could be recomrnenlkd for immediate uloe inmonItoring pn'grammes in an) reginn. These included f«ding fate. bodycondition lI>dex. ~ope for l'''''''lh. ox)'gen nmogen ralio. I)......."al .labihty.lIurine alyane ralio. Ii~er .. fraction of body weight. and ,ncidenee of ulc¢rs andfin erosion.

It can be argued tbat mOSl melhods t«ommend«l b)' GESAMP are 100complex for ule by the a.'ora,e regulalory biological laboratories. especially if,bey lack research cap.>b~ity In many cases. il may "ill be n«ess.ary '0 resort '0Ibe s'andard iKUl< assay. preferably witb a 80""'lbrough sy's'em. and allow for alonser exposure than the usual 48 or 96 hours. Animals ...',b shon·gencrallonlim< ean be us«! for fulllifHO)"le les,in" A bigb<ahbre chemical analylicalcapabilny is essenlial in order 10 mea,ure accuralely Ibe ~oncen,ra,ions ofcbemical'. salinity, dissol,·.,j oxygen. pH and Wa'er bardness

A queslion i. always rais«! aboul lhe applicabilily of labora'ory teSts 10 field.i,ua,ions. Coupled wllh taboral(1)' expenment<, It i. wme,imes prudenl 10carry OUlllmple field 'esU, Ifcbern,cal. are already be,ni released 10 lhe aqua'icen~ifOnment, caged anImals can b<- used 10 ",euure effeclS 10 ,he field. It isposs,ble 10 measure in Ihis way acu'e toxicity and sucb effectS as behaviouralchanges

4,4 MULTISPECIES TESTS

Mullispecies 'eslS represent Ic~el 4 lesling. TeslS al ,be lo...·er Ie~els of ,he StepappfOiKh are largely conducted on indl~idual. and SIngle ,,,,,Iated populations.Altbough single species lem are ~ery useful and at presen' are the major and onlyacapl«l mean, of e"imalin, lhe imp.>ct of chemicals on indi~iduab andpopulatIons as well as on bl&her lenl. ofceot""..1 orpntZlltlon. 11 is not knownb"",' reliable Ibese leslS a re for pred icunl elfecls on communi 'ies and =)"le"",.Sueceffiln, Ie>'el. of ecologICal organtZll,>on h.a~e umque propertie<; lherefore,reaction of",osySlemS 10 Stress should no' be..pected to b<- merel}' a SummaUOnof Ibe reaction' of indi~idual component species. There IS "'id"""" 'ha' somechemICals canJ«Ip.>rdiU a population, community or "'osy'tem bu"hese elf..,,,are nol predictable from 10xlCity lests on ,inale spec1es (e.g. F,sher tl <Z1., 1974;Van Voris eI "L 1980). Sucb elfec15 depend on interaction. of populations, andcan be as oil"ilicantto ,he funclion,o, of an ecosYS'em as are ,he more easilymeasured direct elfects. For lhese reawns, ,uiuble laboratory t.." 'bould bede>'eloped '0 pr«licl the inlerlra'ed reaclions to chemical mess of biololicalcommunilie, and 'be eco'y"em a, a ... hole,

Tesls on mulllspecies sY"ems.... hich ean often b<- done a, microcosm studies.are call«l for If Ihere is ..,denee of chronic effecls in Sln&le SpecIeS tests or lheability of the chemICal 10 accumulate m lhe orpnisms. Chan..,. in beba~iour

that could lead to altenltion~ in ~u!\'i\"1\l and thus \n comman,ty SIru<tU....changes in a~ailabilily of one componenl of lhe ecosyslem or tbe abllily of a

92 Merlwds!or EsnfIWlinj/ RiJk o!C~mi<:<11Injl/ry

!~e~ 10 ~Ignilleantly alter Iheslructure of theehemlcal un also indicate a needfor rnullll('«lel''''II.

Methods llull examine competilive. predalOry and llrazinll relation,hips andmUlualistic interactions. such as those I;>(twttn planl. and rnycorchiUl. arefea.. ble wilhin a laboratory or in limiled scale field studi"'. For example. manyalgae are more sen,iti to lo"ic chemicals when competitors are presenl thanthe~ are in pure culture (Mosoer er <1/" 1972. Fieldinll and Rusoelt 1976). PCBs atcon<entralions as low as 0.1 II I inhIbited nUlrienl uplake of the marine diatom.T~(JI<U'WJir(J p.",donana. when alpl competitors for Iimitinll nUlrienlS "..erepresent but not in pure culture (Fisher ~I <1/" 1974), In an «olopcal come"t. su<:hte'" are more u&eful for predietioll changes in ph)'toplanklon composition andthe structure and d)'nam;C'$ of a nalural aUlotrophic communny than are manyindivtdlUll loucily I"'IS (Calamar; n 0/" thi' "olume).

Ahhough mult;,('«ies laboralOry tes" afe potentially valuable ....arch tool,.they have >orne hmna'ions as regards changes m ecosystems, Moll of the... l<S1$

are ;n the initial stages of dev'eloprnem, A ba,ic 'tumbling block to theirfefinement il ....complet. und.rslandinll of the sillnificance of disruption ofs('«ilk imeraction phenomena for the overall etTec\lveneS$ of an ecosystem tofunclion adequately, [n addition. v.ry lin[e work has been done on the ..n,itiv;tyof these teSt. foc dlStmguishlng real change, induced by chemical stress [romfiuctuation. inherent in natural and model eco.),.tem•. Reproducibility of te"results and thcll apphcabillt~ In predicting changes in various habita" orecologICal sllUations will require extensiv" funh.r "udies.

A recent fev';.,,- oommi"ioned by th. US Env;ronmental f'rQ\ection Agency(Hammon. el al" 1981) pointed outth. n«<l lO bettec dwne:

(I) the limits lO ,,'hich a blo[ogical sy.lem Can I;>( stressed before it. reco"ery tothe ,nltial Slat. is no longer possible.

(2) mea,urabl< properties mdicating adverse commUn;I)' and ecosystem elf«ts...,(3) cro..·.}'.tem gcnerality of such pfopem",.

Multis('«tes labofatOry t",1S of potential utility in risk a,scsSmem ha"e 'ottne"tenSlvely ",,'iewed by Win el al, (1979). Gie.y (1980). Hams (1980). Hammons(l98[) and Hammon> 01 ai, (1981). Hence. only a f.w &elect m.thods willI;>(discuned in this section 10 iUustrate the range of tem being dev'eloped.

