Glyphosate and its formulated product Roundup Transorb R® affect locomotor activity and reproductive and developmental parameters in Jenynsia lineata fish: An intergenerational study.

Glyphosate is the most widely utilized herbicide worldwide due to its effectiveness in controlling agricultural weeds. However, its persistence in aquatic ecosystems has raised concerns about the well-being of non-target organisms such as fish. This study aimed to evaluate the effects of chronic exposure (21 days) to glyphosate or its formulated product Roundup Transorb R®, at an environmentally relevant concentration permitted by regulations in certain countries (65 µg/L of glyphosate), on the locomotor activity and reproductive success of the fish Jenynsia lineata, as well as on the morphology/development and locomotor activity of its offspring, as intergenerational effects. Neither the pure nor formulated herbicide altered the distance traveled and velocity of adult fish exposed to the herbicide (F0), but they negatively affected reproductive success, decreasing the percentage of positive response to the presence of the female, reducing the number of gravid females, causing abortions, and lowering offspring survival (F1). In the F1 generation, a decrease in weight and length was noted along with developmental abnormalities in both treatment groups (pure or formulated glyphosate), with the formulation causing more harm. Observed developmental abnormalities included muscle atrophy, ascites, pigmentary disorders, vertebral agenesis, spinal deviation, and exophthalmia. Furthermore, parental exposure to pure glyphosate led to an increase in the distance traveled and velocity of F1 (hyperlocomotion), whereas exposure to the formulated product resulted in a decrease in these behaviors (hypolocomotion) of F1. These findings highlight the toxic effects of glyphosate at very low concentrations, although varying between pure and formulated, and demonstrate the intergenerational consequences of herbicide exposure, underscoring the risk to the survival of fish offspring in glyphosate-contaminated environments.

Integrated ecotoxicological assessment of the complex interactions between chlorpyrifos and glyphosate on a non-target species Cnesterodon decemmaculatus (Jenyns, 1842).

Pesticide mixtures are frequent in freshwaters systems around the world, threatening the biota exposed to these conditions. The aim of this study was to determine the single and joint effect of two widely used pesticides in southern South America on a widely distributed fish species. In a 96-h assay, individuals of Cnesterodon decemmaculatus were exposed to 0.84 nL/L and 8.4 nL/L of Clorfox and 0.2 mg/L and 2 mg/L of Roundup Max, commercial formulations of chlorpyrifos and glyphosate, respectively. Also, there were four mixture treatments with all the possible combinations of both pesticides. A multi-level approach was carried out to assess their effects covering the following relevant biomarkers: behavior (immobile time, line crossings and average speed), somatic conditions (Fulton condition factor and hepatosomatic index), serum parameters (cortisol levels, lactate dehydrogenase (LDH), and creatine phosphokinase activity (CPK)), brain and muscle acetylcholinesterase and cytological characteristics (micronuclei frequency and nuclear abnormalities in erythrocytes). Our results showed that Clorfox exposures affect behavioral parameters, serum cortisol, and nuclear characteristics of erythrocytes. Roundup Max affects only the cortisol levels whereas mixture treatments have an effect on behavioral parameters, cortisol levels, LDH and CPK activities, and nuclear characteristics of erythrocytes. Potentiation was the main interaction at the lowest concentrations of both pesticides whereas antagonism occurred at the highest concentrations of both pesticides. These results are highly significant since they arise from an integrated ecotoxicological assessment at several levels of biological organization but even more important is that the potentiated effects of the mixtures we registered are environmentally relevant concentrations.

Single and joint effects of chronic exposure to chlorpyrifos and glyphosate based pesticides on structural biomarkers in Cnesterodon decemmaculatus.

Worldwide freshwater bodies that cross agricultural or urban areas are exposed to mixtures of xenobiotics. In particular, pesticides are usually part of these mixtures and could come into direct or indirect contact with biota and therefore, organisms have to cope with this altered scenario and the detrimental effects of these substances. Commercial formulations of chlorpyrifos and glyphosate, and their mixtures were evaluated using a set of biomarkers in the native fish C. decemmaculatus exposed to relevant environmentally pesticides concentrations. The biomarkers measured were: histopathological indices and tissue ultrastructure in liver and nuclear abnormalities and micronuclei in erythrocytes. During 42 days adult females were exposed to the following concentrations of Clorfox and Roundup Max (chlorpyrifos and glyphosate, respectively): 0.84 nl/l and 8.4 nl/l of Clorfox (CF), 0.2 and 2 mg/l of Roundup Max (RM) and all the combinations of these concentrations. Being the low concentrations of both pesticides environmentally relevant. Nuclear abnormalities of erythrocytes were registered under CF, RM and only one mixture. Histological inflammatory alterations increased in individuals exposed to CF and two mixtures. Finally, some pesticide combinations increased the circulatory alterations in liver. Ultrastructural changes in hepatocytes were registered at all the pesticide treatments. The different biomarker responses showed in the mixtures treatments reflected complex interactions, showing the mixture of the low concentrations of both pesticides (the environmentally relevant), potentiated effects. According to our results the presence of these substances in freshwaters could impose important risks for natural populations by causing deleterious effects on the native fish species C. decemmaculatus.

