O peixe zebra como uma ferramenta para avaliação de ecotoxicidade

Mestrado em Toxicologia e EcotoxicologiaO peixe-zebra (Danio rerio) é um vertebrado modelo grandemente utilizado em investigação científica em todo o mundo. Nas últimas décadas, foram desenvolvidos protocolos e técnicas que permitem o uso deste organismo na avaliação de ecotoxicidade. Com o presente trabalho pretendeu-se aplicar múltiplas técnicas para avaliar a toxicidade de diferentes químicos em Danio rerio a nível letal e subletal (embriotoxicidade, genotoxicidade e biomarcadores) realizando testes com embriões e adultos. Numa primeira fase foram selecionados químicos representativos de diferentes tipos de contaminação ambiental. O dicromato de potássio (metal pesado), o triclosão (bactericida) e as δ-endotoxinas (toxinas Bt) foram escolhidos com base nos seus diferentes modos de ação, uso e potecial risco ecológico. Foram realizados ensaios ecotoxicológicos utilizando o peixe-zebra nos diferentes estádios do seu ciclo de vida. Os protocolos da OECD “Fish, Acute Toxicity Test” e “Fish Embryo Test” foram adoptados para avaliação da toxicidade aguda, alterações comportamentais e embriotoxicidade (no caso dos testes com embriões). Foram ainda incluidos ensaios para determinação da actividade de algumas enzimas (colinesterase (ChE), glutationa-S-transferase (GST) e lactato desidrogenase (LDH)) e da genotoxicidade (teste do micronúcleo, anomalias nucleares e teste do cometa). Os resultados mostraram que os testes com embriões são muito mais informativos que os testes com adultos visto que incluem uma gama de endpoints mais vasta, permitindo obter um “perfil de toxicidade” específico para cada químico testado. Os efeitos a nível do desenvolvimento embrionário (anomalias e atrasos) e os biomarcadores mostraram ser os endpoints testados mais sensíveis. O teste com embriões de zebrafish mostrou assim ser uma óptima ferramenta para avaliação da toxicidade contribuindo para uma melhor compreensão dos efeitos dos tóxicos abordados e uma melhor perspectiva do seu risco ambiental.The zebrafish (Danio rerio) is a model vertebrate extensively used in scientific investigation worldwide. In the last decades, protocols and techniques have been developed in order to evaluate the effects of chemicals at different levels of biological organization of this species and to characterize the lethal and sublethal effects of pollutants. The main aim of this study was to assess the toxicity of different classes of chemicals to Danio rerio at lethal and sublethal levels applaying multiple techniques (embryotoxicity, genotoxicity and biomarkers) with adults and embryos. In the first phase of this study was the selection of chemicals in order to contemplate different type of contaminants. Potassium dichromate (heavy metal), triclosan (biocide) and δ-endotoxins (Bt toxinsbiopesticide) were selected due to their different modes of action, use and potential environmental risk. Ecotoxicological assays were performed using different life-stages of zebrafish. The OECD protocols “Fish, Acute Toxicity Test” and “Fish Embryo Test” were followed. Acute toxicity, behavioral alterations and embryotoxicity were evaluated. Adaptations in OECD protocols were performed in order to enable tissue sampling to be used in enzymatic activity assays (acetylcolinesterase – ChE; glutatione-S-trasferase – GST, lactate desidrogenase – LDH) in genotoxixity assays (micronuclei and COMET) and in citotoxicity assays (nuclear abnormalities). Results obtained showed that Danio rerio early-life stages test is much more informative than test with adult fish as they include a wide range of endpoints allowing the obtaining of a more specific toxicity profile for each chemical tested. Effects on embryonic development (anomalies and delays) and biomarkers seemed to be the most sensitive endpoints. Zebrafish embryo test showed to be an excellent tool for toxicity evaluation contributing for a better understanding of the effects of the selected chemicals and for a more accurate prespective of their environmental risk

