Lethality and Acetylcholinesterase Inhibition in a Native Invertebrate Species Exposed to Water Samples of an Impacted Stream (Reconquista River Basin, Argentina).

The study of multiple biomarkers in bioindicator species is a useful tool to evaluate water quality in addition to physicochemical analysis. The aim of this work was to study the toxicity of water samples from two sites with different anthropogenic impacts (R: near a residential area and FP: close to horticultural farms and industrial waste treatment plants) from Las Catonas sub-basin (Reconquista River basin) in the native gastropod Biomphalaria straminea. Some physicochemical parameters and chlorpyrifos concentration were measured in water samples. Snails were exposed in laboratory conditions 48 h to the water samples and neurotoxicity, behavior, lethality and acetylcholinesterase, carboxylesterase, glutathione S-transferase, glutathione reductase and catalase activities were measured. In water from FP, chlorpyrifos was detected and conductivity and pH were higher than in R. Lethality (60%) and a decrease (30%) in acetylcholinesterase were observed in snails exposed to FP indicating that water contamination causes high toxicity in B. straminea.

Toxicity evaluation of the active ingredient acetamiprid and a commercial formulation (Assail® 70) on the non-target gastropod Biomphalaria straminea (Mollusca: Planorbidae).

Neonicotinoids emerged as an environmentally safe alternative to previous generations of insecticides becoming one of the most widely applied in modern agriculture. Nevertheless, they have been reported to affect several non-target organisms. Most toxicity studies focus on the effects on pollinators or terrestrial invertebrates and evaluate either the active ingredient or the commercial formulation. In the present study, we aimed to assess the long-term effects of the active ingredient acetamiprid and a broadly used commercial formulation (Assail® 70) on the non-target freshwater gastropod Biomphalaria straminea using a battery of biomarkers. A 14 day-exposure of adult organisms to both active ingredient and commercial formulation increased carboxylesterase activity and glutathione content, inhibited superoxide dismutase activity and decreased reactive oxygen species levels. The commercial formulation additionally increased glutathione S-transferase activity and inhibited catalase activity. The results indicate a greater toxicity of the commercial formulation than that of the active ingredient alone. Cholinesterase activity, development and offspring survival of B. straminea were not impaired. We conclude that the toxicity of acetamiprid on this gastropod species is mainly related to effects on detoxification and oxidative metabolism responses. This study provides novel information about the adverse effects of the active ingredient and a commercial formulation of a widely used neonicotinoid on a non-target aquatic species.

Effects of paralytic shellfish toxins on the middle intestine of Oncorhynchus mykiss: Glutathione metabolism, oxidative status, lysosomal function and ATP-binding cassette class C (ABCC) proteins activity.

We studied the absorption, cytotoxicity and oxidative stress markers of Paralytic Shellfish Toxins (PST) from three extracts from Alexandrium catenella and A. ostenfeldii, in middle Oncorhynchus mykiss intestine in vitro and ex vivo preparations. We measured glutathione (GSH) content, glutathione-S transferase (GST), glutathione reductase (GR) and catalase (CAT) enzymatic activity, and lipid peroxidation in isolated epithelium exposed to 0.13 and 1.3 µM PST. ROS production and lysosomal membrane stability (as neutral red retention time 50%, NRRT50) were analyzed in isolated enterocytes exposed to PST alone or plus 3 µM of the ABCC transport inhibitor MK571. In addition, the concentration-dependent effects of PST on NRRT50 were assayed in a concentration range from 0 to 1.3 µM PST. We studied the effects of three different PST extracts on the transport rate of the ABCC substrate DNP-SG by isolated epithelium. The extract with highest inhibition capacity was selected for studying polarized DNP-SG transport in everted and non-everted intestinal segments. We registered lower GSH content and GST activity, and higher GR activity, with no significant changes in CAT activity, lipid peroxidation or ROS level. PST exposure decreased NRRT50 in a concentration-depend manner (IC50 = 0.0045 µM), but PST effects were not augmented by addition of MK571. All the three PST extracts inhibited ABCC transport activity, but this inhibition was effective only when the toxins were applied to the apical side of the intestine and DNP-SG transport was measured at the basolateral side. Our results indicate that PST are absorbed by the enterocytes from the intestine lumen. Inside the enterocytes, these toxins decrease GSH content and inhibit the basolateral ABCC transporters affecting the normal functions of the cell. Furthermore, PST produce a strong cytotoxic effect to the enterocytes by damaging the lysosomal membrane, even at low, non-neurotoxic concentrations.

Environmental concentrations of azinphos-methyl cause different toxic effects without affecting the main target (cholinesterases) in the freshwater gastropod Biomphalaria straminea.