Female masculinization and reproductive success in Cnesterodon decemmaculatus (Jenyns, 1842) (Cyprinodontiforme: Poeciliidae) under anthropogenic impact.

Aquatic organisms are exposed to a myriad of chemical compounds, with particular concerns focused on endocrine disruptors. Growing scientific evidence indicates that these compounds interfere with normal endocrine function and could affect the reproductive system of humans and wildlife. We analyzed the proportion of masculinized females, defined by elongation and fusion of the anal fin rays, and the extent of masculinization, masculinization index, defined by anal fin length divided by the standard length, of Cnesterodon decemmaculatus resident to areas of agricultural and urban-industrial activities in the Arroyo Colorado basin. Additionally, a bioassay was carried out to assess the potential effects of masculinization on reproductive success, measured as the number of viable progenies, using pregnant females from the site downstream of the urban-industrial zone. Masculinized females were observed in all sampling sites, particularly downstream of the urban-industrial area, where over 80% of females presented abnormal sexual characteristics and the highest masculinization index was registered. In the laboratory, masculinized adult females showed male mating behavior, and survival of their progeny was lower than those of normal females. To our knowledge, this is the first report of endocrine disruption in field-collected C. decemmaculatus, and the first evaluation of the reproductive success of masculinized females. Finally, our results support C. decemmaculatus as an excellent sentinel species due to its wide distribution, easy culture in laboratory conditions, and its potential capability to respond to sources of pollution, particularly endocrine disruptors.

Effects of water quality on aspects of reproductive biology of Cnesterodon decemmaculatus.

The Suquía River basin (Córdoba, Argentina) is under a strong negative impact due to multiple sources of anthropic pollution. The main goal of our study was to evaluate if variations in the water quality of Suquía river basin affect the reproductive biology of Cnesterodon decemmaculatus and determine if the responses provided by the species can be considered as biomarkers of river quality. This assessment was performed through the measurement of morphological, histological and somatic parameters in adult males collected at four sampling sites during the beginning and the end of the breeding season. The water quality evaluation carried out through the estimation of a water quality index (WQI) and pesticides concentrations in water, revealed a pollution gradient along the studied basin. The same variation pattern was registered for the somatic index. In addition, the analysis of the morphology of the male copulatory organ (gonopodium) showed that individuals collected at Córdoba city had the lowest Gonopodium-Somatic Index (Gonop-SI) value, while those sampled at the most polluted site showed abnormalities in the small structures of the gonopodium. On the other hand, few histological alterations were found in the liver whereas no alterations were found in gonads along the river. The results obtained allowed us to characterize the environmental conditions of the studied basin and demonstrated the water quality deterioration along the Suquía River.

Environmental relevant concentrations of a chlorpyrifos commercial formulation affect two neotropical fish species, Cheirodon interruptus and Cnesterodon decemmaculatus.

The increase of cultivated areas together with the intensive use of pesticides have greatly contributed to impair the quality of aquatic systems along different areas of South America. The main goal of the present study was to assess the effects of a commercial formulation of chlorpyrifos at environmentally relevant concentrations on two native fish species, Cheirodon interruptus and Cnesterodon decemmaculatus. Adult individuals were exposed during 48 h to the following concentrations: 0.084 nl/l (Ci-Cf 1) and 0.84 nl/l (Ci-CF 2) in C. interruptus (Ci) of Clorfox (CF), and 0.84 nl/l (Cd-CF 1) and 8.4 nl/l (Cd-CF 2) in C. decemmaculatus (Cd). Fish behavior was evaluated through locomotor activity and space usage variables. The activity of acetylcholinesterase (AChE) in brain and muscle, catalase (CAT) and glutathione-S-transferase (GST) in brain, liver, muscle and gills, and aspartate amino-transferase (AST), alanine amino-transferase (ALT), AST/ALT ratio and alkaline phosphatase (ALP) in liver, were measured. Both locomotor activity and space usage varied between the two species studied and between CF treatments. The enzyme activities showed significant variations in CAT for C. interruptus and in CAT, GST, AChE, AST, and AST/ALT for C. decemmaculatus under the exposure conditions. Given that both species responded to CF and the concentrations we tested are environmentally relevant, the presence of this pesticide in freshwater systems could impose a risk for populations of both native fish studied at field.

