Baseline activities of four biomarkers in three life-stages of the amphipod, Leptocheirus plumulosus

This study investigated differences in baseline levels of four cellular biomarkers (glutathione (GSH), lipid peroxidation (LPx), acetylcholinesterase (AChE), and cholesterol (CHL)) in the larval, juvenile, and adult stages of the estuarine amphipod, Leptocheirus plumulosus. Glutathione, LPx, and AChE exhibited the same pattern of decreasing levels with increasing developmental stage. Cholesterol showed an inverse relationship of increasing levels with increasing developmental stages. This research provides valuable background information that may be used in future assessments of amphipod biomarker research.     

Lethal and sublethal effects of simvastatin,irgarol, and PBDE-47 on the estuarine fish,Fundulus heteroclitus

This study investigated the effects of simvastatin, a lipid-regulating drug; irgarol, an antifouling biocide; and PBDE-47, a brominated flame retardant, on the estuarine fish, Fundulus heteroclitus. Sublethal effects (changes in glutathione (GSH), lipid peroxidation (LPx), acetylcholinesterase (AChE), and cholesterol (CHL) levels) and lethal effects (survival) were determined after individual exposure to the three compounds. There were no significant differences in GSH or CHL levels in fish exposed to any of the test compounds. LPx levels significantly decreased with increasing irgarol concentrations. AChE levels were significantly lower in fish exposed to simvastatin at the 1.25 mg/L concentration and significantly higher at the PBDE-47 concentration of 0.0125 mg/L. The LC50 values were 2.68, 3.22, and > 0.1 mg/L for simvastatin, irgarol and PBDE-47, respectively.     

Toxicity of the mosquito control insecticide phenothrin to three life stages of the grass shrimp (Palaemonetes pugio)

Phenothrin is a synthetic pyrethroid used as a contact insecticide in mosquito control programs. This study compared the toxicity of phenothrin to adult, larval and embryonic grass shrimp (Palaemonetes pugio) and examined oxidative stress responses in adult and larval grass shrimp. The adult 24-h LC50 was 0.341 μg/L (95 % confidence intervals 0.282–0.412) and the 96-h LC50 was 0.161 μg/L (95 % CI 0.128–0.203 μg/L). The larval 24-h LC50 was 0.50 μg/L (95 % CI 0.441–0.568) and the 96-h LC50 was 0.154 μg/L (95 % CI 0.139–0.170 μg/L). In the presence of sediment, the 24-h LC50 was 6.30 μg/L (95 % CI 5.00–7.44 μg/L) for adults and 0.771 μg/L (95 % CI 0.630–0.944) for larvae. The sublethal biomarkers glutathione and lipid peroxidase (LPx) were examined after 96-h phenothrin exposure at five concentrations, and there were no statistically significant differences in these levels in adults or larvae compared to controls. There was a significant downward trend in larval LPx levels. This research confirms that phenothrin is highly toxic to grass shrimp and suggests that both adult and larval grass shrimp are appropriate life stages for risk assessments.     

Toxicity of the mosquito control insecticide phenothrin to three life stages of the grass shrimp (Palaemonetes pugio)

Phenothrin is a synthetic pyrethroid used as a contact insecticide in mosquito control programs. This study compared the toxicity of phenothrin to adult, larval and embryonic grass shrimp (Palaemonetes pugio) and examined oxidative stress responses in adult and larval grass shrimp. The adult 24-h LC50 was 0.341 μg/L (95 % confidence intervals 0.282-0.412) and the 96-h LC50 was 0.161 μg/L (95 % CI 0.128-0.203 μg/L). The larval 24-h LC50 was 0.50 μg/L (95 % CI 0.441-0.568) and the 96-h LC50 was 0.154 μg/L (95 % CI 0.139-0.170 μg/L). In the presence of sediment, the 24-h LC50 was 6.30 μg/L (95 % CI 5.00-7.44 μg/L) for adults and 0.771 μg/L (95 % CI 0.630-0.944) for larvae. The sublethal biomarkers glutathione and lipid peroxidase (LPx) were examined after 96-h phenothrin exposure at five concentrations, and there were no statistically significant differences in these levels in adults or larvae compared to controls. There was a significant downward trend in larval LPx levels. This research confirms that phenothrin is highly toxic to grass shrimp and suggests that both adult and larval grass shrimp are appropriate life stages for risk assessments.     

