Assessment of the occurrence and potential risks of pharmaceuticals and their metabolites in fish and water using liquid chromatography mass spectrometry

A comprehensive method for the analysis of 11 target pharmaceuticals representing multiple therapeutic classes was developed for biological tissues (fish) and water. Water samples were extracted using solid phase extraction (SPE), while fish tissue homogenates were extracted using accelerated solvent extraction (ASE) followed by mixed-mode cation exchange SPE cleanup and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Among the 11 target pharmaceuticals analyzed, trimethoprim, caffeine, sulfamethoxazole, diphenhydramine, diltiazem, carbamazepine, erythromycin and fluoxetine were consistently detected in reclaimed water. On the other hand, caffeine, diphenhydramine and carbamazepine were consistently detected in fish and surface water samples. In order to understand the uptake and depuration of pharmaceuticals as well as bioconcentration factors (BCFs) under the worst-case conditions, mosquito fish were exposed to reclaimed water under static-renewal for 7 days, followed by a 14-day depuration phase in clean water. Characterization of the exposure media revealed the presence of 26 pharmaceuticals while 5 pharmaceuticals including caffeine, diphenhydramine, diltiazem, carbamazepine, and ibuprofen were present in the organisms as early as 5 h from the start of the exposure. Liquid chromatography ultra-high resolution Orbitrap mass spectrometry was explored as a tool to identify and quantify phase II pharmaceutical metabolites in reclaimed water. The resulting data confirmed the presence of acetyl-sulfamethoxazole and sulfamethoxazole glucuronide in reclaimed water. To my knowledge, this is the first known report of sulfamethoxazole glucuronide surviving intact through wastewater treatment plants and occurring in environmental water samples. Finally, five bioaccumulative pharmaceuticals including caffeine, carbamazepine, diltiazem, diphenhydramine and ibuprofen detected in reclaimed water were investigated regarding the acute and chronic risks to aquatic organisms. The results indicated a low potential risk of carbamazepine even under the worst case exposure scenario. Given the dilution factors that affect environmental releases, the risk of exposure to carbamazepine will be even more reduced.

Efeitos do cobre na macrófita potamogeton pectinatus L. e sua possível utilização o monitoramento ou remediação de ambientes aquáticos contaminados por este metal

O cobre é um metal essencial para as plantas, porém considerado tóxico quando em elevadas concentrações na água. No caso de macrófitas aquáticas já foi demonstrado que este metal inibe o processo fotossintético e provoca alterações pigmentares. Neste contexto, expusemos (96h) a macrófita aquática Potamogeton pectinatus (L.) à diferentes concentrações de cobre 1, 10, 100 e 1000 µM para avaliar o potencial bioacumulador da planta, e às concentrações de 1, 10 e 100 µM de Cu para verificar os possíveis efeitos do metal na taxa fotossintética (24 e 96h) em diferentes intensidades luminosas (17, 100, 300 e 500 µmol/m2 /s), no teor pigmentar (96h) e no crescimento das macrófitas (30 dias). Para os experimentos de bioacumulação mantivemos um grupo de plantas controle (sem adição de cobre no meio), enquanto que para os outros testes mantivemos um grupo controle e um grupo em solução nutritiva de Hoagland 100%, que contem cobre e outros micronutrientes em concentrações ideais para sobrevivência e crescimento de P. pectinatus. Nossos resultados mostram que a macrófita P. pectinatus é capaz de acumular altas concentrações de cobre, sendo que este acúmulo aumenta com a elevação dos níveis do metal na água. Apesar de basicamente não haver diferença estatística entre a concentração do metal nos diferentes órgãos da planta, as raízes mostraram-se capazes de acumular mais cobre que as folhas e caule com base no fator de bioconcentração. Com relação aos teores de clorofila “a”, “b” e carotenoides, estes foram menores nas folhas das plantas controle em comparação com as plantas em solução de Hogland, mas esta diferença só foi significativa nas plantas expostas ao cobre, que apresentaram menor concentração dos teores pigmentares já à 1 µM de Cu. Quanto à fotossíntese, em 24h de exposição, novamente observamos um efeito negativo da ausência e presença de cobre nas concentrações de 1, 10 e 100 µM, bem como, um efeito da luminosidade, de forma que as plantas em solução de Hoagland apresentaram maior taxa fotossintética quando em 100 µmol/m2 /s. Em virtude de um aumento na respiração em 96h, a fotossíntese, quando ocorreu, foi menor que em 24h e não diferiu entre os grupos e luminosidade. Em relação ao crescimento, as plantas perderam biomassa, mas mantiveram seus comprimentos e apenas aquelas em solução de Hoagland aumentaram seu número de folhas. Ainda, verificou-se clorose e necrose nas plantas controle e expostas ao cobre. Diante do exposto, concluímos que a macrófita P. pectinatus acumula altas concentrações de cobre, principalmente na raiz, sendo capaz e refletir as concentrações do metal no meio. Esta condição, sugere seu uso no biomonitoramento e fitorremediação de locais contaminados por cobre. Por outro lado, elas mostram-se sensíveis ao metal pela redução no teor pigmentar e fotossíntese, sugerindo estes como mecanismos toxicidade do cobre.

