Acute toxicity assessment of azadirachtin-based pesticides using murine hybridoma and oyster cells

In vitro acute toxicities of azadirachtin-containing pesticides (Neemix and Bioneem), formulated with neem tree extracts, and pure azadirachtin (AZA), the believed active ingredient, were studied using hybridoma and oyster cells and were compared to results obtained using the standard in vivo Daphnia pulex toxicity assay. Neem-based pesticides showed relatively high toxicity to both hybridoma and oyster cells at concentrations of 1 microg AZA/mL and higher. The IC50 values for hybridoma cells were 2.15 microg AZA/mL for Neemix and 1.67 pg AZA/mL for Bioneem. Oyster cells had IC50 values of 2.18 microg AZA/mL for Neemix and 9.46 pg AZA/mL for Bioneem. Purified AZA, however, did not appear to be as toxic as the formulations. D. pulex was also more sensitive to neem-based pesticide exposure than that of pure AZA. The applications and limits of these two in vitro models for testing the acute toxicity of AZA-based pesticides are discussed in comparison with the in vivo D. pulex test.     

Ecological risk assessment of neem-based pesticides

A tiered process was used to evaluate the risks of pure azadirachtin (AZA) and two neem-based insecticides (Neemix and Bioneem) on six aquatic animals [crayfish (Procambarus clarkii), white shrimp (Penaeus setiferus), grass shrimp (Palaemonetes pugio), blue crabs (Callinectes sapidus), water fleas (Daphnia pulex), and mosquito larvae (Culex quinquefasciatus)] through short term acute toxicity tests. The risk was calculated using the level of concern endpoints (Q values) and relative hazard index (RHI) for acute and chronic exposure scenarios. The Q values of Neemix, Bioneem, and pure AZA derived from acute exposure tests indicated that D. pulex is the only sensitive species to the test pesticides. Furthermore, the RHI values of Neemix and Bioneem for D. pulex were above the critical limit of 10 indicating that these pesticides may pose a moderate hazard to this species and related crustaceans in acute exposure scenarios. The RHI values of the two pesticides and pure AZA were all below the critical limit of 10 for P. clarkii, P. setiferus, P. pugio, C. sapidus, and C. quinquefasciatus. The aquatic risk assessment process showed that the risk values of tested pesticides did not exceed the criteria, and therefore, no ecological hazard is likely to result from their use.     

Effects of the parasiticide ivermectin on the cladoceran Daphnia magna and the green alga Pseudokirchneriella subcapitata

Although widely used for the treatment of endo- and ectoparasites in livestock and pets, very few data on chronic effects on aquatic organisms are available for the parasiticide ivermectin. In the present study, toxicity of ivermectin to two freshwater organisms, the cladoceran Daphnia magna and the green alga Pseudokirchneriella subcapitata was investigated. For D. magna, a mean LC(50) 48 h of 5.7 ngl(-1) was derived from 10 acute tests. Chronic toxicity of ivermectin to D. magna was extremely high: with 0.001 and 0.0003 ngl(-1), respectively, nominal LOEC and NOEC based on growth and reproduction were far below the analytical limit of detection for this compound. P. subcapitata was considerably less sensitive to ivermectin than D. magna. For both growth rate and yield, EC(50) was >4,000 microgl(-1), LOEC was 1,250 microgl(-1) and NOEC 391microgl(-1). In view of the high toxicity to D. magna, the use of ivermectin might pose a risk to local aquatic ecosystems. Further studies should be carried out to investigate the effects of ivermectin and its degradation products on pelagic and benthic freshwater invertebrates.     

