Mycotoxins in Rice Correlate with Other Contaminants? A Pilot Study of the Portuguese Scenario and Human Risk Assessment.

Rice is the second most important cereal crop and is vital for the diet of billions of people. However, its consumption can increase human exposure to chemical contaminants, namely mycotoxins and metalloids. Our goal was to evaluate the occurrence and human exposure of aflatoxin B1 (AFB1), ochratoxin A (OTA), zearalenone (ZEN), and inorganic arsenic (InAs) in 36 rice samples produced and commercialized in Portugal and evaluate their correlation. The analysis of mycotoxins involved ELISA, with limits of detection (LODs) of 0.8, 1 and 1.75 µg kg-1 for OTA, AFB1, and ZEN, respectively. InAs analysis was carried out by inductively coupled plasma mass spectrometry (ICP-MS; LOD = 3.3 µg kg-1). No sample showed contamination by OTA. AFB1 was present in 2 (4.8%) samples (1.96 and 2.20 µg kg-1), doubling the European maximum permitted level (MPL). Concerning ZEN, 88.89% of the rice samples presented levels above the LOD up to 14.25 µg kg-1 (average of 2.75 µg kg-1). Regarding InAs, every sample presented concentration values above the LOD up to 100.0 µg kg-1 (average of 35.3 µg kg-1), although none surpassed the MPL (200 µg kg-1). No correlation was observed between mycotoxins and InAs contamination. As for human exposure, only AFB1 surpassed the provisional maximum tolerable daily intake. Children were recognized as the most susceptible group.

Mycotoxins Exposure in Cabinda, Angola-A Pilot Biomonitoring Survey of Breastmilk.

Breast milk is considered the ideal form of nutrition for newborns and infants. However, it can carry over contaminants, namely mycotoxins, with biological effects to which this population is particularly vulnerable. Human biomonitoring and surveillance programs are particularly scarce in low-income countries, where food security is a more urgent priority in comparison with food safety. This pilot survey aims to assess exposure of breastfed infants to aflatoxin M1 (AFM1), zearalenone (ZEN), and ochratoxin A (OTA) in Angola, and to evaluate the main socio-demographical and food consumption determinants of lactating mothers. All 37 breast milk samples analyzed are found to be contaminated with ZEN and OTA, although none are found contaminated with AFM1. Contamination levels are lower than previously reported for ZEN but higher in the case of OTA. A significant association between ZEN levels in breast milk and the consumption of cookies by the lactating mothers is found. As for OTA, higher levels are observed in the milk from mothers with younger infants, for which high estimated daily intake (EDI) is determined. As far as the authors are aware, this is the first survey of the occurrence of mycotoxins in breast milk in Angola, so further human biomonitoring works should follow, given that mycotoxins are a global health issue that directly impact the health of populations.

Carmines (E120) in coloured yoghurts: a case-study contribution for human risk assessment.

Carmines (E120) are used worldwide as natural food colouring agents of animal origin, with a widespread application, including yoghurts. Despite being considered safe for human health, carmines are known to cause allergic reactions. Our goal was to evaluate the presence of carmines in different yoghurts with a label declaration of E120, purchased in Portugal, and, for the first time, to assess the human risk. The analytical methodology, recommended by JECFA, was based on acid hydrolysis of the samples followed by spectrophotometric UV-Vis analysis at 494 nm. This methodology allowed for a limit of quantification (LOQ) of 39.0 mg/kg and recovery rates higher than 97.7%. All the samples had carmines at levels above the LOQ, ranging between 43.8 and 193.8 mg/kg, with an average of 125.2 ± 34.5 mg/kg. In total, 8 (26.7%) samples exceeded the European Union (EU) maximum permitted level (MPL) established for carmines in this foodstuff, 150 mg/kg. Solid yoghurts presented higher average levels, 137.2 mg/kg, when compared to liquid samples, 107.2 mg/kg, with a significant statistical difference (p= 0.0236) being observed. No significant statistical difference was observed between white and private labels, whose average levels were very similar, 125.4 vs 125 mg/kg, respectively. Although some samples were above the allowable values, the estimated daily intake (EDI), designed for the different scenarios of different yoghurt types, did not exceed the established ADI, 5 mg/kg bw/day. According to the obtained results, carmine ingestion through the consumption of yoghurt poses low risk to the Portuguese consumers. However, children were the most vulnerable population group with a calculated risk value of up to 10% considering the mean content scenario. These first findings point out the need to reinforce surveillance programmes and monitoring studies, contributing to an increased awareness regarding carmine exposure, however it must be emphasised that yoghurt has evident nutritional benefits depending on a healthful consumer choice.

