Science of the Total Environment 637–638 (2018) 1363–1371 Contents lists available at ScienceDirect Science of the Total Environment j ourna l homepage: www.e lsev ie r .com/ locate /sc i totenv Ecotoxicological effects of losartan on the brownmussel Perna perna and its occurrence in seawater from Santos Bay (Brazil) Fernando Sanzi Cortez a,b, Lorena da Silva Souza c, Luciana Lopes Guimarães a, João Emanoel Almeida d, Fabio Hermes Pusceddu a, Luciane Alves Maranho a,b, Luciana Gonçalves Mota d, Caio Rodrigues Nobre b, Beatriz Barbosa Moreno d, Denis Moledo de Souza Abessa b, Augusto Cesar a,d, Aldo Ramos Santos a, Camilo Dias Seabra Pereira a,d,⁎ a Unisanta - Universidade Santa Cecília, Santos, SP, Brazil b Unesp - Universidade Estadual Paulista Julio de Mesquita, São Vicente, SP, Brazil c UCA - Universidad de Cádiz, Spain d Unifesp - Universidade Federal de São Paulo, Santos, SP, Brazil H I G H L I G H T S G R A P H I C A L A B S T R A C T • Losartan concentrations in seawater from Santos bay ranged from 0.2 to 8.6 ng/L. • Reproductive parameters were altered after acute exposure up to 75 mg/L. • Cyto-genotoxic effects observed after short-term exposure (48–96 h) to ng/L • Perna perna is a sensitive model for assessing losartan toxicity. • Lysosomal membrane stability was the most sensitive endpoint. ⁎ Corresponding author at: Departamento de Ciências d E-mail address: camilo.seabra@pq.cnpq.br (C.D.S. Pere https://doi.org/10.1016/j.scitotenv.2018.05.069 0048-9697/© 2018 Elsevier B.V. All rights reserved. a b s t r a c t a r t i c l e i n f o Article history: Received 8 March 2018 Received in revised form 4 May 2018 Accepted 5 May 2018 Available online 22 May 2018 Editor: D. Barcelo The antihypertensive losartan (LOS) has been detected inwastewater and environmentalmatrices, however fur- ther studies focused on assessing the ecotoxicological effects on aquatic ecosystems are necessary. Considering the intensive use of this pharmaceutical and its discharges into coastal zones, our study aimed to determine the environmental concentrations of LOS in seawater, as well as to assess the biological effects of LOS on thema- rine bivalve Perna perna. For this purpose, fertilization rate and embryolarval development were evaluated through standardized assays. Phase I (ethoxyresorufin O deethylase EROD and dibenzylfluorescein dealkylase DBF) and II (glutathione S-transferase GST) enzymes, glutathione peroxidase (GPx), Cholinesterase (ChE), lipoperoxidation (LPO) and DNA damage were used to analyze sublethal responses in gills and digestive gland of adult individuals. Lysosomal membrane stability was also assessed in hemocytes. Our results showed the oc- currence of LOS in 100% of the analyzed water samples located in Santos Bay, Sao Paulo, Brazil, in a range of 0.2 ng/L–8.7 ng/L. Effects on reproductive endpoints were observed after short-term exposure to concentrations up to 75 mg/L. Biomarker responses demonstrated the induction of CYP450 like activity and GST in mussel gills ex- posed to 300 and 3000 ng/L of LOS, respectively. GPx activity was also increased in concentration of exposure to 3000 ng/L of LOS. Cyto-genotoxic effects were found in gills and hemocytes exposed in concentrations up to 300 ng/L. These results highlighted the concern of introducing this class of contaminants into marine Keywords: Antihypertensive Seawater Emerging contaminants Pharmaceuticals Ecotoxicology o Mar, UNIFESP, Campus Baixada Santista, Maria Maximo st. 168, PC 11030100, Brazil. ira). https://doi.org/10.1016/j.scitotenv.2018.05.069 camilo.seabra@pq.cnpq.br Journal logo https://doi.org/10.1016/j.scitotenv.2018.05.069 Unlabelled image http://www.sciencedirect.com/science/journal/00489697 www.elsevier.com/locate/scitotenv http://crossmark.crossref.org/dialog/?doi=10.1016/j.scitotenv.2018.05.069&domain=pdf 1364 F.S. Cortez et al. / Science of the Total Environment 637–638 (2018) 1363–1371 environments, and pointed out the need to include antihypertensive compounds in environmental monitoring programs. © 2018 Elsevier B.V. All rights reserved. 1. Introduction The current number of elderly people in theworld is estimated to be approximately 901 million (equivalent to 12.3% of the world popula- tion) but this number is continuously increasing. The scenario for 2050 is that the population of the elderly surpasses two billion, representing about 22% of the global population (Francisco, 2017). The increasing age of the population, stress, sedentary lifestyle, diet habits are directly associated with high blood pressure (Brazilian Archives of Cardiology, 2016), which makes the use of antihypertensive drugs more frequent. Nowadays it is estimated that over a billion adults worldwide are hypertensive and this figure is projected to reach 1.56 billion by 2025 (Jarari et al., 2016). In this sense, the angiotensin II receptor antagonist class (ARA) has been widely prescribed (Bayer et al., 2014; Knopf and Grams, 2013). In number of prescriptions in United States of America (USA), antihy- pertensive drugs were classified as “Top 1” from 2011 to 2015 (IMS, 2016). Gu et al. (2012) performed a compilation of data from The Na- tional Health and Nutrition Examination Surveys (NHANES - USA) and verified that between 2001 and 2010 there was a 100% increase in the use of ARA. In this period the pharmaceuticals valsartan and losartan (LOS)were classified as the seventh and ninthmost used antihyperten- sive drugs. LOS has also had a significant increase in the consumption in Brazil (23.34%) since 2010, emerging as the main hypertensive drug freely distributed throughout the public health network (Silva et al., 2017). Taking into account thewide use of this therapeutic class, it is impor- tant to point out thewastewater as amain source of aquatic contamina- tion due to the absence or inefficacy of wastewater treatment plants (WWTPs) (Larsson et al., 2007; Bayer et al., 2014). Gurke et al. (2015) determined for LOS a removal rate within a range of 50% to 80% in a sewage treatment plant (STP) including coarse and fine screens, a grit chamber with integrated fat trap, primary clarifiers, biological nitrogen removal and chemical precipitation of phosphorus. The occurrence of Losartan (LOS), an antihypertensive of the ARA class, has been detected in effluents of WWTPs, water supply and environmental matrices (Huerta-Fontela et al., 2011; Godoy et al., 2015a). In samples of amunic- ipal effluent in India, which receives wastewater from bulk drug manu- factures, LOSwas detected in concentrations ranging from 2400 to 2500 μg/L (Larsson et al., 2007). A study carried in Portugal by Santos et al. (2013) revealed the occurrence of LOS in concentration ranging from 59 to 910 ng/L in hospital effluents while the maximum concentration detected in samples of a STP was 364 ng/L. Gros et al. (2017) detected concentration of LOS varying from 705 to 980 ng/L in effluents of a me- dium scaleWWTP in Sweden. In the same study, in a large scaleWWTP, concentrations of LOS of 450 ng/L and 270 ng/L in influent and effluent samples were detected, respectively. Gurke et al. (2015) found a maxi- mum concentration of 333 ng/L in municipal effluent samples in Germany. LOS was also detected in supply water (Spain) with a maxi- mum concentration of 620 ng/L (Huerta-Fontela et al., 2011). In a Brazilian coastal region (São Paulo), Pereira et al. (2016) found LOS in concentrations ranging from 11.8 ng/L to 32 ng/L. With regard to ecotoxicological studies of antihypertensives, Godoy et al. (2015a) pointed out a lack of data related to this therapeutic class, with 60% of the studies conducted until 2014 employing only acute standardized toxicity tests to assess biological effects. The same authors concluded that there was a need of more studies on the potential risk of antihypertensives in marine/estuarine ecosystems. Filter feeding organisms with sessile habits and wide distribution have been used as sentinel organisms in ecotoxicological studies and marine biomonitoring (Gerges, 1994). In Brazil, the brown mussel Perna perna has beenwidely used both as seafood and as sentinel organ- isms in monitoring of anthropogenic pollution trends in coastal waters (Cortez et al., 2012; Trevisan et al., 2014; Pereira et al., 2014; Ortega et al., 2018). Based on previous studies, LOS monitoring in aquatic environments should be considered, taking into account factors such as (i) its occur- rence inwastewater and environmentalmatrices; (ii) it is one of the an- tihypertensive drugs mostly used in different regions; (iii) increasing density of the elderly population leading to higher environmental con- centrations in near future scenarios. In this scenario of high consumption and previous detection of LOS in São Paulo coastal zone (Pereira et al., 2016), our study measured en- vironmental concentrations of LOS in Santos Bay and employed ecotox- icological assays to elucidate metabolism and biological responses in different life stages of the brown mussel Perna perna. 2. Methods 2.1. Chemical Standard of LOS 2 Butyl 4 chloro 1 {[2′ (1H tetrazol 5 yl) (1,1′ biphenyl) 4 yl]methyl} 1H imidazole 5 methanol monopotassium salt, (CAS number 124750-99-8, purity ≥98%) as well as all other chemicals employed in this study were purchased from Sigma-Aldrich (Steinheim, Germany). 