toxicity of neem-based insecticides on aquatic animals and
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LSU Historical Dissertations and Theses Graduate School
1999
Toxicity of Neem-Based Insecticides on Aquatic Animals and Cell Toxicity of Neem-Based Insecticides on Aquatic Animals and Cell
Lines. Lines.
Ipek Goktepe Louisiana State University and Agricultural & Mechanical College
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TOXICITY OF NEEM-BASED INSECTICIDES ON AQUATIC ANIMALSAND CELL LINES
A Dissertation
Submitted to the Graduate Faculty of the Louisiana State University and
Agricultural and Mechanical College In partial fulfillment of the
requirements for the degree of Doctor of Philosophy
in
The Department of Food Science
byIpek Goktepe
B.S. University of Istanbul, 1993 M.S. Louisiana State University, 1996
December, 1999
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DEDICATION
To MY DEAR FATHER
whose dedication to learning is a continuing inspiration to me
11
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ACKNOWLEDGMENTS
My deepest appreciation and thanks go to my advisor. Dr. Plhak, for all
of her guidance, encouragement, help, and friendship throughout my Ph.D.
study and for giving me the opportunity to prove myself. 1 would not be at this
point without her support. I would also like to thank my advisory conunittee
members. Dr. Park, Dr. Portier, Dr. Romaire, Dr. Wilson, and Dr. Chen for their
input, support, valuable time, and advice.
Special thanks go to Ms. Monica Sharp, Mr. James Durbin, and Mr.
Daniel Strecker, C.K. Associates, for providing Daphnia piilex (free of charge),
and for their technical assistance throughout my research. Sincere thanks are
extended to Dr. Romaire, LSU Department of Forestry, Wildlife and Fisheries,
for his assistance and for helping me obtain crayfish from Dr. Huner, University
of Southwestern Louisiana. Appreciation is also offered to Dr. Ottea and Ms.
Stacy. LSU Department of Entomology, for their help and knowledge in the
experimental part o f my research.
1 would like to express my sincere appreciation to Dr. Supan, LSU Office
of Sea Grant Development, for providing oyster eggs and oysters, for sharing
his experience, and also for his assistance during toxicity experiments on this
species. Special appreciation is extended to Dr. Jerome LaPeyre and Dr.
Cooper, LSU Department of Veterinary Science, for allowing me to conduct
oyster cell culture experiments in their lab, for their assistance and answers
111
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involving the technical and practical aspects of oyster cell culture. I wish to
express my sincere appreciation to Dr. Rugutt, LSU Department of Chemistry,
for his help to analyze azadirachtin using NMR spectroscopy and guidance in
the interpretation of the data obtained and to Dr. McCarley, LSU Department of
Chemistry, for her help in analyzing azadirachtin using FAB Mass spectroscopy
and assistance in the interpretation of the data.
Furthermore, I would like thank to the Department o f Food Science
graduate students, faculty, and staff for their support during this research.
I wish to express my sincere thanks to the U.S. Geological Survey and
the Louisiana Water Resources Research Institute (LWRRI) for funding this
research under the project number 1434 HQ 96-GR-02673.
Finally, I would like to express my deepest feelings to my parents,
mother Fatma Goktepe, father Recep Goktepe for their full support, endless
love and trust in me. I would also like to express my infinite gratitude to my
brothers, sisters, nephews, and nieces for their encouragement, endless support
and love.
No words can express my feelings to my husband. Dr. Mohamed
Ahmedna, who has been always there to help me through the hardest moments,
to share my happiness and sadness, to support and understand me in any
occasion, and to believe in me for everything I do.
IV
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TABLE OF CONTENTS
DEDICATION..................................................................................................... ii
ACKNOWLEDGMENTS................................................................................ iii
LIST OF TABLES............................................................................................ vii
LIST OF FIGURES.........................................................................................viii
ABBREVIATIONS.............................................................................................x
ABSTRACT........................................................................................................ xi
INTRODUCTION............................................................................................... 1
LITERATURE REVIEW................................................................................... 5Chemistry of Azadirachtin.....................................................................7Mode of Action of Azadirachtin in Insects.........................................10
1) Antifeedant Effects...............................................................102) Endocrine Effects.................................................................133) Physiological Effects............................................................19
Toxicity of Azadirachtin to Non-target Organisms........................... 20Environmental Fate of Azadirachtin....................................................26Biology of Selected Species................................................................ 28
1) Mollusca............................................................................... 28a) Freshwater snails {Physella virgata)....................... 28b) Oysters (Crassostrea virginica)...............................29
2) Crustaceans.......................................................................... 30a) Red Swamp Crayfish {Procambanis clarkii) 30b) Blue Crab {Callinectes sapidus)............................ 31c) Grass Shrimp (Palaemonetes pugio)...................... 32d) White Shrimp {Penaeus setifenis)......................... 33e) Clodocera {Daphnia pulex)..................................... 34
3) Insecta...................................................................................34Southern House Mosquito {Culex quinquefasciatus) .34
Molting Behavior of Crustaceans........................................... 35
MATERIALS AND METHODS.....................................................................37Chemicals................................................................................................37Test Organisms...................................................................................... 38
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Mutagenicity........................................................................................... 39Bioassays Procedures............................................................................. 40
In vivo Acute Toxicity............................................................... 40Crayfish {Procambanis clarkii).................................... 41Blue Crab {Callinectes sapidus)................................... 42Grass Shrimp {Palaemonetes pugio) ............................42White Shrimp {Penaeus setiferus)................................ 43Water Fleas {Daphnia pulex)........................................ 44Freshwater Snails {Physella virgata)...........................44Oyster {Crassostrea virginica).................................... 45Mosquito (Culex quinquefasciatus Say) .................... 46
In vitro Acute Toxicity............................................................... 47Hybridoma Cells.............................................................47Oyster Cells......................................................................49
Stability o f Toxicity................................................................................51Fractionation ofNeemix^’ and Bioneem™ ........................................52Chemical Analysis..................................................................................53
NMR Spectroscopy.................................................................... 53Mass Spectroscopy......................................................................54
Data Analysis.......................................................................................... 54
RESULTS........................................................................................................... 56Mutagenicity of Neem-based Pesticides.............................................. 56In vivo Acute Toxicity o f Neem-based Pesticides and Pure AZA....56Water Quality Parameters......................................................................71In vitro Acute Toxicity o f Neem-based Pesticides and Pure AZA...71Stability o f Toxicity o f Neem-based pesticides.................................. 78Fractionation of Neem-based Pesticides............................................. 81Chemical Tests........................................................................................ 83
High Field NMR Spectroscopic Study of A ZA ................83Mass Spectra o f AZA..................................................................86
DISCUSSION.....................................................................................................90
CONCLUSIONS...............................................................................................110
REFERENCES................................................................................................. 113
APPENDIXES: RAW DATA..........................................................................128
VITA.................................................................................................................. 145
VI
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LIST OF TABLES
1. Nuclear Magnetic Resonance (NMR) Spectra of Azadirachtin at250 M H z........................................................................................................ 11
2. Effectiveness of neem products against non-target organisms.............. 25
3. Examples o f A2A equivalence in Neemix™ and Bioneem™38
4. LC50 values of Neemix™ for eight aquatic species........................ 61
5. LC50 values of Bioneem™ for six aquatic species.......................... 65
6 . LC50 values (pg/mL) of pure AZA for four aquatic species............68
7. LC50 values of Neemix™ treated with light under air at 24°C after1, 3, 6 . and 9 days of exposure for D. pu lex ..............................................80
8 . LC50 values of Bioneem™ treated with light under air at 24°C after1, 3, 6 , and 9 days of exposure for D. pulex ........................................... 80
9. LC50 values of Neemix™ treated with light under air at 37°C after1, 3, 6 , and 9 days of exposure for D. pu lex ............................................ 81
10. LC50 values of Bioneem™ treated with light under air at 37°C after1, 3, 6 , and 9 days of exposure for D. pulex ............................................81
11. LC50 values of Volatiles and Nonvolatiies obtained from Neemix™for D. pu lex ................................................................................................... 82
12. LC50 values of Volatiles and Nonvolatiies obtained from Bioneem™ for D. pu lex .................................................................................................. 82
13. 1H NMR (Nuclear Magnetic Resonance) spectral data (5) for Azadirachtin (400 MHz, Solvent CDCI3, 7.26 ppm ).......................... 84
14. Diagnostic ^H-^H COSY (Correlation Spectroscopy) Cross Peaksof Azadirachtin at 400 M H z.................................................................... 86
15. Accurate mass data o f Azadirachtin........................................................ 88
vu
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LIST OF FIGURES
I. Chemical structure of azadirachtin type A ................................................. 6
2a. Other bioactive compounds in neem............................................................8
2b. Other bioactive compounds in neem............................................................9
3. Chemical structure of ecdysone.................................................................14
4. Azadirachtin analogs with molt inhibitory activity .................................15
5. Effects of Azadirachtin on ecdysone-mediated pathways.......................18
6 . Mutagenic potential o f Neemix™ on S. typhimurium types TA-98and TA -100................................................................................................... 57
7. Mutagenic potential o f Bioneem™ on S. typhimurium types TA-98and TA-100................................................................................................... 58
8 . Mutagenic potential o f AfB [on S. typhimurium types TA-98 andTA-100............................................ .'............................................................ 59
9. Percent mortality and molting for mosquito larvae exposed to Neemix™...................................................................................................... 62
10. Percent mortality and molting for mosquito larvae exposed to Bioneem™.................................................................................................... 63
II . Percent mortality and molting for mosquito larvae exposed topure AZA...................................................................................................... 64
12. Percent mortality and molting for blue crab exposed to Neemix™.......66
13. Percent mortality and molting for crayfish exposed to Neemix™........ 69
14. Percent mortality and molting for crayfish exposed to Bioneem™...... 70
15. Mortality curves for Hybridoma cells exposed to Neemix™................72
16. Mortality curves for Hybridoma cells exposed to Bioneem™..............73
vni
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17. Mortality curves for Hybridoma cells exposed to pure Azadirachtin ..75
18. Mortality curves for oyster cells exposed to Neemix™........................ 76
19. Mortality curves for oyster cells exposed to Bioneem™...................... 77
20. Mortality curves for oyster cells exposed to pure Azadirachtin...........
21. Nuclear Magnetic Resonance (NMR) Spectra of Azadirachtin..............85
22. Correlation Spectra (COSY) o f Azadirachtin..........................................87
23. Mass spectra of Azadirachtin..................................................................... 89
IX
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ABBREVIATIONS
AZA Azadirachtin
LD50 Lethal dose necessary to kill 50% o f a given population
LC50 Lethal concentration necessary to kill 50% of a given population
IC50 Inhibition concentration
EC50 Effective concentration necessary to kill 50% of a given
population
ppm parts per million (mg/L or pg/mL)
%o Salinity ratio, parts per thousand (mg/mL or pg/pL)
DO Dissolved oxygen
DI Deionized water
NMR Nuclear Magnetic Resonance
MS Mass Specroscopy
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ABSTRACT
The impact o f pure azadirachtin (AZA) and neem-based insecticides
(Neemix™ and Bioneem^^ on eight aquatic animals (crayfish, white shrimp,
grass shrimp, blue crab, water fleas, oyster, freshwater snails, and mosquito)
and two cells (hybridoma and oyster) were assessed by short term acute toxicity
tests of 48 and 96 h. The LC50 {in vivo bioassays) and IC50 (in vitro bioassays)
were determined for each species. Stability o f Neemix™ and Bioneem™ was
tested under light, air, and heat (24 and 37°C) for 1, 3, 6 , and 9 days.
Neemix™ and Bioneem™ were fractionated into two fractions (volatiles and
nonvolatiies) and toxicities o f each were tested on water fleas. AZA showed
less toxicity than Neemix™ and Bioneem™ on all species tested. The toxic and
molt inhibitory activity o f the insecticides were species, dose and time
dependent. Among the test animals, water fleas was the most sensitive to
Neemix™, Bioneem™, and pure AZA with LC50 o f 0.071, 0.034, and 0.382 |ig
AZA/mL, respectively. Next to water fleas, mosquito, oyster and blue crab
were found to be very sensitive to Neemix™ and Bioneem™ with low LC50
values. Crayfish and freshwater snails showed the least sensitivity to Neemix™,
Bioneem™ and pure AZA. The LC50 values o f Neemix™ were 4.705 pg
AZA/mL for crayfish and 4.257 pg AZA/mL for snails. Neemix™ and
Bioneem™ were foimd to be toxic to both hybridoma and oyster cells at
concentration 1 pg AZA/mL and higher. The toxicity o f both insecticides
xi
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decreased with higher temperature, light, and time, but Bioneem^’ remained
more toxic at 37°C than Neemix™ and appears to be less sensitive to
environmental factors. Nonvolatile fractions exhibited significantly lower LC50
values (1.023 |iL fraction/mL) than the full formulations o f both insecticides.
The volatile fraction of Bioneem™ showed lower toxicity than that of
corresponding nonvolatile fractions. Whereas, ± e volatile fraction from
Neemix^’ was not toxic to water fleas.
These results suggest that increased use of neem-based insecticides,
resulting in increased agricultural run-off, may have direct adverse effects on
aquatic organisms, contrary to the common belief that plant-derived insecticides
(e.g. Neemix™ and Bioneem™) pose no risk to the ecosystem.
xii
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INTRODUCTION
Agricultural use of synthetic pesticides has played an essential role in the
production of an abundant food supply during the last 45 years. Approximately
501,000 tons of pesticide were used for agricultural uses in the U.S. between
1994 and 1996 (USDA. 1998). However, the extensive use of some pesticides
has resulted in environmental pollution and caused the development of
resistance to pesticides by some insect species and negative effects on nontarget
organisms (Gary and Mussen, 1984; Frank et a i. 1990). Consequently, interest
in alternatives to synthetic pesticides has greatly increased in the last decade.
Among these alternatives, natural pesticides, particularly plant derived
chemicals, have received considerable attention.
An example of a plant derived pesticide is azadirachtin (AZA). a
limonoid, which is a component of the Neem tree, Azadirachta indica A. Juss.
In 1968, Butterworth and Morgan observed that desert locusts were not able to
eat the leaves of the neem tree which led to the isolation and identification of
AZA as the repelling agent. Since then. AZA has been shown to have repellent,
antifeedant, molt regulating, and insecticidal activity against a large number of
insect species and some mites (Schmutterer et a i, 1981; Jacobson, 1989). AZA
was also found to have nematicidal, fungicidal, bactericidal, anti-inflammatory,
antitumor, and immunostimulating activities (Ara et a i, 1989; Van Der Nat et
a i. 1991; Randhawa and Parmar 1993; Williams et a i, 1998).
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The first commercial product of neem, Margosan-0® (W.R. Grace &
Company, Columbia, MD) was registered by the U.S. Environmental Protection
Agency for nonfood crop insect pest control in 1985 (Stark et a i, 1992).
Several commercial and semi-commercial preparations are now available
including Azatin-EC^*^ (Agridyne Tech., Salt Lake, UT), Bioneem^'^ (Ringer
Corp., Minneapolis, MN), and Neemix™ (Thermo Trilogy, Columbia, MD).
AZA and AZA-based pesticides have been widely used on many insect
species. Insects are classified in the phylum of Arthopoda. Arthopods are
characterized by the presence of an exoskeleton which must be shed in order to
grow (Lachaise et a i. 1993). The animal will undergo discontinuous growth
through molting. Molting is regulated by the hormone ecdysone (Brown and
Cunningham, 1939). Many studies showed that 20-hydroxyecdysone is the
physiologically active form of the insect molting hormone (Borst and
Engelmann, 1974). The insecticidal performance of neem products has been
assessed in terms of both antifeedancy and insect growth regulatory effects, but
the predominant effect in a species often varies with dose. There are numerous
example o f laboratory and greenhouse studies o f the pest control potential of
AZA (Luntz and Blackwell, 1993). Jilani et al. (1988) reported that the
application of Margosan-O® (containing 0.3% AZA) on filter paper strips at
200, 400, or 800 pg Margosan-0®/cm^ reduced adult feeding o f the red
flourbeetle. In another study, 3 mL of Margosan-O® in 1 liter o f sugar syrup
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significantly reduced the population of adult citrus red mites and greatly
reduced the number of honey-bee tracheal mite eggs (Liu, 1995).
Like insects, crustaceans are classified as Arthopoda. There is little
information regarding the toxic effects of AZA and neem-based pesticides on
aquatic crustaceans. Most of data are from the standard toxicity tests on water
fleas and fish that are part o f toxicological screening process for the commercial
development o f pesticides (Kreuzweiser, 1997). Schmutterer (1995) reported
that Daphnia magna was susceptible to various neem formulations at
concentrations from 10 to 100 mg/L. Zebitz (1987) studied the toxicity o f the
commercial neem-based insecticide Margosan-O® on rainbow trout and
bluegills and found 96-h LC50 values of 8.8 mg/L for rainbow trout and 37
mg/L for bluegills.
When compared with other commonly used insecticides, AZA has
relatively low LC50 values. Examples of commonly used insecticides are
acephate (an organophosphate), dimilin (a diflubenzuron), and pyrethrins
(natural insecticide). The 96 h LC50 for rainbow trout exposed to acephate is
>1,000 mg/L, 2,050 mg/L for bluegill, 1,725 mg/L for largemouth bass, 2,230
mg/L for channel catfish, and 9,550 mg/L for goldfish (Worthing, 1987).
Dimilin® has LC50 values of >0.2 mg/L, 135 mg/L, 7.1 ng/L, and 500 mg/L
tested on rainbow trout, bluegill sunfish, Daphnia magna, fathead minnow,
respectively (Dost et a i, 1985). These LC50 values are much higher than the
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LC50 values of AZA. Therefore, the relatively low LC50 values o f AZA
suggest that toxicity studies along with chemical analysis are necessary to
understand the impact o f the neem-based pesticides (Neemix™ and Bioneem™)
on aquatic species in areas where agricultural runoff waters may carry
significant amounts o f AZA to the environment.
The objectives o f this study were to 1) test the mutagenecity of
Neemix™ and Bioneem^''^ in order to determine the potential contamination
with aflato.xin; 2) conduct in vivo acute toxicity tests on selected species of
mollusks (freshwater snails and oyster eggs) and crustaceans (crayfish, grass
shrimp, white shrimp, blue crab, and water fleas) to provide information about
the direct toxic effects o f these insecticides, find the most sensitive species to
these insecticides and compare the results of acute toxicity tests to an insect
species [Culex quinquefasciatus, mosquito larvae); 3) develop cell culture
bioassays (mammalian and oyster cells) and compare the results to whole
animal bioassays results; 4) measure the sensitivity of Neemix™ and
Bioneem™ to air, heat and light using an animal bioassay; and 5) confirm the
identity of pure AZA used in our toxicological tests by mass (MS) and nuclear
magnetic resonance (NMR) spectroscopy.
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LITERATURE REVIEW
The neem tree, Azadirachta indica. is native to southern Asia,
subtropical and tropical Africa (Schumutterer, 1995). The neem tree is a
member of the Melicaceae family, a family characterized by the occurrence of
bitter triterpenoids, such as the limonoids (Schumutterer, 1995). The neem tree
produces a wide range of complex terpenoids, o f which the best known is AZA.
one of "the most powerful plant derived insecticides” (Jacobson. 1989). This is
because it is toxic towards insects and other invertebrates. Over a dozen
analogues of AZA were identified, but only AZA-A (Figure I) and AZA-B (3-
tigloylazadirachtol) have received significant importance. These compounds
typically occur in a ratio of 3:1. the remaining analogues rarely constitute more
than 5% of the total AZA content of seed extracts. It is interesting that AZA-A
shows more activity in the desert locust and tobacco worm than AZA-B,
whereas AZA-B is more active compound against Mexican bean beetle and
tobacco cutworm (Isman, 1997). Although other limonoids, such as salannins
and nimbins, are claimed to make an important contribution to the bioactivity of
neem extracts, data from several insects show a strong correlation between
bioactivity and total AZA content (Kraus, 1995). In these cases, AZA accounts
for 75-90% of the variation in bioactivity between samples of neem, whereas
salannins and nimbins can be effective antifeedants against certain insects
(Isman, 1997).
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OH
AcODH
Figure 1. Chemical structure of azadirachtin type A (AZA-A, determined by Kraus et al., 1987).
The other known components of the neem tree (Figures 2a and 2b) are
nimbln. salannin. nimbolide. azadiron. vilasinin. and azadirachtol (Kraus et al.,
1993). The toxicity of azadiron was studied on the instar larvae of
Epilachna varivestis (Mexican bean beetle) and the EC50 (effective
concentration where 50% of the tested animals died) values was found to be
5.500 ppm (Schwinger et al., 1984). Nimbin was the first isolated constituent
from the neem tree. Insect antifeedant activity of nimbin was observed on E.
varivestis. The EC50 value was found to be 50 ppm for this species (Kraus et
al., 1993). Compounds like nimbolide showed antifeedant activity’, cytotoxicity,
and growth inhibition effects when they were tested on Popillia japonica larvae
(Japanese beetle) and Plasmodium falciparum (Protozoan) with EC50 values
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of 1000 ppm and 0.95 ppm, respectively (Rochanakij et a i, 1985). Salannin
structure is characterized by two oxygen bridges C-26/28 and C-7/14. Its
activity was tested on several insect species among which Spodoptera
frugiperda larvae (fall army worm) was found to be the most sensitive insect
species among the others with an EC50 of 13 ppm (Rajab et a/., 1988). The
group known as vilasinin is the parent compound o f limonoids which are
widespread in Melicaceae. Vilasinin showed antifeedant activity on the 4 h
instar of E. varivestis and its EC50 value was 10 ppm (Kraus et al., 1993). In
1984. azadirachtol was reported by Kubo and coworkers as a new neem
constituent. Its structure is very similar to AZA except azadirachtol does not
contain two hydroxyl groups which exist in AZA’s structure (Figure 2a).
