Long-term carbaryl exposure leads to behavioral abnormalities and reproductive toxicity in male marine medaka through apoptosis-mediated HPA and HPG axes dysregulation.

Carbaryl is a widely used carbamate pesticide that has been detected in the marine environment, but its effects on marine fish are still unknown. This study was aimed to investigate the effects of long-term exposure of carbaryl on male marine medaka. For this purpose, we set up five exposure concentration groups of 0, 0.1, 1, 10, and 100 µg/L for 180 days. On the one hand, we observed increased aggression and decreased ability to avoid predators in males after exposure, which was affected by the levels of HPA-axis hormones, especially decreased cortisol level. On the other hand, after exposure, HPG axis hormone levels and gene transcription levels were disturbed. Males exhibited a decreased gonadosomatic index and a notable reduction in mature sperm proportion and the F1 generation displayed a significant increase in malformation rate. Additionally, the number of apoptotic cells and the transcription level of apoptosis-related genes in the brains of male marine medaka substantially increased after exposure. Apoptosis of brain cells may be responsible for the disturbance of HPA and HPG axes, consequently leading to behavioral and reproductive abnormalities. These findings provide novel insights into evaluating the toxic effects of carbaryl on male marine medaka and emphasizing the criticality of exploring the potential environmental risks posed by carbaryl in the marine environment, thus providing toxicity value basis for further strengthening marine environmental monitoring and the protection of biological resources.

Dietary deprivation reduces the deleterious effects of carbaryl on the survival and activity of both catalase and acetylcholinesterase in earthworms.

Dietary restriction (DR) and dietary deprivation (DD) have been shown to be significantly beneficial in terms of lifespan gains and stress alleviation in invertebrate and vertebrate species. Such beneficial effects, however, have yet to be clearly assessed in the presence of chemical stressors. We conducted a comparative evaluation of the toxicity of carbaryl in Eisenia fetida individuals subjected to a full diet (FD), DR and DD. For 14 days, groups of ten worms subjected to FD received 5 g oatmeal, those subjected to DR received 2.5 g oatmeal, and those subjected to DD received 0 g oatmeal weekly. We evaluated concentrations of 0, 7, 14 and 28 mg carbaryl.kg-1 soil and measured effects on survival, reproduction, biomass and biomarkers (Catalase- CAT and acetylcholine esterase- AChE). Carbaryl caused a total inhibition of reproduction in all the treatments. For each diet level, the 14-day LC50 s were higher than 28 mg.kg-1, but the 14-day LC20 s for the earthworms subjected to FD, DR, and DD were 11.24, 20.51 and > 28 mg.kg-1, respectively. This showed that the toxicity of carbaryl consistently decreased with the reduction in nutrients. Carbaryl caused a significant weight loss in the worms subjected to FD in the 7 mg.kg-1 treatment (P = 0.0065). Such weight loss was not found in any of the other treatments and diets. Both CAT and AChE were significantly inhibited in the two highest treatments (P = 0.0071 and P = 0.0073, respectively). Interestingly, the earthworms subjected to DD showed relatively lower biomarker inhibition, indicating a greater tolerance to oxidative and neurotoxic stresses in these starved earthworms. For all endpoints investigated, aside from reproduction, the starved earthworms fared better under carbaryl toxicity than those given the other diets. Overall, a positive correlation was observed between the amount of food and chemical toxicity as mortality rates, AChE and CAT inhibition increased with the increased amount of nutrients given to the worms. These results show that, in the presence of a chemical stressor, the beneficial effects of DR and DD were variably manifest for select lifecycle parameters and biomarker responses, further suggesting dietary reduction as a non-genetic intervention that could help extend lifespan and alleviate stress even under a chemical insult.

UDP-glucuronosyltransferase is involved in susceptibility of Chironomus kiiensis Tokunaga, 1936 (Diptera: Chironomidae) to insecticides.