4.4.1 POlenll.l T"'IS for C..tD~titi ..nAt Ih. [."el of the pfimary producers_ van<>us m<lhodl for examiningcompetit..'e tnt«actions for esoential nutri.nts ha... 'ottn developed for a[pe incontinuous cuhure (see Fi.her 01 al.• 1974: Fi.1dtnll and Rus",lI, 1976), and forpasture gra"'" and legumes in g",enhouse and .maU field plol "udies (see

MCG3U'i"1j Chemicallnjury in N()It-!ruman Bio'" and Ecosystem.! 93

Bonnell and Runed:lO$. 1977: Kochhar el al.. 1980). Other candida1O$ for plantcompelition tem could be reprO$entauvO$ of d,fferent hfe form,. ta~onomic

ifOUP< or commun,ly Iyl"". Becau.. autotwphslack a di"er$<' ceJ.Qurct ba.. andthe "ariety of behavioural mpon..sofa hel.rotropbic community, compelilionis inten.. and ils mechani'ms are limiled. Sbifls in populatIOn dominance ofplant ,peeies competing under lhe slrO$S of a tox,c cbemical are of ecologicalimportance to the consumer community beeau... for graze .... lhe cboice andnUlritional valu. of food chang. witb popula"on shift" Th.re i' also obviouscommercial importance in unwanled sbift' associaled wilh str..s spurred bycompelilive cbang.. in pa'lure and crop lands. Furtber asses,m.nt of tbeSIgnificance of altered planl slruclure 10 ""'Y'lem funclion (for ..ample.primary aod SCl'ondary prodUCllon) .. O$..ntial for v.lidaling tbe predictivecbaracter of the.. ,imple, ..n,ilive and r.producible 1..1 'ystems.

Variou, compel it ion tem 10 eumine the interaclion oflooplankton spee,.. inaquatic ,y'tems (see for example. Marsball. 1969; Goulden and Hnrnig. 1980)and arthropod 'pee," ,n 'ermtnai S)'SI.ms have been d.,'.loped bUl bav. nolbeen used 10 any greal e~tenl in .....'ing Ibe effects er chemical,. U.. ofcompeling D'OJophilo populalion, in 'Iudies ef gen.lic fitn..s is particUlarlyhighly develeped (..e H.mmens u ai" 1981) as are the smdie' wilb TribaJiumbeedO$ (Park, 1948; Mem tt al.• 1976), lbe.. interaclion' 'he"" a finecompeliliYe balance. may be highly ..n';t;ve lecbemlCals. and could be lhereforeimportant m develepmg th.ery and mooels of cempelilion in the tomelogyef ece'yslems, How.,·.r, they de nol appear Ie be parlicularly repr....mali". efImportant con,umer_level compelitlVe lnleract,ons ,n lhe field. and lh.refore lackecolegical realism ....mial for predicting community leYel cbanges.

Structural chang" in the communily ofsoil arthropods could be lmportanl indecemposilion and nutrient cycling precesses, but the.. lilter communili..display h,gh ,pecies d"e",ty and low feeding ,peclficit~, lbus impeding ,hede"elepment of compelilive t..t systems. Cempetili,'e imer.ctie", of lenge'·lived .pecies have generall}' nel been considered a••n .pprepriale focus ferecolomelegical I.,IS becau.. ef the time required fer manifestatien efsignificant chang.. (.."eral g.neralion. may be required),

4.4.2 Pol.nll,1 Pr••btor-PIOY Ttsl Sy,tOll'.

Fer aqualic ec"'ystem" predater-prey te,t. incorporatinli fi'b a, lbe predalorand looplankten.•hrimp or fi'h .' tlie prey, and tests .umining looplank\on~

looplanklen feeding bav. jXllential u.. in haurd u ..,srnenl. Me.l inv"li·gatie", ef effect. ef chemICal' on predator-prey ,nterac,iens ha"e beenbehaviourai sludies. Boba"ioural re.ponse, 10 cbemlcal' .re lienerally qUII•..n,iti,'e, and nalural predate" <an often discern small anomalies in preybellnioul. Both O\l!oef\lalions !UUesl jlOlemial inciUlion of predalor-preyinleracliens ,n ecotosiceiegicaltO$ling programmes. He",,,,..r. lhere are pro\>-

94 M.11tods fOl Emmal"" RIJk of CIw",it:tJ /IIJI"J'

It1m "itb iocll teilS. pamcull!ty 10 repro to lish. GftImllDIIQOi aboullQ<tJO<lS of. SJItO/io: pttdalor-p~ comblllaloon {Jmerally based on~cx~)... d'lI"onnl lU _marued by GKidinp (1980).'" cffm obw"'N 1Ron< 5I1""loon ao only ",dlCllu potmuaJ "lI"eou on OIber prcdlllO<-preyin~ bUI tbo mall'madll. d,rccuoo or C'U1 <lCCIlI"fftIa ofcffecu '0 other>pccin; canDOt be KJl:Unld) p...bacd. I.. ~lJOo. fllC10B IMber lhan preda~of= ha"" ara.lcr m111>ftKlO on ~uoa .hnM.N'!!' aDd dJstnbul_. aDd.tbcrd"ont. &N.tlcd ",formalloa on populatioa dylWl>lCll and ,topluc SlNCtUte........., be eoxauaJ III " ........... appropnau <nI comb....I.......

Obbplo<} fioh pRdaUOll on to0pbok\Oll aDd zoopIaak1Oll prcdal_ onzooplank:ton may PfO\-.doc US! rocnbtn2lJOaS ..,til f...",. faaorf coafOlllld""!heir pmIlCU~" nubl) ta thcot: tnt ')-u<m$. CXpoJW'l: of tbo prcdalon 10

dloemJatb ....y ptO'o-.doc • Iopeal u,,"litDtolll 'pp'0.l,- Botb med••",SIIUC.~ sudI Il lCIn'vc dlSllO"" aDd captl.lte __ (Gemum andSinctlcr, Im)aDd popuIaUOlllppn:Jlothcs ... feasible("", DrmIlll1" ,1111. 1978).T~ In: rrlall''d) SImple, I1Ipd aDd reproducible. Pt-cdanoll-lndllCrld ctIIn,es '0zooplankton "omnllllllUn 1ft1ft tOlponanl ecoIop:al pile................."bSlP­ClItl,mpact 0fI ,I\( don ofCOaln'lClttll .nd pm< fisb and on 1M t:OlIlpollloon oftho phytoplanktoo <:ommlllllly, T1Ia< <nI ')>ltm$ ha,~ nO' been adcqllliety..ooted undtt d"'ll'l.,aJ Slrfl,I,.

