André M.P.T. Pereira

Environmental impact of pharmaceuticals from Portuguese wastewaters: geographical and seasonal occurrence, removal and risk assessment

The occurrence, fate, geographical and seasonal influence and environmental risk assessment of eleven of the most consumed pharmaceuticals in Portugal were studied in wastewater treatment plants (WWTPs) influents and (WWI) and effluents (WWE). WWI and WWE samples, from two sampling campaigns (spring and summer), in 2013, were evaluated in 15 different WWTPs across the country, by solid phase extraction (SPE) and liquid chromatography coupled with tandem mass detection (LC-MS-MS). Lipid regulators were the most frequently found in WWI and WWE (184.1 and 22.3mg/day/1000 inhab., respectively), followed by anti-inflammatories (1339.4 and 15.0mg/day/1000 inhab., respectively), and antibiotics (330.7 and 68.6 mg/day/1000 inhab., respectively). Anxiolytics were the least detected with 3.3 and 3.4 mg/day/1000 inhab. in WWI and WWE, respectively. The mass loads, both in WWI and WWE, were higher in summer than those found during the spring season, being remarkable the high values registered in a region where population triplicates in this time of the year. The mean removal efficiency achieved was of 94.5%, nonetheless, between the different therapeutic groups, as well as within each group, important variations in removal were observed, going from not eliminated to 100%. In the summer higher efficiencies were observed regarding lipid regulators and antibiotics. Furthermore, an important outcome was the evaluation, by means of risk quotients (RQs), of the potential ecotoxicological risk posed by the selected pharmaceuticals to different aquatic organisms, exposed to the effluents studied. Ciprofloxacin, bezafibrate, gemfibrozil, simvastatin and diclofenac showed RQs higher than one, being expected that these pharmaceuticals might pose a threat to the three trophic levels (algae, daphnids and fish) evaluated. These results highlight the importance of these monitoring studies, as required by the Directive 2013/39/EU, in order to minimize their aquatic environmental contamination and support future prioritization measures.

Reviewing the serotonin reuptake inhibitors (SSRIs) footprint in the aquatic biota: Uptake, bioaccumulation and ecotoxicology

Selective serotonin re-uptake inhibitors (SSRIs) antidepressants are amongst the most prescribed pharmaceutical active substances throughout the world. Their presence, already described in different environmental compartments such as wastewaters, surface, ground and drinking waters, and sediments, and their remarkable effects on non-target organisms justify the growing concern about these emerging environmental pollutants. A comprehensive review of the literature data with focus on their footprint in the aquatic biota, namely their uptake, bioaccumulation and both acute and chronic ecotoxicology is presented. Long-term multigenerational exposure studies, at environmental relevant concentrations and in mixtures of related compounds, such as oestrogenic endocrine disruptors, continue to be sparse and are imperative to better know their environmental impact.

A one-year follow-up analysis of antidepressants in Portuguese wastewaters: Occurrence and fate, seasonal influence, and risk assessment

The occurrence, fate, seasonal influence and environmental risk assessment of four selective serotonin re-uptake inhibitors (SSRIs) antidepressants, citalopram, fluoxetine, paroxetine and sertraline, were studied in 15 different wastewater treatment plants (WWTPs) across Portugal. Influent and effluent samples from four sampling campaigns, in 2013, were extracted through Oasis HLB cartridges, and quantified through liquid chromatography with tandem mass spectrometry (LC-MSn).Results showed that citalopram was the SSRI most frequently found, both in influents and in effluents, with mean mass loads ranging between 14.56 and 9.51 mg/day/1000 inhabitants, respectively. Fluoxetine and sertraline were only detected in influent samples, in lower mean mass loads (14.60 and 1.36 mg/day/1000 inhab., respectively), whereas paroxetine was found in influent and effluent samples (12.61 and 18.90 mg/day/1000 inhab., respectively). WWTPs were not capable of completely removing these pharmaceuticals; nonetheless, the mean removal efficiency was 82.24%. Removal efficiency was lower in winter (74.21%), summer (72.02%), and autumn (81.19%), when compared to spring (100%). Our results translate the variations in SSRI prescription and use between the five Portuguese regions in study. Influent contaminated samples were found in WWTPs from Lisbon, Alentejo, Center and North (28.25, 19.01, 16.55 and 6.98 mg/day/1000 inhab., respectively). In the Algarve region no contaminated samples were found. A seasonal pattern in the presence of SSRIs in influent wastewaters was observed. The SSRIs mass loads in influent wastewaters were higher in autumn, followed by spring, winter, and summer. Finally, the potential ecotoxicological risk posed by SSRIs to different trophic levels of aquatic organisms, exposed to the effluent wastewaters studied was evaluated by means of risk quotients (RQ). Citalopram and paroxetine, the only SSRIs found in these samples, presented RQ lower than 1. According to the results, algae appeared to be the most sensitive followed by fish and daphnids.

