Evaluation of the Acute Effects of Arsenic on Adults of the Neotropical Native Fish Cnesterodon decemmaculatus Using a Set of Biomarkers.

Neotropical fish Cnesterodon decemmaculatus were exposed to different sublethal concentrations (0.5, 1.0 and 5.0 mg As/L) of sodium arsenite (As III) to determine the median lethal concentration (LC50; 96 h) and to evaluate the response of a set of biomarkers (genotoxic, behavioral, biochemical, and metabolic). At the end of the exposure (96 h), fish were video-recorded for behavior assessment. We used the micronucleus and nuclear abnormality tests and the comet assay in peripheral blood as genotoxicity biomarkers. In regard to biochemical and metabolic biomarkers, we dissected the brain for acetylcholinesterase (AChE) activity; the liver for glutathione-S-transferase (GST) and catalase (CAT) activity and glutathione content (GSH); the gills for GSH content; and muscle for AChE, energy metabolism of lipids, carbohydrates, and proteins, and the electron transport system activity of the mitochondrial chain. We calculated an index using metabolic biomarkers, to determine the cellular energy allocation. The LC50 value was 7.32 mg As/L. The As affected some swimming parameters in females. No significant differences in micronucleus were found compared with the control, whereas nuclear aberrations increased significantly at 1.0 and 5.0 mg As/L. The genomic damage index and the percentage of cells with DNA damage (measured by the comet assay) showed a significant increase in the As-treated groups, and this technique was the most sensitive for detecting genotoxic damage. The As affected the antioxidant system (mainly GSH, CAT, and GST) and reduced the lipid content. A preliminary baseline was generated for the response of C. decemmaculatus exposed to sublethal concentrations of As, when it alters swimming behavior and the antioxidant system, has genotoxic effects, and reduces lipid content. Environ Toxicol Chem 2022;41:1246-1259. © 2022 SETAC.

First baseline for bioenergetic biomarkers in Cnesterodon decemmaculatus as test organism in ecotoxicological studies.

Cnesterodon decemmaculatus is a Neotropical teleost fish frequently used in ecotoxicological evaluations, whose biology has been thoroughly studied. Although there is considerable information on its response to different toxicants, no range of reference values has been so far established for the different biological parameters proposed as biomarkers of effect or exposure. Moreover, no study has yet examined the possible influence of the metabolic status of the exposed animals on their response to toxic stress. Therefore, the aim of this work was to provide a first baseline for a set of bioenergetic biomarkers in C. decemmaculatus adults exposed to a control medium under previously standardized conditions, and to assess their possible intrinsic seasonal variability. The responses of the biomarkers obtained from the controls were contrasted with those from the reference toxicant (Cadmio-Cd) and receiving waters (surface waters of the Reconquista River RR, Buenos Aires Province, Argentina). We conducted four 12-day assays (one in each season) of exposure to control media, (reconstituted moderate hard water, MHW) and two assays of exposure to Cd in MHW and surface river water (RR) in both summer and autumn. The variables recorded were: Food intake (In), fecal production (F), specific assimilation (A) and cumulative mortality, oxygen extraction efficiency (OEE), specific metabolic rate (SMR), ammonia excretion (N), ammonia quotient (AQ) and scope for growth (SFG). The seasonal variation shown by some physiological parameters, points to the need for establishing a baseline obtained from standardized media, preferably on a seasonal basis. Moreover, SFG and A appeared as the most sensitive biomarkers, emphasizing the importance to consider the metabolic status of the test organisms for the appropriate interpretation of results from ecotoxicological studies performed under controlled experimental conditions. The obtained results provide useful information on C. decemmaculatus as model species in ecotoxicological bioassays involving biomarkers of early effect.

Auxinic herbicides induce oxidative stress on Cnesterodon decemmaculatus (Pisces: Poeciliidae).

Pesticides might increase the production of reactive oxygen species (ROS). Dicamba (DIC) and 2,4-dichlorophenoxyacetic acid (2,4-D) are auxinic herbicides commonly applied in agroecosystems to control unwanted weeds. We analysed the oxidative damage exerted on the fish Cnesterodon decemmaculatus by an acute exposure to DIC- and 2,4-D-based herbicides formulations Banvel® and DMA®, respectively. The Endo III- and Fpg-modified alkaline comet assay was employed for detecting DNA damage caused by oxidative stress, whereas enzymatic and non-enzymatic biomarkers such as the activities of catalase (CAT), glutathione-S-transferase (GST), acetylcholinesterase (AChE), and glutathione content (GSH) were used to assess antioxidant response to these two herbicides. At the DNA level, results demonstrate that both auxinic herbicides induce oxidative damage at purines level. An increase on CAT and GST activities were detected in 48 h- and 96 h-treated specimens with both auxinics. GSH content decreased in fish exposed to DIC during 48 h and to 2,4-D after 96 h of exposure. Additionally, a diminished AChE activity in specimens treated with DIC and 2,4-D was observed only after 96 h. Total protein content decreased in fish exposed to both auxinics during 96 h. These results represent the first evaluation of oxidative damage related to DIC and 2,4-D exposure on a fish species as the Neotropical freshwater teleost C. decemmaculatus.

