Silver nanoparticles and silver salts : internal concentration and behavioural effects on marine amphipods

Orientadores: Gisela de Aragão Umbuzeiro, Solange CadoreTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A prata vem sendo incorporada nos nanomateriais devido à sua ação antimicrobiana de amplo espectro e, consequentemente, vem sendo descartada no meio ambiente em quantidades preocupantes. Seus efeitos nos organismos marinhos, sobretudo como nanopartícula, ainda não são totalmente conhecidos. Dentre os organismos marinhos, anfípodes são amplamente distribuídos e um importante link ecológico em seus ecossistemas. Os objetivos deste trabalho foram desenvolver e aplicar metodologias para a determinação da concentração interna de prata ¿ presente na hemolinfa ¿ e avaliar as alterações comportamentais em anfípodes marinhos quando expostos a nanopartículas (AgNP) e sais de prata, via água e alimento. Métodos para determinação de prata na hemolinfa foram desenvolvidos com sucesso e aplicados em experimentos de exposição utilizando o anfípode marinho de regiões tropicais, Parhyale hawaiensis. A absorção de Ag foi proporcional ao tempo de exposição e a concentração do metal na água, variando de 0,4 ng mg-1 a 13 ng mg-1. Porém, a concentração de Ag sofreu regulação tempo e concentração-dependente, não sendo observado um aumento significativo de Ag na hemolinfa para uma mesma concentração após 48 horas de exposição nem entre as concentrações de 50 e 100 µg L-1. Nos experimentos via alimento, a taxa de absorção de prata foi 2,8 vezes maior quando os organismos foram expostos à AgNP do que ao AgCl, sugerindo que as nanopartículas foram absorvidas pelo organismo e distribuídas na hemolinfa, possivelmente liberando prata iônica (Ag+) após a sua internalização; ou que as NP ficaram aderidas à parede do intestino, liberando Ag+ ao longo do tempo, mesmo após o período de alimentação. A avaliação do comportamento de natação foi estudada no anfípode marinho de regiões temperadas, Echinogammarus marinus, exposto às mesmas concentrações e pelo mesmo período que P. hawaiensis. Este endpoint comportamental foi relevante para estudos de exposição à metais, uma vez que alterações significativas na velocidade de natação em reposta à luz foram observadas em experimentos via água. O presente trabalho fornece novas ferramentas e informações que contribuem para o entendimento dos efeitos e potenciais riscos da Ag proveniente das AgNPs lançadas no meio ambienteAbstract: Silver has been incorporated in nanomaterials due to its broad-spectrum antibacterial property and, consequently, it has been released into the environment in worrying amounts. Silver effects in marine organisms, especially as nanoparticles, are not totally known yet. Among marine organisms, amphipods are widely distributed and an important ecological link in their ecosystems. The aims of this work were to develop and to apply methodologies to determine silver internal concentration ¿ present in the haemolymph ¿ and to evaluate behavioural alterations in marine amphipods when they are exposed to silver nanoparticles (AgNP) and silver salts via water and via feeding. Methods to determine silver in the haemolymph were successfully developed and applied in exposure experiments using the tropical marine amphipod Parhyale hawaiensis. Silver absorption was proportional to the exposure time and silver concentration in water, ranging from 0.4 ng mg-1 to 13 ng mg-1. However, Ag concentration was regulated in a time/concentration-dependent way, since no significant increase of Ag in the haemolymph was observed for a same concentration after 48 of exposure, neither between the concentrations of 50 and 100 µg L-1. In the feeding exposure, absorption rate of silver was 2.8 times higher when the organisms were exposed to AgNP than AgCl, suggesting that nanoparticles were absorbed by the organisms and distributed in the haemolymph, possibly releasing ionic silver (Ag+) after their internalization; or that the NPs were trapped in the gut, releasing Ag+ over time, even after feeding period. The evaluation of the swimming behaviour was studied in the marine amphipod from temperate zones, Echinogammarus marinus, exposed to the same concentrations and for the same period as P. hawaiensis. This behavioural endpoint was relevant for metal exposure studies, once significant behavioural alterations in swimming velocity in response to light were observed for exposure via water. The present work provides new tools and information that contribute to the understanding of the effects and potential risks of Ag from AgNP released in the environmentDoutoradoRelações Antrópicas, Meio Ambiente e ParasitologiaDoutora em Biologia Animal2013/26301-7, 2016/19635-4552120/2011-1FAPESPCNP