Avaliação dos efeitos de químicos usados na aquacultura

Doutoramento em BiologiaAquaculture provides food and income for millions of families worldwide being an activity with a high growth rate and with a strong potential for further expansion. Both producers and consumers are interested in a sustainable model of aquaculture development covering social, economic and environmental aspects. Such model implies to cope with the environmental impacts generated by aquacultures. The main objective of this work was to evaluate the use of chemicals applied in aquaculture farms and their harmful effects to aquatic organisms. Since more than 80% of global production of aquatic organisms is concentrated in Southwest Asia, special attention was given to the aquaculture in this region, particularly Thailand. Three different types of aquaculture scenarios were studied: shrimp farms and tilapia farms in cages and in earth ponds. Surveys and monitoring campaigns were conducted in several farms and the fate, use and application patterns of chemicals were identified. In cage farms the results indicated overuse/misuse of antibiotics, in shrimp farms the major group of chemicals used were disinfectants whereas in earth pond farms 1,7α-methyltestosterone (MT) was the most used. The results from the monitoring campaigns showed that the excessive use of chemicals was correlated to contamination of the surrounding environment namely with the antibiotics oxytetracycline (OTC) and enrofloxacin (ENR) in the cage farms, and with MT in the earth pond farms. In the laboratory, ecotoxicity assays were performed with antibiotics, disinfectants and pesticides using organisms belonging to different trophic levels. The disinfectants benzalkonium chloride (BKC), chlorhexidine gluconate (ChD) and glutaraldehyde (GA) were tested. BKC and ChD demonstrated to be very toxic to primary and secondary consumers. Moreover, an approach based on the species sensitivity distributions (SSD) was used to estimate the hazardous concentrations for 5 and 50 % of the species after BKC and GA exposure highlighting the sensitivity of primary producers to BKC exposure (HC5 = 10.8 μg/l). In the case of GA, different trophic levels showed similar sensitivities and a general HC5 = 300 μg/l was obtained. Additionally, a probabilistic environmental risk assessment was performed indicating a PAF (potentially affected fraction) of species > 5% for adverse effects of BKC in effluents and surface water. Concerning pesticides, both trichlorfon (TCF) and ivermectin (IVM) were studied. TCF showed to be extremely toxic to daphnids (48h- LC50 = 0.29 μg/l) and affected the acetilcolinesterase activity in D. magna and D rerio. IVM showed to be acutely toxic to D. rerio life stages (96h-LC50 = 18.5 μg/l for juveniles) and affected their development and biomarkers. In chronic exposures, IVM led to changes in the behaviour and growth of D. rerio juveniles (21d-LOEC 5% em efluentes e águas superficiais. Os pesticidas estudados foram o trichlorfon (TCF) e a ivermectina (IVM). O TCF foi extremamente tóxico para dafnídeos (48h-LC50 = 0,29 μg/l) e afectou também a actividade de acetilcolinesterase em D. magna e D. rerio. A IVM foi tóxica para adultos, juvenis e embriões de D. rerio (96h- CL50 = 18,5 μg/l para os juvenis) afectando o seu desenvolvimento e actividade de biomarcadores. Em exposições crónicas, IVM causou alterações no comportamento e crescimento em D. rerio juvenis (21d-LOEC <0,25 μg/l). Os antibióticos amoxicilina e OTC apresentam baixa toxidade para os organismos aquáticos, mas ambos induziram enzimas relacionadas estresse oxidativo em adultos e embriões de D. rerio. Em geral, a maioria dos químicos testados apresentaram toxicidade para algum grupo de organismo da cadeia trófica ou tiveram efeito a nível de parâmetros sub-letais, sendo portanto recomendados estudos de exposição crónica para que se possa refinar a avaliação de risco ambiental destes compostos. Os efeitos letais e sub-letais observados nos organismos não-alvo sugerem que pode haver uma perda da biodiversidade nos ecossistemas aquáticos, e assim, comprometer os serviços prestados pelos ecossistemas num futuro próximo. Sendo o sucesso da aquacultura dependente directo destes serviços (por exemplo, água de alta qualidade), há uma necessidade urgente de medidas de controlo/educação, tanto no uso de produtos químicos na aquacultura e monitoramento/mitigação dos impactos negativos destes nos ecossistemas naturais