Reproductive impairment of a viviparous fish species inhabiting a freshwater system with anthropogenic impact.

The potential threat to animal reproduction by contaminated freshwater systems posed the necessity to identify and develop bioindicators and biomarkers to be used for screening and evaluation of the effects in organisms. The main goal of this work was to determine, through histological analyses and changes in gonopodium morphology, whether a freshwater system polluted by anthropogenic activities-sewage, agricultural, and industrial-could cause alterations at the organ level. We also propose the live-bearing fish, Jenynsia multidentata, as a species suitable to study the effects of contaminated aquatic environments. We compared male fish sampled at two different stations in Suquía River basin (Córdoba, Argentina), both differing in degree of pollution, through liver and testis histology and gonopodial morphometric parameters. The water quality, based on the physicochemical characteristics of the studied stations, varied markedly with a decrease in water quality at the downstream site (station 2). At the highest polluted area, detrimental effects on liver and testis were evidenced on histological analysis. Male individuals from station 2 also presented noticeable structural changes of the anal fin, such as a straight gonopodium and abnormal tip area. The present results demonstrate that a freshwater system polluted by the impacts of anthropogenic activities has detrimental effects to J. multidentata. The alterations registered in individuals from the polluted station indicate an impairment of male reproductive performance and imply a risk for other live-bearing species as well as the entire biodiversity. We consider J. multidentata a sentinel species that is useful to evaluate the potential risk present in the studied basin not only to itself but to other species as well.

Bioindicators and biomarkers of environmental pollution in the middle-lower basin of the Suquía River (Córdoba, Argentina).

The Suquía River middle-lower basin (Córdoba, Argentina) is subject to a strong anthropogenic impact because it receives pollutants from different sources. Recent studies have shown the importance and the need of approaching the monitoring process of water quality from integral perspectives through the use of chemical as well as biological methods. The main goal of this study was to evaluate the fish species Jenynsia multidentata as a bioindicator of environmental pollution in the middle-lower basin of the Suquía River using biotransformation and antioxidant enzymes as well as gill and liver histopathology as biomarkers. We also studied the fish-assemblage characteristics through the fish species pattern variation and the application of a biotic index based on fish data. Our study also included the analysis of a water-quality index, heavy-metal concentrations in sediment and water, and pesticide concentrations in sediment. The chemical analyses of the middle-lower Suquía River basin showed a water-quality degradation gradient. Fish-assemblage structure changed with increasing water pollution, showing a simpler structure at the most polluted area. According to the biotic index, the variation pattern of fish assemblages reflected the aquatic environmental deterioration. Both molecular and histopathological biomarkers reflected the same trend in relation to water quality. However, enzymes varied with more acute precision between seasons. In addition, each enzyme presented with different sensibility. At tissue level, the histopathological analysis detected chronic contamination at both stations and seasons. The present work, which comprises different levels of biological organization together with chemical analyses, generated particular although complementary information, thus evidencing the same trend of aquatic contamination. Thus, the development of integral investigations gives a comprehensive approach and becomes the most effective tool to construct policies both preventive and palliative. Our study constitutes a good model to be applied in other endorheic basins of South America.

Exposure to a commercial glyphosate formulation (Roundup®) alters normal gill and liver histology and affects male sexual activity of Jenynsia multidentata (Anablepidae, Cyprinodontiformes).

Roundup is the most popular commercial glyphosate formulation applied in the cultivation of genetically modified glyphosate-resistant crops. The aim of this study was to evaluate the histological lesions of the neotropical native fish, Jenynsia multidentata, in response to acute and subchronic exposure to Roundup and to determine if subchronic exposure to the herbicide causes changes in male sexual activity of individuals exposed to a sublethal concentration (0.5 mg/l) for 7 and 28 days. The estimated 96-h LC50 was 19.02 mg/l for both male and female fish. Gill and liver histological lesions were evaluated through histopathological indices allowing quantification of the histological damages in fish exposed to different concentrations of the herbicide. Roundup induced different histological alterations in a concentration-dependent manner. In subchronic-exposure tests, Roundup also altered normal histology of the studied organs and caused a significant decrease in the number of copulations and mating success in male fish exposed to the herbicide. It is expected that in natural environments contaminated with Roundup, both general health condition and reproductive success of J. multidenatata could be seriously affected.