High frequency of larval abnormalities of the toad Rhinella arenarum (Anura: Bufonidae) in an Argentinian agroecosystem

Introduction: The frequent use of pesticides is currently considered a cause of environmental pollution due to the high rate of entry of these substances into agroecosystems. This constitutes a risk for the species that inhabit these ecosystems, in particular anurans whose characteristics make them prone to exposure to and interaction with environmental pollutants. Objective: To report the occurrence of abnormalities in larvae of the common toad Rhinella arenarum inhabiting ponds surrounded by agroecosystems. Methods: In two consecutive springs (2015 and 2016), reproductive events of common toads were monitored in temporary pond systems in agricultural and non-agricultural areas, located near the city of La Plata (Buenos Aires, Argentina). The physicochemical parameters of the ponds were measured, and the stage of each reproductive event was recorded, such as the numbers of adult toads, amplexus and clutches. In the laboratory, the larvae were measured and photographed, their stage of development was recorded, and their morphology was examined under a stereomicroscope. Representative samples (normal and abnormal) from each pond studied were processed for histopathological analysis. Results: In the field studies carried out on a population of R. arenarum collected in an agroecosystem, a lower number of reproductive adults and clutches were observed in relation to the population of a non-agricultural pond. A total of 1 910 larvae were collected: 529 and 1 381 larvae from ponds located in non-agricultural and agricultural areas, respectively. Larvae from the agroecosystem showed two types of abnormalities: severe tail flexure and abdominal bloating. In addition, five degrees of severity could be determined in relation to abdominal bloating. Conclusions: This work reports the high frequency and severity of abnormalities observed in the early stages of R. arenarum larvae living within an agroecosystem, providing evidence of the negative impact that agricultural activities cause on aquatic ecosystems surrounded by farming areas.

Introducción: El uso frecuente de plaguicidas es considerado actualmente una causa de contaminación ambiental debido a las altas tasas de ingreso de estas sustancias a los agroecosistemas. Esta situación es un riesgo para las especies que habitan en estos ecosistemas, en particular los anuros cuyas características los hacen propensos a la exposición e interacción con contaminantes ambientales. Objetivo: Informar la presencia de anormalidades en larvas del sapo común Rhinella arenarum que habitan en estanques rodeados por un agroecosistema. Métodos: En dos primaveras consecutivas (2015 y 2016), se monitorearon los eventos reproductivos del sapo común proveniente de sistemas de estanques temporales ubicados en zonas agrícolas y no agrícolas, cerca de la ciudad de La Plata (Buenos Aires, Argentina). Se midieron los parámetros fisicoquímicos de los estanques y se registraron las etapas de cada evento reproductivo como el número de sapos adultos, amplexos y nidadas. En el laboratorio, las larvas fueron medidas y fotografiadas, se registró su estado de desarrollo y se examinó la morfología de cada una bajo microscopio estereoscópico. Se procesaron muestras representativas (normales y anormales) de cada estanque estudiado para análisis histopatológico. Resultados: En la población de R. arenarum que vive dentro de un agroecosistema, se observó un menor número de adultos reproductores y puestas en relación con la del estanque en la zona no agrícola. Se recolectaron un total de 1 910 larvas: 529 y 1 381 larvas de estanques ubicados en zonas no agrícolas y agrícolas, respectivamente. Las larvas del agroecosistema mostraron dos tipos de anormalidades: severa flexión de la cola y distensión abdominal. Además, se pudo determinar cinco grados de gravedad en relación con la distensión abdominal. Conclusiones: Una alta frecuencia y severidad de anormalidades en los estadios tempranos de larvas de R. arenarum que viven dentro de un agroecosistema proporciona evidencia del impacto negativo que las actividades agrícolas causan en los ecosistemas acuáticos rodeados por áreas de cultivo.

Disruptive effects of chlorpyrifos on predator-prey interactions of Ceratophrys ornata tadpoles: Consequences at the population level using computational modeling.