2.2. Study area and water sampling The estuarine region of Santos and São Vicente is located in São Paulo coastal zone, southeastern Brazil. It has an industrial complex; the largest port in Latin America and it is a touristic and densely popu- lated area, where domestic sewage is collected, preconditioned and discharged via submarine outfalls 4.5 kmaway from the beach in Santos Bay (Fig. 1). Water samples from the water column (surface — S and bottom — B) were collected at each sampling station in March 2017, in the vicini- ties of the submarine sewage outfall in Santos Bay, considering the pos- sibilities of effluent plume dispersion. The sampling stations were defined according to the study carried out by Pereira et al. (2016), ex- cept station 6. At each sampling station, 3 L of S (−1 m) and B water (−8 m) were collected by using a Van Dorn bottle. The samples were placed into amber glass bottles previously cleaned with HNO3, metha- nol and distilled water and then transported to the laboratory in an in- sulated box with ice (b6 °C) and placed in a freezer at −20 °C until processing time. 2.3. Sample preparation and LC–MS/MS analysis Seawater samples (field) andwater samples frombioassays (test so- lutions) were prepared according to Pereira et al. (2016). Briefly, the pH of each sample was adjusted to 7.0 ± 0.5 prior to extraction, using an HCl solution (1 M) and then samples were filtered through Whatman filter paper (GF/C diameter 47 mm, particle retention 1.2 μm, Merck, Darmstadt, Germany). The filters were washed with 2 mL of methanol and the methanol extract collected was added to the filtered sample. The samples were then submitted to solid phase extraction using Chromabond HR-X cartridges (3 mL, 200 mg, Macherey-Nagel, Düren, Germany). The SPE cartridgeswere pre-conditionedwith 5mL ofmeth- anol and 5 mL of Milli-Q water and the filtered samples (mixed with Fig. 1. Sampling stations in Santos Bay (São Paulo, Brazil). 1365F.S. Cortez et al. / Science of the Total Environment 637–638 (2018) 1363–1371 methanol extract) were loaded into the cartridges. After the samples were loaded, the cartridges were rinsed with 5 mL of Mili-Q water (2×) and then driedunder vacuum for 30min. The elution stepwas per- formed with 5 mL of acetone and twice with 5 mL of methanol. After elution, the samples were dried under nitrogen flow, resuspended in 1 mL with a solution of water/acetonitrile (95:5, v/v) and filtered in a 0.45 μM filter (Millipore) before MS analysis. 2.4. LC–MS/MS analysis For analyses of field seawater samples, 10 μL of each sample were analyzed by anHPLC Agilent 1260 (Agilent Technologies, CA, USA) com- bined with a 3200 QTRAP hybrid triple quadrupole/LIT (linear ion trap) mass spectrometer ABSciex, Ontario (Canada). Seawater samples were analyzed by an Agilent Eclipse XDB-C18 4.6 × 50 mm, 1.8 μm column at 25 °C, and the mobile phase was in 0.1% formic acid (Sigma-Aldrich LC–MS Grade) in water (solvent A) and acetonitrile (J.T. Baker LC–MS Grade) (solvent B). A linear gradient of 0.7mL·min−1was used, starting with amixture of 95% solvent A and 5% solvent B. The solvent A percent- age was decreased linearly from 95% to 5% over the course of 5 min and this conditionwasmaintained for 1min. Themixturewas then returned to the initial conditions over the course of 2 min, as described in Pereira et al. (2016). LOSwas detected and quantified using ESI ionization (pos- itivemode) inMultiple ReactionMonitoring (MRM)mode (Table 1), ac- cording Pereira et al. (2016). The ion source parameters were (i) curtain gas - 20 a.u; (ii) collision gas - 8 a.u; (iii) ion spray voltage - 5500; (iv) Table 1 Parameters of multiple reactions monitoring for the positive ion mode, limit of detection, limit of quantification and retention time. Compounds Q1 Q3 DP (V) CE (V) CXP (V) LOD (ng/L) LOQ (ng/L) RT (min) Losartan 423.2 207.2 21 31 6 0.01 0.04 4.84 405.2 21 17 4 Q1 (first quadrupole); Q3 (last quadrupole); DP (declustering potential); CE (collision en- ergy); CXP (collision exit potential); LOD (limits of detection); LOQ (limits of quantifica- tion); RT (retention time); MIM (multiple ion monitoring). In Q3, in the upper cell is the quantifier ion and in the lower cell is the qualifier ion. source gas temperature - 650 °C; (v) ion source gas 1–45 a.u; (vi) ion source gas 2–65 a.u. Amatrix-matched calibration curvewas employed, as described by Wille et al. (2010). For determining the LOS concentration in the test solutions from bioassays, 1 L of the spiked water was collected at the beginning of the experiment, then the same procedures adopted for the field seawa- ter samples were applied, and mass spectrometry analyses were per- formed using a Varian 310 Triple-Quadrupole mass spectrometer (Varian Inc., Walnut Creek, CA) with an ESI source (ESI-MS), by direct infusion. Data acquisition was controlled with Varian MS Workstation version 6.9 (Varian Inc.). Sample analysis was carried out in positive ESI mode with a needle voltage of 20 kV. The capillary temperature was 200 °C, the drying gas pressure was 20 psi and the nebulizing gas pressurewas 40psi. LOSwas detected and quantified usingMultiple Re- action Monitoring (MRM) mode, with the selection of a precursor ion (423.2 m/z) and two ion products to quantify and qualify LOS (207.2 and 405.2 m/z, respectively). A matrix-matched calibration curve was employed, as described by Wille et al. (2010). 