Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans.

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that improve not only the interpretation of the effects of environmental stressors on organisms but also the determination of their possible impact. It is well known that the use of ecotoxicological biomarkers at different levels of organization allows for the prediction of the biological responses of organisms to environmental stressors, which is useful in environmental risk assessment. Nevertheless, it is necessary to consider the optimization of basic procedures, to generate historical data in control groups, and to employ specific bioassays to evaluate responses in organs and tissues in order to elucidate the nature and variation of the effects observed. Therefore, the present work aims to describe several ecotoxicological methodologies employed in all stages of neotropical anurans at different ecological levels and to validate them as useful biomarkers to be used both in wildlife and in laboratory conditions. In this work, these biomarkers were applied at the individual/organismic level (body condition index), histological/physiological level (histopathology, histometric, and pigmentary analyses), biochemical level (oxidative stress enzymes), and genetic level (direct and oxidative damage in DNA by comet assay). Although these methodologies have small variations or modifications depending on the species, these techniques provide effective biomarkers for evaluating the effect of xenobiotics on anurans, which possess certain characteristics that make them useful indicator species of aquatic and terrestrial ecosystems. In conclusion, the battery of biomarkers employed in the present study has proven to be adequate for estimating toxic responses in Neotropical anurans and can be further recommended as bioindicators for identifying the impact of pollutants on the aquatic ecosystems of the region. Finally, it is recommended to achieve the standardization of these important biomarkers for anurans in specific regions as well as to possibly include them in risk assessments and decision-making.

The problem with implementing fish farms in agricultural regions: A trial in a pampean pond highlights potential risks to both human and fish health.

The safety of creating fish farms in agricultural settings was evaluated by growing Piaractus mesopotamicus in a pond, while crops where cultivated in a nearby field under a pesticide application regime typical of the Pampa region. Atrazine, glyphosate and its metabolite, aminomethylphosphonic acid (AMPA), were detected in the water of the pond at concentrations ranging between 92 and 118 µg/L for atrazine, 12 and 221 µg/L for glyphosate and 21 and 117 µg/L for AMPA. Atrazine and malathion were detected in fish muscles at concentrations ranging between 70 and 105 µg/kg for atrazine and 8.6 and 23.7 µg/kg for malathion. Compared to fish raised in a pisciculture, fish from the agricultural pond presented reduced values of pack cell volume, hemoglobin, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration, together with significantly greater cholinesterase activity in both plasma and liver and reduced glutathione-S-transferase activity in the liver. A comet assay also demonstrated that P. mesopotamicus from the agricultural pond presented a significantly greater level of DNA damage in both erythrocytes and gill cells. Overall, the present study demonstrates that pisciculture ponds established in an agricultural setting may receive pesticides applied to nearby cultures and that these pesticides may be taken up by the fish and affect their physiology and health. The accumulation of pesticides residues in fish flesh may also present a risk to human consumers and should be closely controlled.

Toxicity to Rhinella arenarum tadpoles (Anura, Bufonidae) of herbicide mixtures commonly used to treat fallow containing resistant weeds: glyphosate-dicamba and glyphosate-flurochloridone.

Glyphosate (GLY)-dicamba (DIC) and GLY-flurochloridone (FLC) are herbicide mixtures which are widely used for treating fallow containing glyphosate resistant weeds. The aim of this study was to evaluate the acute toxic effects and the prevailing interactions on stage 36 tadpoles of the anuran species Rhinella arenarum when exposed to equitoxic and non-equitoxic combinations of these herbicide combinations. Experiments were realized using the following combinations of commercial formulations: 48% GLY-based Credit® + 57.71% DIC-based Banvel® and 48% GLY-based Credit® + 25% FLC-based Twin Pack Gold®. GLY-DIC and GLY-FLC equitoxic mixtures were assayed mixing each constituent with an equivalent individual toxicity able to induce the same lethality effect. After 96 h of exposure, GLY-DIC and GLY-FLC equitoxic mixtures presented toxic unit 50 values (TU50 96h) of 1.74 (confidence interval: 1.58-1.92) and 1.54 (confidence interval: 1.46-1.62) respectively, indicating the presence of a weak antagonistic interaction as TU values were greater than 1. For their part, most non-equitoxic combinations of GLY-DIC and GLY-FLC tested did not significantly differ from additivity, the only exception being when DIC and FLC were fixed at 0.33 TUs, where a weak antagonism was observed. Overall, results indicate that the toxicity of both GLY-DIC and GLY-FLC mixtures to R. arenarum tadpoles vary from additive to slightly antagonistic, depending on the proportion of constituting herbicide formulations present in the mixture.