Fluoroquinolones and Tetracycline Antibiotics in a Portuguese Aquaculture System and Aquatic Surroundings: Occurrence and Environmental Impact

The growth of aquaculture over the past few years is widely recognized as one of the main sources of antibiotics, mainly fluoroquinolones (FQ) and tetracyclines (TC), in the aquatic environment, consequently, increasing the risk of the emergence of antibiotic bacterial resistance and promoting the spread of resistant genes. This study aimed to (1) develop and validate a multiresidue method for determination and quantification of ciprofloxacin (CIP), difloxacin (DIFL), enrofloxacin (ENR), norfloxacin (NOR), sarafloxacin (SARA), and oxytetracycline (OXY) in aquaculture waters and surrounding water bodies and (2) provide the first Portuguese data to utilize in assessment of risk of adverse effects. In addition, the potential environmental impact posed by these antibiotics to aquatic organisms, belonging to different trophic levels, when exposed to the studied aquaculture waters was also assessed. The analytical strategy comprised of solid-phase extraction (SPE) through Oasis HLB cartridges, and detection and quantification by liquid chromatography with tandem mass spectrometry (LC/MSn). Method detection limits (MDL) and method quantification limits (MQL) were in the range of 0.7–3 ng/L and 2.4–10 ng/L, respectively. Recoveries varied between 57.4 and 122.8%. The method was applied to 31 water samples collected from an aquaculture and surrounding water bodies located in north of Portugal. Residues of all antibiotics, except SARA and DIFL, were detected at concentrations ranging from 3 to 75.1 ng/L. Norfloxacin was the antibiotic present at highest frequency and concentration. Regarding the environmental impact assessment (EIA), a risk quotient higher than 1 was observed for NOR.

Assessing environmental risk of pharmaceuticals in Portugal: An approach for the selection of the Portuguese monitoring stations in line with Directive 2013/39/EU.

In line with the Directive 2013/39/EU the most representative surface waters, regarding pharmaceuticals contamination, were selected based on a Portuguese nationwide monitoring exercise. To meet this purpose, and given that wastewater treatment plants (WWTPs) are regarded as the major point sources of pharmaceuticals environmental contamination, the occurrence, fate and environmental risk assessment (ERA) of eleven of the most consumed pharmaceuticals, belonging to several therapeutic classes were assessed in 15 WWTPs (influents (WWIs) and effluents (WWEs)), from five different regions during one year (4 sampling campaigns). Results showed that all samples were contaminated with at least 1, and up to 8 from the 11 targeted pharmaceuticals. The highest concentrations observed were 150 and 33 μg L(-1) for WWI and WWE, respectively. Regarding temporal and spacial influence, winter, Alentejo, Algarve and Center regions presented higher mass loads. The ERA posed by 7 of the selected pharmaceuticals presented a risk quotient higher than 1 to the three trophic levels. Our findings highlighted that the rivers Mondego, Tagus, Ave, Trancão, Fervença and Xarrama should be selected as surface water monitoring stations. This study gives a good overview on pharmaceuticals contamination in WWTPs and its impact on surface waters in Portugal. Thus, a more integrative approach to rank and prioritize pharmaceuticals, based on an integrated assessment of ERA and exposure of surface water, was provided to support the future selection of the 6 most representative monitoring stations in Portugal, as required by the above mentioned directive.

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 Portugals 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.

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.