UDP-glucuronosyltransferases (UGTs) could transform various exogenous and endogenous compounds, which help detoxification of pesticides in insects. To investigate the role of UGTs in the detoxification metabolism of insecticides in Chironomus kiiensis, CkUGT302M1, CkUGT302N1, CkUGT308N1 and CkUGT36J1 genes were identified with 1449-1599 bp encoding 482-532 amino acids. Four UGT genes shared 40.86∼53.36% identity with other homologous insect species, and expressed in all developmental stages, notably in the larval and adult stages. Expression of CkUGTs was higher in the gastric caecum, midgut and head. Moreover, CkUGTs expression and activity were significantly increased in C. kiiensis larvae in exposure to sublethal concentrations of carbaryl, deltamethrin and phoxim, respectively. To further explore the functions of UGT genes, the CkUGT308N1 was effectively silenced in 4th instar C. kiiensis larvae by RNA interference, which resulted in the mortality of dsCkUGT308N1 treated larvae increased by 71.43%, 111.11% and 62.50% under sublethal doses of carbaryl, deltamethrin and phoxim at the 24-h time point, respectively. The study revealed that the CkUGT308N1 gene in C. kiiensis could contribute to the metabolism of pesticides and provide a scientific basis for evaluating the water pollution of pesticides.

Cytotoxicity and genotoxicity of methomyl, carbaryl, metalaxyl, and pendimethalin in human umbilical vein endothelial cells.

Pesticides have adverse effects on the cellular functionality, which may trigger myriad of health consequences. However, pesticides-mediated toxicity in the endothelial cells (ECs) is still elusive. Hence, in this study, we have used human umbilical vein endothelial cells (HUVECs) as a model to quantify the cytotoxicity and genotoxicity of four pesticides (methomyl, carbaryl, metalaxyl, and pendimethalin). In the MTT assay, HUVECs exposed to methomyl, carbaryl, metalaxyl, and pendimethalin demonstrated significant proliferation inhibition only at higher concentrations (500 and 1000 µM). Likewise, neutral red uptake (NRU) assay also showed proliferation inhibition of HUVECs at 500 and 1000 µM by the four pesticides, confirming lysosomal fragility. HUVECs exposed to the four pesticides significantly increased the level of intracellular reactive oxygen species (ROS). Comet assay and flow cytometric data exhibited DNA damage and apoptotic cell death in HUVECs after 24 h of exposure with methomyl, metalaxyl, carbaryl, and pendimethalin. This is a first study on HUVECs signifying the cytotoxic-genotoxic and apoptotic potential of carbamate insecticides (methomyl and carbaryl), fungicide (metalaxyl), and herbicide (pendimethalin). Overall, these pesticides may affect ECs functions and angiogenesis; nonetheless, mechanistic studies are warranted from the perspective of vascular biology using in vivo test models.

Use of computational toxicology (CompTox) tools to predict in vivo toxicity for risk assessment.

Computational Toxicology tools were used to predict toxicity for three pesticides: propyzamide (PZ), carbaryl (CB) and chlorpyrifos (CPF). The tools used included: a) ToxCast/Tox21 assays (AC50 s µM: concentration 50% maximum activity); b) in vitro-to-in vivo extrapolation (IVIVE) using ToxCast/Tox21 AC50s to predict administered equivalent doses (AED: mg/kg/d) to compare to known in vivo Lowest-Observed-Effect-Level (LOEL)/Benchmark Dose (BMD); c) high throughput toxicokinetics population based (HTTK-Pop) using AC50s for endpoints associated with the mode of action (MOA) to predict age-adjusted AED for comparison with in vivo LOEL/BMDs. ToxCast/Tox21 active-hit-calls for each chemical were predictive of targets associated with each MOA, however, assays directly relevant to the MOAs for each chemical were limited. IVIVE AEDs were predictive of in vivo LOEL/BMD10s for all three pesticides. HTTK-Pop was predictive of in vivo LOEL/BMD10s for PZ and CPF but not for CB after human age adjustments 11-15 (PZ) and 6-10 (CB) or 6-10 and 11-20 (CPF) corresponding to treated rat ages (in vivo endpoints). The predictions of computational tools are useful for risk assessment to identify targets in chemical MOAs and to support in vivo endpoints. Data can also aid is decisions about the need for further studies.