镉对泥鳅幼鱼的急性和亚急性毒性研究

在实验条件下研究了镉对泥鳅幼鱼的急性和亚急性毒性。试验从刚孵出的幼鱼开始,采用静水换水法。亚急性试验历时20d。镉对泥鳅幼鱼24、48h的LC50值分别为1．22、0．85mg/L。亚急性试验中,镉对泥鳅幼鱼存活有明显影响的可观察效应浓度（LOEC）最低是O．16mg/L;根据对幼鱼现存量的影响,镉对泥鳅的最低可观察效应浓度为0．08mg/L,而无可观察效应浓度（NOEC）为0．04mg/L。     

Acute toxicity tests with Daphnia magna, Americamysis bahia, Chironomus riparius and Gammarus pulex and implications of new EU requirements for the aquatic effect assessment of insecticides

Threshold concentrations for treatment related effects of 31 insecticides, as derived from aquatic micro-/mesocosm tests, were used to calibrate the predictive value of the European Tier-1 acute effect assessment on basis of laboratory toxicity tests with Daphnia magna, Chironomus spp., Americamysis bahia and Gammarus pulex. The acute Tier-1 effect assessment on basis of Daphnia (EC(50)/100) overall was protective for organophosphates, carbamates and most pyrethroids but not for neonicotinoids and the majority of insect growth regulators (IGRs) in the database. By including the 28-day water-spiked Chironomus riparius test, the effect assessment improves but selecting the lowest value on basis of the 48-h Daphnia test (EC50/100) and the 28-day Chironomus test (NOEC/10) is not fully protective for 4 out of 23 insecticide cases. An assessment on basis of G. pulex (EC(50)/100) is sufficiently protective for 15 out of 19 insecticide cases. The Tier-1 procedure on basis of acute toxicity data (EC(50)/100) for the combination of Daphnia and A. bahia and/or Chironomus (new EU dossier requirements currently under discussion) overall is protective to pulsed insecticide exposures in micro-/mesocosms. For IGRs that affect moulting, the effect assessment on basis of the 48-h Chironomus test (EC(50)/100) may not always be protective enough to replace that of the water-spiked 28-day C. riparius test (NOEC/10) because of latency of effects.     

The toxicity and behavioural effects of neem limonoids on Cnaphalocrocis medinalis (Guenée), the rice leaffolder

Meliaceae plant products have been shown to exert pesticidal properties against a variety of insect species. In agricultural pest control programs, such products may have the potential to be used successfully as botanical insecticides. The effect of the neem (Azadirachta indica) limonoids azadirachtin, salannin, deacetylgedunin, gedunin, 17-hydroxyazadiradione and deacetylnimbin on the biology and mortality of rice leaffolder larvae was investigated. In laboratory experiments, treatment with neem limonoids suppressed leaf folding behaviour of C. medinalis. Biological parameters (larval duration, pupal duration adult longevity and fecundity) were also affected by the treatment. Azadirachtin, salannin, and deacetylgedunin showed high bioactivity at all doses, while the rest of the neem limonoids were less active, and were only biologically active at high doses. Azadirachtin was most potent in all experiments and produced almost 100% larval mortality at 1 ppm concentration. These results indicate neem limonoids affect the larval behaviour. These effects are most pronounced in early instars.     

Herbicide effects on typha latifolia (Linneaus) germination and root and shoot development

Aqueous 7-d germination and growth experiments were performed to compare responses of T. latifolia to exposures of atrazine (2-chloro-4-ethylamino-6-isopropylamine-s-atrazine) and paraquat dichloride (1,1′-dimethyl-4,4′-bipyridinium dichloride). T. latifolia seed germination was < 50 % in concentrations ≥ 1.0 mg/L of paraquat dichloride. No observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for paraquat and root growth were 0.001 and 0.01 mg/L, respectively, while NOEC and LOEC for paraquat and shoot growth were 0.01 and 0.1 mg/L, respectively following 7-d exposures. Greater than 72 % of seeds germinated in each concentration up to 30 mg/L atrazine. After 7-d exposure, NOEC and LOEC for atrazine and root growth were 0.1 and 1.0 mg/L, while atrazine and shoot growth NOEC and LOEC values were 15 and 30 mg/L, respectively. This research provides data concerning relative sensitivity of T. latifolia seedlings to the herbicides atrazine and paraquat, as well as the potential use of T. latifolia as a representative plant test species.     