Wide-scope screening of pharmaceuticals, illicit drugs and their metabolites in the Amazon River

Only a limited number of households in the Amazon are served by sewage collection or treatment facili- ties, suggesting that there might be a significant emission of pharmaceuticals and other wastewater contaminants into freshwater ecosystems. In this work, we performed a wide-scope screening to assess the occurrence of pharmaceuticals, illicit drugs and their metabolites in freshwater ecosystems of the Brazilian Amazon. Our study included 40 samples taken along the Amazon River, in three of its major tributaries, and in small tributaries crossing four important urban areas (Manaus, Santarém, Macapá, Belém). More than 900 compounds were investigated making use of target and suspect screening approaches, based on liquid chromatography coupled to high-resolution mass spectrometry with ion mobility separation. Empirical collision-cross section (CCS) values were used to help and confirm identifications in target screening, while in the suspect screening approach CCS values were predicted using Artificial Neural Networks to increase the confidence of the tentative identification. In this way, 51 compounds and metabolites were identified. The highest prevalence was found in streams crossing the urban areas of Manaus, Macapáand Belém, with some samples containing up to 30 - 40 compounds, while samples taken in Santarém showed a lower number (8 - 11), and the samples taken in the main course of the Amazon River and its tributaries contained between 1 and 7 compounds. Most compounds identified in areas with significant urban impact belonged to the analgesics and antihypertensive categories, followed by stimulants and antibiotics. Compounds such as caffeine, cocaine and its metabolite benzoylecgonine, and cotinine (the metabolite of nicotine), were also detected in areas with relatively low anthropogenic impact and showed the highest total prevalence. This study supports the need to improve the sanitation system of urban areas in the Brazilian Amazon and the development of follow-up studies aimed at quantifying exposure levels and risks for Amazonian freshwater biodiversity

Effects of water and nutrient availability on morphological, physiological, and biochemical traits of one invasive and one native grass of a Neotropical savanna

The cerrado is a Neotropical savanna characterized by a soil and vegetation mosaic where plants endure dystrophic soils and seasonal drought. Dry spells or flooding are the main environmental stress native species face in their growth period. African grasses are common invasive species, jeopardizing the biodiversity by displacing native species and outgrowing them. Invasive species may benefit from human interventions that increase nutrient availability in natural areas and may respond differently than natives to environmental conditions. Therefore, we compared the performance of one native (Schizachyrium microstachyum) and one invasive (Melinis minutiflora) grass in different conditions of water and nutrient availability simulating possible cerrado scenarios. Five-week-old seedlings were submitted to different irrigation treatments (simulating dry spells, normal rainfall, and flooding) and fertilization treatments (high or low nutrient availability) for four weeks, and were analyzed for morphological (leaf area, length of the shoot, number of tillers, seedling dry weight, and root:shoot ratio) and physiological parameters (chlorophyll fluorescence, pigment concentration, nutrient content, and biochemical assays). There was a trend for the invasive species to show better responses to water stress by growing more profusely, showing an even higher effect when the soil was richer in nutrients. The invasive species may outcompete the native species by using nutrients and water more efficiently, showing a weaker oxidative response to drought and fertilization. The native species would perform at a similar pace to the invasive species in conditions of less water and nutrient availability, whereas unnatural fertilization inputs and high-water availability would benefit the invasive species.publishe