2.5. Mussel acclimation and maintenance conditions Adult mussels (average size 6.2 ± 1,3 cm) were purchased from a mussel farming located in Cocanha Beach (Caraguatatuba, SP, Brazil), due to its good environmental quality, where nowater or sediment con- tamination have been reported by the State Environmental Agency (CETESB, 2016). The organisms were acclimatized for one week (300 L tank), receiving food supply (microalgae), and kept in tanks under con- stant temperature (24 ± 2 °C), aeration and filtration system. 2.6. Toxicity assays 2.6.1. Fertilization assay Fertilization assay was performed following USEPA (1991) protocol adapted to Perna perna according to Zaroni et al. (2005). The gametes (eggs and sperm) were obtained by thermal stimulation (from 10 °C to 30 °C) of 20 individuals during 30 min. As soon as the organisms started releasing the gametes, they were removed from the tray to Image of Fig. 1 1366 F.S. Cortez et al. / Science of the Total Environment 637–638 (2018) 1363–1371 prevent fertilization. The gametes from 3males and 3 females were col- lected separately and transferred to glass beakers. A stock solution of 1000 mg/L was prepared in filtered seawater (0,22 μm membrane) and from this solution all the LOS tested concen- trations were prepared. The sperm was exposed to concentrations of 31.25; 62.5; 125; 250 and 500 mg/L for 60 min, in quadruplicate. After this period, a suspension containing approximately 2000 ovules was added to the test recipients. Forty minutes after adding the eggs, the assay was finished by adding 0.5 mL of formaldehyde in each replicate. The first 100 eggs from each replicate were analyzed and fertilization was identified by observation of the occurrence of themembrane of fer- tilization or first cellular divisions. The results were expressed by the concentration that inhibited the fertilization rate in 50% of exposed or- ganisms (IC50; 1 h). 2.6.2. Embryo-larval development assay In order to assess the embryolarval development rate in mussels zy- gotes exposed to LOS, experiments were performed according to the protocol recommended by ASTM (1992) for mussels, with minor adap- tations proposed by Zaroni et al. (2005) concerning salinity, which was elevated to 35 ± 1 ppm. Twenty adult individuals were induced to spawn by thermal stimulation. The gametes from 3males and 3 females were collected separately and transferred to glass beakers. The fertiliza- tion was obtained by adding 2 mL of sperm solution to the 200 mL of ovules solution. The rate of fertilized eggs was estimated with the sup- port of Sedgwick-Rafter chamber, and about 500 embryos were trans- ferred to glass tubes containing different nominal concentrations of LOS (5; 10; 25; 50; 75 and 100 mg/L), for a period of 48 h at a temper- ature of 25 °C and salinity of 35 ppm. After the exposure period, the assaywasfinished by adding 0.5mLof formaldehyde, and the first 100 larvaewere analyzed for each replicate. Larvae developed to D-phase were considered normal and a mean percentage of normal development was obtained for each tested con- centration. Thereafter were calculated: (i) the concentration of LOS that caused inhibition of the development of the embryos to 50% of the exposed organisms (IC50; 48 h); (ii) the highest concentration tested of LOS that did not cause adverse biological effects on exposed or- ganisms - “NOEC” (No Observable Effect Concentration); and (iii) the lowest LOS concentration tested that caused significant adverse biolog- ical effects on exposed organisms - “LOEC” (Lowest Observable Effect Concentration). 2.7. Biomarkers assay 2.7.1. Mussel exposure The mussels were acclimatized for one week to clean seawater under controlled conditions. After this period, the organisms (n = 21) were exposed in aquaria with different concentrations of LOS (30; 300 and 3000 ng/L) and water control, in triplicate, for 96 h. These concen- trations were set according to studies that detected LOS in effluents and surface waters. In each replicate of all treatments, 7 organismswere exposed in each aquarium containing 10 L of the test solution. Since LOS has been con- sidered a stable molecule with low hydrolysis and biodegradation pro- cess (FDA, 2002), controls and test solutions were renewed each 48 h. The physico-chemical parameters were controlled during the experi- ment without significant changes (salinity 35 ppm, dissolved oxygen 8 ± 0.5 mg/L, pH 8.3–8.5). The natural seawater used in the assay was filtered through a membrane of 200 μm in order to filter higher partic- ulates and maintain a food supply (phytoplankton) because during the experiment no other type of food was provided. 