Ecological Risk Assessment of Neem-Based Pesticides

Abstract

A tiered process was used to evaluate the risks of pure azadirachtin (AZA) and two neem-based insecticides (Neemix? and Bioneem?) on six aquatic animals [crayfish (Procambarus clarkii), white shrimp (Penaeus setiferus), grass shrimp (Palaemonetes pugio), blue crabs (Callinectes sapidus), water fleas (Daphnia pulex), and mosquito larvae (Culex quinquefasciatus)] through short term acute toxicity tests. The risk was calculated using the level of concern endpoints (Q values) and relative hazard index (RHI) for acute and chronic exposure scenarios. The Q values of Neemix?, Bioneem?, and pure AZA derived from acute exposure tests indicated that D. pulex is the only sensitive species to the test pesticides. Furthermore, the RHI values of Neemix? and Bioneem? for D. pulex were above the critical limit of 10 indicating that these pesticides may pose a moderate hazard to this species and related crustaceans in acute exposure scenarios. The RHI values of the two pesticides and pure AZA were all below the critical limit of 10 for P. clarkii, P. setiferus, P. pugio, C. sapidus, and C. quinquefasciatus. The aquatic risk assessment process showed that the risk values of tested pesticides did not exceed the criteria, and therefore, no ecological hazard is likely to result from their use.     

Toxicity of hexahydro-1,3,5-trinitro-1,3,5-triazine to larval zebrafish (Danio rerio)

Hexahydro-1,3,5-trinitro-1,3,5-triazine, a cyclonitramine commonly known as RDX, is used in the production of military munitions. Contamination of soil, sediment, and ground and surface waters with RDX has been reported in different places around the world. Acute and subacute toxicities of RDX have been relatively well documented in terrestrial vertebrates, but among aquatic vertebrates the information available is limited. The objective of this study was to characterize the acute toxicity of RDX to larval zebrafish. Mortality (LC50) and incidence of vertebral column deformities (EC50) were two of the end points measured in this study. The 96-h LC50 was estimated at 22.98 and 25.64 mgl(-1) in two different tests. The estimated no-observed-effective-concentration (NOEC) values of RDX on lethality were 13.27+/-0.05 and 15.32+/-0.30 mgl(-1); and the lowest-observed-effective-concentration (LOEC) values were 16.52+/-0.05 and 19.09+/-0.23 mgl(-1) in these two tests, respectively. The 96-h EC50 for vertebral deformities on survivors from one of the acute lethality tests was estimated at 20.84 mgl(-1), with NOEC and LOEC of 9.75+/-0.34 and 12.84+/-0.34 mgl(-1), respectively. Behavioral aberrations were also noted in this acute toxicity study, including the occurrence of whirling movement and lethargic behavior. The acute effects of RDX on survival, incidence of deformities, and behavior of larval zebrafish occurred at the high end of the most frequently reported concentrations of RDX in aquatic environments. The chronic effects of RDX in aquatic vertebrates need to be determined for an adequate assessment of the ecological risk of environmental RDX.     

Toxicity of water and sediment from stormwater retarding basins to Hydra hexactinella

Hydra hexactinella was used to assess the toxicity of stormwater and sediment samples from three retarding basins in Melbourne, Australia, using an acute test, a sublethal test, and a pulse test. Stormwater from the Avoca St retarding basins resulted in a LC50 of 613 ml/L, NOEC and LOEC values of 50 ml/L and 100 ml/L, while the 7 h pulse exposure caused a significant increase in the mean population growth rate compared to the control. Water samples from the two other retarding basins were found non-toxic to H. hexactinella. This is the first study to employ sediment tests with Hydra spp. on stormwater sediments and a lower population growth rate was observed for organisms exposed to sediment from the Avoca St retarding basins. The behavioral study showed that H. hexactinella tended to avoid the sediment-water interface when exposed to sediment from all retarding basins, compared to the reference sediment. Further work is needed to determine the long-term effects of stormwater polluted sediments and acute effects due to organism exposure to short-term high concentrations during rain events.     