In I.rmlnll ecoJ) Item., 1M relal,,~ly h'ab st1l$lUwy of Insec-t P~IIO" 10I""ticKles lulitStslhallMst 1p«1CS m-lly be matched ....ilh appropnat" prey (ptSt'p«ies) 10 m.ke up. ,.nSI1lVO 1<11 Iy't"m. Much of III< labol1l10ry teS<1n:h inthis 'rea hal focwtd on prodalor "lfecti".n.", in b,ol"i'cal oonlrol of PrtYpopulalion, and l"Ii nOI deall 1p«llkally wIth ohemic.1 .lrelS. althouJ,h this"'ould appear 10 be fnlible. Most 'tudin h.~o oxaminod para"totd rolallonship.Searchinlcapacity hal b<en cnod IS til< mosl imporunt mdicalor ofI paraSltold'lIbility 10 act as a b,olo.><;al control faaor (Huff.kor tllll,. t9(3).nd Ihu.provid<lan indicator ofpr<d.a" p~·lnteract;on.However. Illl' of IlICb cmenamould be ,upportod "'nil popuJI"on tbu.

111< besl nOOICd lIOfI·paralitOl<! f<ed,n. ,nteracllO<lS an bel"'ffn lad)blrdbeetles and aphid,. n" plmn. bas und........ COIlIoIderabl< labol1ltOlY(Murdocb.nd Milk,. (913) and 1Ie1cl (HlFn., ai.. 1971) ,n,'nltpllOn•. Th<n:spons< tbtl for. predator-aphld .)....m "ouId be I"""I1I11y R''''''&l\t 10 manyag",:",hu.-.! .nd home ptdnl ')>lnns.

Other lD'=obnue predator-prey <nI ')'SInns an: ,n 'lIf1OU$ IlIF' ofde-"Iop.....n. B) fll lhe _ od'lIl1Ced lD'oI,... predalOry till'". M,u leSt

f)'Sttt1'4 an: luahQ·ll'1lSlIl''IIIO~ chon. ph)-..caI 0ll0Il1I_ boll oharx_terigics and lD""t ralJO ofpmlalO< to pi'<') (HulI"ahr rllll., 19(3). S)'IlnrIS 10dIII !bose ..........'Old b) Suter (1980) "'hodl_lin<ly balollCed sIIouIcI be luahJy......tivc to dl"ln....l· and ha'" • $bon n:spome ume (I<u thaa a _til).Trampooiuc. of rau1u from IIIIIU testS 10 othtr~ II qunuoaable

A1tboua:h Iabon.lOf) Sludlfll u:iUl, '=b<au predaton ha.~ been p .........fut

Mel1swring CJwm;c"llnjwry in NOit-lt1<1tUl1f BWI" ""d uosyslmu ~5

(_ Holl,ng. 1~59 for dttr mice and ..."..f1ies), ""nebnl. predalors I"nenllyhay. complex feed,ng mal.pes (''''1chmg) and on.n ~U1r. an .XlenSIV. leslarea. lher.b,., compllea"ng Ih. de,'.lopm.n' of a ver'.brale-ar'hropod ory.nebral.-y.rlebrale lesl syslem.

4.4.3 Gruing Tn"

GrUing of phy,oplanklon by zooplanklon is an imponan' palhway for lhemo"ernenl of .n.rgy and nuln.nll In aqualic syslem. and, in addilion. il is apossible del.rminanl of plI,.,10plankl"n communi'y OI)mpo<lllon. MaJO' prob­lem. In d.....'lop'ng ll1lZ1ng les, procedu"" a", poorly developed laboralOry andin S;IW lechnique, fOl m.a,uring g,ulng rales.

H.rbi,'ore gruIng on flo".ring l.rreslnal plan,s i' of parhcular inl......l in"'Ial,on 10 tbose mleraclions lhal dimlni'b commercial crops. Insecl-planl pairssPC'h .. aphid_alfalfa, wbll. fly-planl and lICale ,osecn-planl hay'. beeo closely.xamined in greenhouse and field stud,., (ahhough nol .. r.gard. response 10d,emgl st.....s). Th. pLonl and grazer inIOraClioos In lhese 'y'lems a", .asilyestablished arid changes in n.1 planl prodPC'uon can be aehieved in some Cases ina ",la",'.ly shon penod. Mo'" Ulen,,,'. stud,.. may be necessary 1o accouol forre-p,odPC'liY. capacily of bolb planl5 and insects. Que-sllon. remain .. '0 lherel.,'anee of b.rbivore insecl---<!omeSl'" planl inl.raellon. 10 lhe mo", highlyevolyed and perh.ps more sen.i,,,'. msec,-planl gruing ,nICraellon.,n naluralcommunilies. These candidal. les, .,.,..l.m. ha,-e polenual ulilil)' hul need funherd.,'elopmenl and validali"n befo'" 'h.y ,,'ouk! be ",adily a=pted as les' .y".m,In lhe loxicology ecosyst.m•.

Legume-rhizobia and m,..corrhiza-plan' inl.raClion. provide an ""01"gICa1lyr.levanl ba'i' for l.slS of symbiolic relauon,bips. hecau.. primary producli,ilycan be ulum..ely alf""'ed. Tes' dev'e1opmem should includ. e..minalion oflheinfluence of""il ')l'" and Iqume-rbizobium Of planl-fungus species dllf.rences.Tbe oM'ious ben.fil of Ib" """""al,on ca.n be measurtd ,n the q...nhly andquali'y of plant P'odPC'lion. Time course studies have 'ho"n tlull sen,i,ivilY of,h. 'y.lem may dimlOi'h wilh lime owing to degndalion of lb. chemical orselect,on of a res,stanl symb,on, SI,",n. Shorl-(.rm 'esll may. lberefore.eIf...,li,'.I,. predi<1 poICnu.1 inlerf.",nce problem•.

4.4.5 koors'elll Tesl Model.

Two general approaches haye emefl"d in lhe OI)urse of d.""lopttl<'1>l oflabor. tory ecosystem lesl models. One. ,he ~Ileric approach, is bUed on thecon«pt of a '.Sl .yst.m which .xhibu$ uni''¢rSa1 ecos,.-stem propenies (for

96 Mo'ltods fM UlimtJlinq R,,1e 0/ Ck,.,ioaJ INJUry

example, pnmal)' produo;tll'ity, organIC decomposlllon, nuuiell! C)l:linl) but i~

not OXpecied 10 mimic a panicular CCOS},.lom, The oth" approach mloh'eo ar<alis'iIo s,mulat,on of a .peclfic CCOSy:licm.

