The neonicotinoid insecticide imidacloprid (IMI) affects the insect central nervous system and is successfully applied to control pests for a variety of agricultural crops. In the current study, ...acute toxicity and genotoxicity of the IMI-containing commercial formulation insecticide Glacoxan Imida (35 percent IMI) was evaluated on Hypsiboas pulchellus (Anura: Hylidae) tadpoles exposed under laboratory conditions. A lethal effect was evaluated as the end point for lethality, whereas micronucleus (MN) frequency and DNA single-strand breaks evaluated by the single cell gel electrophoresis (SCGE) assay were employed as end points for genotoxicity. Sublethal end points were assayed within the 12.5–37.5mg/L IMI concentration range. Experiments were performed on tadpoles at stage 36 (range, 35–37) according to the classification proposed by Gosner. Lethality studies revealed an LC50 96h value of 52.622mg/L IMI. Increased frequency of MNs was only observed when 25.0mg/L was assayed for 96h, whereas no other nuclear abnormalities were induced. Increase of the genetic damage index was observed at 48h of treatment within the 12.5–37.5mg/L concentration range, whereas an increased frequency of DNA damage was observed only in tadpoles treated with 37.5mg/L IMI for 96h. This study represents the first evidence of the acute lethal and genotoxic effects exerted by IMI on tadpoles of an amphibian species native to Argentina under laboratory conditions.
•IMI lethal and genotoxic properties were evaluated on Hypsiboas pulchellus.•Determination of LC50 96h was employed as lethal end point.•IMI induced micronuclei and DNA single-strand breaks in circulating blood cells.•First evidence of genotoxicity exerted by IMI on an Argentinean amphibian species.
•Effects of imidacloprid (IMI) were evaluated on Hypsiboas pulchellus tadpoles.•Mortality studies revealed a LC5096h value of 84.91mg/L of IMI.•IMI induced both micronuclei and DNA strand breaks in ...circulating blood cells.•Binucleated, blebbed, and notched cells were induced after 48 and 96h of treatment.•First evidence of deleterious effects of IMI on an Argentinean amphibian species.
Acute toxicity and genotoxicity of imidacloprid (IMI) was evaluated on Hypsiboas pulchellus (Anura: Hylidae) tadpoles exposed under laboratory conditions. A lethal effect was used as the end point for lethality, whereas the frequency of micronuclei (MNs) and DNA single-strand breaks evaluated by the single cell gel electrophoresis assay were employed as end points for genotoxicity. Experiments were performed on tadpoles at stage 36 (range, 35–37) according to the classification proposed by Gosner. Mortality studies revealed an LC50 (96h) value of 84.91mg/L IMI (95% confidence limits, 77.20–93.04). While increased frequency of MNs was observed when 15 and 30mg/L were assayed for 48h, only 15mg/L increased the frequency of MNs in tadpoles exposed for 96h. Furthermore, other nuclear abnormalities, i.e., binucleated cells and blebbed and notched nuclei, were induced in tadpoles exposed for both 48h when treated with 15mg/L and 96h when treated with 15 and 30mg/L. An increase in the genetic damage index was observed in tadpoles treated with 30mg/L for 48 and 96h. This study represents the first evidence of acute lethal and sublethal effects exerted by IMI on tadpoles of an amphibian species native to Argentina. Finally, our findings highlight the hazardous properties of this insecticide for nontarget living species exposed to this agrochemical.