Glutathione reductase and catalase as potential biomarkers for synergistic intoxication of pesticides in fish.

Synergy occurs when chemicals give pronounced effect on combination in contrast to their individual effect. The objective of this study was to investigate the synergistic effect of pesticides carbaryl (C) and methyl parathion (MP) on oxidative stress biomarkers viz catalase (CAT), glutathione reductase (GSSG-R) including different enzymes like lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and acetyl cholinesterase (AChE) in different tissues of carps Catla catla. Fishes were exposed to 6.25 mg/L of MP and 2.3 mg/L of C in mixture (one-third of LC50 value). CAT and GSSG-R were studied in gills, brain, liver and muscle of carp were found to be elevated significantly (p < 0.005). LDH activity increased significantly (p < 0.005) in synergistic group, there was a seven-fold (748%) increase in LDH activity in muscle compared to individual studies with same pesticides. Contrary to LDH, sudden decrease in SDH activity was accounted. Significant (p < 0.005) decrease in AChE activity after initial 24 h was remarkable addressing to the shift in neurotransmission pathway in organism. Significant increase was observed in activity of CAT and GSSG-R in all tissues compared to control fishes in individual as well as synergistic (MP + C) group suggesting that CAT and GSSG-R can be a potential biomarker of oxidative stress when studied in combination.

Cholinesterases characterization of three tropical fish species, and their sensitivity towards specific contaminants.

Cholinesterases are frequent targets for toxic effects, namely by insecticides derived from phosphoric and carbamic acids. This effects allows the use of cholinesterase inhibition as a biomarker for contamination of aquatic environments by these specific chemical agents. However, cholinesterases are differently responsive to environmental contaminants, according to their different forms and locations. In addition, cholinesterases seem also to be inhibited by metals, so their use as an environmental criterion requires the prior characterization of their specific forms in each species and tissues, and the study of their sensitivity. The objective of this study was to characterize the cholinesterase isoenzymes present in the brain and dorsal muscle of three tropical fish species, namely Phalloceros harpagos (Lucinda, 2008), Pterygoplichthys pardalis (Castelnau, 1855) and Astyanax altiparanae (Garutti and Britski, 2000). In vitro assays were conducted to quantify the effect of pesticides (dimethoate and carbaryl) and metals (lead and copper) on cholinesterases activity. Although acetylcholinesterase seems to be the most prevalent and abundant form, as commonly described in vertebrates, the here-obtained results showed that three cholinesterase isoenzymes occur in tissues of the three fish species. In addition, the pesticide carbaryl caused a stronger inhibition than dimethoate. Copper caused a significantly higher cholinesterasic inhibition than lead, which is also in line with most results concerning the anticholinesterasic effects by these metals. The here obtained results allowed to conclude that acetylcholinesterase is the predominant form in all tissues from the three analyzed species. In addition, cholinesterases of these three fish were responsive to common environmental contaminants, namely metals and pesticides, similarly to what was already described for fish of temperate areas. This allows using the here proposed fish species in environmental studies for the assessment of the presence of neurotoxicants under neotropical conditions.

Soluble esterases as biomarkers of neurotoxic compounds in the widespread serpulid Ficopomatus enigmaticus (Fauvel, 1923).