Azadirachtol inhibits growth of E. varivestis larvae at a very low concentration
(EC50 = 0.08 ppm) and feeding o ï Spodoptera littoralis (cotton leaf worm) at 1
ppm (Rembold. 1989; Ley e/a/., 1993).
Chemistry of Azadirachtin
AZA is a yellow green powder with a strong garlic-sulfur odor (Bilton et
ai, 1987). The structure of AZA was established by Kraus and coworkers
(1987) on the basis o f and NMR analyses. They isolated AZA by
extraction of neem seeds with acetone followed by petroleum ether and
methanol solutions and performed and NMR analyses at 250 MHz and
62.89 MHz, respectively. The results o f their investigation of the NMR spectra
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PH
HO Azadirachtol
OAc
Nimbin
SalanninAcO
'-0
Figure 2a. Other bioactive compounds in neem.
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OAcA z a d i r o n e
HO
HO OH■=— O
V i l a s i n i n
t -
N i m b o l i d e
Figure 2b. Other bioactive compounds in neem.
9
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are listed in table I. The chemical structure of AZA was found to be
C35H44O 16 AZA is a 16 oxygen atom containing molecule that includes
epoxide rings at positions 13 and 14, a terminal dihydrofuran ring, a tigloyl side
chain at position 1. three free hydroxyl groups at positions 7,11, and 20, and
possession of 16 chiral carbon centers (Kraus et ai, 1987). AZA’s molecular
weight is approximately 720.7 daltons (Kraus et a i, 1987).
Mode of Action of Azadirachtin in Insects
AZA shows three specific modes of action in insects (Luntz and
Blackwell, 1993). First, it has strong antifeedant activity due to its effects on
chemoreceptors. Second, it affects ecdysteroid and juvenile hormone titers
through a blockage of morphogenetic peptide hormone release, resulting in a
severe growth inhibition and molting aberrations. Third, it has direct detrimental
and histopathological effects on most insect tissues, e.g.. muscles, body fat. and
gut epithelial cells (Wilps etal., 1992; Rembold, 1995).
I) Antifeedant Effects: Crude, refined neem extracts, neem enriched extract,
and pure AZA have been applied to asses AZA’s antifeedant activity. These
formulations have been applied to control more than 200 species of insects.
Lepidoptera are the species which are extremely sensitive to AZA and show
antifeedant activities from <1-50 ppm, depending on the species (Meisner et ai,
1981; Blaney et a i, 1990; Isman et a i, 1990; Thomas et al., 1992). Coleoptera,
Hemiptera, and Homoptera have been found to be less sensitive to AZA with up
10
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Table 1. Nuclear Magnetic Resonance (NMR) Spectra of Azadirachtin at 250 MHz (Kraus et a i , 1987)
Position Chemical Shift (5, ppm)1-H 4.75 (dd, 2.9; 3.1)2 -H a 2.34 (ddd, 16.7; 2.9; 2.7)2 - H p 2.13 (ddd. 16.7; 3.1; 2.9)3 -H 5.50 (dd, 2.7; 2.9)5 -H 3.35 (d, 12.5)6 -H 4.60 (dd, 12.5; 2.7)7 -H 4.75 (d, 2.7)9 - H 3.34 (s)
15- H 4.67 (d, 3.4)16- H a 1.73 (ddd. 13.0; 3.4; 5.1)16-Hp 1.31 (d, 9.6)17- H 2.38 (d, 5.1)18- H 2.01 (s)19- Ha 3.63 (d, 9.6)19-Hb 4.15 (d, 9.6)21 -H 5.65 (s)2 2 - H 5.05 (d, 2.9)2 3 - H 6.46 (d. 2.9)28 - Ha, p 4.08 (d, 9.0), 3.76 (d, 9.0)2 9 - H -
3 0 - H 1.74 (s)1 - OH -
3 - OH -
7 - OH 2.89 (br, s)11 - OH 5.05 (s)14- OH -
2 0 - OH 2.92 (br, s)11 - OCH3 -
12 - OCH3 3.68 (s)2 3 -O C H 3 -
29 - OCH3 3.76 (s)CH3COO 1.95 (s)3’ - H 6.93 (qq, 7.0; 1.5)4’ - H 1.78 (dq, 7.0; 1.1)5 -H 1.85 (dq, 1.5; 1.1)
11
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to 100% antifeedant activity at 100-600 ppm, whereas Orthoptera is the most
sensitive species to AZA at 0.05 ppm (Kraus et ai, 1987; Nisbet et a i, 1992).
Isman (1993) tested the antifeedant activity o f 95% pure AZA on six species of
noctuid larvae, Actebia fennica (black army cutworm), Mamestra configurate
(Bertha army worm), Peridroma sauica (Variegated cutworm), Trichoplusia ni
(cabbage looper), Melanchra picta (zebra caterpillar), and S. litiira (Asian army
worm) at concentrations from 0.05 to 0.4 mg/kg. EC50 values following 10
days of feeding ranged from 0.12 to 0.24 mg/kg without significant differences
between species. Topical treatment of 4 ^ instar larvae of each species with
50 or 100 ng of AZA resulted in significant inhibition of subsequent growth,
diet consumption, and dietary utilization. S. litura was found to be the most
sensitive to the antifeedant effects of AZA with a EC50 value of 1.25 ng/cm-.
whereas A. fennica was the least sensitive to AZA with a EC50 value of 40.7
ng/cm-. These results suggest that the efficacy of AZA depends on the species
and dose.
Inhibition of feeding behavior results from either blockage of the input
from receptors which normally respond to phagostimulants, or from stimulation
of specific deterrent (Deither, 1982). Neurophysiological responses from the
medial and lateral taste sensilla of the maxillae, using different concentrations
of sucrose and AZA either singly or together, revealed in most cases a different
population of receptors responsive either to sucrose (sugar cell) or AZA
12
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(deterrent cell) (Waladde et ai, 1989). When these sensilla were stimulated
with a solution containing sucrose and AZA, the response from the respective
sucrose and AZA neurons was lower than when the sensilla were stimulated
with solutions containing only sucrose or AZA at the same concentration. This
phenomenon has been called interaction, and the magnitude of this effect varies
between species (Mordue et al., 1998). The consequences of this phenomenon is
that the conflict of information between the phagostimulant and the deterrent is
at least partly resolved at a peripheral neural level and it is the outcome of the
discord that is reported to the central nervous system (Simmonds and Blaney.
1984).
2) Endocrine Effects: The insect brain produces a protein, prothoracotropic
hormone (PTTH). which stimulates prothoracic glands to initiate synthesis of a
major class of insect endocrine hormones, ecdysteroids and juvenile hormones
(Rembold, 1995). The relative titers of ecdysteroids and juvenile hormones
during the larval or pupal phases are responsible for the molting process.
Although ecdysone (Figure 3) can be found throughout the insect, biochemical
studies have shown an abundance in gut tissues and in developing cuticular
discs, where it is converted to the more active isomer, 20-P-hydroxyecdysone
(Simmonds and Blaney, 1984). Insects control their internal ecdysteroid titers
by diet changes, or binding of ecdysteroid hormone to highly sensitive and
specific carrier and receptor (Rembold 1995).
13
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OH
OH Î
OH
HO
OH
HO
Figure 3. Chemical structure of ecdysone (adapted from Lachaise et al., 1993).
Both AZA and ecdysone analogs lose activity when they are extensively
modified or are hindered at the 20-hydroxy position (Rembold et al., 1987).
Hydroxyl substitution at C-17 greatly reduces insect molt-inhibiting activity
(Figure 4). The double bond at positions C-22-23 of the dihydrofiiran ring does
not appear to contribute to biological activity. However, stability is greatly
improved when the double bond at positions 22-23 has been saturated (Bamy et
ai, 1989). It has been found that AZA may adhere to insect proteins through
hydrogen bonding similar to that formed by 20-p-hydroxyecdysone and that 20-
hydroxy and 3-hydroxy positions o f ecdysone are critical for biological activity
(Schumutterer, 1995). The C-3 and C-20 distance in both AZA and ecdysone is
about 11Â and oxygen atoms at positions C-3, C-6 , and C-20 have a distance of
14
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0.15 Â. Such similar positioning suggests that AZA and ecdysone may bind to
one or more receptor sites in insects (Schumutterer, 1995).
R.O
'OR
R( = H, Tiglate, 2-methylbutyrate. cinnamateRt = H, Acetate. Tiglate. 2-methylbutyrateR 3 = Me. CO^MeR4 = H. MeR 5 = H. CO.M eR(, = H. OH. GAc. OMeR 7 = H. MeX. Y = CH=CH. CH2 -CH2 . CHi-CHRg, CH-alpha-Br-CHRg Rg = Alpha or beta-0 Me. OEt. 0-/-Pr. OAc
Figure 4. Azadirachtin analogs with molt inhibitory activity (adapted from Barny et al., 1989).
AZA causes a decrease of insect weight gain, disrupts and delays molting
regulated by ecdysone. and often causes death at both larval and pupal stages
(Mordue et al., 1985). It also disrupts the proper shedding of old body capsules
during the molting process. Ingestion by feeding or hemolymph injections of
AZA reduces hemolymph ecdysteroid titers and delays the appearance of
ecdysteroid peaks in many insect species (Rembold et al., 1988). This is
thought to occur because PTTH production by the brain is inhibited (Figure 5).
15
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Ecdysone 20-monc-oxygenase from the midgut and body fat is the insect
cytochrome P-450 dependent hydroxylase responsible for the conversion of
ecdysone to its more active metabolite, 20-hydroxyecdysone. Formation of 20-
hydroxyecdysone by 20-mono-oxygenase is affected within 1 h of exposure to
AZA (Smith and Mitchell, 1988). Cytochrome P-450 levels are also
significantly decreased in midguts of 5 ^ instar of an Orthopteran species,
Schistocerca gregaria'whta X.0 25 pg AZA (Bidmon er a/., 1987).
Biological activity of AZA and three of its derivatives was tested on the
5^ instar o f v / > * e 5ce«5, tobacco budworm (Bamby er a/., 1989). At a
dose of 0.5 pg/g of insect tissue. AZA. 22. 23- dihydroazadirachtin, and 2’, 3 \
22. 23-tetrahydroazadirachtin extended the pupation time. At higher doses (2
and 4 pg/g of insect tissue), all four compounds including 3-deactylazadirachtin
caused 100% larval death. The effects of AZA upon juvenile hormone levels
are not easy to define due to close interrelationship between Juvenile hormone
and ecdysone levels and neurosecretions in the molting process.
Malczewska et al. (1988) used chilled Galleria mellonella (greater wax
moth) larvae to investigate the effects of AZA on Juvenile hormone and
ecdysone titers; chilled larvae undergo numerous molts, due to increase in
Juvenile hormone titer. AZA inhibits such molts of last instar larvae by-
blocking the synthesis and release of Juvenile hormone. This block causes a
rapid decrease in whole body Juvenile hormone titers which is maintained for
16
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several days. This result clearly supports the argument that the AZA depresses
the synthesis of neurohormones from the brain as well as their release of
prothoracotrophic hormone, PTTH (Rembold, 1995).
Ecdysteroid depletion by AZA was tested in Tenebrio molitor (dark
lean beetle) pupae (Marco et a l, 1990). Freshly ecdysed pupae of T. molitor
were injected with 1 pL o f the 1 mg/mL solution of AZA/pupae and were
observed for a period of 8 days. The maximum ecdysteroid level was found on
day 4 (4083 ng/mL) in the hemolymph of untreated T. molitor, whereas values
for immunoreactive ecdysteroids present in hemolymph of 1 fiL o f the 1 mg/mL
solution of AZA/pupae treated T. molitor averaged 1762 ng/mL. ITtis result
suggests that ecdysoid depletion has occurred as a result of AZA treatment.
The effects of Neemix™ (4.5% AZA) on Coccinella spetempunctata
(lady beetles) larvae were determined after direct spray exposure by Banken and
Stark (1997). C. sptempiinctata l^t instar larvae were treated by direct
application with 40, 100, 200, 400. 600, and 1000 ppm AZA and 4^ instar
larvae of C. sptempmctata were treated with 400, 600, 800, and 1000 ppm
AZA. Neemix’ *'* was more toxic to instar than to l^t instar at LC50 value
of 520 ppm AZA. Response to the pesticide was dose, stage, and age
dependent. Symptoms included an extended larval stage, loss o f appetite,
lethargy, inability to complete pupal ecdysis, and deformation of wings.
17
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CD■DOQ .C
gQ .
■DCD
C/)C/)
8■D
CD
3.3"CD
CD"OOQ .
o oo3"OO
CDQ .
■DCD
C/)C/)
PTl H from brain secretory cells
iPrStlStimulates Prothoracic Glands
ECD precursors ---------► a-ECDTransport to
Specific tissuesA-""" """A
Enzyme modulation Protein synthesis modulation \C hitin synthetase E C D -20-M on ooxygen ase
1. Chitin precursor ^ Chitin a-ECD ^ 20-p-OH-ECD
2. Effect of AZA on other / Protein bindingenzymes modified by ►ECD not yet studied ECD receptor protein
PTTH - Prothoracotropic hormone DNA bindingECD = Ecdysone /AZA = Azadirachtin ECD protein-DNA complex /
Not AZA blocked I /AZA blocked Protein production method ‘
> Not yet reported
Figure 5. Efiects of Azadirachtin on ecdysone-mediated pathways (adapted from Hansen et ai., 1994)
Disruption of ecdysone hormone level appeared to be more critical
before metamorphosis than the early instars when insects treated with
Neemix™.
3) Physiological Effects: AZA has subtle effects on a variety of tissues which
form part of the overall toxic syndrome of poisoning and which may provide
clues as to its cellular mode of action. For example, adult locusts treated with
AZA become sluggish and show reduced locomotory and flight activity (Wilps
et al., 1992). Such a reduced tendency to fly results in a significantly reduced
elevation of blood lipids after flight activity compared with untreated locusts
(Wilps et al., 1992). The body fat is also known to be affected by AZA with
necrosis of scattered cells occurring after treatment. Insect muscle has been
shown to be affected by AZA (Luntz and Blackwell, 1993). Histological studies
of midgut muscle o f S. gregaria (Army worm) show that the muscles become
swollen and disrupted in a dose and time dependent manner after AZA
treatment (Luntz and Blackwell, 1993).
The effects o f AZA on the reproduction of insects have been known
since 1975, when it was reported for the first time that the number of eggs of
Coleoptera decreased after the intake o f active principles from neem seed
kernels and AZA (Rembold, 1995). Subrahmanyam and Rembold (1989)
treated the locusts with dihydroazadirachtin to find the target effects o f AZA on
the neurosecretory system. The brain and corpus cardium (A tunnel which
19
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carries PTTH to the organs) turned out to be the most important target for AZA
in neurosecretary system. The effects of AZA and a commercial AZA-based
pesticide (Margosan-0®) were studied on oviposition o f the diamondback moth
(Plutella xylostella) by Qiu and coworkers (1998). Margosan-0® (containing
0.3% AZA) significantly inhibited oviposition at the doses tested. AZA was
inactive as a pure compound at the concentration equivalent to that present in
5% Margosan-0®.
Toxicity of Azadirachtin to Non-target Organisms
AZA is relatively harmless to spiders, butterflies, and insects such as
bees that pollinate crops and trees, ladybugs that consume aphids, and wasps
that act as parasites on various crop pests (Spollen and Isman, 1996). Neem-
seed extract was not completely harmless to bees, but serious damage to them
in the field appears if spraying is carried out prior to flowering (Schmutterer and
Holst, 1987). Adverse effects of AZA against an hymenopteran parasitoid of
tobacco worm were studied by Beckage et al. (1988). The suppression of
ecdysis and 100% mortality of the parasitoid were observed with the injection
of 10 pg AZA into the larvae.
The 96-h LC50 o f Margosan-0® in water using Juvenile rainbow trout
was found to be 8.8 mg/L (Larson, 1989). In laboratory trials conducted by
Zebitz (1987), guppies tolerated up to 100 ppm of neem seed extracts. Wan et
al. (1996) tested AZA, neem extracts, and formulated material against juvenile
20
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Pacific Northwest salmon and foimd the highest LC50 to be 4 mg/L from the
neem seed extract. Various neem products, such as aqueous neem seed kernel
(5-10%). neem oil (3%) were tested in semi-field trials against tilapia by
Fernandez et al. (1992). For both concentrations of neem seed kernel extracts,
20% mortality was recorded on the 17 h jay after the first application. In the
3% neem oil treatment, 20% mortality occurred. Acute lethal effects o f two
neem-based formulations (Azatin^^, containing 3% AZA, and an experimental
formulation, containing 1.75% AZA) on three species of caddisfly, two species
of mayfly, two species of stonefly, one species of cranefly. and one species of
amphipoda in flow through screening tests were studied at different
concentrations (Kreutzweiser, 1997). The only species that showed significant
mortality was one of the mayfly species with a LC50 of 1.12 mg/L Azatin™.
The experimental formulation along with Azatin^’' did not show toxicity to any
of remaining species tested. Sadagopan et al. (1981) conducted two feeding
trials to study the toxic effect o f neem seed meal on the performance o f chicks
and its effect on their internal organs throughout the 14 day experimental
period. Inclusion of neem seed meal in the diet at a low concentration (2.5%)
lowered the chicks performance, with mild to severe changes in the kidneys,
liver, spleen, intestine, and hearts. Daily oral administration of Margosan-0®
to mallard ducks at dose levels o f 1-16 mg/kg, induced no negative effects over
21
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a 14-day test period. Bobwhite quail fed a daily basic diet with an added 1,000-
7,000 ppm of Margosan-0®, showed no negative effects over a 5-day test
period (Larson, 1989).
Rats dosed once with Margosan-0® and observed for 14 days showed
no obvious negative effects. The acute oral toxicity to rats was greater than 5
mg/kg (Larson, 1989). In an inhalation tests, albino rats exposed to 15.8 g of
Margosan-0® (estimated concentration of 43.9 mg/L/h) for 4 h showed an
LC50 greater than 43.9 mg/kg (Larson, 1989). Mahboob et al. (1998) studied
the acute and subacute toxicity of a neem based pesticide (Vepacide™ with
12% AZA concentration) in male Wistar rats orally for 7 and 90 days,
respectively. The LD50 value was determined to be 1566.85 mg/kg indicating
that Vepacide^’ is moderately toxic to rat by the oral route. Subacute toxicity
results gave three different doses to rats by the oral route during 90 days
exposure. In high dose (320 mg/kg), 10% of rats died after 90 days and there
was a significant decrease in concentration of Cytochrome-P-450 in liver and
lung tissues. The rats treated with medium dose (160 mg/kg) showed toxic signs
which were less severe than the high dose. Rats treated with low dose (80
mg/kg) did not exhibit any toxic signs. Nimbolide, a limonoid in neem seed
extracts, was cytotoxic in a bioassay against murine neuroblastoma with IC50
values o f 20-200 fig extract/mL culture medium and human osteosarcoma cells
with IC50 values o f 10-20 pg extract/mL culture medium (Cohen et a i, 1996).
22
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The cells were exposed to nimbolide at concentrations from 0.1 to 500
pg extract/mL culture medium for 24 h. Sharma and Saksena (1959) published
the first report of possible spermicidal activity by neem components in humans.
The lethal concentration of neem-based products for human sperm was 1,000
mg/kg (Devakumar et a i, 1990). However, the AZA is not the active compound
in these products. The spermicidal activity of a volatile component of neem oil
coded as NlM-76 was studied in vitro using human semen at concentrations of
5, 10, 15, 20. 25, and 30 mg NIM-76/mL of semen (Riar et a i, 1990). A
concentration of 25 mg/mL of NIM-76 was required to achieve total
spermicidal effect in 20 seconds. NIM-76 was also investigated for its
anti fertility activity in vivo in rats, rhesus monkeys, and rabbits at
concentrations between 1 and 100 mg NIM-76 (Riar et a i , 1991). Intrauterine
administration of 1 mg of NIM-76 in rats resulted in 100% inhibition of
implantation in the injected rat. When applied intravaginally on 1 to 6 days of
implantation, NIM-76 caused 50% inhibition of the number of implants in
rabbits at concentration of 7.5 mg. At concentrations 10 and 20 mg, there was
100% inhibition in the number o f implants. When 100 mg of NIM-76 was
applied on days 7-10 of the menstrual cycle, and the monkeys mated during the
ovulation days, none of the animals conceived.
Immunomodulatory effects o f neem oil emulsion were studied in mice
at concentration of 150 pL by the intraperitoneal route (Upadhyay et a i, 1992).
23
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Spleen cells were removed after 24 h, cultured in vitro for another 24 h, then
the supernatants were tested for the presence of gamma interferon (y-IFN). The
production of y-IFN was induced by neem oil treatment. Spleen cells o f neem
oil treated animals also showed a significantly higher lymphocyte proliferative
response. In another study, effects of aqueous extracts of neem leaves were
evaluated on biochemical, immunological, and visceral parameters in normal
and stressed rats (Sen et a i, 1992). Aqueous extract at a concentration of 100
mg/kg lowered blood glucose, triglyceride, and SCOT levels in normal rats, and
attenuated stress-induced elevations of cholesterol and urea levels. In stressed
rats, aqueous extract significantly attenuated the stress-induced suppression of
humoral immune response and gastric ulcerogenesis. The immunomodulatory
effects of NIM-76 was investigated by SaiRam et a i (1997) on Sprague-Dawley
albino rats. The rats received 120 and 300 mg/kg body weight NIM-76 once.