Amphibians are the most threatened group of vertebrates because they have certain biological and ecological characteristics that make them sensitive to environmental changes. The aim of this study ...was to evaluate the health status of field-collected adult frogs of Leptodactylus luctator (Amphibia, Anura) living in sites with different anthropogenic disturbances (florihorticulture, petrochemical industry and sewage discharges) and a reference site without any detectable influence of such activities. To this end, a battery of 21 biomarkers (hematological, biochemical and individual biomarkers) was studied using a multivariate approach that allows us to evaluate the relationship between them and provide information on their usefulness. The frogs at the florihorticulture, petrochemical and sewage discharges sites exhibited several biomarkers far from homeostasis. In addition, we identified 11 of 21 biomarkers that were useful indicators of the health status of the frogs and allowed discrimination between study sites in the following order: lymphocytes (98 %), neutrophils (45 %), hemoglobin (42 %), monocytes (41 %), fat body index (35 %), eosinophils (35 %), hepatosomatic index (33 %), mean corpuscular hemoglobin (32 %), thrombocytes (27 %), catalase in liver (26 %), and GST in liver (26 %). The results suggest that hematological biomarkers contribute the most to site separation, whereas biochemical biomarkers contribute the least. The integral interpretation of the results also allowed us to diagnose the different health status of L. luctator: The frogs from the petrochemical industry were the most negatively affected, followed by the frogs from the sewages discharges and finally the frogs from the florihorticulture and reference sites. This is the first field study with anurans in which so many biomarkers were examined.
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•Integral analysis of 21 biomarkers allowed diagnosing diverse frog health statuses.•Frogs in disturbed sites showed biomarkers far from homeostasis.•We identified 11 biomarkers indicative of frog health that distinguished each site.•We selected 9 sensitive biomarkers that require non-invasive methods.
The presence of pharmaceutically active compounds (PhACs) in surface water is well known, whereas their natural occurrence in biota is much less explored. The aim of this work was to evaluate the ...bioaccumulation of PhACs in adult toads of the neotropical species Rhinella arenarum. Three sites were selected in Buenos Aires (Argentina): a reference site (Site 1), a site with direct discharge from a secondary wastewater treatment plant (WWTP) (Site 2) and a site 300 m downstream of the WWTP discharge (Site 3). Surface water samples, as well as muscle, liver and fat bodies of toads were collected, extracted and analyzed by LC-MS/MS. Highly significant differences in total PhACs concentration in surface water (p < 0.005) were detected between Site 2 and the other sites. These concentrations ranged from 0.37 to 52.46 ng/L at Site 1, 0.71–6950.37 ng/L at Site 2, and 0.12–75.45 ng/L at Site 3. In general, bioaccumulation of PhACs in toad tissues was similar between sites and tissues of each site. The highest concentrations were detected in the muscle of toads from Site 3 (1.06–87.24 ng/g dw), followed by liver (1.77–38.10 ng/g dw) and fat bodies (0.68–20.59 ng/g dw) from Site 1. Ibuprofen (6950 ng/L), acetaminophen (3277 ng/L) and valsartan (2504 ng/L) were the compounds with the highest concentrations in surface water from Site 2, whereas acetaminophen (87.2 ng/g dw, muscle from Site 3), desloratadine (38.1 ng/g dw, liver from Site 1), and phenazone (25.9 ng/g dw, liver from Site 1) were the ones that showed the highest concentrations in biota. This is the first time a field study has examined the environmental bioaccumulation of PhACs in anurans, demonstrating their potential for monitoring the status of natural ecosystems.
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•Analgesics/anti-inflammatories showed the highest concentrations.•Antibiotics showed the highest frequencies of detection.•Twenty pharmaceuticals detected for the first time in surface water from Argentina.•Nearly thirty pharmaceuticals detected for the first time in adult anurans.•First field study to evaluate the bioaccumulation of pharmaceuticals in toads.
Imazethapyr, a post-emergent herbicide used in worldwide soybean and corn crops, induces genetic and biochemical alterations in aquatic vertebrates. This study examined the relationship between ...biomarkers at different organization levels and imazethapyr real-life route exposure in
Boana pulchella
adults. Frogs were exposed to imazethapyr-based formulation Pivot
®
H (10.59%) at concentrations representing possible acute routes: field runoff (S1:10 mg.L
−1
), exposure after direct foliar application (S2:100 mg.L
−1
) and during direct foliar application (S3:1000 mg.L
−1
). Post-exposure, endpoints levels were evaluated: organism alterations, biochemical activities and cytogenetic assays. Forty-eight hours post-exposure, antioxidant enzymes decrease, micronuclei induction and DNA damage were observed in all scenarios, while cholinesterase activity increase and body condition reduction were observed in frog-exposed to S3. Ninety-six hours post-exposure, frogs showed glutathione-S-transferase inhibition in S1, micronuclei induction in S2 and S3, and DNA-damage increase in S3. Herbicides routes of exposures in real-life could indicate that authorized applications have a risk to amphibian populations.
