Chemosphere,Vol. 39, No. 13, pp. 2347-2356, 1999
© 1999ElsevierScienceLtd. All rightsreserved 0045-6535/99/$ - see front matter
TOXICOLOGICAL STUDY OF TWO NOVEL PESTICIDES ON EARTHWORM EISENIA FOETIDA
YU LUO, Y U ZANG, YUAN Z H O N G and Z H I M I N G KONG* National Key Laboratory of Pollution Control and Resource of China, Department of Environmental Science & Engineering, Nanjing University, Nanjing 210093, People's Republic of China (Receivedin Germany25 February 1999;accepted22 March 1999)
Abstract: In this paper, several studies were conducted to evaluate the toxicity of two pesticides, Imidacloprid and RH- 5849 on earthworm (Eisenia foet~da) in terms of their acute toxicity, biochemical toxicity and effects on sperm morphological deformity. LCs0for earthworms indicated that these two pesticides had different effects in different exposure systems. The results of biochemical toxicity tests showed that lower concentrations of Imidacloprid (< 0.2mg/l ) and RH-5849 (<25rag/1) could be observed to inhibit the activity of cellulase. As to the influence to SOD, they both expressed effects of promotion to a certain degree, except that lower concentrations of lmidacloprid (0. lmg/1 ) could suppress SOD activity. In this study, sperm deformity test was developed to detect the potential adverse influences of pesticides on the reproduction of earthworms. The results demonstrated that significant induction of sperm deformity (p<0.01) and dose-effect relationship displayed when Imidacloprid concentrations higher than 0.5mg/kg dry soil. However, the sperm deformity frequency of RH-5849-expesed groups did not show significant difference (I)>0.05) from the control until the dose reached 100mg/kg dry soil. © 1999 Elsevier Science Ltd. All rights reserved Keywords: Pesticides, Eiseniafoetida, Acute toxicity, Biochemical toxicity, Sperm deformity test
INTRODUCTION Imidacloprid and RH-5849 are two kinds o f novel pesticides that are getting into use in China. The ecotoxicity studies concerning their effects on soil organisms are needed in order to evaluate their toxicity and to prevent the potential risk o f these environmental pollutants on the terrestrial ecosystems. Some toxicity tests on the two pesticides and studies about the physical-chemical behavior of the two pesticides in soil have been conducted [1,2], however, the effects o f pesticides on earthworms and their ecotoxicity on the terrestrial ecosystems have received relatively little attention. Earthworms are important organisms in the soils and they appear to be the best organisms for use in soil toxicity evaluation. Earthworms are probably the most relevant soil species, representing 60to 80% of the total animal biomass in soil . They also contribute a lot to the organic matter degradation and soil fertility . For these reasons, earthworms have gained acceptance for use in tests to assess the effects of chemicals on soil organisms. Several earthworm protocols have been developed to assess the effects o f chemicals on earthworms (Eiseniafoetida) among which the most well-known is the OECD guideline 207 , a 14-day * Corresponding author.