Five days after NIM-76 treatment, the blood was collected and animals were
killed to collect the peritoneal fluid. At 120 mg/kg body weight, there was an
enhanced macrophage activity and lymphocyte proliferation response. While at
300 mg/kg body weight, there was a stimulation of mitogen-induced
lymphocyte proliferation, but no macrophage activity was found. This study
indicates that NIM-76 acts through cell-mediated mechanisms by activating
macrophages and lymphocytes.
24
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■DOQ .C
gQ .
■DCD
(/)Wo'30CD
8■DV<ë '
13CD
p.3"CD
CD■DOQ .C
aO3■DO
CDQ .
Table 2. EfTectiveness of neem products against non target organisms
N>LA
Organism Pesticide Administration LC5Û or LD50 Effect Reference
Rainbow trout Margosan-O® In water 8.8 mg/L Toxic Larson, 1989
Guppies Neem-seed extract In water 100 mg/L Toxic Zebitz, 1987
Pacific Salmon Neem-seed extract In water 4 mg/L Toxic Wan et a/., 1996
Mayfly Azatin’"* In water 1.12 mg/L Toxic Kreutzweiser, 1997
Rats Margosan-O® Oral >5 mg/kg Toxic Larson, 1989
Rats Margosan-O® Inhalation >43.9 mg/kg Toxic Larson, 1989
Wister rats Vepacide™ Oral 1566 mg/kg Toxic Mahboob et al., 1998
Daphnia magna Margosan-O® In water 13 mg/L Toxic Larson, 1989
■DCD
(/)(/)
Environmental Fate of Azadirachtin
There is no available literature on the stability of AZA regarding its
activit)'. However, the stability and the half life of AZA were determined by
several authors. The stability of AZA when exposed to light in field and
laboratory conditions has been explored by Sundaram and Curry (1996). They
showed that AZA is susceptible to photodegradation and hydrolysis on the
surfaces and in water. Analytical grade AZA and the two commercial neem
insecticides (Margosan-O® and Azatin-EC^*'^) were exposed to light at one-
quarter the intensity of sunlight on glass plates (Sundaram and Curry, 1996).
Standard solutions (2.5 mg AZA/mL) of pure AZA and the commercial
insecticides were prepared with the three UV absorbers at ratios of 1:0 (no UV
absorber) and 1:2 (AZA:UV absorber, w/w) in methanol. Then, 100 pL (250 pg
AZA) of each solution was applied onto the surface of a 75x25 mm glass slide.
Slides were kept in an environmental chamber at 20°C and 80% RH. The
chamber was illuminated with 4000 W metal halide lamps and a high pressure
sodium lamp providing 0.0142 w/cm- of irradiant energy on the surface of
slides. Three slides per sampling interval were collected at 0, I, 2, 3, 4, and 8
day. The stability of AZA was analyzed by a HPLC based on the concentration
of AZA. Pure AZA had a half life of 3.87 days, whereas AZA in the
insecticides had a half life o f 5 days. Hull et al. (1993) reported that
methanolic solution of AZA (l.O mg AZA/mL) stored at -20®C was stable for at
26
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least six months. Stability of methanolic solution o f AZA was also determined
by a HPLC analysis based on the concentration of AZA. Early isolation and
structure determinations indicated that AZA was very sensitive to acid and
alkali conditions (Sundaram et a i, 1995; Szeto and Wan, 1996). AZA’s
stability in natural and buffered waters has been studied for its survival after
agricultural and forestry uses (Sundaram et a i, 1995). The half-life of AZA
between pH 4 and 7 was 19 and 12.9 days at 20°C, respectively. In a later
study by Szeto and Wan (1996), it was found that stability of AZA fell rapidly
at higher temperatures. At pH 4 to 6 , the half life was 11.6 days to 8.6 days at
35°C, while at pH 7 the half life was only 20.5 hours at 45°C. By extrapolation
of these results, the half life of AZA was found to be 24 days at pH 7 and 20°C.
Jarvis et a i (1998) studied the stability of AZA in aqueous and organic
solvents. The half lives of AZA at pH 2 to pH II at room temperature ranged
from 15 days to 6 minutes. They concluded that AZA is more stable in acidic
solutions than the alkaline solutions. AZA solutions in some organic solvents
are stable at room temperature (Jarvis et ai, 1998). The stability of AZA in
normal soil is 20 days at 25°C, but is 32 days in autoclaved soil, indicating that
microorganisms are involved in its degradation (Stark and Walter, 1995).
3-DesacetyIAZA was obtained by saponification of AZA with methanol
under basic conditions (Rembold, 1989). 3-Desacet>'lAZA has growth
inhibition action on E. varivestis and H. virescens with EC50 o f 0.38 ppm and
27
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0.09 ppm, respectively (Rembold, 1989). Another breakdown product of AZA
is 1-DetigloylAZA (AZA E), a hydroylsis product of AZA (Kraus et a i, 1991).
AZA E has also growth inhibition action on E. varivestis (Kraus et a i, 1991).
Hydrogenation of AZA yielded the product named 22,23-dihydroAZA which is
an efficient inhibitor of metamorphosis in E. varivestis and Locusta migratoria
(Rembold, 1989). Ley et al. (1988) studied the oxidation products of AZA
with pyridinium dichromate (PDC). The resulting derivatives included 22,23-
dihydro-11-methoxy AZA and 22.23-dihydro-11,20-dimethoxyAZA.
There have been many investigations on the biological activities o f AZA
and its derivatives. These investigations are difficult to compare with each
other because of different experimental conditions, such as test organisms, test
arrangements, test type, and other test conditions as well as purity and
application of test materials. There are certain species which are highly
sensitive to AZA itself, but in certain cases AZA derivatives are more active.
The EC50 value of AZA on E. varivestis is 13 ppm, while 22,23-dihydro-11-
methoxyAZA has a EC50 value of 0.5 ppm on the same species (Kraus et a i,
1987; Ley etai , 1988).
Biology of Selected Species
1) Mollusca: a) Freshwater snails {Physella virgata): Freshwater snails are
pulmonate snails that either rely on surface breathing or have a limited capacity
for oxygen transfer across their epithelial tissues (McMohan, 1983). They live
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in a wide range of temperature, 0 to 40°C, at a pH of 6 .8-8, and hardness of
below 3 mg/L CaCOg. They have a worldwide distribution and are ubiquitous
in North America (Brown, 1991). Their shells are small, sinistral, their tentacles
and feet are slender. They have fingerlike mantle extensions and lay soft,
crescent-shapes eggs masses. Freshwater snails are oviparous hermaphrodites
(Brown, 1983). Around spring, sperm and eggs are produced in the ovotestis
and exit via the hermaphroditic duct. Eggs are fertilized in the hermaphroditic
duct by the same individual's sperm or by sperm from another individual. Eggs
are laid in gelatinous egg cases and attached to plants or rocks (Duncan, 1975).
They reach maturity after the larval stage.
Freshwater snails are sensitive to pollutants, such as acid rain, heavy
metals, pesticides, and oil pollution. They have therefore been used as a
bioindicator species in metal, pesticide, and oil toxicity tests under laboratory
conditions (Brown, 1991).
b) OystersXCrassostrea virginica): Crassostrea virginica is known as the
American oyster. Crassostrea virginica has a significant importance on the
Louisiana's economy with a total production of 3,239,261 sacks/year (LCES,
1997). Oysters grow subtidally from Maine to the Gulf o f Mexico. Large
interdial beds of oysters are also present on the eastern shore of Virginia and
south o f Cape Fear, North Carolina, to northeast Florida (Haven and Burrell,
1982). The American oyster grows well in a salinity range of 5-30 96o with an
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optimum salinity of 18 %o. They can live in temperatures ranging from 0 to
30°C, but temperatures greater than 19.5°C are required for spawning (Clime
and Hammil, 1979). Spawning occurs from May through October in the Gulf of
Mexico. Larval development takes place externally, and free-swimming stages
last 2-3 weeks (Burrell. 1985). Larvae develop best in salinities between 17.5
and 22.5 %o (Castagna and Chanley, 1973). The first true larval stage, the
trochophore, is motile, being propelled by a ring of beating cilia. The following
stage, the veliger. possesses a strong swimming organ, the velum. With
continued larval development the shell becomes more prominent, and a foot
appears, which helps the larvae to crawl about the substrate in search of an
attachment site. When a suitable hard surface is found, the young oyster
permanently cements itself to it and loses its velum and foot. The process of
attachment is called spatfall, after that the oysters remain in the same spot and
mature (Burrell, 1985).
Oysters are filter feeders, drawing phyto and zooplankton and other
organic particles into the mantle cavity via an inhalant current. Suitable food is
passed into the digestive system through the mouth, and unsuitable material is
rejected by the mouth palps and expelled as pseudofeces (Koringa, 1976).
2) Crustaceans: a) Red Swamp Crayfish (Procambarus clarkii): Crayfish is a
freshwater crustacean. Red swamp crayfish live naturally in northern Mexico,
eastern Florida, and throughout the Mississippi River valley (Lee and Wickins,
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1992). Crayfish are successfully cultured in the United States. In 1997, the
commercial production of farm-raised and wild crayfish was 46.9 million lbs
and 30.2 million lbs, respectively (LCES, 1997). Environmental requirements
for their survival are water temperature of 10-33°C, pH of 6 .5-8.5, dissolved
oxygen greater than 3 ppm. salinity less than 5 %o, total hardness of 100 mg/L
and ammonia less than 1 mg/L (Huner and Barr, 1984). Females lay eggs at
temperatures of 20°C (Avault and Huner, 1985). Fertilized eggs are attached to
the female swimmerets and incubation begins, and lasts for about 7 to 180 days
depending on the environmental conditions (Lee and Wickins, 1992). Once eggs
hatch, the larvae resemble yolk-swollen cephalothoraxes. They undergo 2 molts
in 2 weeks, then they leave the mother and fend for themselves (Avault and
Huner, 1985). A young or juvenile crayfish goes through 11 molts to reach
maturity which takes about 6-12 months depending on the environmental
conditions (Lee and Wickins, 1992). In general, the molting cycle is divided
into five stages: A, soft; B, postmolt; C. intermolt; D, premolt; and E, the molt
itself. Juvenile crayfish complete the cycle in 6-10 days (Huner and Avault,
1977). Crayfish become reluctant to molt when environmental conditions are
unfavorable, the environment is polluted, or they are reproductively active (Lee
and Wickins, 1992).
b) Blue Crab {Callinectes sapidiis): The blue crab supports a large
commercial fishery in Louisiana. In 1997, the total production of blue crab
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reached 39.2 million lbs which had a very significant impact on the state’s
economy (LCES, 1997). Blue crabs live at temperatures of 10-35°C and
salinities of 1-35 %o (Heard, 1982). Female blue crabs mate 1 or 2 times in their
life time. Mating takes place in the lower salinity waters of estuaries (Williams,
1965). Spawning occurs at temperatures of20-25°C and salinities o f 23-28 %o.
The eggs are carried 7-14 days under the mother's abdomen attached to
swimmerets (Oesterling and Provenzano, 1985). The blue crabs go through
different metamorphoses. The zoeae stage, after hatching, includes 4 to 7 stages
and takes 12-70 days. Megalopal stage, which has both benthic and planktonic
features, is only one stage and takes place in 6-20 days. At the end of this
period, the megalopa metamorphoses into the first crab, the crab form is seen at
this stage. The crab reaches maturity in 120-540 days, depending on the
environmental conditions (Sulkin, 1974; Lee and Wickins, 1992). They eat a
large variety of food including snails, oysters, clams, other crabs, detritus, and
decaying animal matter (Heard, 1982).
c) Grass Shrimp {Palaemonetes pugio): Grass shrimp are small
crustaceans, about 5 cm long when mature. They are almost transparent. They
can survive in fresh, brackish, and salt water habitats that have grassy bottoms
(Iversen et a i. 1993). Palaemonetes pugio is a year-round resident in the Gulf
o f Mexico coast estuaries. They can live at salinities of 1 to 30 96o and
temperatures o f 9 -35°C (Heard, 1982). Grass shrimps eat a variety of animal
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and plant matter, including detritus, algae, and dead animal matter. Female
grass shrimps carry fertilized eggs on their pleopods. A planktonic nauplius
stage is absent, and zoea hatch directly from the eggs (Heard, 1982). A female
spawns more than once each year and they may reach adult size in 2-4 months
(Iversen et a i. 1993). Grass shrimps have been used widely in toxicity testing
(metallic and petroleum contaminants) under laboratory conditions (Iversen et
a i, 1993).
d) White Shrimp {Penaeus setifenis): White shrimp are very important
for Louisiana's fishery. Total production in 1997 was 98.2 million lbs with a
value of $207 millions (LCES, 1997). In the U.S., white shrimp are distributed
from New York to Texas coasts. Their optimal environmental survival
conditions are 23-32°C water temperatures, dissolved oxygen above 3 mg/L, pH
near 8.0, and a salinity between 10-30 %o (Lawrence et a i. 1985). Mating and
spawning occurs in offshore waters at temperatures of 24-31 °C with a salinity
of 26-35 %o, but the eggs are released into the open ocean. Eggs usually hatch
within 18-24 h at 28°C, and the larval shrimp goes through five naupliar stages
(2-3 days), three protozoea stages (3-4 days), and three mysis stages (3-5 days)
before reaching the postlarval stage which takes approximately 3-35 days,
depending on the temperature and food availability (Lee and Wickins, 1992).
The postlarval shrimp migrates from offshore waters into estuary systems that
serve as nursery grounds. A juvenile white shrimp reaches maturity in 182-300
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days (Lawrence et a l, 1985). The culture of white shrimp is disturbed in the
presence of pesticides, petroleum, and heavy metals (Iversen et a l, 1993).
Water quality is very critical for their survival especially during larval stages
(Lee and Wickins, 1993).
e) Cladocera {Daphnia pulex): Water fleas are generally considered a
clean water species being dominant in nature during periods of low turbidity
(Pennak, 1989). Daphnia populations are generally sparse in winter and early
spring but population density declines during summer months. Four distinct
periods are recognized in the life of Daphnia: 1) egg, 2) juvenile. 3) adolescent,
and 4) adult (Pennak, 1989). The average life span is 50 days at 20°C. They live
in a temperature range of 18-26°C, pH of 7-8, and a dissolved oxygen level
above 5 mg/L. The time required to reach maturity is 6-10 days. Growth occurs
immediately after each molt (Pennak, 1989). Daphnia pulex feed on algae and
bacteria (Borsheim and Olsen, 1984). They are used in several toxicity tests
because o f their tolerance to pollutants and their availability throughout year.
3) Insecta: Southern House Mosquito {Culex quinquefasciatus Say): Culex
quinque- fasciatus is abundant throughout the southern states in the U.S. The
southern house mosquito feeds on the blood of birds, dogs and rarely on man
(Clements, 1992). Larvae breed most prolifically in water that contains a high
level of organic matter, bacteria, and algae (Meek, 1999). They occur in
roadside, septic ditches, sewage oxidation ponds, and water contaminated with
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wastes from food processing plants (Clements, 1992). Female adult lays 50 -
500 eggs at one time, depositing the eggs on water. The eggs hatch in 1-3 days
depending on the temperature (Meek, 1999). The optimum temperature for
their survival is ~27°C (Meek, 1999). The larvae live in water and pass through
4 instars to reach the adult stage (Clements, 1992). After the larval stage, they
become pupae, then adult in a few days (Meek, 1999). They have been used in
several insecticide toxicity tests because their resistance to insecticides and
pollution (Al-Sharook er a/., 1991: Sharma e /«/.. 1993; Rao et a/., 1995).
Molting Behavior of Crustaceans
The development cycles of growth and molting are regulated by
hormones in both insects and crustaceans. The hormones which stimulate
growth and molting are ecydsones (Watson et al., 1989). As discussed earlier,
the ecdysial glands of insects are the prothoracic glands, whereas in crustaceans,
ecdysteroids are secreted by the Y-organs (Smith et al., 1985). In insects, the
control is positive; the PTTH stimulates the secretion of ecdysteroids. In
crustaceans, the control is negative; the molt inhibiting hormone (MIH, a
peptide manufactured in the eyestalk by the X-organ) suppresses production of
ecdysteroids (Smith et al., 1985). MIH designates the well-established
hormonal activity o f a heat stable peptide released by secretory neurons in
crustaceans eyestalks (Rao, 1965). MIH suppression o f ecdysteroid production
by Y-organs is accompanied by an increase in intracellular cAMP (c-Adenosine
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Mono Phosphate) levels. The effect of cAMP is dose-dependent and precedes
the onset o f ecdysteroid inhibition (Mattson and Spaziani 1985). Therefore, Y-
organs are unique among known steroidogenic glands in that raising the level
of intracellular cAMP depresses hormone production (Cymbrorowski, 1984).
Calcium also plays an important role in regulating ecdysteroidogenesis by
decreasing cAMP levels. Thus calcium has opposite effects on the cAMP levels,
but stimulates steroidogenesis (Mattson and Spaziani, 1986). The rise in
ecdysteroids late in premolt might be due to positive stimulation of Y-organs
by the high calcium levels that result from carapace demineralization (Hopkins,
1986). Another pathway of steroidogenesis stimulation is Protein Kinase C
(PKC) activation. PKC activators seem to stimulate steroidogenesis of Y-organ
via the activation of protein biosynthesis, but without affecting cAMP or
calcium concentration (Mattson and Spaziani, 1987).
The inhibitory mechanism of action of MIH on Y-organs has been
extensively investigated but it still remains controversial concerning the
transduction mechanism involved, and the regulated steps are unknown
(Lachaise et a i, 1993).
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MATERIALS AND METHODS
Chemicals
Neemix^’ (0.025% AZA or 2,500 |ig/mL AZA, active compound, and
99.975% inert ingredients) was provided by Thermo Trilogy Corp., Columbia,
MD. Bioneem^^ (0.009% AZA or 900 pg/mL AZA and 99.991% inert
ingredients. Safer® Inc., Bloomington, MN) was purchased from a local
nursery house in Baton Rouge, LA. Azadirachtin (-95% purity) was purchased
from the Sigma Chemical Co., St Louis, MO. Pure AZA was dissolved in
ethanol (100% purity, Aaper Alcohol and Chemical Co.. Shelbyville, KY) prior
making dilution for desired concentrations.
AZA content in Neemi.x^’' and Bioneem^’' was expressed as weight
per volume (pg/mL) ratio and the concentrations o f Neemi.x^^ and Bioneem™
were calculated using the following formula for AZA equivalence:
V[ x C iC2 =
Vo
Where: Co = Desired concentration of Neemix™ and Bioneem^^ (pg/mL)
VI = Volume of stock solution of Neemix™ and Bioneem™ (mL)
C \ = AZA content of N eem ix^ and Bioneem™ (pg/mL)
V] = Volume of the DI water needed to reach the desired
concentrations of Neemix^''* and Bioneem™ (mL)
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TMTable 3 shows a typical example of AZA equivalence calculation in Neemix
and Bioneem™. For pure AZA, desired concentrations were prepared directly
from the stock solution using appropriate dilutions with DI water.
Table 3. Examples of AZA equivalence in Neemix™ and Bioneem™*
Desired AZA Needed Pesticide Volume (pL/20 mL of water)
Concentration Neemix™ Bioneem™
(pg /mL )
0 0 0
0.625 5 (0.25) 13.9 (0.69)
1.25 10 (0.50) 27.8 (1.38)
2.5 2 0 (1.00) 55.6 (2.76)
5 40 (2.00) 111.2 (5.55)
10 80 (4.00) 222.4(11.10)
20 160(8.00) 444.8 (22.20)
‘ This example is for crayfish experiment Values in parenthesis represent the corresponding concentrations o f Neemix™ and Bioneem™ (pL/mL)
Test Organisms
Eight species of aquatic animals were used for bioassays during the two
year period (March 97-99), including crayfish, water fleas, blue crab, white
shrimp, grass shrimp, freshwater snails, mosquitoes, and oysters. Juvenile
crayfish (-3-4 weeks old) were obtained from Dr. Himer at the University of
Southwestern Louisiana Aquaculture Facility (Lafayette, LA). Blue crab
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megalopes (~2 months old) and juvenile grass shrimp (-1-1,5 months old) were
collected from the Port Fouchon, LA wetland area near the shore using a light
trap. Juvenile white shrimp (-2 months old) were collected from the Grand
Chenier. LA wetland area using a dip net. Freshwater snails (-1 month old)
were collected by hand from a runoff drainage canal in Baton Rouge, LA.
Water fleas (<24 h old) were obtained commercially from the C.K. Associates,
Baton Rouge, LA. Oysters were provided by the Louisiana State University Sea
Grant Oyster Hatchery (Grand Isle, LA). Mosquito eggs were collected with a
dipper from a runoff canal in Denham Springs, LA. Environmental conditions
for each species were adjusted according to their natural environmental
conditions, e.g. temperature, salinity, dissolved oxygen (DO), and pH, with a
photoperiod of 16 h light : 8 h dark (L:D). These species were chosen because
of the following reasons: a) their representation of different orders and classes
of aquatic animals, b) their representation of different functional feeding
groups, c) their importance on the seafood economy of the state of Louisiana,
and d) their varying sensitivities to environmental pollution.
Mutagenicity
The Ames test was conducted using Salmonella typhimuriiim types TA-
98 and TA-100 according to method of Maron and Ames (1983). Salmonella
typhimurium types TA-98 and TA-100 were provided by Dr. Bruce Ames,
Berkley, CA. Mutagenicity of Neemix™ and Bioneem™ were tested at 0,
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0.001, 0.01, 0.1, 1, and 10 |ig AZA/plate (0, 0.0004, 0.004, 0.04, 0.4, and 4 pL
Neemix^’ /plate or 0, 0.0011, 0.011, 0.11, 1.11, and 11.1 uL Bioneem™/plate).