2.7.2. Tissue extraction and storage The hemolymph, gill and digestive gland tissues were extracted for analysis at T0, T48 h and T96 h. Organisms were removed from each replicate of the different treatments, totaling 10 mussels for T48 h and 10 mussels for T96 h. Immediately after removal of the hemolymph, the slides were prepared for analysis of the lysosomalmembrane stabil- ity (LMS). After this procedure, gills and digestive glands were dis- sected, separated into microtubes and stocked in ultrafreezer (−80 °C) until the biomarker analyses were carried out. 2.7.3. Neutral red retention time assay (NRRT) NRRT assaywas performed following themethod described by Lowe and Pipe (1994) to assess the lysosomal membrane stability (LMS). Themusselswere removed from the aquariums andwith the aid of a syringe containing physiological saline solution (pH 7.3) 40 μL of hemo- lymph from each organism was withdrawn and placed on glass slides, whichwere transferred to a dark and humid chamber for 15min to pro- mote cell attachment. Then, the excess liquid was removed and 40 μL of the neutral red dye (NR) were added onto all the slides. After another 15 min of incubation the slides were analyzed periodically (every 15 min). The endpoint was the time when at least 50% of the examined cells by optical microscopy (400×) exhibited dye loss from the lyso- somes to the cytosol or structural abnormalities. 2.7.4. Tissue preparation Gills and digestive glands from each organismwere defrosted on ice and homogenized with 4 times the volume of 100 mM NaCl buffer, 25 mM HEPES-NaOH, 0.1 mM EDTA, 0.1 mMDTT, pH 7.5, in a homoge- nizer (Tissue Tearor). After homogenization, the extractwas centrifuged at 4 °C at 15,000g for 20 min, thus obtaining the supernatant fraction (15,000g) where the activities of CYP450 like (Ethoxyresorufin O deethylase - EROD and Dibenzylfluorescein dealkylase - DBF), Glutathione-S-transferase (GST), Glutathione peroxidase (GPX), Cho- linesterase (ChE) were analyzed, as well as the concentration of pro- teins in the cytoplasmic cell fraction, according to the method of Bradford (1976). An aliquot of homogenized tissue was separated for lipoperoxidation (LPO) and DNA damage analyzes, and the protein con- centration in this aliquot was also evaluated by the method of Bradford (1976). 2.7.5. Ethoxyresorufin O deethylase (EROD) The EROD activity was evaluated by the adapted test of Gagné and Blaise (1993). The transformation of 7 hydroxyresorufin in resorufin (EROD activity) was determined fluorometrically using 520 nm (excitation) and 590 nm (emission) filters. The determination of 7 hydroxyresorufin in the samples was performed using a standard calibration curve of 7 hydroxyresorufin. The results were expressed as pmol/min/mg protein. 2.7.6. Dibenzylfluorescein dealkylase (DBF) The determination of DBF activity was performed according the method described by Gagné et al. (2007) using as substrate 10 μM dibenzylfluorescein and incubated with a solution of 1 mM NADPH in a test solution (50 mM NaCl containing 10 mM HEPES-NaOH, pH 7.4). The fluorescence of the sample wasmeasured with a 485 nm excitation and 516 nm emission filter. Results were expressed in pmol/min/mg protein. 2.7.7. Glutathione S-transferase activity (GST) The method used to determine GST activity was adapted from Mc Farland et al. (1999). The activity was analyzed using 42 mM 1 chloro 2.4 dinitrobenzene (CDNB), 1 mM GSH as substrate and measured at 340 nm every 30 s for 3 min. Results were expressed as OD/min/mg proteins. 2.7.8. Glutathione peroxidase activity (GPx) Themethodology used to determine GPx activity was developed ac- cording to the method proposed by Mc Farland et al. (1999). GPx activ- ity was measured at 340 nm every 2 min for 10 min, using 1 mM cumene hydroperoxide as the substrate. The decrease in absorbance of Table 3 Nominal and measured concentrations of LOS in fertilization and embryo-larval assays (T0). Nominal concentration (mg/L) Measured concentration (mg/L) 0 bLOD 5.0 4.72 25.0 23.43 75.0 71.13 125.0 118.36 250.0 232.92 500.0 469.36 LOD - limits of detection. 1367F.S. Cortez et al. / Science of the Total Environment 637–638 (2018) 1363–1371 NADPHmeasured at 340 nmduring the oxidation of NADPH to NADP+ was indicative of GPx activity. Results were expressed as nmol/min/mg protein. 2.7.9. DNA damage DNA damage was evaluated by the Olive (1988) alkaline precipita- tion assay, using fluorescence to quantify traces of DNA (Gagné and Blaise, 1993). Fluorescence was measured using 360 nm filter (excitation) and 450 nm (emission) and a salmon sperm genomic DNA standard (Sigma) was employed for calibration. The results were expressed in μg/mg protein. 2.7.10. Lipid peroxidation Analysis of lipid peroxidation was performed by the thiobarbituric acid method (Wills, 1987). This determination was employed by fluo- rescence using 516 nm (excitation) and 600 nm (emission). The tetramethoxypropane standards were prepared in homogenization so- lution. The results were expressed in μM TBARs/mg proteins. 