Can a mixture of agrochemicals (glyphosate, chlorpyrifos and chlorothalonil) mask the perception of an individual chemical? A hidden trap underlying ecological risk.

As aquatic environments associated with conventional agriculture are exposed to various pesticides, it is important to identify any possible interactions that modify their effects when in a mixture. We applied avoidance tests with Danio rerio, exposing juveniles to three relevant current use pesticides: chlorpyrifos (CPF), chlorothalonil (CTL) and glyphosate (Gly), individually and in binary mixtures (CPF-Gly and CTL-Gly). Our goal was to identify the potential of contaminants to trigger the avoidance response in fish and detect any changes to that response resulting from binary mixtures. Avoidance was assessed for three hours using an open gradient system with six levels of increasing concentrations. Fish avoided environmentally relevant concentrations of the three compounds. The avoidance of CPF [AC50 = 7.95 (3.3-36.3) µg/L] and CTL [AC50 = 3.41 (1.2-41.6) µg/L] was evident during the entire period of observation. In the case of Gly, the response changed throughout the experiment: initially (until 100 min) the fish tolerated higher concentrations of the herbicide [AC50 = 52.2 (12.1-2700) µg/L] while during the later period (after 100 min) a clearer avoidance [1.5 (0.8-4.2) µg/L] was observed. The avoidance recorded using CPF and CTL alone was attenuated by the presence of Gly. Applying an additive concentration model, Gly initially acted synergistically with the other two compounds, although this interaction was not observed during the later period. Avoidance gives us an idea of how the distribution of populations may be altered by contamination, our results suggest that in some mixtures this response may be inhibited, at least temporarily, thus masking the ecological risk of the exposure.

Short-term exposure to benzalkonium chloride in bacteria from activated sludge alters the community diversity and the antibiotic resistance profile.

The continuous introduction of cleaning products containing benzalkonium chloride (BAC) from household discharges can mold the microbial communities in wastewater treatment plants (WWTPs) in a way still poorly understood. In this study, we performed an in vitro exposure of activated sludge from a WWTP in Costa Rica to BAC, quantified the changes in intI1, sul2, and qacE/qacEΔ1 gene profiles, and determined alterations in the bacterial community composition. The analysis of the qPCR data revealed elevated charges of antibiotic resistance genes in the microbial community; after BAC's exposure, a significant increase in the qacE/qacEΔ1 gene, which is related to ammonium quaternary resistance, was observed. The 16S rRNA gene sequences' analysis showed pronounced variations in the structure of the bacterial communities, including reduction of the alpha diversity values and an increase of the relative abundance of Alphaproteobacteria, particularly of Rhodospseudomonas and Rhodobacter. We confirmed that the microbial communities presented high resilience to BAC at the mg/mL concentration, probably due to constant exposure to this pollutant. They also presented antibiotic resistance-related genes with similar mechanisms to tolerate this substance. These mechanisms should be explored more thoroughly, especially in the context of high use of disinfectant.

The effect of the insecticide diazinon on the osmoregulation and the avoidance response of the white leg shrimp (Penaeus vannamei) is salinity dependent.

Diazinon is one of the insecticides that represent a high risk for Costa Rican estuarine environments due to its widespread use in pineapple plantations. In estuaries, organisms are frequently submitted to stress caused by natural factors (e.g., continuous changes in salinity levels) and, additionally, to stress due to contamination. Therefore, the driving question of this study was: will organisms be more susceptible to suffer the deleterious effects caused by diazinon because of the stress resulting from the salinity changes? The estuarine shrimp Penaeus vannamei was used as the model organism and two responses were measured: osmoregulation (the physiological effect after a forced and continuous 24 h-exposure) and avoidance [the behavioural effect after a short (3 h) non-forced, multi-compartmented exposure]. Juveniles were exposed to diazinon (0.1, 1, 10 and 100 µg/L) at three different salinities (10, 20 and 30). Disruption in the capacity to regulate the haemolymph osmotic pressure was observed at a salinity of 30 in individuals exposed to diazinon and methanol (used as vehicle). At that salinity, the ability of shrimps to detect and avoid the highest diazinon concentrations was impaired. P. vannamei juveniles inhabit environments with a high variation in salinity, but with an optimum osmotic point close to a salinity of 20; therefore, the higher the salinity, the greater the vulnerability of shrimps to the effects of diazinon. From an ecological point of view, this combined effect of salinity and contamination might also limit the spatial distribution of the organisms.

