Behavioral and biochemical consequences after chronic exposition to the herbicide atrazine in tadpoles.

Atrazine (ATZ) is the third most sold herbicide in Brazil, occupying the seventh position between most widely used pesticides. Due to its easy outflow, low reactivity and solubility, moderate adsorption to organic matter and clay, and long soil persistence, residual herbicide can be identified after long periods following application, and its usage has been prohibited in diverse countries. Amphibians are important bioindicators to assess impact of pesticide like atrazine, due to having a partial aquatic life cycle. This study had as objective to assess the response of bullfrog (Lithobates catesbeianus) tadpoles when exposed to this herbicide. Animals were exposed for a total of 168h to following concentrations: negative control, 40 µg/L, 200 µg/L, 2000 µg/L, 20000 µg/L of ATZ. Analysis of swimming activity was performed, and biochemical profile was assessed by analysis of blood and plasma glucose levels, urea, creatinine, cholesterol, HDL, triglycerides, glutamic pyruvic transaminase (GPT), alkaline phosphatase (AP), calcium, total proteins, phenol, peroxidase and polyphenol oxidase activity. Results exhibited malnutrition, anemia, likely muscle mass loss, and hepatic damage, indicating that ATZ can lead to an increase in energy to maintain homeostasis for animal survival.

Atrazine contaminated sites and bullfrog tadpoles: evasive trends and biochemical consequences.

Atrazine (ATZ) is one of the most used active principles in agricultural systems. This pesticide has the ability to easily accumulate in terrestrial and aquatic environments, causing impacts with chronic adverse effects. Avoidance tests are tests that seek to assess the concentration from which a given organism escapes, that is, migrates to another habitat. They are being used as a modality of innovative and minimally invasive ecotoxicological tests. Our objective was to evaluate the sensitivity and possible toxic effects of ATZ in bullfrog tadpoles (Lithobates catesbeianus), through avoidance tests and oxidative stress analyses. We performed the behavioral avoidance test lasting 12 h, with observations every 60 min in a linear multi-compartment system with seven compartments. Each compartment corresponded to a concentration: negative control, 1, 2, 20, 200, 2000, 20,000 µg L-1. After the selection of habitat, organisms were forcedly maintained in the chosen concentrations for 48 h and then, metabolic effects were measured assessing the blood plasma amino acid profile and liver protein degradation. We also determined the effective concentrations of ATZ tested at 0 h and 48 h. The results showed that there was an effect of the treatment on the distribution of tadpoles, but not on the hours or on the combined effect (interaction). The biochemical analyses also showed a concentration-dependent relationship which caused significant toxic effects even in a short period of time. In conclusion, these frogs were able to avoid places with high concentrations of ATZ in the first hours of exposure, which suggests that in the natural environment these animals can migrate or avoid areas contaminated by this herbicide; however, depending on the selected concentration, serious biochemical consequences can occur.

Genomic sequencing of a frog virus 3 strain from cultured American bullfrogs (Lithobates catesbeianus) in Brazil.

Frog virus 3 (FV3) was detected in cultured bullfrogs in Southeast Brazil. Phylodynamic analysis revealed recombination events in this strain that were nearly identical to those detected in North American and Brazilian FV3 strains. These data suggest that international trade of live bullfrogs has spread recombinant strains of FV3.

Toxic effects in Aphanomyces brasiliensis and zebrafish embryos caused by oomyceticides.