2.7.11. Cholinesterase (ChE) The analysis of the ChE activities of the gills and digestive glands were performed according to the method described by Ellman et al. (1961) using a concentration of 0.3 mM acetylcholine iodide in the en- zyme assay. The variation of absorbance per minute at 412 nm at 25 °C was recorded in a spectrophotometer. The results were expressed in μmol DNTB/min/mg protein. 2.8. Statistical analysis For the fertilization assay, an EC50 was calculated by Trimmed Spearman-Karber. The linear interpolation method was used to calcu- late the IC50 (48 h) for the embryo-larval development assay. t-Test was employed to assess differences between T0 and water controls. Since no difference was detected, water controls were used as refer- ences for 48 h and 96 h. One-way ANOVA followed by the Dunnett's test was used to identify the concentrations significantly different of water controls. Statistical differences were considered significant when p ≤ 0.05. The software Prism v.7 was employed for ANOVA and pos hoc analysis. 3. Results 3.1. Environmental concentrations Table 2 shows the environmental concentrations of LOS. This phar- maceutical compound was detected in all sampling stations including the reference area. 3.2. Fertilization rate and embryo-larval development assays The measured concentrations of LOS at the beginning of the expo- sure experiment for the fertilization and embryo-larval assays (T0) are shown in Table 3. The EC50 1 h for the rate of fertilization of P. pernawas calculated as 219.2 mg/L, with a confidence interval ranging from 208.3 to 231.8mg/L. The normal embryo-larval development of 50% exposed zy- goteswas inhibited in the concentration of 84.6mg/L (IC50 48 h)with a Table 2 Environmental concentration of LOS in surface and bottom water samples (1–6 sampling stations) from Santos Bay. LOS concentration (ng/L) 1 2 3 4 5 6 Surface 8.70 3.89 2.10 0.295 3.62 0.60 Bottom 2.46 1.59 1.50 1.18 1.07 1.79 confidence interval ranging from 62.8 to 87.5 mg/L, while NOEC and LOEC were 50 mg/L and 75 mg/L, respectively. 3.3. Biomarkers responses The nominal and the measured concentrations of LOS at time T = 0 are reported in Table 4. The nominal concentrations were similar to those determined at T = 0. The lysosomal membrane stability (LMS) showed a concentration- time response, with a significant decrease in the NR retention time after exposure to 3000 ng/L in 48 h, and down to 300 ng/L after 96 h (Fig. 2). The activities of EROD, DBF, GST, GPX and ChE as well as the DNA damage and LPO in the gill tissue are shown in Fig. 3. When gills were evaluated, the activity of EROD and LPO did not show significant difference in relation to the control in any of the ana- lyzed times. With regard to the activity of the DBF, only the concentra- tion of 300 ng/L showed significant difference, with induction of the activity after 96 h. Significant increase of GST, GPx activities, as well as DNA primary damages were found after 48 h exposure to 3000 ng/L. With regard to the ChE, an induction of the activity of this enzyme in the concentration of 3000 ng/L at 96 h of exposure was observed. The activities of EROD, DBF, GST, GPX and ChE as well as the DNA damage and LPO in the digestive glands are shown in Fig. 4. Significant differences were only detected to EROD (inhibition of activity) in mus- sels exposed to 300 ng/L and 3000 ng/L after 96 h. 4. Discussion The pharmaceuticals represent a major group of emerging pollut- ants, which have been found in freshwater and marine environments (UNESCO, 2017), representing a global challenge to water quality in terms of environmental status and human supply. Their occurrence in the aquatic compartment leads to the need of knowledge about possible harmful effects of this class of substances on the biota. LOS was quantified in different rivers of the Iberian Peninsula in a concentration ranging from 0.17 ng/L to 220.63 ng/L (Osorio et al., 2016). In the marine environment, Moreno-González et al. (2015) de- tected concentrations of 104 ng/L and 6.47 ng/g in samples of water and sediment from the Spanish coast, respectively. In a tropical coastal zone (Santos Bay, Brazil) LOS concentrations varied from 11.8 ng/L to 32 ng/L in marine water (Pereira et al., 2016). In the present study, the concentrations of LOS detected in surface and bottom samples ranged from 0.29 ng/L to 8.70 ng/L. These concentrations were lower than those reported in the study performed by Pereira et al. (2016); Table 4 Nominal and measured concentrations of LOS (T0) in the experiments with biomarkers. Nominal concentration (ng/L) Measured concentration (ng/L) 30 27.3 300 276.9 3000 2811.2 Fig. 3. Biomarker responses in gill tissues (mean ± SE). An asterisk indicates Fig. 2. Neutral red retention time assay (mean ± SE). An asterisk indicates a significant difference from the control (ANOVA - Dunnett's, p b 0.05). 