Biomonitoring of Alterations in Fish That Inhabit Anthropic Aquatic Environments in a Basin from Semi-Arid Regions.

Industrial, agricultural, and urban areas can be sources of pollution and a cause of habitat fragmentation. The Conlara River located in the northeast of San Luis Province suffers different environmental pressures along its course from urban to agro-industrial areas. The present study aims to assess the water quality of the Conlara basin by evaluating how metals and pesticide contamination as well as physicochemical parameters relate to physiological stress in Jenynsia multidentata. Samplings were carried out in four sites characterized by a growing gradient of anthropic impact from the springs to the final sections of the river, starting with tourism passing through urban areas and ending with large agricultural areas (from S1 to S4) during both the dry and wet seasons. A total of 27 parameters were determined (11 physicochemical, 9 heavy metals, and 7 pesticides) in surface waters. Biomarkers (CAT, TBARS, ChE, and MN) showed significant physiological and cytological alterations in J. multidentata depending on the hydrology season. The combination of physicochemical parameters, metals, and pesticide levels allowed typification and differentiation of the sites. Some metal (Cr, Mn, Pb, and Zn) and pesticide (α-BHC, chlorpyrifos, permethrin and cypermethrin, and endosulfan α) levels recorded exceeded the recommended Argentinian legislation values. A principal component analysis (PCA) allowed detection of differences between both seasons and across sites. Furthermore, the differences in distances showed by PCA between the sites were due to differences in the presence of physicochemical parameters, metals, and pesticides correlated with several biomarkers' responses depending on type of environmental stressor. Water quality evaluation along the Conlara River shows deterioration and different types of environmental stressors, identifying zones, and specific sources of pollution. Furthermore, the biomarkers suggest that the native species could be sensitive to anthropogenic environmental pressures.

Comprehensive evaluation of the toxicity of the flame retardant (decabromodiphenyl ether) in a bioindicator fish (Gambusia affinis).

In recent years, concerns have increased about the adverse effects on health and the environment of polybrominated diphenyl ethers (PBDEs), especially BDE-209, the most widely PBDE used globally. These pollutants derive from e-waste and present different adverse effects on biota. In this work, a toxicological study on mosquitofish (Gambusia affinis) using BDE-209 (2,2',3,3',4,4',5,'5',6,6'-decabromodiphenyl ether) was carried out. Acute toxicity bioassays were conducted with daily renewal of solutions, using different concentrations of environmental relevance, ranged between 10 and 100 µg L-1 of BDE-209. At 48 and 96 h of exposure, several parameters were evaluated, such as mortality, individual activity (swimming), biochemical activity (catalase; thiobarbituric acid-reactive substances; and acetylcholinesterase), and cytotoxic responses (micronucleus frequencies). In addition, integrated biomarker response and multivariate analyses were conducted to study the correlation of biomarkers. The calculated Lethal Concentration-50 remained constant after all exposure times (24 to 96 h), being the corresponding value 27.79 µg L-1 BDE-209. Furthermore, BDE-209 induced effects on the swimming activity of this species in relation to acetylcholine, since BDE-209 increased, producing oxidative damage at the biochemical level and genotoxicity after 48 h of exposure to 10 and 25 µg L-1 BDE-209. The results indicate that BDE-209 has biochemical, cytotoxic, neurotoxic, and genotoxic potential on G. affinis. In addition, mosquitofish could be used as a good laboratory model to evaluate environmental stressors since they could represent a risk factor for Neotropical species.

Tissue distribution and sublethal effects of imidacloprid in the South American grayish baywing (Agelaioides badius).

