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Moluscos bivalves e impacte ambiental: cafeína e biomarcadores de efeito
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Este estudo avaliou a qualidade de diversas espécies de moluscos bivalves, com diferentes estratégias alimentares, de dois ecossistemas marinhos, Ria de Alvor e Ria Formosa, através da avaliação morfométrica (índice de condição), fisiológica (determinação de biomarcadores) e nutricional (água, matéria gorda, proteínas, hidratos de carbono e minerais). Para essa avaliação também se quantificou a cafeína nestes organismos (biomarcador de exposição) e no seu habitat (contaminante ambiental), assim como, alguns biomarcadores de efeito, nomeadamente, acetilcolinesterase (AChE), peroxidação lipídica (LPO) e o sistema de transporte de eletrões (ETS). A quantificação de cafeína nos moluscos bivalves e nas águas marinhas foi realizada por cromatografia líquida de alta eficiência com detetor de ultra-violeta-visível (HPLC-UV), após pré-tratamento das amostras pelo método QuEChERS (Quick Easy Cheap Effective Rugged Safe) e de extração em fase sólida (SPE), respetivamente. O método foi aplicado à monitorização de ambas as amostras após otimização das condições de ensaio. Os resultados foram confirmados por cromatografia líquida de alta eficiência associada à espectrometria de massa tandem (HPLC-MS/MS). Os métodos de QuEChERS-HPLC-UV e de SPE- HPLC-UV não apresentam sensibilidade adequada à deteção da cafeína nos moluscos bivalves (< 0,36 µg/g) e nas águas marinhas (< 0,26 mg/L), respetivamente. Deste modo, a cafeína foi quantificada nos extratos de ambas as amostras por HPLC-MS/MS. Nas águas marinhas as concentrações de cafeína variaram entre os 18 ng/L (Alvor #1) e 83 ng/L (Olhão). A maioria dos moluscos bivalves (82%) apresentou cafeína, cuja concentração variou entre 0,27 ng/g para o mexilhão (Ludo) e 5,7 ng/g para o lingueirão (Alvor #1). Os dados de cafeína na água refletem somente as condições do momento da amostragem enquanto os de concentração nos tecidos dos bivalves reflectem a integração da sua bioacumulação ao longo da vida dos indivíduos. O teor em proteínas nos moluscos bivalves variou entre 6% (berbigão) e 11% (ostra). A matéria gorda variou entre 0,6% (lingueirão) e 2,1% (ostra). O mexilhão é a espécie com maior teor em minerais (3,6%) e o lingueirão corresponde à espécie com maior teor em hidratos de carbono (3,2%). O teor de água (humidade) variou entre os 80% e 87%. O índice de condição (IC) médio variou entre 18% (ostra) e 68% (lingueirão). Os biomarcadores de efeito apresentaram uma resposta diferenciada para as diferentes espécies: a amêijoa-boa de Olhão registou a menor atividade da AChE, o mexilhão da Praia de Faro os maiores níveis de LPO e lingueirão de Alvor #1 a maior atividade da ETS. Globalmente, observou-se uma correlação positiva entre a cafeína e o IC, na qual os indivíduos mais robustos bioacumulam mais cafeína nos seus tecidos. Porém a cafeína isoladamente não é um fator de stress e não justifica as variações do IC para os vários moluscos bivalves. O IC médio intra-específico foi superior nos bivalves pertencentes à Ria Formosa. Na Ria Formosa, registaram-se níveis de AChE e LPO inferiores mas níveis de ETS superiores comparativamente com a Ria de Alvor. Os níveis de cafeína nas águas marinhas e nos moluscos bivalves sugerem que as concentrações não foram suficientemente elevadas para inibir a AChE e verificou-se que os indivíduos maiores desenvolvem uma maior atividade da AChE. Os dados também sugerem que o aumento deste biomarcador pode dever-se exclusivamente ao aumento das suas dimensões. A correlação entre a cafeína e a LPO e entre o IC e o LPO não é significativa. Em relação à atividade da ETS verificou-se que os indivíduos maiores e com maiores concentrações de cafeína, também apresentaram maior atividade da ETS, o que pode sugerir que estes indivíduos estão sob maior stress fisiológico. A avaliação global dos três biomarcadores de efeito sugere que a cafeína isoladamente não está diretamente associada ao aumento do stress oxidativo destes organismos e que este stress também é provocado por outros fatores, nomeadamente outros contaminantes, fatores ambientais (temperatura, oxigénio e salinidade) e/ou bióticos.