Insecticides biomarker responses on a freshwater fish Corydoras paleatus (Pisces: Callichthyidae)

This study was undertaken to investigate the effects of sublethal concentration of three different classes of insecticides (carbamate, organophosphate, and pyrethroid compounds) on the freshwater fish Corydoras paleatus. For this purpose, fish were exposed for 96 hours to commercial pesticides. Different biomarkers were analyzed as levels of lipid peroxidation (LPO), piscine micronucleus test, and enzymatic activities of catalase (CAT), glutathione S-transferase (GST), and acetylcholinesterase (AChE). The brain AChE was inhibited with carbaryl and methyl parathion, but no inhibition was observed with deltamethrin. The insecticides did not cause oxidative stress or genotoxic effects at the tested concentrations. Further studies are needed to elucidate the biotransformation of Corydoras paleatus insecticides and a possible resistance mechanism.     

Toxicity assessment of municipal sewage treatment plant effluent by an integrated biomarker response in the liver of crucian carp (Carassius auratus)

In this study, crucian carp (Carassius auratus) was exposed to the increasing concentrations of municipal sewage treatment plant effluent (MSTPE) for 15 days, and the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and acetylcholinesterase (AChE), together with the contents of malondialdehyde (MDA) and glutathione (GSH) in the liver of C. auratus were investigated. Moreover, the integrated biomarker response (IBR) approach was applied to assess the adverse effects of MSTPE in freshwater. The aim of the study was to provide an effective biological indicator for evaluating the toxicity effects and ecological risks of MSTPE in the freshwater environment quantitatively. Results showed that MSTPE could cause oxidative damage to the liver of C. auratus, which reflected through the increasing MDA content over the exposure period. MSTPE also led to the biochemical responses of antioxidant defense in C. auratus liver, such as the enhancement of SOD, CAT, and GPx activities, as well as the inhibition of AChE activity and GSH content. It was found that MDA, SOD, GPx, and GSH could be used as the biomarkers for reflecting the adverse effects of MSTPE in the receiving freshwater on the 12th day of exposure. A significant increase of IBR values was observed as the increasing concentration of MSTPE, and the IBR values presented a significant positive correlation (r?=?0.891, P?<?0.05) with the increasing concentrations of MSTPE, indicating that IBR approach is a promising tool for assessing the toxicity effects of MSTPE in environmental freshwater.

     

Alteration of acetylcholinesterase activity in Semele solida (MOLLUSCA: SEMELIDAE) as a biochemical response to coastal anthropogenic impact

Acetylcholinesterase (AChE) activity was analyzed as a molecular marker indicative of exposure to organophosphorus pesticide residues in individuals of the endemic clam species Semele solida in selected coastal locations of Chile's VIII Region. AChE activity was assayed in clams from (i) Penco Beach near the mouth of the Andalién River (Concepción Bay), (ii) Lenga Beach, near the mouth of the Lenga Estuary (San Vicente Bay), and (iii) Coliumo Beach, near the mouth of the Coliumo Estuary (Coliumo Bay). We also analyzed variations in protein content of clam hemolymph, and variability in the activity of AChE in relation to the sizes of the individual clams sampled. Collection of the clams was done using routine methods, during the spring of 2005, the period during which the use of pesticides is typically intensified in the surrounding forestry and agriculture. The results showed no significant correlation of AChE activity with either the size of the clam, or with the concentration of proteins in the hemolymph. The lowest AChE activity was observed in clams from mouth of the Andalién River (187.5 ± 34.9 Umin^?1) which was significantly less than that measured in specimens collected near the mouth of the Coliumo Estuary and the mouth of the Lenga Estuary. A very close relation was observed between the degree of anthropogenic inputs, ocean dynamics, and alterations in AChE activity in S. solida. This clam appears to be a useful indicator species, and AChE activity a sensitive marker for the presence of xenobiotics.     

