Liliana J.G. Silva

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.

Occurrence of fumonisins B1 and B2 in Portuguese maize and maize-based foods intended for human consumption.

Fumonisin B1 (FB1) and fumonisin B2 (FB2) are mycotoxins mainly produced by Fusarium verticillioides and Fusarium proliferatum, which are field pathogens of maize. A survey was conducted on the incidences of FB1 and FB2 in both maize and derived products purchased in Portugal. The analytical method involved extraction with methanol–water, clean-up by immunoaffinity column and derivatization with naphthalene-2,3-dicarboxaldehyde. Determination was carried out by high-performance liquid chromatography (HPLC) with spectrofluorimetric detection, with liquid chromatography/mass spectrometry (LC/MS) confirmation. The presence of FB1 and FB2 was determined in 67 samples of maize and maize-based foods, such as flour, semolina, starch, sweet maize, cornflakes and other breakfast cereals, and snacks collected in 2005. FBs were found in 15 samples at concentrations ranging from 113 to 2026 µg kg−1. Two of the samples showed higher contamination levels than the limits established by the European Commission Regulation. None of the samples contained levels of fumonisins that would lead to an exposure exceeding the tolerable daily intake (TDI).

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.

Ochratoxin A survey in Portuguese wine by LC-FD with direct injection

Wine and grape juices were identified as one of the most important sources of ochratoxin A (OTA), a mycotoxin with diverse toxic effects that naturally appears in food and foodstuffs all over the world. The aim of this study was to assess the OTA levels in Portuguese wines through the application of a simple and accurate method based on liquid chromatography (LC) with direct injection, followed by fluorescence detection (FD). Randomly selected wine samples were used to evaluate the performance of direct injection as efficient, fast, inexpensive and safe sample preparation method. The proposed method was successfully validated. The limit of quantification (LOQ) was 1.0 microg/L and OTA recoveries from wine samples, spiked at the three fortification levels, were higher than 85.4%, with RSDs lower than 9.6% for both red and white wines. The presence of OTA was confirmed by methyl ester derivatization followed by LC analysis. Data on OTA levels were obtained for 60 Portuguese red and white wine samples. OTA was found in 12 samples, nine (26%) red wine samples and three (12%) white wine samples. Only one red wine sample and one white wine sample presented a contamination level above the LOQ, with 1.23 and 2.4 microg/L, respectively. It should be pointed out that this white wine sample exceeded the EC maximum permitted level of 2.0 microg/L. The safe dose established as 120 ng/kg body weight/week was not exceeded by the weekly intake estimated for the samples contaminated above the LOQ.

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.

Sphinganine-sphingosine ratio in urine from two Portuguese populations as biomarker to fumonisins exposure

Fumonisins (FBs), mycotoxins specially found in maize, interfere with sphingolipid metabolism, altering the sphinganine (Sa) to sphingosine (So) ratio. In humans, FBs have been epidemiologically associated with esophageal cancer. The exposure evaluation of different populations is of primordial interest for public health maintenance. This might be accomplished through the determination of biomarkers, such as the Sa/So ratio, in biological samples. This paper presents the optimization of an analytical methodology for the determination of the Sa/So ratio in human urine. Isolation of exfoliated cells from urine, followed by extraction with ethyl acetate, derivatisation with naphthalene-2,3-dicarboxaldehyde (NDA), and detection and quantification by liquid chromatography (LC) with fluorescence detection (FD) allowed adequate sensitivity, accuracy, and precision. The application of this analytical procedure to a rural and an urban population from the Central zone of Portugal demonstrated that the Sa/So ratio was 0.43+/-0.22 and 0.42+/-0.17, respectively. Regarding the rural population, the Sa/So ratio presented mean levels of 0.48+/-0.22 for females, and 0.30+/-0.16 for males. Concerning the urban population, the Sa/So ratio presented a mean of 0.44+/-0.18 for females, and was 0.29 in the only male sample where its establishment was possible. The statistical analysis showed that no significant differences were found between the different studied populations.

Comparison of three solid‐phase extraction processes in quantification of ciprofloxacin and enrofloxacin in pork meat

Due to strong implications for food safety, control of fluoroquinolones residues in swine meat should be undertaken to verify compliance of the contamination levels with the maximum residue limits recently updated by Commission Regulation (EU) No. 37/2010 of 22 December 2009. Solid-phase extraction is widely used in antibiotic analysis in food of animal origin. In this study, the results of a comparative study using different types of solid-phase extraction columns, HLB, MCX, and MAX, for ciprofloxacin and enrofloxacin analysis, in pork meat, are presented. In addition, diverse sample treatments for defatting, precipitate proteins, eliminate cations, and increase the ionic strength, were used to obtain the most suitable method of analysis. Only the MCXs use followed by liquid chromatography with fluorescence detection resulted in chromatograms that allow the quantification at maximum residue limits. The validation method, in terms of CCα and CCβ, recovery and precision determination, was according to the EU Decision 2002/657/EC. This procedure was used in the analysis of 50 samples of pork meat of Portuguese origin. Only two samples presented residues of enrofloxacin at 30 and 42 μg/kg, values under the legal maximum residue limit.

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.