Higher silver bioavailability after nanoparticle dietary exposure in marine amphipods

On release into surface waters, engineered silver nanoparticles (AgNPs) tend to settle to sediments and, consequently, epibenthic fauna will be exposed to them through diet. We established Ag uptake and accumulation profiles over time in the hemolymph of a marine amphipod fed with a formulated feed containing AgNPs or AgCl. Silver bioavailability was higher in organisms exposed to AgNPs, indicating that the nanoparticles pose a higher risk of toxicity compared to similar concentrations of AgCl384806810CAPES - Coordenação de Aperfeiçoamento de Pessoal e Nível SuperiorCNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulo2013/26301‐7sem informação400362/2014‐7; 552120/2011‐

Determinação seletiva de tributilestanho na presença de Sn(IV) em amostras ambientais usando HG-ICP OES e Saccharomyces cerevisiae como material sorvente

A method for selective determination of tributyltin in the presence of Sn(IV) by combining hydride generation inductively coupled plasma optical emission spectrometry (HG-ICP OES) and solid phase extraction (SPE) using baker's yeast is proposed. The procedure is based on selective retention of TBT by the yeast at pH = 6. Detection limit of 1.9 µg L-1 and quantification limit of 6.3 µg L-1 were obtained. TBT and San(IV) were determined in the range of 0-25 µg L-1 and the proposed approach was applied to analyze river water, sea water and biological extracts, with recoveries of 114, 101 and 86%, respectively

Toxicity responses for marine invertebrate species of brazilian occurrence

To assess the risk of chemical contaminants it is desirable to derive toxicity data from representative species of the ecosystem intended to be protected. However, species of occurrence in Brazil are rarely used in toxicity tests, especially for marine and estuarine environments. To address this need, we evaluated the toxicity of different toxicants such as metals and organic compounds to marine invertebrates of Brazilian occurrence, representative from tropical regions and cultivated in laboratory. We used two epibenthic test species of Brazilian occurrence, Parhyale hawaiensis, a deposit feeding amphipod and Nitocra sp, a harpacticoid copepod. Nitocra sp. was more sensitive than other copepods to zinc, and more sensitive than P. hawaiensis to disperse dyes. Sensitivity species distribution revealed that Nitocra sp. and P. hawaiensis were similarly responsive as other marine species to zinc, and Nitocra sp. was among of the most sensitive species. Moreover, our study highlighted that organic compounds are poorly explored in toxicity evaluation with marine organisms; therefore, more studies need to be carried out to assess the toxicity of different substances using marine and estuarine organisms representative of tropical ecosystems

Biodisponibilidade de metais na baía da Ilha Grande, RJ: avaliação pelas técnicas de bivalves transplantados (Nodipecten nodosus) e difusão de filmes finos por gradiente de concentração