1368 F.S. Cortez et al. / Science of the Total Environment 637–638 (2018) 1363–1371 however, these authors detected LOS only in 30% of samples collected three years before, whereas the present study has found LOS in 100% of samples, including the farthest station of the effluent discharged (sta- tion 6). Gros et al. (2012) also detected LOS in marine surface water (Spain) in a similar range found in the present study. The frequent oc- currence of this antihypertensive in Santos bay could be related to aging population, which according Bersusa et al. (2010), 79.3% of the hypertensive people in Santos are over 40 years old, and according to the Brazilian Institute of Geography and Statistics (IBGE, 2010), the cit- ies of Santos and SãoVicentemake up82.3% of the population at this age group. With regard to adverse biological effects, we have employed stan- dardized ecotoxicology assays and a suite of biomarkers responses of a marine invertebrate. P. perna mussel is considered a key species in Brazilian rocky shores and broadly cultivated for human consumption. The results obtained in the fertilization and embryo-larval assays fit a significant difference from the control (ANOVA - Dunnett's, p b 0.05). Image of Fig. 3 Image of Fig. 2 Table 5 Toxicity assays with LOS and aquatic organisms of different trophic levels. Losartan Species Endpoint LC50/EC50 mg/L NOEC mg/L LOEC mg/L References Daphnia sp. Lethal effects 331 80 nd FDA (2002) Pimephales promelas N1000 100 nd Oncorhynchus mykis N929 N929 nd S. capricornutum Cell growth nd 143 nd Lemna minor Growth rate No data 0.78 1.56 Godoy et al. (2015b)Frond number 63.7 nd nd Total frond area 64.6 nd nd Fresh weight 76.9 nd nd Lytechinus variegatus Embryo-larval development nd 50 70 Yamamoto et al. (2014) nd - no data. Fig. 4. Biomarker responses in digestive gland (mean ± SE). An asterisk indicates a significant difference from the control (ANOVA - Dunnett's, p b 0.05). 1369F.S. Cortez et al. / Science of the Total Environment 637–638 (2018) 1363–1371 into the same order of magnitude (mg/L) in relation to the previous studies (Table 5). The bivalve Perna perna presented values of NOEC (50 mg/L) and LOEC (75 mg/L) similar to those found in a study employing the sea ur- chin Lytechinus variegatus (Yamamoto et al., 2014). Considering all the previous studies, it is possible to note that the aquatic macrophyte Lemna minor shows a relatively higher sensitivity to LOS. Another anti- hypertensive pharmaceutical of the “sartans” group (Valsartan) has been found in the aquatic environment (Klosterhaus et al., 2013; Bayer et al., 2014). Bayer et al. (2014) found EC50 (72 h) of N115 mg/L and NOEC of 85 mg/L to microalgae Desmodesmus subspicatus exposed to Valsartan. In the same study effect concentrations for the fish Onco- rhynchus mykiss and for Daphnia magna of N100 mg/L and N580 mg/L, respectively, were reported. Using the sea urchin Lytechinus variegatus, Yamamoto et al. (2014) conducted a studywith Valsartan and observed NOEC of 12.5 mg/L and LOEC of 25 mg/L. This author related the higher Log Kow of Valsartan (3.65) to its higher toxicity when compared to LOS (Log Kow 1.19), considering that such property determines a higher ca- pacity to bioaccumulate and reach target organs. Image of Fig. 4 1370 F.S. Cortez et al. / Science of the Total Environment 637–638 (2018) 1363–1371 The data obtained through the chemical analysis to determine the real concentrations of LOS in the biomarkers assays were similar to the nominal concentrations. Thus, the nominal concentrations were employed to calculate NOEC and LOEC. Biomarker responses shed light on the metabolism and sub-lethal effects of the antihypertensive LOS in a non-target marine organism. The activity of DBF was induced in the gills after 96 h of exposure at 300 ng/L, whereas GST was induced in gills of mussels exposed to 3000 ng/L after 48 h. The metabolism of this pharmaceutical in verte- brates occurs through the Cytochrome P450 (CYP 450) system specifi- cally through the CYP2C9 and CYP3A4 families (Ripley and Hirsch, 2010), which is in agreement with our results obtained in a marine bi- valve. Phases I and II of the detoxification system produce reactive oxy- gen species (ROS) andmetabolites, which are able to promote oxidative stress and cellular damages. This fact can induce the activity of antioxi- dant enzymes to protect against oxidant damages (Maranho et al., 2014). The activity of GPx was also induced at 3000 ng/L (48 h), which shows a correlation with ROS generated by phase I detoxification sys- tem. The data on sub-lethal effects, especially DNA damage and ChE ac- tivity, suggest a possible overlapping of the antioxidant system, since these effects were estimated in the same concentrations (3000 ng/L). The enzymatic activity of the ChE is considered a biomarker of neuro- toxicity effects and, in bivalve mollusks the alterations in the activity of this enzyme can be related to the control of the closure of the valves, alterations in the muscle movements, in the ciliary