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Gastropod mollusks have achieved an eminent importance as biological indicators of environmental quality. In the present study, we applied a multibiomarker approach to evaluate its applicability for the pond snail Lymnaea stagnalis, exposed to common industrial and agricultural pollutants at environmentally relevant concentrations. The snails were exposed to copper (Cu 2+ , 10 µg L -1 ), zinc (Zn 2+ , 130 µg L -1 ), cadmium (Cd 2+ , 15 µg L -1 ), or the thiocarbamate fungicide "Tattoo" (91 µg L -1 ) during 14 days. Metal treatment and exposure to "Tattoo" caused variable patterns of increase or decrease of metal levels in the digestive gland, with a clear accumulation of only Cd and Zn after respective metal exposure. Treatment with Cu and "Tattoo" caused an increase of cytochrome P450-related EROD activity. Glutathione S-transferase was inhibited by exposure to Cu, Zn, and "Tattoo." Treatment with the "Tattoo" led to an inhibition of cholinesterase activity, whereas Cu and Cd increased its activity. Caspase-3 activity was enhanced by up to 3.3 times in all treatments. A nearly uniform inhibitory effect for oxidative stress response parameters was observed in all kinds of exposure, revealing an inhibition of superoxide dismutase (Mn-SOD) activity, a depression of glutathione (GSH and GSSG) and of protein carbonyl levels. Pollutant-specific effects were observed for the catalase activity, superoxide anion production, and lipid peroxidation levels. Due to the high response sensitivity of Lymnaea stagnalis to chemical impacts, we suggest our study as a contribution for biomarker studies with this species under field conditions.

Thiocarbamates are one of the components of pesticides that target weeds by inhibiting adenosine triphosphate (ATP) synthesis. Orbencarb, one of the isomeric thiocarbamates applied to wheat, maize, and soybean, has been found to have toxic effects on mammals and marine ecosystems. Although the toxicity ranges of orbencarb in different organisms are known, specific studies on the environmental contamination and harmful effects of orbencarb on non-target organisms are scarce. In this study, we observed that orbencarb induced embryotoxicity during zebrafish development as well as apoptosis and reactive oxygen species (ROS) production in the intestine. It was further observed that orbencarb decreased the viability of the embryos and simultaneously affected the heart rate and vessel formation. Orbencarb decreased the mRNA levels of ccnd1, ccne1, cdk2, and cdk6 and induced abnormal development of the eyes, brain, yolk sac, and spinal cord in zebrafish embryos. Orbencarb also hampered vasculogenesis in the zebrafish embryos by inhibiting the mRNA expression of flt1, flt4, kdr, and vegfc. Collectively, these results suggested that orbencarb is embryotoxic and disrupts the normal growth of zebrafish embryos by inducing the generation of ROS and hampering vasculogenesis., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Excessive perfluorooctane sulfonate (PFOS) in natural water ecosystem has the potential to detrimentally affect immune system, but little is known of such effects or underlying mechanisms in fish. In the present study, we evaluated the effects of PFOS on growth performance, organizational microstructure, activities of immune-related enzymes and expressions of immune-related genes in male zebrafish (Danio rerio) exposed to different concentrations of 0, 0.02, 0.04 and 0.08 mg/L of PFOS for 7, 14, and 21 days or cotreatment with PFOS and PDTC to investigate the effects of PFOS on immune system and the potential toxic mechanisms caused by PFOS. The results indicated that PFOS accumulated in livers after exposure, and remarkably elevations were found in three exposure groups compared with the control group at three stages. The growth of the adult zebrafish in the experiments was significantly inhibited, the microstructures of liver were serious damaged. The ROS levels were remarkably increased. The activities of ACP, AKP, and lysozyme were obviously decreased, while the activities of MPO and NF-κB were significantly increased. The expressions of immune-related mRNA were significantly affected. After co-treatment with PFOS and PDTC, the growth inhibition, the morphological damage, the ROS induction, and the expressions of immune-related mRNA were reversed. Taken together, the results indicated that PFOS can significantly inhibit the growth, disturb the immune system by changing the normal structure of liver, the activities of immune-related enzymes, and a series of gene transcriptions involved in immune regulation in liver of male zebrafish. PFOS-induced pro-inflammatory effect of hepatocytes was observed, and the involvement of NF-κB signaling pathway was participated in its action mechanism. These findings provide further evidence that PFOS interferes with the immune regulation of liver of male zebrafish under in vivo conditions., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

