Toxicity of Neem-Based Insecticides on Aquatic Animals and

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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


UMI Number 9960055

UMI'UMI Microform9960055

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DEDICATION

To MY DEAR FATHER

whose dedication to learning is a continuing inspiration to me

11


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


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


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


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


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


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


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


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


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


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


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).


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


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


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.


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).


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


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


PH

HO Azadirachtol

OAc

Nimbin

SalanninAcO

'-0

Figure 2a. Other bioactive compounds in neem.


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


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


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


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


(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


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


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


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


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


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


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


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


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


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


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


■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


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


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

28


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

29


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,

30


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

31


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

32


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

33


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

quinquefasciatus 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

34


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

35


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).

36


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)

37


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

38


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,

39


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

40


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

41


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

42


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.

43


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

44


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™

45


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

46


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.

47


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


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


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


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


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


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


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


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


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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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)

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Concentration (|ig AZA/plate)

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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)

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Figure 8. Mutagenic potential of AfB, on S. typhimurium types TA-98 and TA-100 with metabolic activation

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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


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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200□ % Mortality

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0.0313(0.012)

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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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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)

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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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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


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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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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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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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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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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75


CD■ DOQ .C

gQ .

■DCD

C/)(/)

8"O

CD

3.3"CD

CD■DOQ .C

aO3"OO

CDQ .

■DCD

C/)C /)

Os

ss 48 h24 h

esSaa■ei 0.5.a<

1000.01 100 0 1(0 ) (0.004) (0.04) (0.4) (4) (40)*


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

gQ .

■DCD

C/)C/)

8■D( O '

3.3"CD

CD■DOQ .C

aO3"OO

CDQ .

■DCD

(/)(/)

-J

48 h24 h

Ea

goes

I<

0 0.01 0.1 10 100(0) (0 .011) (0 . 1 1 ) ( 1 . 1 )


( 1 1 . 1 1 ) ( 1 1 1 )*

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


o

dfNm

do o

<BDase2-wS3sou

m a 06^ |B aao sq jo sq y

w2S12

10a.2JS1

S

Iuau

w

0se

23

1

79


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)

80


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

81


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

82


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.

83


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


m

incn

o

ZQ .a

in

into .

ors.

Figure 21. Nuclear Magnetic Resonance (NMR) Spectra of Azadirachtin

85


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

86


[

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


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


o•H

U53U2"vo

JO X3T5U33UI

Figure 23. Mass Spectra of Azadirachtin

89


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.

90


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

91


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).

92


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

93


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

94


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

95


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

96


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

97


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.

98


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.

99


(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

100


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

101


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

102


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

103


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

104


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

105


(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

106


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.

107


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

108


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.

109


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

110


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


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.

112


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127


APPENDIXES:

RAW DATA

128


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APPENDIX A

WATER QUALITY PARAMETERS

For Crayfish Exposed to Neemix


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

CDO .

"OCD

(/)(/)

CD■ DOQ .C

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CD

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CD■ D

OQ .C

aO3" O

O

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■ DCD

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U>O

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


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

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For D. pulex Exposed to NeemixTM

W


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

gQ .

■DCD

C/)C/)

For D. pulex Exposed to Pure Azadirachtin

8■D( O '

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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

CD■DOQ .C

aO3"OO

CDQ .

■DCD

(/)(/)

WWFor Blue Crab Megalopes Exposed to NeemixTM


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

gQ .

■DCD

C/)Wo"3

8■D

CD

3.3"CD

For White Shrimp Exposed to NeemixTM


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

■DO

CDQ .

■DCD

C/)(/)

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

CD■ DOQ .C

gQ .

■DCD

C/)Wo"3

8■D

CD

3.3"CD

CD■DOQ .C

aO3

"OO

CDQ .

■DCD

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)

CD■ DOQ .C

gQ .

■DCD

C/)C/)

8■D

CD

3.3"CD

CD■D0 Q .

1 S■Oo

CDQ .

■DCD

C/)C /)

For Freshwater Snails Exposed to Bioneem"^


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

CD■ DOQ .C

gQ .

■DCD

C/)C/)

For Oyster Larvae Exposed to NeemixTM

8■D

CD

3.3"CD

CD■DOQ .C

aO3"DO

CDQ .

■DCD

C/)C /)

ON


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.

■OCD

C/)(gO=3

CD

85cB'

=3CD

C=TCD

CD■OIC

aO=3

■OO

For Mosquito Larvae Exposed to Neemix TM


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

C/)

o'=3

CD■ DOQ .C

gQ .

■DCD

C/)C/)

For Mosquito Larvae Exposed to Bioneem TM

8■D

3.3"CD

CD"DOQ .C

aO3"Oo

CDQ .

■DCD

C/)C /)

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

CD■ DOQ .C

8Q .

■DCD

C/)C/)

APPENDIX B

ABSORBANCE VALLES

8■D

CD

3.3"CD

CD■DOQ .C

aO3"DO

CDQ .

For Hybridoma Cells Exposed to Neemix

W'O


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

C/)C /)

CD■ DOQ .C

gQ .

■DCD

C/)(/)

8■D( O '

3.3"CD

CD■DOQ .C

aO


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

(/)(/)

CD■ DOQ .C

gQ .

■DCD

(/)C/)

For Hybridoma Cells Exposed to Pure Azadirachtin

8■D( O '

3.3"CD

CD■DOQ .CaO3"Oo


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 .

■DCD

(/)(/)

CD■ DOQ .C

gQ .

■DCD

C/)C/)

For Oyster Cells Exposed to NeemixT M,

CD

8■D( O '

3.3"CD

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 .

■DCD

(/)(/)

CD■ DOQ .C

gQ .

■DCD

C/)C/)

CD

8■D( O '

3.3"CD

CD■DOQ .C

aO3"OO


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

(/)(/)

CD■ DOQ .C

gQ .

■DCD

C/)C/)

8■D( O '

"OCD

(/)(/)

For Oyster Cells Exposed to Pure Azadirachtin"


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


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


Documents

Toxicity of Neem-Based Insecticides on Aquatic Animals and