4.4.5,1 T)j'''' of Tw Modtb

M"'ed na.k cuhur....uo:h a, those of Bt}'ors (1963) and more ,ecenlly of Leffler(1977), of bacteria. alpo and milo'o,nverteblllieo ha>'e been sUlllleSted as lhe...osystem-I<>'c! ',,'hll< rai'. They arc useful at lhe: highe' 1<>'<1. of chcnucal..rccnillj, as Ihc} arc 'mall and replicable and could pro>'id<data on Sy:l1Cm-""'c!prOC<'SSC1 Ql ehaniCS in homoco'ta,i. of Ih< model 'ysl<m, Gen<ric ItllsenSi"...,ty to ch<miloals. as ,..<11 as <",dellC< of cons,Stenl reproduc'bility and·...oloBiIoal rtali.m· of rcsponse, rtmain 1o bt validattd,

Mort rca1i51i1o ...osy.tern model. an: bt,nB developed for ....'<IllI aquallChab,tat> suo:h a. pond< (Glddlnp and Edd1<mon_ 1978), lakcs (Han< tI 01..1980), lIn:ams (Rodgtrs 01 <>1" 1980) and co...tal marin< .},.tems (hull., 01..1977). Only a limntd numbtr of ~r" of elf...ls of chemICals on .uch lCSI.}'tem. "iSI, bUI Ihtle art indication' that m<tabolic ..action, to ptrlu,balion(for <umple. pnmary product,v;ty r«ponscl may bt assc<sed more taSily ihannutrient ..laitd pr"","i... Sc-..ral p,obl<m. ha>'< b<com< appa..nlln de>'<lop­inl lhose lCSU, Man) aqualic "'OS)"I<m. arc ,nhertnlly ,'anab!<: and un­predictable and model 'y:ltem, an: no Its. so' Macrocon,um<rs are difficuh 10mamia,n m small m"'rocosms. and ,f 'lICluded. ofl<n Ottrl an lnOrlhnateinfluence on .y.t<m \truclure and fUllCtion, The 'hallow d<plh and small siZ< ofItst sy.tems exalltlate th< influcncc of sedim<nl and "'stricl llot bth.-'iour ofSOmo microin".nebraics. The largo surface-In-Iolum< ral'O of ieoi S)'S1tmS<nh.ances ',,-aU elf...,,'. primarily expressed as l'0",h of allachtd alpo, Of lhc""riou. microcosm types, tho pond 'imulalion' aft SlrllCtullllly and fUllCllonallycl<»eSltO mimlCinB tho nOlural ""ttl bodi.. and h.-.. been included al 10all inone chemical manufacturer's assessmenl programm.

Scdim.nl and so,l co.... (PolC<lla., 0/.. 1976; Jackson and Hall. 1978) h.a"ebeen used as homoienized or intact ')'lI.m, 10 oxamine milorobial processes.Carbon d,ox,de <lolu"on. mil...rahzat,on of n,tfOFn and leachina of nutncnllh.-.. been examined as mtaSu.... of d...ompos,uon and nulnenl eytlingprocesses. Such lest ')"S1emsarc thouiht 10 bt representative only ofoite-.pc<:il\cpropcrtlcs. The senSiI"',ty ofso,l proces... 10 chemical SIres, i' "..Il-documcntedonly for certain melal" and lhere i. some ",'ideo« indicahnl ,hat soil microbesand microbial proccsses may bt less sen,i""e 10 organ,c compounds than a",lndiv,du.ol macrospccic"

Qucsuon. "'ma,n aboui ,,,lidily of Ben<raluing from t..t dala obtained wilhmICrocosm model. of .pecific aquatic or lerrestrial s)-.terns. AhhouBh the ,-al...of Ihe<c leolS would Increase ifdala could predict 'typical' elT...ts in a numhcr of

M."",,,"fI Chemica/Injury in Non-humon BiOla ond UO'YJlCmS 97

'imilar ecosy,lems. lhe a'a,lable informalion is msufficient to calobmte labora­tory sy"ems for general predIClions of <afe exposure le~el, in nature,

4.4,J.l The MiuQfos,"", as a Tewng Tool in UOJ}"lfm ToxIcology

Many multispecies lesl system, lhal are being de~eloped can be classified a,microcosms. A bnef w,iew ofmicrocosmsa' a toxiCity testing toolthu, pro'idesa posSlb,lity to ,ummarize the SlalUS of muhispecie. lest ,y,tem, in seneral,

Be<ause of the very wide range of j\O$.ibk microcosm de,ign., it has been,UilleSled tbal microcosm. could occupy a primary role tn 'he toxtcology ofecosy'tems, Ho....,,'er. tbis technique is ,tilt too Iomited 10 permit a recommen­dation for routine use. Seler'ion of the experimental paradigm for design of amicrocosm test will perforce be • fUOCllon of the specific lest, Thus, it i,unrealistic to speak of the Slandardiution ofmicrocosms. ex~pt in considering aspecific goal

Six major documents dealong wllh the u", of microcosm. in ecotoxkololl)'have been publi,hed recenlly (Gillett and Will. 1979, Wilt" al" 1979; Giesy.1980; Hammons. 1981; NRC 1981; Le~in er al.. 1984), 5o"eral aUlborsconlrlbuled to more than one of th= revie"'"1, and the lOtal number ofeontributinll scient'''S was ""'eral score, Eyen though the objeclives of eachdocumenl were different. lhere wer< no major d,sagreements about the use ofmicroco'ms ,n ecotoxicololl)', The main conclusion' agreed upon include thefono"'ing

(l) It bas yet to be delennmed whICh o<os)'stem '-ariables au most suilable for,pecifie microcosm application,

(2) Microcosms art limited by scale relationships (s,u dependeney oforganism, and area ~olume ralios) and art un$lable on time

(3) Microcosm des,gn rO<lu"es an ecosystem pe"pectiH.(4) A. complexity of mICroco,m design increase,. realo'm and cost tend 10

,ncrease and precision lend' 10 decrease,(5) There art few 0 prwri rul.. for inclu'ion ofcommon faClol'1 in mICrocosm

design: generic design microcosm' ha"e a very lim,ted use(6) Microcosms occupy a place on a t..red scbeme of en,'ironmenta] hazard

a,,,,,,ment of chemi""l',(7) Mierocosm experiment. are oflimiled u'" unl,l they are ,-alidated by field

exponmenlS(8) Matbematical model' mu" playa central tole on all environmental

hazard a....,mentS because of 'he onherenl limitations of empirical dala,(9) Microcosm. provide onfonnation at the proces' le..el: Ihey are ,ubsets of

ecosystem•. not miniature ecosystems. Processe, ;oclude energy relalionships.nutrient c~ling, .hcmica.1 traOlfolm.1];On! /loo;! orpmsm "SPOOleS al papu­lation or eommunity 1"",,1,

98 Me/hods/or £;wtla/mg Ruk 0/ C/wm!(al Injury

(10) Bo(h ',ymh~(,,' (gno(obioli,) and 'oatu!"..]' (~A'i~ or w;omtrw:IN)microcosm' havt po,emial use in hazard lI$s.t..rntni.

(11) For as....'ing en""on"",n,al hazard,. populanon_ or communi'y-lev.1metabolic pr~,s<, are more approprla,e ,han organi'm·I....l pr~...,

(12) Cri"rla for a'ing "sulls from microcosm tests in ,he regulatol)' processh..·• not be.n dt,·elope<.!.

