Ecotoxicity of veterinary enrofloxacin and ciprofloxacin antibiotics on anuran amphibian larvae.

The ecological risks posed by two ß-diketone antibiotics (DKAs, enrofloxacin, ENR and ciprofloxacin, CPX), characterized by their long persistence in aqueous environments and known deleterious effect on model organisms such as zebrafish were analysed using Rhinella arenarum larvae. Sublethal tests were conducted using environmentally relevant concentrations of both ENR and CPX (1-1000µgL-1) under standard laboratory conditions for 96h. Biological endpoints and biomarkers evaluated were body size, shape, development and growth rates, and antioxidant enzymes (glutathione-S-transferase, GST; Catalase, CAT). Risk assessment was analysed based on ration quotients (RQ). The size and shape measurements of the larvae exposed to concentrations greater than 10µgL-1 of CPX were lower compared to controls (Dunnett post hoc p<0.05) and presented signs of emaciation. Concentrations of 1000µgL-1of CPX induced GST activity, in contrast with inhibited GST and CAT of larvae exposed to ENR. Risk assessments indicated that concentrations greater than or equal to10µgL-1 of CPX and ENR are ecotoxic for development, growth, detoxifying, and oxidative stress enzymes. It is suggested that additional risk assessments may provide evidence of bioaccumulation of CPX and ENR in tissues or organs of amphibian larvae by mesocosm sediment test conditions. Finally, intestinal microbiome studies should be considered to establish the mechanisms of action of both antibiotics.

Toxicity of Bacillus thuringiensis var. israelensis in aqueous suspension on the South American common frog Leptodactylus latrans (Anura: Leptodactylidae) tadpoles.

The effects of commercial formulations of Bacillus thuringiensisvar.israelensis (Bti) on non-target organisms are still a matter of debate; in amphibians, the risks of Bti are little known. To evaluate the toxicity of a commercial liquid (aqueous suspension, AS) formulation of Bti (Introban(®)) on Leptodactylus latrans tadpoles, including median lethal concentration (LC50) and no-and lowest-observed-effect concentrations (NOEC and LOEC, respectively), as well as the possible effects of Bti on oxidative responses, erythrocytes genotoxicity, and histology of the intestines. In the laboratory, tadpoles were exposed to nominal concentrations of 0 (control), 2.5, 5, 10, 20 and 40 mg/L of formulated Bti-AS. Glutathione S-transferase (GST) and catalase (CAT) activities, as well as formation of erythrocyte nuclear abnormalities (ENAs), and histological effect were measured in tadpoles displaying survival rates >85%. L. latrans tadpoles were sensitive to exposure to Bti-AS, reaching 100% mortality after 48 h of exposure at the highest concentration. Bti-AS induced GST and CAT enzymes and genotoxicity (erythrocyte's nuclear abnormalities), and caused intestine's histopathology. Our results demonstrate that toxicity of Bti-AS is dose-dependent for L. latrans tadpoles and that sublethal exposure alters enzymes of oxidative stress, induces genotoxicity, and causes intestine damage. Further research is needed to evaluate the ecotoxicological risk of the massive use of Bti formulations on amphibian populations that commonly used suburban wastewater or urban waterbodies to reproduce and where this biopesticide is frequently applied.

Induction of micronuclei and nuclear abnormalities in tadpoles of the common toad (Rhinella arenarum) treated with the herbicides Liberty® and glufosinate-ammonium.