The characterization of soluble cholinesterases (ChEs) together with carboxylesterases (CEs) in Ficopomatus enigmaticus as suitable biomarkers of neurotoxicity was the main aim of this study. ChEs of F. enigmaticus were characterized considering enzymatic activity, substrate affinity (acetyl-, butyryl-, propionylthiocholine), kinetic parameters (Km and Vmax) and in vitro response to model inhibitors (eserine hemisulfate, iso-OMPA, BW284C51), and carbamates (carbofuran, methomyl, aldicarb, and carbaryl). CEs were characterized based on enzymatic activity, kinetic parameters and in vitro response to carbamates (carbofuran, methomyl, aldicarb, and carbaryl). Results showed that cholinesterases from F. enigmaticus showed a substrate preference for acetylthiocholine followed by propionylthiocholine; butyrylthioline was not hydrolyzed differently from other Annelida species. CE activity was in the same range of cholinesterase activity with acetylthiocholine as substrate; the enzyme activity showed high affinity for the substrate p-nytrophenyl butyrate. Carbamates inhibited ChE activity with propionylthiocholine as substrate to a higher extent than with acetylthiocoline. Also CE activity was inhibited by all tested carbamates except carbaryl. In vitro data highlighted the presence of active forms of ChEs and CEs in F. enigmaticus that could potentially be inhibited by pesticides at environmentally relevant concentration.

Immediate and lag effects of pesticide exposure on parasite resistance in larval amphibians.

Across host-parasite systems, there is evidence that pesticide exposure increases parasite loads and mortality following infection. However, whether these effects are driven by reductions in host resistance to infection or slower rates of parasite clearance is often unclear. Using controlled laboratory experiments, we examined the ability of larval northern leopard frogs (Lithobates pipiens) and American toads (Anaxyrus americanus) to resist and clear trematode (Echinoparyphium sp.) infections following exposure to the insecticide carbaryl. Northern leopard frogs exposed to 1 mg L-1 of carbaryl had 61% higher parasite loads compared with unexposed individuals, while there was no immediate effect of carbaryl on parasite encystment in American toads. However, when tadpoles were exposed to carbaryl and moved to freshwater for 14 days before the parasite challenge, we recovered 37 and 63% more parasites from carbaryl-exposed northern leopard frogs and American toads, respectively, compared with the control. No effects on clearance were found for either species. Collectively, our results suggest that pesticide exposure can reduce the ability of amphibians to resist parasite infections and that these effects can persist weeks following exposure. It is critical for researchers to incorporate species interactions into toxicity studies to improve our understanding of how contaminants affect ecological communities.

Antioxidant enzymes and their role in phoxim and carbaryl stress in Caenorhabditis elegans.

Pesticide exposure can induce oxidative stress and cause changes to antioxidant enzymes in living organisms. In the present study, the effects of phoxim (an organophosphorus insecticide) and carbaryl (a carbamate insecticide) on antioxidant enzyme activity and gene expression were investigated in the model organism Caenorhabditis elegans. The results show that phoxim exposure can induce superoxide dismutase (SOD) and catalase (CAT) activities and decrease glutathione peroxidase (GPx) activity at lower concentrations. The expression levels of sod-3, sod-5, ctl-1, gpx-6, and gpx-8 were up-regulated after treatment with phoxim. The mRNA expression levels of sod-5, ctl-1 and gpx-6 were increased approximately 70-, 170- and 130-fold, respectively, in the 0.25mM treatment group compared to the control group. Carbaryl exposure decreased SOD activity and induced CAT and GPx activities. The addition of carbaryl up-regulated the expression of sod-5, ctl-1, ctl-3 and gpx-8. Specifically, ctl-1 expression increased approximately 10-fold, and gpx-8 expression increased <30-fold in the 0.5mM treatment group relative to the control group. The transcript level of sod-5 increased >20-fold, and ctl-3 increased approximately 10-fold in the 1mM treatment group. The functions of the antioxidant enzymes during oxidative stress caused by the two insecticides were investigated using deletion mutants. The LC50 values phoxim for the of sod-3 (tm760), sod-5 (tm1146), ctl-1 (ok1242), ctl-3 (ok2042) and gpx-8 (tm2108) mutant strains were lower than those observed for the N2 strain. The LC50 values of carbaryl for the ctl-1 (ok1242), ctl-3 (ok2042) and gpx-6 (tm2535) deletion mutant strains decreased in comparison to the N2 strain. The results suggest that these two insecticides caused oxidative stress and changed altered the antioxidant enzyme activities and their gene expressions in C. elegans. The sod-3, sod-5, ctl-1, ctl-3, gpx-6, and gpx-8 encoding enzymes may play roles in defending cells from oxidative stress caused by these two insecticides.