Organophosphate insecticides (OPs) are commonly used in Argentina and around the world for pest control in food crops. They exert their toxicity through the inhibition of the enzyme acetylcholinesterase. In the present study, we aimed to evaluate biochemical and reproductive effects in Biomphalaria straminea, a freshwater gastropod naturally distributed in Argentina, of subchronic exposures to environmental azinphos-methyl concentrations (20 and 200 µg L-1). For biochemical parameters, adult organisms were exposed for 14 days and the activity of cholinesterases (ChEs), carboxylesterases (CEs), glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), the production of reactive oxygen species (ROS), the total antioxidant capacity (TAC), glycogen and proteins were determined. For reproductive parameters, the egg masses of B. straminea were exposed to azinphos-methyl for one month, and the hatching time and success as well as the offspring survival were registered. We found different toxic effects elicited by the insecticide on the studied biomarkers. CEs activity was significantly inhibited while CAT and GST activities, ROS production and TAC were significantly increased, with respect to the solvent control group. ChE and SOD activities and protein and glycogen contents were not altered by azinphos-methyl. The hatching time and success were not statistically different from control. Nevertheless, the offspring survival was severely affected by the insecticide. Our results show that the primary target of the insecticide (ChE) was not inhibited but CEs, GST, CAT, ROS, TAC and offspring survival were sensitive biomarkers and valuable endpoints for subchronic toxicity assessments in this species.

Effects of azinphos-methyl on enzymatic activity and cellular immune response in the hemolymph of the freshwater snail Chilina gibbosa.

The use of a battery of biomarkers, especially those more closely related to species integrity, is desired for more complete ecotoxicological assessments of the effects of pesticide contamination on aquatic organisms. The phosphorodithioate azinphos-methyl has been intensively used in agriculture worldwide and have been found in the habitat of Chilina gibbosa, a freshwater snail endemic to South America. This snail has been proposed as a good model organism for ecotoxicity bioassays on the basis of studies focused mainly on enzymatic responses in whole tissue homogenates. Our aim was to evaluate the effect of an acute 48 h exposure to an environmental concentration of azinphos-methyl on C. gibbosa hemolymph enzymatic activity and cellular immune response. Our results show that cholinesterase activity was strongly inhibited (94%) in hemolymph of exposed snails. Carboxylesterase activity measured with p-nitrophenyl butyrate and glutathione S-transferase activity were augmented 47% and 89% respectively after exposure. No differences were found for hemolymph carboxylesterase activity measured with p-nitrophenyl acetate. These results differ from those reported for whole tissue homogenates and reveal that tissue-specific responses of enzymatic biomarkers exist in this species. Regarding immune cell response, hemocytes were identified for the first time for C. gibbosa. Their viability and phagocytic activity decreased after azinphos-methyl exposure although total number of circulating cells did not differ between treatments. We conclude that concentrations of azinphos-methyl that can be found in the environment can compromise both hemolymph cholinesterase activity and the immune system of C. gibbosa. Furthermore, we propose that carboxylesterase and glutathione S-transferase activities measured in hemolymph and hemocyte viability and phagocytic activity could be incorporated as sensitive biomarkers to evaluate the effects of pesticide exposure on this and related species.

Resistance in cholinesterase activity after an acute and subchronic exposure to azinphos-methyl in the freshwater gastropod Biomphalaria straminea.

Organophosphorous and carbamates insecticides are ones of the most popular classes of pesticides used in agriculture. Its success relies on their high acute toxicity and rapid environmental degradation. These insecticides inhibit cholinesterase and cause severe effects on aquatic non-target species, particularly in invertebrates. Since the properties of cholinesterases may differ between species, it is necessary to characterize them before their use as biomarkers. Also organophosphorous and carbamates inhibit carboxylesterases and the use of both enzymes for biomonitoring is suggested. Azinphos-methyl is an organophosphorous insecticide used in several parts of the word. In Argentina, it is the most applied insecticide in fruit production in the north Patagonian region. It was detected with the highest frequency in superficial and groundwater of the region. This work aims to evaluate the sensitivity of B. straminea cholinesterases and carboxylesterases to the OP azinphos-methyl including estimations of 48 h NOEC and IC50 of the pesticide and subchronic effects at environmentally relevant concentrations. These will allow us to evaluate the possibility of using cholinesterase and carboxylesterase of B. straminea as sensitive biomarkers. Previously a partial characterization of these enzymes will be performed. As in most invertebrates, acetylthiocholine was the preferred hydrolyzed substrate of B. straminea ChE, followed by propionylthiocholine and being butyrylthiocholine hydrolysis very low. Cholinesterase activity of B. straminea was significantly inhibited by the selective cholinesterases inhibitor (eserine) and by the selective inhibitor of mammalian acethylcholinesterase (BW284c51). In contrast, iso-OMPA, a specific inhibitor of butyrylcholinesterase, did not inhibit cholinesterase activity. These results suggest that cholinesterase activity in total soft tissue of B. straminea corresponds to acethylcholinesterase. Carboxylesterases activity was one order of magnitude higher than cholinesterase. A greater efficiency (Vmax/Km) was obtained using acetylthiocholine and p-nitrophenyl butyrate. Acute exposure to azinphos-methyl did not cause inhibition of cholinesterase activity until 10 mg L(-1) used. Carboxylesterases towards p-nitrophenyl butyrate was inhibited by azinphos-methyl being the IC502.20±0.75 mg L(-1) of azinphos-methyl. Subchronic exposure to environmental concentrations of azinphos-methyl (0.02 and 0.2 mg L(-1)) produced a decrease in survival, protein content and carboxylesterases activity despite no inhibition of cholinesterase activity was observed. B. straminea cholinesterase is not a sensible biomarker. On the contrary, carboxylesterases activity was inhibited by azinphos-methyl. Carboxylesterases could be protecting cholinesterase activity and therefore, protecting the organism from neurotoxicity. This work confirms the advantages of measuring cholinesterases and carboxylesterases jointly in aquatic biomonitoring of pesticide contamination. This becomes relevant in order to find more sensitive biomarkers and new strategies to protect non-target aquatic organisms from pesticide contamination.