beating, among others (Viarengo et al., 2007). Previous studieswith pharmaceuticals re- ported the induction of the activity of ChE in bivalvemollusks (Mesquita et al., 2011; Gonzalez-Rey and Bebianno, 2014) a fact that coincides with the data obtained for LOS in the present study. Zhang et al. (2002) reported that the induction of ChE is associated with cellular ap- optosis in several human cells and other mammals, possibly because ChE is released after the rupture of the cellular membrane. It is possible that the induction of the activity of ChE is related to the membranothropic effects and the resulting cellular apoptosis caused by the LOS. The reduced stability of the lysosomal membranes confirms this hypothesis, since dependent concentration-time responses were found after exposures up to 300 ng/L. Furthermore, toxicological studies reported the ability of the LOS to inhibit H+ ATPase activity in rodent kidney cells (Valles and Manucha, 2000) and the capacity of this pharmaceutical to interact with the bi- layer cell membrane altering the fluidity of membrane (Zoumpoulakis et al., 2003). These characteristicsmay also be related to effects detected on the lysosomal membrane stability. This biomarker showed the most sensitive response, which coincides with previous studies on the cyto- toxicity of pharmaceuticals compounds (Cortez et al., 2012; Pusceddu et al., 2018). Aguirre-Martínez et al. (2013) evaluating the toxicity of ibuprofen, carbamazepine and novobiocin on the crab Carcinus maenas, concluded that stability of lysosomal membrane is a good indicator of general stress in organisms exposed to pharmaceuticals at environmen- tally relevant concentrations. With regard to the sensitivity of the different mussel's life stages used in the present study, the endpoints fertilization rate and embryolarval development, although considered phases highly sensi- tive to environmental pollutants (Beiras et al., 2003), were not respon- sive to detect adverse biological effects in environmentally relevant concentrations of LOS ranging from ng/L to μg/L. On the other hand, the data obtained with the use of biomarkers in adult mussels showed some significant biological effects (DNA damage and reduced lysosomal membrane stability) after short-term exposures at concentration closer to those found in WWTPs effluents and marine surface water (Larsson et al., 2007; Moreno-González et al., 2015; Pereira et al., 2016; Gros et al., 2017). Perna perna mussels showed to be a suitable marine model, which could be employed in future environmental assessments. The gill was the most responsive tissue, showing detoxification (DBF and GST activities) and antioxidante defenses (GPx), but thiswas not able to pre- vent mitochondrial DNA damage. 5. Conclusion The pharmaceutical LOS was quantified in 100% of surface and bot- tom water samples from Santos Bay ranging from 0.295 to 8.70 ng/L. Adverse effects of the antihypertensive Losartan on reproductive pa- rameters of the brownmussel Perna pernawere detected in higher con- centrations (mg/L) after short-term exposure. In spite of the high ecological relevance of these endpoints, they are not expected in realis- tic scenarios of aquatic ecosystems. However, detoxification and antiox- idant systems were induced after exposure to concentrations ranging from ng/L to μg/L, as such as cyto-genotoxic effects in gills and hemo- lymph. These results highlighted the concern of introducing this class of contaminant into marine environments, and pointed out the need to include antihypertensive compounds as targets to wastewater treat- ments plants, such as including them in environmental monitoring programs. Acknowledgments This study was funded by CNPq (Processes n° 481358/2012-9 and n°481553/2012-6). Camilo Dias Seabra Pereira, Augusto Cesar, Denis Abessa, João Emanoel Almeida thank CNPq for fellowships. 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brown mussel Perna perna and its occurrence in seawater from Santos Bay (Brazil) 1. Introduction 2. Methods 2.1. Chemical 2.2. Study area and water sampling 2.3. Sample preparation and LC–MS/MS analysis 2.4. LC–MS/MS analysis 2.5. Mussel acclimation and maintenance conditions 2.6. Toxicity assays 2.6.1. Fertilization assay 2.6.2. Embryo-larval development assay 2.7. Biomarkers assay 2.7.1. Mussel exposure 2.7.2. Tissue extraction and storage 2.7.3. Neutral red retention time assay (NRRT) 2.7.4. Tissue preparation 2.7.5. Ethoxyresorufin O‑deethylase (EROD) 2.7.6. Dibenzylfluorescein dealkylase (DBF) 2.7.7. Glutathione S-transferase activity (GST) 2.7.8. Glutathione peroxidase activity (GPx) 2.7.9. DNA damage 2.7.10. Lipid peroxidation 2.7.11. Cholinesterase (ChE) 2.8. Statistical analysis 3. Results 3.1. Environmental concentrations 3.2. Fertilization rate and embryo-larval development assays 3.3. Biomarkers responses 4. Discussion 5. Conclusion Acknowledgments References