RNA interference of acetylcholinesterase in the Asian citrus psyllid, Diaphorina citri, increases its susceptibility to carbamate and organophosphate insecticides.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Lividae) transmits the Candidatus Liberibacter asiaticus, which causes citrus greening disease or Huanglongbing, (HLB). To date, there is no efficient cure for HLB disease and the control of D. citri using insecticides became the most important tools for the management of HLB. However, the extensive use of insecticides could increase D. citri resistance to these insecticides. The objective of this study was to investigate the effect of RNA interference of acetylcholinesterase (AChE) on the mortality and susceptibility of D. citri to the four major insecticides used in Florida. In this study, we used a consensus sequence derived from the two AChE genes and cholinesterase 2-like (ChE-2-like) gene to target all of the three genes. Treatment with dsRNA-AChE increased the mortality percentages of both nymphs and adults of D. citri. The mortality percentage increased with the increase in the concentration of applied dsRNA-AChE, and the highest mortality (> 60%) was observed at the highest applied concentration (125ng/µl). Treatments of nymphs or adults with dsRNA-AChE down-regulated the expression of the three targeted genes of D. citri. Silencing of AChE and ChE in D. citri nymphs increased the susceptibility of emerged adults to chlorpyrifos and carbaryl, which act as AChE inhibitors. However, treatment with dsRNA-AChE did not increase the susceptibility of emerged adults to imidacloprid, which acts as an agonist of nicotinic acetylcholine receptors. In the same manner, treatment of adults with dsRNA-AChE increased their susceptibility to chlorpyrifos and carbaryl, but did not affect their susceptibility to imidacloprid. The ANOVA did not show any significant increase in susceptibility of D. citri adults to fenpropathrin after treatment with dsRNA-AChE, either as nymphs or as adults. However, simple linear regression showed that treatment with dsRNA-AChE increased D. citri susceptibility to fenpropathrin, which indicated that AChE could be involved in the metabolism of fenpropathrin. Our results indicated that silencing of AChE and ChE genes in D. citri to increase its susceptibility to insecticides could be a promising tool for the control of this important vector.

Carbaryl-induced ototoxicity in rat postnatal cochlear organotypic cultures.

Carbaryl, a widely used carbamate-based insecticide, is a potent anticholinesterase known to induce delayed neurotoxicity following chronic exposure. However, its potential toxic effects on the cochlea, the sensory organ for hearing that contains cholinergic efferent neurons and acetylcholine receptors on the hair cells (HC) and spiral ganglion neurons has heretofore not been evaluated. To assess ototoxic potential of carbaryl, cochlear organotypic cultures from postnatal day 3 rats were treated with doses of carbaryl ranging from 50 to 500 µM for 48 h up to 96 h. Carbaryl damaged both the sensory HC and spiral ganglion neurons in a dose- and duration-dependent manner. HC and neuronal damage was observed at carbaryl concentrations as low as 50 µM after 96-h treatment and 100 µM after 48-h treatment. Hair cell was greatest in the high frequency basal region of the cochlea and progressively decreased towards the apex consistent with the majority of ototoxic drugs. In contrast, damage to the spiral ganglion neurons was of similar magnitude in the basal and apical regions of the cochlea. Carbaryl damage was characterized by soma shrinkage, nuclear condensation and fragmentation, and blebbing, morphological features of programmed cell death. Carbaryl upregulated the expression of executioner caspase-3 in HC and spiral ganglion neurons indicating that cellular damage occurred primarily by caspase-mediated apoptosis. These results suggest that chronic exposure to carbaryl and other carbamate anticholinesterases may be ototoxic. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 956-969, 2017.