Os ecossistemas marinhos próximos ao continente são locais muito sensíveis às mudanças ambientais. Nestas áreas, a biodiversidade é extremamente expressiva, com diversas espécies de importância econômica. A determinação de metais nos ecossistemas marinhos que se interceptam com atividades humanas é importante para avaliar as interferências e modificações que o homem causa nestes ambientes. A determinação de metais em ambientes aquáticos utilizando organismos vem sendo muito empregada, principalmente por meio de bivalves, proporcionando dados importantes sobre a biodisponibilidade destes contaminantes na água. A técnica de difusão por filmes finos em gradientes de concentração (DGT) também tem sido empregada para avaliar a concentração de metais em águas doces e marinhas, determinando a fração lábil destes potencias poluentes. Nesta pesquisa foram realizadas amostragens in situ na baía da Ilha Grande, RJ, utilizando Nodipecten nodosus (“vieira”) transplantados juntamente com dispositivos da técnica DGT para a determinação de Al, Cd, Co, Cu, Mn, Ni, Pb e Zn; visando uma comparação das duas técnicas e a obtenção de dados sobre a labilidade e biodisponibilidade destes metais. As vieiras foram obtidas de uma fazenda de criação próxima ao local de estudo. Os dispositivos DGTs foram do tipo convencional: resina Chelex-100 como ligante, gel difusivo de poliacrilamida-agarose e membrana filtrante de acetato de celulose. As vieiras e os dispositivos DGT ficaram imersos em três pontos de amostragem no interior da baía durante Julho a Setembro/2012 (Campanha de Inverno) e Dezembro/2012 a Fevereiro/2013 (Campanha de Verão). As determinações das concentrações dos metais foram feitas por espectrometria de massas com plasma acoplado indutivamente (ICP-MS) e espectrometria de emissão óptica (ICP OES)...Marine ecosystems near to the continent are very sensitive to local environmental changes, where biodiversity is extremely important. Also in this areas, there are several species of economic importance and used for human consumption. Determination of metals in areas where the marine ecosystem and human activities are connected is important to evaluate interference and modifications that the man causes in these environments. The determination of metals in aquatic organisms has been widely used, mainly by using bivalves, providing important data on the bioavailability of these contaminants in the water. The technique of Diffusive Gradients in Thin Films (DGT) has also been employed to assess the concentration of metals in freshwater and marine water, determining the lability these potential pollutants. Aiming to compare different techniques, this research was carried out in the Ilha Grande bay, RJ, using transplanted Nodipecten nodosus (scallop) and the DGT technique for the determination of Al, Cd, Co, Cu, Mn, Ni, Pb and Zn. The scallops were obtained from a breeding farm near to the study site. The DGTs were assembled with Chelex-100 resin as binding agent, diffusion gel of agarose-polyacrylamide and membrane filter of cellulose acetate. The scallops and DGTs were immersed in three sampling points within the bay during July to September/2012 (Winter campaign) and December/2012 to February/2013 (Summer campaign). The metals were determined by inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectrometry (ICP OES). The DGT technique was successfully used to determine the concentration of most the metals, except Al, Zn and Pb when short deployment time were used. The results obtained using different porosities of diffusive gels showed no significant presence of metal organic forms... (Complete abstract click electronic access below)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Bioacumulação de tributilestanho em siri azul (Callinectes sapidus)

Não disponíve

Behavioural effects on marine amphipods exposed to silver ions and silver nanoparticles

Behavioural responses to contaminants are an important endpoint in ecotoxicology because they link effects at biochemical or cellular levels to impacts on individual fitness. Due to the increasing use of silver in nanomaterials, studies of its effects on the behaviour of aquatic organisms are important to assess the risks of silver nanoparticles (AgNP) released into the environment. The aim of this work was to evaluate the behavioural effects of silver on the marine amphipod Echinogammarus marinus after exposure to AgNO3 via water and AgCl or AgNP via food. Swimming activity of the amphipods was tracked during 6 min alternating dark and light conditions. Animals swam slower and responded less to light at higher concentrations of silver in the water. No differences were found in the behaviour of animals exposed via feeding up to 28 days, hence, longer exposure times may be required for the observation of effects. This is the first work to appraise behaviour effects of silver ions and AgNP on marine amphipods. Although the protocol has been successfully developed for this purpose, specimens appeared to habituate to test conditions during the experiments. Therefore, the need for further understanding of baseline behaviours in these model organisms is discussed. (C) 2019 Elsevier Ltd. All rights reserved25210511058COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2013/26301-7; 2016/19635-4Sem informaçã

Speciation of lead in seawater and river water by using Saccharomyces cerevisiae immobilized in agarose gel as a binding agent in the diffusive gradients in thin films technique

