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.

Ecotoxicological assessment of complex environmental matrices from the lower Paraná River basin.

Sediments of aquatic ecosystems constitute the fate of most atmospheric and terrestrial pollutants. Since aquatic organisms, such as amphibians, interact with sediments, the presence of pollutants may affect their survival, growth and reproduction. So, the aim of this study was to evaluate, the sediment and water quality of five sites from the lower basin of the Paraná River (Buenos Aires, Argentina) with different anthropic impacts: Morejón stream (S1), de la Cruz stream upstream (S2) and downstream (S3), Arrecifes river (S4), tributary stream of Arrecifes river (S5). Physicochemical parameters were measured in situ (water) and in laboratory (water and sediment samples). Also, a screening of metals and pesticides was performed. Chronic (504 h) lethal toxicity bioassays were performed exposing Rhinella arenarum larvae to sediment and water samples. Oxidative stress (catalase, superoxide dismutase, glutathione S transferase, reduced glutathione and lipid peroxidation) and genotoxicity (micronuclei test) biomarkers were analyzed at acute (96 h) exposure. According to the calculated water quality index, S1 and S3 showed excellent quality, S2 good quality and, S4 and S5 poor quality. Dissolved oxygen was low in all sites (2.26-5.63 mg/L) and S5 had the highest organic matter content. Copper levels exceeded the limit for the protection of aquatic life in S2 and S4; arsenic levels exceeded its limit in S4; and selenium levels exceeded its limit in S4 and S5. Pesticides were mainly detected in water samples. Sediment from S5 showed higher sulfide and organic matter concentrations. At 504 h, no significant mortality was observed in the control group while S5 caused the greatest mortality (80%), followed by S2 (66.67%), S1 (63.33%), S3 (46.67%) and S4 (43.4%). All samples caused oxidative stress and lipid peroxidation, and samples from S4 also caused genotoxicity. The analysis of sediment and water samples was a suitable approach to assess the effects of water bodies on a native amphibian species.

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.

Comprehensive assessment of water quality through different approaches: Physicochemical and ecotoxicological parameters.

Traditionally, water quality was assessed by physicochemical parameters. However, a more comprehensive analysis is needed to study the effects of polluted water bodies on key species over time. So, the aim of this study was to monitor through physicochemical and ecotoxicological indicators the surface water quality of four study sites with different land uses from the lower Paraná river basin (Argentina) during spring and summer of two years: Morejón stream (S1), De la Cruz stream upstream (S2), downstream (S3) and Arrecifes river (S4). Physicochemical parameters were measured in situ and in laboratory, and a Water Quality Index (WQI) was calculated. Chronic toxicity bioassays were performed with surface water samples using Rhinella arenarum embryos and larvae. Also, oxidative stress (catalase, superoxide dismutase, glutathione S-transferase, reduced glutathione and lipid peroxidation), neurotoxicity (butyrylcholinesterase) and genotoxicity (micronuclei frequency) biomarkers were measured at acute exposure, and an Integrated Biomarkers Response (IBR) index was calculated. The water quality varied between excellent and bad in S1, good and bad in S2 and S3, and bad and marginal in S4. S1 presented the greatest variability of pesticides and S4 the highest number of metals exceeding the limits for the local protection of aquatic life. Mainly, S4 caused lethality in R. arenarum larvae, reaching a maximum mortality of 83.3% at 504 h of exposure. The lethal toxicity of S1 and S2 varied between periods. Water samples from all sites altered the oxidative stress, neurotoxicity and genotoxicity biomarkers, and the IBR was negatively correlated with the WQI. The IBR reflected the effects of the degraded water quality on the exposed organisms. So, the importance of evaluating both physicochemical and ecotoxicological parameters to analyze integrally the water quality of polluted areas is highlighted. A degradation of the studied water bodies and its negative impact to the native amphibian R. arenarum were observed.

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.

Integrated analysis of the quality of water bodies from the lower Paraná River basin with different productive uses by physicochemical and biological indicators.