Positive controls (pure Aflatoxin at concentrations o f 1. 10, 100, and 1000
ng/plate) were included in all assays. Dimethyl Sulfoxide (DMSO, Sigma
Chemical Co., St Louis, MO) was used as a negative control. The plates were
incubated at 37°C for 48 h. All experiments were repeated twice and each
experiment was triplicated. The number of revenant colonies for each treatment
was calculated from the means of triplicated plates. Samples that presented
over double the number of natural revenant colonies were considered
mutagenic.
Bioassay Procedures
In vivo Acute Toxicity
Acute, lethal effects of the neem-based pesticides and pure AZA on eight
species were determined in static non-renewal acute toxicity tests during a
period of 96 hours (h) for crayfish, white shrimp, grass shrimp, mosquito, and
freshwater snails, and blue crab, and 48 h for water fleas and oyster eggs (EPA,
1993). Juvenile crayfish, white shrimp, grass shrimp, freshwater snails and blue
crab megalopes were transported in water in an ice box from the place they
were obtained to the Louisiana State University, Department o f Food Science
Building (Baton Rouge, LA). Upon arrival, they were kept in an aquarium and
fed with shrimp pellets for 3 days before the toxicity tests started. Blue crab
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and freshwater snails were fed with ground shrimp pellets. Mosquito eggs were
carried in a small bucket to the LSU Food Science Building and kept in a glass
container in deionized (DI) water until they hatched. The hatched eggs were
then fed with ground dog food until they reached the 3*" instar. Water fleas
were kept in a white plastic container after arriving to the LSU Food Science
Building for 2 h before the toxicity tests started. Live oysters were carried in
ice from Grand Isle to Baton Rouge and left in the cooler over-night.
Crayfish (Procambariis clarkii): Each test concentration included
twenty crayfish and tests were carried out in triplicates. The duration o f acute
toxicity tests was 96 h. During toxicity experiments crayfish were kept in a
separate glass containers to avoid cannibalism. Shortly before starting
exposure, seven different concentrations, such as 0, 0.625, 1.25, 2.5, 5, 10, and
20 jig AZA/mL (0, 0.25, 0.5, 1.0. 2.0, 4.0, and 8.0 p,L Neemix™/mL or 0, 0.69.
1.38, 2.76, 5.55, 11.10. and 22.20 |iL Bioneem^'^^/mL), were prepared in 20 mL
of DI water. The pH, DO, hardness, temperature, and conductivity of DI water
were monitored daily. The pure AZA concentrations were 0.001, 0.01, 0 .1, and
I fig AZA/mL. Animals were placed in glass containers at various pesticide
dilutions. Observations, such as mortality and molting, were made at 0, 24, 48,
72, and 96 h. Test animals were considered dead if they showed no movement
after being agitated for 5 sec. Animals were not fed during exposure. Water
parameters including pH, temperature, and DO were monitored and recorded at
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the time of observations. After 96 h of exposure, live crayfish were placed in
clean DI water to be observed for 30 days to accommodate the delayed molting
and feeding behavior. During the 30 days of observation experiment, crayfish
were fed with shrimp pellets.
Blue Crab (Callinectes sapidus): Salinity and temperature of water
where blue crab were collected were 30 %o and 26°C. Salt water at the same
salinity was prepared using DI water and sea salt (Instant Ocean Salt,
Aquarium Systems, Mentor. OH). The concentrations o f Neemix™ were 0,
0.25. 0.5. 1. 2. and 4 pg AZA/mL (0, 0.1. 0.2. 0.4, 0.8, and 1.6 pL
Neemi.x^^/mL) in 10 mL of DI water. B i o n e e m ^ M and pure AZA were not
used for this experiment. After preparing the solutions, twenty blue crabs per
concentration were placed in separate glass containers to avoid cannibalism.
The pH. temperature, salinity, and DO parameters were monitored and recorded
at 0. 24. 48. 72 and 96 h. Animals were observed daily for mortality and
molting. During 96 h o f exposure, blue crabs were not fed. Shortly after
finishing the experiment, surviving crabs were placed in clean salt water and
separate containers to be observed for delayed molting for one week.
Grass Shrimp (Palaemonetes pugio): The concentrations o f Neemix™
and Bioneem™ used in this experiment were 0, 0.625, 1.25, 2.5, 5, and 10 pg
AZA/mL (0. 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 pL Neemix™/mL or 0, 0.69,
1.38, 2.76, 5.55, 11.II pL Bioneem™/mL). Saltwater was prepared using DI
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water and sea salt (Instant Ocean Sait, Aquarium Systems, Mentor, OH) at 22
96o. Dissolved oxygen, temperature, pH, conductivity, hardness, and alkalinity
of salt water were monitored every day. Pure AZA was not used for this
experiment. Shortly after preparing the dilutions, twenty animals per
concentration were placed in separate glass containers to avoid caimibalism.
Each experiment was repeated twice and animals were not fed during the 96 h
of exposure.
White Shrimp (Penaeiis setiferus): Salinity of water where white shrimp
were caught was 31 %o. Salt water with the same salinity was prepared using DI
water and sea salt (Instant Ocean Salt, Aquarium Systems, Mentor, OH).
Dissolved oxygen, temperature, pH. conductivity, and alkalinity of salt water
were monitored at 0, 24. 48, 72, and 96 h. The concentrations of Neemix^’'
were 0, 0.625, 1.25, 2.5, 5, and 10 pg AZA/mL (0, 0.25, 0.5, 1.0, 2.0, and 4.0
pL Neemix™ /mL). Bioneem™ and pure AZA were not used for this
experiment. After preparing the solutions, ten animals were placed in separate
glass containers at varying concentrations of the pesticide. The reason for
placing animals in separate containers was to avoid caimibalism. White shrimp
were not fed during exposure. The pH, temperature, salinity, and DO
parameters were monitored and recorded at 0, 24, 48, 72, and 96 h. Animals
were observed daily for mortality.
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Water Fleas {Daphnia pulex): Seven dilutions of Neemix^” , Bioneem^^
and pure AZA were prepared at concentrations of 0, 0.0156, 0.0313, 0.0625,
0.125. 0.25, and 0.5 pg AZA/mL (0, 0.0062, 0.0124, 0.0248, 0.0496, 0.0998,
0.196 pL Neemix™ /mL or 0.0173, 0.0346, 0.0692, 0.138, 0.276, and 0.552 pL
Bicneem^” /mL) using DI water. The initial temperature, pH, DO, hardness,
conductivity, and alkalinity of DI water were measured. Shortly after preparing
the dilutions, ten less than 24 h old water fleas per concentration were placed in
glass vials. Each concentration was tested in triplicate and each experiment was
repeated twice. Water parameters including pH, temperature, and DO were
monitored at 24, and 48 h. Water fleas were observed daily for mortality and
not fed during 48 h of exposure.
Freshwater Snails (Physella virgata): Pure AZA dilutions at
concentrations of 0. 0.15, 0.30. 1.50, 3, and 30 pg/mL were prepared using 20
mL of DI water. The concentrations of Neemix™ and Bioneem™ were 0,
0.313, 0.625, 1.25, 2.5. 5 and 10 pg AZA/mL (0, 0.125, 0.25. 0.5, l.O, 2.0, and
4.0 pL Neemix™ /mL or 0, 0.34, 0.69, 1.38 2.76, 5.55, and 11.10 pL
Bioneem™/mL) in 20 mL of DI water. Parameters including temperature, DO,
pH, hardness, alkalinity, and conductivity of DI water were recorded initially.
After preparing the dilutions, twenty freshwater snails were placed in glass
containers. Each experiment was carried out in triplicates and was repeated
twice. Animals were observed daily for mortality. During 96 h of exposure, the
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pH, temperature, and DO parameters were monitored and recorded daily at 24,
48, 72, and 96 h. Freshwater snails were not fed during 96 h of exposure.
Oyster (Crassostrea virginica): Oysters were taken from the cooler and
cleaned with tap water. Shells were opened with a knife and muscles on top of
gonads were cut. The sex o f the oysters were checked and females and males
were placed in different trays. Gonads were then taken from the shell into a
small beaker and rinsed with saltwater. The eggs were filtered first through 75
pm. then 10 pm filters. Eggs retained by a 10 pm filter were collected, rinsed
with saltwater, placed in a 1 L beaker, and the volume was brought up to 200
mL with saltwater. The sperm were filtered first through 75 pm, then 15 pm
filters. The sperm that passed through the 15 pm filter were collected in a
beaker and the volume was brought up to 200 mL with saltwater. Eggs and
sperm were left in saltwater for 1 hour. After 1 h, 5 mL of sperm suspension
was added into the egg suspension and mixed with a plunger six times. Eggs
were left to be fertilized for 1 h with continuous aeration. Afterwards, 1 mL of
sample was taken from the egg suspension and put on a depression slide to
check fertilization status under a microscope. Fertilization was determined to be
positive when polar body occurred (Galtsoft, 1964). Fertilized eggs were
counted 3 times and the average concentration (eggs/mL) was calculated.
One hundred eggs per mL of Neemix™ and Bioneem^” were placed in
24 well tissue culture plates. The concentrations o f Neemix™ and Bioneem™
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were prepared in 22 %o saltwater at 0, 0.0625, 0.125, 0.25, 0.5, 1, and 2 pg
AZA/mL (0, 0.0124, 0.0248, 0.0496, 0.0992, 0.198, and 0.396 pL
Neemix^^/mL or 0, 0.034, 0.068, 0.136, 0.272, 0.544, and 1.08 pL
Bioneem™/mL). Each concentration was used in triplicate and each
experiment was repeated twice. DO, pH, temperature, hardness, salinity,
alkalinity, and conductivity measurements were recorded at the beginning o f the
test. Eggs were observed for mortality, mobility, and development at 0, 24, and
48 h. At the end o f 48 h of exposure, survivors and eggs that reached the D
stage were counted (Labare et a i, 1997).
Mosquito (Culex quinquefasciatus Say): The third instar larvae of
mosquitoes were used to test acute toxicity of Neemix^'^, Bioneem™ and pure
AZA. The concentrations of Neemix™,and Bioneem™ were 0, 0.0313, 0.0625.
0.125, 0.25, 0.5. and 1 pg AZA/mL (0, 0.0124, 0.0248. 0.0496, 0.0992, 0.198,
and 0.396 pL Neemix™/mL or 0, 0.034, 0.068, 0.137, 0.274, 0.551, and 1.10
pL Bioneem™/mL) in 20 mL of DI water. For every concentration twenty
larvae were placed in glass containers. Each concentration was tested in
triplicate and each experiment was repeated twice. Larvae were exposed to the
pesticides named above for 96 h. During 96 h of exposure, the larvae were fed
daily with 5 mg o f ground dog food. The concentrations of pure AZA were
prepared using 20 mL of DI water at concentrations of 0, 1.25, 2.5, 5, and 10 pg
AZA/mL. Shortly after preparing the solutions, 20 mosquito larvae were placed
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in glass containers. Each concentration was tested in triplicate. Animals were
exposed to pure AZA for 192 h and were fed every day with 5 mg o f ground
dog food during exposure. Parameters including pH, temperature, and DO were
monitored and recorded every day for each experiment. Observations of
mortality and molting were also made and recorded at 24, 48, 72, and 96 h for
Neemix™ and Bioneem™ experiments, 24, 48, 72, 96, 120, 144, 168, and 192
h for pure AZA experiment.
In vitro Acute Toxicity
Toxicity of Neemix™, Bioneem™, and pure AZA was tested in vitro
using hybridoma and oyster cell cultures.
Hybridoma Cells: Cells of hybridoma cell line (anti-glycoalkaloid),
developed by Plhak and Spoms (1994), were cultured in RPMI 1640 medium
(Sigma Chemical Co., St Louis, MO) which was supplemented with 10 mL of
heat-inactivated fetal bovine serum (Gibco BRI, Burlington, ON), 1 mL of
glutamine. 1 mL of pyruvic acid, and 1 mL of streptomycin-penicillin. This cell
culture medium was referred to R-10. Cells were cultured in cell culture plates
containing 10 mL o f R-10 and incubated at 37°C under 5% CO2 . After 3 days,
plates were removed from the incubator and cells were centrifuged for 10 min
at 540xg using an International Centrifuge (Model Hn, International Equipment
Co., Needham Hs., Mass). The desired cell density was 2 .5x 10^ cells/mL which
was obtained by dilution with R-10 media.
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Cell culture suspension (45 |iL/well) was placed in flat bottom 96 well
tissue culture plates and 5 p i of six different concentrations of Neemix^” ,
Bioneem™, or pure AZA were added to each well. Neemix™, Bioneem™, and
pure AZA concentrations were prepared in R-IO media at concentrations of 0,
0.01, 0.1, I, 10, and 100 pg AZA/mL (0, 0.004, 0.04. 0.4. 4, and 40 pL
Neemix™/mL or 0, 0.011, 0.11, 1.11, 11.10, and 111.1 pL Bioneem™/mL).
Each plate also contained blank background control wells containing an
appropriate amount o f media (45 pL) and 5 pL of Neemix™, Bioneem^*^, or
pure AZA, with no cells. Plates were incubated at 37°C in 5% CO? for 24, 48,
72 h, and 96 h.
At the end of 24. 48, 72 h, and 96 h of incubation, plates were removed
from the incubator and 10 pL of 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT, Sigma Chemical Co., St Louis, MO) was
added to each well. MTT solution was prepared at a concentration o f 5 mg
MTT per mL of phosphate buffer saline (PBS), filter sterilized and stored at 4°C
in the dark. After incubation for an additional 4 h at 37°C, the formazan
crystals were dissolved by addition of 150 pL of 0.04 N HCl in isopropanol
(Mosmann, 1983). The absorbance was read at a test wavelength of 550 nm and
a reference wavelength of 630 nm using a SpectraMax Plus ELISA reader
(Molecular Devices, Suimyvale, CA).
48
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Oyster Cells: Oysters were provided by the Louisiana State University
Sea Grant Oyster Hatchery (Grand Isle, LA). They were depurated for a week
in the depuration tank in the LSU Veterinary Science Building. The shells o f 14
depurated oysters were scrubbed using a brush with 5% bleach solution and
rinsed with tap water. Then, they were dried at room temperature in a biosafety
hood. Oysters were opened with a knife and rinsed again with sterile saltwater.
The pericardial membrane around the heart was cut and two parts of the heart
(ventricules and atria) were removed with sterile forceps. The ventricule is the
right side of the heart and has a yellow color. The atrium has brown color and it
is located on the left side of the heart. The vetricules and atria were then
placed in different test tubes containing 10 mL of sterile saltwater and shaken
10 times. The tissues were removed from the tubes and put in clean tubes
containing 30 mL of sterile saltwater and shaken again 10 times. The tissues
were then transferred to sterile test tubes filled with 30 mL of decontamination
solution, and shaken for 30 min. The decontamination solution consisted of
several antibiotic solutions (penicillin/ streptomycin/gentamycin/kanamycin/
neomycin in sterile saline buffer). The heart tissues were rinsed with sterile
saltwater and placed in sterile glass petri dishes. Then they were minced to a
puree-like consistency using a sterile single-edged razor blade. The minced
tissues were added to sterile beakers containing 10 mL o f oyster dissociation
solution containing 1.0 mg/L Pronase (CalBiochem, LaJolla, CA) in a saline
49
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solution (0.635 g/l CaCl2 .2H2 0 , 1.46 g/L MgSO^, 2.18 g/L M gCb.6H2 0 ,
0.310 g/L KCL 11.61 g/L NaCl, and 0.35 mg/L NaHCOg) and stirred for 1 h
at high speed. After 1 h, the cell suspensions were removed from the beaker
with a sterile pipet and placed into sterile 15 mL test tubes. The cell suspension
was centrifuged for 5 min at 200xg. The supernatant was transferred to a new
sterile test tube and centrifuged again at 200xg for 10 min and the supernatants
were discarded. Cells were resuspended in 2 mL of oyster media (Medium JL-
0DRP-4A). Concentrations and viability of the cells were determined using
trypan blue exclusion staining (0.4% Trypan blue in phosphate buffer solution,
PBS) and counting in a Neubauer hemocytometer. The desired cell density for
ventricule and atria cells was 2.5x10^ cells/mL which was diluted with the
addition of oyster media (Buchanan et a i, 1999).
Oyster cell cultures (90 |iL/well) were placed in flat bottom 96 well
tissue culture plates and 10 fiL of six different concentrations of Neemix™,
Bioneem™, or pure AZA was added to each well. Neemix™ or Bioneem™
concentrations were prepared in oyster media at concentrations of 0 , 0 .01 , 0 .1,
1, 10, and 100 pg AZA/mL ((0, 0.004, 0.04, 0.4, 4, and 40 pL Neemix™/mL or
0,0.011, 0.11, 1.11, 11.10, and 111.1 pL Bioneem™/mL) and pure AZA
concentrations were 0, 0.1, 1, 10, and 100 pg/mL . Plates were then incubated at
28°C for 24,48, 72 h, and 96 h.
50
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At the end of 24 h incubation, plates were removed from the incubator
and 20 |iL of 3-(4,5-dimethylthiazol-2-yi]-5-(3-carboxymethoxy- phenyl)-2-(4-
sulfophenyI)-2H-tetrazolium inner salt (MTS, Promega Corp., Madison, WI)
and phenazine methosulfate (PMS, Promega Corp., Madison, WI) mixture was
added to each well (Buttke et a i. 1993). The plates were incubated for an
additional 4 h. After incubation for an additional 4 h at 28°C, the absorbance
was read at a test wavelength of 490 nm and a reference wavelength o f 550 nm
using a microplate reader (MR5000. Daynatech Lab. Inc., Chantilly, VA).
Stability of Toxicity
Neemi.x™ and Bioneem™ were exposed to light, heat, and air to
measure their stabilities. One mL of Bioneem™ (900 pg/mL o f AZA) or
Neemix™ (2500 pg/mL of AZA) was put in a glass container and placed in a
Judge® II light box (GretagMacbeth^M, New Windsor, NY) without any
cover for air exposure. First, they were treated with light (6500K, northern sky
daylight includes LTV) at temperature of 24°C for 1, 3, 6 , and 9 days. Then,
another set of 1 mL of Neemix™ and Bioneem™ was treated with light at the
same density, but at 37°C for 1, 3 ,6 , and 9 days. Samples treated for I day with
light, heat, and air were removed from the light box, mixed with 100 mL of DI
water to simulate environmental conditions. By diluting the treated samples
with DI water, the initial concentration of AZA in these pesticides was
decreased to 25 pg AZA/mL for Neemix™ and 9 pg AZA/mL for Bioneem™.
51
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Diluted samples were sonicated for 45 min using an ultrasonic cleaner (Mettler
Electronics Corp., Anaheim, CA) to mix the solutions thoroughly. Afterwards,
seven different concentrations of treated Neemix^''* and Bioneem^” were
prepared in 15 mL of DI water. The concentrations of Neemix^"^ and
Bioneem™ were 0, 0.0156. 0.0313, 0.0625, 0.125, 0.25, and 0.5 pg AZA/mL
(0, 0.62, 1.24, 2.48, 4.96, 9.92, and 19 pL Neemix™/mL or 0, 1.7, 3.4, 6 .8,
13.6, 27.2, and 55.4 pL Bioneem™/mL). Shortly after preparing the dilutions,
10 water fleas per concentration were placed in glass vials. Each experiment
was evaluated in triplicate and repeated twice. Water fleas were exposed to the
treated pesticides for 48 h. They were not fed during 48 h of exposure and were
observed for mortality. The same procedure was followed for each pesticide at
days 3. 6 . and 9.
Fractionation of Neemix^ and Bioneem”*
Ten mL of Neemix™ and Bioneem™ was placed in round bottom flasks.
A rotary evaporator (Rotavapor Model RE 121, Biichi Laboratororiums-Technik
Ag., Falwil/Schweiz. Switzerland) set at 65°C and a speed o f 240 rpm was used
to evaporate volatiles. Each pesticide took 5-6 min to be fractionated. The
volatiles were collected in a vial and nonvolatiles were kept in round bottom
flasks. The nonvolatiles were mixed with 100 mL o f DI water and sonicated for
45 min using a ultrasonic cleaner (Mettler Electronics Corp., Anaheim, CA) to
mix the solutions thoroughly. Solutions were prepared from these fractions at
52
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concentrations of 0, 0.0625, 0.125, 0.25, 0.5, 1, and 2 |iL fraction/mL water.
Neemix™ and Bioneem™ volatile fractions were diluted to the same
concentrations as above directly from the vials in which volatiles were
collected. These were compared to controls (full formulations, AZA plus inert
ingredients) prepared as pL/mL. After preparing the dilutions, 10 water fleas
per concentration were placed into the vials to test the acute toxicity of
fractions. The DO, pH, temperature, alkalinity, and hardness of the DI water
were measured initially. Water fleas were exposed to the fractions for 48 h.
During 48 h of exposure, they were not fed.
Chemical Analysis
Chemical structure confirmation of pure AZA was carried out using
NMR and mass spectroscopy.