Use of integrated biomarker indexes for assessing the impact of receiving waters on a native neotropical teleost fish.

In the field of aquatic ecotoxicology, indexes obtained from a battery of biomarkers have proved to be a useful tool for assessing quantifiable and integrated health responses of organisms exposed to pollutants. The objective of this work was to evaluate the effects of exposure to the Reconquista River water (RR) on adults of Cnesterodon decemmaculatus using different integrated indexes. We conducted a 12-d laboratory assay involving the exposure of fish to RR, a negative control (moderately hard water - MHW medium), and a positive control (for genotoxicity with MHW + Cyclophosphamide, CP). There were measured metabolic (food intake and specific assimilation, specific metabolic rate, oxygen extraction efficiency, ammonia excretion, and ammonia quotient), genotoxic (comet assay, micronucleus test, and nuclear abnormalities), morphological variables (total length, body and liver weight) and biochemical variables (Electron Transport System - ETS, Acetylcholinesterase activity - AChE, Catalase - CAT, Glutathione-S-transferase - GST, Glutathione content - GSH and tissue proteins). These variables were grouped into different indexes: morphological (Condition Factor - K and Liver Somatic Index-LSI), metabolic (Scope for Growth-SFG), genetic damage (GDI) and integrated biomarker response - IBR (AChE brain, CAT, GST and GSH liver, GSH gills, ETS muscle) indexes. Results indicated that RR water induced metabolic, biochemical and genetic damages. The SFG, GDI and IBR were suitable to assess the effects of exposure to an environmental sample in an integrated approach, reducing uncertainty due to inherent biomarker variability. These indexes have emerged as promising tools for environmental monitoring studies.

Cadmium effects on some energy metabolism variables in Cnesterodon decemmaculatus adults.

This work is focused on the responses of some energy metabolism variables in Cnesterodon decemmaculatus adults exposed to cadmium under controlled laboratory conditions. This species has been used as bioindicator for evaluating the effects of different chemicals on diverse biological processes and is frequently used as test organism in ecotoxicity studies that include cadmium as reference toxicant. Animals were exposed for 12 days to the following concentrations: 0, 0.45, and 0.8 mg Cd/L. Food intake, fecal production, specific assimilation, condition factor, mortality percentage, oxygen consumption, oxygen extraction efficiency, specific metabolic rate, ammonia excretion, and ammonia quotient were measured. The overall balance was expressed as scope for growth (SFG). Cadmium-exposed groups showed a significant decrease in food assimilation and condition factor at the end of the exposure. There was an increase in specific metabolic rate and a decrease in SFG in the group exposed to 0.8 mg Cd/L. The condition factor and the SFG appeared as sensitive biomarkers of health status and growth of the animals, respectively. Cadmium-exposed fish reduced food intake, which was reflected in a decreased assimilation with concomitant decline in the external energy supply from feeding. Our results highlight the importance of considering the metabolic status of the test organisms when analyzing the responses of the biomarkers usually used as effect parameters in ecotoxicological evaluations under experimental conditions.

Toxicity of waterborne Copper in premetamorphic tadpoles of Lithobates catesbeianus (Shaw, 1802).

Toxicity parameters of Copper to pre-metamorphic larvae of Lithobates catesbeianus were evaluated in laboratory conditions. The acute toxicity (as LC-50 96 h) was 3.96 mg Cu(2+) L(-1) (95% confidence interval: 3.21-4.89); the bioconcentration of the metal after 96 h exposure followed an exponential increase. The potential genotoxicity effect evaluated with Micronucleus Test showed a reduced sensitivity of the animals to the assayed concentrations of the metal, exhibiting only a modest increase in the frequency of erythrocytes micronuclei, meanwhile larvae exposed to cyclophosphamide (positive control) showed significant increases. The Condition Factor was significantly reduced while the Hepatosomatic Index remained unaltered.