We investigated toxic effects of the antifouling biocide polycarbamate (PC) on marine fish by conducting acute, early-life stage toxicity (ELS), and embryo toxicity tests. Mummichog (Fundulus heteroclitus) 96-h LC 50 values for hatched larvae (body weight about 2.0 mg) and juveniles (660 ± 36 mg) were about 12 and 630 μg/L, respectively. The ELS test using mummichog embryos yielded a lowest-observed-effect concentration of 3.9 μg/L and a no-observed-effect concentration of 2.1 μg/L with growth as the most sensitive endpoint. The embryo toxicity test for spotted halibut (Verasper variegatus) revealed a 10-d EC 50 of 8.1 μg/L with abnormality as an endpoint. During the ELS and embryo toxicity tests, morphological abnormalities (notochord undulation) were induced in the embryos. Biochemical and gene-expression analysis suggest that PC-induced morphological abnormalities involve disruption of lysyl oxidase-mediated collagen fiber organization, essential for notochord formation, and inhibition of gene expression related to notochord formation ., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The present study investigated how principal environmental factors such as temperature and light intensity change the toxicological properties of thiobencarb (TB) herbicide to the green alga, Raphidocelis subcapitata. At first, we investigated the inhibitory effect of TB (0, 15.6, 31.2, 62.4, and 125 μg L -1 ) on growth of R. subcapitata at five temperatures (10, 15, 20, 25, or 30 °C) for 144 h exposure and calculated 72- and 144-h effective concentration values (EC10, 20, and EC50) for growth rate. All EC values significantly decreased with an increasing temperature. The maximum quantum yield of photosystem II in R. subcapitata exposed to 125 μg L -1 of TB was also significantly inhibited with increased temperature. These physiological effects could explain the lower EC values at high temperatures. Then, single and interactive effects of TB, temperature, and light intensity on growth rate were investigated by three-way of analysis of variance. As a result, single and interactive effects were detected in all explanatory variables. These results suggest that temperature and light intensity change the acute toxicity parameter in R. subcapitata exposed to TB and must be considered in evaluating the risk of TB.

Thiobencarb and propanil are two of the most extensive used herbicides worldwide in rice cultivation. Especially scanty is the available information regarding the effect of herbicides on fish energy resources. In the present study, the effect of sublethal exposure to these herbicides on the energy reserves of juvenile eel Anguilla anguilla was compared. Eels were exposed to 72 h to the herbicide thiobencarb (0.22 mg L(-1)) or Propanil (0.63 mg L(-1)), and allowed to recover in clean water (144 h). Caloric content was determined in liver and skeletal muscle. Fish exposed to thiobencarb rapidly mobilized energy. Reserves from liver were depleted (21%) compared to control values (2.50 kcal g(-1)) at 2 h, whereas in muscle diminished between 12 and 72 h (35%) (control value 0.89 kcal g(-1)). Energy reserves from liver normalized after 144 h in water while in the skeletal muscle were still depleted (24%). Major harmful effects were induced by propanil. Caloric content in liver diminished from the first hours (depletion of 70% at 48h exposure) and in skeletal muscle a 60% (72 h). At the end of the recovery period, energy reserves in pre-exposed eels represented less than 50% compared to control animals. The study indicated that thiobencarb and propanil would constitute a great risk to animals inhabiting freshwater bodies nearby fields of application. Judging from the results, herbicides resulted toxic enough to mobilize fish energy stores. On the other hand, a period of six days in herbicide-free water was not enough time to allow fish to restore energy budgets., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The aim of the present study was the investigation of the effect of in situ exposure history on the responses of freshwater mussels to thiocarbamate fungicide. Male bivalve mollusks Anodonta anatina (Unionidae) from polluted (A) and unpolluted (F) sites were subjected to 14 days of exposure to fungicide Tattoo (mixture of propamocarb and mancozeb, 91 μg L(-1)). When unexposed mussels were compared, chronic effect of toxic environment in site A was confirmed by oxidative stress indices (high levels of superoxide dismutase and catalase activities, lipid peroxidation, protein carbonyls and oxyradical production, low level of total glutathione (GSH)), genotoxicity (high levels of DNA-strand breaks and caspase-3 activity in digestive gland), and cytotoxicity (low lysosomal membrane stability in hemocytes), elevated vitellogenin-like proteins (Vtg-LP) concentration in gonads, high levels of Cu, Zn, Cd, metallothionein (MT)-bound metals (MT-Me) and MT-related thiol (MT-SH), and low ethoxyresorufin-O-deethylase (EROD) activity in digestive gland. The major differences in the responses of the two exposed groups were related to antioxidant defense and MT: in the group A, prominent oxidative stress response with the participation of MT-SH and GSH in the gills, EROD activation, but decrease of MT-Me level was shown, whereas in group F exposure provoked the elevation of MT-Me, caspase-3 and Vtg-LP values. Carbamate did not cause cholinesterase depletion and cytotoxicity. However, genotoxic and pro-oxidant effects (increased levels of hemocytes with micronuclei and nuclear abnormalities, DNA-strand breaks and oxyradical in digestive gland), were common responses for both the exposed groups.

