Effects of glyphosate, 2,4-D, chlorantraniliprole, and imidacloprid formulations, separately and in mixtures in Caiman latirostris hatchlings.

The present study demonstrated the potential of glyphosate (GLY), 2,4-dichlorophenoxyacetic acid (2,4-D), imidacloprid (IMI) and chlorantraniliprole (CAP) separately and in mixtures to induce oxidative stress and DNA damage in Caiman latirostris hatchlings. Under controlled condition, an embryonic exposure to these pesticides was done at concentrations recommended for soybean crops. Treatments were: negative control, GLY, 2,4-D, IMI, CAP, mixture 1 (M1): GLY + 2,4-D, M2: IM I + CAP and M3: GLY + 2,4-D + IMI + CAP. At hatching, blood samples were taken for the evaluation of genotoxicity, oxidative damage to lipids and DNA, the enzymatic activity of catalase (CAT) and superoxide dismutase (SOD), and the expression level of their corresponding genes (catalase: cat and superoxide dismutase: sod). It has been shown that IMI, M2 and M3 induced a significant inhibition of CAT activity while no effect was observed on SOD. In turn, lipid peroxidation was significantly higher in individuals exposed to IMI, and to all the mixtures. Besides, genotoxicity and oxidative DNA damage were observed in all exposed groups. The results of mRNA expression showed no difference at transcription levels. In the same way, no alterations in growth parameters were recorded at hatching. Regarding to the mixtures, we observed a potentiating action of IMI on M3 in lipid peroxidation as well as independent action on oxidative DNA damage and genotoxicity parameters. Our results highlight the importance of investigating the effect of pesticides and their mixtures considering the potential consequences to caimans living in natural environments.

How the exposure to environmentally relevant pesticide formulations affects the expression of stress response genes and its relation to oxidative damage and genotoxicity in Caiman latirostris.

This study aimed to analyze the molecular stress responses thought the expression levels of catalase (cat), superoxide dismutase (sod) and heat shock protein 70 (hsp70) genes, and how these relate with cellular stress response considering oxidative damage to lipids, DNA and genotoxicity in blood of Caiman latirostris hatchlings exposed to pesticide formulations under ex situ conditions. Treatments were: negative control (NC-tap water), glyphosate 2% (GLY), cypermethrin 0.12% (CYP), chlorpyrifos 0.8% (CPF), and their ternary mixture (Mx3). The concentrations and schedule of application were those recommended in soybean crops. Soil and water showed pesticides residues in all exposed groups. Results showed a statistically significant increase in the micronucleus frequency and DNA damage, with an important oxidation in all exposed groups. The expression level of cat gene was significantly higher in CYP while the expression of hsp70 was significantly lower in GLY, CYP and Mx3, compared to NC. Pesticides tested showed alterations in expression levels, growth parameters, DNA damage and base oxidation under realistic exposure conditions, and can threaten, in the long term, the health status of wild populations.

Identification and evaluation of antioxidant and reference genes for quantitative real-time PCR in blood of Caiman latirostris.

The quantitative real-time polymerase chain reaction (qPCR) has been one of the most promising approaches to perform rapid and accurate quantification of DNA in various biological systems. The aim of this study was to standardized the qPCR technique for the analysis of important genes involved in the main routes of antioxidant defense against reactive oxygen species (catalase: cat and superoxide dismutase: sod) and evaluate the stability of different reference genes in blood of Caiman latirostris hatchlings. The stability of the reference genes, ß-actin, glyceraldehyde 3-phosphate dehydrogenase (gapdh) and ribosomal protein L8 (rpl8) was determined using the comparative ΔCt, NormFinder, geNorm, BestKeeper and RefFinder. Then, cat and sod genes were normalized with each reference gene and their mRNA abundances were determined through the qPCR. Stability of genes was ranked through the different methods in the following order: ß-actin, rpl8 and gapdh , under normal physiological conditions. The results reveal that cat and sod genes present a similar relative mRNA abundance with ß-actin and rpl8. This is the first report of the analysis of antioxidant mRNA as potential biomarkers of oxidative stress in blood for all crocodilians species. Besides, we determined the stability of different reference genes that can be used for normalization of mRNA abundance patterns in blood of C. latirostris, without the need to sacrifice the animals.

