Biochemical responses of the golden mussel Limnoperna fortunei under dietary glyphosate exposure.

The aim of this study was to analyze the biochemical alterations in the golden mussel Limnoperna fortunei under dietary glyphosate exposure. Mussels were fed during 4 weeks with the green algae Scenedesmus vacuolatus previously exposed to a commercial formulation of glyphosate (6 mg L-1 active principle) with the addition of alkyl aryl polyglycol ether surfactant. After 1, 7, 14, 21 and 28 days of dietary exposure, glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), acetylcholinesterase (AChE), carboxylesterases (CES) and alkaline phosphatase (ALP) activities, glutathione (GSH) content and damage to lipids and proteins levels were analyzed. A significant increase (72%) in the GST activity and a significant decrease (26%) in the CES activity in the mussels fed on glyphosate exposed algae for 28 days were observed. The ALP activity was significantly increased at 21 and 28 days of dietary exposure (48% and 72%, respectively). GSH content and CAT, SOD and AchE activities did not show any differences between the exposed and non exposed bivalves. No oxidative damage to lipids and proteins, measured as TBARS and carbonyl content respectively, was observed in response to glyphosate dietary exposure. The decrease in the CES activity and the increases in GST and ALP activities observed in L. fortunei indicate that dietary exposure to glyphosate provokes metabolic alterations, related with detoxification mechanisms.

Toxicokinetic and toxicodynamic studies of carbaryl alone or in binary mixtures with azinphos methyl in the freshwater gastropod Planorbarius corneus.

Carbamate insecticides such as carbaryl and organophosphates such as azinphos-methyl share the ability to inhibit the activity of B-esterases. This study aimed to (1) assess the inhibitory effects of carbaryl on B-esterase activity in soft tissues and hemolymph of Planorbarius corneus; (2) establish whether binary mixtures of carbaryl and azinphos-methyl depart or not from a model of concentration addition on the inhibition of cholinesterase activity; (3) determine the bioconcentration and elimination of the pesticides. The results showed that exposure of gastropods to increasing concentrations of carbaryl (0.1-5 mg L-1) for 48 h inhibited cholinesterase activity in a concentration-dependent manner, with an EC50 of 1.4 ±â€¯0.3 mg L-1 and 1.2 ±â€¯0.1 mg L-1 for soft tissue and hemolymph, respectively. Carboxylesterase activity, measured with the substrates p-nitrophenyl butyrate and p-nitrophenyl acetate, was between 2.3 and 25 times more sensitive to carbaryl inhibition than cholinesterase activity. Binary mixtures corresponding to 0.5 EC50 carbaryl + 0.5 EC50 azinphos-methyl and 0.75 EC50 carbaryl + 0.75 EC50 azinphos-methyl produced inhibitions of cholinesterase activity similar to those of individual pesticides, following a model of concentration addition. Bioconcentration was analyzed using a one-compartment model. The absorption kinetics (k1) for both pesticides alone (1.4 mg L-1 of carbaryl or 1.8 mg L-1 of azinphos-methyl) or mixed (1.4 mg L-1 of carbaryl + 1.8 mg L-1 of azinphos-methyl) were similar. The elimination kinetics ratio (k2) estimated for the pesticides alone or in the mixtures showed that carbaryl was eliminated 3.5 times faster than azinphos-methyl. These results suggest that exposure of Planorbarius corneus to binary mixtures of carbaryl and azinphos-methyl for 48 h follow a concentration addition model on inhibition of cholinesterase activity and that the pesticide mixtures do not change the toxicokinetic parameters of the parent compounds.

Evaluation of biochemical markers in the golden mussel Limnoperna fortunei exposed to glyphosate acid in outdoor microcosms.

In this study, the impact of technical grade glyphosate acid on Limnoperna fortunei was assessed employing outdoor microcosms treated with nominal glyphosate concentrations of 1, 3 and 6 mg L(-1). At the end of the experiment (26 days), catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST), acetylcholinesterase (AChE), carboxylesterases (CES) and alkaline phosphatase (ALP) activities, and lipid peroxidation levels were analyzed. GST and ALP activities and lipid peroxidation levels showed a significant increase with respect to controls in the mussels exposed to glyphosate (up to 90, 500 and 69 percent, respectively). CES and SOD activities showed a significant decrease in glyphosate exposed bivalves with respect to controls (up to 48 and 37 percent, respectively). CAT and AChE did not show differences between exposed and no exposed bivalves. The increase in lipid peroxidation levels and the decrease in SOD and CES activities observed in L. fortunei indicate that glyphosate had adverse effects on the metabolism of this bivalve. The results of the present study also indicate that a "multibiomarker approach" provides a more precise knowledge of the impact of glyphosate on L. fortunei.

