Metals, pesticides, and emerging contaminants on water bodies from agricultural areas and the effects on a native amphibian.

In the Paraná River lower basin, an important agro-productive area of Argentina, crop fields and cattle breeding activities are common and may affect water quality. So, the aim of this study was to analyze the impacts of cattle breeding and agricultural activities on a stream from Buenos Aires, through physicochemical parameters (metals, pesticides, and emerging contaminants) and ecotoxicological parameters with Rhinella arenarum larvae, a native amphibian species. Three sites were selected on an ordinary plain stream that goes through agricultural fields and a cattle breeding establishment (upstream -S1-, near -S2- and downstream -S3- the establishment). Physicochemical parameters were measured in situ (in water) and in laboratory (in water and sediment samples: metals, pesticides, ivermectin and oxytetracycline). A semi-static chronic toxicity bioassay (504 h) was performed with water samples, and neurotoxicity, oxidative stress and genotoxicity biomarkers were measured after acute exposure (96 h). According to the index, a degradation in the water quality was observed in all sites. Ivermectin (8.03 mg/kg) and oxytetracycline (1.9 mg/kg) were detected in sediment samples from S2. Pesticides were detected in all sites, mainly in water samples: S1 presented the highest variability (7 residues) and in S3 AMPA, glyphosate and acetochlor concentrations were higher (10.3, 22.4 and 23.8 µg/L). Also, all sites significantly produced lethality at chronic exposure. Lethality at 504h was 40% for S1, 56.66% for S2 and 93.33% for S3. At acute exposure, the oxidative stress biomarkers were altered on R. arenarum larvae exposed to all sites and the neurotoxicity biomarkers were altered on larvae exposed to S1 and S3. Water quality was severely degraded by the surrounding agricultural and cattle breeding activities, which may represent a threat to the ecosystems.

Synergistic effects of glyphosate- and 2,4-D-based pesticides mixtures on Rhinella arenarum larvae.

Glyphosate and 2,4-D are two herbicides commonly used together. Since there is little information about the interactions between these pesticides, the aim of this study was to evaluate the single and joint lethal toxicity of the glyphosate-based herbicide (GBH) ATANOR® (43.8% of glyphosate, isopropylamine salt) and the 2,4-D-based herbicide (2,4-DBH) Así Max 50® (602000 mg/L of 2,4-D) on Rhinella arenarum larvae. Equitoxic and non-equitoxic mixtures were prepared according to the recommendation for their combination and analyzed with a fixed ratio design at different exposure times and levels of lethality (LC10, LC50, and LC90). GBH (504h-LC50=38.67 mg ae/L) was significantly more toxic than 2,4-DBH (504h-LC50=250.31 mg ae/L) and their toxicity was time-dependent. At 48h, the equitoxic mixture toxicity was additive and from the 96h was antagonistic at LC10 and LC50 effect level. The non-equitoxic mixture toxicity was additive at LC10 effect level from the 48h to the 168h, and synergistic from the 240h. At LC50 and LC90 effect level, the mixture interaction resulted synergistic for all exposure times. This is the first study to report the synergistic interactions between GBH and 2,4-DBH on amphibians, alerting about its negative impact on aquatic ecosystems.

In situ exposure of amphibian larvae (Rhinella fernandezae) to assess water quality by means of oxidative stress biomarkers in water bodies with different anthropic influences.

In situ bioassays provide valuable information about the environment and offer more realistic results than usual laboratory experiments. The aim of this study was to evaluate the quality of water bodies from the lower Paraná River basin, the second most important in South America, through analysis of physiochemical parameters, metals and pesticides and in situ exposure of Rhinella fernandezae larvae to assess oxidative stress biomarkers. The sites were: S1(Morejón stream, reference); S2, S3(De la Cruz stream upstream and downstream, respectively) and S4(Arrecifes River). In all sites, dissolved oxygen was low, atrazine was detected and Cu was higher than the limit for aquatic life protection. According to the water quality index, S2, S3 and S4 presented bad water quality, while S1 good water quality. Larvae were exposed in situ for 96h in order to analyze: lipid peroxidation(TBARS) as oxidative damage, antioxidant enzymatic (catalase-CAT-, superoxide dismutase-SOD- and glutathione s-transferase-GST-) and non-enzymatic defenses (reduced glutathione-GSH-). Larvae exposed in the most impacted sites (S2, S3 and S4) presented oxidative stress since the levels of TBARS were around 2 times higher than in S1. Also, the other oxidative stress biomarkers were altered in larvae exposed at S2, S3 and S4. These results highlight the importance of analyzing oxidative stress biomarkers during in situ exposures since they are useful tools for documenting the extent of exposure at sublethal levels. The complex pollution of the water bodies affected the exposed larvae, which may jeopardize the native populations.

Monitoring of toxicity of As(V) solutions by AMPHITOX test without and with treatment with zerovalent iron nanoparticles.

Changes in toxicity of As(V) solutions from acute to chronic exposure have been evaluated by the AMPHITOX test. This test employs Rhinella arenarum, a widely distributed toad in Argentine areas. LOEC values were 6.37 and 1.88 mg L-1 for embryos and larvae, respectively, and serious sublethal effects have been observed. Toxicity of As(V) solutions has been also evaluated after treatment with zerovalent iron nanoparticles (nZVI). After 60 min of treatment with nZVI, As(V) removal was 77%, and neither lethal nor sublethal effects were observed. However, nZVI had to be eliminated before the bioassay because they caused adverse effects in both embryos and larvae. This work highlights the high sensitivity of R. arenarum to As(V), the relevance to assess toxicity on different periods of the lifecycle, and the need to expand exposure to As(V) to chronic times. The utility of the test for monitoring toxicity changes in As(V) solutions after nZVI treatment has been also shown.