The possible identification and introduction of metallothionein in Persian Sturgeon (Acipenser persicus)

Due to anthropogenic activities, toxic metals still represent a threat for various marine organisms. Metallothionein (MT) and cadmium concentration in gills, liver, and kidney tissues and cadmium partitioning in soluble (cytosol) and insoluble fractions of mentioned tissues of Persian sturgeon (Acipenser persicus) were determined following exposure to sub-lethal levels of waterborne cadmium (Cd) (50, 400 and 1000 μg L-1) after 1, 2, 4 and 14 days. The increases of MT from background levels in comparison to controls were 4.6-, 3-, and 2.8-fold for kidney, liver, and gills, respectively after 14 days. The matallothionein concentration in liver was in the range of 56.89-168.44 μgL-1 and for kidney and gills, 39.78-189.30 and 28.15-91.20 μgL-1, respectively. The results showed that MT level change in the kidney is time and concentration dependent. Also, cortisol measurement revealed elevation at the day 1 of exposure and that followed by MT increase in the liver. Cd concentrations in the cytosol of experimental tissues were measured and the results indicated that Cd levels in the cytosol of liver, kidney, and gills increased 240.71-, 32.05-, and 40.16-fold, respectively 14 days after exposure to 1000 μgL-1 Cd. The accumulation of Cd in cytosol of tissues is in the order of liver > gills > kidney. Spearman correlation coefficients showed the MT content in kidney is correlated with Cd concentration, the value of which is more than in liver and gills. Thus, kidney can be considered as a tissue indicator in Acipenser persicus for waterborne Cd contamination. Also, tissue metal accumulations (gills, liver, kidney and muscle) in Persian sturgeon (Acipenser persicus) were compared following exposure to sublethal levels of waterborne Cd (50, 400 and 1000 μg L-1) after periods of 1, 2, 4 and 14 days. Meanwhile, the trends of Cd concentration increase in different tissues during the exposure periods and concentrations were modelled as equations. The obtained results indicate that at the end of 4 and 14 days of exposure, total tissue cadmium concentration followed the pattern: liver> gill> kidney> muscle. Calculation of bioconcentration factor (BCF) after 14 days exposure showed that at low and high concentrations, highest BCFs were found in kidney and liver, respectively. According to the results, the accumulation capacity of muscle was the lowest at all exposure concentrations. The hematological parameters including osmolarity, total protein, cortisol and glucose of plasma were measured, too. Total protein of plasma was in the range of 416.90-1068.10 mg dl-1 plasma.Total protein decreased not significantly (P≥0.05) after exposure to Cd. Cortisol increased after 1 day exposure that followed by significant (P≤0.05) elevation of glucose. The range of cortisol was very vast and it was determined between 0.03 to 16.21 ng mL-1. The content of plasma osmolarity was in the range of 282.33-294.20 mOsmol L-1.Osmolarity of treated fish plasma showed no significant decrease (P≥0.05). Total protein in gills, liver, and kidney showed that at high concentrations of metal, protein content decreased significantly (P≤0.05) in the liver after 4 and 14 days exposure. Thus, total protein of liver and glucose of plasma can be used as general biomarkers of exposure to Cd. Also, the metallothionein and cadmium were measured in gills, kidney and liver of 8 wild Persian sturgeon caught in coast of Guilan Province. According to the results, the concentration of metallothionein was in the range of 45.87-154.66 microgram per liter with the maximum and minimum concentrations in liver and gills, respectively. The trend of cadmium concentration in cytosol of tissues was: liver> kidney> gills. The results of Spearman correlation test showed that there was a significant positive correlation between metallothionein and cadmium in cytosol of liver (r2= 0.850, p≤ 0.01). In the kidney, the correlation between cadmium and metallothionein was significantly positive (r2= 0.731, p≥ 0.05). But there was not such significant correlation in the gills (p≥ 0.05).