The Paraná River basin is one of the most important in South America and is affected by human activities that take place on its margins. In particular, the De la Cruz stream flows through an industrial pole and the Arrecifes River goes mainly through agricultural fields. The aim of this study was to evaluate the water quality of the De la Cruz stream (S1) and the Arrecifes River (S2) by means of physicochemical parameters, including metals and pesticides concentrations. Since amphibians are good indicators of environmental quality, bioassays with Rhinella arenarum were carried on. For lethal and sublethal parameters, embryos and larvae were exposed to a dilution gradient of water samples and AMPHITOX Solution (AS) as negative control for 504 h. For the determination of oxidative stress biomarkers (Catalase -CAT-, Glutathione S-Transferase -GST-, Reduced Glutathione -GSH-, and lipid peroxidation -TBARS-), embryos and larvae were exposed to undiluted water samples and AS. For the determination of micronuclei, larvae at hind limb bud stage (S.28) were exposed to undiluted water samples, simultaneously with negative and positive controls (AS and cyclophosphamide 40 mg/L, respectively). Dissolved oxygen was low in both sites and the copper levels exceeded the Argentine limit for the protection of aquatic life. In embryos exposure, water sample from S1 caused lethal effects (504h-LC50 = 49 (28-71.6)%), increased TBARS levels, and GST and CAT activities. In larvae exposure, water sample from this site decreased CAT activity, while the water sample from S2 caused important lethal effects (504h-LC50 = 98.72 (60.60-302.52)%), low GSH levels and increased GST activity. Water samples from both sites induced higher micronuclei frequency than the negative control. This study alerts about the degradation of water quality of the studied sites including lethal and sublethal effects in R. arenarum that can jeopardize the native populations of this species.

Assessment of environmental quality of water bodies next to agricultural areas of Buenos Aires province (Argentina) by means of ecotoxicological studies with Rhinella arenarum.

Little is known about the effects of polluted water bodies from Buenos Aires Province on the development of native fauna. Ecotoxicological quality of water bodies from agricultural sites was evaluated by means of standardized laboratory bioassays with embryos and larvae of the native amphibian Rhinella arenarum. The organisms were acutely and chronically exposed to surface water samples from streams of Arrecifes (A), Pergamino (P) and Salto (S) districts that represent the most important agricultural core from the region. Lethal, sublethal and genotoxic effects were assessed. Water sample from (A) caused chronic toxicity (LC50:45.35%) in embryos, followed by (S) and the water sample from (P) was not toxic. In larvae, an inversion of the toxicity pattern was found. Thus, the 504 h-LC50s were 28.12%, 39% and 61% for (S), (P) and (A), respectively. A stage-dependent sensitivity was registered, being larvae more affected than embryos. Significant genotoxic effects, estimated by micronucleus test were observed in the larvae exposed to water samples from all sites. The present study warns about environmental degradation of surface waters next to agricultural areas of Buenos Aires Province. This fact jeopardizes R. arenarum populations in this area.

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.

Combined endosulfan and cypermethrin-induced toxicity to embryo-larval development of Rhinella arenarum.

The combined effects of two widely used pesticides, endosulfan and cypermethrin, on survival of embryo-larval development of the South American toad (Rhinella arenarum) were examined. The toxicity bioassays were performed according to the AMPHITOX test. Embryos and larvae were exposed to mixtures of these pesticides at equitoxic ratios from acute or chronic exposure to evaluate interaction effects. The results were analyzed using both Marking's additive index and combination index (CI)-isobologram methods. Acute (96-h) and intermediate (168-h) toxicity of endosulfan-cypermethrin mixtures remained almost constant for larvae and embryos, but when exposure duration was increased, there was a significant elevation in toxicity, obtaining chronic (240-h) no-observed-effect concentrations (NOEC) values of 0.045 and 0.16 mg/L for embryos and larvae, respectively. These are environmentally relevant concentrations that reflect a realistic risk of this pesticide mixture to this native amphibian species. The toxicity increment with the exposure duration was coincident with the central nervous system development on embryos reaching the larval period, the main target organ of these pesticides. The interactions of the pesticide mixtures at acute and chronic exposure were antagonistic for embryo development (CI > 1), and additive (CI = 1) for larvae, while chronic exposure interactions were synergistic (CI < 1) for both developmental periods. Data indicated that endosulfan-cypermethrin mixtures resulted in different interaction types depending on duration and developmental stage exposed. As a general pattern and considering conditions of overall developmental period and chronic exposure, this pesticide mixture usually applied in Argentine crop fields is synergistic with respect to toxicity for this native amphibian species.

Abatement of toxicity of effluents containing Cr(VI) by heterogeneous photocatalysis. Toxicity assessment by AMPHITOX assay.