Multi-biomarker approach to evaluate the toxicity of chlorpyrifos (active ingredient and a commercial formulation) on different stages of Biomphalaria straminea.

Biomphalaria straminea is a freshwater gastropod native to South America and used in toxicological assessments. Our aim was to estimate 48 h-LC50 and sub-chronic effects after the exposure to low concentrations of chlorpyrifos as commercial formulation (CF) and active ingredient (AI) on B. straminea adult, embryos and juveniles. Concentrations between 1 and 5000 µg L-1 were chosen for acute exposures and 0.1 and 1 µg L-1 for the sub-chronic one. After 14 days biochemical parameters, viability and sub-populations of hemocytes, reproductive parameters, embryotoxicity and offspring' survival were studied. Egg masses laid between day 12 and 14 were separated to continue the exposure and the embryos were examined daily. Offspring' survival and morphological changes were registered for 14 days after hatching. 48 h-LC50, NOEC and LOEC were similar between CF and AI, however the CF caused more sub-lethal effects. CF but not the AI decreased carboxylesterases, catalase and the proportion of hyalinocytes with respect to the total hemocytes, and increased superoxide dismutase and the % of granulocytes with pseudopods. Also CF caused embryotoxicity probably due to the increase of embryos' membrane permeability. Acetylcholinesterase, superoxide dismutase, hemocytes sub-populations, the time and rate of hatching and juveniles' survival were the most sensitive biomarkers. We emphasize the importance of the assessment of a battery of biomarkers as a useful tool for toxicity studies including reproduction parameters and immunological responses. Also, we highlight the relevance of incorporating the evaluation of formulations in order to not underestimate the effects of pesticides on the environment.

Different effects of subchronic exposure to low concentrations of the organophosphate insecticide chlorpyrifos in a freshwater gastropod.

Chlorpyrifos is an organophosphate insecticide used for pest control on a number of food crops in many parts of the world. In recent years, there has been an important decrease in the number of organisms of Planorbarius corneus. Since the presence of pesticides in the water can be one of the reasons for this decrease, it is very important to study the effect of subchronic exposure to environmental concentrations of pesticides on these organisms. The aim of the present work was to investigate different effects of the subchronic exposure to low concentrations of the organophosphate chlorpyrifos in P. corneus and the possibility to use these as biomarkers. To this end, we have exposed the organisms to 0.4 and 5 µg L(-1) of chlorpyrifos for 14 days and recorded the number of egg masses, the number of eggs per mass, the number of eggs without embryo, the time for hatching, and the % of hatching and survival. We have also determined the activities of cholinesterases, carboxylesterases and glutathione S-transferase in whole organism soft tissue and in the gonads. A 14 days exposure to 0.4 µg L(-1) caused an increase in the number of egg masses without eggs and a decrease in carboxylesterases measured with p-nitrophenyl butyrate. However the exposure to 5 µg L(-1) also caused an increase in the time for hatching, a decrease in the % of hatching and survival and also inhibition of cholinesterases and carboxylesterases with p-nitrophenyl acetate and butyrate. In contrast, the glutathione S-transferase has not been modified with the tested concentrations. We concluded that when P. corneus exposed to chlorpyrifos for 14 days, the CES determined with p-nitrophenyl butyrate proved to be the most sensitive biomarker. However, exposure to environmental concentrations showed a decrease in the reproduction ability which could cause a decrease in the number of organisms of this species.