Urine biomonitoring of glyphosate in children: Exposure and risk assessment.

The use of glyphosate has been increasing over the years, making it one of the most consumed herbicides in the world. Although children are considered a vulnerable population, only four previous published studies determined glyphosate in the urine of non-occupationally exposed children. The paucity of epidemiological data and biomonitoring surveys are considered major gaps, that hinder the implementation of science driven policies in the protection of public health. The aim of the present study was to determine glyphosate in the urine of 41 Portuguese children (2-13 years old) and identify potential determinants of exposure. Glyphosate was detected in 95.1% of the samples (1.77 ± 0.86 µg/L), up to a maximum value of 4.35 µg/L. Glyphosate concentrations were higher in the urine of children aged 7-9 years, living near agricultural areas (<1 km), with a higher percentage of consumption of home-produced foods, and whose parents applied herbicides in the backyard. Risk assessment revealed an exposure representing 1-5.58% of the established Acceptable Daily Intake (ADI) of glyphosate (0.5 mg/kg bw/day). The results should be further analyzed considering the age of the participants, for which no adjusted ADI exists. This was the first published report of glyphosate exposure in the urine of Portuguese children.

Human Biomonitoring of Selected Hazardous Compounds in Portugal: Part II-Lessons Learned on Mycotoxins.

Human biomonitoring (HBM) data provide information on total exposure regardless of the route and sources of exposure. HBM studies have been applied to quantify human exposure to contaminants and environmental/occupational pollutants by means of determining the parent compounds, their metabolites, or even their reaction products in biological matrices. HBM studies performed among the Portuguese population are dispersed and limited. Thus, to overcome this knowledge gap, this work reviews the published Portuguese HBM information concerning mycotoxins detected in the urine, serum, milk, hair, and nails of different groups of the Portuguese population. This integrative approach to the available HBM data allows us to analyze the main determinants and patterns of exposure of the Portuguese population to the selected hazardous compounds, as well as to assess the potential health risks. We also aimed to identify the main difficulties and challenges of HBM through the analysis of the enrolled studies. Ultimately, this study aims to support national and European policies in promoting human health by summarizing the most important outcomes and lessons learned through the HBM studies carried out in Portugal.

Human Biomonitoring of Selected Hazardous Compounds in Portugal: Part I-Lessons Learned on Polycyclic Aromatic Hydrocarbons, Metals, Metalloids, and Pesticides.

Human biomonitoring (HBM) data provide information on total exposure regardless of the route and sources of exposure. HBM studies have been applied to quantify human exposure to contaminants and environmental/occupational pollutants by determining the parent compounds, their metabolites or even their reaction products in biological matrices. HBM studies performed among the Portuguese population are disperse and limited. To overcome this knowledge gap, this review gathers, for the first time, the published Portuguese HBM information concerning polycyclic aromatic hydrocarbons (PAHs), metals, metalloids, and pesticides concentrations detected in the urine, serum, milk, hair, and nails of different groups of the Portuguese population. This integrative insight of available HBM data allows the analysis of the main determinants and patterns of exposure of the Portuguese population to these selected hazardous compounds, as well as assessment of the potential health risks. Identification of the main difficulties and challenges of HBM through analysis of the enrolled studies was also an aim. Ultimately, this study aimed to support national and European policies promoting human health and summarizes the most important outcomes and lessons learned through the HBM studies carried out in Portugal.

Citrinin in Foods and Supplements: A Review of Occurrence and Analytical Methodologies.

Citrinin (CIT) deserves attention due to its known toxic effects in mammalian species and its widespread occurrence in food commodities, often along with ochratoxin A, another nephrotoxic mycotoxin. Human exposure, a key element in assessing risk related to food contaminants, depends upon mycotoxin contamination levels in food and on food consumption. Commercial supplements, commonly designated as red rice, usually used in daily diets in Asiatic countries due to their medicinal properties, may pose a health problem as a result of high CIT levels. In addition to the worldwide occurrence of CIT in foods and supplements, a wide range of several analytical and detection techniques with high sensitivity, used for evaluation of CIT, are reviewed and discussed in this manuscript. This review addresses the scientific literature regarding the presence of CIT in foods of either vegetable or animal origin, as well as in supplements. On what concerns analytical methodologies, sample extraction methods, such as shaking extraction and ultrasonic assisted extraction (UAE), clean-up methods, such as liquid-liquid extraction (LLE), solid phase extraction (SPE) and Quick, Easy, Cheap, Effective, Rugged and Safe (QuECHERS), and detection and quantification methods, such as thin layer chromatography (TLC), high performance liquid chromatography (HPLC), capillary electrophoresis (CE), biosensors, and ELISA, are also reviewed.