2348 artificial-soil test. Although these tests are useful in assessment of the toxicity of pesticides on earthworm, they have limited ecological significance  because many sublethal effects such as changes in behavior, reproduction and inhibition on enzyme activities are not addressed in standard acute toxicity tests . In this paper, we reported the acute toxicity tests in different exposure systems( in solution, in artificial soil and on filter paper), the biochemical toxicity in terms of effects on enzyme activities of cellulase and SOD, and effects of pesticides on sperm morphological deformity. Our objective is to get a more comprehensive understanding on the effects of pesticides on earthworms and provide more information about the potential ecological risk of chemicals on soil ecosystem. MATERIALS AND METHODS
Test organism and test chemicals The earthworm species Eiseniafoetida was selected as the test organism. It is widely available, easily reared in laboratory culture and reproduces rapidly and steadily relative to other earthworm species. Healthy earthworms of about 120 days old and weighed about 350 mg were used for all experiments. lmidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitro-imidazolidin-2-ylideneamine] and RH-5849 [2'-benzoyl-l'-tert-butylbenzoylhydrazine]were obtained from Jiuangsu Institute of Pesticides and were over 95% pure. Imidacloprid was dissolved in distilled water to different concentration and RH-5849 was dissolved in a littleTween-80 first and diluted in distilled water to different concentration
Acute toxicity tests Earthworms (Eiseniafoetida) were exposed to different exposure systems: in solution, in artificial soil and on filter paper. In solution: As described by Belfroid [9,12], earthworms were half immersed in distilled water in beakers and were incubated at 20+ Ic' for 48h to make them purge their gut contents. Earthworms were put individually in glass tubes (3X8cm) containing lml of different concentrations of pesticides solution. Six earthworms were used for every concentration. According to the result of the preliminary tests, four concentrations of each compound were designed: 0.24rag/L, 0.48rag/L, 0 96mg/L and 2.00mg/L for Imidacloprid and l1875mg/L, 237.50mg/L, 475.00mg/L and 950.00mg/L for RH-5849. For the controls, earthworms were put in 1 ml of distilled water for lmidacloprid and lml of 1% Tween-80 solution for RH-5849 respectively. The tubes with worms were incubated at 20+1°Cfor 48h and earthworms were counted after 24h and 48h respectively. Worms were considered dead when they did not respond to gentle touching The data was calculated by TSK computer program and the values of LC~, were finally given. Onfilter paper: According to the standard test procedure recommended by the EPA of China  and method of Zhang Chenwu [1 I], after the earthworms empty gut, they were rinsed in distilled water and dried on filter paper. The compound was dissolved in acetone and lml of solution was added to the paper in glass tube (3xScm). After evaporating the acetone, lml of demineralized water was added to keep moisture and one earthworm was placed in the
2349 tube. Every six earthworms were used to each concentration. Exposure concentrations were designed according to the results of preliminary tests: 0.004ug/cm 2, 0.020 ug/cm 2, 0.100 ug/cm 2 and 0.500 ug/cm 2 for Imidacloprid. For Rh-5849, no worms were observed dead even under 480 ug/cm 2 which is the highest concentration of RH-5849 that could be absorbed by filter paper. For the controls, lml of acetone was used. Earthworms were incubated and counted as described above. In artificial soil: Artificial soil according to OECD with a water content of 35% on wet weight basis was used in these experiments. The soil was contaminated with pesticides by mixing the solution of the compounds with the test soil to make the final concentrations of Imidacloprid are 1, 2, 4, 8,16 mg/kg dry soil and 125, 250, 500, 1000, 2000, 4000mg/kg dry soil for RH-5849. Earthworms were incubated in uncontaminated artificial soil for 24h and ten of them were added to contaminated soils of different concentration contained in broadopen bottles(lL). Earthworms in the bottle were incubated at 20+1 °c for 14 days. Water was added regularly in order to keep the moisture of 80%o. Worms were counted at the 3th, 7th and 14th days respectively. They were considered dead when they did not respond to gentle touching and were removed immediately.
Effects on enzyme activi(y Earthworms were exposed in solution of pesticides. Enzyme activities of cellulase and SOD were determined under three different concentrations of each compound and during different exposure time. Six earthworms were used for each concentration and exposure time. Enzymes were extracted following the method of Mishra [ 13] with modification. Earthworms were rinsed in distilled water and dried on filter paper. Homogenates of earthworms were made in cold distilled water and were centrifuged at 2500rpm for 10min. The supernatant fluid was removed into another centrifuge tube and were centrifuged at 3000rpm for 5min.The supernatant fluid was collected and was use to test enzyme activity. The protein content was determined as the method ofZhang Longxiang  using bovine serum albumin as standard. 0.5ml of enzyme preparation was added into 0.5ml of distilled water and 4ml of biuret solution and the reaction system was incubated at 20-25°C for 30 min. The A540 was tested and the content of protein was determined according to the standard curve. The method of Zhang Dean [ 15] was followed to determine cellulase activity. 0.5ml of enzyme preparation was added into test-tube containing 1.0ml preheated CMC-Na and the mixture was incubated at 50°c for 30rain. After reaction for 30min, the concentration of glucose was determined by adding DNS and test As~,,. The enzyme activities are expressed in mg of glucose per mg of protein per hour. The SOD activity was determined by the method of Yang T.B. et al. . 0.1 ml of enzyme preparation was kept at 50°C for 10 min and was added into test tube containing 3.5ml of phosphorous buffer, 0.5ml of EDTA(lumol/I) and 0.5ml of Met. 0.2ml of lactochrome and 0.2ml of NBT were added into the test tube in dark and the reaction system was moved into 10 cm below 2 × 4 0 W fluorescence lamp for 20 min. As60 was tested immediately and the unit of enzyme activity was pressed as 50% inhibition of the photochemical reaction of NBT.
