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.

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.

Immune and biochemical responses in hemolymph and gills of the Patagonian freshwater mussel Diplodon chilensis, against two microbiological challenges: Saccharomyces cerevisiae and Escherichia coli.

Immune cell characterization, immunological response and the associated gill oxidative balance were studied in the Patagonian freshwater mussel, Diplodon chilensis, using two microbiological immunostimulant models: Saccharomyces cerevisiae and Escherichia coli. Mussels were collected out of the breeding season in Paimún Lake and acclimated in the laboratory. Two exposure experiments were performed during two consecutive weeks: (1) mussels challenged with 500 yeast cells mL-1; and (2) mussels challenged with 1000 bacteria cells mL-1. Microorganisms were added in the water every two days, alternating with 6000 lyophilized cells of the green algae Scenedesmus vacuolatus mL-1. A control group, fed with S. vacuolatus, was set for each treatment. Morphological cell characterization was carried out in adherent hemocytes of D. chilensis hemolymph under control conditions. The most important cell type observed were the hyalinocytes (representing ca. 98% of the circulating cells), agranular cells with non-central polymorphic nucleus surrounded by cytoplasm; granulocytes (cells with cytoplasmic granules and non-central rounded nucleus) represented ca. 2%. Another two cell types were occasionally detected, binucleated hyalinocytes and hemoblast-like cells but were not considered for the analyses. Both adherent hyalinocytes and granulocytes exhibit phagocytic activity towards Congo red stained yeast, which was two-fold higher in granulocytes than in hyalinocytes, regardless of the applied challenge. Total hemocyte counts were diminished in mussels challenged with S. cerevisiae or E. coli. Hydrolytic and defense cellular enzyme activities were analyzed only for hyalinocytes. Both, S. cerevisiae and E. coli increased acid phosphatase activity. E. coli challenge diminished hemocyte lysosomal membrane stability and increased humoral phenoloxidase activity, while S. cerevisiae challenge did not affect any of these variables. Mussels challenged with E. coli showed increased gill antioxidant response without oxidative damage, while those challenged with S. cerevisiae showed no change in these variables.

Modulation of immune and antioxidant responses by azinphos-methyl in the freshwater mussel Diplodon chilensis challenged with Escherichia coli.

The aim of the present study was to characterize the immune response-total hemocyte number, cell type proportion, hemocyte viability, lysosomal membrane stability, phagocytic activity, cellular acid and alkaline phosphatase activity, and humoral bacteriolytic and phenoloxidase activity--in Diplodon chilensis exposed to 0.2 mg/L of azinphos-methyl (AZM), using Escherichia coli as immunological and pro-oxidant challenges. In addition, glutathione-S-transferase and lipid peroxidation thiobarbituric acid reactive substances were analyzed in gill tissue. Mussels from an unpolluted site were treated for 3 d as follows: 1) experimental control; 2) solvent effects control (acetone 0.01%); 3) bacterial challenge effects control (E. coli, 5 cells/mL × 104 cells/mL); 4) pesticide effects control (AZM in acetone); 5) control for combined effects of solvent and bacterial challenge; and 6) exposed to AZM, then challenged with E. coli. The results showed increased granulocyte proportion and phagocytic activity. Partial reversion of deleterious effects of E. coli on lysosomal membranes was observed in mussels exposed to AZM and then challenged with E. coli. Total hemocyte number and humoral bacteriolytic activity were increased only by E. coli challenge. Acid phosphatase activity was increased by both E. coli and AZM, whereas the stimulating effect of E. coli on alkaline phosphatase activity was negatively modulated by AZM. Azinphos-methyl inhibited phenoloxidase activity regardless of the E. coli challenge. Gill glutathione-S-transferase activity was increased by E. coli treatment either alone or pretreated with acetone or AZM and by AZM alone. Thiobarbituric acid reactive substance levels were reduced by AZM alone or combined with the E. coli challenge and by acetone followed by the E. coli challenge. Both acetone and AZM seem to be important modulators of immune and antioxidant responses in D. chilensis. Environ Toxicol Chem 2017;36:1785-1794. © 2016 SETAC.

Comparative study of toxicity and biochemical responses induced by sublethal levels of the pesticide azinphosmethyl in two fish species from North-Patagonia, Argentina.

Biochemical effects of azinphosmethyl (AZM), an organophosphate pesticide, were determined in gill, brain and muscle tissues of Odontesthes hatcheri and Jenynsia multidentata. The 96-h toxicity was first assessed, estimating lethal concentrations fifty (LC50) of 7 and 30µgL(-1) AZM for O. hatcheri and J. multidentata, respectively. Considering the LC50, sublethal 96-h static exposures were designed for O. hatcheri (0.1-0.5µgL(-1) AZM) and J. multidentata (5-10µgL(-1)AZM) to determine biochemical endpoints. Brain acetylcholinesterase (AchE) was inhibited by AZM in both species, while the buffer enzyme carboxylesterase (CarbE) was not affected in this tissue. Conversely, muscular AchE was not affected but CarbE was augmented by AZM. The enzymes glutathione reductase, glutathione-S-transferase and CarbE were significantly inhibited in O. hatcheri gills but none of them was affected by AZM in J. multidentata gills compared to control. GSH levels were augmented in gills of both species in exposed fish compared to controls and in addition, lipid peroxidation was significantly increased in O. hatcheri gills. Ex vivo histochemical analysis of ROS by fluorescence microscopy was also performed in J. multidentata gills, indicating a significant increase upon exposure to 10µgL(-1) AZM. Principal component analyses (PCA) were applied, both to the species together or separately. The general analysis demonstrated a clear separation of responses in the two species. For O. hatcheri, the variable that explains the major variation in PC1 is gill catalase and brain AchE in PC2. In J. multidentata in turn, the variable that explains the major variation in PC1 is brain AchE and total oxyradical scavenging capacity in PC2. The toxicity data and biomarker responses obtained for both species were compared to environmental concentrations of AZM detected in superficial water from different points in the Alto Valle region and risk quotients (RQ) were calculated. This approach indicated probable acute effects for O. hatcheri in river and irrigation channels (RQ>0.1), while the risk was unacceptable in drainage superficial water (RQ>1). In contrast, J. multidentata showed minimal risk in river or channel water (RQ<0.1) and probable risk in drainage water (RQ=0.75). We conclude that not only the differential susceptibility of both species to AZM is environmentally relevant, but also that the different biomarkers responding in each case underlie particular pathways stressed by this agrochemical.