Artificial sweeteners in non-alcoholic beverages: Occurrence and exposure estimation of the Portuguese population.

Artificial sweeteners such as acesulfame-potassium (ACE-K). saccharin (SAC), and aspartame (ASP), play a vital role in the food industry. Despite apparent benefits, several studies report adverse reactions on consumers. EFSA is currently re-evaluating several individual sweeteners, including ASP, Ace-K and SAC that shall be completed by 31 December 2020. Our goal was to evaluate, by liquid chromatography with ultraviolet detection (LC-UV), their levels in non-alcoholic beverages from the Portuguese market (soft drinks, sport drinks, energy drinks, and nectars), and estimate the exposure of the Portuguese adolescent/adult population. ACE-K was found in 85.7% of the samples, in mean levels of 97.4 mg/L. ASP and SAC, detected in 37.5% and 33.9% of the samples, were found in averages of 161.5 and 55.0 mg/L, respectively. The maximum limits set for ASP were exceeded in one energy drink. SAC exceeded the maximum permitted level (MPL) level in 3 soft drinks based on tea extracts. In 4 samples SAC was found but not mentioned in the label, and in one of these samples exceeded the MPL. The estimated daily intake (EDI), calculated in mg per kilogram of body weight per day, of ACE-K, SAC and ASP was found to be below the acceptable daily intake (ADI), both for Portuguese adolescents and adult population. Traditional soft drinks accounted for the highest EDIs representing 1.8%, 2.3% and 0.6% for ACE-K, SAC and ASP, respectively, of the ADI. Considering the previous report in Portugal it is observed that the population is currently exposed to a greater amount of artificial sweeteners, which may indicate a trend for more artificial sweeteners' usage in order to reduce sugar intake.

Reviewing the Analytical Methodologies to Determine the Occurrence of Citrinin and its Major Metabolite, Dihydrocitrinone, in Human Biological Fluids.

Until now, the available data regarding citrinin (CIT) levels in food and the consumption of contaminated foods are insufficient to allow a reliable estimate of intake. Therefore, biomonitoring configuring analysis of parent compound and/or metabolites in biological fluids, such as urine or blood, is being increasingly applied in the assessment of human exposure to CIT and its metabolite, dihydrocitrinone (DH-CIT). Most studies report urinary levels lower for the parent compound when compared with DH-CIT. A high variability either in the mean levels or in the inter-individual ratios of CIT/DH-CIT between the reported studies has been found. Levels of DH-CIT in urine were reported as being comprised between three to seventeen times higher than the parent mycotoxin. In order to comply with this objective, sensitive analytical methodologies for determining biomarkers of exposure are required. Recent development of powerful analytical techniques, namely liquid chromatography coupled to mass spectrometry (LC-MS/MS) and ultra-high-performance liquid chromatography (UHPLC-MS/MS) have facilitated biomonitoring studies, mainly in urine samples. In the present work, evidence on human exposure to CIT through its occurrence and its metabolite, in biological fluids, urine and blood/plasma, in different countries, is reviewed. The analytical methodologies usually employed to evaluate trace quantities of these two molecules, are also presented. In this sense, relevant data on sampling (size and pre-treatment), extraction, cleanup and detection and quantification techniques and respective chromatographic conditions, as well as the analytical performance, are evidenced.

Selected Pharmaceuticals in Different Aquatic Compartments: Part II-Toxicity and Environmental Risk Assessment.

Potential risks associated with releases of human pharmaceuticals into the environment have become an increasingly important issue in environmental health. This concern has been driven by the widespread detection of pharmaceuticals in all aquatic compartments. Therefore, 22 pharmaceuticals, 6 metabolites and transformation products, belonging to 7 therapeutic groups, were selected to perform a review on their toxicity and environmental risk assessment (ERA) in different aquatic compartments, important issues to tackle the water framework directive (WFD). The toxicity data collected reported, with the exception of anxiolytics, at least one toxicity value for concentrations below 1 µg L-1. The results obtained for the ERA revealed risk quotients (RQs) higher than 1 in all the aquatic bodies and for the three trophic levels, algae, invertebrates and fish, posing ecotoxicological pressure in all of these compartments. The therapeutic groups with higher RQs were hormones, antiepileptics, anti-inflammatories and antibiotics. Unsurprisingly, RQs values were highest in wastewaters, however, less contaminated water bodies such as groundwaters still presented maximum values up to 91,150 regarding 17α-ethinylestradiol in fish. Overall, these results present an important input for setting prioritizing measures and sustainable strategies, minimizing their impact in the aquatic environment.