(13) The us.tfuln... of microcosm' for roatin. screening of pot.ntiallyhazardous substances is not d.ar

(14) No labora'ory system or microc""m i' currently ready for us< as a 'es'protOCol or for prediction of .n,'ironm"'ltal hazards of chemical•.

o TESTS AT THE ECOSYSTEM LEVEL

This is ,h. la" 10',.1 of leSling (1......1 S). T.m conducted in nataral teosyslem,may in ",m. case' be ,h. only way to d.termin. the dfecu on ecosj"lemprocess<s. becaus< of compl.x int.rconnte'e<! abiotic and biotIC phtnomenaassocia,e<! "'ith ,peclfic compounds and ,h••xposed ecosj"'enl. For in"ance.Col.. and Ramspolt (l982)d.monma'e<! that laboralory ..perim.nts falle<!topre<!ict tlte migratIon of radionuelidn in iroundwater_ possibly o"-ing todifference' in chemical speciatIon of ruthenium under field and laboralorycondi'ion', l'amerou, <lud,e. have al", sho"'n that ,peci.. at the lOp ofa trophic"'eb are often sen,it"'. 10 certain compounds and can exerl regulalOry inlluence,on OCOSj"lem homoeo.tasi" bUI macroconsumer Imerac,ions cannol be .a,ily,ncorporated into labora,ory or .mall-scale field 'tudies. The impact onteosystem function of the eliminatIon of lariOT Con,a"",.. i. al'" diffical' '0demonSlrale in lhe labora,ory.

Large-.eale field tem can be used al'" to vahda,e resalls from multi.pecie.labora,ory test 'y't.ms whIch are no' s.tlf-vahdating. Tests at lhi' level are th.most realistic and .hoald provide lhe best assess"",nt of change, in eco,ystemdynamic,. HOI"."er. technIcal and ewnomic problems associale<! Wllh uper­iments at this level prohibil exten&lvt ase of oc""j"tem-scale 'tOOIe<.Con..derable attention rna" be &i'-en to lhe proper chOlet: of experimentaldesign••ite and pollulants 10 maximize tnformatlon and to pro"ide bench-marksfor <ro...,ile and cros...,htm;eal compari",n•.

EcOSj"lem-levell..'ini 'hoald incorporate rnonltonng of the toxic <hemical inthe e"'ironment and biota and dtl.nn,nauon of biologi<al efleer. at s<veralI"'els including changes tn:

(I) popalation dynaml<$.(2) commanily 'lmcture, and(3) ewsy>ltm function

Th. uillmal< foea, 'hoald be al the Ie,-.J of ecosyslem proee..... (.uch a,proda<li"ity. organic decompo'ition, elemcnt cycling) and on the dynamic<

(hom<>rostuis) of dominant commun,t,es. A proces,-ori.nted approach besladdresseslhe question of ho'" seriou.ly the «'>SYStem function " di,rupted by.xperimental exposure. M.du.nl'lm, ofcompensation inherent In the ecosystem(for.xample. abihtyoftol.rant populations to increa", the u.. of resources 1h.l1beeom<' available ,.,hen ..n,,"v. population. are lost) are accounted for ""'th thiSchoice of .ndpoint. ,.,herea. th.) a.. nOt obviOus In stnctly structure-orientedapproach.

Th. time bet"...n initial ",,-posure and ",,-peI'ted r.""tlon In .xpenments at tbi'Ie>-el isoflen relal",'.ly Ion. (..v.ral monlhs to )urs) depend in. on Ih. "",.rity of""-posure and type of effect> ""-amined. For Inst.a""", redu<110nS In the rate ofphotosynthesis may be sho,.,n In the shon t.rm. Ho...-e,·er. redu<1ion. in primaryproductivity may not be obVIOUS until completIon of the .ro,.,th period and".t"ficant change, ImIy not be d.monstrated for ",v.ral y.ars If the dommantplant Spel'les have a normal slo,., ..-o,.,th rat. (ImIture tree,).

The fate and etTects of chem;ca,1 pollUt.aDlS have been exanuned only in alimned number of ...·bole-')·>I.m .xperiment>. The number of 'uch .xperimentS islarae If Othe' dramallc perturbations (sucb 10$ clear,ut \oaglna and nUlncnladdiuon. 10 aquati< ,yst.m.) are considered .

Schindler and bi. collea.ues (1973) .xamined ... hole-Iake .tTects nf nutncntaddition in the hperimentallakes Area (ELA) ofth. Canadian Shield. In thelauer ILalfof the 19Ws, ScbindlereloJ. (1980) applied Ibe "'bole-lak. apprl»Ch 10"""mlOC acidification of a poorly butT.red lake in tbl. area (ELA lake 223).These Studies ".re con»<:l.red as an ""-penment.al a=1.ration of acidifJ<-at,onproetW':S occumn. ove, ""rta.ln reJlons ofU>lern North Amenca. AC>dificationo".r a 3-y... period from 1916 to 1978 decreased pH from 6,7-7.0 to ~. 7~S.9.

However, sulphurIC acId addition did not decreM<' pH as ",,-pe<:ted owina tocomplex Int.raction. of the iron mobilizalion and the .ulphat. redUCIion cyclesin butTenn. a6d addition•. Soluble concentration. of .lement> sucb ... zinc andaluminium did inc se substanually ,n a ment ....,Ib both Iaboralory andmonllonna ohse" "on•. There was. ho...· no evidence 10 .upport the Grahnand Hultbera (l974) self-a=I.ralinl olilotrophic hypothesis. beeause nochanl.S in chlorophyll or pnlmlry producti"ity .....r••vld.nt (Schtndler. 1980)This Itudy cmpha$lzed several importanl a.pect> of these type of teslS:

(1) lb. importance of considenn. and cxamlnina mIcrobIal p,oetW':S.(2) the problem of .xperimenlally mimickinl .xposure observed in contami·

naled .ystems. and(3) th. uniqu."... of site-,pectfic propertie',

Hall and Likens (198<1) and Hall U oJ, (1980) des<::rihed .xperiment.alact<lifJ<-atlon of Norri. Brook in lhe Hubbard Brook Expenmental Fo....'- WIlhincreased acidity of .tream "'at.,, lhe concentration' of aluminium. calcium.magnesium and pot.auium increa>cd >tgmficantly. Emer••DCC of adult mallli~.Slo".fl,es and true flie' decr.ased at low pH (4), Expenment.al acidIfication

100 Mellw<b fo' Emmali>!9 Ri3k of Chemical /njWFy

11~~~ mil\:roiJl\'~rt~br~t~ l1i~mny ano ~ompluity of the r(XX! ~haiD,

Periphyton biomass incc¢MW, and this wa' partially anribmed to reduction innumbtrs of scraping and collecling in>CC1s which generall}' f«<l on ~fiphylon.