I examined the toxicity of rice paddy herbicides simetryn, mefenacet, and thiobencarb to amphibians by conducting acute toxicity tests with tadpoles of Silurana tropicalis, which has attracted as a new model species instead of Xenopus laevis. The tadpoles at stages 49 and 50 were exposed to the herbicides at several concentrations during 96 h, and median lethal concentration (LC50) values were calculated at 24-h intervals. The LC50 values of simetryn, mefenacet, and thiobencarb were 16.9-3.70 mg/L (79.3-17.4 microM), 3.06-2.70 mg/L (10.3-9.04 microM), and 1.77-0.752 mg/L (6.85-2.92 microM), respectively. The most toxic herbicide was thiobencarb followed by mefenacet and simetryn. As for thiobencarb, the sensitivity of S. tropicalis was similar to that of X. laevis reported previously. This suggests that S. tropicalis as well as X. laevis can act as a model species in acute toxicity tests. The LC50 values of the three herbicides were very close to or at most two orders of magnitude higher than the maximum concentrations likely to occur in paddy water. Therefore, the three herbicides can be harmful to amphibian larvae living in paddy water., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Exposures of Limnodynastes tasmaniensis tadpoles to atrazine (0.1, 1, 3 and 30microgL(-1)), metolachlor (0.1, 1 and 10microgL(-1)) and thiobencarb (90, 180 and 360microgL(-1)) from Gosner stage 28 to 42 under controlled laboratory conditions gave no significant effects on tadpole growth, development and sex ratios. A binary mixture of atrazine and thiobencarb as well as a ternary mixture of all three herbicides also had no significant effects on the developing larvae to show no evidence of interactive toxicity. Abnormal gonad morphology was observed on two occasions; both from 0.1microgL(-1) atrazine treatments with one tadpole observed with testicular ovarian follicles. The low frequencies of abnormal gonadal morphology and testicular ovarian follicles did not indicate a concentration associated response to herbicide exposure. No significantly unbalanced sex ratios were observed to suggest any evidence of chemically induced feminisation. These observations suggest that environmentally relevant concentrations of atrazine, metolachlor and thiobencarb do not present a significant threat to the normal development of L. tasmaniensis larvae in surface waters of irrigated agricultural areas., (2009 Elsevier Ltd. All rights reserved.)

Cartap is a widely used insecticide which belongs to a member of nereistoxin derivatives and acts on nicotinic acetylcholine receptor site. Its effects on aquatic species are of grave concern. To explore the potential developmental toxicity of cartap, zebrafish embryos were continually exposed, from 0.5 to 144h post-fertilization, to a range of concentrations of 25-1000microg/l. Results of the experiment indicated that cartap concentrations of 100microg/l and above negatively affected embryo survival and hatching success. Morphological analysis uncovered a large suite of abnormalities such as less melanin pigmentation, wavy notochord, crooked trunk, fuzzy somites, neurogenesis defects and vasculature defects. The most sensitive organ was proved to be the notochord which displayed defects at concentrations as low as 25microg/l. Both sensitivity towards exposure and localization of the defect were stage specific. To elucidate mechanisms concerning notochord, pigmentation, and hatching defects, enzyme assay, RT Q-PCR, and different exposure strategies were performed. For embryos with hatching failure, chorion was verified not to be digested, while removing cartap from exposure at early pre-hatching stage could significantly increase the hatching success. However, cartap was proved, via vitro assay, to have no effect on proteolytic activity of hatching enzyme. These findings implied that the secretion of hatching enzyme might be blocked. We also revealed that cartap inhibited the activity of melanogenic enzyme tyrosinase and matrix enzyme lysyl oxidase and induced expression of their genes. These suggested that cartap could impaired melanin pigmentation of zebrafish embryos through inhibiting tyrosinase activity, while inhibition of lysyl oxidase activity was responsible for notochord undulation, which subsequently caused somite defect, and at least partially responsible for defects in vasculature and neurogenesis.