Aquatic Contamination in Lugano Lake (Lugano Lake Ecological Reserve, Buenos Aires, Argentina) Cause Negative Effects on the Reproduction and Juvenile Survival of the Native Gastropod Biomphalaria straminea.

Lugano Lake is located in an Ecological Reserve of Buenos Aires City. Biomonitoring of its water quality is essential due to its importance as a place for recreation and protection of native species. Biomphalaria straminea is a native hermaphrodite aquatic gastropod that inhabits different freshwater bodies of Argentina and was recently selected as a potential bioindicator. We propose this study as a first approach to assessing specific organisms' use in biomonitoring of urban wild reserves, and the usefulness of reproduction assays. B. straminea survival, behavior, reproduction success and offspring survival after the exposure to water samples from Lugano Lake (L1, L2, and L3) were evaluated. Temperature, pH, conductivity and dissolved oxygen were registered in situ. Samples were transported to the laboratory and chemical analysis and bioassays were performed using 20 snails per site. A control group with tap water was added. Egg masses were separated, exposed individually and observed daily using a stereoscopic microscope. After hatching, juveniles were placed in tap water and offspring survival was registered at the first, second, third and fourth months after the beginning of the assay. High levels of conductivity, turbidity and nutrients were obtained. Ammonium and nitrite were higher than the guideline level for the protection of aquatic life. During the bioassay 20% of the snails (L2 and L3) showed abnormally protruding of the head-food region. The number of eggs and embryonated eggs per mass did not differ between treatments. Egg masses exposed to water samples from the lake presented overlapping and abnormal eggs and arrested embryos. Besides, low % of hatching (L1: 33%, L2: 42%, and L3: 16%) and juvenile survival after the first (L1:14%; L2:78%) and second month (L1: 60%) were noted. In the control group, 85% of hatching and 100%-90% of survival were observed. Our results suggests the presence of pollutant in the lake. B. straminea seems to be a sensitive local species. Biomphalaria spp. reproduction assays can provide a valuable endpoint for toxicity and risk assessments and a usefulness tool for biomonitoring water quality.

Acute neurotoxicity evaluation of two anticholinesterasic insecticides, independently and in mixtures, and a neonicotinoid on a freshwater gastropod.

Neurotoxic insecticides are ubiquitous in aquatic ecosystems, frequently as part of complex mixtures. Freshwater gastropods are generally underrepresented in neurotoxicity evaluations and cumulative toxicity testing. This study investigates the behavioural and biochemical effects of acute exposures to the carbamate carbaryl, the organophosphate chlorpyrifos, and the neonicotinoid acetamiprid on the freshwater gastropod Chilina gibbosa. First, we evaluated behavioural neurotoxicity and cholinesterase (ChE), carboxylesterase (CE), and glutathione S-transferase (GST) activities in acute (48h) single-chemical exposures to increasing concentrations of carbaryl (0.5-500 µg L-1), chlorpyrifos (10-7500 µg L-1), and acetamiprid (1-10000 µg L-1). We then studied the effects of acute (48h) exposures to binary mixtures of carbaryl and chlorpyrifos equivalent to 0.5, 1, and 1.5 ChE 48h-IC50. None of the insecticides caused severe behavioural neurotoxicity, except for a significant lack of adherence by 5000 µg L-1 chlorpyrifos. Carbaryl caused concentration-dependent inhibition of ChEs (NOEC 5 µg L-1; 48h-IC50 45 µg L-1) and CEs with p-nitrophenyl butyrate as substrate (NOEC 5 µg L-1; 48h-IC50 37 µg L-1). Chlorpyrifos caused concentration-dependent inhibition of ChEs (NOEC 50 µg L-1; 48h-IC50 946 µg L-1) but did not affect CEs (NOEC ≥7500 µg L-1). Carbaryl-chlorpyrifos mixtures inhibited ChEs additively, inhibited CEs with p-nitrophenyl butyrate, and did not affect behaviour. GST activity was not affected by single or mixture exposures. Acute exposure to acetamiprid did not affect any of the endpoints evaluated. This study provides new information on carbaryl, chlorpyrifos, and acetamiprid toxicity on C. gibbosa, relevant to improve gastropod representation in ecotoxicological risk assessment.

Azinphos-methyl causes in Planorbarius corneus toxic effects on reproduction, offspring survival and B-esterases depending on the exposure time.