The quality of several bivalve molluscs species, with different feeding strategies from two marine ecosystems, Ria de Alvor and Ria Formosa, was evaluated through morphometric (condition index), physiological (biomarker determination) and nutritional (water, fat, protein, carbohydrate and mineral) determinations. Caffeine was quantified in bivalves molluscs (exposure biomarker) and also in waters from mollusc´s habitat (environmental contaminant), simultaneous with some effect biomarkers, mainly, acetylcholinesterase (AChE), lipid peroxidation (LPO) and electrons transport system (ETS). Caffeine quantification in bivalve molluscs and in marine waters was performed by high performance liquid chromatography with ultraviolet-visible detector (HPLC-UV) after samples pre-treatment by QuEChERS method (Quick Easy Cheap Effective Rugged Safe) or solid phase extraction (SPE), respectively. This method was applied to the monitoring of both samples after optimization of the experimental conditions. Both results were confirmed by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The limit of determination of QuEChERS-HPLC-UV method (< 0.36 µg/g) and SPE-HPLC-UV method (0.26 mg/L) were not enough for caffeine detection in bivalve molluscs) and marine waters, respectively. Therefore, caffeine was quantified in both samples extracts by HPLC-MS/MS. Caffeine concentrations in marine waters ranged from 18 ng/L (Alvor #1) to 83 ng/L (Olhão). Most bivalve molluscs (82%) showed caffeine with concentrations between 0.27 ng/g (mussels from Ludo) and 5.7 ng/g (razor clam from Alvor #1). The concentration of caffeine in water samples only represents a snapshot of its distribution at the sampling sites, while caffeine concentration in bivalve tissues represents their bioaccumulation time integrated over lifetime of individuals. The protein content in bivalve molluscs ranged between 6% (cockles) and 11% (oysters). Fat content ranged from 0.6% (razor clam) to 2.1% (oyster). Mussel was the species with the highest mineral content (3.6%) and razor clam corresponded to the species with the highest carbohydrate content (3.2%). The water content (humidity) ranged from 80% to 87%. The average condition index (CI) ranged from 18% (oyster) to 68% (razor clam). The effect biomarkers showed a discriminated response for different species: the clams from Olhão exhibited lowest AChE activity, Faro Beach mussels presented the highest LPO levels and the razor clam from Alvor #1 showed the highest ETS activity. Overall, a positive correlation was observed between caffeine and CI and the results showed that higher robust individuals bioaccumulated higher caffeine concentrations in their tissues. However, caffeine alone did not show to be a stress factor and did not justify the variations in CI for the different bivalve molluscs. The average intra-specific CI values were higher for Ria Formosa bivalves. In Ria Formosa AChE and LPO levels were lower than Ria de Alvor but ETS levels were higher than Ria de Alvor. Caffeine levels in marine waters and bivalve molluscs indicate that concentrations were not high enough to inhibit AChE. It was verified that larger individuals developed greater AChE activity, which leads to the conclusion that the increase of this biomarker may be exclusively due to the increase of their size. The correlation between caffeine and LPO and between CI and LPO was not significant. Regarding ETS activity, larger individuals, even with higher caffeine concentrations, also presented higher ETS activity, which may suggest that these individuals were under greater physiological stress. The overall assessment of the three effect biomarkers may indicate that caffeine alone is not directly associated with the increased of oxidative stress of these organisms as well as stress be induced by other factors rather than caffeine, including other contaminants, environmental factors (temperature, oxygen and salinity ) and/or biotic factors.