Aged garlic extract ameliorates immunotoxicity, hematotoxicity and impaired burn-healing in malathion- and carbaryl-treated male albino rats.

Malathion and carbaryl are the most widely used organophosphate and carbamate insecticides, respectively, especially in developing countries; they pose a potential health hazard for both humans and animals. Here, we evaluated the protective effects of an odorless (free from allicin) Kyolic aged garlic extract (AGE, containing 0.1% S-allylcysteine; 200 mg/kg body weight) on the toxicity induced by 0.1 LD50 of malathion (89.5 mg/kg body weight) and/or carbaryl (33.9 mg/kg body weight) in male Wistar rats. Doses were orally administered to animals for four consecutive weeks. The present study showed that AGE completely modulated most adverse effects induced by malathion and/or carbaryl in rats including the normocytic normochromic anemia, immunosuppression, and the delay in the skin-burning healing process through normalizing the count of blood cells (erythrocytes, leucocytes and platelets), hemoglobin content, hematocrit value, blood glucose-6-phosphodehydrogenase activity, weights and cellularity of lymphoid organs, serum γ-globulin concentration, and the delayed type of hypersensitivity response to the control values, and accelerating the inflammatory and proliferative phases of burn-healing. In addition, AGE completely modulated the decrease in serum reduced glutathione (GSH) concentration and the increase in clotting time in malathion alone and carbaryl alone treated rats. Moreover, AGE induced a significant increase (P < 0.001) in serum GSH concentration (above the normal value) and accelerating burn-healing process in healthy rats. In conclusion, AGE was effective in modulating most adverse effects induced in rats by malathion and carbaryl, and hence may be useful as a dietary adjunct for alleviating the toxicity in highly vulnerable people to insecticides intoxication. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 789-798, 2017.

The Degree of Impairment of Foraging in Crayfish (Orconectes virilis) due to Insecticide Exposure is Dependent upon Turbulence Dispersion.

As toxicants move into aquatic systems, the concentration at any point in space or time is heavily influenced by the flow dynamics. The dispersion of these chemicals creates a toxicant concentration that fluctuates widely in time and is highly dependent on the spatial heterogeneity of turbulence. Despite this knowledge on the movement of toxicants in natural systems, most ecotoxicological studies use static exposure paradigms that ignore the spatio-temporal dynamics of toxicants in aquatic systems. Although recent studies have begun to use pulsed paradigms in an attempt to mimic natural conditions, the heterogeneity of real concentrations in natural systems rarely is considered for use in these tests. Thus, understanding how organisms are impaired by naturally distributed toxicants is relatively unknown. The purpose of this experiment was to determine how turbulent dispersion of a toxicant negatively impacts a behavioral task and if altering the nature of turbulence will change the negative impact of the toxicant. Crayfish were exposed to a turbulent plume of carbaryl, an insecticide, under two different turbulent conditions and two different spatial conditions. Turbulence was altered by placing an obstruction within the flow which mimics a natural obstruction in lentic systems. Crayfish were exposed to sublethal concentrations of carbaryl for 48 h under these different dynamic conditions. After toxicant exposure, crayfish foraging ability was measured in a flow-through Y maze. We hypothesized that crayfish exposed to the toxicant under more turbulent conditions would exhibit more detrimental responses due to the increased variation in chemical fluctuations. The fine-scale chemical distribution of the toxicant and the three-dimensional velocity profile were characterized for each of the turbulent conditions and each of the spatial locations. Analyses of these data showed that changes in turbulence or spatial location created a unique exposure condition. Particularly, significant variations in the rise time, intermittency, and slope of toxicant pulses were quantified, whereas average concentration of the peaks remained constant across locations. Deficits in the foraging ability of crayfish exposed under these dynamic conditions paralleled the differences quantified in parameters of the turbulent toxicant plume. Given these results, the concept of toxicant exposure needs revision and needs to incorporate the more temporally based measures of toxicant dispersion. In addition, static and pulsed exposure models do not duplicate natural exposure and may not reflect behavioral or physiological impairments that occur under more realistic exposure conditions.