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.

Genotoxicity induced by widely used pesticide binary mixtures on Caiman latirostris (broad-snouted caiman).

In this study, we investigated the effects of three binary mixtures of pesticide formulations commonly used in soybean crop: Cypermethrin Atanor® (25% -CYP), Chlorpyrifos Lorsban 48E® (48% -CPF) and Glyphosate Roundup® Full II (66.2% -GLY) on broad-snouted caiman (Caiman latirostris) hatchlings exposed by voluntary immersion under controlled condition. Genotoxicity was evaluated in peripheral blood erythrocytes through the micronucleus (MN) test and other nuclear abnormalities (NAs) and besides, growth of caiman was analyzed in each experimental group. The results showed that pesticide formulations tested, at concentrations similar to those recommended for application in the field, induced an increase in the frequency of micronucleus (FMN; p = 0.001) and Notched nuclei (NN; p = 0.010) in the mixture CYP + CPF, while an increase in the frequency of buds and NN was observed in the mixture of GLY + CYP (pbuds = 0.016 and pNN = 0.021), compared to the vehicle control (VC). On the contrary, a possible antagonistic action was observed between the components in the mixture GLY + CPF. Growth was not affected in any exposed groups (p > 0.05). There was a clutch effect in the frequency of binucleated erythrocytes (BiN; p = 0.011), total length (TL; p = 0.001) and snout-vent length (SVL; p = 0.031). Biomarkers used in this study are considered important predictive tools for the evaluation of xenobiotics. In this study, we demonstrated genotoxicity of pesticide mixtures under conditions that simulate the real situation of exposure suffered by caiman and other wild species in Argentina.

Effects of cypermethrin (pyrethroid), glyphosate and chlorpyrifos (organophosphorus) on the endocrine and immune system of Salvator merianae (Argentine tegu).

Several geographical areas where Salvator merianae is distributed in Argentina are included in regions with agricultural activity and exposed to pesticide formulations. Some pesticides could affect defense mechanisms being able alter structures of some components of immune and endocrine systems. To assess the potential effects of pesticides in this reptile under seminatural conditions, on the immune system and endocrine responses in S. merianae we analyzed several blood parameters. Total (TWBCC), differential (DWBCC) white blood cells count, heterophils/lymphocytes index (H/L), lobularity index (LI), natural antibodies (NAbs) titres, complement system (CS), and corticosterone concentration were analyzed in animals exposed to a mixture of cypermethrin (25%), glyphosate (66.2%) and chlorpyrifos (48%) formulations. In addition, body size was considered in these analyzes. TWBCC and NAbs revealed lower values in organisms exposed to pesticides respect to a control indicating a possible immunosuppression effect. Besides, the LI showed a greater number of lobes in organism exposed demonstrating symptoms of chronic infection. In addition, we observed a reduced growth in these animals possibly related to a less energy investment in body mass to maintain an active defense against pesticides. Finally, we found high levels of plasma corticosterone in animals exposed to mix formulation that could demonstrate neuroendocrine axis activation. Other parameters like DWBCC, H/L index and activity of CS showed no differences in treated animals respect to control group, which could indicate low sensibility of these parameters to the concentration of pesticides used. Our results provide evidence of the toxic effects of pesticides on different immune system parameters, but also a trade-off among these parameters, corticosterone levels and growth. In this way, we can conclude that the formulated pesticides applied widely and constantly in the areas occupied by S. merianae, would be affecting its immune and endocrine systems and therefore its ability to defend against external agents. This kind of studies is of great interest to know the possible responses of wild species to anthropogenic disturbances such as pesticide contamination.

Total and differential white blood cell counts in Caiman latirostris after in ovo and in vivo exposure to insecticides.