Toxicity of a Cr(VI) solution before and after treatment by TiO2 heterogeneous photocatalysis (HP) was performed with AMPHITOX bioassay. Changes in toxicity on Rhinella arenarum larvae for 10-d were monitored after exposure to an untreated Cr(VI) solution and to the same solution after HP treatment. The HP treatment of a 41.60 mg L(-1) Cr(VI) solution reduced to 37.5% the concentration of the metal ion. A 10-fold reduction in toxicity at acute exposure (72 h) and 150-fold reduction in toxicity after 240 h was found. Further, the LOEC value increased from 0.001% for the untreated solution to 0.153% after HP treatment. Moreover, the safe concentration in untreated solution corresponded to 0.0001% sample, and it was 0.01% after the treatment, i.e., 100 times higher. A saving of water of about 100,000 L per L of effluent would be possible through dilution to allow safer concentrations for discharge; the saving would reach the highest value (1,000,000 L per L) at 240 h. Sub-lethal effects were completely absent in larvae exposed to the treated solution. The AMPHITOX test allowed to detect chronic effects at low Cr concentrations, i.e. at environmentally relevant levels.

Differential uptake of endosulfan in the South American toad under sublethal exposure.

Agroecosystems are usually polluted with a wide variety of contaminants with pesticides being very frequently detected. Endosulfan, an organochlorine pesticide, has been shown to cause both lethal and sublethal effects on aquatic organisms such as amphibians and especially on its early developmental stages. In this context, the aim of this study was to evaluate the uptake of environmentally relevant concentrations of endosulfan and its correlation with differential sensitivity in the early development stages of the common South American toad, Rhinella arenarum. Embryos and larvae were exposed to sublethal concentrations of endosulfan for several periods of exposures. According to the developmental stage at which they were exposed, the uptake rate was different. Bioconcentration factors (BCFs) for embryos significantly decreased with exposure time and concentration (p < 0.05) reaching a BCF of ≤1679 for embryos at 96 h of exposure to 0.001 mg endosulfan L(-1). BCFs for larvae significantly increased with exposure time (p < 0.05) obtaining a maximum of 40 at 504 h. In our previous study, we reported that embryos were less sensitive to the effects of endosulfan than larvae, which is in line with the main tendency of embryos to bioconcentrate endosulfan as observed in this study.

Stage-dependent toxicity of bisphenol a on Rhinella arenarum (anura, bufonidae) embryos and larvae.

The acute and chronic toxicity of bisphenol A (BPA) was evaluated on the common South American toad Rhinella arenarum embryos and larvae by means of continuous and pulse exposure treatments. Embryos were treated continuously from early blastula (S.4) up to complete operculum (S.25), during early larval stages and by means of 24 h pulse exposures of BPA in concentrations ranging between 1.25 and 40 mg L(-1) , in order to evaluate the susceptibility to this compound in different developmental stages. For lethal effects, S.25 was the most sensitive and gastrula was the most resistant to BPA. The Teratogenic Index for neurula, the most sensitive embryonic stage for sublethal effects was 4.7. The main morphological alterations during early stages were: delayed or arrested development, reduced body size, persistent yolk plug, microcephaly, axial/tail flexures, edemas, blisters, waving fin, underdeveloped gills, mouth malformations, and cellular dissociation. BPA caused a remarkable narcotic effect from gill circulation stage (S.20) onwards in all the organisms exposed after 3 h of treatment with 10 mg L(-1) BPA. After recovering, the embryos exhibited scarce response to stimuli, erratic or circular swimming, and spasmodic contractions from 5 mg L(-1) onwards. Our results highlight the lethal and sublethal effectsof BPA on R. arenarum embryos and larvae, in the last case both at structural and functional levels.

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.

Sublethal effects of atrazine on embryo-larval development of Rhinella arenarum (Anura: Bufonidae).