Large-scale ecotoxicological studies have technical and ethical limitations, both related to the need to expose large numbers of individuals to potentially harmful compounds. The computational modeling is a complementary useful and predictive tool that overcomes these limitations. Considering the increasing interest in the effects of pesticides on behavioral traits, the aim of this study was to evaluate the effects of chlorpyrifos (CPF) on intra- and inter-specific interactions of anuran tadpoles, complementing traditional ecotoxicological tools with a theoretical analysis verified by computational simulations. Experiments were developed under two consecutive phases: a first phase of exposure (treated and control group), and a second phase of interactions. The second phase consisted of evaluating the effects of CPF on intra- and inter-specific interactions of exposed C. ornata (Co) tadpoles acting as predators and unexposed Rhinella fernandezae (Rf) tadpoles acting as prey (Experiment I), under different predator-prey proportions (0/10 = 0Co-10Rf, 2/8, 4/6, 6/4, 8/2, 10/0). Also, intraspecific interactions of three Co tadpoles under different conditions of exposure were evaluated (Experiment II: 3 exposed Co, 2 exposed Co/1 non-exposed, 1 exposed Co/2 non-exposed). During the exposure phase, chlorpyrifos induced significant mortality from 48 h (48 h: p < 0.05, 72 h-96 h: p < 0.001), irregular swimming, tail flexure, and the presence of subcutaneous air. Also, it induced effects on the sounds emitted after 96 h of exposure, registering a smaller number of pulses and higher dominant frequencies, and altered intra- and inter-specific interactions. During the interaction phase, the larvae continued to show sound effects, however, the antipredator mechanism continued to be operating and efficient. Finally, it was possible to model the behavior of the larvae under the effects of chlorpyrifos. We conclude that experimental data and computational modeling matched. Therefore, computational simulation is a valuable ecotoxicological tool that provides new information and allows prediction of natural processes.

Sensitivity of Boana pulchella (Anura: Hylidae) Tadpoles to Environmentally Relevant Concentrations of Chlorpyrifos: Effects at the Individual and Biochemical Levels.

We report sublethal effects of environmentally relevant concentrations of chlorpyrifos at the individual (swimming alterations) and biochemical (esterase activities and antioxidant enzymes) levels in the Montevideo tree frog Boana pulchella larvae. The 50% lethal concentration at 96 h (LC50-96h) for chlorpyrifos in stage-29 B. pulchella tadpoles was 0.98 mg/L, which was close to the 65th percentile of published anuran species sensitivity. In B. pulchella, chlorpyrifos disrupted biochemical processes: tadpoles showed a significant inhibition of esterase activity and a significant induction of antioxidant enzymes, indicating a response to an environmental challenge causing oxidative stress. Using principal components analysis, we could associate chlorpyrifos reduction in esterase activity with swimming alterations at 0.5 mg/L of the toxicant. The biochemical biomarkers reported in the present study respond at levels 20 times lower than the LC50-96h and were associated with a biologically important response-swimming behavior. The link of responses across different levels of biological organization was demonstrated. The species is suitable as a model for ecotoxicological studies at different levels, including the individual and biochemical levels, and may be considered a good reference organism in environmental control programs. Environ Toxicol Chem 2020;39:834-841. © 2020 SETAC.

Embryonic ontogeny of three species of Horned Frogs, with a review of early development in Ceratophryidae.

Horned Frogs of the family Ceratophryidae are conspicuous anurans represented by three endemic South American genera. Most ceratophryids inhabit semiarid environments, but three species of Ceratophrys occupy tropical or temperate humid areas. Several morphological and behavioral characters of larvae and adults are conserved across the family. Based on examination of specimens and accounts in the literature, the embryonic development of C. ornata, C. cranwelli, and the monotypic genus Chacophrys are described and compared with that of species of Lepidobatrachus. Ceratophryid embryos share a suite of morphological features and heterochronic shifts during development. Most features, such as gill structure, ciliation, early hatching, and precocious differentiation of the gut and hind limbs, are shared by all the species regardless the differences in the habitats that occupy. This is consistent with previous observations of some adult characters, and likely supports the hypothesis of an early diversification of ceratophryids in semiarid environments. Other embryonic features, such as the morphology and ontogeny of the oral disc and digestive tract, are correlated with larval feeding habits and vary within the family. The evolutionary and ecological significance of some conserved characters (e.g., gastrulation pattern, Type-A adhesive glands) and other taxon-specific features (e.g., nasal appendix) remain to be explored in the group.