An imazethapyr-based herbicide formulation induces genotoxic, biochemical, and individual organizational effects in Leptodactylus latinasus tadpoles (Anura: Leptodactylidae).

Genotoxic, biochemical, and individual organizational effects on Leptodactylus latinasus tadpoles were evaluated after exposure to an imazethapyr (IMZT)-based commercial herbicide formulation, Pivot® H (10.59% IMZT). A determination of the value of the lethal concentration (LC50) was determined as a toxicological endpoint. Alterations in animal behavior and morphological abnormalities as well as cholinesterase (ChE), catalase (CAT), and glutathione S-transferase (GST) activities were employed as individual sublethal endpoints. Micronuclei frequencies (MNs), binucleated cells (BNs), blebbed nuclei (BLs), lobed nuclei (LBs), notched nuclei (NTs), erythroplastids (EPs), and evaluation of DNA strand breaks were employed as genotoxic endpoints. All biomarkers were evaluated after 48 and 96 h of exposure to concentrations of IMZT within 0.07-4.89 mg/L. LC5096h values of 1.01 and 0.29 mg/L IMZT were obtained for Gosner stages 25 and 36, respectively. Irregular swimming, diamond body shape, and decreased frequency of keratodonts were detected at both sampling times. Results showed that IMZT increased GST activity and MN frequency at 48 and 96 h of exposure. Other nuclear abnormalities were also observed in the circulating erythrocytes of tadpoles, i.e., NT and BL values after 48 h, and LN, BL, and EP values after 96 h. Finally, results showed that IMZT within 0.07-0.22 mg/L increased the genetic damage index in tadpoles exposed for both exposure times (48 and 96 h). This study is the first to report the sublethal biochemical effects of IMZT in anurans and is also the first report using L. latinasus tadpoles as a bioindicator for ecotoxicological studies.

Oral acute toxicity of imidacloprid, thiamethoxam and clothianidin in eared doves: A contribution for the risk assessment of neonicotinoids in birds.

Neonicotinoids have recently been demonstrated to cause direct negative impacts on birds from North America and Europe. To further understand the impact of these compounds on bird species and to improve risk assessment capacities, the current study determined the acute toxicities of imidacloprid, clothianidin, and thiamethoxam formulations on South American eared doves (Zenaida auriculata). Insecticides were administered by gavage to adult doves to determine median lethal doses (LD50) according to a standardized sequential procedure. The acute toxicity of formulated imidacloprid (LD50=59mg active ingredient, a.i./kg body weight, b.w.) was much higher than that of the tested formulations of clothianidin (LD50=4248mga.i./kg b.w.) and thiamethoxam (LD50=4366mga.i./kg b.w.). Imidacloprid also differed from the other two neonicotinoids in terms of the onset and intensity of intoxication signs and the times of death and recovery. All three insecticides induced a reduction in food consumption that led to body weight loss. An average weight dove of 127g would obtain a dose equivalent to the LD50 of imidacloprid by consuming 1.7g of treated sorghum seeds. As eared doves offered non-treated sorghum seeds 5h per day consumed on average 6.4±1.8g (mean±S.D.), it is concluded that these doves could feasibly be exposed to lethal doses in the field. This work is the first to describe intoxication signs and report oral neonicotinoid LD50s in a wild South-American bird species.

Environmentally-relevant concentrations of atrazine induce non-monotonic acceleration of developmental rate and increased size at metamorphosis in Rhinella arenarum tadpoles.

Despite of the various studies reporting on the subject, anticipating the impacts of the widely-used herbicide atrazine on anuran tadpoles metamorphosis remains complex as increases or decreases of larval period duration are almost as frequently reported as an absence of effect. The aim of the present study was to examine the effects of environmentally-relevant concentrations of atrazine (0.1, 1, 10, 100, and 1000µg/L) on the timings of metamorphosis and body size at metamorphosis in the common South American toad, Rhinella arenarum (Anura: bufonidae). None of the atrazine concentrations tested significantly altered survival. Low atrazine concentrations in the range of 1-100µg/L were found to accelerate developmental rate in a non-monotonic U-shaped concentration-response relationship. This observed acceleration of the metamorphic process occurred entirely between stages 25 and 39; treated tadpoles proceeding through metamorphosis as control animals beyond this point. Together with proceeding through metamorphosis at a faster rate, tadpoles exposed to atrazine concentrations in the range of 1-100µg/L furthermore transformed into significantly larger metamorphs than controls, the concentration-response curve taking the form of an inverted U in this case. The no observed effect concentration (NOEC) was 0.1µg atrazine/L for both size at metamorphosis and timings of metamorphosis. Tadpoles exposed to 100µg/L 17ß-estradiol presented the exact same alterations of developmental rate and body size as those treated with 1, 10 and 100µg/L of atrazine. Elements of the experimental design that facilitated the detection of alterations of metamorphosis at low concentrations of atrazine are discussed, together with the ecological significance of those findings.