This obst:rva,ion illu'traled the importance of using ex~rimentaltechniques

"'hleh allow euminalion of consumer int~raclion in lhe conlrol of prodlXerbiomass and producli"ty, phenomena generally' l1ifficuh to ao:ount for inmulmpecie\ laboralory syslems.

The conlrolled dosing ofa Imall Ohio Slrtam. Shayer Run, w'lh copper by lheUS En~ironm.nlalProlection Agency (Gectler rl al" 1976) is a good rumple ofa 'est for .xamining ,Iream <cosy$lem$, Th'$ extrci.. "'as undertaken specifically10 ,,,Iidale mult$ of laboralory l"ling, The ""uh. oblained subslan'ialed theimportanco of con,iderinll avoidance bthaVlour In fi'h and directinll tbeassessm.nt lowards chanll" in Ie.. mobile communll... (macroin".nebrat..jand lhe oc"'y"$lem as an entity. Studi•• by Winner n ai. (I97~, 1980) on ,heShayle< Run macroin'-"lebrale communit}' follOWIng 2 ytars of experimenu.lexpo,ure to copptr at 20-120 iii I d.monllfa,ed lhal changes in communi'y>lrutlure dunng lh" ecolOgically sbort period " ..'" in agreem.nt "'tb findingsmulting from loni-lerm e'JIOSurt 10 metals of ,tream communilies (Sheehanand Winner. 1984)

Suter (1982) describtd sev.ral largt_scale field 'echniq"", fOf e,amming~nurballons m terreSlrial ')"Stems. The scale of e1periments in ler""trial')"Slem, is nOt as well--ddined U Ibat for tbe aqua'ic Itudies previou,ly described.Tb. 'tudyarea may be a parI ohn agricuhural field, gras.land or fo",st, 0< II maybt as large as an entiu watershed, an approach sU<:tt$sfully used hy Bormannand Likens (1979) and othe,.,. in lbe Hubbard Brook Experimental Fomt. The>eale of ,lXb Sludits depend, on communit}' d,,,",,,,y. produ<:ti"il}' andpalchiness; wilh ocO$j"$tem$ of low producti"ty, or billh prodlXlIvity and highpatch,ne... laraer plots are required (Suter, 1982). Tbt s= oflhe penurbed arealimits the ranlle of propeJ'he$ lhal can bt monilored. SUle< (1980) Inl1,coted lhal.hon-term .ffecl. 'u<:h a, leaf chloros;" change< in gas .xchanie rates andbehaviour 'hould be monilOrtli and relaled to medium- and long_'erm change<in population. {reproduction $IlOO:5$)_ communilit. (dominance shifts) andeco.)'Slems (primar)' productIvity).

Pesticide< and berhicide' bave betn tested for ad"N" dTecls in controlled fieldexperiments (Barrell, 1968; Moull1ing, 1976), and so ba.... ",riou. ai< pollutants(lJ"lingand M'ller, 1977; Van Voris and Tolle, 1979).

Several hmitallonl ha.. bt.n noted for t..1> al lht ecosy'tem level.Reproducibility and precision are dIfficult 10 ass«' for large-scale e.periments.A recent disc"""on Oflhi' problem (Levin el aI., 1984) provIdes some in.i"'t intolhe .patialileneralll}' of 'uch uperim.ntal eKlrapcia,ions. although there i. linle,pecific information in ulation to cbemICal pertu<bations. Nutrient loss«associated wilb clear CUlling in lhe Hubbard Brook FOml "-ere conmtent Wilhtbe effocls of sim,lar 10Uing lechnique, applied to Olher fou'ted land in lhe

Meaturiny Chemica/ln)l/r,. in NM-hllMan Biolo and £eo')'Slmu 101

regien but did not quantitati>'ely predict nutrient lones In Oregon forests, Inaddition. labour and finan<:ial expendItures a'sociated WIth lalge-scale field test<or ecosystem studie, can also be substantial.

In .ummary. ecosystem I"'eltests are essential and necessary to demon matecenain long-term effects. to validate laboratory data and to prOVide apprepnateinformation for predicti'.. test models. Teshng at thIS Ie"el is complex, costly anddIfficult. \f not Impossible. to rep!rcate.

4.6 ECOTOXICOLOGICAL MODELLl1'iG

Thedne!opment and use of mathematICal models ,n ecotoxicology can provide ameans for ,ntograting test and mon,tonng <!ata as weU a, SImulating populationand ecosj"tem response, to chemical perturbations, In a gi'-en case. ifone ~no"'s

the quan",y ef the toxlCantln question in each compartment of the ecosystem(such as. water. soil. plants and animals). the size of each compartment and Iherates of tlansfer betw«n the compartomnts. one can calculat. Ihe quantity andth....fore the expo,ure to the tOXICant that the tarllCt orpni,m "''ill experience, Ifthe damage function is ~newn (I.e. the exposure to the texicant that wIn prodU<:ean effect). then an evaluation of the toxicant in the envltonment Can be made,T.. e rec.nt.xample, of the 'uccess ofthi< approach a ... the Ottawa R,wI Proj=(M,ller, 1977) and the [ntegrated lake Wate"'hed Proje<:t (Chen. 1982).

Thi' ecosj"tem approach reqUites funher development ofappropriate modelx.Model calculations 'heuld identIfy the mOSI cnucal areas te be examined inspecific pollution eases, Once the ob)ectjves are so identified. the methods withwhich to conduct the wor~ can be selected (b,oassays. microcosms. field studIes)Hopefully this process will enable the selection ofa small number of 'ituations tobe <tudlCd \n detail.

4.7 CONCLUSI01'iS

(1) E<;osystems vary greatl)' in their complexity rang'ng. for example. from afield of corn to a tropical forest. There ha' been some progress ,n acquinng theability to measure the 'mpact of pollutant< on ecosystems but the state-of-the-artvanes greatly from ecosystem to ecosystem. For compl.. ecosystems moststudies of the effects of pollutant< end with effect' on populatlens but co""enedeffort' 'hould be made to advance towards the eff...,ts on whole ecosystems,

(2) Under envltonmental condi:ions only in f.,.· cases is there exposure tosingle chemicals, The usual 'ituation " thaI a wid( Varlety of ChemlCa" arepresenl \n \"al)lng concentration, under '·a[)'inll condition•. It is imponantto bear in mind the ;nt(raction, bet"·een chemicals and ph)""ca; condihons.