The toxic effects of neem extract and azadirachtin on the brown planthopper, Nilaparvata lugens (Stål) (BPH) (Homoptera: Delphacidae)

Extracts of neem (Azadirachta indica A. Juss) are used in the developing world for many purposes including management of agricultural insect pests. The effects of different neem extracts (aqueous (NSKEaq), ethanol (NSKEeth) and hexane (NSKEhex)) on mortality, survival and weight of the brown planthopper, Nilaparvata lugens (St?l) (BPH) (Homoptera: Delphacidae) third and fourth nymphal instars were investigated. When fed rice plants treated with neem derivatives in bioassays, the survival of BPH nymphs is affected. Comparisons were made with the pure neem limonoid, azadirachtin (AZA) to ascertain its role as a compound responsible for these effects. AZA was most potent in all experiments and produced almost 100% nymphal mortality at 0.5 ppm and higher concentrations. When higher concentrations were applied, the effects appeared shortly after treatment and mortality was higher. Many insects died after remaining inactive for several days or during prolonged moulting. At lower concentrations, if moulting was achieved, disturbed growth and abnormalities were then likely to occur in the moulting process. Nymphs that were chronically exposed to neem extract showed a reduction in weight (45-60%). The results clearly indicate the simple NSKE (aqueous, ethanolic or both), containing low concentrations of AZA, can be used effectively to inhibit the growth and survival of BPH.     

Toxicity of copper in an oxic stream sediment receiving aquaculture effluent.

Sediments were collected from a stream (upstream, outfall and downstream) receiving copper laden catfish pond effluent to assess toxicity to non-target biota. No significant reduction in Hyalella azteca survival or growth (10 d), or Typha latifolia germination and root and shoot growth (7 d) were observed after exposure to upstream and outfall sediments. A significant reduction in H. azteca survival was observed after exposure to the downstream sediment sample; however, no reduction in T. latifolia germination or seedling growth was detected. Bulk sediment copper concentrations in the upstream, outfall and downstream samples were 29, 31, and 25 mg Cu/kg dry weight, respectively. Interstitial water (IW) concentrations ranged from 0.053 to 0.14 mg Cu/l with 10 d IW toxicity units > or = 0.7. Outfall samples were amended with additional concentrations of copper sulfate so that bulk sediment measured concentrations in the amended samples were 172, 663, 1245, and 1515 mg Cu/kg dry weight. Survival was the most sensitive endpoint examined with respect to H. azteca with a no observed effects concentration (NOEC) and lowest observed effects concentration (LOEC) of 1245 and 1515 mg Cu/kg, respectively. NOEC and LOEC for T. latifolia root growth were 663 and 1245 mg Cu/kg, respectively. IW copper concentrations were > or = 0.86 mg Cu/l with H. azteca intersitial water toxicity unit (IWTU) concentrations > or = 1.2. Sequential extraction qualitatively revealed the carbonate and iron oxide fractions which accounted for a majority of the copper binding. In this instance, the copper which was applied to catfish ponds does not appear to be adversely impacting the receiving stream system.     

Comparative study of the effects of MCPA, butylate, atrazine, and cyanazine on Selenastrum capricornutum