Dithiocarbamates (DTCs) are sulfhydryls (thiol)-containing compounds, often associated with metals, and have both antioxidant and pro-oxidant abilities depending on the compound, experimental system and condition. In this study we investigated whether cell death plays a role in the manifestation of DTC-induced notochord distortions in the developing zebrafish and if thiol-containing compounds or antioxidants could modify this developmental toxicity. Sodium metam (NaM) induced notochord distortions could not be protected with the antioxidants ascorbic acid, trolox (synthetic vitamin E) or lipoic acid. However, NaM-induced distortions could be protected with co-exposure to glutathione or N-Acetyl Cysteine. Staggering the NaM and glutathione exposures in consecutive 10h developmental windows also resulted in protection. There were no discernable changes in TUNEL positive cells, a marker of apoptotic cells, at 24h post-fertilization (hpf) in NaM, dimethyl-dithiocarbamate, carbon disulfide, or neocuproine exposed embryos. Live NaM-exposed embryos incubated with acridine orange, a general stain for cell death, for 1h beginning at 11, 18 and 24hpf showed clusters of stained nuclei near the somitogenic front but not in the cells making up the notochord. Overall, induction of apoptotic pathways and widespread cell death are not involved in the manifestation of the adverse developmental outcomes following NaM exposure. However, cellular thiol status or critical sulfhydryl moieties are important considerations in the mechanisms of DTC developmental toxicity.

Exposures to sodium metam (NaM) within the developmental period of somitogenesis (10- to 18-h postfertilization [hpf]) results in easily detectable distortions of the notochord by 24 hpf in the developing zebrafish. We hypothesized that NaM-induced transcriptional changes during somitogenesis would reveal the major molecular targets in the zebrafish embryo. Embryos were exposed to NaM beginning at 4 hpf (1000 cells) and total RNA was isolated from embryos at the 3 somite (11 hpf), 10 somite (14 hpf), 18 somite (18 hpf), and larval (24 hpf) stages of development. Using the Affymetrix zebrafish gene array we observed relatively few mRNAs differentially regulated at least twofold at each time point (11 hpf, 101 genes; 14 hpf, 151; 18 hpf, 154; 24 hpf, 33). The transcriptional profiles reveal neurodevelopment and myogenesis as the two primary targets of NaM developmental exposure. Quantitative PCR of several muscle and neuronal genes confirmed the array response. We also followed the structural development of the peripheral nervous system under NaM exposure using antibodies against neuronal structural proteins. Although there was no change in the onset of antibody staining, profound alterations became apparent during the period in which the notochord becomes distorted (> 18 hpf). Motor neuron development observed with the Tg(NBT:MAPT-GFP)zc1 transgenic zebrafish and a primary motor neuron specific antibody showed similar timing in the structural alterations observed in these cell types. Further study of the interactions of dithiocarbamates with the regulatory elements of fast muscle development and neurodevelopment is warranted.

The mobility of the rice pesticides thiobencarb (S-[(4-chlorophenyl) methyl] diethylcarbamothioate) and fipronil ([5-amino-3-cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]pyrazole) were investigated in the glasshouse under flooded conditions using two Australian rice-growing soils. When using leakage rates of 10 mm day(-1), less than 20% of applied thiobencarb and fipronil remained in the water column after 10 days due to rapid transfer to the soil phase. Up to 70% and 65% of the applied thiobencarb and fipronil, respectively, were recovered from the 0-1 cm layer of soils. Only 5-7% of each pesticide was recovered from the 1-2 cm layer, and less than 2% was recovered from each 1 cm layer in the 2-10 cm region of the soils. Analysis of the water leaking from the base of the soil cores showed between 5-10% of the applied thiobencarb and between 10-20% of the applied fipronil leaching from the soil cores. The high levels of pesticide in the effluent was attributed to preferential flow of pesticide-laden water via soil macropores resulting from the wetting and drying process, worm holes and root channels.

Assessing the toxicity of chemicals in treated laboratory water may not accurately represent the toxicity of chemicals in natural aquatic systems. In natural water, dissolved organic matter, suspended particulate matter, and sediment play key roles in the sorption of contaminants from the water. Our previously published series of papers illustrated that the presence of sediment in aquatic toxicity testing systems significantly (p < 0.05) reduced the bioavailability of the herbicides atrazine and molinate to five Australian freshwater organisms. It is not clear whether the reduced bioavailability means that the trigger values (TVs) in the current Australian and New Zealand water quality guidelines, which are calculated using toxicity data from water-only toxicity tests, provide appropriate environmental protection. Several new sets of TVs were derived in the present study and were compared to each other and to the current Australian and New Zealand TVs for atrazine and molinate. The current Australian and New Zealand TVs for atrazine and molinate provided appropriate protection to Australian freshwater species. Australian freshwater species have a sensitivity distribution similar to those of overseas species to atrazine and molinate.