L'étain et les organoétains en milieu marin. Biogéochimie et écotoxicologie.

This report synthesizes the knowledge on the biogeochemical cycle, bioconcentration processes and ecotoxicity of tin and organotins in the marine environment. Concentrations levels, biomethylation mechanisms and exchanges between sediments and water are assessed. Emphasis is given on tributyltin sublethal effects, toxicity mechanisms and no effect levels for the most sensitive organisms. Regulations on the use of organotins as antifoulants in paints are examined. Conclusions and recommendations for environmental management and research are given.

Determination of thresholds in marine molluscs as an alternative to the Environmental Quality Standards in marine water defined in the Water Framework Directive

The Water Framework Directive (WFD) establishes Environmental Quality Standards (EQS) in marine water for 34 priority substances. Among these substances, 25 are hydrophobic and bioaccumulable (2 metals and 23 organic compounds). For these 25 substances, monitoring in water matrix is not appropriate and an alternative matrix should be developed. Bivalve mollusks, particularly mussels (Mytilus edulis, Mytilus galloprovincialis), are used by Ifremer as a quantitative biological indicator since 1979 in France, to assess the marine water quality. This study has been carried out in order to determine thresholds in mussels at least as protective as EQS in marine water laid down by the WFD. Three steps are defined: - Provide an overview of knowledges about the relations between the concentrations of contaminants in the marine water and mussels through bioaccumulation factor (BAF) and bioconcentration factor (BCF). This allows to examine how a BCF or a BAF can be determined: BCF can be determined experimentally (according to US EPA or ASTM standards), or by Quantitative Activity-Structure Relationship models (QSAR): four equations can be used for mussels. BAF can be determined by field experiment; but none standards exists. It could be determined by using QSAR but this method is considered as invalid for mussels, or by using existing model: Dynamic Budget Model, but this is complex to use. - Collect concentrations data in marine water (Cwater) in bibliography for those 25 substances; and compare them with concentration in mussels (Cmussels) obtained through French monitoring network of chemicals contaminants (ROCCH) and biological integrator network RINBIO. According to available data, this leads to determine the BAF or the BCF (Cmussels /Cwater) with field data. - Compare BAF and BCF values (when available) obtained with various methods for these substances: BCF (stemming from the bibliography, using experimental process), BCF calculated by QSAR and BAF determined using field data. This study points out that experimental BCF data are available for 3 substances (Chlorpyrifos, HCH, Pentachlorobenzene). BCF by QSAR can be calculated for 20 substances. The use of field data allows to evaluate 4 BAF for organic compounds and 2 BAF for metals. Using these BAF or BCF value, thresholds in shellfish can be determined as an alternative to EQS in marine water.

Results of a round-robin exercise on read-across

A round-robin exercise was conducted within the CALEIDOSLIFE project. The participants were invited to assess the hazard posed by a substance, applying in silico methods and read-across approaches. The exercise was based on three endpoints: mutagenicity, bioconcentration factor and fish acute toxicity. Nine chemicals were assigned for each endpoint and the participants were invited to complete a specific questionnaire communicating their conclusions.The interesting aspect of this exercise is the justification behind the answers more than the final prediction in itself. Which tools were used? How did the approach selected affect the final answer?

Cytogenotoxicity biomarkers in fat snook Centropomus parallelus from Cananeia and Sao Vicente estuaries, SP, Brazil

The aquatic environment receives many contaminants that can induce damages at the molecular, biochemical, cellular and physiological levels. Centropomus parallelus, an important food resource for local populations, is a predator fish that feeds on small fishes and benthic invertebrates, thus being vulnerable to the bioconcentration and biomagnification processes. This study aimed to evaluate cytogenotoxic responses in erythrocytes from C. parallelus juveniles collected in the Cananeia and Sao Vicente estuaries, both in winter and in summer. After anesthesia, blood samples were collected by caudal puncture. Blood smears were prepared on glass slides and stained with May-Grunwald-Giemsa dye. Two thousand cells were analyzed per slide (1000x), and nuclear abnormalities (NA) and micronuclei (MN) were scored. The Sao Vicente sample showed MN and NA frequencies (%/1000 cells) of 0.325 and 3.575, in winter, and of 0.125 and 2.935 in summer respectively; the Cananeia sample showed frequencies of 0.0325 and 0.03, in winter, and of 0.065 and 0.355 in summer, respectively. The rates found in Sao Vicente were significantly higher than those found in Cananeia, evidencing that the levels of pollution in that estuary were high enough to induce genetic damages.