Ochratoxin A in Beers Marketed in Portugal: Occurrence and Human Risk Assessment.

Ochratoxin A (OTA) is produced by fungi present in several agricultural products with much relevance to food safety. Since this mycotoxin is widely found in cereals, beer has a potential contamination risk. Therefore, it was deemed essential to quantify, for the first time, the levels of OTA in beer, a cereal-based product that is marketed in Portugal, as well as to calculate the human estimated weekly intake (EWI) and risk assessment. A total of 85 samples were analyzed through immunoaffinity clean-up, followed by liquid chromatography-fluorescence detection (LC-FD). This analytical methodology allowed a limit of quantification (LOQ) of 0.43 µg/L. The results showed that 10.6% were contaminated at levels ranging between

Selected Pharmaceuticals in Different Aquatic Compartments: Part I-Source, Fate and Occurrence.

Potential risks associated with releases of human pharmaceuticals into the environment have become an increasingly important issue in environmental health. This concern has been driven by the widespread detection of pharmaceuticals in all aquatic compartments. Therefore, 22 pharmaceuticals, 6 metabolites and transformation products, belonging to 7 therapeutic groups, were selected to perform a systematic review on their source, fate and occurrence in different aquatic compartments, important issues to tackle the Water Framework Directive (WFD). The results obtained evidence that concentrations of pharmaceuticals are present, in decreasing order, in wastewater influents (WWIs), wastewater effluents (WWEs) and surface waters, with values up to 14 mg L-1 for ibuprofen in WWIs. The therapeutic groups which presented higher detection frequencies and concentrations were anti-inflammatories, antiepileptics, antibiotics and lipid regulators. These results present a broad and specialized background, enabling a complete overview on the occurrence of pharmaceuticals in the aquatic compartments.

Ochratoxin A and Portuguese children: Urine biomonitoring, intake estimation and risk assessment.

Ochratoxin A (OTA) is a mycotoxin detected worldwide. Urine biomonitoring is a more realistic and non-invasive way to assess exposure when compared to traditional methods based on food occurrence and consumption data. Few studies have investigated children's exposure to OTA, although it is a more susceptible population. Our main goal was the OTA biomonitoring in urine of Portuguese children to better characterize the health risk of this population. The validated analytical methodology was based on an immunoaffinity clean-up, followed by LC-FD. First morning samples were collected, between 2018 and 2019, from 85 healthy children, 41 boys and 44 girls, aging between 2 and 13 years old. Overall, from the 85 analysed samples 79 (92.94%) were found positive. The average OTA concentration level was of 0.020 ng/mL (29.41 ng/g of creatinine), with a maximum value of 0.052 ng/mL (114.45 ng/g of creatinine). No statistical differences were found between the OTA contamination levels, the anthropometric data and dietary habits considered. Depending on the excretion rate considered for the PDI calculation, the risk assessed for the average OTA contamination varied from 10% to 194%, indicating a potential concern. This is the first study regarding OTA exposure in Portuguese children, showing their clear exposure.

Ochratoxin A in the Portuguese Wine Market, Occurrence and Risk Assessment.

Ochratoxin A (OTA) is mainly found in cereals and cereal-based foodstuffs, but also in wine. Being one of the most consumed alcoholic drinks in Portugal and one of the main sources of human exposure to OTA, wine monitoring and exposure studies are essential. The analytical methodology consisted of the direct injection of the filtered samples into the liquid chromatograph, equipped with fluorescent detection (LC-FLD). Linearity was adequate, both in mobile phase and in matrix-matched solutions, with R2 values higher than 0.997. The limits of detection were 0.08 and 0.39 µg/L for white and red wine, respectively and recoveries were above 91.9%. One hundred wine samples acquired on the Portuguese market were investigated. In 5 samples the OTA was detected, with the red wine presenting higher frequency of contamination. Regarding the risk to human health it was observed that the estimated weekly intake (EWI) is considerably lower than the established tolerable weekly intake (TWI).