Ecotoxicological evaluation of aquaculture and agriculture sediments with biochemical biomarkers and bioassays: antimicrobial potential exposure.

Inappropriate practices and lack of regulations regarding antimicrobial use in agricultural production of developing countries increase the risk of exposure to aquatic ecosystems. Sediments may act as sink of antimicrobial compounds and can provide a historical record of pollution. In the present study, toxic potential of sediments receiving effluents from a fish farm (TIL1), rice farm (AZ) and swine farm (RD2) and from a reference natural wetland (PV) in a tropical dry region was evaluated. According to local surveys of antimicrobials and national product registries, sites were classified from highest to lowest potential exposure as following: RD2>TIL1>AZ>PV. Both, whole sediment and interstitial water tests, showed a high toxicity of pig farm sediments to the behavior of Anodontites luteola and the survival of Daphnia magna (EC50 -48hrs: 2.4 -11.8%) (ANOVA, p < 0.05). Integrated responses from Cholinesterase activity (ChE), Gluthathion-S-transferase (GST) and Lipoperoxidation (LPO) measured in A. luteola tissue pointed at the pig and rice farms as sites influenced by activities with an intensive use of xenobiotic substances. The assessment of toxicity pointed at the need of more research on sub-lethal effects of antimicrobials on aquatic invertebrates. With this purpose, we analyzed biomarker response of A. luteola to oxytetracycline in vitro and found a decrease of ChE and GST in concentrations of 100 microg I(-1).

Ecological consequences when organisms avoid a contaminated environment: A study evaluating the toxicity of fipronil.

The ability of aquatic organisms to sense the surrounding environment chemically and interpret these signals correctly is crucial to their survival and ecological niche. This study applied the Heterogenous Multi-Habitat Assay System - HeMHAS to evaluate the avoidance potential of Daphnia magna to detect fipronil-contaminated habitats in a connected landscape after a short (48 h), previous, forced exposure to an environmentally relevant concentration of the same insecticide. The swimming of daphnids was also analyzed by recording the total distance covered. D. magna preferred areas with less contamination, although the effect of fipronil on their swimming ability (a decrease) was observed for all the concentrations tested. The application of non-forced multi-compartment exposure methodologies is a recent trend and is ecologically relevant as it is based on how contamination can really produce changes in an organism's habitat selection. Finally, we consider the importance of more non-forced exposure approaches where Stress Ecology can be aggregated to improve systemic understanding of the risk that contaminants pose to aquatic ecosystems from a broader landscape perspective.

Dissimilar behavioral and spatial avoidance responses by shrimps from tropical and temperate environments exposed to copper.

Behavioral changes associated with exposure to pollutants represent the earliest response for organisms confronted by perceivable chemical signals. This study was carried out with the objective of evaluating behavioral responses associated with different scenarios of exposure to pollutants (non-forced vs forced) in two shrimp species (Penaeus vannamei and Palaemon varians), representative of different latitudes and using copper as a model contaminant. The effects on locomotion were evaluated by exposing the shrimps to a range of copper concentrations (0, 0.5, 5, 50, and 250 µg/L) in the forced scenario. After exposure, the movement patterns for each shrimp were recorded and used to estimate changes in the shrimps' locomotion. For the non-forced scenario, the avoidance response was assessed by placing shrimps in a multi-compartment system where they were able to move freely along a gradient of copper (0, 0.5, 5, 50, and 250 µg/L). In terms of locomotion, an opposite trend was observed between the species: movements were significantly reduced in P. varians with concentrations above 50 µg/L, while hyperactivity was observed for P. vannamei. When exposed to a gradient of copper in the multi-compartment system, both species significantly avoided the highest concentrations of copper, although the repellence of copper was stronger for P. vannamei. In summary, both species of shrimps were able to recognize and avoid copper; however, in terms of locomotion, they showed an opposite behavioral reaction. These results show that a contamination event can have different behavioral outcomes depending on the species and complementing forced and non-forced exposure with species-specific information can be helpful to characterize and predict the effects of contaminants at higher biological levels.