NMR Spectroscopy
Proton ( 1H) and correlated spectroscopy (COSY) spectra were recorded
on a Bruker AMX 400 MHz (Bruker Instruments Inc., Billerica, MA)
spectrometer at room temperature. All spectra were recorded in
deuteriochloroform (CDCI3). Five himdred microgram o f AZA was dissolved in
0.5 mL of CDCI3 (99.8% isotopically pure in deuterium, from Aldrich
Chemical Company Inc., Milwaukee, WI) in a 5 mm diameter NMR tube. The
spectrometer was locked on the deuterium signal o f CDCI3 . All chemical shifts
are expressed in parts per million (ppm) using tetramethylsilane (TMS) as an
53
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internal standard. Chemical shifts are reported in ppm on the Ô (ppm) scale
(ÔTMS ~ 0 ppm). The standard pulse programs used for acquisition and
processing were those provided by Bruker Instruments. The following
parameters were employed in ^H-^H COSY: Relaxation delay (RD) of 2
seconds, 512 ti increments; 1024 to 2048 t? points. Sine bell squared and
shifted (ti/4, ti/6 , and ti/8 ) apodization functions were used for processing.
Mass Spectroscopy
Pure AZA was mixed with 10 pL of methanol (MeOH, Sigma Chemical
Co.. St. Louis, MO). From this solution. 1 pL was taken and mixed with 3 pi of
glycerol in a small tube. The mixture was then poured on a stainless steel probe
and the probe was placed in a fast atom bombardment spectroscopy (FAB,
Finnignn MAT 900 Double Focusing Mass Spectrometer, Bremen, Germany).
The masses of pure AZA were obtained between 450 and 800 m/z (mass to
charge ratio).
Data Analysis
Statistical analyses included the calculation o f lethal concentration for
mortality of 50% (LC50) the bioassayed individuals, average, standard error
o f the parameters recorded daily, and 95% confidence intervals of LCgg. LC50
values on all mortality toxicological data and corresponding 95% confidence
limits were calculated using a computer-based Sperman Kerber Procedure
(SAS, 1995). The differences among treatment levels in molting experiments
54
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were tested by two-way analysis of variance (ANOVA). One way ANOVA
was used to determine differences among the different treatments in Ames test.
An IC50 (inhibition concentration index, the concentration o f the pesticides
needed to cause 50% inhibition of cell growth) was calculated using
Excel/Solver computer package (Microsoft Corp., Redmond, WA). The
inflection point (IC50) was determined after fitting the data to a 4-parameter
sigmoidal curve.
55
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RESULTS
Mutagenicity of Neem-based Pesticides
Neemix™ and Bioneem™ did not show mutagenicity to Salmonella
typhimurium types TA-98 and TA-100 at the concentrations tested (Figures 6
and 7). The number of revenants per plate was significantly (p<0.05) higher for
type TA -100 than for type TA-98 in both Neemix™ and Bioneem™
experiments. Metabolic activation (S9 (rat liver) addition) also had a significant
effect (p<0.05) on the growth of bacteria strains. Aflatoxin Bj (AfB%) tested
on the same strains of S. typhimurium was chosen to be the positive control in
this study. These samples were tested with S9 addition because AfBi does not
become a mutagen without metabolic activation. Both strains o f S. typhimurium
showed higher mutagenicity with increased concentrations of AfBi (Figure 8).
Dimethyl sulfoxide (DMSO) solvent was used as a negative control to test
mutagenecity. 5. typhimurium strains TA-98 and TA-100 exposed to DMSO
had 29 and 149 revenants per plate, respectively.
In vivo Acute Toxicity of Neem-based Pesticides and Pure AZA
LC50 values of Neemix™ for eight aquatic species are listed in Table 4.
Water fleas (D. pulex) had the lowest LC50 value, 0.07 pg AZA/mL (0.03 pL
Neemix™/mL) for Neemix™ followed by mosquito and blue crab with LC50
of 0.57 pg AZA/mL (0.29 pL Neemix™/mL) and 1.15 pg AZA/mL (0.46 pL
Neemix™/mL), respectively. Bioneem™ also showed the higher toxicity to
56
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■ o
IcgÛ.
■oCO
C/)
o'3
CD
8 5c5'
3CD
Cp.3"CD
CD■oICaO3
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LA
T A - 9 8 + S 9
- I - T A - 9 8 - S 9
■ l é i i T A - 1 0 0 + S 9
■■All T A - 1 0 0 - S 9
2 5 0
200
a 1 5 0
100
0 100.10.001 0.01 1.0(0) ( 0 .0 0 0 4 ) ( 0 .0 0 4 ) ( 0 .0 4 ) ( 0 .4 ) (4)*
Concentration (^g AZA/plate)
Figure 6. Mutagenic potential of Neemix '^ on S. typhimurium types TA-98 and TA 100
TA -98 +S9 = TA-98 strains tested with metabolic activation TA-98 -S9 = TA-98 strains tested without metabolic activation TA-100 +S9 = T A -100 strains tested with metabolic activation TA-100 -S9 = TA-100 strains tested without metabolic activation DMSO (Negative control) = 294 revertants/plate for type TA-98, 1498 revertants/plate for type TA-100 o f .S', typhimurium
’Values in parenthesis represent the corresponding Neemix^** concentrations (p L / plate)
CD■ DOQ .C
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■■èii T A - 1 0 G + S 9 T A - 1 0 0 - S 9
200
1 5 0
100
5 0
00.01 0.1 100 0.001
(0) ( 0 . 0 0 1 1 ) ( O .O l l ) ( 0 . 1 1 ) ( 1 .1 1 )
Concentration (|ig AZA/plate)
( 1 1 1 )*
■DCD
(/)C/)
Figure 7. Mutagenic potential of Bioneem^'^ on S. typhimurium types TA-98 and TA-100
TA-98 +S9 = TA-98 strains tested with metabolic activation TA-98 -S9 = TA-98 strains tested without metabolic activation TA-100 +S9 = TA-100 strains tested with metabolic activation TA-100 -S9 = TA-100 strains tested without metabolic activation DMSO (Negative control) = 294 revertants/plate for type TA-98, 1498 revertants/plate for type TA-100 o f .9. typhimurium
* Values in parenthesis represent the corresponding Bioneem™ concentrations (JIL/ plate)
CD■ DOQ .C
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2 5 0 0
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1 5 0 0
1000
5 0 0
0
10 5 0 100 2 5 0 5 0 0 1000
Concentration (ng/plate)
"OCD
Figure 8. Mutagenic potential of AfB, on S. typhimurium types TA-98 and TA-100 with metabolic activation
(/)(/)
D. pulex than all other species tested (Table 5). LC50 of Bioneem™ for D.
pulex was 0.03 jig AZA/mL (0.04 |iL Bioneem™/mL). In the present study,
even though AZA concentrations were equivalent in the neem-pesticides and
pure AZA preparations, pure AZA was less toxic to D. pulex with an LC50
value of 0.382 pg AZA/mL.
Neemix^*' was an effective insecticide against mosquito larvae at
concentrations higher than 0.25 pg AZA/mL (O.IO pL Neemix^"'*/mL). Toxicity
of Bioneem™ to mosquito larvae was similar to that of Neemix™ (Tables 4 and
5). The LC50 values o f Bioneem™ and Neemix™ for the larvae were 0.14
(0.12 pL Bioneem™/mL) and 0.57 pg AZA/mL (0.29 pL Neemix™/mL),
respectively. In contrast, pure AZA did not show significant toxicity to
mosquito larvae. The toxicity of pure AZA remained lower than neem-based
pesticides after an extended duration of exposure of 7 days. The LC50 value for
pure AZA was not determined because there was insufficient number of deaths
at the highest concentrations used.
Molting fi-equency decreased in mosquitoes as the concentrations of
Bioneem™ and Neemix™ increased. This is expected since the number of
deaths were also high at higher concentrations. There was no significant
difference (p>0.05) between the molting rate at concentrations lower than 0.5
pg AZA/mL or 0.2 pL Neemix™/mL (Figure 9).
60
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Table 4. LCgq values of Neemix^ for eight aquatic species'
Species Estimated LC50 LC50 upper limit
LC50 lower limit
Crayfish 6.60h (2.64) 7.56^5 (3.06) 5.75*5 (2.30)
White shrimps 2.68^(1.19) 5.03*5 (1.56) 1.426*5 (0.907)
Grass shrimps 3.8lb (2.00) 4.75*5 (2.43) 3.06*5 (1.65)
Blue crabs 1.15*5(0.46) 1.39*5 (0.56) 0.95*5 (0.38)
Snails 4.26*5(1.87) 5.37*5 (2.35) 3.37*5 (1.48)
Oysters 0.12*5 (0.05) 0.13*5(0.06) 0.12*5 (0.04)
Water fleas 0.07*5 (0.03) 0.08*5 (0.04) 0.06*5 (0.05)
Mosquitoes 0.57*5 (0.29) 0.65*5 (0.31) 0.50*5 (0.28)
* Data generated with Sperman Kerber Analysis (EPA , 1993)Concentrations based on AZA equivalence (pg AZA/ mL)Values in parenthesis represent the corresponding Neemix™ concentrations { \iU mL)
The same was observed for Bioneem^''^ (Figure 10). The molt Inhibitory
action o f both neem pesticides was expressed at concentrations higher than 0.5
jig AZA/mL. On the other hand, after 7 days of exposure to pure AZA (Figure
11 ). mosquito larvae molted less trequently than mosquito larvae treated with
Neemix^^ and Bioneem™. In this experiment, there was no significant
difference (p>0.05) among treatments, except in 1% EtOH. EtOH at i%
concentration reduced molting of mosquito larvae by about 5%, Based on the
results discussed above, AZA caused a sharper decrease in molting of mosquito
larvae than the neem-based pesticides. Chemical analyses (NMR and MS) were
61
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250n
200□ % Mortality
■ % Molting
0.0313(0.012)
0.0625(0.024)
0.125(0.048)
0.25(0.096)
Concentration (fig AZA/mL)
Figure 9. Percent mortality and molting for mosquito larvae exposed to NeendxTw
‘Values in parenthesis represent the corresponding concentrations o f N eem ix ™ (flL / mL)
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^ % Mortality
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0 0 . 0 3 1 3 0 .0 6 2 5 0 . 1 2 5( 0 ) ( 0 . 0 3 4 ) ( 0 .0 6 8 ) ( 0 . 1 3 6 )
0 .2 5 0 .5 1( 0 .2 7 2 ) ( 0 . 5 4 4 ) ( 1 .1 0 ) '
Concentration (|ag AZA/mL)
Figure 10. Percent mortality and molting for mosqnito larvae exposed to Bioneem^^
‘ V alues in parenthesis represent the corresponding concentrations o f Bioneem'*^ (^IL/ mL)
CD■ DOQ .C
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□ % Mortality
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Concentration (pg AZA/mL)
10
Figure 11. Percent mortality and molting for mosquito larvae exposed to pure Azadirachtin■DCD
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performed to confirm the identity o f the AZA used in this study and the results
o f these analyses are discussed under the chemical tests section of this chapter.
Table 5. LCso values of Bioneem " for six aquatic species"
Species Estimated LC50 LC5Q upper limit LC50 lower limitCrayfish 4.7lb (12.25) 5 .75b (16.29) 3.85b (9.21)
Grass shrimps 3 .19b (4.71) 3.92b (5.67) 2 .59b (3.91)
Snails 1.62b (1.82) 1.80b (2 .02 ) 1.46b (1.63)
Oysters O.I7 I (0.19) 0.18b (0 .2 0 ) 0.16b (0.18)
Water fleas 0 .03b (0.04) 0 .04b (0.05) 0 .03b (0.03)
Mosquitoes 0 .14b (0 .12) 0 .15b (0.14) 0 .12b (0 . 11)
* Data generated with Sperman Kerber Analysis (EPA . 1993)Concentrations based on AZA equivalence (pg AZA/ mL)Values in parenthesis represent the corresponding Bioneem™ (pL/ mL)
Toxicity of Neemix™ on blue crab was also dose and time dependent.
During 96 h o f exposure, blue crab showed higher mortality when exposed to
Neemix™ at 2 and 4 pg AZA/mL (0.8 and 1.6 pL Neemix^‘'^/mL). The percent
molting decreased with increased concentration of Neemix^” . The molting
rates at concentrations of 0.25, 0.5 and I pg AZA/mL (0.1, 0.2, and 0.4 pL
Neemix™/mL) were not significantly different (p>0.05) firom the control
reatment (Figure 12). It was observed that treatments using 2 and 4 pg
AZA/mL caused animals to die immediately after molting. Mortality appeared
to be more likely immediately after molting than during pre-molting.
65
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1 0 0 - I
I"Oaes
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0(0)
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0 .5(0 .2) ( 0 .4 ) (0 .8)
Concentration (|ig AZA/mL)
□ % Mortality
■ % Molting
4( 1.6 )
Figure 12. Percent mortality and molting for blue crab exposed to Neemix^*^
‘Values in parenthesis represent the corresponding concentrations o f N eem ix^ '^ tflL / mL)
White shrimp were found to be the fourth most sensitive species to
Neemix^^ of those tested (Table 4). During 96-h exposure, almost 100%
mortality occurred within the first 24 h of exposure to high concentrations (0.5,
1, and 2 pg AZA/mL) of Neemix^” . The LC50 o f Neemix™ for white shrimp
was 2.68 pg AZA/mL (1.19 pL Neemix™/mL). Susceptibility of white shrimp
to Neemix™ was about 0.7 times higher than that of grass shrimp. Mortality
criteria were easy to check in shrimp experiments because the translucent body
of the live shrimp changed to a dark pink color when animals died. However,
the main difficulty o f using white shrimp as test organisms was their handling
because they are difficult to keep in test containers.
The effects of Neemix^^ and Bioneem™ on grass shrimp were similar to
those of white shrimp (Tables 4 and 5). Grass shrimp showed more sensitivity
to Bioneem™ than Neemix™. Even though the LC50 values of Neemix™ and
Bioneem™ were similar, mortality occurred in animals exposed to Bioneem™
in a shorter time (at 24 h) than for Neemix™. The LC50 values of Neemix™
and Bioneem™ for grass shrimp were 3.81 pg AZA/mL (2.00 pL
Neemix™/mL) and 3.19 pg AZA/mL (4.71 pL Bioneem™/mL), respectively.
Pure AZA did not show significant toxicity to crayfish at the tested
concentrations (Table 6). It was observed that most crayfish died immediately
after molting at the tested concentrations of Neemix™ and Bioneem™ during
96-h o f exposure. Crayfish exposed to Neemix™ at a concentration of 2.5 pg
67
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
AZA/mL (1.0 iL Neemix^^/mL) had a significantly (p<0.05) higher molting
rate than at the lower concentrations (Figure 13). There was no significant
difference (p>0.05) between concentrations of 0.625 and 1.25 pg AZA/mL
(0.25 and 0.50 pL Neemix^^/mL) compared to the control. Using Bioneem™,
the molting rate o f crayfish was about 3 times lower than that for Neemix™.
There was also no significant difference between concentrations lower than 5
pg AZA/mL (5.55 pL Bioneem™/mL) and the control (Figure 14). The effect
of AZA on crayfish was similar to its effects on mosquito. Pure AZA delayed
molting in crayfish at concentrations above 1.25 pg AZA/mL.
Table 6 . LCgg values (pg AZA/mL) of pure AZA for four aquatic species"
Species Estimated LCgo^ LC50 upper limith LC50 lower limit*’Crayfish >1 - -
Snails >30 - -
Water fleas 0.382 0.463 0.315
Mosquitoes >10 - -
' Data generated with Sperman Kerber Analysis (EPA . 1993)" Concentrations based on pg AZA/mL
Exposure to various concentrations of Neemix™ and Bioneem™ caused
mortality and abnormality in oyster eggs. The larval development of oyster
eggs was inhibited at concentrations higher than 0.0625 pg AZA/mL (0.0248
pL Neemix™/mL) for both Neemix™ and Bioneem™ (Tables 4 and 5). The
LC50 values for oyster eggs were 0.12 and 0.17 pg AZA/mL for Neemix™ and
68
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en
IS
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0 % Mortality
■ % Molting
0.625(0.25) (2.0)
Concentration ()ig AZA/mL)
10 20 (4.0) (8.0)'
Figure 13. Percent mortality and molting for crayfish exposed to Neemix^*^
‘V alues in parenthesis represent the corresponding concentrations o f N eem ix™ (JiL / mL)
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4 0 0
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° % Mortality
y ■ % Molting
i J j i r i n0
(0)0 . 6 2 1 .25 2 .5 0 5 . 0 0 1 0 .0 2 0 . 0
( 0 6 9 ) ( 1 . 3 8 ) ( 2 .7 6 ) ( 5 .5 5 ) ( 1 1 .1 0 ) ( 2 2 . 2 0 ) ’
Concentration (|ig AZA/mL)
Figure 14. Percent mortality and molting for crayfish exposed to Bioneem^"^
‘Values in parenthesis represent the corresponding concentrations o f Bioneem ™ ( jiL / mL)
Bioneem^“ , respectively. Laboratory evaluation of Neemix and Bioneem
toxicity indicated that the molluscicidal activity against freshwater snails
{Physella virgata) was both dose and time dependent. The toxicity of
Bioneem™ was higher than the other neem-based pesticide (Neemix™) used.
LC50 values for freshwater snails were 4.26 [ig AZA/mL (1.87 p.L
Neemix™/mL) for Neemix™ and 1.62 jig AZA/mL (1.82 pL Bioneem™/mL)
for Bioneem^"'^. In contrast, pure AZA was not toxic to P. virgata at the
concentrations tested.
Water Quality Parameters
For all toxicity tests, water quality parameters did not differ significantly
(p>0.005) between test initiation and test termination in any of the experiments
(See appendix A).
In vitro Acute Toxicity of Neem-based Pesticides and Pure AZA
Neem-based pesticides showed variable cytotoxicities to the mammalian
and mollusk cells assayed. However, pure AZA does not appear to contribute
to the cytotoxicity. Neemix™ caused dose and time dependent inhibition of cell
proliferation and viability of hybridoma and oyster cells. Neemix™ and
Bioneem™ at concentration I pg AZA/mL (0.4 pL Neemix™/mL) and higher
showed significant (p<0.05) cytotoxic effects on hybridoma cells after 24, 48,
and 72 h o f exposure (Figures 15 and 16). The IC50 values of Neemix™ were
71
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Concentration (^g AZA/mL)
(4) ( 4 0 ) '
Figure 15. Mortality curves for Hybridoma cells exposed to Neemix^^
‘Values in parenthesis represent the corresponding concentrations o f N e e m ix '^ (( iL / mL)
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0 0.01 0.1 10 100(0) (0 .011) (0 . 1 1 ) ( 1 1 . 1 )( 1 . 1 1 )
Concentration (^g AZA/mL)
Figure 16. Mortality curves for Hybridoma cells exposed to Bioneem^'^
‘ V alues in parenthesis represent the corresponding concentrations o f Bioneem ™ (fiL /n iL )
(111)*
estimated to be 2.61, 2.23, and 1.60 |ig AZA/mL for 24, 48, and 72 h exposure,
respectively. Overall IC50 value of Neemix^'^ was 2.15 pg AZA/mL (0.86 pL
Neemix^^/mL). Similarly, ± e IC50 values of Bioneem^*^ were 1.54, 2.13, and
1.35 pg AZA/mL for 24, 48, and 72 h exposure, respectively. These values
show that Bioneem^’' was more toxic than Neemix™ to hybridoma cells with
an overall IC50 of 1.67 pg AZA/mL (1.86 pL Bioneem™/mL). However, after
96-h exposure of hybridoma cells to Neemi.x^’' and Bioneem™, the results were
similar for the two pesticides. There was no significant difference (p>0.05) in
the MTT signal of hybridoma cells among different concentrations of pure AZA
(Figure 17).
The cytotoxic effects of neem-based pesticides on oyster cells were
evaluated at various concentrations for 24 and 48 h. Oyster heart cell cultures
displayed a time and dose dependent viability. Toxicity of both neem pesticides
to oyster cells was expressed at 10 and 100 pg AZA/mL (4 and 40 86 pL
Neemix™/mL or 11.11 and 111.1 86 pL Bioneem™/mL) for 24 and 48 h
exposure (Figures 18 and 19). There was no significant difference (p>0.05)
between concentrations lower than 10 pg AZA/mL and the control for both
pesticides. There was a significant (p<0.05) decrease in the IC50 o f Neemix™
with increase in exposure time fi-om 24 h to 48 h. The IC50 decreased fi-om 2.91
pg AZA/mL following 24-h exposure to 1.46 pg AZA/mL after 48-h exposure.
The reverse was observed with Bioneem™. When oyster cells were exposed to
74
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esSaa■ei 0.5.a<
1000.01 100 0 1(0 ) (0.004) (0.04) (0.4) (4) (40)*
Concentration (^g AZA/mL)
Figure 18. Mortality curves for oyster cells exposed to Neemix^^^
* V alues in parenthesis represent the corresponding concentrations o f N eem ix™ ()lIL/ mL)
CD■ DOQ .C
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Figure 19. Mortality curves for oyster cells exposed to Bioneem^"^
‘V alues in parenthesis represent the corresponding concentrations o f Bioneem*'^’ ( f i U mL)
Bioneem^^ for 24 h, the IC50 was found to be 3.19 fig AZA/mL, whereas after
48 h, the IC50 was 15.72 fig AZA/mL. At 24-h exposure time, the
susceptibility of oysters cells to Bioneem™ was about 10 times lower than that
of Neemix™. As observed with hybridoma cells, the data showed that pure
AZA was not toxic to oyster cells (Figure 20).
Stability of Toxicity of Neem-based Pesticides
The toxicity of Neemix™ and Bioneem™ to D. pulex decreased over
time of exposure to light at 24°C. Neemix™ was found to remain stable for 6
days at 24°C as shown by the similar LC50 values (Table 7). No significant
effect (p>0.05) in mortality of D. pulex was observed when exposed to
Neemix™ treated with light under air at 24°C for 48 h. Bioneem™ treated with
light and heat at 24°C did not show toxic effects on D. pulex after 3 days of
exposure (Table 8). When treated with the same temperature for 1 day, the
LC50 values of Neemix™ and Bioneem™ were similar, 0.10 and 0.09 pg
AZA/mL (2.53 pL Neemix™/mL and 5.59 pL Bioneem™/mL), respectively.