Effects of glyphosate and its commercial formulation, Roundup® Ultramax, on liver histology of tadpoles of the neotropical frog, Leptodactylus latrans (amphibia: Anura).

In the last years, the agricultural expansion has led to an increased use of pesticides, with glyphosate as the most widely used worldwide. This is also the situation in Argentina, where glyphosate formulations are the most commercialized herbicides. It is known that glyphosate formulations are much more toxic than the active ingredient, and this difference in toxicity can be attributed to the adjuvants present in the formula. In this context, the aim of the present study was to evaluate and compare sub-lethal histological effects of the glyphosate formulation Roundup Ultramax and glyphosate active ingredient on Leptodactylus latrans tadpoles at Gosner-stage 36. Semi-static bioassays were performed using 96 h of exposure with Roundup Ultramax formulation (RU; 0.37-5.25 mg a.e./L), glyphosate (GLY; 3-300 mg/L), and a control group. RU exposure showed an increment in the melanomacrophagic cells (MMc) and melanomacrophagic centers (MMCs) from 0.37 mg a.e./L. GLY exposure showed a significant increment in the number of MMc from 15 mg/L, and of MMCs from 3 mg/L. Also, histopathological lesions were observed in the liver of tadpoles exposed to both, GLY and RU. These lesions included: lipidosis and hepatic congestion, but only RU showed significant differences respect to control, with a LOEC value of 2.22 mg a.e./L for both effects. In sum, this study represents the first evidence of adverse effects of glyphosate and RU formulation on the liver of anuran larvae at concentrations frequently found in the environment.

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.

Flurochloridone-based herbicides induced genotoxicity effects on Rhinella arenarum tadpoles (Anura: Bufonidae).

Acute toxicity and genotoxicity of the flurochloridone (FLC)-containing commercial formulation herbicides Twin Pack Gold(®) (25 percent a.i.) and Rainbow(®) (25 percent a.i.) were evaluated on Rhinella arenarum (Anura: Bufonidae) tadpoles exposed under laboratory conditions. Lethal effect was evaluated as end point for lethality, whereas frequency of micronuclei (MN) and single cell gel electrophoresis (SCGE) were employed as end points for genotoxicity. Lethality studies revealed equivalent LC-5096 h values of 2.96 and 2.85 mg/L for Twin Pack Gold(®) and Rainbow(®), respectively. Twin Pack Gold(®) did not induce DNA damage at the chromosomal level, whereas Rainbow(®) increased the frequency of MN only when the lowest concentration (0.71 mg/L) was used. However, all concentrations of Twin Pack Gold(®) and Rainbow(®) increased the frequencies of primary DNA lesions estimated by alkaline SCGE. This study represents the first evidence of the acute toxic and genotoxic effects exerted by two FLC-based commercial formulations, Twin Pack Gold(®) and Rainbow(®), on tadpoles of an amphibian species native to Argentina under laboratory conditions. Finally, our findings highlight the importance of minimizing the impacts on nontarget living species exposed to agrochemicals.

Influence of existing site contamination on sensitivity of Rhinella fernandezae (Anura, Bufonidae) tadpoles to Lorsban®48E formulation of chlorpyrifos.