The assessment of micronucleated erythrocytes (ME) in blood represents a widely used method for the detection of chromosomal damage by chemical agents, such as herbicides that may occur as water contaminants. We investigated the changes in some circulating blood-cell parameters of tadpoles of the common toad (Rhinella arenarum) that were exposed during 48 or 96 h to three sub-lethal concentrations (3.75, 7.5, and 15 mg/L) of a commercial formulation of a glufosinate-ammonium (GLA)-based herbicide (Liberty(®), LY(®)) as well as to the corresponding active ingredient GLA. The frequency of ME and other erythrocyte nuclear abnormalities (ENA, i.e., lobed nuclei, binucleates or segmented nuclei, kidney-shaped nuclei, notched nuclei, and picnotic nuclei) were evaluated and compared with positive (cyclophosphamide, CP, 40 mg/L) and negative (de-chlorinated tap water) controls. The results indicate that the exposure of R. arenarum tadpoles to LY(®) induces a concentration-dependent increase in ME frequency. The ENA frequency at 48 h was also significantly higher than that in the negative control group for all the chemicals assayed (CP, LY(®) and GLA) whereas at 96 h, increases in ENA over the negative control group were found only for CP and GLA (7.5 mg/L). Our study demonstrates that the commercial formulation of a GLA-based herbicide induces micronucleus formation in R. arenarum tadpoles, in contrast to the active ingredient. According to these results, the inert ingredients of the commercial formulation played an important role in the production of genotoxic damage in erythrocytes of amphibian tadpoles.

Effect of exposure to contaminated pond sediments on survival, development, and enzyme and blood biomarkers in veined treefrog (Trachycephalus typhonius) tadpoles.

Sediments are important elements of aquatic ecosystems and in general sediments accumulate diverse toxic substances. Amphibians potentially have a greater risk of exposure to contaminants in sediments, and the test of sediments provides first lines of evidences. Sediment outdoor microcosm experiments were conducted to analyze biological endpoints (survival, development, growth, and morphological and organ malformation), enzyme activity (butyrylcholinesterase, BChE; glutathione-S-transferase, GST; and catalase, CAT) and blood biomarkers in veined treefrog Trachycephalus typhonius tadpoles, a widespread neotropical species. Hatching (stage 23) of T. thyphonius was exposed until they reached metamorphosis (stage 46). Sediment tests were performed and four different treatments were used: three ponds (LTPA, ISP, and SSP) influenced by industrial and agricultural activities and a reference treatment from a forest (RFS). Physical and chemical variables and concentration of nutrients, pesticide residues, and metals were determined. One treatment was metal-rich (LPTA) and two were nutrient-rich (ISP and SSP). Sediment treatments had no significant effect on survival; in contrast they had significant sublethal effects on T. typhonius larval development and growth rates, and affected overall size and shape at stage 38. Principally, in LPTA animals were significantly larger than in RFS, exhibiting swollen bodies, tail muscles and tail fin. In addition, metamorphs from LPTA, ISP, and SSP were smaller and showed signs of emaciation by the end of the experiment. Statistical comparisons showed that the proportions of each type of morphological abnormalities (swollen bodies and diamond shape, gut uncoiling, diverted gut, stiff tails, polydactyly, and visceral and hindlimb hemorrhaging) were significantly greater in metal- and nutrient-rich sediment treatments. Moreover, activities of BChE, GST and CAT, as well as and presence of micronuclei, immature, mitotic, anucleated erythrocytes varied significantly among treatments. Our biological effects-based sediment study highlights the use of different biological endpoints and biomarkers on anuran larvae at sites where pond sediment is risky and sediment management should be considered. Finally, the information of those biological endpoints and biomarkers would be useful as a management tool to decide if there are sufficient exposures of tadpoles to suspected pollutants on sediment.

Cholinesterase activities and behavioral changes in Hypsiboas pulchellus (Anura: Hylidae) tadpoles exposed to glufosinate ammonium herbicide.

In this study, amphibian tadpoles of Hypsiboas pulchellus were exposed to herbicide Liberty®, which contains glufosinate ammonium (GLA), for 48 h to the following concentrations: 0 (control), 3.55, 4.74, 6.32, 8.43, 11.25, 15, 20, 26.6, and 35.5 mg GLA L(-1). Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities, as well as swimming capabilities (swimming speed and mean distance) were measured in tadpoles whose concentrations displayed survival rates > 85 %. Our results reveal that sublethal concentrations of GLA significantly inhibited both AChE and BChE activities in tadpoles with respect to the control, showing a concentration-dependent inhibitory effect. The highest inhibition percentages of AChE (50.86%) and BChE (53.02%) were registered in tadpoles exposed to 15 mg GLA L(-1). At this concentration, a significant increase of the swimming speed and mean distance were found in exposed tadpoles with respect to the control, as well as a negative and significant correlation between swimming speed and BChE activity, thus suggesting that this enzyme inhibition is related to an increase in swimming speed. Therefore, exposure of tadpoles to GLA in the wild at concentrations similar to those tested here may have adverse consequences at population level because neurotransmission and swimming performance are essential for tadpole performance and survival.