Atrazine (ATR), one of the most widely used herbicides in the world, affects not only target organisms but also the biota in general. Here, the teratogenic and neurotoxic effects of ATR on Rhinella arenarum (South American toad) embryos, and larvae were evaluated by means of standardized bioassays during acute and chronic exposures. The herbicide had a significant incidence of malformations, with a Teratogenic Index (TI) of 3.28. The main effects were delayed development, reduced body size, microcephaly, axial flexures, wavy tail and edema. In addition, delayed development, reduced development of forelimbs, and edema were recorded at metamorphosis stages. Scanning electron microscopy allowed observing different degrees of cellular dissociation and persistent cilliar cells in specific regions like the adhesive structure and tail fin. Results obtained by ATR 24 h pulse exposures at six developmental stages pointed out blastula as the most susceptible developmental stage both for immediate and delayed adverse effects. A noteworthy recovery capacity from acute toxic effects was recorded from the neural plate stage onwards. Regarding neurotoxic effects, abnormal, and erratic swimming and spasmodic contractions were recorded. Both the teratogenic and neurotoxic effects reported in this study demonstrate the importance of evaluating sublethal effects in non-target organisms as they could imply reduced fitness of individuals and eventually a population decline. The Hazard Quotients (HQ) for ATR ranged from 0.14 to 10.80, and the fact that some of these values are above USEPA's level of concern indicate that ATR is likely a risk to R. arenarum.

Stage-dependent toxicity of 2,4-dichlorophenoxyacetic on the embryonic development of a South American toad, Rhinella arenarum.

The acute and short term chronic toxicity of both the herbicide butyl ester of 2,4-Dichlorophenoxyacetic acid (2,4-D) and a commercial formulation (CF) were evaluated on Rhinella (= Bufo) arenarum embryos at different developmental stages. Adverse effects were analyzed by means of the isotoxicity curves for lethality, malformations, stage-dependent susceptibility, and ultrastructural features. For all experimental conditions, the CF was more toxic, up to 10 times, than the active ingredient, being the open mouth stage (S.21) the most susceptible to the herbicide. For continuous treatment conditions, the early embryonic development was the most susceptible to 2,4-D and the LC50s for 96 and 168 h were 9.06 and 7.76 mg L(-1) respectively. In addition, both the active ingredient and the CF were highly teratogenic, resulting in reduced body size, delayed development, microcephaly, agenesis of gills, and abnormal cellular proliferation processes as the main adverse effects. According to US EPA, 2,4-D in agricultural scenarios may be up to three times higher than the NOEC values for teratogenic effects reported in this study. Therefore, they might represent a risk for amphibians. This study also points out the relevance of reporting the susceptibility of embryos at different developmental stages to both the active ingredient and the CF of agrochemicals in order to protect nontarget organisms.

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.

Comparative susceptibility to atrazine of three developmental stages of Rhinella arenarum and influence on metamorphosis: non-monotonous acceleration of the time to climax and delayed tail resorption.

Acute and subchronic toxicity of atrazine was evaluated in embryos (stage 4) and in premetamorphosis (stage 25) and prometamorphosis (stage 38-39) larvae of the common South American toad Rhinella arenarum (Anura: bufonidae). The influence of atrazine on the last stages of metamorphosis was also examined by exposing prometamorphosis larvae until completion of metamorphosis. Results obtained revealed that larvae in premetamorphosis are more sensitive than larvae in prometamorphosis and that these are, in turn, more sensitive than embryonic stages. Indeed, concentrations of atrazine as high as 30 mg/L had little effects on embryonic stages, the embryos surviving and developing in a similar manner as controls. LC50s of premetamorphosis larvae equaled 27.16, 7.03 and 2.32 mg/L of atrazine after 4, 14 and 21 days of exposure, respectively, compared to LC50s values of 18.27 and 14.43 mg/L after 14 and 21 days of exposure for larvae in prometamorphosis. In experiments with premetamorphosis larvae, the range of tested concentrations was extended to very low concentrations (down to 0.0001 mg/L) to examine whether recent findings of greater mortality at lower doses than at higher doses were also observed in R. arenarum but no such pattern was found. Exposure of prometamorphosis larvae to concentrations of atrazine of 10 mg/L and above widely prevented completion of metamorphosis and caused important mortality. Alternatively, whereas all animals eventually completed metamorphosis when exposed to concentrations of atrazine between 0.1 and 5 mg/L, the timings of metamorphosis were altered starting from 0.1 mg/L, the lowest concentration tested. Indeed, a significant decrease in the time needed for 50% of the larvae to reach the metamorphic climax (stage 42) was observed within this range of atrazine concentrations, the response presenting a U-shaped non-monotonic dose-response curve. Larvae exposed to these concentrations of atrazine also needed significantly more time for completing tail resorption, this effect being equivalent at all concentrations. Overall, the combination of these two different facets of atrazine influence on metamorphosis resulted in a significant acceleration of metamorphosis at 1 mg/L and a significant increase in the duration of metamorphosis at 5 mg/L, whereas no significant difference was observed with 0.1 mg/L.

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.