This work aimed to study in the freshwater gastropod Planorbarius corneus the effects of acute (2 days) and subchronic (14 days) exposures to an environmental concentration of the organophosphate azinphos-methyl on different reproductive parameters, offspring survival and B-esterase activities in gonads and in the whole organism soft tissue. The acute exposure inhibited only carboxylesterase activity in both tissues while the subchronic exposure also inhibited cholinesterase activity, decreased the number of hatched-eggs and increased offspring lethality (92%). On the other hand, B-esterases in gonads were more effective biomarkers than B-esterases in the whole organism due their inhibition appeared earlier in time (cholinesterase activity) and their activity remained inhibited for a longer time (carboxylesterase activity) when recovery studies were performed. We concluded that B-esterases and reproductive parameters can be used as effect biomarkers of aquatic contamination with azinphos-methyl. Our studies showed that a 14 days exposure to an environmental concentration of azinphos-methyl produced severe signs of toxicity in adult organisms, egg masses and juveniles that could cause negative effects at the population level in contaminated environments.

Effects of azinphos-methyl exposure on enzymatic and non-enzymatic antioxidant defenses in Biomphalaria glabrata and Lumbriculus variegatus.

Azinphos-methyl is an organophosphate insecticide used for pest control on a number of food crops in many parts of the world. The oligochaete Lumbriculus variegatus and pigmented and non-pigmented specimens of the gastropod Biomphalaria glabrata are freshwater invertebrates that have been recommended for contamination studies. Recently, it has been shown that L. variegatus worms exhibit a higher cholinesterase (ChE) activity and a greater sensitivity to in vivo ChE inhibition by azinphos-methyl than pigmented B. glabrata snails. The aims of the present study were (1) to investigate if, in addition to its anticholinesterase action, azinphos-methyl has also pro-oxidant activity in L. variegatus and B. glabrata, and (2) to examine if species that are highly susceptible to the neurotoxic effects of organophosphates also suffer a greater degree of oxidative stress. Therefore, total glutathione (t-GSH) levels and activities of cholinesterase (ChE), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glucose 6-phosphate dehydrogenase (G6PDH) were measured in the whole body soft tissue of organisms exposed for 48 and 96 h to a level of azinphos-methyl that produces 50% of inhibition on ChE. Results showed different patterns of antioxidant responses between the gastropods and the oligochaetes, and even between the two phenotypes of gastropods: (1) in exposed L. variegatus t-GSH levels increased and CAT and SOD activities decreased with respect to control organisms, (2) in pigmented gastropods, SOD decreased while CAT transiently diminished, and (3) in non-pigmented gastropods, SOD activity showed a biphasic response. GST and G6PDH were not altered by azinphos-methyl exposure. Of note, t-GSH levels were 4-fold times higher in L. variegatus than in both phenotypes of B. glabrata. This may suggest that GSH could play a more important role in antioxidant defense in L. variegatus than in B. glabrata.

Inhibition of cholinesterase activity by azinphos-methyl in two freshwater invertebrates: Biomphalaria glabrata and Lumbriculus variegatus.

In this study, some biochemical features and the extent of inhibition induced by the organophosphorous pesticide azinphos-methyl on the cholinesterase (ChE) activity present in whole soft tissue of two freshwater invertebrate species, the gastropod Biomphalaria glabrata and the oligochaete Lumbriculus variegatus were investigated. Both invertebrate organisms presented marked differences in ChE activity, type of enzymes and subcellular location. Acetylthiocholine was the substrate preferred by B. glabrata ChE. The enzyme activity was located preferentially in the supernatant of 11,000 x g centrifugation and was inhibited by increasing concentrations of substrate but not by iso-OMPA. Results showed that there were progressive inhibitions of the enzyme activity, with values 21%, 59%, 72%, 76%, and 82% lower than the control at levels of 1, 10, 50, 100 and 1000 microM of eserine, respectively. In contrast, L. variegatus ChE activity was distributed both in the supernatant and pellet fractions, with values approximately 6 and 20 times higher than B. glabrata, respectively. Studies with butyrylthiocholine and iso-OMPA suggested that about 72% of the activity corresponded to butyrylcholinesterase. A strong enzyme inhibition (88-94%) was found at low eserine concentrations (1-10 microM). ChE activity from L. variegatus and B. glabrata was inhibited by in vivo exposure to azinphos-methyl suggesting that both species can form the oxon derivative of this pesticide. However, both invertebrate species showed a very different susceptibility to the insecticide. The NOEC and EIC50 values were 500 and 1000 times lower for L. variegatus than for B. glabrata, reflecting that the oligochaetes were much more sensitive organisms. A different pattern was also observed for the recovery of the enzymatic activity when the organisms were transferred to clean water. The recuperation process was faster for the oligochaetes than for the gastropods. Mortality was not observed in either of the experimental conditions assayed, not even at concentrations that induced 90% of ChE inhibition. The differences in substrate specificity, sensitivity to inhibitors, and subcellular location between the ChEs of B. glabrata and L. variegatus could be the main factors contributing to the differential susceptibility to azinphos-methyl ChE inhibition found in the present study.

In vitro and in vivo studies of cholinesterases and carboxylesterases in Planorbarius corneus exposed to a phosphorodithioate insecticide: Finding the most sensitive combination of enzymes, substrates, tissues and recovery capacity.