Large-scale monitoring and risk assessment of microplastics in the Amazon River

Microplastics (MPs) are one of the most widespread contaminants worldwide, yet their risks for freshwater ecosystems have seldom been investigated. In this study, we performed a large monitoring campaign to assess the presence and risks of MPs in Amazonian freshwater ecosystems. We investigated MP pollution in 40 samples collected along 1500 km in the Brazilian Amazon, including the Amazon River, three major tributaries, and several streams next to the most important urban areas. MPs in the 55–5000 µm size range were characterized (size, shape, color) by microscopy and identified (polymer composition) by infrared spectroscopy. Ecotoxicological risks were assessed using chronic Species Sensitivity Distributions for effects triggered by food dilution and tissue translocation using data alignment methods that correct for polydispersity of environmental MPs and bioaccessibility. This study shows that MPs are ubiquitous contaminants in Amazonian freshwater ecosystems, with measured concentrations (55–5000 µm) ranging between 5 and 152 MPs/m3 in the Amazon River and its main tributaries, and between 23 and 74,550 MPs/m3 in urban streams. The calculated Hazardous Concentration for the 5% of species (HC5) derived from the SSDs for the entire MP range (1–5000 µm) were 1.6 × 107 MPs/m3 (95% CI: 1.2 × 106 – 4.0 × 108) for food dilution, and 1.8 × 107 MPs/m3 (95% CI: 1.5 × 106 – 4.3 × 108) for translocation. Rescaled exposure concentrations (1–5000 µm) in the Amazon River and tributaries ranged between 6.0 × 103 and 1.8 × 105 MPs/m3, and were significantly lower than the calculated HC5 values. Rescaled concentrations in urban streams ranged between 1.7 × 105 and 5.7 × 108 MPs/m3, and exceeded both calculated HC5 values in 20% of the locations. This study shows that ecological impacts by MP contamination are not likely to happen in the Amazon River and its major tributaries. However, risks for freshwater organisms may be expected in near densely populated areas, such as the cities of Manaus or Belem, which have limited wastewater treatment facilities.Large-scale monitoring and risk assessment of microplastics in the Amazon RiverpublishedVersio

Exposure to tricyclic antidepressant nortriptyline affects early-life stages of zebrafish (Danio rerio)

Psychiatric drugs are among the leading medications prescribed for humans, with their presence in aquatic environments raising concerns relating to potentially harmful effects on non-target organisms. Nortriptyline (NTP) is a selective serotonin-norepinephrine reuptake inhibitor antidepressant, widely used in clinics and found in environmental water matrices. In this study, we evaluated the toxic effects of NTP on zebrafish (Danio rerio) embryos and early larval stages. Developmental and mortality analyses were performed on zebrafish exposed to NTP for 168 h at concentrations ranging from 500 to 46,900 µg/L. Locomotor behaviour and acetylcholinesterase (AChE) activity were evaluated by exposing embryos/larvae to lower NTP concentrations (0.006–500 µg/L). The median lethal NTP concentration after 168 h exposure was 2190 µg/L. Although we did not identify significant developmental changes in the treated groups, lack of equilibrium was already visible in surviving larvae exposed to ≥ 500 µg/L NTP. The behavioural analyses showed that NTP was capable of modifying zebrafish larvae swimming behaviour, even at extremely low (0.006 and 0.088 µg/L) environmentally relevant concentrations. We consistently observed a significant reduction in AChE activity in the animals exposed to 500 µg/L NTP. Our results highlight acute toxic effects of NTP on the early-life stages of zebrafish. Most importantly, exposure to environmentally relevant NTP concentrations may affect zebrafish larvae locomotor behaviour, which in turn could reduce the fitness of the species. More studies involving chronic exposure and sensitive endpoints are warranted to better understand the effect of NTP in a more realistic exposure scenario