Sperm deformity test Earthworms were exposed in artificial soil. The exposure concentrations were designed according to the results of acute toxicity tests. Distilled water was used as negative control and cyclophosphamide as positive control. Six earthworms were exposed at each concentration for 10 days. After anesthetized in 12% ethanol, the earthworm was dissected and spermatheca was removed. Spermatheca was placed between two glass slides, and by exerting pressure the sperm were forced out of the sacs. Sperms were then fixed in methanol and stained in Giemsa. The slides were observed under optical microscopy (×400) to detect abnormality in sperm morphology. 1000 sperms were detected for every slide and sperm deformity was calculated. RESULTS AND DISCUSSION
Acute toxicity tests Table 1. LCs0of Imidacloprid and RH-5849 to earthworms in different exposure systems Exposure method Exposure time LCs,~ of Imidacloprid LCs0 of RH-5849 In solution (mg/L)
On filter paper (ug/cm2)
In artificial soil (m$/k$ dr3' soil)
7 days 14 days
LCs0 of Imidacloprid and RH-5849 to earthworms in different exposure systems were calculated by TSK computer program and were shown in Table 1. It can be seen from the table above that the acute toxicity of lmidacloprid to earthworms (Eiseniafoetida) was higher than that of RH-5849. This is easy to understand because these two pesticides are different in their physical and chemical characters. Although the two pesticides did not have much difference in their molecular weight, they do have significant difference in their ability to dissolve in water, which will affect the bio-availability of compounds in environment. In the solution and filter paper exposure systems, the pesticides were absorbed mainly by skin, and in the artificial soil, the pesticides were absorbed mainly by gut. Because RH-5849 was more difficult to dissolve in water and organic dissolvant and is more difficult to be absorbed by skin, the toxicity of Imidacloprid was more than 550 times higher than the RH-5849 in solution and filter paper. However, the difference was reduced to about 200 times in the artificial soil, which showed that RH-5849 was easier to be absorbed by gut than by skin. For the RH-5849, the highest concentration that could be absorbed by filter paper was 480ug/cm 2 and no worms were observed dead under this concentration. Thus we concluded that the LCs,, of RH-5849 on filter paper was higher than 480ug/cm 2, Although the acute toxicity tests can provide only limited information on the true potential risk to soil organisms and it is difficult to give correlation between the results in different exposure systems, the acute toxicity tests can serve to classify chemicals according with their ecotoxicity [ 17]. Analyzing the sublethal
2351 effects of pesticides on earthworms and their transportation and degration in soils will help us better understand their risk to the soil fauna and environment.
Biochemical toxicity The cellulase and SOD activities in E. F under different exposure concentrations and over different exposure time of Imidacloprid and RH-5849 were shown in table 2-5. The results (Table 2.) showed that the Imidacloprid could be observed to inhibit the activity of cellulase. The cellulase activities in Eisenia foeada were decreased with an increase in the exposure concentrations of Imidacloprid and had especially significant reduction (P<0.01) when the concentration of Imidacloprid reached 0. lmg/l. From the results of exposure to Imidacloprid at the concentration of 0.2mg/l over different time, we can find the same inhibition effect. The cellulase activities in Eiseniafoetida were decreased with an increase in the exposure time of Imidacloprid (Table 3.) and had significant reduction (P<0.05) when the exposure time reached 3 hours. However, when the concentration of Imidacloprid was high as the 1/2 LCs0 (0.5mg/l), the ceUulase activity o f EJsenia foeada repromoted to be higher than that of exposed to 0.1mg/1 and lower than the control, which may be caused by the death of earthworm and auto-lysis of earthworms. RH-5849 showed the similar inhibition effects on the cellulase activity of Eisenia foetida, but the results did not show significant statistical differences. Table 2. Effects of Imidacloprid and RH-5849 on the cellulase activity in EiseniafoetTdaunder different exposure concentrations. (exposure time: 4 hours) Imidaeloprid RH-5849 Concentrations Cellulase activity Concentrations (mg/l) Mean£+SD (mg/1) 0 0.352_+0.053 0 0.1 0.253_+0.037 5 0.2 0.233_+0.025 25 0.5 0.265+_0.031 50 (the unit of enzyme activity: mg of glucose per mg of protein per hour).