Effectiveness evaluation of glyphosate oxidation employing the H(2)O(2)/UVC process: toxicity assays with Vibrio fischeri and Rhinella arenarum tadpoles.

The H(2)O(2)/UVC process was applied to the photodegradation of a commercial formulation of glyphosate in water. Two organisms (Vibrio fischeri bacteria and Rhinella arenarum tadpoles) were used to investigate the toxicity of glyphosate in samples M(1,) M(2), and M(3) following different photodegradation reaction times (120, 240 and 360 min, respectively) that had differing amounts of residual H(2)O(2). Subsamples of M(1), M(2), and M(3) were then used to create samples M(1,E), M(2,E) and M(3,E) in which the H(2)O(2) had been removed. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in tadpoles to determine possible sub-lethal effects. In V. fischeri, M(1,E), which was collected early in the photodegradation process, caused 52% inhibition, while M(3,E), which was collected at the end of the photodegradation process, caused only 17% inhibition. Survival of tadpoles was 100% in samples M(2), M(3), and in M(1,E), M(2,E) and M(3,E). The lowest percentages of enzymatic inhibition were observed in samples without removal of H(2)O(2): 13.96% (AChE) and 16% (BChE) for M(2), and 24.12% (AChE) and 13.83% (BChE) for M(3). These results show the efficiency of the H(2)O(2)/UVC process in reducing the toxicity of water or wastewater polluted by commercial formulations of glyphosate. According to the ecotoxicity assays, the conditions corresponding to M(2) (11 ± 1 mg a.e. L(-1) glyphosate and 11 ± 1 mg L(-1) H(2)O(2)) could be used as a final point for glyphosate treatment with the H(2)O(2)/UV process.

[Hematology and blood cell cytochemistry of Rhinella fernandezae (Amphibia: Anura) from Espinal and Delta-Islands of Paraná River, Argentina]. / Hematología y citoquímica de las células sanguíneas de Rhinella fernandezae (Anura: Bufonidae) en Espinal y Delta-Islas del río Paraná, Argentina.

The description of amphibian hematology is scarce and most of these studies have been done in species from North America, Asia and Europe. With the purpose to obtain basic hematological information of Rhinella fernandezae, 23 blood samples from Santa Fe and Entre Rios natural reserves were studied. Blood of each individual was extracted by cardiac puncture and hemograms were carried out. Morphological and cytochemical description of blood cells were analyzed in slides and were inspected for extra and intra cellular parasites. Five leucocytes types were observed, being lymphocytes the predominant ones followed by basophiles. Heterophils and eosinophils were positive to PAS, Sudan B and peroxidase. The erythrocytes and its precursors were negative for cytochemical reactions. Micronuclei and nuclear alterations frequencies were scarce. No significant differences (p > 0.05) were observed between sexes neither in hemograms nor in blood cells morphology. Microfilarias were the only hemoparasites found with a relative low prevalence and infection intensity. The hematological characteristics studied were similar to those reported for other amphibians, suggesting that R. fernandezae individuals present optimal nutritional and immunological status.

Hematología y citoquímica de las células sanguíneas de Rhinella fernandezae (Anura: Bufonidae) en Espinal y Delta-Islas del río Paraná, Argentina

Hematology and blood cell cytochemistry of Rhinella fernandezae (Amphibia: Anura) from Espinal and Delta-Islands of Paraná River, Argentina. The description of amphibian hematology is scarce and most of these studies have been done in species from North America, Asia and Europe. With the purpose to obtain basic hematological information of Rhinella fernandezae, 23 blood samples from Santa Fe and Entre Ríos natural reserves were studied. Blood of each individual was extracted by cardiac puncture and hemograms were carried out. Morphological and cytochemical description of blood cells were analyzed in slides and were inspected for extra and intra cellular parasites. Five leucocytes types were observed, being lymphocytes the predominant ones followed by basophiles. Heterophils and eosinophils were positive to PAS, Sudan B and peroxidase. The erythrocytes and its precursors were negative for cytochemical reactions. Micronuclei and nuclear alterations frequencies were scarce. No significant differences (p>0.05) were observed between sexes neither in hemograms nor in blood cells morphology. Microfilarias were the only hemoparasites found with a relative low prevalence and infection intensity. The hematological characteristics studied were similar to those reported for other amphibians, suggesting that R. fernandezae individuals present optimal nutritional and immunological status. Rev. Biol. Trop. 59 (1): 17-28. Epub 2011 March 01.