Organophosphate insecticides (OPs) continue to be an important class of agrochemicals used in modern agriculture worldwide. Even though these pesticides persist in the environment for a relatively short time, they show a high acute toxicity that may represent a serious hazard for wildlife. Sub-lethal effects on non-target species are a focus in pest management programs and should be used as biomarkers. Cholinesterases (ChEs) are the most used biomarker of OP exposure in vertebrate and invertebrate species. However, the combined monitoring of ChE and carboxylesterase (CE) activities may provide a more useful indication of exposure and effect of the organisms. The objective of the present work was to find the most sensitive combination of enzyme, substrate, tissue and capacity to recovery of B-esterases in the freshwater gastropod Planorbarius corneus exposed to the OP azinphos-methyl. For this purpose, ChE and CE activities in different tissues of P. corneus (head-foot, pulmonary region, digestive gland, gonads and whole organism soft tissue) were studied. Measurements of ChE activity were performed using three substrates: acetylthiocholine, propionylthiocholine and butyrylthiocholine and CE activity using four different substrates: p-nitrophenyl acetate, p-nitrophenyl butyrate, 1-naphthyl acetate, and 2-naphthyl acetate in control and exposed organisms. Finally, the recovery rates of ChE and CE activities following 48h exposure to azinphos-methyl were analyzed. Our results show a preference for acetylthiocholine as substrate, a high inhibition with eserine (a selective ChE inhibitor) and inhibition with excess of substrate in all the analyzed tissues. The highest ChE and CE activity was found in the pulmonary region and in the digestive gland, respectively. The highest CE Vmax was obtained with 1 and 2-naphthyl acetate in all the tissues. CEs were more sensitive than ChE to azinphos-methyl exposure. The highest sensitivity was found using p-nitrophenyl acetate and butyrate as substrates. On the other hand, CEs of the digestive gland and the pulmonary region were more sensitive than CEs of the whole organism soft tissue. Regarding the recovery of enzyme activities after 48h exposure, ChE and CEs with p-nitrophenyl butyrate reached control values after 14days in the digestive gland and after 21days in the pulmonary region. Our results show marked differences in P. corneus basal ChE and CE activities depending on substrates and the tissue. Also, both tissue-dependent and substrate-dependent variations in sensitivity to azinphos-methyl exposure and recovery were obtained. CEs measured with p-nitrophenyl butyrate in the pulmonary region were the best combination to be used as biomarker of exposure to azinphos-methyl due to their sensitivity and low recovery capacity. Environmental concentrations of azinphos-methyl inhibited CE activity so they could be used as effective biomarkers of aquatic contamination.

Recovery study of cholinesterases and neurotoxic signs in the non-target freshwater invertebrate Chilina gibbosa after an acute exposure to an environmental concentration of azinphos-methyl.

Azinphos-methyl belongs to the class of organophosphate insecticides which are recognized for their anticholinesterase action. It is one of the most frequently used insecticides in the Upper Valley of Río Negro and Río Neuquén in Argentina, where agriculture represents the second most important economic activity. It has been detected in water from this North Patagonian region throughout the year and the maximum concentration found was 22.48 µg L(-1) during the application period. Chilina gibbosa is a freshwater gastropod widely distributed in South America, particularly in Patagonia, Argentina and in Southern Chile. Toxicological studies performed with C. gibbosa in our laboratory have reported neurotoxicity signs and cholinesterase inhibition after exposure to azinphos-methyl for 48 h. Recovery studies together with characterization of the enzyme and sensitivity of the enzyme to pesticides can improve the toxicological evaluation. However, little is known about recovery patterns in organisms exposed to organophosphates. The aim of the present work was to evaluate the recovery capacity (during 21 days in pesticide-free water) of cholinesterase activity and neurotoxicity in C. gibbosa after 48 h of exposure to azinphos-methyl. Also, lethality and carboxylesterase activity were registered during the recovery period. Regarding enzyme activities, after a 48-h exposure to 20 µg L(-1) of azinphos-methyl, cholinesterases showed an inhibition of 85% with respect to control, while carboxylesterases were not affected. After 21 days in pesticide-free water, cholinesterases continued to be inhibited (70%). Severe neurotoxicity signs were observed after exposure: 82% of the snails presented lack of adherence to vessels, 11% showed weak adherence, and 96% exhibited an abnormal protrusion of the head-foot region from shell. After 21 days in pesticide-free water, only 15% of the snails presented severe signs of neurotoxicity. However, during the recovery period significant lethality (30%) was registered in treated snails. C. gibbosa is a very sensitive organism to azinphos-methyl. These snails play an important role in the structure and function of aquatic food webs in this region. Thus, a decline of this species' population would probably have an impact on aquatic and non-aquatic communities. Our results show that C. gibbosa is a relevant sentinel species for studying exposure and effects of azinphos-methyl using behavioral and biochemical biomarkers. Neurotoxic behavioral signs are very sensitive, non-destructive biomarkers, which can be easily detected for about one week after acute exposure. Cholinesterse activity is a very useful biomarker showing a high sensitivity and a slow recovery capacity increasing the possibility to indirectly detect organophosphates for long periods after a contaminant event.