Cellulase activity MeanA+SD 0.359+-0.089 0.295+-0.026 0.271_+0.069 0.363_+0.062
Table 3. Effects of Imidaeloprid and RH-5849 on the cellulase activity in EisenJafoet~daover different exposure time. Imidaeloprid (0.2mg/1) RH-5849 (25rag/l) Time
Cellulase activity Time Mean~+SD 0h 0.352_+0.053 0h 1h 0.325_+0.057 1h 2h 0.298_+0.045 2h 3h 0.255_+0.051 3h 4h 0.233_+0.025 4h (the unit of enzyme activity: mg of glucose per mg of protein per hour).
Cellulase activity Mear~+SD 0.359+_0.089 0.339_+0.064 0.320+0.069 0.284+-0.059 0.271+-0.073
Table 4-5 showed the effects of the two pesticides on the SOD activity in Eisenia foetida. It can be seen from Table 4. that Imidacloprid showed inhibition effect on the SOD
2352 activity at low concentration and promotion effect at higher concentrations. After 4 hours' exposure under the concentration of 0.1mg/1, the SOD activity decreased especially significantly (P<0.01) and as the concentrations increased (0.2mg/1, 0.5mg/1) the SOD activities began to increase and were higher than the control. As to the effect of Imidacloprid over various exposure time, it showed inhibition effects on the SOD activity of earthworms in lh, 2h and 3h, but promoted the SOD activities when the exposure time reached 4 hours. RH5849 did not have inhibition effect on the SOD activity. The SOD activities were observed to have especially significant increase under the exposure concentration of 5mg/1 and continued to promote with the increase of concentrations of RH-5849 Shown from the results of different exposure time under 25mg/l RH-5849, the SOD activities increased when the exposure time extended and the results showed the dose-effect relationship. Table 4. Effects of lmidacloprid and RH-5849 on the SOD activity in Eiseniafoetida under various exposure concentrations. (exposure time: 4 hours) Imidacloprid RH-5849 Concentrations SOD activity (mg/l) Mean_+SD 0 0.0585_+0.0084 0.1 0.0394_+0.0036 0.2 0.0588_+0.0078 0.5 0.0674+0.0087 (the unit of enzyme activity: U per mg of protein ).
Concentrations (mg/l) 0 5 25 50
SOD activity Mean~+SD 0.0572+0.0080 0.0822_+0.0191 0.1033_+0.0197 0.0979_+0.0091
Table 5. Effects of Imidaclopnd and RH-5849 on the SOD activity in Eisenia foetida over different exposure time. Imidaeloprid (0.2mg/l) RH-5849 (25mg/1) Time 0h 1h 2h 3h 4h
SOD activity Mean_+SD 0.0585+0.0084 0,0505_+0.0075 0,0463_+0.0064 0.0440+0.0072 0.0588_+0.0078
Time 0h 1h 2h 3h 4h
SOD activity Mean_+SD 0.0572+0.0080 0.0693-+0.0173 0.0667_+0.0960 0.0812_+0.0141 O.1033_+0.0197
(the unit of enzyme activity: U per mg of protein). Occurrence of number of enzymes, particularly cellulase in the gut of earthworms indicate their role in the decomposition of plant litter and other cellulosic materials . This study shows clearly that sub-lethal exposure of the insecticide reduced the cellulase activity, which indicates harmful effect of the pesticides on the biochemical metabolism of earthworm. As to the SOD activity, the two pesticides both expressed effects of promotion to a certain degree, except that imidacloprid could suppress SOD activity at low concentration of 0. lmg/l. Imidacloprid is a nitro-methylene insecticide and is the receptor of nicocholinesterase, while RH-5849 is a entomological growth regulating agent and will make the entomology ecdysis earlier than normal. According to the theory of active oxygen toxicology, organisms have the ability to respond with the change of environment. When the pollutant induced the amount of
2353 02- in vivo increased, the amount of SOD in organisms will increase to a certain degree respectively. Patnaik  has reported that the urease activity of was greater in earthworms exposed to organophosphorus insecticide than the untreated worms and Robert  and Vig  have founded that the SOD activity in aquatic organisms promoted when exposed to organic pesticides. Hence increased SOD activity in earthworms (Eiseniafoetida) due to the two insecticides exposure is also not good for the earthworms. Sperm deformity test