Tidal influence on the distribution of hydrophobic organic contaminants in the Seine Estuary and biomarker responses on the copepod Eurytemora affinis

To elucidate tidally related variations of hydrophobic organic contaminant (HOC) bioavailability and the impact of these contaminants on estuarine ecosystems, both PCB and PAH concentrations were investigated in the dissolved phase and in the suspended particulate material (SPM) of the Seine Estuary. Both PAH and PCB highest levels were observed in surface and bottom water when SPM remobilizations were maximum, in relation to higher speed currents. In parallel, acetylcholinesterase (AChE) and glutathione-S-transferase (GST) activities were investigated in the copepod Eurytemora affinis. Significant decreasing AChE levels were measured during the tidal cycle and between surface and bottom copepods related to salinity and to HOC concentration variations. Significant increasing GST levels were also observed when HOC concentrations in the water column were the highest. This study underlined the need to standardize sampling procedures for biomonitoring studies in order to avoid interfering factors that could modify biomarker responses to chemical exposure.     

Differences in response of two model estuarine crustaceans after lethal and sublethal exposures to chlorpyrifos

This study assessed the in vitro and in vivo effects of an acetylcholinesterase enzyme inhibitor (chlorpyrifos) in two estuarine crustaceans: grass shrimp (Palaemonetes pugio) and mysid (Americamysis bahia). The differences in response were quantified after lethal and sublethal exposures to chlorpyrifos and in vitro assays with chlorpyrifos-oxon. Results from the in vitro experiments indicated that the target enzyme, acetylcholinesterase (AChE), in the two species was similar in sensitivity to chlorpyrifos inhibition with IC50s of 0.98 nM and 0.89 nM for grass shrimp and mysids, respectively. In vivo experiments showed that mysids were significantly more sensitive to chlorpyrifos-induced AChE inhibition after 24 h of exposure. The in vivo EC50s for AChE inhibition were 1.23 μg L^?1 for grass shrimp and 0.027 μg L^?1 for mysids.

Median lethal concentrations (24h LC50 values) were 1.06 μg L^?1 for grass shrimp and 0.068 μg L^?1 for mysids. The results suggest that differences in the response of these two crustaceans are likely related to differences in uptake and metabolism rather than target site sensitivity.     

The effect of pesticides and metals on acetylcholinesterase (AChE) in various tissues of blue mussel (Mytilus trossulus L.) in short-term in vivo exposures at different temperatures

The purpose of the study was to assess the impact of short-term exposure to selected toxicants as well as metal accumulation upon acetylcholinesterase (AChE) in the blue mussel, Mytilus trossulus L., in laboratory in vivo experiments. Mussels were exposed for up to 48 hours to a mixture of copper (Cu^{2 +}, 400 μ g L^?1) and cadmium (Cd^{2 +}, 200 μ g L^?1), to dichlorvos (DDVP, 100 μ g L^?1), and to carbaryl (100 μ g L^?1) at two temperatures: 5°C and 20°C. Samples were collected after 0, 6, 12, 24, and 48 hours of exposure, and AChE activity and metal concentration (where applicable) were analysed in gills, digestive gland, mantle+muscles, and the whole soft tissue. Very strong AChE inhibition was observed in response to the dichlorvos treatment, mainly in gills. Carbaryl and the metals caused a short-term inhibition effect. Considerable differences in AChE activity between the two temperatures were noticed. In particular, the metals were accumulated much faster at 20°C than at 5°C, especially in gills. No correlation between AChE activity and metal concentration was found. Gills turned out to be the optimal tissue for AChE activity analysis in short-term studies.     