Acute lethal and sublethal toxicity of the pirimicarb-based commercial formulation Aficida® were evaluated on Boana pulchella tadpoles. Whereas mortality was used as end point for lethality, ...frequency of micronuclei and other nuclear abnormalities as well as alterations in the frequency of erythroblasts in circulating blood as biomarkers for genotoxicity and cytotoxicity, respectively. Swimming, growth, developmental and morphological abnormalities were also employed as sublethal end points. Results show that the species is within the 13th percentile of the distribution of acute sensitivity of species to pirimicarb for aquatic vertebrates. Results revealed values of 23.78 and 101.45mg/L pirimicarb as LC5096h for GS25 and GS36 tadpoles, respectively. The most evident effects were related with the swimming activity with NOEC and LOEC values within the 0.005–0.39mg/L pirimicarb concentration range. Aficida® induced DNA damage at the chromosomal level by increasing micronuclei frequency and other nuclear abnormalities, i.e., lobbed and notched nuclei and binucleated cells. Cellular cytotoxicity was found after Aficida® treatment. The presence of abdominal oedemas in exposed organisms and thus flotation response of organisms could be proposed as a new sensitive exposure parameter. The multiple end point assessment approach used allowed a complete understanding the multi level of effects occurring by exposure to pirimicarb, at least in B. pulchella.
•Pirimicarb-based formulation Aficida® jeopardizes anuran tadpoles.•Acute lethal and sublethal toxicity of Aficida® were evaluated on Boana pulchella.•The LC5096h for GS25 and GS36 tadpoles were 60.95 and 260.02mg/L Aficida®, respectively.•Flotation of organisms is proposed as a new sensitive biomarker.•Aficida® induced DNA damage at the chromosomal level.
Acute lethal and sublethal toxicity of the imidazolinone imazethapyr (IMZT)-based commercial formulation herbicide Pivot H® (10.59% IMZT) was evaluated on Hypsiboas pulchellus tadpoles. Whereas ...mortality was used as the end point for lethality, frequency of micronuclei (MNs) and other nuclear abnormalities as well as DNA single-strand breaks evaluated by the single cell gel electrophoresis assay were employed to test genotoxicity. Behavioral, growth, developmental, and morphological abnormalities were also employed as sublethal end points. Mortality studies revealed equivalent LC50 (96h) values of 1.49mg/L (confidence limit, 1.09–1.63) and 1.55mg/L (confidence limit, 1.51–1.60) IMZT for Gosner stage (GS) 25 and GS36, respectively. Behavioral changes, i.e., irregular swimming and immobility, as well as a decreased frequency of keratodonts were observed. The herbicide increased the frequency of MNs in circulating erythrocytes of tadpoles exposed for 48h to the highest concentration assayed (1.17mg/L). However, regardless of the concentration of the herbicide assayed, an enhanced frequency of MNs was observed in tadpoles exposed for 96h. The herbicide was able to induce other nuclear abnormalities, i.e., blebbed and notched nuclei, only when tadpoles were exposed for 96h. In addition, we observed that exposure to IMZT within the 0.39–1.17mg/L range increased the genetic damage index in treatments lasting for both 48 and 96h. This study represents the first evidence of acute lethal and sublethal effects exerted by IMZT on amphibians. Finally, our findings highlight the properties of this herbicide that jeopardize nontarget living species exposed to IMZT.
•Lethality and genotoxicity of imazethapyr were used as endpoints in H. pulchellus.•Imazethapyr-based formulations induced DNA primary lesions in blood cells.•First evidence of genotoxicity exerted by imazethapyr-based formulations on tadpoles.