Saccharomyces cerevisiae immobilized in agarose gel is proposed as a binding agent for the diffusive gradients in thin films (DGT) technique for determination of Pb in river water and seawater. DGT samplers were assembled with the proposed binding agent (25-mm disk containing 20 %, m/v, S. cerevisiae and 3.0 %, m/v, agarose) and a diffusive layer of cellulose (3MM Chr chromatography paper of 25-mm diameter). The effects of some DGT parameters (e.g., immersion time, ionic strength, and pH) were evaluated. Elution of Pb from the binding agent was effectively done with 1.75 mol L-1 HNO3. The deployment curve (between 2 and 24 h) was characterized by a significant uptake of Pb (346 ng Pb h(-1)) and good linear regression (R (2) = 0.9757). The experimental results are in excellent agreement with the predicted theoretical curve for mass uptake. Consistent results were found for solutions with ionic strengths of 0.005 mol L-1 or greater and within a pH range of 4.5-8.5. Interferences from Cu (20:1), Mn (20:1), Fe (20:1), Zn (20:1), Ca (250:1), and Mg (250:1) in Pb retention were negligible. Determination of Pb in spiked river water samples (from the Corumbatai and Piracicaba rivers) performed using the proposed device was in agreement with total dissolved Pb, whereas measurements in seawater suggest that of the various species of Pb present in the samples, only cationic Pb species are adsorbed by the agarose-yeast gel disks. The in situ concentration of Pb obtained at two different sites of the Rio Claro stream (Corumbatai basin) were 1.13 +/- 0.01 and 1.34 +/- 0.04 mu g L-1. For 72-h deployments, a detection limit of 0.75 mu g L-1 was calculated. The combination of inductively coupled plasma optical emission spectroscopy and in situ deployments of DGT samplers during the 72-h period makes possible the determination of labile Pb in river water.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Bioaccumulation of Tributyltin by Blue Crabs