Effects of triclosan on zebrafish early-life stages and adults

The biocide triclosan (TCS) is commonly used in personal care, acrylic, plastic, and textiles products. TCS has been detected in surface water in several countries, and its ecological impact is largely unknown. In this work, the toxicity of TCS in zebrafish (Danio rerio), embryos and adults was studied. Several lethal and sub-lethal endpoints were analysed in organisms exposed to TCS such as mortality, embryo development and behaviour, hatching, micronuclei and biochemical markers (cholinesterase (ChE), glutathione S-transferase (GST) and lactate dehydrogenase (LDH)). Embryo/larvae assay followed the OECD guideline on Fish Embryo Toxicity Test. Embryos were exposed at nominal concentrations of 0.1, 0.3, 0.5, 0.7 and 0.9 mg/l of TCS for 6 days and were inspected daily with the help of a stereomicroscopy for mortality, developmental parameters (otolith formation, eye and body pigmentation, somite formation, heart beat, tail circulation, detachment of the tail-bud from the yolk sac) and hatching. A similar test was run to obtain larvae for ChE, GST and LDH analysis. The adult test followed the OECD Guideline TG 203 in semi-static conditions. Adult zebrafish of similar length and age were exposed to nominal concentrations of 0.1, 0.2, 0.3, 0.4 and 0.5 mg/l of TCS for 96 h and were inspected daily for mortality and behaviour alterations. A second test was run to obtain organs for biomarkers analysis: Heads, muscles and gills were isolated and snap-frozen in eppendorfs and used for ChE, LDH and GST determinations, respectively. Adult zebrafish testing also comprised a third test for micronucleus analysis in which the nominal concentrations of 0, 0.175 and 0.350 mg/l were used. Peripheral blood was obtained by cardiac puncture and used for the analysis. TCS showed acute toxicity for embryo/larvae (96 h LC(50) = 0.42 mg/l) and delayed hatching. Moreover, embryo toxicity was evident: Delay on the otolith formation and eye and body pigmentation were found, and malformations were also evident, including spine malformations, pericardial oedema and undersize. Biomarkers levels were affected: ChE and LDH activity were increased in larvae exposed to 0.25 mg/l, and GST activity was increased in larvae exposed to 0.25 and 0.35 mg/l. TCS also demonstrated acute toxicity to adult zebrafish (96 h LC(50) = 0.34 mg/l). However, TCS did not change biomarkers levels and did not elicit a micronucleus in adults. Despite the fact that similar 96 h LC(50) values have been found for D. rerio embryos and adults (0.42 and 0.34 mg/l, respectively), the embryo assay was much more informative, showing important effects at several levels, including teratogenic response, hatching delay and alteration of biomarker levels. TCS does not seem to be genotoxic for adult fish or to interfere with biomarkers levels at the concentrations tested. TCS has deleterious effects on zebrafish adults and during early stages, (including embryotoxicity, hatching delay and alterations of biomarkers levels). The range of endpoints used on the embryo test allows an integrated analysis that contributes to a better understanding of the toxicity and mode of action of TCS. Future works should focus on a deeper investigation of TCS modes of action on zebrafish early-life stages. As embryo testing was revealed to be so informative, a refinement of the test could be made, including other endpoints such as different biochemical markers as well as DNA microarrays to assess a gene expression level for the effect of exposure to TCS. In the perspective of risk assessment, these endpoints should be explored in order to assess their usefulness as early warning signs and links should be sought between these short-term tests and effects of long-term exposures as it is observed in more realistic scenarios.FCT - SFRH/BPD/31752/200

Prochloraz effects on biomarkers activity in zebrafish early life stages and adults