In fish farming, high losses occur during egg incubation and larviculture due to diseases caused by oomycetes. This study aimed to identify the oomycete species that occurs in zebrafish Danio rerio eggs and to evaluate the oomyceticidal effect of copper sulfate, bronopol and methylene blue on the mycelial growth of this organism, as well as to determine the lethal and sublethal toxicity of these compounds in embryos of D. rerio. The isolates were cultivated in yeast-starch medium to determine the concentration necessary to inhibit mycelial growth by 50% (IC50) and 100% (minimum oomyceticidal concentration) after a 96 h exposure to these compounds. In addition, tests with D. rerio eggs were conducted to determine the lethal concentrations for 50% of the organisms (96h-LC50), and the concentrations that inhibited 17% of the eggs hatching (96h-IC17) after 96 h. The organism responsible for the mortality of D. rerio eggs was classified by classical and molecular methods as Aphanomyces brasiliensis, representing the first report of this pathogen in zebrafish eggs. IC50 values could be determined for both bronopol and copper sulfate, whereas methylene blue had low effectiveness against the oomycete. Copper showed high toxicity to D. rerio eggs at low concentrations, while methylene blue and bronopol toxicity was low and similar to each other. The use of bronopol at a concentration of 4.8 mg l-1 for the treatment of zebrafish eggs allows controlling the pathology without causing deleterious effects to the treated organisms.

Evaluation of the potential teratogenic and toxic effect of the herbicide 2,4-D (DMA® 806) in bullfrog embryos and tadpoles (Lithobates catesbeianus).

The objective of this work was to evaluate the potential teratogenic and toxic effect of the herbicide 2,4-D (DMA® 806) on bullfrog embryos and tadpoles (Lithobates catesbeianus). We used the FETAX (Frog Embryo Teratogenesis Assay Xenopus) assay for embryos, and for tadpoles, we used acute (96 h) and chronic (49 days) toxicity tests and evaluated aspects of healthiness, hematology, and histopathology. The LC50-144h (Median Lethal Concentration), EC50-144h (Median Effective Concentration), MCIG (Minimum Concentration to Inhibit Growth) and TI (Teratogenic Index) for embryos were 792 mg/L, 593 mg/L, 150 mg/L of 2,4-D (DMA) and 1.34, respectively. For tadpoles, the LC50-96h was 700 mg/L of 2,4-D (DMA) and chronic test indicated an inflammatory process and erythrocytosis (with possible polycythemia), with consequent reduction of the spleen. This demonstrates physiological stress probably due to dehydration, which can be proven by the gill tufts widening intercellular space and gill tuft fusions. We also found injuries to the kidneys and skin of the animals even in the lowest concentration tested. Our results indicated that this pesticide is minimally teratogenic and has a low toxicity on L. catesbeianus embryos and tadpoles, but it can inhibit embryo growth in concentrations lower than those tested in this study. We hypothesized that the herbicide 2,4-D (DMA® 806) may be a respiratory allergen for L. catesbeianus tadpoles and recommend precautionary measures for prolonged exposure of aquatic organisms to this pesticide.

Experimental Frog Virus 3 infection using Brazilian strain: amphibians susceptibility / Infecção experimental utilizando estirpe brasileira de Frog Virus 3: suceptibilidade de anfíbios

An alarming number of global warnings concerning amphibian mortality outbreaks have been released in recent years. Emerging diseases stand out as the main potential causes. Ranavirus is a worldwide-spread highly infectious disease capable of affecting even other ectothermic animals such as fish and reptiles. One major issue regarding this pathology is the lack of clinical signs before it leads up to death. Aiming at having a better understanding of anurans susceptibility, this study analyzed bullfrog (Lithobates catesbeianus) survival rate, when challenged with three doses of a Brazilian strain of Frog Virus 3 (FV3). The qPCR analysis indicated a low infectivity rate in these animals both as larvae and as adults. To elucidate the results, the following hypothesis was performed: 1) The amount of inoculum used on the frogs was insufficient to trigger an infection; 2) For the FV3 to produce clinical signs in this species, there is the need for a cofactor; 3) The animals did undergo FV3 infection but recovered in the course of the experiment, and 4) The inoculum utilized might have been low-virulence. Finally, the presence of actual clinical signs of ranavirus is discussed, with the more likely hypothesis.(AU)