Biotransformation and oxidative stress responses in fish (Astyanax aeneus) inhabiting a Caribbean estuary contaminated with pesticide residues from agricultural runoff.

The estuarine ecosystem of Madre de Dios Lagoon (MDL), in the Caribbean Coast of Costa Rica, is exposed to contamination with pesticide residues coming from the upstream agricultural areas. Biomarkers can provide a better indication of the fitness of biota in real mixture exposure scenarios than traditional lethal dose toxicity measurements. Here, we measured biomarkers of biotransformation, oxidative stress, and neurotoxicity on Astyanax aeneus, an abundant fish species in MDL. Glutathione S-transferase activity (GST), catalase activity (CAT), lipid peroxidation (LPO), and cholinesterase activity (ChE) were measured in fish collected during seven sampling campaigns, carried out between 2016 and 2018. Pesticide residues were analyzed in surface water samples collected every time fish were sampled. Residues of 25 pesticides, including fungicides, insecticides, and herbicides, were detected. The biomarkers measured in A. aeneus varied along the sampling moments, with biotransformation and oxidative stress signals showing a coupled response throughout the assessment. Furthermore, significant correlations were established between three biomarkers (GST, LPO, and CAT) and individual pesticides, as well as between GST and LPO with groups of pesticides with shared biocide action. Among pesticides, insecticide residues had a major influence on the responses observed in fish. This work demonstrates the chronic exposure to pesticide residues in MDL and how such exposure is related to physiological responses in fish that can affect their health and potentially, the trophic networks. This early warning information should be considered to improve the protection of estuarine ecosystems in the tropics.

Comparison of the Sensitivity of Tetragonisca angustula (Apidae-Meliponini) and Apis mellifera (Apidae-Apini) to Three Insecticides (Malathion, Imidacloprid, and Fipronil) Used in Costa Rica.

The decline of insect pollinators is a significant concern within the current biodiversity crisis. The paradox between the benefits that these animals represent to humans and the evidence of human activities driving their extinction calls for the urgent protection of bees. To address the role of chemical pollution in this scenario, we assessed the acute toxicity as well as four biomarker responses (cholinesterase [ChE], glutathione S-transferase, catalase, and lipid peroxidation [LPO]) elicited by dietary 24-h exposure to three insecticides (malathion, imidacloprid, and fipronil) on the stingless neotropical bee Tetragonisca angustula and the honeybee Apis mellifera. Malathion was the most toxic substance to both species, with 48-h median lethal doses (LD50s) of 0.25 ng/bee to A. mellifera and 0.02 ng/bee to T. angustula. Fipronil was also highly toxic and presented a similar toxicity to both species, with 48-h LD50s of 0.5 ng/bee (A. mellifera) and 0.4 ng/bee (T. angustula). Imidacloprid had the lowest acute toxicity with a 48-h LD50 of 29 ng/bee for A. mellifera, whereas T. angustula tolerated exposure higher than 35 ng/bee. Apparent biomarker responses were observed in bees of both species that survived exposure to higher concentrations of malathion (ChE inhibition) and fipronil (increased LPO). Our results suggest that specific sensitivity to insecticides varies greatly among compounds and pollinator species, but the use of different representative species can facilitate the prioritization of substances regarding their risk to pollinators. Further research is necessary to better characterize the risk that pesticides represent in neotropical agricultural landscapes. Environ Toxicol Chem 2023;42:1022-1031. © 2023 SETAC.