O presente estudo avaliou a bioacumulação de tributilestanho (TBT) pelo siri azul (Callinectes sapidus). Os animais foram alimentados com comida contaminada com 30 µg g -1 de TBT, expresso como Sn. Os analitos foram determinados nas brânquias, hepatopâncreas e músculo. Realizou-se uma digestão ácida para determinação da concentração total de Sn, e a técnica de extração em fase sólida foi utilizada para determinação seletiva de TBT. Obteve-se limites de detecção de 44,6 e 4,46 ng g -1 para HG-ICP OES (geração de hidretos (HG) por espectrometria de emissão óptica com plasma indutivamente acoplado) e ICP-MS (ICP-espectrometria de massas), respectivamente. Os resultados para os tecidos dos animais não contaminados foram inferiores a 50 ng g -1 , enquanto os submetidos à alimentação contaminada mostraram elevadas concentrações de Sn (até 6229 ng g -1 ) e TBT (até 3357 ng g -1 ) relacionadas aos dias de exposição. De acordo com os resultados, Sn é acumulado pelo siri azul em elevadas concentrações no hepatopâncreas. Para a maioria dos animais, os resultados sugerem que o Sn é bioacumulado como TBT. This study evaluated the bioaccumulation of tributyltin (TBT) by the blue crab (Callinectes sapidus). Animals were fed with contaminated food containing 30 µg g -1 of TBT expressed as Sn. The analytes were determined in the gills, hepatopancreas and muscle. Acid digestion was used in the total Sn determination, and a solid-phase extraction technique was used for the selective determination of TBT. Limits of detection of 44.6 and 4.46 ng g -1 were found for HG-ICP OES (hydride generation-inductively coupled plasma optical emission spectroscopy) and ICP-MS (ICP-mass spectrometry), respectively. The results for non-contaminated animals were below 50 ng g -1 , while the animals subjected to the contaminated food showed higher tissue concentrations of Sn (until 6229 ng g -1 ) and TBT (until 3357 ng g -1 ) related to the number of exposure days. According to the results, Sn is bioaccumulated by the blue crab in higher concentrations in the hepatopancreas. For most of these animals, the results suggest that Sn is bioaccumulated as TBT. Keywords: tin bioaccumulation, speciation, HG-ICP OES, ICP-MS Introduction Bioaccumulation is the result of absorption (by surface, breathing and diet) and the excretion processes (by breathing, defecation, metabolic biotransformation and dilution) of substances in organisms. For crustaceans and marine bivalves, accumulation can occur from an aquatic environment, ingested food and sediment. The presence of antifouling paints and biocides containing tributyltin (TBT) in coastal ecosystems is of great environmental concern. Although TBT can persist in sediments for years 7 Many studies attempting to evaluate the TBT bioaccumulation process and its effects using amphipods, 8 fish 9-11 and molluscs 12-17 can be found in the literature. Even at low concentration level in sediments, TBT causes imposex in molluscs. 18 For fish, TBT can lead to a bias of sex toward males. 19 However, few studies using crabs exposed to TBT have been reported. Weis and Kim 20 studied U. pugilator crabs exposed to 0.5 mg L -1 TBT and concluded that the presence of the compound in the organism can delay tissue regeneration and ecdysis. In addition, anatomical abnormalities were found during tissue regeneration and basal growing, indicating that the compound has teratogenic effects on tissue development. Botton et al. Rouleau et al. Although there have been several studies evaluating metallic elements bioaccumulation (Cd, Cr, Cu, Hg, Pb and Zn) by the Callinectes sapidus crab, The main objective of this work was to investigate the TBT bioaccumulation process in C. sapidus. Additionally, the objectives focused on evaluating which form (organic or inorganic) the metallic element is stored and in which tissue the bioaccumulation predominates (gill, hepatopancreas or muscle). Thus, the animals were subjected to TBT-contaminated food, and after exposure, the total Sn and TBT concentrations in different tissues were determined. Several variables, such as time of exposure (days) and amount of ingested food, were evaluated. Experimental Materials Instruments In this study, an Agilent 7500ce inductively coupled plasma mass spectrometer (ICP-MS) and a GBC model Integra XL inductively coupled plasma optical emission spectrometer (ICP OES) were used. Operations conditions for the instruments and for hydride generation (HG) are described below: ICP-MS condition: plasma RF power of 1500 W; sample depth from load coil of 7.5 mm; carrier gas flow of 0.8 L min ; spray chamber temperature at 2 °C; sample flow rate of 0.6 µL min -1 ; concentric micromist nebulizer; nickel sample and skimmer cones interface; m/z 118; 120; integration time of 0.1000 s; reaction/collision cell without gas; detector mode in pulse HV. HG-ICP OES condition: forward power of 1200 W; plasma gas flow of 10 L min -1 ; auxiliary gas flow of 0.5 L min ; and carrier gas flow of 0.6 L min -1 . Reagents and solutions Deionized water (18.2 MW cm) was produced in a Milli-Q system (Millipore, Bedford, MA, EUA). The nitric acid used for the ICP-MS analysis was purified by distillation below its boiling