La descripción de la hematología de los anfibios anuros es escasa, habiéndose realizado la mayoría de los trabajos en especies de Norteamérica, Asia y Europa. Con el propósito de obtener datos hematológicos para Rhinella fernandezae, fueron estudiados 23 especímenes provenientes de zonas protegidas de las provincias de Santa Fe y Entre Ríos. Se les extrajo sangre por punción cardíaca y se realizaron hemogramas. En los extendidos sanguíneos, se efectuaron la descripción morfológica y citoquímica de las células sanguíneas y búsqueda de parásitos. Se observaron cinco tipos de leucocitos, donde predominaron los linfocitos pequeños. Heterófilos y eosinófilos resultaron positivos para PAS, Sudan B y peroxidasa; contrariamente, los eritrocitos y sus precursores fueron negativos. Los puntajes de las reacciones citoquímicas fueron variables para basófilos, linfocitos, monocitos y trombocitos. Las frecuencias de micronúcleos y de alteraciones nucleares fueron escasas. No se observaron diferencias significativas (p>0.05) entre sexos en el hemograma ni en la morfología sanguínea. Los únicos hemoparásitos encontrados fueron microfilarias (Nematoda: Filaroidea), cuya prevalencia e intensidad de infección fueron bajas. Las características hematológicas estudiadas fueron semejantes a los valores reportados para otros anfibios, pudiendo inferir que los individuos de R. fernandezae estudiados se encuentran en un buen estado nutricional e inmunológico.

Toxicity of four herbicide formulations with glyphosate on Rhinella arenarum (anura: bufonidae) tadpoles: B-esterases and glutathione S-transferase inhibitors.

In this study, amphibian tadpoles Rhinella arenarum were exposed to different concentrations of Roundup Ultra-Max (ULT), Infosato (INF), Glifoglex, and C-K YUYOS FAV. Tadpoles were exposed to these commercial formulations with glyphosate (CF-GLY) at the following concentrations (acid equivalent [ae]): 0 (control), 1.85, 3.75, 7.5, 15, 30, 60, 120, and 240 mg ae/L for 6-48 h (short-term). Acetylcholinesterase (AChE), butyrylcholinesterase (BChE), carboxylesterase (CbE), and glutathione S-transferase (GST) activities were measured among tadpoles sampled from those treatments that displayed survival rates >85%. Forty-eight-hour LC(50) for R. arenarum tadpoles exposed to CF-GLY in the static tests ranged from ULT = 2.42 to FAV = 77.52 mg ae/L. For all CF-GLY, the LC(50) values stabilized at 24 h of exposure. Tadpoles exposed to all CF-GLY concentrations at 48 h showed decreases in the activities of AChE (control = 17.50 ± 2.23 nmol/min/mg/protein; maximum inhibition INF 30 mg ae/L, 71.52%), BChE (control = 6.31 ± 0.86 nmol/min/mg/protein; maximum inhibition INF 15 mg ae/L, 78.84%), CbE (control = 4.39 ± 0.46 nmol/min/mg/protein; maximum inhibition INF 15 mg ae/L, 81.18%), and GST (control = 4.86 ± 0.49 nmol/min/mg/protein; maximum inhibition INF 1.87 mg ae/L, 86.12%). These results indicate that CF-GLY produce a wide range of toxicities and that all enzymatic parameters tested may be good early indicators of herbicide contamination in R. arenarum tadpoles.

Predator-prey interactions between Synbranchus marmoratus (Teleostei: Synbranchidae) and Hypsiboas pulchellus tadpoles (Amphibia: Hylidae): importance of lateral line in nocturnal predation and effects of fenitrothion exposure.