Um número alarmante de notificações globais sobre surtos de mortalidade de anfíbios tem sido realizado nos últimos anos. As doenças emergentes destacam-se como as principais causas potenciais. O ranavírus é uma doença altamente infecciosa disseminada em todo o mundo, capaz de afetar até outros animais ectotérmicos como peixes e répteis. Uma questão importante em relação a essa patologia é a falta de sinais clínicos antes de levar à morte. Com o objetivo de compreender melhor a suscetibilidade dos anuros, o presente trabalho analisou a taxa de sobrevivência de rãs-touro (Lithobates catesbeianus), desafiadas com três doses de uma estirpe brasileira do Frog virus 3 (FV3). A análise de qPCR indicou baixa taxa de infectividade nesses animais, tanto como larvas quanto como adultos. Procurando esclarecer os resultados, foram formuladas as seguintes hipóteses: 1) A quantidade de inóculo aplicada nas rãs foi insuficiente para desencadear uma infecção; 2) Para que o FV3 dê sinais clínicos nesta espécie, é necessário um cofator; 3) Os animais sofreram infecção por FV3, mas se recuperaram no decorrer do experimento, e 4) O inóculo utilizado pode ter sido de baixa virulência. Finalmente, foi discutida a presença de sinais clínicos reais de ranavírus e levantada a hipótese mais provável(AU)

Genome analysis of Ranavirus frog virus 3 isolated from American Bullfrog (Lithobates catesbeianus) in South America.

Ranaviruses (family Iridoviridae) cause important diseases in cold-blooded vertebrates. In addition, some occurrences indicate that, in this genus, the same virus can infect animals from different taxonomic groups. A strain isolated from a Ranavirus outbreak (2012) in the state of Sao Paulo, Brazil, had its genome sequenced and presented 99.26% and 36.85% identity with samples of Frog virus 3 (FV3) and Singapore grouper iridovirus (SGIV) ranaviruses, respectively. Eight potential recombination events among the analyzed sample and reference FV3 samples were identified, including a recombination with Bohle iridovirus (BIV) sample from Oceania. The analyzed sample presented several rearrangements compared to FV3 reference samples from North America and European continent. We report for the first time the complete genome of Ranavirus FV3 isolated from South America, these results contribute to a greater knowledge related to evolutionary events of potentially lethal infectious agent for cold-blooded animals.

Acute toxic and genotoxic effects of formalin in Danio rerio (zebrafish).

Formalin is a readily soluble chemical used as a sanitizing agent in the home and hospital. Formaldehyde solutions are routinely used in aquaculture for the prophylaxis and treatment of parasites and fungi, but the adverse effects of their application need to be further investigated. Danio rerio or zebrafish has characteristics favorable to its handling and breeding, and it is highly sensitive to various chemicals, being an ideal experimental model for this type of investigation. Thus, the objective of this study was to verify the toxic and genotoxic effects of formalin and to determine the lethal concentrations of this chemical to support its safe use in disinfection processes. Acute and chronic tests were performed using methods in accordance with international protocols. The genotoxic effect of formalin was evaluated with the micronucleus test using blood samples, which were collected at 96 and 192 h of exposure. The LC50-96h of formalin in D. rerio was 45.73 mg L-1, demonstrating its high resistance compared to other species. Regarding the genotoxic effect, the sublethal concentrations of formalin showed a positive correlation with micronuclei according to the increase in its concentration independent of the time of exposure. The incidence of micronuclei increased with concentration, and the addition of 1 mg L-1 formalin corresponded to an increase of 2.9% in the average number of micronuclei. In other words, formalin at even sublethal concentrations caused genotoxic effects in peripheral blood erythrocytes of D. rerio. Therefore, we recommend further studies and other tests involving this chemical for its use at environmentally safe concentrations.

Acute toxicity and ecotoxicological risk assessment of rice pesticides to Lithobates catesbeianus tadpoles.

The objective of this study was to determine the acute toxicity of some pesticides used in irrigated rice farming to Lithobates catesbeianus tadpoles. The LC 50-96h for commercial formulations containing bentazon, penoxsulam, vegetable oil, permethrin and carbofuran, separately and their mixtures, were determined at the proportions commonly used in the field. The limits of risk concentrations of these products for the studied species were also established. The LC 50-96h for tadpoles was 4,530 mg L(-1) for bentazon; 7.52 mg L(-1) for penoxsulam + 145.66 mg L(-1) of vegetable oil; 81.57 mg L(-1) for vegetable oil; 0.10 mg L(-1) for permethrin; 29.90 mg L(-1) for carbofuran (active ingredients), and 38.79 times the dose used in the field for the mixture of these products. The environmental risk was determined only for permethrin, and care should be taken when using the vegetable oil.