Pesticide use in banana plantations in Costa Rica - A review of environmental and human exposure, effects and potential risks.

Biodiversity is declining on a global scale. Especially tropical ecosystems, containing most of the planetary biodiversity, are at risk. Agricultural monocrop systems contribute to this decline as they replace original habitats and depend on extensive use of synthetic pesticides that impact ecosystems. In this review we use large-scale banana production for export purposes in Costa Rica as an example for pesticide impacts, as it is in production for over a century and uses pesticides extensively for more than fifty years. We summarise the research on pesticide exposure, effects and risks for aquatic and terrestrial environment, as well as for human health. We show that exposure to pesticides is high and relatively well-studied for aquatic systems and humans, but hardly any data are available for the terrestrial compartment including adjacent non target ecosystems such as rainforest fragments. Ecological effects are demonstrated on an organismic level for various aquatic species and processes but are not available at the population and community level. For human health studies exposure evaluation is crucial and recognised effects include various types of cancer and neurobiological dysfunctions particularly in children. With the many synthetic pesticides involved in banana production, the focus on insecticides, revealing highest aquatic risks, and partly herbicides should be extended to fungicides, which are applied aerially over larger areas. The risk assessment and regulation of pesticides so far relies on temperate models and test species and is therefore likely underestimating the risk of pesticide use in tropical ecosystems, with crops such as banana. We highlight further research approaches to improve risk assessment and, in parallel, urge to follow other strategies to reduce pesticides use and especially hazardous substances.

Transient exposure to sublethal concentrations of a pesticide mixture (chlorpyrifos-difenoconazole) caused different responses in fish species from different trophic levels of the same community.

The assessment of early effects caused in biota by sublethal exposure to pesticide mixtures should enhance the realism in the ecological risk assessment for agricultural landscapes. This study aimed to evaluate sub-individual responses in fish, which can be linked with outcomes at higher levels of biological organization and affect their trophic relationships. A multilevel biomarker approach was applied to assess the effects of a 48 h exposure of two freshwater mesoamerican fish species (Parachromis dovii and Poecilia gillii) to a mixture of sublethal concentrations of chlorpyrifos (5 µg/L) and difenoconazole (325 µg/L). Transcriptomic induction of cyp1A and the activities of 7-ethoxy-resorufin-O-distillase (EROD) and glutathione S-transferase (GST) were measured as biotransformation-related biomarkers; cholinesterase activity (ChE) was assessed as a neurotoxicity biomarker; resting metabolic rate (RMR) was measured as a physiological biomarker; and the movement of fish in a dark-light environment as a behavior biomarker. The exposure to the mixture had evident effects on P. gillii, with significant induction of cyp1A transcription, increased EROD activity, ChE inhibition in muscle, and increased permanence in the light side of the dark-light environment. Meanwhile, P. dovii only showed significant induction of cyp1A, without evidence of neurotoxicity or changes in behavior. This study demonstrates that the severity of the effects caused by the exposure to a mixture of pesticides can differ among species from the same trophic chain. The potential impairment of predator-prey relationships is a relevant effect that pesticide pollution can cause and it should be considered for the risk assessment of such contaminants.

Effects of the insecticide ß-endosulfan on tadpoles of Isthmohyla pseudopuma (Anura: Hylidae).

Conventional agriculture uses pesticides intensively. Once pesticides are released into the environment, they can be toxic to non-target organisms. Exposure of amphibians to pesticides can be lethal and affect their growth, development and behavior. ß-endosulfan is a persistent organochlorine that has been detected in environmental samples within protected sites in Costa Rica, far from agricultural areas. The aim of this study was to evaluate the lethal and sublethal effects, as well as changes in three biomarkers (Cholinesterase activity [ChE], glutathione S-transferase activity [GST] and lipid peroxidation [LPO]) in tadpoles of Isthmohyla pseudopuma exposed to ß-endosulfan. A 96-h acute test (20, 40, 60, 80, 100 and 200 µg/L) was performed in order to calculate the median lethal concentration (LC50), while effects on growth and development were assessed during a 4-weeks chronic test (10, 20, 30 and 50 µg/L). In addition, we measured the aforementioned biomarkers in tadpoles exposed to concentrations below the LC50. The 96-h LC50 for this species was 123.6 µg/L. We found no evidence of ß-endosulfan influencing any of the three biomarkers evaluated. At 50 µg/L, both length and total weight of tadpoles decreased with respect to the control. Also, at 30 and 50 µg/L we observed that individuals showed a slower development. Therefore, we demonstrated that at sublethal concentrations, ß-endosulfan negatively affects I. pseudopuma at early stages causing tadpoles to develop slower and smaller than normal.