Cholinesterases and neurotoxicity as highly sensitive biomarkers for an organophosphate insecticide in a freshwater gastropod (Chilina gibbosa) with low sensitivity carboxylesterases.

In the Upper Valley of Río Negro and Río Neuquén in Argentina, agriculture represents the second most important economic activity. Azinphos-methyl has been found in water from this region throughout the year at a maximum concentration of 22.48 µg L(-1) during the application period. Toxicological studies on local non-target species have been performed mostly on vertebrates, while mollusks, which could be more sensitive, have not been studied so far. This work aims to characterize cholinesterase (ChE) and carboxilesterase (CE) activities of Chilina gibbosa, a freshwater gastropod native to southern Argentina and Chile. These enzymes, together with neurotoxicity signals, are evaluated herein after as sensitive biomarkers of exposure to azinphos-methyl at environmentally relevant concentrations. Effects of azinphos-methyl on antioxidant defenses: glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione S-transferase (GST) are also studied in order to complete a set of biomarkers with different sensitivity and specificity, to propose C. gibbosa as a sentinel species. The highest specific activity was obtained with acetylthiocholine as substrate, followed by propionylthiocholine (83% in comparison to acetylthiocholine) and butyrylthiocholine (19%).The lowest Km and the highest efficiency for ChE were obtained with acetylthiocholine. Regarding CEs activities, a higher efficiency was obtained with p-nitrophenyl butyrate than with p-nitrophenyl acetate. Eserine produced significant inhibition of ChE activity (81% with 0.001 mM and 98% with 1mM) while iso-OMPA did not produce any significant effect on ChE. Our results show that C. gibbosa ChE is very sensitive to azinphos-methyl (CI50 0.02 µg L(-1)) while CEs are inhibited at higher concentrations (CI50 1,000 µg L(-1)). CEs have been reported to be more sensitive to OPs than ChEs in most of the aquatic invertebrates protecting the organisms from neurotoxic effects. In contrast, C. gibbosa, has ChE which are much more sensitive to azinphos-methyl than CEs and shows marked signs of neurotoxicity. Regarding antioxidant defenses, GSH levels were significantly increased by 0.02 and 20 µg L(-1) azinphos-methyl (80 and 103%, respectively), CAT activity was increased 85% only at 0.02 µg L(-1) and SOD and GST did not show any significant response. Since ChE activity, neurotoxicity signs, GSH and CAT are sensitive biomarkers of acute exposure to azinphos-methyl at environmental concentrations C. gibbosa could be included as sentinel species in monitoring programs of pesticide hazard in regions of Argentina and Chile.

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.

In vivo studies on inhibition and recovery of B-esterase activities in Biomphalaria glabrata exposed to azinphos-methyl: analysis of enzyme, substrate and tissue dependence.

Cholinesterases and carboxylesterases belong to the group of B-esterases, the serine superfamily of esterases that are inhibited by organophosphorus compounds. It is now generally accepted that before using the B-esterases as biomarkers of exposure to organophosphorus and carbamates in a given species, the biochemical characteristics of these enzymes should be carefully studied. In this study, the enzyme/s and the tissue/s to be selected as sensitive biomarkers of organophosphorus exposition in the freshwater gastropod Biomphalaria glabrata were investigated. Firstly, the substrate dependence of cholinesterase and carboxylesterase activities in whole organism soft tissue and in different tissues of the snail (head-foot, pulmonary region, digestive gland, and gonads) was analyzed. Measurements of cholinesterase activity were performed using three substrates: acetylthiocholine (AcSCh), propionylthiocholine (PrSCh), and butyrylthiocholine (BuSCh). Carboxylesterase activity was determined using four different substrates: 1-naphthyl acetate (1-NA), 2-naphthyl acetate (2-NA), p-nitrophenyl acetate (p-NPA), and p-nitrophenyl butyrate (p-NPB). Regardless of the tissue analyzed, the highest specific activity was obtained when using AcSCh, followed by PrSCh. Cholinesterase activity measured with BuSCh was very low in all cases. On the other hand, the highest cholinesterase activity was measured in head-foot and in pulmonary region, representing in the case of AcSCh hydrolysis 196% and 180% of the activity measured in whole organism soft tissue, respectively. In contrast, AcSCh hydrolysis in digestive gland and gonads was 28% and 50% of that measured in whole organism soft tissue. Regarding carboxylesterase activity, although all tissues hydrolyzed the four substrates assayed, substrate preferences varied among tissues. In particular, digestive glands showed higher carboxylesterase activity than the other tissues (299%, 359% and 137% of whole organism soft tissue activity) when measured with 1-NA, 2-NA and p-NPA as substrates, respectively. In contrast, with p-NPB as substrate, the highest carboxylesterase activity was observed in pulmonary region. Exposure of the snails for 48 h to azinphos-methyl concentrations in the range of 0.05-2.5 mg L⁻¹ resulted in different degrees of inhibition of cholinesterase and carboxylesterase activities, depending on the enzyme, pesticide concentration, the substrate, and the tissue analyzed. In general, carboxylesterase activity measured with p-NPA and p-NPB was much more sensitive to azinphos-methyl inhibition than cholinesterase activity. The results also showed that while B-esterase activities in whole organism soft tissue and pulmonary region recovered completely within 14 days, carboxylesterase activity in digestive glands remained highly inhibited. On the whole, the results of the present study emphasize how important it is to characterize and measure cholinesterase and carboxylesterase activities jointly to make a proper assessment of the impact of organophosphorus pesticides in non-target species.