Risk assessment of fluoroquinolones from poultry muscle consumption: Comparing healthy adult and pre-school populations.

Antibiotics, especially fluoroquinolones (FQs), have been largely used in animal husbandry namely poultry production. Therefore, this study aimed to identify, quantify and estimate the daily intake for adult and 3-year-old populations of the FQs norfloxacin (NOR), ciprofloxacin (CIP) and enrofloxacin (ENR) through poultry muscle consumption. The results showed detection frequencies of 78% and 62% in supermarket and school canteen samples, respectively. Of the 182 analysed samples, 4 did not comply with ENR maximum residue level (MRL), and 9 were contaminated with NOR, not allowed as a veterinary medicine of food-producing animals. The highest estimated daily intake value was obtained for the 3-year-old population regarding the sum of ENR and CIP (0.46 µg kg-1 day-1); value substantially lower than the established acceptable daily intake (2.0 µg kg-1 day-1). Although the low risk found, the high detection frequencies support the apprehension of the different international organizations, towards the emergence of human bacterial resistances to FQs originated from poultry production.

The mycoestrogen zearalenone in Portuguese flowing waters and its potential environmental impact.

This study provides the first environmental risk assessment (ERA) for zearalenone (ZEN), the only known mycoestrogen, based on a broad-scale investigation on its occurrence in rivers and creeks from Portugal. Water sample filtration and immunoaffinity columns (IAC) clean-up followed by liquid chromatograph with tandem mass spectrometry (LC/MS n ) provided an analytical method with good analytical performance. ZEN levels were determined for seven Portuguese rivers and one creek, during two different seasons, in a total of 38 samples collected upstream wastewater treatment plants (WWTPs). Overall, 23.7% were contaminated with ZEN at levels ranging between 5.6 and 82.6 ng/L. The highest concentration was observed during spring, although no statistically significant differences were observed between spring and autumn sampling campaigns. The potential ecotoxicological risk from ZEN to different trophic levels of aquatic organisms was evaluated by means of risk quotients (RQs) calculation. Although all the RQs obtained were lower than 1, our results confirm that ZEN is a relatively frequent contaminant in flowing waters in Portugal and might contribute to the overall estrogenic activity in the environment.

Human pharmaceuticals in Portuguese rivers: The impact of water scarcity in the environmental risk.

Pharmaceuticals occurrence and environmental risk assessment were assessed in Portuguese surface waters, evaluating the impact of wastewater treatment plants (WWTPs) and river flow rates. Twenty three pharmaceuticals from 6 therapeutic groups, including metabolites and 1 transformation product, were analysed in 72 samples collected from 20 different sites, upstream and downstream the selected WWTPs, in two different seasons. Analysis was performed by solid phase extraction followed by liquid chromatography coupled to tandem mass spectroscopy. Pharmaceuticals were detected in 27.8% of the samples. Selective serotonin reuptake inhibitors (SSRIs), anti-inflammatories and antibiotics presented the highest detection frequencies (27.8, 23.6 and 23.6%, respectively) and average concentrations (37.9, 36.1 and 33.5ngL-1, respectively). When assessing the impact of WWTPs, an increase of 21.4% in the average concentrations was observed in the samples located downstream these facilities, when compared with the upstream samples. Increased detection frequencies and concentrations were observed at lower flow rates, both when comparing summer and winter campaigns and by evaluating the different rivers. Risk quotients (RQs) higher than one were found for two pharmaceuticals, concerning two trophic levels. However, since Iberian rivers are highly influenced by water scarcity, in drought periods, the flow rates in these rivers can decrease at least ten times from the lowest value observed in the sampling campaigns. In these conditions, RQs higher than 1 would be observed for 5 pharmaceuticals, additionally, all the detected pharmaceuticals (11) would present RQs higher than 0.1. These results emphasize that the river flow rate represents an important parameter influencing pharmaceuticals concentrations, highlighting the ecotoxicological pressure, especially due to water scarcity in drought periods. This should be a priority issue in the environmental policies for minimizing its impact in the aquatic environment.

A critical evaluation of different parameters for estimating pharmaceutical exposure seeking an improved environmental risk assessment.