Novel, panzootic and hybrid genotypes of amphibian chytridiomycosis associated with the bullfrog trade.

Global amphibian declines are linked with the presence of specific, highly virulent genotypes of the emerging fungal disease chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) known as the global panzootic lineage (Bd-GPL). The global trade in amphibians for human consumption is suspected to have facilitated emergence of the disease, but evidence to support this is largely lacking. Here, we investigated the role the Lithobates catesbeianus (North American bullfrog) trade in spreading Bd genotypes by comparing strains associated with L. catesbeianus to a global panel using 36 sequenced loci from multiple chromosomal regions. Most bullfrogs were infected with Bd-GPL genotypes, but we also detected novel, highly divergent Bd genotypes (Bd-Brazil) from a live bullfrog in a US market and from native Brazilian anurans in the Atlantic Forest where bullfrogs are widely farmed. Sexual reproduction was also detected for the first time in Bd in the form of a hybrid genotype between the Bd-GPL and Bd-Brazil lineages in the Atlantic Forest. Despite the demonstration that ribosomal RNA types in Bd fail to undergo concerted evolution (over 20 sequence types may be found in a single strain), the Bd-GPL and Bd-Brazil lineages form largely separate clusters of related internal transcribed spacer (ITS) RNA sequences. Using ITS sequences, we then demonstrate the presence of Bd-Brazil in Japan, primarily on invasive L. catesbeianus. The finding that Bd is capable of sexual reproduction between panzootic and endemic genotypes emphasizes the risk of international wildlife trade as a source of additional Bd epizootics owing to hybridization.

Hematological parameters in Nile Tilápia, Oreochromis niloticus exposed to sub-letal concentrations of mercury

Mercury toxicity in tilapia, Oreochromis niloticus, (Linnaeus, 1758) was investigated by the hematological parameters after long-term (14 days) exposure to various Hg concentrations (0.02, 0.002, 0.0002mg/L Hg). Test groups were set up with three replicates for each concentration, plus the control group. Blood samples were collected from six individuals for each concentration at 0, 3, 7, 10 and 14 days of exposure. The hematological parameters analyzed were: total red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), total white blood cell count (WBC) and differential leukocyte counts and total thrombocyte count (Tr). There were no significant differences among the mean hematological values at the different Hg concentrations indicating that Hg at the concentrations studied was not toxic to tilapia.

A toxicidade do mercúrio foi avaliada em tilápia, Oreochromis niloticus (Linnaues, 1758) através da análise dos parâmetros hematológicos após exposição a diferentes concentrações sub-letais, durante um período de 14 dias. O bioensaio foi conduzido no laboratório de toxicologia do Instituto de Pesca, SP. Foram utilizados alevinos (12.44 ± 0.84 cm, e 27.13 ± 4.67 g) e aquários com capacidade para 50 litros e preenchidos com água declorada e mais a quantidade de solução de mercúrio (HgCl2) correspondendo as seguintes concentrações: 0,02; 0,002; 0.0002 mg.L-1 Hg. Foram utilizadas 3 repetições de cada concentração e grupo controle. Amostras de sangue foram coletadas de seis animais de cada concentração nos tempos 0, 3, 7, 10 e 14 dias de exposição. Foram avaliados: a contagem de eritrócitos (RBC), concentração de hemoglobina (Hb), hematócrito (Ht), volume corpuscular médio (VCM), hemoglobina corpuscular média (HCM) e concentração de hemoglobina corpuscular média (CHCM), trombócitos totais (Tr), contagem diferencial e total de leucócitos (Lc). Os resultados demonstram que as concentrações de Hg testadas, não alteraram significativamente os parâmetros hematológicos, permitindo concluir que a quantidade de Hg na água não foram suficientes para afetar o quadro hematológico de Oreochromis niloticus.