Environmental contaminants can disrupt interactions between aquatic species by altering community structure. We explored predator-prey interactions between marbled swamp juvenile eels (Synbranchus marmoratus; predator) and anuran tadpoles (Hypsiboas pulchellus; prey) in relation to two aspects: the importance of lateral line in the predator and whether the absence of light modifies predation rates; and the effect of a sub-lethal concentration of fenitrothion on both predator and prey. Eels were tested under two sensory conditions (lateral line intact and lateral line blocked by cobalt chloride) in dark conditions. Predation rates were evaluated using different treatments that combined predator and prey exposed or not to insecticide. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were also measured in muscle samples of eels and tadpoles to explore whether fenitrothion affects predator and prey differentially. Marbled swamp eels were more efficient in feeding on tadpoles during the night than during the day, showing that lateral line makes an important contribution to prey detection and capture. Regarding pesticide effects, short-term (6 h) exposure to an ecologically relevant fenitrothion dose of 2.5 mg L(-1) altered the predator-prey relationship by changing prey behaviour, reducing prey detection and therefore increasing tadpole survival. At this concentration, the outcome of the predator-prey relationship appears biased in favor of the exposed tadpoles, which were released from predation risk, despite their altered behaviour and the higher inhibition percentages of tail BChE (70%) and AChE (51%) than in control individuals. Our study involving these model species and agrochemicals demonstrates that fenitrothion affected the outcome of a predator-prey relationship. Further studies are needed, in these species and other native amphibians, to investigate the nature of the mechanisms responsible for the adverse effects of pesticides on antipredator behaviour and predation efficiency.

Activity levels of B-esterases in the tadpoles of 11 species of frogs in the middle Paraná River floodplain: implication for ecological risk assessment of soybean crops.

Soybean fields provide habitats for many species of amphibians. However, the persistence and health of amphibian populations may be at risk from the increasing use of pesticides and other agricultural chemicals. We examined the activities of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterases (CbEs) in 11 syntopic species of larval anurans. In vitro effects of malaoxon causing 50% BChE inhibition (IC(50)) were also studied. In addition, we calculated a relative risk index (RI) based on the geographic distributions of the anurans, the phenology of soybean cultivation, and basal enzymatic values related to potential pesticide detoxification. Among the 11 species, AChE activity varied from 17.5 ± 1.6 to 68.2 ± 4.7 nmol min(-1) mg(-1) protein (PT). BChE activity also varied significantly, ranging from 3.3 ± 0.4 to 7.5 ± 0.4 nmol min(-1) mg(-1)PT. Both measures of CbE activities varied widely (CbE α-NA: 2.1 ± 0.5-12.4 ± 1.1 nmol min(-1) mg(-1) PT; CbE-4NPV: 21.8 ± 1.8-102.6 ± 7.9 nmol min(-1) mg(-1) PT). We also corroborate that lower BChE activity levels for the tadpoles were associated at minor IC(50) values. The results of this study demonstrate significant variation in enzymatic levels among several tadpole species and intermediate to high RI values for 7 species. Based on these results, it appears that a conversion of native ecosystems to soybean crops may lead to increased ecological risk for anuran amphibians.

Inhibition and recovery of cholinesterases in Odontophrynus americanus tadpoles exposed to fenitrothion.

We determined the levels of brain acetylcholinesterase (AChE) and tail butyrylcholinesterase (BChE) activities in tadpoles of Odontophrynus americanus exposed to a commercial formulation of fenitrothion. The mean brain AChE activities in the controls tadpoles varied from 6.91 to 6.39 micromol min(-1) mg(-1) protein, whereas tail BChE activities ranged among 0.26 to 0.17 micromol min(-1) mg(-1) protein; the two sublethal concentrations of fenitrothion assayed produced AChE and BChE inhibition (p < 0.01). Brain AChE recovered a substantial level of activity with a maximum of 93.2%; after the transference of tadpoles to a free-pesticide solution, whereas tail BChE recovery showed a smaller increase (39%) in the activity at 168 hr after to transference to clear water. According with our results, we suggest that tadpole's tail BChE presents higher sensibility than brain AChE.