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.

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.

Plasma sampling and freezing procedures influence vitellogenin measurements by enzyme-linked immunoassay in the fathead minnow (Pimephales promelas).

The present study compared three different methods for measuring plasma vitellogenin (VTG) in fathead minnow (FHM; Pimephales promelas): A procedure using liquid chromatography with electrospray ionization combined with tandem mass spectrometry (LC/ESI-MS/MS), and two commercial enzyme-linked immunoassay (ELISA) kits using either anti-carp or anti-FHM antibodies. The influence on plasma VTG measurements of using the protease-inhibitor aprotinin during blood sampling and of submitting the plasma samples to a freeze-thaw cycle before analysis also was evaluated. The addition of aprotinin to the blood during sampling significantly reduced the plasma VTG concentrations measured by ELISA, whereas the VTG values measured after plasma samples were submitted to a freeze-thaw cycle were significantly higher than those measured before freezing. This inflating effect of freezing on VTG measurements made by ELISA could be prevented if plasma samples were frozen diluted in citrate buffer containing 16 mg/ml of polyethylene glycol (PEG). In contrast, measurements of VTG made by LC/ESI-MS/MS were unaffected by freezing and, conceptually, are independent from enzymatic degradation. Although the use of aprotinin and PEG effectively reduced the influence of enzymatic and physical degradation caused by freezing and thawing on VTG measurements made by ELISA, it did not improve agreement between the three analytical techniques evaluated. More information is needed regarding the molecular structure and the existence of possible multiple forms of VTG before this protein can be measured adequately in FHM.

Potentiation of the vitellogenic response to 17alpha-ethinylestradiol by cortisol in the fathead minnow Pimephales promelas.

The effects of elevated plasma cortisol levels on vitellogenin (VTG) induction were examined in the fathead minnow (Pimephales promelas) in an attempt to evaluate the potential influence of stress on this commonly used biomarker of estrogenicity. Two separate experiments were conducted in which fish plasma cortisol was elevated to various levels for 14 d by noninvasive additions of cortisol to the aquaria water. Fathead minnows were exposed to either cortisol alone, 17alpha-ethinylestradiol (EE2) alone, or a combination of the two hormones, and plasma levels of VTG as well as liver expression of VTG mRNA were measured. Both experiments gave similar results, with an exposure to 4 ng/L of EE2 resulting in significantly greater levels of plasma VTG in the presence of, compared to that in absence of, cortisol, whereas exposure to cortisol alone at concentrations between 144 and 800 microg/L had no effect on plasma VTG levels. This potentiation of the EE2-induced vitellogenesis by cortisol was dose-dependent, with plasma VTG reaching 125, 167, and 295% of the values obtained with EE2 alone when 144, 360, and 800 microg/L of cortisol, respectively, were added to the water. Liver mRNA results were consistent with plasma VTG, although they generally were more variable. The present study demonstrates that cortisol does not independently induce vitellogenesis but can potentiate estrogen-induced VTG synthesis in fathead minnow. The implications of these findings for the use of VTG as a biomarker of estrogenicity are discussed.

Quantitation of 17alpha-ethinylestradiol in aquatic samples using liquid-liquid phase extraction, dansyl derivatization, and liquid chromatography/positive electrospray tandem mass spectrometry.

A rapid and sensitive method for the analysis of 17alpha-ethinylestradiol (EE2) in environmental aqueous samples has been developed. Aquatic samples were extracted using liquid-liquid extraction, and organic phase extracts were concentrated and derivatized with dansyl chloride. Analysis was performed using high-performance liquid chromatography with positive electrospray ionization and tandem mass spectrometry (HPLC/ESI-MS/MS). Deuterated 17alpha-ethinylestradiol was used as internal standard and was added to samples before extraction. A limit of quantitation of 1 ng/L was obtained using a 25 mL aqueous sample. The average recovery of EE2 spiked into a 25 mL tapwater sample was 100%. This highly sensitive quantitation method is useful for measuring low levels of EE2 in aqueous environmental samples.

Quantitative measurement of fathead minnow vitellogenin by liquid chromatography combined with tandem mass spectrometry using a signature peptide of vitellogenin.