The herbicides MCPA, butylate, atrazine and cyanazine are extensively used in Canadian agriculture and information regarding their effects on indigenous biota is scarce. Phytotoxicity assessments were conducted in the laboratory on the common green alga Selenastrum capricornutum using both the active ingredient of the herbicides and their formulated products (for MCPA and butylate). Endpoints determined after the 96 h exposure included algal population growth inhibition (IC50-cell counts), percent lethality (LC50-flow cytometry derived) and photosynthetic electron transport inhibition (EC50-fluorescence induction). Pesticide formulations had greater toxic effects than the active ingredient alone. The 96 h IC50 (50% Inhibition Concentration) and LOEC (Lowest Observable Effects Concentration) using cell counts of S. capricornutum were 18.4 and 8.9 mg l(-1) respectively for MCPA-active ingredient and for MCPA-formulated, these were 0.62 and 0.0062 mg l(-1) respectively. Those for butylateactive ingredient were 61.0 and 8.3 mg l(-1) and for butylate-formulated 1.46 and 0.17 mg l(-1), respectively. The triazines active ingredient, which are photosynthetic inhibitors, had greater effects than either the MCPA or butylate. The IC50 for cyanazine and atrazine were 0.059 and 0.026 mg l(-1), respectively. By comparing the IC50 and LC50 values for the tested active ingredients, it was found that the effects of atrazine were algicidal, whereas those of cyanazine, butylate and MCPA were algistatic.     

Investigation of acute toxicity of chlorpyrifos-methyl on guppy Poecilia reticulata

Static bioassays were made to determine acute toxicity of chlorpyrifos-methyl, a wide spectrum organophosphorus insecticide and potential toxic pollutant of aquatic ecosystem, Guppy fish (Poecilia reticulata). Bioassays were made at a regulated temperature of 22+/-1 degrees C and were repeated three times. Lethal doses of the insecticides were determined using LC50 software programme of U.S. EPA based on Finney's Probit Analysis statistical method. The 96 h LC50 value and 95% confidence limit of chlorpyrifos-methyl for Guppy was estimated as 1.79 (1.47-2.10) mg/l. The fish exposed to chlorpyrifos-methyl exhibited behavioral changes in the form of neurotoxin toxicity: less general activity than control group, loss of equilibrium, erratic swimming and staying motionless at a certain location generally at mid-water level for prolonged periods. The 1 mg/l (lowest) concentration had similar behavior (NOEC) with the control group.     

Chronic toxicity of tributyltin to development and reproduction of the European freshwater snail Lymnaea stagnalis (L.)

Chronic toxicity, growth and reproduction were measured in the freshwater gastropod Lymnaea stagnalis exposed to waterborne bis(tri-n-butyltin) oxide (TBTO) over a range of four nominal concentrations (0-10microg TBTl(-1)). Egg development was completely inhibited at 10microg TBTl(-1), whilst abnormal embryonic development was observed at 1microg TBTl(-1). For the solvent control and the 0.01microg TBTl(-1)treatment group, normal development of L. stagnalis was observed. Survivorship of hatchlings was significantly reduced by TBT at 1microgl(-1) while inhibition of shell growth of L. stagnalis was also observed at this concentration. The data were used to determine intrinsic growth rates (r) using two theoretical approaches (the Euler-Lotka equation and a Leslie Matrix). Both approaches showed that survival, fecundity and population growth rate were reduced at 1microg TBTl(-1). Interestingly, at 0.01microg TBTl(-1) snails showed a higher fecundity and growth rate than in the solvent control. The TBT concentration at which the r would equal zero (ECr(0)) and the population NOEC (No Observed Effect Concentration) were estimated. The population NOEC was defined as either the lower 95% confidence or lower 95% pointwise percentile limit of the ECr(0). Values obtained using the two different approaches were similar and thus a geometric mean was calculated to obtain a final representative population NOEC value for L. stagnalis of 2745ng TBTl(-1). The present data together with chronic toxicity TBT data for freshwater organisms, obtained from peer-reviewed literature, were used to construct a species sensitivity distribution (SSD). A predicted no effect concentration was then derived from the SSD (hazardous concentration at 5%, i.e., HC5 or 95% protection level). This SSD was compared with the SSD derived from saltwater species datasets. The HC5 value for saltwater species (3.55ng TBTl(-1); lower confidence limit: 1.93ng TBTl(-1)) was significantly lower than that for freshwater species (30.13ng TBTl(-1); lower confidence limit: 9.23ng TBTl(-1)), indicating that saltwater species are probably more susceptible to TBT than their freshwater counterparts.     