Both Neemix^^ and Bioneem™ did not show toxic effects to D. pulex when
treated with light under air at 24°C for 6 days.
Toxicity of the two pesticides treated with light under air at 37°C were
significantly (p<0.05) different. Neemix™ showed toxic activity on D. pulex
after 1 day of treatment at 37°C with an LC50 o f 0.16 pg AZA/mL (4.09 pL
Neemix™/mL), almost double of the LC50 value o f Neemix™ treated at 24°C
78
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79
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for I day (Table 9). This result indicates that the toxic activity of Neemix^^ can
be reduced by heat treatment. Neemix^''^ appeared to loose its activity after 3
days of treatment at 37°C. In contrast, Bioneem^^ remained active under light
for I, 3, 6 , and 9 days at 37°C (Table 10). Its toxicity was higher than that of
Bioneem^'^ exposed to light under air at 24°C.
Table 7. LC50 values of Neemix™ treated with light under air at 24°C after 1,3 , 6 , and 9 days of exposure for D. pulex '
Treatment Estimated LC50 LC50 upper limit LC50 lower limit1 day 0 . 10b (2.53) 0.1 lb (3.00) 0 .09b (2.13)
3 days 0 .25b (5.59) 0 .29b (6.51) 0 .21b (4.80)
6 days >0.5b (>19.92) - -
9 days >0.5b (>19.92) - -
* Data generated with Sperman Kerber Analysis (EPA , 1993)Concentrations based on AZA equivalence (pg AZA/ mL)Values in parenthesis represent the corresponding Neemix™ concentrations (pL/mL)
Table 8 . LCjo values of Bioneem treated with light under air at 24* after 1, 3, 6, and 9 days of exposure for D. pulex^
Treatment Estimated LC50 LC50 upper limit LC50 lower limit1 day 0.09b ( 5 .6 5 ) 0 . 10b (6.70) 0.08b (4.77)
3 days 0.1 ib (6.39) 0 .12b (7.65) 0.10b(5.34)
6 days >0.5b (>55.40) - -
9 days >0.5b (>55.40) - -
* Data generated with Sperman Kerber Analysis (E P A , 1993)*’ Concentrations based on AZA equivalence (pg AZA/mL)
Values in parenthesis represent the corresponding Bioneem™ concentrations (pL/mL)
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Table 9. LC50 values of Neemix™ treated with light under air at 37°C after 1,3, 6 , and 9 days of exposure for D. pule:^
Treatment Estimated LC50 LC50 upper limit LC50 lower limitI day 0.1615 (4.09) 0.19*5(4.79) 0 . 15b (3.49)
3 days >0.5b (>19.92) - -
6 days >0.5b (>19.92) - -
9 days >0.5b (>19.92) - -
* Data generated with Sperman Kerber Analysis (EPA , 1993)" Concentrations based on AZA equivalence (pg AZA/'mL)
Values in parenthesis represent the corresponding Neemix™ concentrations (pL/mL)
Table 10. LC50 values of Bioneem^^' treated with light under air at 37°< after 1, 3 ,6 , and 9 days of exposure for D. pulex^
Treatment Estimated LC50 LC50 upper limit LC50 lower limit1 day O.Ogh (5.28) 0.10*5 (6.45) 0.07*5 (4.32)
3 days 0.10b(5.75) 0.1 lb (6.83) 0 .09b (4.84)
6 days 0 .22b (9.32) 0 .25b (10.83) 0 . 19b (8.01)
9 days 0.38b (15.22) 0 .45b (17.73) 0 .33b (13.03)
' Data generated with Sperman Kerber Analysis (EPA , 1993)*’ Concentrations based on AZA equivalence (pg AZA/mL)
Values in parenthesis represent the corresponding Bioneem"* concentrations (pL/mL)
Fractionation of Neem-based Pesticides
The effects o f fractionated Neemix™ and Bioneem™ on D. pulex are
summarized in tables II and 12, respectively. The nonvolatile fractions from
Neemix™ and Bioneem™ were oily residues with a brownish yellow color.
Volatiles from Neemix™ and Bioneem^^ were colorless, but had a strong
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solvent odor. Nonvolatiles from both Neemix^^ and Bioneem™ exhibited the
same LC50 value, 1.023 fiL fraction/mL, for D. pulex. Volatiles from Neemix™
were not toxic (LCgQ>2.0 (xL fraction/mL) to D. pulex at the concentrations
tested (Table 11). However, volatiles derived from Bioneem™ were more toxic
than the corresponding nonvolatile fraction with an LC50 of 0.97 |xL
fraction/mL (Table 12).
Table 11. LCso values (|iL fraction/mL) of Volatiles and Nonvolatiles obtained from Neemix " for D. pulexf^
Treatment Estimated LC5q* LC50 upperlimitb
LC50 lower limits
Volatiles >2.0 - -
Nonvolatiles 1.02 1.21 0.87
NeemixTM 0.17 0.20 0.14
* Data generated with Sperman Kerber Analysis (EPA , *’ Concentrations based on pL fraction/mL
,1993)
Table 12. LCso values (fiL fraction/mL) of Volatiles and Nonvolatiles obtained from Bioneem"^ for D. pulex*
Treatment Estimated LCgQ ^ LC50 upper limit^
LC50 lower limitb
Volatiles 0.97 1.03 0.91
Nonvolatiles 1.02 1.18 0.88
BioneemTM <0.063 - -
‘ Data generated with Sperman Kerber Analysis (EPA , 1993) " Concentrations based on pL fraction/mL
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D. pulex appeared to be more sensitive to the full formulations o f both
pesticides. When the full formulation o f Neemix^''^ was tested at the same
concentrations as the fractionated Neemix™, the LC50 dropped to 0.17 pL
Neemix™/mL versus >2.0 pL fraction/mL for volatiles and 1.02 pL
fraction/mL for nonvolatiles, indicating greater toxicity in the full formulation.
The toxicity of full formulation of Bioneem™ was severe in comparison to the
nonvolatile and volatile fractions from the same pesticide. All animals died at
the lowest concentration (0.063 pL Bioneem™/mL); therefore, LC50 value was
not estimated.
Chemical Tests
High Field ‘H NMR Spectroscopic Study of AZA
The ^H-NMR spectra of AZA (Table 13) confirmed the presence of 44
hydrogen atoms which resonate in the chemical shift range between 1.32 and
6.46 ppm (Figure 21). Signals at 5 1.33 and 1.30 correspond to the geminal
protons, 16-Ha 16-Hy. The signals at 2.32 and 2.25 ppm were assigned to
2-H(x and 2-Hp, respectively. The chemical shifts at 2.75 and 5.05 ppm belong
to the hydroxyl groups (7-OH and 11-OH) o f AZA. The assignment of the
diastereotopic protons (28-Hq and 28-Hp) was achieved using ^H-^H COSY
experiments. The signal 4.08 ppm was assigned to 28-Hot, while the signal at
3.77 ppm was assigned to 28-Hp.
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Table 13. ‘H-NMR (Nuclear Magnetic Resonance) spectral data (5) for Azadirachtin (400 MHz, Solvent: CDCI3, 7.26 ppm)
'H Signals Chemical shift (5, ppm)1
~a 2.32(m)2.25(m)
3 5.5 l(t)4 -
5 3.34(d)6a 4.59(dd)6b 4.62(dd)7 4.76(m)8 -
9 3.32(s)15 4.67(d)16-Hb 1.33(d)16-Ha 1.30(d)17 2.38(d)18 2 .01(d)19-Ha 3.61(d)19-Hb 4.14(d)20 -
21 5.64(s)22 -
23 6.46(d)28-Ha 3.77(d)28-Hp 4.09(d)3-OH 1.77(s)7-OH 2.75(s)220H; 11 OH 5.05(d,s)CH3COO 1.95(s)I2 -OCH3 3.69(s)29-OCH3 3.79(s)4 ’ 1.78(dd)5' 1.85(s)
84
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m
incn
o
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in
into .
ors.
Figure 21. Nuclear Magnetic Resonance (NMR) Spectra of Azadirachtin
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The COSY (Correlation SpectroscopY) spectrum (Figure 22)
showed that 16-Ha resonating at 1.33 ppm was coupled only with Hy proton
resonating at 1.30 ppm. Cross peaks between 19-Ha 19-Hy occurred in the
COSY spectrum (Table 14). The protons with chemical shifts o f 6.46 and
5.05 ppm coupled to each other. Tigloyl groups, 3’-H, 4 ’-H, and 5’-H showed
resonances at 6.46, 1.77, and 1.85 ppm.
Table 14. Diagnostic 'H -‘H COSY (Correlation Spectroscopy) Cross Peaks of Azadirachtin a t 400 MHz
Proton is coupled to Proton16 Hb 16 Ha
17 16 HaCH3OH 12
4’ 176 2a
2a 2P7 2a33 173* 16 Ha23 11
Mass Spectra of AZA
Mass spectroscopy analysis showed that AZA may have lost substituents,
possibly during the analysis. Fast atom bombardment spectroscopy (FAB)
results confirm that AZA produced peaks at m/z 721.1, m/z 703.3, and m/x
685.2 (Figure 23). The peak at m/z 721.1 corresponded to the pronated AZA
(M'^H). Strong fragment ions at m/z 685.2 corresponded to composite losses of
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[
2 . 0
3 . 0
4 . 0
5 . 0
5 . 0
7 . 0
PPM1' ' " 1 ' I ' I ' ' ' ' ' i ' ' ' ' ' I ■ ■ ■ I
7 . 0 5 . 0 5 . 0 4 . 0 3 . 0 2 . 0PPM
Figure 22. Correlation Spectra (COSY) of Azadirachtin
87
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water. The ion m/z 619.1 may be the result of the loss of C4H5O2 . The more
accurate data is listed in table 15. The oxygen in the epoxide containing half of
the molecule was lost in the low mass region of the spectra giving m/z at 603.2.
The additional losses of four hydrogen atoms give rise to m/z 563.1 in AZA.
Table 15. Accurate Mass data of Azadirachtin
m/z Formula
721.2 C35H45O 16
703.3 C35H43O 15
685.3 C35H41O 14
619.2 C30H35O 14
585.1 C30H33O 12
567.1 C30H31O 11
543.2 C2 8 H3 1 O 11
475.1 C24H27O 10
88
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o•H
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JO X3T5U33UI
Figure 23. Mass Spectra of Azadirachtin
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DISCUSSION
Mutagenicity of neem-based insecticides was determined using the Ames
test with two bacterial strains of S. typhimurium. TA-98 and TA-100 strains
were chosen to detect ffame-shift and base-pair alterations in DNA due to
exposure to neem-based insecticides. In addition. Salmonella tester strains were
tested with 89 fraction (the livers of rats induced with Aroclor 1254 contains a
combination of mixed function oxidases and cytochromes capable of converting
many promutagens into ultimate mutagens, that is, direct-acting compounds
which cause mutation of the test strains of bacteria) because & typhimurium
strains multiply faster in the presence of 89 fraction (Maron and Ames, 1983).
Both Neemix^^ and Bioneem^"'^ elicited no mutagenicity at the
concentrations tested. This finding is in agreement with the results reported by
Jongen and Koeman (1983) who found neem oil to be non-mutagenic in strains
TA-98 and TA-100 of Salmonella typhimurium. Non-mutagenicity was also
shown using Margosan-0® (National Research Council, 1992). Other neem
compounds, such as salannin and nimbolide also failed to cause mutagenicity by
the Ames test using TA-98 and TA-100 strains of S. typhimurium (Jacobson,
1981; Uwaifo, 1984). Although neither TA-98 nor TA-100 strains showed
alterations in their DNA when exposed to Neemix™ and Bioneem™, the
number of revertants for TA-100 strain was higher than that for TA-98 strain.
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This was expected because S. typhimurium type TA-100 proliferates faster than
type TA-98 (Maron and Ames, 1983).
Accurate prediction of chemical hazard to organisms requires knowledge of
chemical fate and bioavailability in the environment, behavior and physiology
of the organism, and route, duration, and frequency of exposure. Exposure
assessment provides critical information for establishing dose-effect
relationships that determine chemical toxicity to an organism (Landrum et a i,
1992). The toxicity of Neemix^''^. Bioneem™, and pure AZA was determined
based on the aqueous exposure of the above insecticides to aquatic organisms.
Toxicity of Neemix^’' and Bioneem™ was more pronounced against water fleas
{Daphnia pulex) than crayfish, blue crab, freshwater snails, oyster, grass
shrimp, white shrimp, and mosquitoes probably because water fleas are much
smaller and would have greater metabolic rates than the other animals used in
this study. The wide range of responses exhibited by various aquatic animals
can be attributed to many factors. The individual test organisms used in this
study were different species with different genetic, physiological, and
behavioral characteristics. Both Neemix™ and Bioneem™ were more toxic
than pure AZA to all animals tested. The high LCgg's of neem-based
insecticides suggests that AZA is not the only active compound N eem ix^ and
Bioneem^’'* or the inert ingredients enhance the potency of AZA. The high
toxicity o f neem-based pesticides to D. pulex might be due to the high surface to
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volume ratio of D. pulex. Dosdall and Lehmkuhl (1989) reported that small
aquatic animals with high surface area to volume ratios are generally more
susceptible to applications of contact insecticides than larger aquatic animals.
It was observed that different formulations of neem-based insecticides
showed different effects on the same and similar species. For example.
Neemix'^’ (0.025% AZA) and Bioneem'^’ (0.009% AZA) had LC50 of 0.07
and 0.03 fig AZA/mL for D. pulex (<24 h old), respectively. In contrast, Larson
(1989) studied the toxicity of Margosan-0® (contains 0.3% AZA) to D. magna
and found an LC50 o f 13 fig Margosan-0®/mL using D. magna (<20 h old)
and no observed effects at concentration of 10 fig Margosan-0®/mL.
Comparison of the relative toxicity of Neemix™ or Bioneem^’ with two
commonly used organophosphate pesticides (OP), namely Chlorpyrifos® and
Malathion®, indicates that these neem-based insecticides are as toxic as
organophosphate pesticides against water fleas. LCgg values of Chlorpyrifos®
and Malathion® for D. magna were reported by Leight and Dolah (1999) to be
0.001 and 0.033 fig/mL, respectively. Tomlin (1994) found that EC50 of
Dimilin® (a chitin inhibitor insecticide) for Daphnia spp. was 0.0021 fig/mL.
D. magna was also found to be very sensitive to a pyrethroid insecticide
(Permethrin®) with a 48-h LC50 in the range of 0.0002-0.0006 fig/mL (Stratton
and Corke, 1981).
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Among all the species tested, crayfish was found to be the least sensitive
to Neemix^’ and Bioneem™. LCggs of the above pesticides for crayfish ranged
from 4.71 to 6.60 fig AZA/mL (2.64 jiL Neemix™/mL or 12.25 jiL
Bioneem™/mL), while the LC50 for pure AZA was greater than the highest
concentration used. This result suggests that toxicity of Neemix™ and
Bioneem™ to crayfish resulted from the formulation components or from
synergistic effects of the formulation components that enhanced the toxicity of
AZA. Most crayfish which molted within 30 days died immediately after the
molt. This might be due to the greater sensitivity of post-molt animals to
pesticide formulations. Similar results were found by Banken and Stark (1997)
who studied the toxic effects of Neemix™ on aphid Coccinelle septempunctata
ist and 4^h instar. They reported that 4^^ instar (after 3 molt) aphids were more
sensitive to the growth disrupting effects of acute exposure to Neemix^"^ than
the l^t instar (before molt). AZA effects on insects have been investigated by
many researchers. Wilps et al. (1992) reported a significant reduction in the
hatching rate of Phormia terraenovae (fly) eggs after contamination o f the
substrate (minced meat) with 50 fil o f neem seed kernel extract with 1.7%
AZA/g. When crayfish previously exposed to <5 fig AZA/mL were observed
for 30 days following acute toxicity tests, they showed aberrant behavior and
feeding disorders. This might lead to mortality of crayfish if such exposure
occurs in their natiual habitat. LC50 values of neem-based pesticides were
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lower than those of Terbufos® (OP, 0.0059 ^ig/mL), Endosuifan®
(organochiorine pesticide, 0.009 pg/mL), Chlorpyrifos® (OP, 0.021 pg/mL),
and Malathion® (OP, 5 pg/mL) (Fomstrom et a i, 1997; Cheah et a i, 1980;
Cebrian et a i, 1992). Even though toxicity of neem-based pesticides was lower
than the most commonly used organophosphate and organochiorine pesticides,
they are potentially harmful to the ecosystem through their negative impact on
crayfish. Crayfish is an aquatic keystone species, uniquely capable of
converting unavailable nutrients into energy for other aquatic organisms,
controlling macrophyte and invertebrate populations, and providing food for
higher trophic level organisms. Decreases in crayfish populations may cause
changes in energetics, nutrient cycling, and community structure and function
(Odum, 1985).
Blue crab megalopes exposed to Neemix^"'^ for 96 h showed sensitivity
similar to that of water fleas and crayfish. Mortality increased with increased
concentrations o f Neemix™. Megalopes that survived to juvenile at
concentration lower than 1 pg AZA/mL (0.4 pL Neemix™/mL) were able to
molt and develop normally. This might be due to the fact that survivors were
able to recover from the short term toxic effects of Neemix™. Similar toxic
effects have been observed with other pesticides such as Malathion® and
Dimilin®. Bookhout and Monroe (1977) reported increased larval development
time and increased death at high concentrations o f Malathion®. Blue crab
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larvae were exposed to 0.0005-0.006 pg/mL Dimilin® for 96 h and survival
was always <5% at Dimilin® concentrations higher than 0.003 pg/mL (Costlow,
1979). These results suggest that blue crab is highly sensitive to exposure of
any pesticide at megalope and larval stages, possibly due to the frequency of
molting cycles (Heard. 1982). Thus, in cases of spills or considerable
agricultural run-off contaminated with neem-based insecticides, blue crabs may
be one of the most susceptible species to direct exposure of these pesticides in
the aquatic environment.
White shrimp were more sensitive to Neemix™ than grass shrimp at the
same pesticide concentrations. The exact reason for the different responses
between the two shrimp species (white and grass) to Neemix™ are not yet
known; however, previous studies indicated that the effectiveness o f neem-
based pesticides may vary among life stages, species, and formulations (Isman,
1997). There was no significant differences between the LC50 values for
Neemix™ and Bioneem™ for grass shrimp. The LC50 values for grass shrimp
found in this study are close to literature LC50 values o f some commonly used
pesticides such as Dimilin®, Chlorpyrifos®, Nonylphenol®, Malathion®, and
Endosuifan®. Lussier et a i (1996) found the 96-h LC50 value of
Nonylphenol® to be 0.071 pg/mL. Grass shrimp larvae had a 96-h LC50 o f
0.00044 pg/mL when exposed to Chlorpyrifos® at concentrations o f 0.0001 -
0.0016 pg/mL (Key and Fulton, 1993). Key (1995) studied the toxic effects of
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Malathion® on grass shrimp at different life stages and found that 96-h LC50
values varied from 0.009 to 0.038 pg/mL with different age of the animal. The
premolt grass shrimp was the most sensitive species to Dimilin® exposure with
a 96-h LC50 o f 0.0011 pg/mL. This sensitivity was due to Dimilin®’s chitin
inhibition effect. Mayer (1987) reported 48-h EC50 of 0.0002 pg/mL for P.
azteciis and 0.0024 pg/mL for P. duoranim when exposed to chlropyrifos.
These studies show that in larval life cycle the effects of pesticide exposure
might be severe and sublethal effects might result from continuous exposure to
pesticides. In the present study, only juvenile shrimp were exposed to
Neemix^''^ and Bioneem^^ and the exposure was discontinuous to simulate field
run-off conditions. Although not studied here, a continuous exposure
potentially could result in abnormal growth and developmental effects in the
Juveniles.
Neemix^"^ and Bioneem^''^ exhibited similar toxicity to oyster embryos
(tables 4 and 5). During 48-h o f exposure, most oyster embryos died at high
concentrations of Neemix™ and Bioneem™ by 12 h. Aqueous exposure to
both pesticides at lower concentrations caused abnormal development in oyster
embryos. This might be due to slow depuration rate of the embryos or fast
accumulation of neem-based pesticides in oyster embryos. Mature oysters are
able to depurate many chemicals to a limited extent. For example, adult pacific
oysters {Crassostrea gigas) metabolized Pentachlorophenol® (a general
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biocide) at 11.2% of total depurated material to an undetermined hydrophilic
product (Shofer and Tjeerdema, 1993). Although adult oysters metabolize and
potentially detoxify chemicals to a great degree, there is no evidence to prove
the same is true for oyster larvae. Therefore, the observed toxic activity of neem
pesticides against oyster larvae demonstrates that oysters are at risk if their
environment is contaminated with neem products, which may impact the overall
harvest of oysters in areas receiving large quantities o f agricultural run-off.
The second species of mollusk exposed to neem-based pesticides and
pure AZA was freshwater snails. Freshwater snails showed higher sensitivity to
Bioneem™ than Neemix™. The high toxicity of Bioneem™ may be due to the
presence of other toxic compounds (e.g., limonoids or solvents) in its
formulation. Bhatnagar and Nama (1990) tested the toxic effects of aqueous
extracts of neem fruit and of ethanolic extracts of leaves at concentrations of
0.5, 1.2, and 5% against adults of Lymnaea acuminata, Indoplanorbis exustus,
and Viviparus bengalensis, all dangerous vector snails in India. Among the
aqueous and alcoholic extracts, the 5% alcoholic extract, obtained by Soxhlet
extraction, was the most effective, causing 100% mortality o f L acuminata after
4 h, whereas I. exustus and V. bengalensis died after 9 h. Application of a 5%
aqueous extract of leaves resulted in 100% mortality o f L acuminata after 11 h,
while I. exustus and V. bengalensis were exterminated after 17 h.