SUSPECTED CARBARYL TOXICITY IN A CAPTIVE COLONY OF STRAW-COLORED FRUIT BATS ( EIDOLON HELVUM).

Carbaryl was the first carbamate insecticide produced and remains the most widely used due to its perceived low level of toxicity in nontarget species. This report describes the management and evaluation of a group of straw-colored fruit bats, Eidolon helvum, that were exposed to carbaryl. Cholinesterase activity of blood, retina, and brain was evaluated to further investigate whether carbaryl was the causative agent. Decreased whole blood and retinal cholinesterase activity coupled with the response to atropine suggests that the cause of the clinical signs in this bat colony was due to carbaryl exposure. Whole blood and retinal tissue may be the best samples for confirming carbamate exposure in this species.

Population-specific toxicity of six insecticides to the trematode Echinoparyphium sp.

The ubiquitous use of pesticides has increased concerns over their direct and indirect effects on disease dynamics. While studies examining the effects of pesticides on host-parasite interactions have largely focused on how pesticides influence the host, few studies have considered the effects of pesticides on parasites. We investigated the toxicity of six common insecticides at six environmentally-relevant concentrations to cercariae of the trematode Echinoparyphium from two populations. All six insecticides reduced the survival of cercariae (overall difference between mortality in control vs pesticide exposure = 86·2 ± 8·7%) but not in a predictable dose-dependent manner. These results suggest that Echinoparyphium are sensitive to a broad range of insecticides commonly used in the USA. The lack of a clear dose-dependent response in Echinoparyphium highlights the potential limitations of toxicity assays in predicting pesticide toxicity to parasites. Finally, population-level variation in cercarial susceptibility to pesticides underscores the importance of accounting for population variation as overlooking this variation can limit our ability to predict toxicity in nature. Collectively, this work demonstrates that consideration of pesticide toxicity to parasites is important to understanding how pesticides ultimately shape disease dynamics in nature.

Taxonomic applicability of inflammatory cytokines in adverse outcome pathway (AOP) development.

Cytokines, low-molecular-weight messenger proteins that act as intercellular immunomodulatory signals, have become a mainstream preclinical marker for assessing the systemic inflammatory response to external stressors. The challenge is to quantitate from healthy subjects cytokine levels that are below or at baseline and relate those dynamic and complex cytokine signatures of exposures with the inflammatory and repair pathways. Thus, highly sensitive, specific, and precise analytical and statistical methods are critically important. Investigators at the U.S. Environmental Protection Agency (EPA) have implemented advanced technologies and developed statistics for evaluating panels of inflammatory cytokines in human blood, exhaled breath condensate, urine samples, and murine biological media. Advanced multiplex, bead-based, and automated analytical platforms provided sufficient sensitivity, precision, and accuracy over the traditional enzyme-linked immunosorbent assay (ELISA). Thus, baseline cytokine levels can be quantified from healthy human subjects and animals and compared to an in vivo exposure response from an environmental chemical. Specifically, patterns of cytokine responses in humans exposed to environmental levels of ozone and diesel exhaust, and in rodents exposed to selected pesticides (such as fipronil and carbaryl), were used as case studies to generally assess the taxonomic applicability of cytokine responses. The findings in this study may aid in the application of measureable cytokine markers in future adverse outcome pathway (AOP)-based toxicity testing. Data from human and animal studies were coalesced and the possibility of using cytokines as key events (KE) to bridge species responses to external stressors in an AOP-based framework was explored.

Effect of carbaryl (carbamate insecticide) on acetylcholinesterase activity of two strains of Daphnia magna (Crustacea, Cladocera).