Full life-cycle toxicity assessment using rotifer resting egg production: implications for ecological risk assessment

The majority of standardized toxicity tests incorporate only a fraction of the test organism's life-cycle. However, in natural ecosystems, organisms may be exposed at various times during their life-cycle or throughout their life-cycle. Thus, ecotoxicological data from standardized toxicity tests is of limited ecological relevance. Existing standardized toxicity tests using the freshwater rotifer Brachionus calyciflorus utilize 24-h survival or 48-h asexual reproduction as endpoints, despite evidence that sexual reproduction is more sensitive. A 96-h B. calyciflorus resting egg toxicity test was developed and used to estimate the toxicity of pentachlorophenol (PCP) and copper. Results were compared to a variety of acute and sublethal endpoints for both toxicants. The B. calyciflorus 96-h resting egg production NOEC for PCP of 10 micrograms/l was 20 times lower than the 48-h asexual reproduction no observed effect concentration (NOEC) and 120 times lower than the 24-h acute lethal concentration 50%. The 96-h resting egg production NOEC for copper of 2.8 micrograms/l was 7 times lower than the 48-h asexual reproduction NOEC and nine times lower than the 24-h acute LC50. Resting egg production was a more sensitive indicator of toxicity than several other sublethal endpoints as well. These results indicate that partial life-cycle toxicity tests are not sufficiently sensitive to detect ecologically relevant adverse effects.     

Sustainable soil management practices and quality of potato grown on erodible lands

Land productivity can decline when top soil is lost. In Kentucky, limited resource farmers often produce vegetable crops on erodible lands. The objectives of this study were 1) to quantify the impact of three soil management practices (SMPs) on quantity of potato produced on erodible land, 2) to evaluate the impact of pyrethrin and azadirachtin insecticides on potato tuber quality, and 3) to assess the impact of yard waste compost on the chemical composition (ascorbic acid, free sugars, phenol contents) of potato tubers. Potatoes (Solanum tuberosum L. cv. Kennebec) were grown in a silty loam soil of 10% slope. Plots (n= 18) were 3.7 m wide and 22 m long (10% slope), with metal borders of 20 cm above ground level. Two botanical insecticides, Multi-Purpose Insecticide (containing pyrethrin 0.2%) and Neemix 4EC (containing 0.25% azadirachtin) were sprayed twice on potato foliage during each of two growing seasons (1997 and 1999) at the recommended rates of 6 lbs and 2 gallons.acre(-1), respectively. The SMPs were tall fescue strips (FS) intercropped between each two potato rows, soil mixed with yard waste compost (COM) and no-mulch (NM) treatment (roto-tilled bare soil). The experimental designwas a 2 x 3 x 3 factorial with main factors of two insecticides and three SMPs replicated three times. Average potato yield was lowest in NM and FS and highest in COM treatments. Yield obtained from the bottom of the plots was greater than that obtained from the top of plots. Tuber defects (rot, scab, sun green, hollow heart, necrosis, and vascular discoloration) were significantly different between the two growing seasons. The two insecticidal treatments did not have much influence on potato yield or tuber defects. Tubers obtained from tall fescue treatments had low levels of ascorbic acid and reducing sugars compared to compost treatments.     

Effect of neem limonoids on lactate dehydrogenase (LDH) of the rice leaffolder, Cnaphalocrocis medinalis (Guenée) (Insecta: Lepidoptera: Pyralidae)

Neem is derived from the neem tree Azadirachta indica A. Juss. (Meliaceae), and its primary insecticidal component is the tetranortriterpenoid azadirachtin and other limonoids. The effect of neem limonoids azadirachtin, salannin, deacetylgedunin, gedunin, 17-hydroxyazadiradione and deacetylnimbin on enzyme lactate dehydrogenase (LDH) activity of the rice leaffolder (RLF) Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) larvae was investigated. There was a decrease in enzyme activity relative to the control at all concentrations tested. When fed a diet of rice leaves treated with neem limonoids in bioassays, gut tissue enzyme, LDH levels in rice leaffolder larvae are affected. These results indicate neem limonoids affect LDH activity. These effects are most pronounced in early instar larvae. Azadirachtin was the most potent in of all the limonoids in all experiments indicating strong enzyme inhibition. Clear dose-response relationships were established with respect to LDH activity.     