Agricultural activities associated mainly with soybean crops affect the natural environment and wildlife by habitat destruction and the extensive use of agrochemicals. The aim of this study was to evaluate immunotoxic effects of the insecticides cypermethrin (CYP) and endosulfan (END) in Caiman latirostris analyzing total blood cell count (TWBC) and differential white blood cell count (DWBC) after in ovo and in vivo exposure. Eggs (in ovo) and hatchlings (in vivo) from nests harvested in natural habitats were artificially incubated and reared under controlled conditions in the Proyecto Yacaré (Gob.Santa Fe/MUPCN) facilities. Exposure of embryos was performed by topication on the eggshell during the first stage of development. The treatments were distilled water (negative control; NC), ethanol (vehicle control; VC), four groups treated with different concentrations of CYP and four groups with END. In vivo exposure was performed by immersion; treatments were NC, VC, two groups exposed to CYP and two to END. After embryonic exposure to the insecticides, no differences were found in TWBC or DWBC among the neonates exposed to pesticides versus controls. In the in vivo scenario, similar results were obtained for TWBC, but DWBC data showed differences between NC hatchlings and CYP-1 hosts for heterophil, lymphocyte and monocyte levels, and between NC and END-1 hosts for lymphocyte and monocyte levels. Research on the effects of pesticide exposure on this species is of special interest not only to assess the impact on caiman populations, but also to further characterize the species as a potential sentinel of ecosystem health.

Genotoxicity induced by Roundup® (Glyphosate) in tegu lizard (Salvator merianae) embryos.

Environmental contaminants produce multiple adverse consequences at individual, population and ecosystem levels. High volumes of agrochemicals applied to great variety of crops, together with agricultural expansion, generate great concerns due to the impact for the environment and large risk implicated for wildlife. The lack of data on these threats is striking. The tegu lizard (Salvator merianae) is one of the species that live in environments under contaminant effects. Several characteristics allow proposing this species as a potential sentinel organism for the monitoring of pesticides in their habitat. The present study is the first report about genotoxicity in tegu lizard neonates after embryonic exposure to Roundup® (glyphosate 66.2%). The micronucleus test (MN), nuclear abnormalities (NAs) assay and comet assay (CA) were used as biomarkers of genotoxic effects induced in erythrocytes by topical exposure of the eggs to the glyphosate commercial formulation Roundup® (RU), in laboratory controlled conditions. A total of 96 eggs were distributed in six groups exposed to RU (50, 100, 200, 400, 800, 1600µg/egg), one positive control (PC; 200µg cyclophosphamide/egg) and one negative control (NC; distilled water). No teratogenic effects were observed in any of the exposed or control neonates. A significant increase in DNA damage was observed in all concentrations higher than 100µg/egg with respect to NC (p<0.05). However, no statistical differences were found in the frequencies of MN and NAs in any group exposed to RU compared to the NC. No statistically significant differences were found in the size of the lizards at birth or after six months post-exposure (p>0.05). Our results provide new information about the undesirable effects of the glyphosate-based herbicide formulations RU on this lizard species that inhabits areas permanently exposed to several pesticide formulations. We consider of utmost necessity a strict regulation of the agrochemical application conditions in those environments near to places where wild populations of terrestrial and aquatic species live, in order to minimize the adverse effects on ecosystems.

Biomarkers of oxidative damage and antioxidant defense capacity in Caiman latirostris blood.

Several xenobiotics, and among them pesticides, can produce oxidative stress, providing a mechanistic basis for their observed toxicity. Chronic oxidative stress induces deleterious modifications to DNA, lipids and proteins that are used as effective biomarkers to study pollutant-mediated oxidative stress. No previous report existed on the application of oxidative damage and antioxidant defense biomarkers in Caiman latirostris blood, while few studies reported in other crocodilians were done in organs or muscles of dead animals. The aim of this study was to characterize a new set of oxidative stress biomarkers in C. latirostris blood, through the modification of conventional techniques: 1) damage to lipids by thiobarbituric acid reactive substances (TBARS), 2) damage to DNA by comet assay modified with the enzymes FPG and Endo III, and 3) antioxidant defenses: catalase, superoxide dismutase and glutathione; in order to apply them in future biomonitoring studies. We successfully adapted standard procedures for CAT, SOD, GSH and TBARS determination in C. latirostris blood. Calibration curves for FPG and Endo III showed that the three dilutions tested were appropriate to conduct the modified comet assay for the detection of oxidized bases in C. latirostris erythrocytes. One hour of incubation allowed a complete repair of the damage generated. The incorporation of these biomarkers in biomonitoring studies of caiman populations exposed to xenobiotics is highly important considering that this species has recovered from a serious endangered state through the implementation of sustainable use programs in Argentina, and represents nowadays a relevant economic resource for many human communities.