Acute effects of copper and mercury on the estuarine fish Pomatoschistus microps: Linking biomarkers to behaviour

The main objective of the present study was to investigate possible links between biomarkers and swimming performance in the estuarine fish Pomatoschistus microps acutely exposed to metals (copper and mercury). In independent bioassays, P. microps juveniles were individually exposed for 96 h to sub-lethal concentrations of copper or mercury. At the end of the assays, swimming performance of fish was measured using a device specially developed for epibenthic fish (SPEDE). Furthermore, the following biomarkers were measured: lipid peroxidation (LPO) and the activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), glutathione S-transferases (GST), 7-ethoxyresorufin-O-deethylase (EROD), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx). LC₅₀s of copper and mercury (dissolved throughout metal concentrations) at 96 h were 568 μg L⁻¹ and 62 μg L⁻¹, respectively. Significant and concentration-dependent effects of both metals on swimming resistance and covered distance against water flow were found at concentrations equal or higher than 50 μg L⁻¹ for copper and 3 μg L⁻¹ for mercury (dissolved throughout metal concentrations). These results indicate that SPEDE was efficacious to quantify behavioural alterations in the epibenthic fish P. microps at ecologically relevant concentrations. Significant alterations by both metals on biomarkers were found including: inhibition of AChE and EROD activities, induction of LDH, GST and anti-oxidant enzymes, and increased LPO levels, with LOEC values ranging from 25 to 200 μg L⁻¹ for copper and from 3 to 25 μg L⁻¹ for mercury (dissolved throughout metal concentrations). Furthermore, significant and positive correlations were found between some biomarkers (AChE and EROD) and behavioural parameters, while negative correlations were found for others (LPO, anti-oxidant enzymes and LDH) suggesting that disruption of cholinergic function through AChE inhibition, decreased detoxification capability due to EROD inhibition, additional energetic demands to face chemical stress, and oxidative stress and damage may contribute to decrease the swimming performance of fish. Since a reduced swimming capability of fish may reduce their ability to capture preys, avoid predators, and interfere with social and reproductive behaviour, the exposure of P. microps to copper and/or mercury concentrations similar to those tested here may decrease the fitness of wild populations of this species.     

Biochemical effects of clomazone herbicide on piava (Leporinus obtusidens)

This study aims to verify the effects of the clomazone concentration used in rice fields on acetylcholinesterase (AChE), thiobarbituric acid reactive substances (TBARS), protein carbonyl and catalase activity in tissues of piava (Leporinus obtusidens). LC(50)-96h was 5.0 mg L(-1) and the fish were exposed to 1/10 of LC(50)-96 h: 0.5 mg L(-1) of clomazone for 96 and 192h. The same parameters were also assayed after a recovery period of 192 h in clean water. AChE activity was reduced only in the brain and heart of fish exposed for 96 h. AChE activity was decreased in the brain, muscle and heart tissues after 192 h of exposure. After 192 h of recovery period, AChE activity remained diminished in brain and muscle and showed a decrease in eye. However, after 192 h of recovery, AChE activity in heart was recovered. Fish showed increased TBARS levels in brain at all experimental periods. TBARS levels decreased in liver and muscle tissues after 192 h of exposure. The increase in muscle TBARS persisted in fish transferred to clean water. Protein carbonyl in the liver was increased in all periods studied including the recovery period. Catalase activity was reduced during all periods. The present study demonstrates the occurrence of disorders in AChE, TBARS, protein carbonyl and catalase activity in piava. The results also show changes in fish after exposure to an environmentally relevant concentration of clomazone. Most effects observed persisted after the recovery period. Thus, these parameters may be used to monitor clomazone toxicity in fish.     

Effect of phenobarbital and polychlorinated biphenyls on the toxicity and disposition of 1,2,4,5‐tetrachlorobenzene in the rat

Soil avoidance by earthworms has been generally considered a relevant and sensitive endpoint for assessing soil contamination by xenobiotics. However, when pesticide ecotoxicological assessment is concerned, the sensitivity of the recently standardized avoidance assay has been questioned. We hypothesized that this controversy may be due to the specific pesticide mode of action of the chemicals used rather than reveal inconsistencies in the test feasibility, i.e. provided that no pesticides interfering with neuronal pathways are tested, this bioassay should keep expected high levels of sensitivity. In this study, the avoidance behaviour of the earthworm Eisenia andrei under exposure to the carbamate insecticide methomyl [S-methyl N-(methylcarbamoyloxy)thioacetimidate] was linked to the corresponding acetylcholinesterase (AChE) inhibition. Significant AChE inhibition occurred at lower concentrations (from 0.86 mg Kg^?1 onwards) than significant avoidance of spiked soil (from 5.62 mg Kg^?1 onwards). This indicates that assessments regarding pesticides that have neurotoxic activity may be biased if behavioral endpoints are selected. Despite theoretical hypothesis that have been raised, this should be the first study providing preliminary experimental evidence on such a link between avoidance behavior and neuronal impairment levels in earthworms. Further studies are ongoing that should refine conclusions of this study.     