Zebrafish early life stages (ELS) assays have been increasingly used to assess the toxicity of chemicals and waste waters, showing a great potential due to the wide variety of endpoints that can be incorporated in the test. Moreover, zebrafish ELS test have been proposed, mainly for ethical reasons, as a surrogate for tests with adults. In this work, the fungicide Prochloraz (PCZ) was used as a chemical model to compare sensitivities of embryos/larvae and adult zebrafish focusing on lethality and biomarkers as endpoints. Effects on embryo development were also assessed. Organization for economic cooperation and development (OECD) guidelines were followed and adapted to include the analyses of the biomarkers cholinesterase, glutathione S-transferase, and lactate dehydrogenase. The acute toxicity of PCZ for zebrafish ELS and adults seemed to be similar with 96h-LC50 values of 8.5 and 4.6 mg/L, respectively. However, biomarkers were only responsive in larvae indicating a higher sensitivity of this life stage at sublethal level. PCZ also proved to be teratogenic: at intermediate concentrations effects on embryo development could be noticed, including spine deformations, edemas, lack of pigmentation, slower heart rate, and complete hatching failure. At the lowest concentrations, the more relevant effects (edemas and abnormal absorption of the yolk sac) were observed after hatching. The measurement of biomarkers in the zebrafish ELS assay seems to a useful tool in the detection of chemical effects showing higher sensitivity than adults. Further research should focus on the link between biomarkers responses in larvae and effects at later life stages of zebrafish.publishe

Lethal and sub-lethal effects of nitrofurantoin on zebrafish early-life stages

Antibiotics are among the most extensively used pharmaceuticals worldwide. They are natural or synthetic drugs with the capacity to kill or inhibit the growth of microorganisms. Several antibiotics have been detected in aquatic environments, but little is known about their effects on non-target organisms, especially fish. The aim of this study was to evaluate the effects of the antibiotic nitrofurantoin (NTF) using zebrafish embryos as model organisms. To assess mortality and development effects, the embryos were exposed to 0, 4, 9, 44, 100, 223 and 500 mg/L of NTF. A sub-lethal range of concentrations (0, 0.001, 0.02, 0.32, 5.62 and 100 mg/L) was used for biomarker analyses, namely cholinesterase, lactate dehydrogenase, glutathione S-transferase and catalase. The results indicated low toxicity of NTF to zebrafish, with a 168 h-LC50 value of 129.2 mg/L. The main effect on development was the loss of equilibrium related to the uninflated swim bladder (168 h-EC50 = 96.72 mg/L). Biomarker activity was induced in concentrations as low as 0.02 mg/L (cholinesterase, lactate dehydrogenase, glutathione S-transferase). Exposure to NTF induced no significant effects on zebrafish larvae behaviour. In summary, short-term exposure of zebrafish embryos to NTF induced developmental alterations only at high concentrations. However, biochemical changes occurred at lower levels of exposure, suggesting long-term effects on fish populations2312CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPFUNDAÇÃO DE APOIO À PESQUISA DO DISTRITO FEDERAL - FAPDFSem informaçãoSem informação2018/03108-0edital da água—05/201

Genotoxic evaluation of different delta-endotoxins from Bacillus thuringiensis on zebrafish adults and development in early life stages

The use of toxins from Bacillus thuringiensis as biopesticides in the biological control of insects and in transgenic plants has increased their availability in the environment. Ecotoxicological evaluations of four delta-endotoxins cry 1Aa, cry 1Ab, cry 1Ac, and cry 2A from B. thuringiensis were carried out on zebrafish Danio redo to explore the adverse effects on their genome and embryos. Cry 1Aa increased the micronucleus (MN) frequency in peripheral erythrocytes of adult D. rerio, while cry I Ab, cry 1Ac and cry 2A did not show genotoxicity, after 96-h of exposure at concentration of 100 mg/L Exposures to binary mixtures (cry 1Aa + cry 1Ac, 50:50 mg/L) and (cry 1Aa + cry 2A, 50:50 mg/L) for 96-h showed also significant increased MN frequency. Other evaluated binary mixtures did not show genotoxicity. Activity of glutathione S-transferase measured in the gills did not seem to be altered after exposure to all tested toxins as well as to the six combinations of binary Mixtures. In the embryo-larval study, all tested 8-endotoxins showed embryo toxicity and developmental delay after exposure to the concentrations of 25,50, 100 and 150 mg/L for 96-h. However, each toxin presented a different pattern of toxic response suggesting that a specific approach should be taken into account to its toxicological evaluations. (c) 2008 Elsevier B.V. All rights reserved.FCT - SFRH/BPD/31752/200