Effects of the organophosphate insecticide azinphos-methyl on the reproduction and cholinesterase activity of Biomphalaria glabrata.

Azinphos-methyl is an organophosphate insecticide used for pest control on a number of food crops in many parts of the world. The snail Biomphalaria glabrata is a freshwater gastropod widely distributed in South America, Central America and Africa. The aim of the present work was to investigate whether azinphos-methyl causes alterations in the reproduction of B. glabrata. To this end, gastropod pigmented specimens were exposed to various concentrations of the insecticide (0.021, 0.5, 2.5, and 5 mg L(-1)) for either 2 or 14 d. Along 14 d, several reproduction parameters and cholinesterase (ChE) activity were evaluated. In each group, the number of egg masses, the number of eggs per mass, the number of hatchings, the time to hatching, and the survival of the offspring after one month of treatment was evaluated. The results showed that, depending on the concentration and time of exposure, azinphos-methyl induced alterations in the reproduction of B. glabrata. These alterations were mainly represented by a decrease in the number of egg masses, and, in some cases, by a lower number or even the total absence of hatchings. Thus, the gastropods exposed to 2.5 and 5 mg L(-1) of azinphos-methyl for 14 d showed ChE inhibitions higher than 35% along time and completely lost their ability to reproduce. On the other hand, exposure to high acute concentrations or exposure to low concentrations for 14 d resulted in ChE inhibition equal to or lower than 35% between 7 and 14 d of treatment and similar alterations in reproduction. These were represented by a decrease in the number of egg masses. At low pestice levels, the number of egg masses and the number of offspring resulted to be more sensitive biomarkers than ChE inhibition. It is concluded that the insecticide azinphos-methyl can cause a decline in the reproductive performance of B. glabrata.

Inhibition of cholinesterases and carboxylesterases of two invertebrate species, Biomphalaria glabrata and Lumbriculus variegatus, by the carbamate pesticide carbaryl.

In this study, the effects of sublethal concentrations of the carbamate carbaryl on the cholinesterase (ChE) and carboxylesterase (CES) activities present in the oligochaete Lumbriculus variegatus and in the pigmented Biomphalaria glabrata gastropod were investigated. The results showed that ChE activity from both species was inhibited by in vivo and in vitro exposure to carbaryl, with EC(50) and IC(50) values approximately 20 times lower for the oligochaete than for the gastropod. On the other hand, the recovery process in uncontaminated media was more efficient in oligochaetes than in snails. Thus, in only 2h the oligochaetes showed no inhibition with respect to control values whereas the snails did not reach control values even after 48h of being in pesticide-free water. CES activity was investigated in whole body soft tissue homogenates using three different substrates: p-nitrophenyl butyrate, 1-naphthyl acetate (NA) and 2-NA. In addition, the presence of multiple CES isozymes in L. variegatus and B. glabrata extracts, with activity towards 1- and 2-NA, was confirmed by native polyacrylamide electrophoresis. In both species, the activities measured using the naphthyl substrates were higher than the activity towards p-nitrophenyl butyrate. In addition, B. glabrata showed a higher CES activity than L. variegatus independently of the substrate used. In L. variegatus, in vivo CES activity towards the different substrates was less sensitive to carbaryl inhibition than ChE activity. In contrast, in B. glabrata, CES activity towards p-nitrophenyl butyrate was inhibited at lower insecticide concentrations than ChE. The results of this study contribute to the knowledge of the sensitivity of non-target freshwater invertebrate Type B-esterases towards pesticides.