Synergistic effects caused by atrazine and terbuthylazine on chlorpyrifos toxicity to early-life stages of the zebrafish Danio rerio

This study examined the effects of three widely used pesticides that have been previously detected in aquatic systems neighbouring agricultural fields on the early-life stages of the zebrafish Danio rerio. Tests involving single exposures and binary combinations of the s-triazine herbicides (atrazine and terbuthylazine) and the organophosphate insecticide chlorpyrifos were performed. Several endpoints, such as swimming behaviour, morphological abnormalities and mortality, were studied. In addition, the inhibition of acetylcholinesterase (AChE) activity was investigated in order to evaluate the mode of action and toxicity of chlorpyrifos in the presence of these herbicides. Results indicate that both binary mixtures elicited synergistic responses on the swimming behaviour of zebrafish larvae. Moreover, although the herbicides were not effective inhibitors of the AChE on their own, a synergistic inhibition of the enzyme activity was obtained by exposure to mixtures with chlorpyrifos. We observed a correlation between impairment of swimming behaviour of the larvae and inhibition of AChE activity. This study supports previous studies concerning the risk assessment of mixtures since the toxicity may be underestimated when looking only at the single toxicants and not their mixtures.     

Development and application of long-term sublethal whole sediment tests with Arenicola marina and Corophium volutator using Ivermectin as the test compound

Short-term whole sediment tests using the amphipod Corophium volutator and the polychaete Arenicola marina are now routinely used in Europe to assess the acute toxicity of marine sediments. However, there is still a need to develop longer-term assays which measure effects on sublethal endpoints that are more relevant to predicting impacts at the population level. The effect of increasing exposure times and measuring additional endpoints such as growth, on the sensitivity of these assays was investigated. The test compound used was the chemotherapeutant Ivermectin (IVM), used in aquaculture to treat sea lice infestations. IVM was found to be acutely toxic to both test organisms. Extending the lugworm test to 100 days increased sensitivity of survival by a factor of three; a significant reduction in casting rate was observed at concentrations an order of magnitude lower. This assay shows potential for detecting the sublethal effects of low concentrations of sediment contaminants. Increasing the exposure time did not seem to affect the sensitivity of the amphipod, but further method development is required.     

Effects of four rice herbicides on some metabolic and toxicology parameters of teleost fish (Leporinus obtusidens)

Effects of different herbicides on acetylcholinesterase (AChE), catalase and TBARS formation in teleost fish (Leporinus obtusidens) were studied. Fish were exposed during 30 days at concentrations of herbicides used in rice field. AChE activity in the brain decreased significantly after exposure to the herbicides clomazone and quinclorac. However, AChE activity increased significantly in muscle tissue after exposure to clomazone, propanil and metsulfuron methyl. Fish exposed to quinclorac, propanil and metsulfuron methyl showed TBARS decreased levels in brain and muscle tissues. However, TBARS and catalase activity increased in liver tissue after clomazone and propanil exposure. This study pointed out long-term effects on AChE activity, oxidative stress and antioxidant enzyme catalase in tissues of L. obtusidens after exposure to environmentally relevant concentrations of rice field herbicides. These parameters have been used to monitor fish toxicity in rice field system.     

Effect of coumaphos on cholinesterase activity,hematology, and biochemical blood parameters of bovines in tropical regions of Mexico