Previous studies reported that some species of the family Ceratophryidae are able to produce sounds during premetamorphic tadpole stages. We have now determined the effects of the ...cholinesterase-inhibiting insecticide chlorpyrifos (CPF) on sounds emitted by tadpoles of Ceratophrys ornata. Tadpoles were exposed individually in order to evaluate the progression of effects. Effects on sound production were complemented with common ecotoxicological endpoints (mortality, behavior, abnormalities and growth inhibition). C. ornata was found to be more sensitive than other native (= 67%, 50%) and non-native species (= 75%, 100%) considering lethal and sublethal endpoints, respectively. Effects on sounds appear along with alterations in swimming, followed by the presence of mild, then severe abnormalities and finally death. Therefore, sound production may be a good biomarker since it anticipates other endpoints that are also affected by CPF. Ceratophrys ornata is a promising new model species in ecotoxicology.
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•First investigation using C. ornata to assess the effects of a pollutant.•Environmentally-relevant concentrations of chlorpyrifos are disrupting key behaviors.•Tadpole sounds may be a good biomarker since it anticipates other endpoints.•The species is more sensitive than other native and most non-native species.
Environmentally-relevant chlorpyrifos concentrations are disrupting key behaviors in C. ornata, including sound emission and locomotion.
The occurrence of abnormalities in amphibians has been reported in many populations, and its increase could be related to environmental pollution and habitat degradation. We evaluated the type and ...prevalence of abnormalities in 5 amphibian populations from agroecosystems with different degrees of agricultural disturbance (cultivated and reference areas). We detected 9 types of abnormalities, of which the most frequent were those occurring in limbs. The observed prevalence of abnormality in assessed populations from cultivated and reference areas was as follows: Rhinella fernandezae (37.1 and 10.2%, respectively), Leptodactylus latrans adults (28.1 and 9.2%) and juveniles (32.9 and 15.3%), and Hypsiboas pulchellus (11.6 and 2.8%). Scinax granulatus populations did not show abnormalities. Pseudis minuta, which was only detected in the reference area, exhibited a prevalence of 13.3%. For R. fernandezae, L. latrans, and H. pulchellus, generalized linear mixed models showed that prevalence of abnormalities was significantly higher (p < 0.05) in cultivated than in reference areas. L. latrans juveniles were more vulnerable to abnormalities than adults (p < 0.05). The presence of abnormalities in some species inhabiting different agroecosystems suggests that environmental stress factors might be responsible for their occurrence. While we detected pesticides (endosulfan, cypermethrin, and chlorpyrifos) and lower dissolved oxygen levels in ponds of the cultivated area, no data are currently available on how other factors, such as injuries from predators and parasite infections, vary by land use. Further research will be necessary to evaluate possible causes of abnormalities detected in the present study mainly in the context of factor interactions.
Imazethapyr is an herbicide that is used in a variety of crops worldwide, including soybean and corn. The aim of the present study was to evaluate the biomarkers responses of adult
Leptodactylus ...latinasus
exposed to the formulation Pivot
®
H (10.59% imazethapyr) in the laboratory at concentrations and under conditions that simulate two potential field exposure scenarios: an immersion in field runoff (Scenario 1: 10 mg/L) and a direct exposure to the droplets emitted by spray noozles (Scenario 2: 1000 mg/L). In both scenarios, the experimental procedure involved completely immersing the frogs over a period of 15 s. Different endpoints were evaluated at several ecotoxicological levels 48 and 96 h after the herbicide exposure. These included individual (biometric indices and behavior alterations), histological (liver pigments and lesions), biochemical (catalase, glutathione system and cholinesterase activities) and genotoxic effects (micronuclei induction and nuclear abnormalities). Forty-eight hours after imazethapyr exposure, frogs submitted to Scenario 1 presented an inhibition of liver glutathione-S-transferase activity, whereas histological alterations and increased hepatic cholinesterase levels were observed in frogs exposed under Scenario 2. Ninety-six hours after exposure to the imazethapyr formulation, frogs from the Scenario 1 treatment presented a decrease in liver melanin and hemosiderin, increased hepatic catalase activity and micronuclei induction. For their part, frogs exposed to Scenario 2 presented a decrease in the hepatosomatic index, an increase in liver alterations, melanin reduction and micronuclei induction. The multivariate analysis enables correlations to be made between biomarkers of different organizational level in exposed anurans. Our result indicates that real exposure to imazethapyr formulations under field conditions may pose a risk to
Leptodactylus latinasus
populations living in the agroecosystems.
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