The quality of several bivalve molluscs species, with different feeding strategies from two marine ecosystems, Ria de Alvor and Ria Formosa, was evaluated through morphometric (condition index), physiological (biomarker determination) and nutritional (water, fat, protein, carbohydrate and mineral) determinations. Caffeine was quantified in bivalves molluscs (exposure biomarker) and also in waters from mollusc´s habitat (environmental contaminant), simultaneous with some effect biomarkers, mainly, acetylcholinesterase (AChE), lipid peroxidation (LPO) and electrons transport system (ETS). Caffeine quantification in bivalve molluscs and in marine waters was performed by high performance liquid chromatography with ultraviolet-visible detector (HPLC-UV) after samples pre-treatment by QuEChERS method (Quick Easy Cheap Effective Rugged Safe) or solid phase extraction (SPE), respectively. This method was applied to the monitoring of both samples after optimization of the experimental conditions. Both results were confirmed by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The limit of determination of QuEChERS-HPLC-UV method (< 0.36 µg/g) and SPE-HPLC-UV method (0.26 mg/L) were not enough for caffeine detection in bivalve molluscs) and marine waters, respectively. Therefore, caffeine was quantified in both samples extracts by HPLC-MS/MS. Caffeine concentrations in marine waters ranged from 18 ng/L (Alvor #1) to 83 ng/L (Olhão). Most bivalve molluscs (82%) showed caffeine with concentrations between 0.27 ng/g (mussels from Ludo) and 5.7 ng/g (razor clam from Alvor #1). The concentration of caffeine in water samples only represents a snapshot of its distribution at the sampling sites, while caffeine concentration in bivalve tissues represents their bioaccumulation time integrated over lifetime of individuals. The protein content in bivalve molluscs ranged between 6% (cockles) and 11% (oysters). Fat content ranged from 0.6% (razor clam) to 2.1% (oyster). Mussel was the species with the highest mineral content (3.6%) and razor clam corresponded to the species with the highest carbohydrate content (3.2%). The water content (humidity) ranged from 80% to 87%. The average condition index (CI) ranged from 18% (oyster) to 68% (razor clam). The effect biomarkers showed a discriminated response for different species: the clams from Olhão exhibited lowest AChE activity, Faro Beach mussels presented the highest LPO levels and the razor clam from Alvor #1 showed the highest ETS activity. Overall, a positive correlation was observed between caffeine and CI and the results showed that higher robust individuals bioaccumulated higher caffeine concentrations in their tissues. However, caffeine alone did not show to be a stress factor and did not justify the variations in CI for the different bivalve molluscs. The average intra-specific CI values were higher for Ria Formosa bivalves. In Ria Formosa AChE and LPO levels were lower than Ria de Alvor but ETS levels were higher than Ria de Alvor. Caffeine levels in marine waters and bivalve molluscs indicate that concentrations were not high enough to inhibit AChE. It was verified that larger individuals developed greater AChE activity, which leads to the conclusion that the increase of this biomarker may be exclusively due to the increase of their size. The correlation between caffeine and LPO and between CI and LPO was not significant. Regarding ETS activity, larger individuals, even with higher caffeine concentrations, also presented higher ETS activity, which may suggest that these individuals were under greater physiological stress. The overall assessment of the three effect biomarkers may indicate that caffeine alone is not directly associated with the increased of oxidative stress of these organisms as well as stress be induced by other factors rather than caffeine, including other contaminants, environmental factors (temperature, oxygen and salinity ) and/or biotic factors.
Descrição
Tese de mestrado, Qualidade Alimentar e Saúde, Universidade de Lisboa, Faculdade de Farmácia, 2019
Palavras-chave
Moluscos bivalves Cafeína Biomarcadores Ria de Alvor Ria Formosa Teses de mestrado - 2019