To assess the effect of coumaphos [O-(3-chloro-4-methyl-2-oxo-2H-1-benzopyran-7-yl) O,O-diethyl phosphorothioate] exposure on physiological responses during bovine production, acetylcolinesterase (AChE) and butyrylcholinesterase (BuChE) activities were measured in whole blood, erythrocytes, and plasma of healthy male steers (Bos Taurus x Bos indicus) sprayed with coumaphos at a non-lethal dose of 1 mg kg^{? 1} body weight per day once every 14 (in vivo group) or 21 days (southern and central groups). Coumaphos topically administered at 1 mg/kg body weight per day to cattle under normal management practices in tropical areas produced a significant inhibition in erythrocyte (RBC) AChE and BuAChE activities when compared to baseline levels. RBC-AChE activity for the in vivo group decreased 71.3% (P < 0.05) and BuChE activity 59.1% (P < 0.05); RBC-AChE activity decreased 55.1% (P < 0.05) (southern group) and 43.4% (P < 0.05) (central group). Compared to the control specimens, steers from in vivo, southern, and central groups after 150 days of exposure had lower (P < 0.05) leukocyte count, absolute lymphocyte, erythrocyte, and platelet counts. Decreases in RBC-AChE activities correlated with decreased lymphocyte (r = 1.000, p = 0.01), erythrocyte (r = 1.000, p = 0.003), and platelet counts (r = 0.841, p = 0.036). Significantly increased BUN levels (P < 0.05) correlated with the decrease in RBC-AChE activities (r = ? 0.997, p = 0.047) and with the decrease in absolute red blood cell (r = ? 0.883, p = 0.020) and lymphocyte (r = ? 0.825, p = 0.043) counts; increased (P < 0.05) total plasma protein levels correlated with the decrease in RBC-AChE activities (r = ?0.998, p = 0.043), absolute red blood cell (r = ? 0.998, p = 0.040), lymphocyte (r = ? 0.893, p = 0.017), and platelet (r = -0.855, p = 0.030) counts. The physiological responses correlated with the erythrocyte acetylcholinesterase inhibition could be considered as early indicators or warning responses of bovine exposures to organophosphorus pesticides (OPs).     

Comparative analysis of selected biomarkers and pesticide sensitivity in juveniles of Solea solea and Solea senegalensis

The common sole, Solea solea (Linneus, 1758), and the Senegalese sole, Solea senegalensis (Kaup, 1858), are two important commercial species that coexist in the NW Mediterranean. In order to assess the species' ability to respond to chemical insults, a comparison of activities on enzymes involved in xenobiotic metabolism was carried out. Juveniles of both species were sampled in winter 2011 from the Ebro Delta region, and activities of selected enzymes such as acetylcholinesterase (AChE), carboxylesterase (CbE), ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) were determined in several tissues. Lipid peroxidation (LP) levels in plasma were measured as a sign of oxidative stress. In vitro exposures to selected pesticides were contrasted, analysing AChE and CbE activities in several tissue homogenates. Overall, enzymatic activities were higher in S. solea except for gill GST and CbE and kidney GST, while plasmatic LP levels were similar. In vitro contrasts revealed lower IC50 values for CbE activities in S. solea, suggesting a greater buffer capacity of this enzyme to potentially reduce pesticide toxicity over AChE.     

Herbicide Roundup® and its main constituents cause oxidative stress and inhibit acetylcholinesterase in liver of Carassius auratus

Roundup® is a glyphosate-based herbicide containing a mixture of surfactants. This paper evaluates the toxic effects of Roundup® and its main constituents on the goldfish, Carassius auratus, after 7 days exposure. Fish were exposed to 0.16, 0.032 and 0.0064 mg/L of Roundup® [containing 41% isopropylamine salt of glyphosate (G.I.S) and 18% polyoxyethylene amine (POEA)], G.I.S, and POEA. Their livers were taken for determining reactive oxygen species (ROS), superoxide dismutase (SOD) activity, malondialdehye (MDA) content and acetylcholinesterase (AChE) activity. Hydroxyl radical (·OH) could be induced by exposing Roundup® at a rate of 43%–111%, G.I.S at 90%–124% and POEA at142%–157%. A decreased SOD activity was observed in fish exposed to G.I.S and POEA. The contents of MDA significantly increased when exposed to Roundup® at all concentrations, 0.16 mg/L G.I.S and 0.032 mg/L POEA. The exposure led to an inhibition of AChE in livers overall during the experimental periods. POEA was more toxic than Roundup® or G.I.S during this experiment. AChE and ·OH are supposed to be sensitive biomarkers of the exposure of Roundup® and its main constituents to C. auratus.