Acute (96 h) semi-static toxicity tests were conducted by exposing the freshwater fish, Melanotenia fluviatilis, to atrazine and molinate in laboratory and river water both with and without sediment. The 96-h EC50 (imbalance) values of atrazine to M. fluviatilis ranged from 5.6 to 10.4 mg L(-1) while the corresponding values for molinate ranged from 7.9 to 14.8 mg L(-1), respectively. Atrazine was classed as having moderate toxicity while molinate had low to moderate toxicity to M. fluviatilis. Neither the presence of river water nor sediment significantly (P<0.05) reduced the bioavailability of either herbicide to M. fluviatilis. A series of other studies by the authors have found that sediment significantly (P<0.05) reduced the bioavailability of these two chemicals to a variety of organisms. Reasons for sediment having no effect for this species were examined. This lack of effect by sediment is most likely due to the relative rates of absorption into the fish and adsorption onto the sediment. However, contributions to this outcome by resuspended sediment, contaminated food and a combined effect of the herbicides and sediment could not be excluded.

The toxicity and bioavailability of molinate to Vibrio fischeri (Microtox((R))) were determined in both laboratory and river water in the absence and presence of sediment after 0, 24, 48, 72 and 96-h exposure. The bioavailability of molinate, expressed as 5min EC50s (bioluminescence) and their fiducial limits calculated using initial measured concentrations, to V. fischeri in laboratory water in the absence and presence of sediment ranged from 1.8 (1.7-2.1) to 3.6 (3.5-3.7) mgL(-1) and 1.3 (1.2-1.4) to 4.2 (3.5-4.5) mgL(-1), respectively. The corresponding values in river water and river water plus sediment were 1.7 (1.6-1.8) to 3.8 (3.6-4.1) and 1.3 (1.3-1.4) to 4.6 (4.2-4.9) mgL(-1), respectively. River water did not significantly (P>0.05) reduce the bioavailability of molinate to V. fischeri compared to that of laboratory water. However, the presence of sediment significantly (P<0.05) reduced the bioavailability of molinate to V. fischeri in both waters. The exposure time also significantly (P<0.05) reduced the bioavailability of molinate to V. fischeri in both waters in the presence and absence of sediment. The type of water did not significantly (P>0.05) affect the loss of molinate during the 96-h exposure period. However, the presence of sediment significantly (P<0.01) increased the loss of molinate from the test solutions, probably by binding to the sediment particles. Exposure period and concentration levels significantly (P<0.05) affected the loss of the herbicides over the 96h.

Effects of thiobencarb on growth, photosynthesis and photoinhibition of Nostoc sphaeroides colonies were examined. Thiobencarb concentrations higher than 2 mg/l led to a significant decrease in phycoerythrin, phycocyanin and allophycocyanin content, but no significant effect on chlorophyll a (chl a) content. Biomass yield, protein content and photosynthetic rate were reduced only in the highest thiobencarb concentration (10 mg/l). Colonies exposed to high thiobencarb dose (10 mg/l) were more sensitive to high light than those exposed to the control and 4 mg/l thiobencarb. Dim light induced recovery from photoinhibition to more great extent in colonies grown in the control and 4 mg/l thiobencarb than 10 mg/l thiobencarb. Darkness resulted in a insignificant recovery regardless of thiobencarb treatments.

Acute (96-h) semistatic toxicity tests were conducted by exposing the freshwater shrimp, Paratya australiensis, to atrazine and molinate in laboratory water and in river water both with and without sediment. The median lethal concentrations (LC50) and 95% fiducial limits of atrazine for P. australiensis in laboratory water in the absence and presence of sediment were 9.9 (8.6-11.5) and 6.8 (5.4-8.5)mg/L, respectively, while the corresponding values in river water were 9.8 (8.5-11.2) and 6.5 (5.4-7.8)mg/L, respectively. For molinate, the LC50 values in laboratory water in the absence and presence of sediment were 9.2 (7.0-12.1) and 9.0 (6.8-12.0)mg/L, respectively and the corresponding values in river water were 8.7 (6.4-11.8) and 8.2 (6.6-10.2)mg/L, respectively. Neither the river water nor the presence of sediment significantly (P<0.05) reduced the bioavailability of either chemical to P. australiensis. This was unexpected, as studies with other aquatic organisms have shown that sediment significantly reduced the bioavailability of these chemicals.