Concentrations lower than 5% caused less mortality. Molluscicidal activity of
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other neem-based pesticides (Achook and Nimbecidine) were tested against two
species o f snails {Lymnaea acuminata and Indoplanorbis exustus) by Singh et
al. (1996). Nimbecidine (0.03% AZA, 90.57% neem oil, 5.0% hydroxyel,
0.50% epichlorohydrate, and 3.0% aromax) was more toxic than Achook
(0.003% AZA, 0.005% azadiradione, 0.02% nimbocinol and epinimbocinol)
against both snail species. The authors concluded that the high toxicity of
Nimbecidine may be due to the formulation, especially its high oil content
(90.57% neem oil).
Pure AZA was less toxic to freshwater snails than Neemix^"'^ and
Bioneem^''^ when used at comparable AZA concentration. The low toxicity of
pure AZA compared to the two neem-based pesticides may be attributed to
synergistic effects between AZA and the inert ingredients in Neemi.x™ and
Bioneem™ or a greater toxicity of other compounds in neem extracts. These
results are in agreement with West and Mordue (1992) who investigated the
toxic and antifeedant effects of pure AZA on snail species including Deroceras
reticulatum, Arion distinctus, Agriolimax caruanae, and Maximus sp. Snails
were fed with AZA treated barley plants. These authors found that pure AZA at
500 ppm neither had a toxic effect nor affected feeding behavior of the snails
investigated. On the other hand, Singh et al. (1996) found pure AZA to be
highly toxic to L. acuminata and I. exustus with an LC50 value o f 0.35 mg/L.
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However, the toxic effect of pure AZA. decreased after 24 h of exposure. These
results indicated that pure AZA was not stable in water and lost part of its toxic
activity.
Both Neemix™ and Bioneem™ had strong toxic action against mosquito
larvae at concentration higher than 0.25 pg AZA/mL (0.096 pL Neemix™/mL)
for Neemi.x™and 0.0625 pg AZA/mL (0.096 pL Bioneem™/mL) for
Bioneem™. The higher toxicity of Bioneem™ might be due to the presence of
other compounds which are absent in Neemix^*^ or caused by synergistic effect
of "inert ingredients" and pure AZA. The toxic effects of other neem
compounds and pure AZA have been tested on different species of mosquito
larvae. Sharma et al. (1993) tested neem oil on mosquito larvae at
concentrations of 0.5, 1. and 2% mixed in coconut oil and reported that it
produced strong toxic action on mosquito larvae. Su and Mulla (1998) tested
the ovicidal activity of Azad™ WPIO (10% AZA as an active agent) and
Azad™ EC4.5 (4.5% AZA as an active agent) on two different mosquito egg
colonies {Culex tarsalis and Culex quinquefasciatus) at 0, 0.1, 0.5, 1, 5, and 10
pg pesticide/mL. The egg colonies of C. quinquefasciatus were more
susceptible to both formulations than Culex tarsalis egg rafts. The neem
suspension at 1 pg/mL produced almost 100% mortality in eggs o f C
quinquefasciatus, while 5 pg/mL neem suspension/mL showed 100% mortality
in eggs o f C. tarsalis. These results differ from those o f Al-Sharook et al.
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(1991) who tested the toxicity and growth inhibitory activity o f 96% pure AZA
on Culex pipiens molestus instar larvae and found an LC50 value of 1-5
|ig/mL for pure AZA on this species. Two possible reasons for the observed
difference are the species used and the AZA extraction method. As discussed
above, different mosquito species (i.e. Culex tarsalis and Culex
quinquefasciatus) show different sensitivity to AZA. Furthermore, the source
and method of extraction used in AZA production may yield AZA with varying
potency. Molt inhibitory action of various concentrations o f pure AZA on
mosquito larvae was not significantly (p>0.005) different from the control at
any concentration used. Pure AZA inhibited or delayed molting of mosquito
larvae at all concentrations used. On the other hand, both neem-based
pesticides did not have the same molt inhibitory effects as pure AZA had. Even
though both Neemix^” and Bioneem '^^ showed high toxic action on mosquito
larvae at lower comparable AZA concentrations, they did not cause the same
effects on molting behavior. This suggests that AZA in the pesticides either lost
its molt inhibitory activity when reacted with other neem compounds or the
solvents used to make the formulations altered its inhibition effects on mosquito
larvae. The molt inhibitory effect of AZA has been studied using many insect
species. In Tenebrio molitor pupae the injection o f 1 pg of AZA was found to
induce a delayed and reduced secretion o f ecdystroid hormones which inhibit
the imaginable molt (Marco et al., 1990). In addition, 50% inihibition of
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ecdysone-20-monooxygenase in Drosophila melanogaster, Aedes aegypti, and
Manduca sexta was brought about by lO"^ to 4x10-4 M concentrations of AZA
(Smith and Mitchell, 1988). The effects of AZA and other neem tree
compounds (salannin, nimbin, and 6-deacteylnimbin) on the third instar larvae
of D. melanogaster, adult females of A. aegypti, and 5^ instar larvae of M,
sexta were studied by Mitchell et al. (1997). At lower concentrations, 10"^ to
IQ-6 M, none of the four compounds showed any significant activity on
ecdysone in any of the species tested. Increasing the compounds concentration
to 10-4 M resulted in increasing inhibition of enzyme activity. Salannin was
found to be the most effective of the four neem seed compounds in inhibiting
ecdysone-20-monooxygenase activity, whereas nimbin was the least effective.
These results indicate that besides AZA, other neem compounds, such as
salannin and 6 -deacteylnimbin can also affect ecdysteroid synthesis which, in
turn, can affect the molting rate of both insects and crustaceans. Similar toxicity
results were obtained by Dunkel and Richards (1998) who found that Align, a
neem-based pesticide, had an LC50 of 2.74 mg/L for an aquatic species, such as
Brachycentrus americanus.
Based on a proposed application rate of 50 g AZA/ha, the expected
environmental concentration of AZA in water would be approximately 0.035
mgL (Canadian Pest Management Regulatory Agency, 1993). The survival and
molting rates o f eight species exposed to pure AZA and neem formulations in
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laboratory conditions indicate that AZA at an environmental concentration o f
0.035 mg/L might not be toxic to these species in long term exposure directly
in the water column or indirectly through consumption of contaminated organic
material in the aquatic environment. However, at these concentrations AZA
may have adverse effects on water fleas, oyster eggs, and blue crab megalopes
whose LC50 values were lower than the estimated 0.035 mg/L. Younger
animals of the 8 species or other small species not tested in this study could also
be affected.
The growth inhibitory and cytotoxic effects of Neemix™. Bioneem^'^^,
and pure AZA were tested on hybridoma and oyster cells. Hybridoma cells were
chosen because they are mammalian and oyster cells were chosen to represent
cells of mollusk origin. Hybridoma cells are able to proliferate in the presence
of nutrients (immortalized cells), whereas these oyster cells (primary cell) do
not proliferate in vitro. Both hybridoma and oyster cells were sensitive to the
pesticides at the higher concentrations tested. The correlation between cell
number and formazan production expressed as absorbance (OD550 nm) was
measured in cultures exposed to Neemix™, Bioneem™, and pure AZA using
tétrazolium salts (MTT and MTS-PMS methods). The MTT and MTS-PMS are
well established methods for detection of cell proliferation and cytotoxicity
(Mosmann, 1983; Cory et a i , 1991). The total outcome of formazan is
dependent on the intracellular metabolism and the number of cells. When the
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metabolism is low, mitochondrial activity in cells is reduced giving light purple
color in MTT and light brown color in MTS-PMS, both characterized by low
absorbance. This situation occurred in hybridoma cells exposed to high
concentrations of pesticides for over 72 h. The absorbance of hybridoma cells
after 96-h exposure were significantly (p<0.005) lower than those of 24, 48,
and 72 h exposure. This might be due to the natural death of hybridoma cells
which normally occurs after about 72 h of incubation depending on the cell
density. When shorter exposure times were used (24, 48, and 72 h), Neemix™
and Bioneem™’ but not pure AZA, were found to be toxic to hybridoma cells.
The graphical estimation of IC50 showed that both neem-based pesticides have
cytotoxic and growth inhibitory effects on hybridoma and oyster cells when
used at concentrations higher than 1 pg AZA/mL. Neemix™ and Bioneem™
showed a greater cytoxicity to oyster cells than to hybridoma cells. Unlike
hybridoma cells, oyster cells are not growing cells; and thus, may not be able to
accumulate or metabolize neem-based pesticides in the same way. Oyster cells
may be more sensitive to these pesticides than hybridoma cells. Similar results
were found by Cohen et al. (1996) who studied the cytotoxic effects of seven
neem-seed extracts, used for preparing commercial neem pesticides, on NIE-
115 murine neuroblastoma cells. Of the seven neem-seed extracts, pure AZA,
nimbin, and deacetylnimbin did not appear to cause cytotoxicity to NlE-115
cells. Epoxy azadiradione, salannin, and deactylsalannin were found to be
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cytotoxic to NlE-115 ceils with IC50 values of 20-200 fig/mL. On the other
hand, the major cause of cytotoxicity to neuroblastoma cells was nimbolide on
the basis of its relative potency and low IC50 value of 15 pg/mL.
Pure azadirachtin, the active compoimd in the neem-based pesticides, did
not have any harmful effects on hybridoma and oyster cells, but may have an
effect when formulated with inert ingredients present Neemix^” and
Bioneem^''^. These findings also indicate that the biological activity of neem-
based pesticides come from either other major constituents, such as limonoids
(nimbolide, salannin, nimbin, and epoxyazadiradione), inert ingredients' used
in the pesticide formulation, or a synergistic effect of both. Rembold and
Annadurai (1993) studied the cytotoxic effect of AZA A on insect cells (Sf9,
Spodoptera frugiperda ovarian cells) and mammalian cell lines (Chinese
Hamster Ovarian cells). AZA showed a remarkably specific and high toxic
effect on insect cells but not on mammalian cells. Based on these results, it was
suggested that AZA has cell specificity. It should be noted that this apparent
specificity may be due to origin (species) as well as phenotype (primary vs.
Immortalized cells). The in vitro effect of neem oil on the development of
mouse two-cell embryos and trophecto-dermal cell attachment and proliferation
was studied by Juneja et al. (1994). Exposure of two-cell embryos to neem oil
concentrations of 0.05-0.5% for 1 h, 0.01-0.25% for 12 h, and 0.005-0.1% for
24 h caused significant inhibition in the formation of total and hatching
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blastocysts, in a dose dependent manner. These investigators showed that
neem oil has toxic effects on early embryos and the embryo attachment process
in vitro. Cytotoxic effect of neem-based pesticides on hybridoma cells is
similar to commonly used pesticides. Hybridoma cell line 1E6 was exposed to
Lindane®. Carbofuran®, Cypermethrine®, and Glyphosate® at different
concentrations (Bertheussen et al.. 1997). IC50 values for hybridoma cells were
0.13 mg/L for Lindane®. 0.081 mg/L for Carbofuran®, 0.016 mg/L for
Cypermethrine®, and 0.14 mg/L for Glyphosate®.
Neemix™ was found to be more susceptible to heat, air, and light
treatments than Bioneem^"'^. Toxicity of Neemix™ to D. pulex decreased by
time under light, air and heat treatments. On the other hand, exposure of
Bioneem™ to light, air. and heat did not reduce its toxicity to D. pulex.
Bioneem^” showed higher toxicity to D. pulex after exposure to light and air at
37°C. Results suggest that there is more degradation in Bioneem™ exposed to
light under air at 37°C than at 24°C. The degradation products might be more
toxic to D. pulex. This is corroborated by the findings of many studies which
showed that biodégradation products of AZA had higher biological activity
than the parent compound itself. Ermel et a/. (1991) compared the toxic effects
of a biodégradation product o f AZA, marrangin or 'AZA L’, to the parent
compound, AZA, by conducting a short term bioassay on Epilachna varivestis.
These authors showed that marrangin had higher toxicity to E. varivestis
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(EC50 o f 0.25 mg/L), than AZA (EC50 o f 1 mg/L). Another breakdown
product o f AZA, 22,23-dihydro-11-methoxyAZA, was found to be more toxic
to E. varivestis than the parent compound. The EC50 value o f AZA for E.
varivestis was 13 ppm, whereas 22,23-dihydro-II-methoxyAZA had an EC5Q
of 0.5 ppm for the same species (Ley et al., 1988). Even though Neemix™ had
high acute toxicity to all animals used in this study, it lost its bioactivity when
exposed to environmental factors. This means that Neemix™ run-off to aquatic
environment would have less adverse effects than Bioneem™. Because the
total composition of these pesticides is unknown, it is difficult to explain the
difference in stabilities between Neemix™ and Bioneem™.
Compounds in the nonvolatile fractions of Neemix™ and Bioneem^"'^
showed the same toxicity to D. pulex. The volatile fraction of Neemix™ did
not show any observable harmful effect on D. pulex during 48 h of exposure,
whereas the volatile fraction of Bioneem™ was toxic to D. pulex with an
LC50 o f 0.97 pL/mL. Since the volatile fraction was trapped by condensation
under a vacuum, it is possible that some volatiles in Neemix™, including
possible bioactive components, were lost. Bioneem™ possibly contained
bioactive components that were stable through the fractionation. It was also
foimd that Bioneem^’'* remained active after 9 days of treatment with light imder
air at 37°C. These results suggest ± a t more toxic compounds that were
relatively nonvolatile were produced when Bioneem™ was exposed to light, air
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or high temperature. The same toxic compounds might exist in either the
volatile or nonvolatile fractions of Bioneem™ given the fact that fractionation
was achieved at 65°C. On the other hand, when recombined full formulations
either Neemix™ or Bioneem™ were tested on D. pulex, the toxicity was
enhanced. These findings suggest that there is a synergistic effect of both
nonvolatile and volatile fractions since their combination was more potent
against D. pulex. Since the ingredients in neem formulations are not known, it
is difficult to fully interpret the results of the present study. However, it was
found that the compounds in Bioneem™ are probably more active than those in
Neemix™ based on the fact that toxicity of Bioneem™ at comparable AZA
concentrations were much higher than for Neemix™. Dunkel and Richards
(1998) indicated that inert ingredients in Align™, a neem-based pesticide, were
as toxic as the full formulation. LC50 values for both full formulations and the
inert ingredients were in the range from 2 to 4 mg/L for macroinvertebrates
assayed, Drunella doddsi, Brachycentrus americanus, B. occidentalis, and
Shwala paralella. Antagonistic effects of inert ingredients were observed by
Wan and Rahe (1998) using Glomus intraradices (a symbiotic fungus).
Compared to full formulation o f Neemix™ (IC$o= 130 mg/L), Neemix™
carriers had a higher toxicity to G. intraradices with an IC50 value of 80
mg/L.
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Chemical structure of pure AZA used in toxicological tests was
confirmed by nuclear magnetic resonance spectroscopy (NMR). The results
show that pure AZA had 35 carbon, 45 hydrogen, and 16 oxygen atoms. In the
structure, pure AZA contained 2 epoxide rings and I tigloyl side chain at carbon
I . These results are in agreement with the results reported by Kraus et al. (1987)
who observed a similar chemical shift pattern for the epoxide rings and tigloyl
group. In addition, the high field ^H-NMR measurements of AZA (Table 13)
indicated marginal differences in coupling constants from those reported in the
literature (Kraus et a i, 1987; Bilton et a i, 1987). These differences might be
attributed to the presence of impurities or the different methods used. Overall,
the AZA (-95% pure) used in this study was found to be type A AZA.
Mass spectroscopy data of pure AZA showed that pure AZA used in this
study had a molecular weight of 721.2 daltons. Bilton and his coworkers ( 1987)
reported that AZA’s molecular weight is 720.2. The differences of 1.0 in
molecular weight in the present study might be due to the presence of one extra
hydrogen atom in the structure. Pure AZA is a heat sensitive molecule;
therefore, heating o f the probe in which pure AZA was placed above 200°C
might have caused fi-agmentations o f the AZA molecule. Strong fragment ions
at m/z 685.2 and m/z 667.2 corresponded to composite losses of water. The ion
at m/z 619.1 may have arisen from the loss o f C4H5O2 group in AZA molecule
as suggested by Bilton et at. (1987) who attributed this type o f phenomenon to
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the loss of a C4 unit from the terminal hydroxylated ring of the AZA molecule.
The ion m/z 643.2 could be the result of a composite loss o f acetic acid. The
fragmentation of AZA at m/z 621.2 which was shifted to m/z 619.1 might be
due to the loss of two hydrogen atoms.
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CONCLUSIONS
Neem extracts have been reported to have inhibitory and toxic effects on
a wide range of animals and microorganisms (Schmutterer, 1995). This study
attempted to determine the toxic effects o f neem-based pesticides and pure
azadirachtin on select aquatic crustaceans and mollusks and in two different in
vitro cell culture systems. Results showed that there is a risk o f direct adverse
effects on aquatic crustaceans, mollusks, and insects resulting from
contamination of water bodies with neem-based Insecticides used in pest
management applications. Toxicity of neem-based pesticides to aquatic animals
was in tlie following order: water fleas > oyster embryos > mosquito larvae >
blue crab megalopes > juvenile white shrimps > Juvenile grass shrimps >
Juvenile freshwater snails > Juvenile crayfish. Bioneem™ was more toxic than
Neemix™ to all the species studied. Differences in the susceptibility of the
aquatic animals to neem-based pesticides depended on the formulation of the
pesticide, the animal species, size and age of the test animals. The toxic effect
o f pure AZA was lower than Neemix™ and Bioneem™ at comparable AZA
concentrations. This suggests that AZA may not be the only active compound in
Neemix™and Bioneem™.
The cytotoxic effects o f Neemix™ and Bioneem™ to hybridoma and
oyster cells increased with increased concentration. Growth inhibitory and
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cytotoxic effects o f both pesticides were detected at concentration as low as I|ig
AZA/mL. Pure AZA did not show toxicity to hybridoma cells and oyster cells.
Biological activity of Neemix™ decreased over time when exposed to
light and heat under air. On the other hand, Bioneem™ remained biologically
active and even became more toxic after exposure to light, heat, and air. These
findings suggested that toxicity of Neemix^^ to the aquatic animals may be
reduced under the effects of environmental factors, such as heat, light, and air,
but the reverse may be true for Bioneem™. Similar results were also obtained
for Bioneem^’' fractionated by condensation into two fractions (volatile and
nonvolatile). Both nonvolatile and volatile fractions of Bioneem^''^ showed
high toxicity to D. piilex at relatively low concentrations. Whereas, only the
nonvolatile fraction of Neemi.x™ was toxic to D. pulex.
Nuclear magnetic resonance and mass spectroscopy data confirmed that
pure AZA used in this study was type A with a molecular weight of 721.1
daltons.
From the ecotoxicological point o f view, it should be emphasized here
that the LC50 and IC50 values of from Neemix™and Bioneem™ in the present
study were determined by exposing test animals to pesticides under laboratory
conditions that would represent the worst case contamination scenario (e.g.
accidental applications and spills). These data should not be directly
extrapolated to represent the toxic effect o f neem compounds occurring in the
1 1 1
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field or ecosystems when they are used under prescribed circumstances. Under
field conditions, soil microbial and chemical activities would influence the
effect of these compounds (Woodcock, 1967). However, in the absence of an
alternative testing method that is rapid, reliable, relatively reproducible, and
cost-effective in determining pesticides toxicity to aquatic crustaceans and
mollusks, LC50 and IC50 values obtained from the techniques described in this
research can be used for the initial screening of pesticides before registration.
Future studies should include the testing of the inert ingredients in
neem-based pesticides. Despite the fact that neem-based pesticides are approved
by the EPA, our laboratory results showed that these pesticides may have
adverse toxic effects on aquatic crustaceans and mollusks. Such an effect may
not be acute and massive but rather slow. Especially of concern are small
animals, such as water fleas, oyster eggs, blue crab megalopes and possibly
crayfish and white shrimp eggs. This may create a long term negative impact
on the fishing industry in areas, such as the river deltas, where agricultural run
off waters meet high densities of aquatic animals, especially those in
reproductive or early stages.
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Van Der Nat, J. M., Van der Sluis, W. G., De Silva, K. T. D., and labadie, R. P. 1991. Ethnopharmacognostical survey of Azadirachta indica. J. Ethnopharmacol. 35: 1-24.
Waladde, S.M., Hassanali, A., and Ochieng, S.A. 1989. Taste sensilla responses to limonoids, natural insect antifeedants. Insect Sci. Appl. 10:301-308.
Wan, M. T. and Rahe, J. E. 1998. Impact o f azadirachtin on Glomus intraradices and vesicular-arbuscular mycorrhiza in root inducing transferred DNA transformed roots oïD aucus carota. Environ. Toxicol. Chem. 17:2041- 2050.
126
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Wan, M.T., Watts, R.G., Isman, M B., and Stmb, R. 1996. Evaluation of the acute toxicity to juvenile Pacific Northwest salmon of azadirachtin, neem extract, and neem-based products. Bull. Environ. Contam. Toxicol. 56:432-439.
Watson, R.D., Spaziani, E., and Boilenbacher, W.E. 1989. Regulation of ecdysone biosynthesis in insects and crustaceans: A comparison. In Ecdysone. (Koolman, J. Ed.). p. 188-201. Thieme medical Pub. Inc., New York.