The neonicotinoids are globally used insecticides, which have been shown to cause negative impacts on birds. The current study aimed to evaluate the distribution of the neonicotinoid imidacloprid (IMI) in the tissues of a songbird and identify related physiological effects. Adults of the grayish baywing (Agelaioides baduis) were administered with a single dose of 35 mg IMI/kg, and the IMI concentration was evaluated in liver, kidney and plasma at 4, 12, 24, and 48 h after dosing. At the same time points, effects on hematological, genetic and enzymatic parameters were assessed. Results showed that IMI was absorbed before 4 h, and eliminated at 48 h, in every tissue, and the highest concentrations were detected in plasma. Baywings showed intoxication signs and reduced mobility within the first 5 min post-dosing. Hematological parameters: red blood cells, packed cell volume, hemoglobin, and their derived indices exhibited a transient elevation 24 h after dosing, which coincided with maximum concentrations of IMI in the tissues. No effects were observed on the genotoxicity parameters evaluated: micronuclei and comet assay. Treated birds exhibited an alteration of cholinesterases activity in the muscle and plasma, and of glutathione-S-transferase (GST) activity in the plasma, brain, liver, and muscle. Based on the results obtained, the combined detection of IMI and inhibition of GST activity in the plasma is suggested as a non-lethal biomarker of IMI exposure in wild birds. As efficient field monitoring depends on the availability of proven biomarkers, the current study provides valuable tools for bird conservation in agroecosystems.

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.

DNA damage exerted by mixtures of commercial formulations of glyphosate and imazethapyr herbicides in Rhinella arenarum (Anura, Bufonidae) tadpoles.

Glyphosate (GLY) and imazethapyr (IMZT) are two herbicides commonly used worldwide, either alone or in mixtures. They represent key pesticides in modern agricultural management. The toxicity that results when employed as mixtures has not been characterized so far. Acute toxicity of the 48% GLY-based herbicide (GBH) Credit® and the 10.59% IMZT-based herbicide (IBH) Pivot® H alone and their binary combinations was analyzed in Rhinella arenarum tadpoles exposed in a semi-static renewal test. Lethal effects were determined using mortality as the end-point, whereas sublethal effects were determined employing the single-cell gel electrophoresis (SCGE) bioassay. Based on mortality experiments, results revealed LC5096 h values of 78.18 mg/L GBH and 0.99 mg/L IBH for Credit® and Pivot® H, respectively. An increase in the genetic damage index (GDI) was found after exposure to Credit® or Pivot® H at 5 and 10% of LC5096 h values. The combinations of 5% Credit®-5% Pivot® H LC5096 h and 10% Credit®-10% Pivot® H LC5096 h concentrations significantly enhanced the GDI in comparison with tadpoles exposed only to Credit® or Pivot® H. Thus, the effect of interaction between GBH and IBH inducing DNA damage in R. arenarum blood cells can be considered to be synergistic.

Biomarkers at different levels of organisation after atrazine formulation (SIPTRAN 500SC®) exposure in Rhinella schineideri (Anura: Bufonidae) Neotropical tadpoles.

Brazil is an important consumer of herbicides. In sugarcane cultivation-the country's most extensive agricultural crop-atrazine-based formulations are the principal form of weed control. Several studies have investigated adverse effects of atrazine or their formulations on anurans, but not specifically on Brazilian species. Our aim was therefore to investigate the lethal and sublethal effects of an atrazine-based herbicide in Rhinella schneideri tadpoles and, in particular, effects on the pigmentation system as a new endpoint in ecotoxicological studies. Rhinella schneideri tadpoles at the Gosner-30 stage were exposed to the atrazine-based herbicide formulation, SIPTRAN 500 SC®, in acute bioassays at concentrations of 1.5-25 mg/L. The lethal and sublethal effects induced were analysed at different ecotoxicological levels: organismal level (alterations in behaviour, growth, development, and body mass; morphologic abnormalities), histological level (liver histopathology), the pigmentation system (melanomacrophages and dermal-melanophores), and cellular level (erythrocyte micronucleus formation and other nuclear-abnormalities). This herbicide induced sublethal effects at the organismal level with alterations in swimming and growth and morphologic abnormalities. These results demonstrated that, in anuran tadpoles, the atrazine-based agrochemical increased the frequency of micronucleus formation and other nuclear-abnormalities in erythrocytes and caused liver damage. In addition, we demonstrated for the first time effects of an atrazine-based formulation on the pigmentation system of anuran tadpoles, specifically an increase in the number of melanomacrophages and dermal melanophores. This study is the first to use several widely differing endpoints at different ecotoxicological levels in a comprehensive manner for assessment of the effects of environmental stressors in order to determine the health status of Neotropical anuran species. In doing so, this study establishes a foundation for future ecological assessments.