Accumulation of heavy metals (Cd, Cr, Cu, Mn, Pb, Ni, Zn) in sediments, macroalgae (Cryptonemia crenulata) and sponge (Cinachyrella kuekenthali) of a coral reef in Moín, Limón, Costa Rica: An ecotoxicological approach.

Moín, on the Caribbean coast of Costa Rica, is a multi-use coastal zone with a variety of human activities that can cause metal pollution. With the purpose of assessing the current environmental burden due to heavy metal presence in the marine environment of Moín, and their bioaccumulation in organisms of the nearby coral reef, we determined seven metals in samples of bottom sediments, macroalgae (Cryptonemia crenulata) and sponge (Cinachyrella kuekenthali). The results were compared with samples from the southern Caribbean, an area with little human activity. Using ICP-MS, results showed a concentration range for sediments Mn > Cu > Zn > Cr > Ni > Pb > Cd, algae Mn > Cu > Zn > Ni > Cr > Pb > Cd and sponge Mn > Cu > Zn > Ni > Cr > Cd > Pb, relatively low concentrations overall and no differences observed between sites. Bioconcentration factor > 1 was determined for Cd, Cu, Ni and Zn, while concentrations in sediments were below the SQG thresholds. Our study provides the first data on metal concentrations in a macroalgae and a sponge from the Costa Rican Caribbean.

Early Toxic Effects in a Central American Native Fish (Parachromis dovii) Exposed to Chlorpyrifos and Difenoconazole.

In Costa Rica, agriculture is one of the most important economic activities. Chlorpyrifos and difenoconazole have been identified as agrochemicals widely used in banana and pineapple crops in the Caribbean area of the country and are constantly recorded in aquatic ecosystems. The toxicity of these pesticides in Parachromis dovii was studied. Median lethal concentrations (LC50s) for each substance were obtained from 96-h acute tests. Then, fish were exposed to sublethal concentrations of both substances (10% of LC50), individually and in mixture, to evaluate biomarker responses. Ethoxyresorufin-O-deethylase (EROD), catalase, and glutathione S-transferase activities as well as lipid peroxidation were measured in liver and gill tissues as markers of biotransformation and oxidative stress processes. Cholinesterase activity in brain and muscle tissue was also quantified as a biomarker of toxicity. The LC50s were 55.34 µg/L (95% confidence interval [CI] 51.06-59.98) for chlorpyrifos and 3250 µg/L (95% CI 2770-3810) for difenoconazole. Regarding the biomarkers, a significant inhibition of brain and muscle cholinesterase activity was recorded in fish exposed to 5.50 µg/L of chlorpyrifos. This activity was not affected when fish were exposed to the mixture of chlorpyrifos with difenoconazole. Significant changes in lactate dehydrogenase activity were observed in fish exposed to 325 µg/L of difenoconazole, whereas fish exposed to the mixture showed a significant increase in EROD activity in the liver. These results suggest harmful effects of chlorpyrifos insecticide at environmentally relevant concentrations. There is also evidence for an interaction of the 2 substances that affects the biotransformation metabolism at sublethal levels of exposure. Environ Toxicol Chem 2021;40:1940-1949. © 2021 SETAC.

Not Only Toxic but Repellent: What Can Organisms' Responses Tell Us about Contamination and What Are the Ecological Consequences When They Flee from an Environment?