Genotoxicity of two novel pesticides for the earthworm, Eisenia fetida

In this paper, several studies were conducted to evaluate the genotoxicity of two pesticides, Imidacloprid and RH-5849, for earthworm (Eisenia fetida). Earthworms were exposed in different exposure systems to evaluate their acute toxicity and the genotoxicity of the two pesticides was evaluated by using the method of sperm deformity assessment, micronucleus test of root tip cells in Vicia faba, a mouse bone-marrow micronucleus test, and comet assay. LC(50) (interpolated concentration at which 50% mortality of test population occurs) for earthworms varied in different exposure systems. The results indicated that Imidacloprid was consistently more toxic than RH-5849 in all exposure systems. In this study, sperm deformity test was used 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 a dose-effect relationship displayed at Imidacloprid concentrations higher than 0.5 mg/kg dry soil. However, the sperm deformity frequency of groups exposed to RH-5849 did not show significant difference (p>0.05) from the control until the dose reached 100 mg/kg dry soil. The results of the V. faba micronucleus tests showed that micronuclei frequency of the exposed group did not show significant difference (p>0.05) from the control until the concentration of Imidacloprid and RH-5849 reached 100 mg/ml. The results of the mouse bone-marrow micronuclei test also indicate that two pesticides did not show significant effects (p>0.05) on the micronuclei frequency in mice bone-marrow cells until the dose reached 100 mg/kg for Imidacloprid and 300 mg/kg for RH-5849 (2/3 LD(50)). Although no genotoxicity was detected by using the micronucleus tests, the results of the comet assay showed that the two pesticides induce significant DNA damage (p<0.01) in earthworms and dose-effect relationships were displayed. The 'earthworm comet assay' is a rapid and sensitive way to screen chemicals or terrestrial environments for their DNA-damaging properties.     

Acute toxicity and genotoxicity of two novel pesticides on amphibian, Rana N. Hallowell

Imidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitro-imidazolidin-2-ylideneamine] and RH-5849 [2'-benzoyl-l'-tert-butylbenzoylhydrazinel] are two pesticides used in China since 1992. In the present study we conducted acute toxicity test, micronucleus (MN) test and comet assay of the two pesticides on amphibian, Rana N. Hallowell, a sensitive organism suitable for acting as the bio-indicator of aquatic and agricultural ecosystems. The values of LC50-48 h of imidacloprid were found to be 165 mg l(-1) for tadpoles of Rana limnocharis and 219 mg l(-1) for tadpoles of Rana N. Hallowell. On the other hand, RH-5849 showed no acute toxicity to tadpoles during the 96 h exposure even it was saturated in the test solutions. There were significant differences in the MN frequencies between the negative controls and the treated groups at the dose of 8 mg l(-1) for imidacloprid (p < 0.05) and 40 mg l(-1) for RH-5849 (p < 0.01). Comet assay found significant differences (p < 0.01) in the distributions of DNA damage grades between the negative controls and groups treated in vitro with 0.05, 0.1, 0.2 and 0.5 mg l(-1) of imidacloprid and 5, 25, 50 and 100 mg l(-1) of RH-5849, respectively. DNA damage scores increased with the exposure levels of the two pesticides and dose-effect relationships were observed for both imidacloprid (r2 = 0.92) and RH-5849 (r2 = 0.98). The MN test and comet assay revealed potential adverse effects of the two pesticides on DNA in the erythrocytes of amphibians in aquatic and agricultural ecosystems.