West, A. J. and Mordue, (Luntz), A. J. 1992. The influence of azadirachtin on the feeding behavior of cereal aphids and slugs. Entomolgia. Exp. Appl. 62:75- 79.
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Williams, J. R., Peng, C. V. S., Chuand, R. Y., Doi, R. H., and Mussen, E. C.1998. The inhibitory effect of azadirachtin on Bacillus subtilus, Escherichia coli, and Paenibacillus larvae, the causative agent of american foulbrood in the honeybee. Apis mellifera L. J. Inverteb. Pathol. 72: 252-257.
Wilps, H., Kirkilionis, E., and Muschenich, K. 1992. The effects of neem oil and azadirachtin on mortality, flight activity, and energy metabolism of Schistocerca gregaria- a comparison between laboratory and field locusts. Comp.Biochem.Physiol. 102:67-71.
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Worting, C. R. 1987. The Pesticide Manual: A World Compendium. 8 ^ Ed. The British Crop Protection Council.
Zebitz, C.P.W. 1987. Potential o f neem seed extracts in mosquito control. In Proceedings, 3^^ International Neem Conference on natural Pesticides from the Neem Tree (Azadirachta indica) and other Plants. (H. Schmutterer and K.R.S. Ascher, Eds.), pp. 537-555. GTZ, Eschbom.
127
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
APPENDIXES:
RAW DATA
128
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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APPENDIX A
WATER QUALITY PARAMETERS
For Crayfish Exposed to Neemix
Concentration (pg AZA/mL)
pH DO(mg/L)
Temperature("O
Hardness(mg/L)
Conductivity(pmhos)
0 6.91 ±0.20 8.8011.20 2010.5 >445 401300.625 (0.25) 6.86±0.46 7.5510.90 2010.5 >445 25115
1.25 (0.5) 6.78±0.31 8.1210.56 2010.5 >445 301202.5 (1.0) 6.49±0.26 7.8010.60 2010.5 >445 351155.0 (2.0) 6.3510.56 7.7610.36 2010.5 >445 3512010(4.0) 6.1410.84 7.9010.58 2010.5 >445 3512020 (8.0) 5.7610.66 8.1210.33
hi2010.5 >445 30120
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Concentration (kig AZA/mL)
00.625 (0.69) 1.25 (1.38) 2.5 (2.76) 5.0 (5.55) 1 0 ( 11. 10) 20 (22 .20)
For Crayfish Exposed to Bioneem TM
pH DO(mg/L)
Temperature(“C)
Hardness(mg/L)
6 .88+ 0.227.2110.337.0210.456.9610.565.5410.644.8110.984.5610.74
7.8010.807.1410.986.9510.636.8610.536.5810.696.4210.856.9810.26
2010.32010.52010.52010.62010.52010.32010.5
>445>445>445>445>445>445>445
Conductivity(pmhos)20110251152012025115201102512020115
Values in parenthesis represent the corresponding concentration o f Bioneem™ (pL/mL)
For Crayfish Exposed to Pure Azadirachtin
Concentration (pg AZA/mL)
pH DO(mg/L)
Temperature(°C)
Hardness(mg/L)
Conductivity(jimhos)
0 6.8210.08 8.3810.12 19.611.12 >445 401350 .0 0 1 6.4810.14 8.3410.22 19.611.12 >445 301300 .0 1 6.6210.20 8.2610.22 19.611.12 >445 301300 .1 6.4610.12 8.4210.08 19.611.12 >445 30130
1 6.6410.16 8.3410.14 19.611.1^ >445 30130
■ooû.cgû.
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8" Ov<ë '
3CD
Cp.
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For D. pulex Exposed to NeemixTM
W
Concentration (|ig AZA/mL)
pH DO(mg/L)
Temperature(°C)
Alkalinity(mg/L)
Hardness(mg/L)
Conductivity(pmhos)
0 6.72±0.24 9.2210.02 1911.0 6 >445 100.0156 (0.0062) 6.53±0.35 9.1810.05 1911.0 9 >445 100.0313 (0.0124) 6.11 ±0.25 8.9610.04 1911.0 12 >445 100.0625 (0.0248) 5.6710.23 8.5610.12 1911.0 6 >445 100.0125 (0.0496) 5.3510.32 8.2610.06 1911.0 6 >445 10
0.25 (0.0998) 5.4210.21 8.1310.04 1911.0 6 >445 100.50 (0.196) 5.3710.29 8.0510.02 1911.0 6 >445 10
Values in parenthesis represent the corresponding concentration o f Neemix™ (pL/mL)
For D. pulex Exposed to Bioneem^*^
Concentration pH DO Temperature Alkalinity Hardness Conductivity(pg AZA/mL) (mg/L) (°C) (mg/L) (mg/L) (pmhos)
0 6.6210.02 9.0610.40 18.610.20 6 >445 100.0156 (0.0173) 5.9610.40 8.9410.22 18.610.20 6 >445 100.0313(0.0346) 5.8410.02 8.7610.01 18.610.20 6 >445 100.0625 (0.0692) 5.7910.01 8.3610.02 18.610.20 6 >445 100.0125 (0.138) 5.5910.02 8.1810.02 18.610.20 6 >445 10
0.25 (0.276) 5.5610.20 8.0910.02 18.610.20 6 >445 100.50 (0.552) 5.5310.02 7.9610.02 18.610.20 6 >445 10
V alues in parenthesis represent the corresponding concentration o f Bioneem"' (pL /niL )
CD■ DOQ .C
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For D. pulex Exposed to Pure Azadirachtin
8■D( O '
3.3"CD
Concentration (pg AZA/mL)
pH DO(mg/L)
Temperature(°C)
Alkalinity(mg/L)
Hardness(mg/L)
Conductivity(pmhos)
0 8.82±0.02 2011.0 6 >445 100.0156 8.53+0.65 2011.0 6 >445 100.0313 8.3710.22 2011.0 6 >445 100.0625 8.1910.09 2011.0 6 >445 100.0125 8.0210.11 2011.0 6 >445 10
0.25 7.6510.32 2011.0 6 >445 100.50 7.2310.52 2011.0 6 >445 10
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WWFor Blue Crab Megalopes Exposed to NeemixTM
Concentration (pg AZA/mL)
pH DO(mg/L)
Temperature(°C)
Salinity(%o)
Hardness(mg/L)
Alkalinity(mg/L)
0 8.0910.08 2511.0 30 >445 2040.25 (0.1) 8.0510.05 2511.0 30 >445 2020.5 (0.2) 8.0610.02 2511.0 30 >445 2321.0 (0.4) 8.1110.03 2511.0 30 >445 2282.0 (0.8) 8.1310.06 2511.0 30 >445 2244.0 (1.6) 8.2110.02 2511.0
___ . Til": ■ -30 >445 234
V alues in parenthesis represent the corresponding concentration o f Neemix (pL/m L)
CD■ DOQ .C
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8■D
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3.3"CD
For White Shrimp Exposed to NeemixTM
Concentration (|ig AZA/mL)
PH DO(mg/L)
Temperature(°C)
Salinity(%o)
0 8.12±0.20 7.80+0.26 25 330.625 (0.25) 8.08±0.02 7.64+0.32 25 33
1.25 (0.5) 8.04±0.23 7.84+0.08 25 332.5 (1.0) 7.99+0.12 7.62±0.22 25 335.0 (2.0) 7.82+0.26 7.24±0.82 25 3310(4.0) 7.86+0.28 6.84+0.28 25 33
Values in parenthesis represent the corresponding concentration o f Neemix'** (pL/mL)
CD■oOQ .Cg.o3
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CDQ .
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For Grass Shrimp Exposed to NeemixTM
Concentration(mg/L)
pH DO(mg/L)
TemperatureC O
Salinity(96o)
Conductivity(pmhos)
Hardness(mg/L)
Alkalinity(mg/L)
0 8.0 8.10+0.2 2611.0 22 33,000 >445 651240.625 (0.25) 8.0 8.0410.3 2611.0 22 33,000 >445 45123
1.25 (0.5) 8.0 7.9410.6 2611.0 22 33,000 >445 631122 5 (1 .0 ) 8.0 7.7210.7 2611.0 22 33,000 >445 681145.0 (2.0) 8.0 7.3710.5 2611.0 22 33,000 >445 5711310(4.0) 8.0 7.4410.9 2611.0
" _____ . V U
22 33,000 >445 5919.0
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enen
For Grass Shrimp Exposed to Bioneem TM
Concentration(mg/L)
pH DO Temperature Salinity (mg/L) (°C) (%o)
Conductivity(pmhos)
Hardness(mg/L)
Alkalinity(mg/L)
0 8.0 8.44+0.4 26+1.0 22 33,000 >445 5212.00.625 (0.69) 8.0 8.16±0.6 26±1.0 22 33,000 >445 4913.01.25 (1.38) 8.0 7.99+0.8 26±1.0 22 33,000 >445 4812.02.5 (2.76) 8.0 7.8310.2 26±I.O 22 33,000 >445 4412.05(5.55) 8.0 7.4610.5 2611.0 22 33,000 >445 5512.0
10(11.10) 8.0 7.3210.6 2611.0 22 33,000 >445 5913.0Values in parenthesis represent the corresponding concentration o f Bioneem™ (pL/mL)
For Freshwater Snails Exposed to Neemix^
Concentration PH DO Temperature Alkalinity Hardness Conductivity(pg AZA/mL) (mg/L) (°C) (mg/L) (mg/L) (pmhos)
0 6.12±0.24 9.1710.32 1911.0 9 >445 100.313(0.125) 6.42±0.21 8.4110.33 1911.0 6 >445 100.625 (0.25) 5.96+0.09 8.1010.62 1911.0 6 >445 101.25 (0.50) 5.78+0.13 7.9510.12 1911.0 6 >445 102.5 (1.0) 5.59+0.26 7.8110.36 1911.0 9 >445 105.0 (2.0) 5.11+0.54 6.9510.27 1911.0 9 >445 1010(4.0 4.75±0.14 6.6510.41 1911.0 9 >445 10
Values in parenthesis represent the corresponding concentration o f Neemix (pL/m L)
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8■D
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1 S■Oo
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For Freshwater Snails Exposed to Bioneem"^
Concentration (|ig AZA/mL)
PH DO(mg/L)
Temperature(°C)
Alkalinity(mg/L)
Hardness(mg/L)
Conductivity(pmhos)
0 6.12±0.24 9.1710.32 1911.0 9 >445 100.313(0.34) 5.08±0.06 8.6510.26 1911.0 6 >445 100.625 (0.69) 4.62±0.23 8.1010.21 1911.0 6 >445 101.25(1.38) 4.43±0.35 7.9510.16 1911.0 6 >445 102.5 (2.76) 4.31 ±0.22 7.7810.29 1911.0 6 >445 105.0 (5.55) 4.0910.19 7.5110.14 1911.0 9 >445 1010(11.10) 4.0110.13 6.8410.28 1911.0 9 >445 10
Values in parenthesis represent the corresponding concentration o f Bioneem™ (pL/mL)
For Freshwater Snails Exposed to Pure Azadirachtin
Concentration PH DO Temperature Alkalinity Hardness Conductivity(|ig AZA/mL) (mg/L) (°C) (mg/L) (mg/L) (pmhos)
0 6.4310.23 8.8010.02 2111.0 6 >445 100.15 6.2910.12 8.6010.21 2111.0 6 >445 100.30 6.2210.34 8.1210.32 2111.0 6 >445 101.50 6.1810.29 8.0610.14 2111.0 6 >445 103.0 6.0510.31 7.9610.06 2111.0 6 >445 1030 5.9110.52 7.1710.13 2111.0 6 >445 10
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For Oyster Larvae Exposed to NeemixTM
8■D
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ON
Concentration (pg AZA/mL)
pH DO(mg/L)
Temperature Salinity (°C) (%*)
Conductivity(pmhos)
Hardness(mg/L)
Alkalinity(mg/L)
0 7.7710.7 7.810.5 2513 20 28 >445 72160.0625 (0.0124) 7.7710.7 7.810.5 2513 20 28 >445 72160.125 (0.0248) 7.7710.7 7.810.5 2513 20 28 >445 72160.25 (0.0496) 7.7710.7 7.810.5 2513 20 28 >445 72160.50 (0.0992) 7.7710.7 7.810.5 2513 20 28 >445 7216
1.0 (0.198) 7.7710.7 7.810.5 2513 20 28 >445 72162.0 (0.396) 7.7710.7 7.810.5 2513 20 28 >445 7216
Values in parenthesis represent the corresponding concentration o f Neemix'" (pL/mL)
For Oyster Larvae Exposed to Bioneem^
Concentrations pH DO Temperature Salinity Conductivity Hardness Alkalinity(pg AZA/mL) (mg/L) (°C) (% o) (pmhos) (mg/L) (mg/L)
0 7.7710.7 7.810.5 2513 20 28 >445 72160.0625 (0.034) 7.7710.7 7.810.5 2513 20 28 >445 72160.125 (0.068) 7.7710.7 7.810.5 2513 20 28 >445 72160.25 (0.136) 7.7710.7 7.810.5 2513 20 28 >445 72160.50 (0.272) 7.7710.7 7.810.5 2513 20 28 >445 72161.0 (0.544) 7.7710.7 7.810.5 2513 20 28 >445 72162.0(1.08) 7.7710.7 7.810.5 2513 20 28 >445 7216
■oIcgQ.
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=3CD
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For Mosquito Larvae Exposed to Neemix TM
Concentration (pg AZA/mL)
PH DO(mg/L)
Temperature(°C)
Hardness(mg/L)
0 6.96+0.04 8.0310.30 2211.0 >4450.0313(0.0124) 6.72+0.61 8.0910.25 2211.0 >4450.0625 (0.0248) 6.63+0.22 8.1010.12 2211.0 >4450.125 (0.0496) 5.8310.10 8.0910.09 2211.0 >4450.25 (0.0992) 5.7210.13 8.1110.27 2211.0 >4450.50 (0.198) 5.6310.24 8.1210.12 2211.0 >4451.0 (0.396) 5.4210.28 8.0810.16 2211.0 >445
Values in parenthesis represent the corresponding concentration o f Neemix (pL/mL)
&
oc
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o'=3
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For Mosquito Larvae Exposed to Bioneem TM
8■D
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woo
Concentration PH DO Temperature Hardness(pg AZA/mL) (mg/L) (°C) (mg/L)
0 6.93±0.10 8.1010.18 2211.0 >4450.0313(0.034) 6.66±0.08 8.0010.24 2211.0 >4450.0625 (0.068) 6.11 ±0.25 7.8110.54 2211.0 >4450.125(0.137) 5.7910.23 7.7110.01 2211.0 >4450.25 (0.274) 5.6410.16 7.6210.33 2211.0 >4450.50 (0.551) 5.3310.31 7.6310.12 2211.0 >445
1.0(1.10) 5.2110.11 75610.36 2211.0 >445Values in parenthesis represent the corresponding concentration o f Bioneem™ (pL/mL)
For Mosquito Larvae Exposed to Pure Azadirachtin
Concentration PH DO Temperature Hardness(|ig AZA/mL) (mg/L) (°C) (mg/L)
0 6.9410.14 8.1010.23 2211.0 >4451.25 6.7510.06 8.0910.02 2211.0 >4452.5 6.3510.32 7.8010.21 2211.0 >4455.0 5.7810.02 7.6910.11 2211.0 >44510 5.3310.11 7.5910.20 2211.0 >445
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APPENDIX B
ABSORBANCE VALLES
8■D
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For Hybridoma Cells Exposed to Neemix
W'O
Concentration (pg AZA/mL)
00.01 (0.004)
0.1 (0.04) 1.0 (0.4) 10(4.0) 100 (40)
Duration (h)
24 48 720.494±0.01?b 0.646±0.009 b 0.646+0.009 b 0.554±0.003 b 0.266+0.007 b 0.20710.009 b
0.51210.002 b 0.59710.010 b0.56710.005 b 0.50510.007 b 0.19210.003 b 0.16810.004 b
* Absorbance was measured at 550 nm " n = 5 , ± S EValues in parenthesis represent the corresponding concentration o f Neemix™ (pL/mL)
0.43110.007 b 0.51710.006 b 0.48510.013 b 0.38510.006 b0.15510.002 b 0.17510.005 b
960.14110.008 b 0.16010.009 b 0.13810.004 b 0.18610.003 b 0.13010.002 b 0.18710.001 b
■DCD
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8■D( O '
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CD■DOQ .C
aO
Concentration (pg AZA/mL)
For Hybridoma Cells Exposed to Bioneem T M,
Duration (h)
24 48 72
n= 5, ± SEValues in parenthesis represent the corresponding concentration o f Bioneem " (pL/mL)
ê
960 0.583±0.009 b 0.47110.010 b 0.45610.006 b 0.10810.002 b
0.01 (0.011) 0.56510.007 b 0.51210.009 b 0.53210.005 b 0.11610.001 b0.1 (0.11) 0.56810.007 b 0.46910.005 b 0.47910.006 b 0 .10210.004 b1.0(1.1) 0.48610.007 b 0.36910.004 b 0.37210.014 b 0 .11510.007 b
10(11.10) 0.21510.004 b 0.16410.002 b 0.17910.003 b 0.13810.003 b100(111 1) 0.26110.009 b 0.24210.002 b 0.20010.005 b 0.16210.004 b
■DO
CDQ .
■DCD
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■DCD
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For Hybridoma Cells Exposed to Pure Azadirachtin
8■D( O '
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CD■DOQ .CaO3"Oo
Concentration (pg AZA/mL)
Duration (h)
24 48 72 960 0.676+0.009 0 0.65110.009 b 0.23810.010 b 0.12210.0009*5
0.01 0.674±0.016b 0.67010.006 b 0.24010.007 b 0.19310.007 b0.1 0.68410.004 b 0.66010.003 b 0.27410.026 b 0.12610.006 b1.0 0.64710.021 b 0.67110.012 b 0.21510.003 b 0.11610.006 b10 0.65010.006 b 0.65710.007 b 0.18510.014 b 0 .11210.005 b
100 0.66910.006 b 0.65010.018 b 0.22110.005 b 0 . 15010.014 b** Absorbance was measured at 550 nm
n = 5 , ± S E
CDQ .
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For Oyster Cells Exposed to NeemixT M,
CD
8■D( O '
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CD■DOQ .CaO3"Oo
Concentration (jig AZA/mL)
Duration (h)
24 480 0.384+0.015 0.35210.018
0.01 (0.004) 0.374±0.027 0.32210.0220.1 (0.04) 0.370±0.015 0.31510.0271.0 (0.4) 0.37310.013 0.18610.01610(4.0) 0.21210.012 0.07310.0012100 (40) 0.20910.0009 0.15110.001
* Absorbance was measured at 550 nm ** n= 5, ± SEValues in parenthesis represent the corresponding concentration o f Neemix " (pL/mL)
to
CDQ .
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CD
8■D( O '
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CD■DOQ .C
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Concentration (pg AZA/mL)
00.01 (0.0 II)
0.1 (0 . 11) 1.0 ( 1. 1)
1 0 ( 1 1 1 0 )
1 0 0 ( 111.1)
For Oyster Cells Exposed to Bioneem T M ,
Duration (h)
241.196±0.270 1.476±0.140 1.295±0.100 1.041 ±0.044 0.369+0.0070.047+0.025
' Absorbance was measured at 550 nm *’ n= 5, ± SEValues in parenthesis represent the corresponding concentration o f Bioneem " (pL/mL)
480.547±0.0300.643±0.0240.669±0.0250.628±000220.428+0.0230.13910.014
CDQ .
■DCD
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CD■ DOQ .C
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8■D( O '
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(/)(/)
For Oyster Cells Exposed to Pure Azadirachtin"
Concentration (pg AZA/mL)
Duration 24 h
0 0.362±0.040.1 0.315+0.041.0 0.334±0.0410 0.351 ±0.05
100 0.403+0.0023. Absorbance^ n= 5, ± SECD■DOQ .CaO 3"Oo
CDQ .
VITA
The author was bom in Istanbul, Turkey, on June 17, 1970. She
graduated from the University of Istanbul, Turkey, with a bachelor of science
degree in Aquacultural Engineering in June 1992. In 1993, she was awarded a
scholarship by the Turkish Higher Education Council to pursue a master of
science degree in Seafood Processing Technology in the United States of
America. In the Fall semester of 1994, she was accepted as a graduate student
in the Department o f Food Science, Louisiana State University, Baton Rouge,
Louisiana, where she received a master’s degree in December 1996. In the same
year, she was awarded an assistantship under the supervision of Dr. Leslie C.
Plhak to pursue a doctoral program in the same department. She is currently a
candidate for the degree of Doctor of Philosophy in Food Science.
Ms. Goktepe is a member of the Institute of Food Technologists (IFT),
Society of Environmental Toxicology and Chemistry, and IFT Student
Association, IFT Gulf Coast Section. She served as a South Central Area
representative of the IFT Student Association during 1998-1999, as a president
of the Food Science Club at LSU from 1998 to 1999, as an associate in the
LSU Union International Committee in 1998-1999, and as a treasurer in the
Turkish American Student Association from 1996 to 1999.
Her future plans include being an enthusiastic researcher and
academician in the area o f food science and environmental toxicology.
145
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
DOCTORAL EXAMINATION AND DISSERTATION REPORT
Candidate: Ipek Goktepe
Major Field: Food Science
Title of Dissertation: Toxicity of Neem-Based Insecticides onAquatic Animals and Cell Lines
Approved:
Kator Professor and/^Zbaimian
EXAMINING COMMITTEE:
Date of Baamination :
June 4, 1999
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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