The ability of aquatic organisms to sense the surrounding environment chemically and interpret such signals correctly is crucial for their ecological niche and survival. Although it is an oversimplification of the ecological interactions, we could consider that a significant part of the decisions taken by organisms are, to some extent, chemically driven. Accordingly, chemical contamination might interfere in the way organisms behave and interact with the environment. Just as any environmental factor, contamination can make a habitat less attractive or even unsuitable to accommodate life, conditioning to some degree the decision of organisms to stay in, or move from, an ecosystem. If we consider that contamination is not always spatially homogeneous and that many organisms can avoid it, the ability of contaminants to repel organisms should also be of concern. Thus, in this critical review, we have discussed the dual role of contamination: toxicity (disruption of the physiological and behavioral homeostasis) vs. repellency (contamination-driven changes in spatial distribution/habitat selection). The discussion is centered on methodologies (forced exposure against non-forced multi-compartmented exposure systems) and conceptual improvements (individual stress due to the toxic effects caused by a continuous exposure against contamination-driven spatial distribution). Finally, we propose an approach in which Stress and Landscape Ecology could be integrated with each other to improve our understanding of the threat contaminants represent to aquatic ecosystems.

Neurotoxicity of organophosphate pesticides could reduce the ability of fish to escape predation under low doses of exposure.

Biomarkers are frequently used in ecotoxicology as they allow to study toxicant effects happening at low concentrations of exposure. However, most sublethal studies only evaluate cellular biomarkers which lack evident ecological relevance. We used a multibiomarker approach to estimate the toxic effects of ethoprophos, an organophosphate insecticide commonly used in banana plantations, on the tropical fish Astyanax aeneus (Characidae). We measured biomarkers at sub-individual (cellular) and individual (metabolism, behavior) levels and examined relationships among these responses. A sublethal exposure to ethoprophos caused a significant (54%) reduction of brain Cholinesterase (ChE) activity, reflecting the pesticide's high neurotoxicity. However, other biomarkers like oxidative stress, biotransformation reactions, and resting metabolic rate were not affected. Exposure to ethoprophos modified antipredator behaviors such as escape response and detection avoidance (light/dark preference): exposed fish escaped slower from a simulated attack and preferred brighter areas in a novel tank. The relationship between ChE activity and reaction time suggests that pesticide-induced ChE inhibition reduces escape ability in fish. Our results provide evidence that impacts of organophosphate pesticides on fish ecological fitness can occur even with short exposures at very low concentrations.

Risk assessment of agriculture impact on the Frío River watershed and Caño Negro Ramsar wetland, Costa Rica.

The Caño Negro Ramsar wetland is a conservation area of great natural and societal value, located in the lower part of the Frío River watershed in the north of Costa Rica. Its aquatic ecosystems may be considered vulnerable to pollution due to recent changes in land use toward agriculture. In 2011 and 2012, quarterly sampling was done at ten sites located in the middle and lower sections of the Frío River Basin that pass through crop areas and later drain into Caño Negro wetland. Pesticide residues, nitrates, sediment concentrations, and diversity of benthic macroinvertebrates and fish biomarkers were studied in the selected sites. Additionally, risk of toxicity was calculated in two different ways: (1) by using a ratio of MEC to hazard concentrations threshold for 5% of species (HC5) to calculate a risk quotient (RQ), and (2) by using a ratio of MEC to available ecotoxicity data of native fish and cladocera for diazinon and ethoprophos, to obtain a risk quotient for native species (RQns). Results indicated that three out of the ten sites (rivers Thiales, Mónico, and Sabogal) showed variable levels of pollution including six different active ingredients (a.i.) of pesticide formulations (herbicides ametryn, bromacil, and diuron; insecticides cypermethrin, diazinon, and ethoprophos). Moreover, potential adverse effects on fishes in Thiales and Mónico rivers were indicated by cholinesterase (ChE) inhibition and glutathione S-transferase (GST) enhancement. Risk evaluations indicated pesticide residues of ametryn, bromacil, and ethoprophos to be exceeding the limits set by MTR, also RQ was high (>1) in 70% of the positive samples for diuron (most frequently found pesticide in water samples), cypermethrin, diazinon, and ethoprophos, and RQns was high for diazinon. Therefore, these substances might be of major concern for the ecological health of aquatic ecosystems in the middle basin of the Frío River. The most critical site was Mónico River, which had the highest pollution (75% detection samples with 3-5 a.i.) and highest calculated risk (RQ > 1 in 75% of the samples). This is also the river that most directly drains into the protected wetland. Even though pesticide pollution in this area is not as severe as in other parts of Costa Rica, it is imperative that measures are taken, particularly in the surroundings of Mónico River, in order to diminish and mitigate possible detrimental effects to biota in Caño Negro Ramsar Site.