Are the damaging effects induced by the imazethapyr formulation Pivot® H in Boana pulchella (Anura) reversible upon ceasing exposure?

In the present study, the damage recovery capabilities of Boana pulchella tadpoles after acute exposure (96h) to 0.39mg/L concentration of the imazethapyr (IMZT)-based herbicide formulation Pivot® H (25% IMZT LC50 value) were assessed during a period of 7 to -21 days. To appraise the recovery capabilities, frequency of micronuclei (MNs), other nuclear abnormalities and DNA single-strand breaks evaluated by single cell gel electrophoresis assay on circulating blood cells were employed as endpoints for genotoxicity. Growth, development, body mass, and morphological abnormalities were also employed as individual endpoints in the recovery assay. Results demonstrated that IMZT induced sublethal effects at both the individual (i.e., loss of keratodonts) and cytogenetic levels (e.g., increase of MN frequency, other nuclear abnormalities and DNA single-strand breaks). At 11 days of the exposure phase, tadpoles recovered their basal levels of frequency of MNs, other nuclear abnormalities, and comets. However, loss of keratodonts, observed at the end of the exposure period, was present up to 21 days thereafter. Finally, axial abnormalities and delay in development stage were observed only during the postexposure phase in IMZT-exposed tadpoles at 18 and 25 days, respectively and were observed until the end of the experiment. This is the first evidence of use the comet assay as cytogenetic biomarker of genotoxicity in evaluating the recovery capabilities of amphibians in general and also those of B. pulchella after exposure to IMZT.

Evaluation of imazethapyr-induced DNA oxidative damage by alkaline Endo III- and Fpg-modified single-cell gel electrophoresis assay in Hypsiboas pulchellus tadpoles (Anura, Hylidae).

Imazethapyr (IMZT) is a selective postemergent herbicide with residual action. Available data analyzing its effects in aquatic vertebrates are scarce. In previous studies, we demonstrated that IMZT induces lesions into the DNA of Hypsiboas pulchellus tadpoles using the single-cell gel electrophoresis (SCGE) assay as a biomarker for genotoxicity. Currently, this assay can be modified by including incubation with lesion-specific endonucleases, e.g., endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), which detect oxidized pyrimidine and purine bases, respectively. The aim of this study was to evaluate the role of oxidative stress in the genotoxic damage in circulating blood cells of H. pulchellus tadpoles exposed to the IMZT-based Pivot H® formulation (10.59% IMZT) at a concentration equivalent to 25% of the LC50 (96h) value (0.39mg/L IMZT) during 48 and 96h. Our results demonstrate that the herbicide induces oxidative DNA damage on H. pulchellus tadpoles at purines bases but not at pyrimidines. Our findings represent the first evidence of oxidative damage caused by IMZT on anuran DNA using the alkaline restriction enzyme-modified SCGE assay.

Effects of glyphosate on hepatic tissue evaluating melanomacrophages and erythrocytes responses in neotropical anuran Leptodactylus latinasus.

Glyphosate (GLY) is the most used herbicide worldwide and its effects on anurans are well known. Pollutants can cause physiological and morphological effects. Therefore, this study evaluated the effects of GLY on hepatic melanomacrophages as a response to environmental stressors. Three treatments were exposed to different concentrations of pure GLY (100, 1000, and 10,000 µg g(-1), respectively), and there was also a control group. After the experimental time, liver and blood were analyzed. Melanomacrophages (MMCs) were located between the hepatocyte cordons, close to sinusoids. GLY increased the melanin area in MMCs of Leptodactylus latinasus exposed since lowest concentration until highest concentration. GLY also changed the occurrence of hepatic catabolism pigments into melanomacrophages and erythrocyte nuclear abnormalities; therefore, it can interfere with the hepatic metabolism. In conclusion, GLY promotes alterations in the hepatic tissue and erythrocyte nuclear abnormalities. Furthermore, MMCs may be useful as morphological responses of GLY effects.