(3) Consid.rablepr~has been mad( to improve th. prediClivecapacity of

teSts. A~ment of tbe ~'alue and limitations ora single melhod. while cenaiolyrelevanl for the method itself, has limited imponance on a "'ider pe",pective on

I02 M~lhod.< 10' EJllmaling Ri<k 01 Ch~mka1 /lljl/fY

environmental pollution. for correcl evaluauon of methods capahle of predict­In,chemical injury 10 ecosystems, only into,nled infonnalion isof real help, n..a=s,menl of the 'mpact ofa chemical .ubslal'lCe on ec"'Y"lem, has 10 b< madeby inle,ralin, "ery d,rrerenl kind. of information induding phy'lCal andchemical properties of molecules, biolic and abiotic de,ndabiiily, err""tS onolher orpnisms at dirreren'lrophic le"e15, mode ofactIon oflhechemical. and soon. M,croco>ms can be used for ecolox,,::oloJicaI 'ludies 10 bridge lhe ppbelwoen ,imple bioassay, and complu field 'ludies,

(4) There is al present a limiled capacity to .valuate quantilalively ,he Impactof chemicals on some oflh. simplerec",)'''em•. In mosl CA$e5, we are more al asta,e of ranl"n, huards ralher Ihan of quanlilat",. evalualion of each huard.Th.re in need 10 use the a....ilabl.data and focus lhe direclion of research SO Ihalwe can move towards the ,oal of ri'k as",,,ment at lhe ""osy.tem level.Unforlunatol)' the a"ailable data on lhe err""" of chemicals on ""o>ystomsoblained f.om rehable lon,-term studies are extremely Iimiled. EcologIcalmodels, hased on high quahty field data, 'hould ueally aSSIS! in advancing ourpred,cti.'e capabilil)',

•.s RECOMMENDATIOSS

(I) Bau/ifU! "aditl. EvaluatIon of chemical hazards In the environmentdepends on the e~"ence of information on the propenies ofa g,ven chemical andother factors lhat determln. us env"onmental behaviour. In most sue-5pec1ficcascs thi' N.ic information i. nol available." ". lherefore. recommended thathaseline studIes be condocted in area> where chemical emuent. are Upecled 10increase, These .tudies should be designed to ..amine change< in groups oforganisms a> ",.11 as in the ph)'sical environment. Soch studIes are of necessityIons lerm. but lhe expen.. and dIfficulty of this "'ork should n01 be permuted toinftoence the priority for carry,ng oul lhe nece<sary ObsefYalions.

(2) Mtxk/ dn'€Iopmem and ",,/idalion. The developmenl of appropriatemodel. will produce a framework for identifyinll aDd guidIng the nece<saryresearch on a given chemical hazard. and will thus provIde an integralion ofexperimental work SO as to yield the mo>t reali'tic and broadly appbcabl. results,Such model' must be "alidated by field data. Vahdat,on is an ....ntial t..t Dfa"umplion, made during model dC"elopment and. therefore, of lhe realism ofthe model. A crilical review of lhe .xi'ling models would be useful.

(3) &/uli"" 01 '05' "",,/mh. h would be inappropriate to suggest specIfic1..1S for the evaluation of all chemical hazards. Rather, tesl methods should beselected from lhe hattery of t..,hni<Ju.. available for obtainins the r"'luiredlnformalion about effects at the appropriate level of ""olog,cal organization. Insome cases. simultaneous tom ,,'ill be needed on Ihe effec" of chemicals Onorpni,ms in the laboralory and on the same orpni,m. in the field, This sbouldp,ovlde a meaSUre of the effects of uncontrolled environmental variables in Ihe

field and how lhe.. variables m'g/I! inftuen" the applica'ion of laboralory dala10 the eco'y'tem

(4) Ecologically I'f'1f't'om inse,' ItJlS. !lttaU$( of di,e"ity and adaptation tospecial eeolOjiical condilions. inSttlS provide ..ositi"e system. for testingenvironmenlal chemical'. To U$( this "",sibilit}. melhods applied in ,..tingpestic,de••hould be adapted. new ecologtCally relevant ,pecies fOf testing 'houldbe identifie<l. and method' for lesting oflong·tenn efreelS ofchemicals <)n insectsat the le"el of indi"idual•• populations. and commuolties should be develol'e<!

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Jack>Ofl. D. R",oo Hall. J M. (1978). uttoClion ofnutti<-n" from ,nact >Oil <ore> toa..... til< impoe! of cbe-mocal to,ocan" 0<1 "'ii, PtJoi>~i<", 18.272-278.

Kena~•• E, E, (1914). Factor> to be consideTtd In Ihe ",·.Iu.tion of the to.i<:ily ofpestICIde> to bi,d. in tbeir eo";'onment. Em'iT. Q""I, 5I>f, 2. 166_191.

Kochhar. M.• Blom. U" and R01n.... R. A. (1980). Err"".. of o<ooe .nd or f<scue onIadino do,'e" ,n"...."on•. C... J 81>,.. 58, 241_249

Kramer. P. L .nd KotlO""h T. T. (1979). P!I)"'~y"f Woody PI""". Acad<mlC Pre".New York: 811 palt",

lefl\e,. J W. (1977), A Mi<rtXoJm Afpr<XlC~ 10 ... E,"II<i>'"," of ,~, lJ;,',,,j';~S",bili'y

Hy",,'''';'. PhD D"..rtotlon. Uoi".,...ty of Georpa. Alhon•• Geol'Ji•.win. S, A.• Klmb.lI. S.. Ktmball. K...od M<Dowell. W. (Ed•.) (19i4). N~Pmpt<ti«'

;n uowxieoJogy. Ecos),,1<m. R<scarcb C<nter, Com<ll Uni-.nity. ltha<.a. N,..' Yor!<(in pre»),

Matsllall. J. S. (1969). Compelitioo bel"'«11 Dap~"io JlI'I•• • nd PophhiQ _110 nm<>dlfied by radiatiOSl m .... EcoloJicaI Soc>ety of Americ•• Anoual M<t'1lnl. Ao~o"17-22. 1969. Unive...ily of Vermont, Borlington, u"/09lc,,1 SQcie'y of A"",reoBu/l.,,,,, 50. 10 (.bslrae! only)

M..... D. B.. Cawthon. D. A., .nd Port. T. (1976) An ..perim<tltol .n.lysi. ofcompetit"'e indctenninanq 10 r,ibcii"",. boc. ..tn, ,Ie"" Sd, U,S.A .. 13. 1*8­1372,

Miller. D, R. (Ed.) (1917). D""iIMioII tmd T,,,,,,,,o" of PoIIo<t""" IJt Flo"'~ War"UaJJ'''"''. 011""'" lI.;,'" Proj<cr. Final ilLro'" Vol. I and 2. Nall""ol R....rchCouf\C,1 of Canada. Ot...... : S8g .nd 178 pqes.

Moose'. J. L. Fisher. N. S.• and Wu.....r. C F, (1912). Polychlorinated bipb<nylo andDDT .I ..r.pec;es compellllon tn m"ed culto.... Sel<""e. ]7~. S33-S3S.

Mouldin~, J. D. (1976), EffOCl. of alow-pe...iste~ Inse<:tioc,de on fo...t bird popolation>.Aule. 93., 692-708

Mordr><b. W W.. and Mort.. J R. (1973). Predation by <O«ioeUid .....~ "pe"ment.on ...otehlD" &o~y, Sol. 1«1-167.