Effects of the widely employed insecticide Lorsban(®)48E formulation of chlorpyrifos (CPF) was studied on Rhinella fernandezae tadpoles, a native species of Argentina, Brazil, Paraguay and Uruguay, under the hypothesis of a differential response of organisms from ponds of two sites with different degree of anthropogenic disturbance: S1 an unpolluted area, and S2 area with high degree of antropogenic disturbance. To collect a representative sample of the genotypic variability of each population, small portions from six clutches were taken randomly from each site when the period of clutching was finished. Embryos and tadpoles were maintained under controlled laboratory conditions. Toxicity tests were conducted under standardized conditions to study acute and chronic lethal (mortality) and sublethal effects (behavior, growth, and abnormalities), within the range of concentrations of 0.010 to 5 mg/L. Chronic effects were assessed with organisms from one of the demes (S1). CPF showed high toxicity on the tadpoles, inducing lethal and sublethal effects at 96 h exposure within a narrow range of concentrations from 0.066 to 0.887 mg/L. Results indicate that R. fernandezae tadpoles are below the 30th percentile in the species sensitivity distribution of existing data. The acute LC50, NOEC, and LOEC values were 0.151, 0.066, and 0.133 mg/L for S1, and 0.293, 0.177, and 0.266 mg/L for S2, respectively. Considering all acute end-points evaluated, the effects of CPF showed no significant differences (p = 0.3484) between the studied populations. CPF has more severe effects at higher concentrations than at higher times of exposure. Contaminants in S2 do not seem to induce local adaptation. Sublethal effects data and measured environmental concentrations indicate potential risk for populations inhabiting agroecosystems.

Reduced body condition and enzymatic alterations in frogs inhabiting intensive crop production areas.

Parameters indicative of general condition and health were compared amongst adult frogs inhabiting uncultivated lands and fields subjected to agricultural practices typical of the humid pampas of Argentina. Whereas no significant differences existed in the parasite load and external malformations prevalence rate of adult frogs from either environments, a reduced condition factor was clearly demonstrated in frogs from agricultural lands. This conclusion was reached for four frog species of different life habits: the terrestrial fossorial Rhinella fernandezae, the terrestrial Leptodactylus latinasus, the semi-aquatic Leptodactylus ocellatus, and the arborescent Hypsiboas pulchellus. A distinct pattern of enzymatic modifications was furthermore observed in L. ocellatus and H. pulchellus from agricultural lands, including elevated hepatic activities of catalase and cholinesterase, and an inhibition of liver GST activity. Further studies should investigate the causes and consequences of the systematically low condition factor observed in frogs from agricultural fields of the humid pampas of Argentina.

Lethal and sublethal effects of cypermethrin to Hypsiboas pulchellus tadpoles.

The study of the effects of the insecticide cypermethrin (CY) technical grade and its Sherpa® commercial formulation on Hypsiboas pulchellus tadpoles assessing lethality, behavior, growth, and abnormalities under standardized laboratory conditions is reported. Observed behaviors were identified and categorized by means of a ranking system according to the loss of mobility. Results of acute lethal effects indicate higher potency for Sherpa® at this level of assessment. All effects on behavior showed an increasing degree of injury as insecticide concentration increased. Organisms exposed to technical grade CY showed lower body length with respect to controls from 3.44 µg CY/L to higher concentrations, whereas those exposed to Sherpa® exhibited lower growth from 0.83 µg CY/L. Both forms of the tested insecticide caused abnormalities between 0.34 and 4.18 µg CY/L, but 100% of malformed individuals was detected from 34.4 µg CY/L for those exposed to the technical grade CY, and from 8.36 µg CY/L for those exposed to Sherpa®. This study proposes the use of easily identifiable and distinguishable sublethal end-points. The high input loads of CY in natural environments, the detected concentrations in the field, in addition to the low levels of this insecticide required to induce sublethal effects (which could eventually lead to death), allow for the conclusion that the insecticide is a risk factor for amphibians inhabiting agroecosystems.

Hormonal induction of spawning in 4 species of frogs by coinjection with a gonadotropin-releasing hormone agonist and a dopamine antagonist.