Multibiomarker assessment of cadmium-based quantum dots effects in the marine mussel Mytilus galloprovincialis

Tese de Doutoramento, Ciências do Mar, da Terra e do Ambiente, Ramo: Ciências e Tecnologias do Ambiente, Especialização em Ecotoxicologia, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

Assessment of the Occurrence and Potential Risks of Pharmaceuticals and their Metabolites in Fish and Water Using Liquid Chromatography Mass Spectrometry

A comprehensive method for the analysis of 11 target pharmaceuticals representing multiple therapeutic classes was developed for biological tissues (fish) and water. Water samples were extracted using solid phase extraction (SPE), while fish tissue homogenates were extracted using accelerated solvent extraction (ASE) followed by mixed-mode cation exchange SPE cleanup and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Among the 11 target pharmaceuticals analyzed, trimethoprim, caffeine, sulfamethoxazole, diphenhydramine, diltiazem, carbamazepine, erythromycin and fluoxetine were consistently detected in reclaimed water. On the other hand, caffeine, diphenhydramine and carbamazepine were consistently detected in fish and surface water samples. In order to understand the uptake and depuration of pharmaceuticals as well as bioconcentration factors (BCFs) under the worst-case conditions, mosquito fish were exposed to reclaimed water under static-renewal for 7 days, followed by a 14-day depuration phase in clean water. Characterization of the exposure media revealed the presence of 26 pharmaceuticals while 5 pharmaceuticals including caffeine, diphenhydramine, diltiazem, carbamazepine, and ibuprofen were present in the organisms as early as 5 h from the start of the exposure. Liquid chromatography ultra-high resolution Orbitrap mass spectrometry was explored as a tool to identify and quantify phase II pharmaceutical metabolites in reclaimed water. The resulting data confirmed the presence of acetyl-sulfamethoxazole and sulfamethoxazole glucuronide in reclaimed water. To my knowledge, this is the first known report of sulfamethoxazole glucuronide surviving intact through wastewater treatment plants and occurring in environmental water samples. Finally, five bioaccumulative pharmaceuticals including caffeine, carbamazepine, diltiazem, diphenhydramine and ibuprofen detected in reclaimed water were investigated regarding the acute and chronic risks to aquatic organisms. The results indicated a low potential risk of carbamazepine even under the worst case exposure scenario. Given the dilution factors that affect environmental releases, the risk of exposure to carbamazepine will be even more reduced.

Estimativa do fator de bioconcentração de pesticidas em maçãs.

O objetivo desse trabalho foi estimar o fator de bioconcentração (BCF) de trinta e seis pesticidas do sistema brasileiro de produção integrada de maçãs (PIM) e indicar quais deles devem ser monitorados em maçãs. Foi assumido um cultivo hipotético de macieiras e usado o modelo de Paraíba (2007) [Pesticide bioconcentration modelling for fruit trees Chemosphere, v.66, p.1468-1475, 2007].O modelo relaciona o BCF com características da planta e do pesticida.

Bioaccumulation and elimination of Endosulfan in zebra fish (Brachydanio rerio).

The bioaccumulation and elimination of endosulfan in zebra fish (Brachydanio rerio) were investigated in a semi-static bioassay. The pesticide mean concentration in water was 03ug litre(-1) and the level of endosulfan residues (x(alfa)+B(beta)-isomers+endosulfan sulfate) in the exposed fish at day 21 was 0.81 (+-0.12)ug g(-1) body weight. The estimated value of the bioconcentration factor (BCF) was 2650 (+-441), the total endosulfan residues being eliminated with a biological half-life of four days. Histopathological studies showed predominantly lipid accumulation in the liver and necrotic focus in the gills of exposed fish.

Uptake and elimination of endosulfan by zebra fish.

1992