Daphnia magna (F0 generation) was exposed to different sublethal molinate concentrations (0, 3.77, 4.71, 6.28, 9.42, and 18.85 mg/L) during 21 days. Chronic toxicity tests, using the same herbicide concentrations, were also carried out during 21 days using neonates of F1 first brood (F1-1st) and F1 third brood (F1-3rd) offspring generations from the parentals (F0) preexposed to the herbicide. Finally, offspring (from F1-1st and F1-3rd broods) were transferred to herbicide-free medium during a 21-day recovery period. The alga Nannochloris oculata (5 x 10(5) cells/mL) was used as food in all the experiments. The effect of molinate on survival, reproduction, and growth was monitored for the selected daphnid generations. The parameters used to evaluate herbicide effect on reproduction were mean total young per female, mean brood size, time to first reproduction, mean number broods per female, and intrinsic rate of natural increase (r). Survival and growth (body length) were also determined after 21 days of exposure to molinate. Reproduction was significantly reduced when molinate concentration was increased in the medium. This effect was higher in the parental (F0) daphnids than in the F1-1st and F1-3rd offspring. In the recovery study, reproduction was still reduced in F1 generation daphnids (1st and 3rd), but only in those animals from parentals exposed to the highest molinate concentrations. The decreased with increasing concentrations of molinate in daphnids from the parental generation (F0). Significant differences were also found in daphnids from the F1 generation (exposure). The growth of the exposed organisms (F0 and F1) decreased, although the greatest decrease was found in the parental animals (F0) (25%) exposed to 9.42 mg/L molinate. F1 daphnids (1st and 3rd broods) from the recovery period did not show any significant difference in their growth after 21 days of study. Finally, survival was not affected after exposure to the selected molinate concentrations except in those daphnids from the F0 generation in which survival decreased 51% and 78% at the highest herbicide concentrations tested (9.42 and 18.85 mg/L). Our results suggest that the offspring daphnids seem to be adaptated to the herbicide molinate, showing more longevity and reproduction than their parentals.

Atrazine and molinate are widely used herbicides and concern has been raised about their potential deleterious impacts on aquatic ecosystems. Although there have been some studies on the toxicity of herbicides to aquatic organisms using laboratory or natural water, information on the effect of sediments, suspended particulates, and dissolved organic matter on their bioavailability is quite limited. This study aims to provide toxicity data that considers these factors and the effect that these factors have on bioavailability. In this study, the toxicity of the test chemicals was calculated following the Organisation for Economic Co-operation and Development (OECD) methods, whereas change in bioavailability was measured using EC50 values based on measured initial concentrations of the test chemicals. The acute (48-h) static toxicity of atrazine and molinate to the freshwater cladoceran Daphnia carinata was determined in cladoceran water and river water in the absence and presence of sediment. The 48-h EC50 (immobilization) values of atrazine to D. carinata ranged from 22.4 to 26.7 mg/L, while the corresponding values for molinate ranged from 18.3 to 33.6 mg/L, respectively. Both chemicals were classed as having low acute toxicity to D. carinata. The presence of dissolved organic matter and suspended particles in river water did not significantly (p > 0.05) reduce the bioavailability (measured as toxicity) of atrazine to D. carinata compared to that tested in cladoceran water. The presence of sediment, however, significantly (p < 0.05) reduced the bioavailability (48-h EC50) of atrazine in cladoceran water, from 24.6 to 30.7 mg/L, and in river water, from 22.4 to 31.0 mg/L. Similarly, the presence of sediment in cladoceran water, significantly (p < 0.05) reduced the bioavailability (48-h EC50) of molinate, from 26.6 to 46.4 mg/L, and in river water, from 22.5 to 45.6 mg/L.

European eels (Anguilla anguilla) were exposed to a sublethal thiobencarb concentration of 0.22 mg/L in a flow-through system for 96 h. Mg(2+) and Na(+)-K(+) adenosine triphosphatase (ATPase) activities were evaluated in gill and muscle tissues at 2, 12, 24, 48, 72, and 96 h of thiobencarb exposure. Gill ATPase activities were rapidly inhibited from 2h of contact onward. Highest inhibition was registered for Na(+), K(+)-ATPase (85%) from 2 to 12h. Both Mg(2+) and total ATPase were inhibited (>73%) during the first hours of toxicant exposure. At the end of the exposure period (96 h) ATPase activities were still different from those of the controls (>50%). Significant inhibition was detected in Na(+), K(+)-ATPase activity (80%) in muscle tissue after 2h and it was maintained over the entire exposure time. However, Mg(2+)-ATPase and total ATPase showed only perturbations after 2 h of exposure. Eels were exposed to 0.22 mg/L of thiobencarb for 96 h and then a recovery period in herbicide-free water was allowed for 192 h. Gill and muscle samples were removed at 8, 24, 72, 96, 144, and 192 h and ATPase activity was evaluated. Following 144 h of recovery, Mg(2+)- and Na(+), K(+)-ATPase activities, as well as total ATPase activity, in gills of those animals previously exposed to 0.22 mg/L of thiobencarb were still significantly different compared to controls. Thiobencarb seems to act to alter the ionic profiles. Since ion-dependent ATPases are known to regulate the influx and efflux of ions across the membrane to maintain the physiological requirements of the cells, the inhibition of Na(+), K(+)-ATPase probably induced osmoregulatory perturbations. On the other hand, thiobencarb exposure causes increases in the muscle water content of A. anguilla. The results indicated that water content increased significantly (>100% higher than the controls) during the first 24 h of exposure.