NRC (1981). rwiJt9 fo' Elfec" ofClt<",icab "" uNJ"e"". A Report by !he Commal«1o Re,1tW Mothod. for EcotoJ.icoIO$:!' COfJlrIliWon on Na'ural R"""""" NationalR....rch Council. Na"on.1 A<ad<my Pm<, WashinJtOll. DC 251 paaa·

OECD (1979). Preliminary 11..""" of ,'" SliP Sysrt", G,O"p. 28-30 N~, 1979,Orpnir.ation for Economl< C<><>penItioo and De"elnpment, Paris: IS palt"

OECD (1980), R,por' "" ,Itt A",,,,,,,n, of P""nriol brri''''''''''''ol Elfec" ofC..",icalJ,TIt< EIf«" or> O'~""i>nu 0,,,,, 'Ium MIl1I i>J!d "" uaJJJI"",, Vol. 2 .nd 3. Publi'l>edfo' th, OrJan"'tion for EcOSlomioc Cooperali"" and De",lopmenl by TN.O.Department of BiniQ5Y, Siudy and Infonn>lIon C""", fo' En"i,onmen..1Resurch.Delfl, "el!>e,londs: 1S7 000 135 pai'l

OECD (I 'lIB). ilLport or> 11.",.... P"".Ior> Di>la Inr"p..'a'ion G"~S for l"il141 H",ardA"""",", ofC..",icolJ. Orpoil:.ltion for Economic COOpt.."on .nd De,..lop...nt.Pari>: 75 pa~.

O<'iau, C, A.• Per02. K. T.. .nd Ni'on, S. W. (1977). Mul"""'1< anal)";' of..peri...otalmllilll< «:r»pll:m•. He/iof4ndt:r, "'jJ" MuwW1Iltrs.. 30. 30-*,

Part. T. (194S), uperi"""tai studios of in"''''poci,,<olDpelition, 1 Competillon bet..'..n

106 Melhods lor £J""""lIIg Jlult "I Clitmic,,1 ("JW'Y

pvpu!l.lWDI of Ill< llvllr becllcs. TrIbl1filfm m1,1K!luPr 0II\1I1.1ll1 TrlbolJwlr C4U_HtrboL Eml M_, II. 26$-lOlI

PeabIL 0, I.II97S) PbIPO':""" tfroctlelchl<>nnat<dhyd........-..._'JI<Od.[n H..,... R- _ ffftd. \' H (&10.) !AM ...~.,1't~'i<"', f!P ~l·

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Ro4p:n.J, J H. C1att.J k.. Dod-." 1..._ CairJIIJ.~J (1_~ >.; .......-Jr- 01 ....... _ r- vL ............._ ..... loaf< -. 101Gx>T.Jr I P fEd .M~,.. .. CtttIarI...t__ l'-'TlSCOSf·711101.f!P CS·60U L".5, 0<panmaI' vL £8rfv. SpofflOllidd, \-__

Scbaftc, L 111 (19"21 "'uaottonl_)vLlmp"'''·f'lal~_odoo<• °k .....;W bordo. Tu. wJ ...._ .. 11. lU-110

SchmdItt. 0 Ill' 11_. u.~~'- vL. __ tate' .... vL lb<~... ~l'P"'. InDnblol.D.. _T-'A-(Edl-)£<! 'f',JJ...,",AcJ "ft. ro_. " ' Ill'''.__C~. PI' )70_l70 n..SMF ptOOJOllOt. 0IJ0. l' ,.

SchmdItt.D W .• K..... H. Sri h. LV.. Prd<_..... J.. FtoIt. \ L. RtIIl, R. A._ Cap<T, M U97l) E~'- 01 Uk< 227 by adcb'- '" p!oclaph.1< _""rate: Ibt__ i.hlrd aDd fooonh ,.... ol<an<llm<fl.. IlnO. [971._ 1972 I Fulo/(n. &Ie- 31.1415 lUQ

Sdlirldler. D W. "".............. 11. .• Coc>Ir.. LB. R""",,-nsk.. T.. aDd Prokoptl'O",h. I(1980). E>.pen......1&1 Kf'llllal_ olW. 22l. Exp<nmnll.ll W .. AIU' lIooct,.-........04 Ibt ftm Ib ).... '" Kf'llllc:o""". C"" I. FisJI. _I. S..... n, ~2-lS4

Sbothon, P. l.. aDd W, II. W (1934). C"",ponoon or Jl"Od....UllWh.OI.. htO''l'.mtw.poIJnt<d ""'.ms. I. S IIo•• P J., M,ller, D. 11..• Butle,. G. C...ad 8oIIrduu. P H(Ed.,) E:jf.." 01 Poll.""",,., ,lot £eM}''''''' ~I. SCOPE 22. s... l. Joh. W,Io) &SM•• Chich..l.,. "co' Yo'k. anoboll<. Toronlo (iII p.....).

Sut.,. G. W. (1980). T.......ltiol 1<"01(1). I.. S.r>d<... F. S, (Ed.) S,,~..gwsI'" £cologlc~1

EjJ"'l> ASH","",,'.' 00£ EAt,gy ,1<11<1')' S,u•. ORSl '11.1·6733, pp. 101- IJ.tI DokRid.. N.liOlloll.obo,otot}". Oa~ R,d... T...",.....

Sut." G. W. (1982). T.....'no1 pe"~rboll"" ""penn...".. fOf: .".irorl.......1.Il ............,&ri,. M~",'. '-4J-~

Voa V"ris,P. O'NnlL 11.. V •Eaw...u. Ill' 11... oodShnpn. H. H. J,. (I'ISO). F~tICl>ODal

camplo ,y _ I<"...yo.... IlabWly, £c"'y, 'I. IJ52-1J60Voa \ P. _ Talit. D (1979). f'ltld .tId tIIlICtOCCOm irlvnllfO.-I af Ibt tfrecu 01

atmaopl>modtpaoo""" f...... f..-J futI cunbwuoa. [0 ruJ' ,1_ ~"IIP,. 19.RP 122.-5. Ilo._ CDI-.boo lobon._ CDI__ Ohoo.

W&rDt1. II.. L.~ t:: K.• _1Iorpwt. L 11966~ lIeIoo.-ral potbolosY '" fislo. aquoat>Uo,,,. OIud) '"1lIbietlIol,...,.... _tJCIft, J.~ £atl ) (SuppLl. llJ-241w....... II.. W~ Ik>oocL M N. _ Fan<II, M. P. C1980). 1""",,__y_...

-.01 .....,. -.aI palIoI..... 1o:MJ< «:00)-' C._I. Full.-' Sn- n.64~­.,",-....... II..W \'... O)~•• l S,Cono,""._FarmLM '.(19751 11._"''''''

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