point. The other reagents were analytical grade. Solutions of 3% NaBH 4 (m/v) (MP Biomedicals, EUA) in 0.05 mol L -1 NaOH (Synth, São Paulo, Brazil) were used in the hydride generation system and prepared immediately before analysis. All solid phase extraction (SPE) tests for the TBT determination were conducted using commercially obtained dehydrated Saccharomyces cerevisiae yeast (Fermix, São José dos Campos, Brazil). Bioaccumulation of Tributyltin by Blue Crabs J. Braz. Chem. Soc. 1644 The working solutions were prepared from stock solutions of 1000 mg L -1 Sn (IV) made from Sn 0 (Aldrich, Milwaukee, EUA) and 1000 mg L -1 TBT made from TBTCl (tributyltin chloride, Aldrich). Methods Field work Callinectes sapidus, known as blue crab, were caught in the Santos city (São Paulo, Brazil) ocean coast (S 23º 54' 750" WO 45º 25' 460") using traps made of plastic mesh and by manual catching. All of the collected individuals were carried to the laboratory (Centro de Estudos Ambientais, Universidade Estadual Paulista). Exposure Four crabs were kept for 14 days and fed non-contaminated food (controls). Nine other crabs were incubated for 40 days and fed contaminated food. The crabs were individually kept in plastic bottles. The seawater used in the experiments was collected at the same place and in the same period in which the crabs were caught. This water was analyzed and it showed no significant amount of tin. The water was cleaned daily by suction of the eventual residue and renewed every 5 days. The concentration of TBT in contaminated food (30 µg g -1 ) was stated by considering a previously related exposure experiment. 22 Hake (Merluccius hubbsi) fillet was cut into pieces and 5 g samples were then separated into flasks. The food was contaminated by adding 150 µL of a TBT stock solution to the 5 g fish samples, resulting in a final TBT concentration of 30 µg g -1 . After this addition, the mixture was mixed into the fish meat using a vortex agitator for 5 min. The mixture visually seemed very homogeneous slurry. The flasks were then stored at 4 ºC. The contamination of the food with Sn from TBT was prepared one day before use. Each animal was fed 3 times a week with small pieces of fish (with or without contamination). The food was given to the animals until they refused to ingest it, and the eaten fish mass was registered for each animal. The mass of ingested food for each contaminated animal is shown in After exposure, all of the animals were euthanized by chilling at −10 ºC and classified by sex (6 females and 10 males), mass (average of 55.9 g), carapace length (3.9-5.2 cm) and width (8.1-11.4 cm). Using stainless materials, the gills, hepatopancreas and muscle tissues were removed from each individual, weighed and stored in 2 mL Eppendorf tubes at −10 ºC until analysis. All of the glassware and plastics used during exposure, tissue dissection and determination of Sn concentrations were decontaminated with 20% HNO 3 (v/v) and rinsed with deionized water before use. Total Sn determination The collected tissues were digested using a nitro-perchloric digestion as previously reported. 33,34 The digestion process was conducted with blank samples to evaluate any occasional contamination due to the reagents and/or the flasks. To evaluate the accuracy of the method, the total Sn concentrations in the digested samples were determined by hydride generation combined with inductively coupled plasma optical emission spectrometry (HG-ICP OES) and inductively coupled plasma mass spectrometry (ICP-MS). The experimental conditions for total Sn determination by HG-ICP OES were previously evaluated. 35 TBT determination TBT determination (and other tin organic compounds) in sediments and biological tissues has been effectively performed by coupling gas chromatography and inductively coupled plasma mass spectrometric (CG-ICP-MS). 35 The tin extraction from biological material was performed with a procedure described by Silva et al. After the extraction of Sn from the tissues, a SPE was performed to separate the organic Sn compound (TBT) from the inorganic forms potentially present in the samples. The resulting suspensions were vigorously agitated and centrifuged. During this procedure, TBT is retained by the yeast (solid phase), while any inorganic Sn remains in the liquid phase. Finally, the solid phase was treated with nitric acid and analyzed by HG-ICP OES Results and Discussion Non-contaminated blue crabs HG-ICP OES and ICP-MS techniques were used to determine the total amount of Sn in the gills, hepatopancreas and muscle of the non-contaminated crabs. The results were similar for both techniques. The limit of detection (LOD) for Sn using ICP-MS (0.055 µg L -1 ) was 10 times lower than the one obtained using HG-ICP OES (0.55 µg L -1 ) and allowed for analyte determination in the majority of samples The accuracy of the results obtained by both techniques was evaluated through recovery tests (by spiking Sn (IV) before the digestion step). The determinations made by ICP-MS presented better recoveries than those obtained by HG-ICP OES, with average values between 73 to 89% and 64 to 87%, respectively. All tissues showed relatively low Sn concentrations (highest value of 45.18 ng g -1 for the hepatopancreas sample), except for the concentration obtained from muscle sample 2 Contaminated blue crabs Total Sn concentration Figures 1 and 2 show the total Sn concentrations found in each animal in relation to the number of days they were exposed to the contaminated food determined by HG-ICP OES and ICP-MS, respectively. The limits of detection (wet basis) for total Sn As observed in the figures, the results from both techniques were similar and yielded graphs with similar patterns. However, higher Sn concentrations (mainly for two hepatopancreas samples) were observed when ICP-MS determinations were used. Signal suppression due to change on stannane generation by the presence of organic material in these samples is a possible interference in HG-ICP OES determination. Due to the large variations observed in both data sets, the results could only be compared statistically by performing a logarithmic standardization of the data. After standardization, an F-test was applied to determine if it was possible to compare the samples. A paired t-test was performed after the F-test (significance level 95%). The t-test showed that both data sets were not significantly different (significance level 95%). Therefore, despite the observed increase in Sn concentration values, a significant difference between the results obtained by the HG-ICP OES and ICP-MS techniques was not statistically confirmed. By studying the mass of the contaminated food eaten by the crabs, an identical pattern was observed compared with the data obtained for the number of exposure days To evaluate the differences between the Sn concentrations in the tissues, a Friedman statistic test was used. This test allows for comparisons between dependent variables (different tissues from the same animal). The results showed significant statistical differences (p < 0.05), i.e., there were differences between the total Sn concentrations found in the different tissues. When comparing the gills and hepatopancreas separately, there was no significant statistical difference between the tissues. In addition, the same behavior (no significant statistical difference) occurred when the gills and muscle were compared. However, the hepatopancreas and muscle samples showed a statistically significant (p < 0.05) difference (12), suggesting that the hepatopancreas bioaccumulated more Sn than the muscle. This characteristic suggests that these animals preferentially store the metallic element in the hepatopancreas. When the differences between the gills and muscle (9), gills and hepatopancreas (3) and hepatopancreas and muscle (12) were analyzed, the data showed the following sequence for Sn concentrations in contaminated crabs: muscle < gills < hepatopancreas. The factors for the total amount of Sn bioaccumulation were calculated for all of the samples by dividing the final concentration of Sn (obtained for each tissue from each animal) by the Sn concentration of the food given during the experiment (30 µg g -1 ). It was observed that BCF increased as the time of exposure increased for all tissues. The highest values for the bioaccumulation factors were found in the TBT concentration The relationships found between the TBT concentrations and number of exposure days determined by HG-ICP OES and ICP-MS are shown in Figures 4 and 5 indicate that the results for the total Sn and TBT concentrations are consistent. The highest TBT concentrations were found in the samples that presented the highest total Sn concentrations. For almost all samples, the TBT concentrations represent a significant part of the total Sn concentrations (especially considering the results in T h e r e s u l t s f r o m t h i s s t u d y s u g g e s t t h a t Callinectes sapidus is a potentially good bioindicator for the presence of TBT in an environment. Therefore, further studies with this species are of great importance because the deleterious effects of Sn on ecosystems, especially in its organic form (TBT), are well defined and related to many research studies. 2-22 Conclusion HG-ICP OES and ICP-MS techniques can be effectively used to evaluate total Sn concentration in contaminated crabs. However, the limit of detection for HG-ICP OES method does not allow the determination of Sn in non-contaminated crabs. Determinations of Sn in crab tissue digest by ICP-MS presented better recoveries values as compared with HG-ICP OES. Also, the results found by HG-ICP OES in some samples were lower than those found by ICP-MS. However, the two data sets were not significantly different (significance level 95%). The analysis of tissue samples from crabs subjected to contaminated food with TBT showed that these animals accumulate Sn in their tissues (gills, hepatopancreas and muscle). According to BCF, it appears that there is no mechanism for the regulation or excretion of TBT. Among the tissues analyzed in this work, the hepatopancreas showed the highest capacity for TBT bioaccumulation. By comparing the total Sn and TBT concentrations found in the tissues, we inferred that most of the accumulated Sn was present as TBT. In this sense, Sn could be used as a biomarker for TBT exposure in environment