The midge Chironomus tepperi was used in laboratory experiments to assess the relative toxicity of formulated molinate, clomazone, and thiobencarb, three herbicides used in Australian rice crops. Static bioassays were initiated with first-instar larvae at herbicide concentrations between 0.0625 and 2 times the anticipated field concentrations (AFCs) expected from the registered application rates. Adult emergence success, development time, and wing length were used as indices of the effect of each herbicide. Clomazone had no effect on any parameters at concentrations up to 0.288 mg/L (p > 0.05). Molinate significantly increased development time at concentrations equivalent to the AFC (3.6 mg/L) and above (p < 0.05). Thiobencarb reduced emergence success of adult C. tepperi at 0.0625 times the AFC (0.1875 mg/L) as well as decreasing male adult size and increasing development time for males and females at 0.125 times the AFC (p < 0.05). Nontarget effects of the herbicides on aquatic invertebrate communities were assessed in shallow experimental ponds using commercial application rates. One week after treatment, only thiobencarb had a significant effect, suppressing populations of chironomids, calanoids, and cyclopoids (p < 0.05). Four weeks later, all populations had recovered, equaling or exceeding control densities.

Metam sodium is the third most commonly used agricultural pesticide (by weight) in the U.S. A spill of 19,000 gallons of metam sodium into the Sacramento River in 1991 clearly demonstrated that a major uncontrolled release can have adverse ecological and human health effects. Furthermore, this incident revealed that estimates of Reference Exposure Levels for the major breakdown product of metam sodium (methylisothiocyanate, MITC) were reasonable with regard to the induction of discomfort. In fact, the irritant properties of MITC seem to account for many of the most commonly reported symptoms in this incident. However, neurotoxicity may also account for some of these symptoms. There is evidence that metam sodium can act as a contact sensitizer in humans, inducing allergic dermatitis. It also may exacerbate or induce respiratory allergy (asthma). The ecological impact of routine use of metam sodium is not clear, but adverse effects on non-target plants have been inferred from modeling studies, and adverse effects on soil microbes have been observed. These issues deserve further study. Human health effects of occupational or routine environmental exposure to metam sodium are not known, but there is limited evidence for immunological (hypersensitivity) and developmental effects as well as irritation and associated symptoms. Animal studies suggest a potential for immunological, developmental, carcinogenic, and atherogenic effects. Metam sodium and some of its breakdown products have a wide variety of molecular and cellular actions that could explain the health effects noted here. However, further studies are needed to relate specific molecular or cellular actions to specific health effects.

Chronic toxicity tests with Daphnia magna were applied for examination of river water quality. Water was sampled from the Maioka River in Yokohama City on May 14, 20, and 27, 1999, and used for the test after solid-phase extraction. The chronic test was carried out according to the OECD method. The duration was 21 days and the total number of live offspring produced per parent animal was counted. The results of the tests showed, survival rates of 100% using river water sampled on May 14 and 20 and the total numbers of live offspring produced per parent animal did not differ from the control. However, the survival rate of the sample collected on May 27 was 0% and the pesticides, fenitrothion, and thiobencarb were detected in the water. In addition to the river water samples, reconstituted water (Elendt M7) with additions of fenitrothion and thiobencarb was prepared to investigate mortality. When the reconstituted water with thiobencarb was applied to the test, the total number of live offspring produced per parent animal did not differ from the control. In contrast, when reconstituted water with fenitrothion was applied to the test, most parents were alive, but the total number of live offspring produced per parent animal was apparently different. The results of the above tests indicate that D. magna was affected not only by fenitrothion in the river water collected on May 27, but also by other factors that were not clarified in this study.

The inhibition of both total and specific acetylcholinesterase activities was measured in the whole eyes of the yellow eel Anguilla anguilla after exposure to the carbamate thiobencarb. In vivo assays were conducted under a constant flow-through system of thiobencarb-contaminated water (1/60 LC(50) 96 h=0.22 ppm for 96 h) followed by a recovery period in clean water (192 h more). The results indicated a measurable level of AChE activity on eyes of control eels, which resulted in a sensitive indicator of the presence of thiobencarb in the water. The pesticide induced significant inhibitory effects on AChE activity ranging from 35% in total AChE activity to 75% in specific AChE activity. Following 1 week of recovery, AChE activity in eel eyes was still different from that of controls. Specific AChE activity remained significantly depressed (35%). On the other hand, protein content in whole eyes of pesticide-treated eels did not exhibit any significant difference from control animals. It has been accepted that a minimum of 20% reduction in brain AChE would be necessary to be indicative of exposure to anticholinergic agents; however, few data are reported about this effect on sense organs., (Copyright 2000 Academic Press.)