Lethal and sublethal toxicity of the industrial chemical epichlorohydrin on Rhinella arenarum (Anura, Bufonidae) embryos and larvae.

Lethal and sublethal toxicity of the major chemical used in epoxide compounds, epichlorohydrin (ECH) was evaluated on the early life cycle of the common South American toad, Rhinella arenarum (Anura, Bufonidae). The stages evaluated were (according to Del Conte and Sirlin): early blastula (S.3-S.4), gastrula (S.10-S.12), rotation (S.15), tail bud (S.17), muscular response (S.18), gill circulation (S.20), open mouth (S.21), opercular folds (S.23) and complete operculum (S.25). The LC50 and EC50 values for lethal and sublethal effects were calculated. The early blastula was the most sensitive stage to ECH both for continuously and pulse-exposures (LC50-24h=50.9 mg L(-1)), while S.20 was the most resistant (LC50-24h=104.9 mg L(-1)). Among sublethal effects, early blastula was also the most sensitive stage (LOEC-48 h=20 mg L(-1)) and it has a Teratogenic Index of 2.5, which indicates the teratogenic potential of the substance. The main abnormalities were persistent yolk plugs, cell dissociation, tumors, hydropsy, oral malformations, axial/tail flexures, delayed development and reduced body size. ECH also caused neurotoxicity including scarce response to stimuli, reduction in the food intake, general weakness, spasms and shortening, erratic or circular swimming. Industrial contamination is considered an important factor on the decline of amphibian populations. Considering the available information about ECH's toxicity and its potential hazard to the environment, this work shows the first results of its developmental toxicity on a native amphibian species, Rhinella arenarum.

Stage-dependent teratogenic and lethal effects exerted by ultraviolet B radiation on Rhinella (Bufo) arenarum embryos.

The adverse effects of ultraviolet B radiation from 547.2 to 30,096 J/m2 on morphogenesis, cell differentiation, and lethality of amphibian embryos at six developmental stages were evaluated from 24 up to 168 h postexposure. The ultraviolet B radiation lethal dose 10, 50, and 90 values were obtained for all developmental stages evaluated. The lethal dose 50 values, considered as the dose causing lethality in the 50% of the organisms exposed, in J/m2 at 168 h postexposure, ranged from 2,307 to 18,930; gill circulation and blastula were the most susceptible and resistant stages, respectively. Ultraviolet B radiation caused malformations in all developmental stages but was significantly more teratogenic at the gill circulation and complete operculum stages. Moreover, at the gill circulation stage, even the lowest dose (547.2 J/m2) resulted in malformations to 100% of embryos. The most common malformations were persistent yolk plug, bifid spine, reduced body size, delayed development, asymmetry, microcephaly and anencephaly, tail and body flexures toward the irradiated side, agenesia or partial gill development, abnormal pigment distribution, and hypermotility. The stage-dependent susceptibility to ultraviolet B radiation during amphibian embryogenesis could be explained in the framework of evoecotoxicology, considering ontogenic features as biomarkers of environmental signatures of living forms ancestors during the evolutionary process. The stage-dependent susceptibility to ultraviolet B radiation on Rhinella (Bufo) arenarum embryos for both lethal and teratogenic effects could contribute to a better understanding of the role of the increased ultraviolet B radiation on worldwide amphibian populations decline.

Nickel tissue residue as a biomarker of sub-toxic exposure and susceptibility in amphibian embryos.

Although low level exposure to physicochemical agents is the most common environmental scenario, their effects on living organisms are very controversial. However, there is an increasing need to integrate low level exposures from risk assessment to remediation purposes. This study focus on the possibility to employ Ni tissue residue values as biomarkers of sub-toxic exposure and susceptibility to this metal in a range of almost pristine to sub-toxic concentrations for Rhinella arenarum embryos. For that purpose, three batches of amphibian embryos were pretreated during 10 days with three increasing concentrations of Ni starting in 2, 8 and 20 microg Ni(2+) L(-1) and ending in 16, 64 and 160 microg Ni(2+) L(-1) (in natural fresh waters this value ranges from 2 to 10 microgL(-1); the LC(50)-24h for R. arenarum is 26.2mg Ni(2+) L(-1)). For the experimental conditions, the Ni tissue residue values at 360 h post exposure were 0.5, 2.1 and 3.6 microg Ni g(-1) embryo w/w, respectively, corresponding to BCFs of 31, 33 and 23. The susceptibility to Ni in those experimental embryos was evaluated by means of challenge exposures to three lethal concentrations of this metal (10, 20 and 30 mg Ni(2+) L(-1)), registering survival during the following 10 days of treatment. As a general pattern, the lower, intermediate and higher pretreatments with Ni resulted in enhanced, neutral and adverse effects on embryonic survival, respectively. Thus, sub-toxic exposure to Ni could modify the resistance of the amphibian embryo to this metal and Ni tissue residue values could be considered as biomarkers of both, exposure and susceptibility.