BACKGROUND: It is well known that many anurans do not reproduce easily in captivity. Some methods are based on administration of mammalian hormones such as human chorionic gonadotropin, which are not effective in many frogs. There is a need for simple, cost-effective alternative techniques to induce spawning. METHODS: Our new method is based on the injection of a combination of a gonadotropin-releasing hormone (GnRH) agonist and a dopamine antagonist. We have named this formulation AMPHIPLEX, which is derived from the combination of the words amphibian and amplexus. This name refers to the specific reproductive behavior of frogs when the male mounts and clasps the female to induce ovulation and to fertilize the eggs as they are laid. RESULTS: We describe the use of the method and demonstrate its applicability for captive breeding in 3 different anuran families. We tested several combinations of GnRH agonists with dopamine antagonists using Lithobates pipiens. The combination of des-Gly10, D-Ala6, Pro-LHRH (0.4 microrams/g body weight) and metoclopramide (10 micrograms/g BWt. MET) was most effective. It was used in-season, after short-term captivity and in frogs artificially hibernated under laboratory conditions. The AMPHIPLEX method was also effective in 3 Argentinian frogs, Ceratophrys ornata, Ceratophrys cranwelli and Odontophrynus americanus. CONCLUSION: Our approach offers some advantages over other hormonally-based techniques. Both sexes are injected only once and at the same time, reducing handling stress. AMPHIPLEX is a new reproductive management tool for captive breeding in Anura.

Sublethal and lethal effects on Rhinella arenarum (Anura, Bufonidae) tadpoles exerted by the pirimicarb-containing technical formulation insecticide Aficida.

Acute toxicity, genotoxicity, and cytotoxicity of the pirimicarb-containing commercial-formulation carbamate insecticide Aficida(R) (50% pirimicarb) were evaluated on Rhinella arenarum (Anura, Bufonidae) tadpoles exposed under laboratory conditions. Lethal and sublethal effects were employed as bioassays for acute toxicity, whereas micronuclei (MNi) induction and alterations in the ratio erythrocytes:erythroblasts were employed as end-points for genotoxicity and cytotoxicity, respectively. Cr(VI) (23 mg L(-1)) and cyclophosphamide (40 mg L(-1)) were employed as positive controls for toxicity and geno-cytotoxicity assays, respectively. In Gosner stage 25 (STD25), the results revealed mean values of 402.0 and 223.6 mg Aficida L(-1) for LC-50(24)(h) and LC-50(96)(h), respectively. When STD37-39 tadpoles were exposed, the LC-50(24)(h) and LC-50(96)(h) reached values of 239.4 and 181.7 mg Aficida L(-1), respectively. Sublethal effects revealed a mean EC-50(96)(h) of 133.85 and 104.2mg Aficida in those STD25 and STD37-39 treated tadpoles, respectively. The results demonstrated that in 48-h-exposed tadpoles, a MNi increase was found only in those 80.0 mg L(-1) Aficida-treated individuals. When tadpoles were exposed to Aficida for 96h, only the 160 mg L(-1)-treated individuals showed a significant increase in MNi frequency. Concentrations ranging from 80.0 to 250.0mg Aficida L(-1) resulted in cellular cytotoxicity, revealed by a decreased proportion of circulating erythrocytes and an enhancement of erythroblasts. Accordingly, this species could provide a suitable and useful experimental model for biomonitoring aquatic ecosystems.

Chytrid fungus parasitizing the wild amphibian Leptodactylus ocellatus (Anura: Leptodactylidae) in Argentina.

The present contribution is the first report of parasitosis by a chytrid fungus in wild anuran amphibians in Argentina, as well as the first case of amphibian mortality documented to date in Argentina. We report the presence of the chytrid fungus in dead adult Leptodactylus ocellatus. It has been suggested that chytridiomycosis is the main cause of death in several amphibian populations worldwide. Our study demonstrates that chytridiomycosis afflicts L. ocellatus, a common widespread amphibian species, and is the first report of chytridiomycosis in the Argentinian lowlands. The occurrence at this latitude would indicate an extended distribution of this fungus in wildlife populations. It is also the first report of amphibian mortality due to chytrid fungus in our country. It is noteworthy that the site of collection is situated very close to sea level in a temperate climate zone and that this represents the southern most record for South American wild amphibians.