Immunotoxicity of commercial-mixed glyphosate in broad snouted caiman (Caiman latirostris).

The expansion and intensification of agriculture during the past 50 years is unprecedented, and thus environmental problems have been triggered at different scales. These transformations have caused the loss of habitat and biodiversity, and disruption of the structure and functioning of ecosystems. As a result of the expansion of the agricultural frontier in the recent past, many areas of the natural geographic distribution of the local wildlife, among them crocodilians and particularly the broad snouted caiman (Caiman latirostris), are being exposed to contaminants. The present study was designed to evaluate the effect of commercially-mixed glyphosate (RU) on some parameters of the immune system of C. latirostris. Two groups of caimans were exposed for two months to different concentrations of RU recommended for its application in the field, while one group was maintained as an unexposed control. The RU concentration was progressively decreased through the exposure period to simulate glyphosate degradation in water. After exposure, total and differential white blood cell (WBC), and complement system activity (CS) were determined. In addition, the animals were injected with a solution of lipopolysaccharide (LPS) from Escherichia coli to trigger an immune response and evaluate the parameters associated with it. The results showed that an effect of the herbicide on CS was observed, as animals exposed to RU showed a lower CS activity than animals from the negative control (NC) but not in total WBC. In the case of leukocyte population counts, differences were only found for heterophils and lymphocytes.

Spontaneous genetic damage in the tegu lizard (Tupinambis merianae): the effect of age.

Several studies indicate that certain factors such as age, sex or nutritional status among others, may affect the level of DNA damage, both induced and spontaneous, so it is very important to consider them for a more accurate interpretation of the findings. The aim of this study was to analyze the influence of age, sex, and nest of origin on spontaneous genetic damage of Tupinambis merianae determined by the comet assay (CA) and the micronucleus (MN) test, in order to improve reference data for future in vivo studies of xenobiotics exposure in this species. Sixty-five tegu lizards of three different ages: newborns (NB), juveniles (JUV) and adults (AD), both sexes and from different nests of origin were used. Blood samples were collected from the caudal vein of all animals and the MN test and CA were applied on peripheral blood erythrocytes to determine basal frequency of MN (BFMN) and basal damage index (BDI). The comparison between age groups showed statistically significant differences in the BFMN and BDI (p<0.05). NB animals showed significantly higher BDI values in relation to JUV and AD (p<0.016), but no statistically differences were found between the latter two. NB showed lower BFMN respect to other age groups, being statistically significant only when compared to AD (p<0.016). BFMN or BDI showed no statistically significant differences between sexes or nests of origin (p>0.05). A weak negative relationship was found only between BFMN and weight of NB tegu lizard (p=0.014; R(2)=0.245). Basal values of genetic damage obtained with both biomarkers in the tegu lizard evidenced that age is an intrinsic factor that should be taken into account to avoid misunderstanding of the results in future biomonitoring studies.

Detection and characterization of phospholipase A(2) (PLA(2)) in Caiman latirostris and Caiman yacare plasma.

Reptiles have proven to have a versatile and efficient nonspecific immune system adapted to the environments in which they commonly live. Phospholipase A(2) (PLA(2)) is important hydrolytic enzyme involved in the regulation of specific types of messengers, with significant roles in the innate immune response. A number of agents that exert effects on cellular receptors emit a series of signals leading to the increased activity of PLA(2). Phospholipase A(2) has been identified and characterized in temperature, plasma concentration, and kinetic dependence in two species of caiman. The results of these studies suggest that the high PLA(2) activities observed in caiman plasma may be an important component of a well-developed innate immunity. Based on the knowledge of their properties, this powerful immunologic component should be evaluated as a possible application in the veterinary or even human therapeutic industry. Additionally, this is another reason to consider these animals excellent models for the study of immune phylogenetic mechanisms.