In situ toxicity and ecological risk assessment of agro-pesticide runoff in the Madre de Dios River in Costa Rica.

The River Madre de Dios (RMD) and its lagoon is a biodiversity rich watershed formed by a system of streams, rivers, channels, and a coastal lagoon communicating with the Caribbean Sea. This basin sustains a large area of agricultural activity (mostly banana, rice, and pineapple) with intensive use of pesticides, continually detected in water samples. We investigated in situ the toxicological effects caused by pesticide runoff from agriculture and the relation of pesticide concentrations with different biological organization levels: early responses in fish biomarkers (sub-organismal), acute toxicity to Daphnia magna (organismal), and aquatic macroinvertebrate community structure. The evaluation was carried out between October 2011 and November 2012 at five sites along the RMD influenced by agricultural discharges and a reference site in a stream outside the RMD that receives less pesticides. Acute toxicity to D. magna was observed only once in a sample from the RMD (Caño Azul); the index of biomarker responses in fish exposed in situ was higher than controls at the same site and at the RMD-Freeman. However, only macroinvertebrates were statistically related to the presence of pesticides, combined with both physical-chemical parameters and habitat degradation. All three groups of variables determined the distribution of macroinvertebrate taxa through the study sites.

Acute, chronic and biochemical effects of chlorothalonil on Agalychnis callidryas, Isthmohyla pseudopuma and Smilisca baudinii tadpoles.

Declines of amphibian populations have been a worldwide issue of concern for the scientific community during the last several decades. Efforts are being carried out to elucidate factors related to this phenomenon. Among these factors, pathogens, climate change, and environmental pollution have been suggested as possible causes. Regarding environmental pollutants, some pesticides are persistent in the environment and capable of being transported long distances from their release point. In Costa Rica, some pesticides have been detected in protected areas, at locations where amphibian populations have declined. Information about toxicity of pesticides used in Costa Rican agriculture to amphibians is still scarce, particularly for native species.Toxicity tests with chlorothalonil, a fungicide intensively used in Costa Rica, were carried out exposing tadpoles of three Costa Rican native species: Agalychnis callidryas, Isthmohyla pseudopuma, and Smilisca baudinii in order to evaluate acute and chronic toxicity as well as the biomarkers cholinesterase activity (ChE), glutathione-S transferase activity (GST), and lipid peroxidation (LPO).96-h LC50: 26.6 (18.9-35.8) µg/L to A. callidryas, 25.5 (21.3-29.7) µg/L to I pseudopuma and 32.3 (26.3-39.7) µg/L to S. baudinii were determined for chlorothalonil. These three species of anurans are among the most sensitive to chlorothalonil according to the literature. Besides, GST was induced in S. baudinii after exposure to sub-lethal concentrations of chlorothalonil while evisceration occurred in S. baudinii and A. callidryas tadpoles exposed to lethal concentrations of the fungicide. Chronic exposure to sub-lethal concentrations accelerated development in S. baudinii and caused lesions in tail of S. baudinii and I. pseudopuma tadpoles. Our results demonstrate that chlorothalonil is highly toxic to native amphibian species and that low concentrations can cause biochemical responses related to phase II of biotransformation and effects on development.