Vitellogenin (VTG) has been proposed as a sensitive biomarker of exposure to environmental estrogenic contaminants that induce VTG production in oviparous species. Enzyme-linked immunosorbent assay (ELISA) methods are currently widely used to measure the VTG levels. In this paper, a new liquid chromatography combined with tandem mass spectrometry (LC/ESI-MS/MS) method for the quantitative analysis of VTG in the plasma of fathead minnows exposed to 17alpha-ethinylestradiol (EE2) has been developed. This method includes, first, the selection of the signature peptide, which involves sodium dodecyl sulfate-polyarylamide gel electrophoresis separation, in-gel digestion, LC/ESI-MS/MS analysis with an ion trap mass spectrometer, and sequence determination with the TurboSEQUEST MS/MS database application; second, optimization of the selected signature peptide in multireaction monitor (MRM) mode with a triple quadrupole mass spectrometer; and third, trypsin digestion of plasma and VTG quantitation via MRM-mode LC/ESI-MS/MS. A series of plasma samples from fathead minnows following exposure to EE2 was assayed. A good correlation was found when EE2-induced plasma samples from fathead minnows were analyzed with ELISA and the described new method. Although used here with fathead minnow, the new LC/ESI-MS/MS method could be easily applied to the analysis of VTG expressed in any other fish species. Quantitation of VTG by this method was found to be highly specific and linear. The absence of potential artifactual measurements of VTG at low exposure levels could also be critical in future studies that evaluate weakly estrogenic compounds in aquatic species.

Myogenic cell cycle duration in Harpagifer species with sub-Antarctic and Antarctic distributions: evidence for cold compensation.

In teleosts, the proliferation of myogenic progenitor cells is required for muscle growth and nuclear turnover. We measured the cell cycle and S-phase duration of myogenic cells in the fast myotomal muscle of two closely related Harpagifer species by cumulative S-phase labelling with 5-bromo-2'-deoxyuridine (BrdU). Harpagifer antarcticus is a stenothermal species from the Antarctic peninsula (experiencing temperatures of -2 degrees C to +1 degrees C) and Harpagifer bispinis is a eurythermal species from the Beagle Channel, Tierra del Fuego (living at +4 degrees C in winter and up to 11 degrees C in summer). Specific growth rates in the adult stages studied were not significantly different from zero. Myogenic progenitor cells were identified using an antibody against c-met. Seventy-five percent of the c-met(+ve) cells were in a proliferative state in both species. Cell cycle time was 150 h at 5 degrees C and 81.3 h at 10 degrees C in H. bispinis (Q(10)=3.4). Cell cycle duration was 35% shorter in H. antarcticus at 0 degrees C (111 h) than in H. bispinis at 5 degrees C. The predicted cell cycle time for H. bispinis at 0 degrees C (based on the Q(10) relationship) was 277 h, which was more than double that measured for the Antarctic species at this temperature. The results obtained are compatible with an evolutionary adjustment of cell cycle time for function at low temperature in the Antarctic species.

Proliferation of myogenic progenitor cells following feeding in the sub-antarctic notothenioid fish Harpagifer bispinis.

Feeding metabolism and the activation of myogenic progenitor cells were investigated in the fast myotomal muscle of the sub-Antarctic fish Hapagifer bispinis acclimatized to either simulated summer (10 degrees C; 18 h:6 h light:dark) or simulated winter (5 degrees C; 6 h:18 h light:dark) conditions. Ingestion of a single meal equivalent to 10% and 15% of body mass in simulated winter and summer groups, respectively, resulted in an average 2.6-fold and 3.6-fold increase in oxygen consumption, declining to 75% of peak values after 63 h and 46 h. In fasted individuals, the number of myogenic progenitor cells, identified by the expression of c-met, was not significantly different between simulated summer and winter fish, representing 6.6% and 5.8% of total myonuclei, respectively. However, the number of cells expressing myogenin was higher whereas the expression of MyoD was lower in winter than in summer groups. The ingestion of a single meal under winter and summer treatment regimes resulted in a significant increase in the number of cells expressing MyoD (51% and 111%) and PCNA (88% and 140%, respectively). This was followed by an increase in the abundance of c-met (74 and 85%) and myogenin (42 and 97%, respectively) positive cells, indicating the production of new myogenic progenitor cells and the commitment to differentiation of a number of them. These results show that the proliferation of myogenic progenitor cells can be induced by feeding in teleost fishes and that temperature and photoperiod influence the expression of myogenic regulatory factors.