Baseline values of micronuclei and comet assay in the lizard Tupinambis merianae (Teiidae, Squamata).

The Micronucleus test (MN) and Comet assay (CA) are currently the most widely used methods that allow the characterization of DNA damage induced by physical and chemical agents in wild species. The continuous expansion of the cultivated areas in Argentina, since the introduction of transgenic crops, mainly soy, in association with the increased use of pesticides, transformed deeply the natural environments where the lizard Tupinambis merianae (tegu lizard) occurs. Despite the fact that reptiles have shown to be excellent bioindicators of environmental contaminants, there is no record of genotoxicity studies in T. merianae. The aim of the present study was to adjust the MN test and CA protocols to be applied in erythrocytes of T. merianae, and determine the baseline values of DNA damage in this species. We used 20 adult lizards (10 males: 10 females) from Estación Zoológica Experimental "Granja La Esmeralda" (Santa Fe, Argentina). Peripheral blood samples were collected from all animals and the MN test and CA applied according to the protocols established for other reptilian species. We test critical parameters of CA protocol (cell density, unwinding and electrophoresis times) using increasing concentrations of H2O2 (10, 25 and 50 µM) as a known genotoxic agent to induce DNA damage. Based on this, we determined the most suitable conditions for the CA in this species: a cell density of 4×10(3) erythrocytes per slide, 10 min of unwinding and 15 min of electrophoresis at 0.90 V/cm approximately. The baseline frequency of micronuclei (BFMN=MN/1000 erythrocytes counted) determined for this species was 0.95±0.27 and the basal damage index (BDI: calculated from 100 comet images classified in arbitrary units)=103.85±0.97. No differences were observed between sexes in the BFMN or BDI (p>0.05), and no relation was found between baseline values and length or weight of the analyzed animals (p>0.05). These results demonstrated the sensitivity of both biomarkers of genotoxicity to be applied in erythrocytes of this species, with baseline values comparable to those reported in other reptilian species. These results allow us to propose the tegu lizard for future in vivo studies to assess the genotoxicity of different agents, including those possibly affecting it in its natural geographic distribution.

Genetic, enzymatic and developmental alterations observed in Caiman latirostris exposed in ovo to pesticide formulations and mixtures in an experiment simulating environmental exposure.

In South America, economic interests in last years have produced a constant increase in transgenic soybean cropping, with the corresponding rise in pesticide formulated products. The aim of this study was to determine the effects of pesticides formulations and mixtures on a South American caiman, Caiman latirostris, after in ovo exposure. We conducted a field-like experiment which simulates the environmental exposure that a caiman nest can receive in neighbouring croplands habitats. Experimental groups were Control group, Treatment 1: sprayed with a glyphosate herbicide formulation, and Treatment 2: sprayed with a pesticide mixture of glyphosate, endosulfan and cypermethrin formulations. Results demonstrated genotoxicity, enzymatic and metabolic alterations, as well as growth delay in caimans exposed in ovo to Treatments 1 and 2, showing a higher toxicity for the mixture. Integral evaluation through biomarkers of different biological meaning is highly informative as early indicators of contamination with pesticides and mixtures in this wildlife species.

Toxicity of waterborne Copper in premetamorphic tadpoles of Lithobates catesbeianus (Shaw, 1802).

Toxicity parameters of Copper to pre-metamorphic larvae of Lithobates catesbeianus were evaluated in laboratory conditions. The acute toxicity (as LC-50 96 h) was 3.96 mg Cu(2+) L(-1) (95% confidence interval: 3.21-4.89); the bioconcentration of the metal after 96 h exposure followed an exponential increase. The potential genotoxicity effect evaluated with Micronucleus Test showed a reduced sensitivity of the animals to the assayed concentrations of the metal, exhibiting only a modest increase in the frequency of erythrocytes micronuclei, meanwhile larvae exposed to cyclophosphamide (positive control) showed significant increases. The Condition Factor was significantly reduced while the Hepatosomatic Index remained unaltered.