The acute toxicity of the herbicide thiobencarb (S-4-chlorobenzyl diethylthiocarbamate) was determined for the European eel (Anguilla anguilla). The 24, 48, 72 and 96 hours median lethal concentrations (LC50) were 25.7, 21.7, 17.0 and 13.2 mg/L, respectively. Fish were also exposed to a sublethal thiobencarb concentration (1/60 LC50-96 hr = 0.22 mg/L) during 96 hours in a flow-through system and then an elimination period of 192 hours in clean water was allowed. Eels were removed and blood samples taken out at each exposure time and recovery period in order to evaluate AChE activity. Thiobencarb induced significant inhibitory effects on plasma AChE activity of A. anguilla from the first contact time with the toxicant. This inhibition (under 50% activity) was maintained during the entire exposure period (96 hours) and even those animals transferred to clean water showed plasma AChE activity different from the controls. Differences between total and specific AChE activity were detected during the exposure period. Total AChE activity in the plasma from animals transferred to a medium free of toxicant recovered its normal value while specific AChE activity remained depressed (< 50%) until five days later.

Thiram, a dithiocarbamate fungicide, is known to evolve to dimethylnitrosamine (DMNA) when associated with nitrites. Conditions of appearance of that carcinogenic compound have been studied in short-term experiments by association of the fungicide, nitrates or nitrites, and species representative of freshwater biota. DMNA has been estimated by GLC equipped with a specific detector. Chlorella vulgaris can rapidly produce nitrites from nitrates and DMNA is obtained in presence of thiram. Daphnia magna can also synthesize DMNA but nitrites have to be added to the medium. Increased toxicity of thiram is observed. The same results are obtained on Cyprinus carpio and for a part on Brachydanio rerio. When the species are associated in a 15-day experimental food chain, and intoxicated algae feed the two other levels, no significant transfer is observed. Nevertheless, some DMNA hazard may exist for particular species exposed to thiram associated with nitrites or even nitrates if algae are present.

Analysis of water samples from the lake in Albufera, Valencia, indicates that the pesticides molinate, benthiocarb, and fenitrothion do not reach levels that are lethal to fish.

The effects in vivo and in vitro of benthiocarb, an organocarbamate, on acetylcholinesterase (AChE) activity and its recovery in vitro in the brain of the freshwater fish Sarotherodon mossambicus have been studied. Findings revealed that the specific activity of AChE was inhibited both in vivo and in vitro in a concentration-dependent manner. Of the different modulators tested, dexamethasone (0.72 mg), prednisolone (0.74 mg), testosterone (0.9 mg), and ADP (9.5 mg) were found to neutralize the inhibitory effect (IC50 = 0.30 microM) of benthiocarb on AChE.

The effects of some common rice field herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D), Machete, and Saturn, on the paddy field nitrogen-fixing cyanobacteria Nostoc linckia, Nostoc calcicola, Nostoc sp., and Anabaena doliolum were studied. These cyanobacteria were found to be more tolerant to 2,4-D (lethal doses 1500-2000 micrograms/ml) than to Machete and Saturn (lethal doses 6-8 micrograms/ml). The effects of these three herbicides on some physiological processes of N. linckia were studied. The 2,4-D stimulated the growth and nitrogen fixation up to 100 micrograms/ml concentration (a dose higher than the field dose, i.e., about 40 micrograms/ml), recommended for field application. However, with Machete and Saturn this type of stimulation was not observed even at lower concentrations. Similarly, the uptake of nutrients, such as NO3- and NH+4, was also inhibited by Machete and Saturn. However, 100 micrograms/ml 2,4-D stimulated the uptake of NO3- but not of NH+4; higher doses of 2,4-D inhibited the uptake of both nutrients. Factors such as pH, organic carbon sources (glucose and acetate), and amino acids were found to regulate the toxicity of all three herbicides to N. linckia. Lower pH enhanced the toxicity of all three herbicides, whereas higher pH (up to 9.0) lowered it. Glucose and acetate (each 500 micrograms/ml) protected against the toxicity of 2,4-D and Saturn, but not against Machete. Whereas glutamine, arginine, serine, and tryptophan conferred upon N. linckia a greater protection against the toxicity of all three herbicides, methionine did not do so, and the presence of methionine with herbicide in the culture medium resulted in greater toxicity to N. linckia than that in the presence of the herbicide alone.