Cholinesterase and carboxylesterase inhibition in Planorbarius corneus exposed to binary mixtures of azinphos-methyl and chlorpyrifos.

Though pesticide mixtures are commonly encountered in fresh water systems, the knowledge of their effects on non-target aquatic species is scarce. The present investigation was undertaken to explore the in vivo inhibition of the freshwater gastropod snail Planorbarius corneus cholinesterase (ChE) and carboxylesterases (CES) activities by the organophosphorus pesticides azinphos-methyl (AZM) and chlorpyrifos (CPF). To this end, snails were exposed for 48 h to different concentrations of AZM and CPF in single-chemical and binary-mixture studies, and ChE and CES activities were measured in the whole soft body tissues and hemolymph. ChE activity was measured with acetylthiocholine as substrate and CES activity was measured with four substrates: p-nitrophenyl acetate, p-nitrophenyl butyrate, 1- and 2-naphthyl acetate. Single-chemical experiments showed that CPF was a more potent inhibitor of ChE activity than AZM (350 and 27 times for the whole soft tissue and hemolymph, respectively). CES were 15 times more sensitive than ChE when the activities were measured in the whole soft tissue of the animals exposed to AZM. Based on a default assumption of concentration addition, P. corneus snails were exposed to mixtures of AZM+CPF designed to yield predicted soft tissue ChE inhibitions of 31% (mixture 1), 50% (mixture 2) and 61% (mixture 3). Results showed that ChE inhibition produced by mixture 1 followed a model of concentration addition. In contrast, the other mixtures showed synergism, both in whole soft tissue and hemolymph. In addition, results obtained when the snails were exposed sequentially to the pesticides showed that the sequence AZM/CPF produced at 48 h a higher ChE inhibition than the sequence CPF/CPF. A range of metabolic pathways and responses associated with bioactivation or detoxification may play important roles in organophosphorus interactions. In particular, the data presented in the present study indicate that CES enzymes would be important factors in determining the effects of the mixtures of OPs on P. corneus ChE activity.

Binary mixtures of azinphos-methyl oxon and chlorpyrifos oxon produce in vitro synergistic cholinesterase inhibition in Planorbarius corneus.

In this study, the cholinesterase (ChE) and carboxylesterase (CES) activities present in whole organism homogenates from Planorbarius corneus and their in vitro sensitivity to organophosphorous (OP) pesticides were studied. Firstly, a characterization of ChE and CES activities using different substrates and selective inhibitors was performed. Secondly, the effects of azinphos-methyl oxon (AZM-oxon) and chlorpyrifos oxon (CPF-oxon), the active oxygen analogs of the OP insecticides AZM and CPF, on ChE and CES activities were evaluated. Finally, it was analyzed whether binary mixtures of the pesticide oxons cause additive, antagonistic or synergistic ChE inhibition in P. corneus homogenates. The results showed that the extracts of P. corneus preferentially hydrolyzed acetylthiocholine (AcSCh) over propionylthiocholine (PrSCh) and butyrylthiocholine (BuSCh). Besides, AcSCh hydrolyzing activity was inhibited by low concentrations of BW284c51, a selective inhibitor of AChE activity, and also by high concentrations of substrate. These facts suggest the presence of a typical AChE activity in this species. However, the different dose-response curves observed with BW284c51 when using PrSCh or BuSCh instead of AcSCh suggest the presence of at least another ChE activity. This would probably correspond to an atypical BuChE. Regarding CES activity, the highest specific activity was obtained when using 2-naphthyl acetate (2-NA), followed by 1-naphthyl acetate (1-NA); p-nitrophenyl acetate (p-NPA), and p-nitrophenyl butyrate (p-NPB). The comparison of the IC(50) values revealed that, regardless of the substrate used, CES activity was approximately one order of magnitude more sensitive to AZM-oxon than ChE activity. Although ChE activity was very sensitive to CPF-oxon, CES activity measured with 1-NA, 2-NA, and p-NPA was poorly inhibited by this pesticide. In contrast, CES activity measured with p-NPB was equally sensitive to CPF-oxon than ChE activity. Several specific binary combinations of AZM-oxon and CPF-oxon caused a synergistic effect on the ChE inhibition in P. corneus homogenates. The degree of synergism tended to increase as the ratio of AZM-oxon to CPF-oxon decreased. These results suggest that synergism is likely to occur in P. corneus snails exposed in vivo to binary mixtures of the OPs AZM and CPF.