The normal sperm of Eiseniafoetida is composed by a bacillary head and a flagellum. The total mean length of the sperm is about 50.47+2.81 um long as measured by light microscopy. The morphology abnormalities of earthworm (Eisenia foetida)sperm were shown in Fig 1. The most usually abnormalities are apical loop (Fig. 1A) and head bending (Fig. 1B), while some of the sperms showed the morphology abnormalities of tail folding (Fig. 1C) and swelling head (Fig. 1D). The deformity percentage were calculated and reported in Table 6. It can be seen that when the dose of Imidacloprid reached 0.5 mg/kg dry soils, the deformity percentage of sperm showed special significant difference with the negative control group and showed remarkable dose-effect relationship when the dose increased. As to the RH-5849, the statistical results did not show significant difference from the negative control until the dose reached 100mg/kg dry soils.
• ':>:: "
,-.i" ,..,~-. ::,
Fig. 1. Eisenia foetida. Micrograph ( × 400) of earthworm sperms showing the morphology abnormalities (indicated by arrow). (A) Apical loop, (B) Head bending, (C) Tail folding and (1)) Swelling head.
2354 Table 6. Effect oflmidacloprid and RH-5849 on sperm deformity percentage of Eiseniafoetida Test chemicals Exposure Dose Deformity (mg/kg dry soils) percent~e(%) negative control 1.69+0.70 (distilled water) 0.1 1.86_+0.57 Imidacloprid 0.2 3.51_+0.53"* 0.5 7.71_+0. 82** RH-5849 Positive control (eyelophosphamide)
*: P<0.05, **:P<0.01 Attempts have been made to develop indexes for predicting toxicities of pollutants . In China, acute toxicity tests are now compulsory as a standard test to evaluate toxicity of new substances before they can be marketed. However, the standard tests to detect the genotoxicity of pollutant on soil ecosystems are not available now and studies in this field are fewer than those in water and air ecosystems. This may be because many organisms in soil are lower creatures such as polymera and arthropod, while the prevailing method using to detect genotoxicities are not proper for these organisms In this study, we are exploring the possibility of using changes in sperm morphology as an indicator of environmental quality. We have studied the mutagenesis of lmidacloprid and RH-5849 using the method of micronucleus test of root tip cells in Vivia Faba and a mouse bone-marrow micronucleus test. The micronucleus percentage of the exposed group( the highest exposure concentration or dose: 2/3 LDs0 or LCs0 ) showed no significant differences from the negative control. In this study, however, we found that the two pesticides have affected the sperm morphology of Eiseniafoetida, especially the Imidacloprid This contrast may be caused by the difference of species and target site of pesticides, at the same time, selecting different endpoint in biomonitoring may result in different conclusion also. Wryobek had reported on the GeneTox programme of the U.S. Environmental Protection Agency (EPA), that the human sperm tests can be used to identify chemicals that affect sperm production and the percentage of normal sperm morphological features has an important role in fertilization in humans . Since the sperm deformity test can assess the extent and the potential reversibility of induced spermatogenic damage and indicate the pressure on the animal's reproductive performance at a certain degree, it can serve a useful purpose to understand the basic effects ofxenobiotics on reproductive processes and to provide some information on the population level . Furthermore it has been reported that the sperm deformity test is valuable in assessing the potential carcinogenicity of test chemicals and can provide an early warning of soil pollution of mutation compounds . As an easy-to-perform, cheap, repeatable, and ecologically relevant test for specific environment, the sperm deformity test is suggestible in testing the potential ecotoxicity of chemicals in specific environment.
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