A critical evaluation of the European Medicines Agency (EMA) Guideline on Environmental Risk Assessment (ERA) was performed on 16 of Portugal's most consumed pharmaceuticals in wastewater effluents (WWEs), the main route for aquatic contamination. The predicted environmental concentrations (PECs) were formulated based on the Guideline, after incorporating several refinements. The best approach was selected by comparing the measured environmental concentrations (MECs) to the PECs in WWEs. Finally, risk was assessed by comparing PECs to predicted no-effect concentrations (PNECs). The results showed that the default value of the penetration factor (Fpen) used by the EMA (0.01) was surpassed and that national consumption and excretion data were the two most important parameters for PEC calculations. The risk quotient between PECs and PNECs was higher than 1 for 12 pharmaceuticals, indicating a risk to all three trophic levels of aquatic organisms (algae, daphnids and fish). To improve the current ERA framework, suggestions were made for incorporating consumption and excretion data, changing the default value of Fpen to 0.04 and adding a safety factor of 10. Moreover, this evaluation should be performed for pharmaceuticals already on the market, and future ERAs should incorporate a risk-benefit analysis, an important risk-management step.

SSRIs antidepressants in marine mussels from Atlantic coastal areas and human risk assessment.

Selective serotonin reuptake inhibitors (SSRIs) are among the most prescribed antidepressants nation and worldwide. Their presence in the aquatic environment is known, causing growing pressures in heavily populated coastal areas. For instance, disruption of invertebrate endocrine systems has been described. Since discrete seawater sampling may not be suitable to evaluate such environmental risk, we aimed to assess SSRIs and metabolites in wild Mytilus galloprovincialis, and assess risk to human health. A total of 1225 mussels were sampled during one year along the Portuguese Atlantic coast. Each freeze-dried pool of 25 specimens was analyzed, in duplicate, by solid phase extraction (SPE) and liquid chromatography with tandem mass spectrometry (LC-MSn). Approximately 70% of the samples were contaminated with at least one, and up to 4 from the 7 targeted compounds. The cumulative SSRIs and metabolites content in mussels ranged between 3.17 and 33.93ngg-1 dry weight, in a mean level of 14.68±9.69ngg-1. Norfluoxetine was the most recurring SSRI and the one with the highest mean concentration, 13.52ngg-1. Although some differences were found between different sampling periods and locations, those were not statistically significant (p>0.05) and no correlation was found between wet weight and mean contamination levels. Regarding risk to human health following consumption of these filter-feeders, when considering the average sample contamination of positive samples and the average Portuguese and European consumption, the ratio between the estimated daily intake (EDI) and the acceptable daily intake (ADI) ranged from 0.45 to 5.50%, and from 1.60 to 19.41%, respectively. Therefore, although the resultant risk is lower for the Portuguese consumer than for the average EU consumer, apparently, no appreciable risk to human health is expected.

Uptake, accumulation and metabolization of the antidepressant fluoxetine by Mytilus galloprovincialis.

Fluoxetine, a selective serotonin re-uptake inhibitor (SSRI) antidepressant, is among the most prescribed pharmaceutical active substances worldwide. This study aimed to assess its accumulation and metabolization in the mussel Mytillus galloprovincialis, considered an excellent sentinel species for traditional and emerging pollutants. Mussels were collected from Ria Formosa Lagoon, Portugal, and exposed to a nominal concentration of fluoxetine (75 ng L(-1)) for 15 days. Approximately 1 g of whole mussel soft tissues was extracted with acetonitrile:formic acid, loaded into an Oasis MCX cartridge, and fluoxetine analysed by liquid chromatography with tandem mass spectrometry (LC-MSn). After 3 days of exposure, fluoxetine was accumulated in 70% of the samples, with a mean of 2.53 ng g(-1) dry weight (d.w.) and norfluoxetine was only detected in one sample (10%), at 3.06 ng g(-1) d.w. After 7 days of exposure, the accumulation of fluoxetine and norfluoxetine increased up to 80 and 50% respectively, and their mean accumulated levels in mussel tissues were up to 4.43 and 2.85 ng g(-1) d.w., respectively. By the end of the exposure period (15 days), both compounds were detected in 100% of the samples (mean of 9.31 and 11.65 ng g(-1) d.w., respectively). Statistical analysis revealed significant accumulation differences between the 3rd and 15th day of exposure for fluoxetine, and between the 3rd and 7th against the 15th day of exposure for norfluoxetine. These results suggest that the fluoxetine accumulated in mussel tissues is likely to be metabolised into norfluoxetine with the increase of the time of exposure, giving evidence that at these realistic environmental concentrations, toxic effects of fluoxetine in mussel tissues may occur.