The present study was designed to investigate the effect of carbaryl (carbamate insecticide) on the acetylcholinesterase activity in two strains (same clone A) of the crustacean cladoceran Daphnia magna. Four carbaryl concentrations (0.4, 0.9, 1.8 and 3.7 µg L(-1)) were compared against control AChE activity. Our results showed that after 48 h of carbaryl exposure, all treatments induced a significant decrease of AChE activities whatever the two considered strains. However, different responses were registered in terms of lowest observed effect concentrations (LOEC: 0.4 µg L(-1) for strain 1 and 0.9 µg L(-1) for strains 2) revealing differences in sensitivity among the two tested strains of D. magna. These results suggest that after carbaryl exposure, the AChE activity responses can be also used as a biomarker of susceptibility. Moreover, our results show that strain1 is less sensitive than strain 2 in terms of IC50-48 h of AChE activity. Comparing the EC50-48 h of standard ecotoxicity test and IC50-48 h of AChE inhibition, there is the same order of sensitivity with both strains.

Acute and chronic ecotoxicity of carbaryl with a battery of aquatic bioassays.

The ecotoxic effects of carbaryl (carbamate insecticide) were investigated with a battery of four aquatic bioassays. The nominal effective concentrations immobilizing 50% of Daphnia magna (EC50) after 24 and 48 h were 12.76 and 7.47 µg L(-1), respectively. After 21 days of exposure of D. magna, LOECs (lowest observed effect concentrations) for cumulative molts and the number of neonates per surviving adult were observed at carbaryl concentration of 0.4 µg L(-1). An increase of embryo deformities (curved or unextended shell spines) was observed at 1.8 and 3.7 µg L(-1), revealing that carbaryl could act as an endocrine disruptor in D. magna. Other bioassays of the tested battery were less sensitive: the IC50-72h and IC10-72h of the algae Pseudokirchneriella subcapitata were 5.96 and 2.87 mg L(-1), respectively. The LC50-6d of the ostracod Heterocypris incongruens was 4.84 mg L(-1). A growth inhibition of H. incongruens was registered after carbaryl exposure and the IC20-6d was 1.29 mg L(-1). Our results suggest that the daphnid test sensitivity was better than other used tests. Moreover, carbaryl has harmful and toxic effects on tested species because it acts at low concentrations on diverse life history traits of species and induce embryo deformities in crustaceans.

Developmental neurotoxic effects of two pesticides: Behavior and biomolecular studies on chlorpyrifos and carbaryl.

In recent times, an increased occurrence of neurodevelopmental disorders, such as neurodevelopmental delays and cognitive abnormalities has been recognized. Exposure to pesticides has been suspected to be a possible cause of these disorders, as these compounds target the nervous system of pests. Due to the similarities of brain development and composition, these pesticides may also be neurotoxic to humans. We studied two different pesticides, chlorpyrifos and carbaryl, which specifically inhibit acetylcholinesterase (AChE) in the nervous system. The aim of the study was to investigate if the pesticides can induce neurotoxic effects, when exposure occurs during a period of rapid brain growth and maturation. The results from the present study show that both compounds can affect protein levels in the developing brain and induce persistent adult behavior and cognitive impairments, in mice neonatally exposed to a single oral dose of chlorpyrifos (0.1, 1.0 or 5mg/kg body weight) or carbaryl (0.5, 5.0 or 20.0mg/kg body weight) on postnatal day 10. The results also indicate that the developmental neurotoxic effects induced are not related to the classical mechanism of acute cholinergic hyperstimulation, as the AChE inhibition level (8-12%) remained below the threshold for causing systemic toxicity. The neurotoxic effects are more likely caused by a disturbed neurodevelopment, as similar behavioral neurotoxic effects have been reported in studies with pesticides such as organochlorines, organophosphates, pyrethroids and POPs, when exposed during a critical window of neonatal brain development.