Science of the Total Environment 514 (2015) 273–280

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Occurrence and fate of select psychoactive pharmaceuticals and antihypertensives in two wastewater treatment plants in New York State, USA Bikram Subedi a, Kurunthachalam Kannan a,b,⁎ a Wadsworth Center, New York State Department of Health, and Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, United States b Biochemistry Department, Faculty of Science and Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia

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

• 16 psychoactive pharmaceuticals were found at 0.98–1220 ng/L in wastewater influent. • Over 50% of total mass of 8 psychoactives were found sorbed to particulate matter. • Influx of psychoactives in WWTPs ranged from 0.91 to 347 mg/d/1000 inhabitants. • Environmental emission of psychoactives ranged from 0.01 to 316 mg/d/1000 inhabitants.

a r t i c l e

i n f o

Article history: Received 24 November 2014 Received in revised form 5 January 2015 Accepted 27 January 2015 Available online xxxx Editor: Adrian Covaci Keywords: Pharmaceuticals Psychoactives Metabolites Fate Removal efficiency Sludge Suspended particulate matter Wastewater treatment plant

a b s t r a c t The fates of psychoactive pharmaceuticals, including two antischizophrenics, six sedative–hypnotic–anxiolytics, four antidepressants, four antihypertensives, and their select metabolites, were determined in two wastewater treatment plants (WWTPs) in the Albany area of New York. All target psychoactive pharmaceuticals and their metabolites were found at a mean concentration that ranged from 0.98 (quetiapine) to 1220 ng/L (atenolol) in wastewater and from 0.26 (lorazepam) to 1490 ng/g dry weight (sertraline) in sludge. In this study, the fraction of psychoactive pharmaceuticals that was sorbed to suspended particulate matter (SPM) was calculated for the first time. Over 50% of the total mass of aripiprazole, norquetiapine, norsertraline, citalopram, desmethyl citalopram, propranolol, verapamil, and norverapamil was found sorbed to SPM in the influent. The mass loadings, i.e., influx, of target psychoactive pharmaceuticals in WWTPs ranged from 0.91 (diazepam) to 347 mg/d/ 1000 inhabitants (atenolol), whereas the environmental emissions ranged from 0.01 (dehydro-aripiprazole) to 316 mg/d/1000 inhabitants (atenolol). The highest calculated removal efficiencies were found for antischizophrenics (quetiapine = 88%; aripiprazole = 71%). However, the removal of some psychoactive pharmaceuticals through adsorption onto sludge was minimal (b 1% of the initial mass load), which suggests that bio-degradation and/or chemical-transformation are the dominant mechanisms of removal of these pharmaceuticals in WWTPs. © 2015 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Wadsworth Center, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, United States. E-mail address: [email protected] (K. Kannan).

http://dx.doi.org/10.1016/j.scitotenv.2015.01.098 0048-9697/© 2015 Elsevier B.V. All rights reserved.

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B. Subedi, K. Kannan / Science of the Total Environment 514 (2015) 273–280

1. Introduction Neurological disorders have emerged as a significant public health concern. The total global burden of diseases that result from neuropsychiatric disorders is projected to be the second most frequent by 2020 (Menken et al., 2000). Globally, psychoactive drugs were among the most prescribed classes of pharmaceuticals in 2008, with over 30 billion doses prescribed daily in that year (INCB, 2010). In the USA, the total usage of psychoactive pharmaceuticals increased from 1998 to 2008 by 78% (Gallini et al., 2013), and psychoactives were the top-selling class of prescription medications, with $14.6 billion in sales in 2009 (IMS, 2009). Most psychoactive pharmaceuticals undergo metabolism in the human body and excrete either unchanged, as metabolites, or as conjugates (Calisto and Esteves, 2009). Metabolites of select psychoactive pharmaceuticals are biologically active, and the conjugates can be further transformed to corresponding parent compounds in wastewater treatment processes (Calisto and Esteves, 2009). Psychoactive pharmaceuticals, their metabolites, and conjugates can enter the environment through the discharge of effluents from wastewater treatment plants (WWTPs) (Ternes, 1998), effluents from pharmaceutical industries (Ruhoy and Daughton, 2008), use of treated wastewater for irrigation (Pedersen et al., 2005), and the application of biosolids in agriculture (Kinney et al., 2008; Subedi et al., 2013). Psychoactive pharmaceuticals were reported to occur in wastewater, surface water, and drinking water at ng/L to μg/L levels and in sludge/biosolids at ng/g to μg/g levels (Benotti et al., 2009; Kolpin et al., 2002; Subedi et al., 2013; Wick et al., 2009; Yuan et al., 2013). Studies have reported that exposure of aquatic organisms to psychoactive pharmaceuticals affect reproduction (Brooks et al., 2003), endocrine function (van der Ven et al., 2006), or photosynthesis (Escher et al., 2006). A mixture of psychoactive pharmaceuticals venlafaxine (VLF), fluoxetine, and carbamazepine (CBZ) at environmentally relevant concentrations induced autism-like gene expression in fathead minnows (Thomas and Klaper, 2012). A recent study showed that exposure of fish and benthic invertebrates to psychoactive drugs altered the behavioral responses (Brodin et al., 2014; Rosi-Marshall et al., 2015). To date, however, very little is known about the ecotoxicologic effects of psychoactive pharmaceuticals in aquatic ecosystems (Alonso et al., 2010; Petersen et al., 2014). Psychoactive pharmaceuticals, such as CBZ and VLF, are recalcitrant, and their removal efficiencies in WWTPs were estimated at b20% (Lajeunesse et al., 2012; Zhang et al., 2008). Estimation of removal rates of pharmaceuticals in the aqueous phase alone can under- or over-estimate the actual removal (Petrovic and Barceló, 2007). In the USA, ~240 kg of biosolids are produced for every million liter of domestic wastewater treated (Kinney et al., 2008). The US Environmental Protection Agency (EPA) estimated that ~ 50% of the annual production (8 × 106 tons in 2006) of biosolids is land-applied (USEPA, 2006). Despite the sorption of psychoactive pharmaceuticals to suspended particulate matter (SPM), studies on the fate of these chemicals have focused only on the analysis in filtered wastewater (Lajeunesse et al., 2012; Radjenovic et al., 2009) with the assumption that pharmaceuticals partition mainly to the aqueous phase. Radjenovic et al. (2009) reported mean emission of 7.1, 41.1, and 3.9 g/d of CBZ, atenolol (ATN), and propanolol (PPN), respectively, based on the concentrations measured in filtered wastewater and treated sludge from nine WWTPs, with an average inflow of 42,000 m3/d. In this study, psychoactive pharmaceuticals, including two antischizophrenics [aripiprazole (APPZ) and quetiapine (QTP)], six sedative– hypnotic–anxiolytics [lorazepam (LZP), alprazolam (APZ), diazepam (DZP), oxazepam (OxZP), nordiazepam (NDZP), and CBZ], four antidepressants [VLF, bupropion (BPP), sertraline (STL), and citalopram (CTP)], four antihypertensives [ANL, PPN, diltiazem (DTZ), and verapamil (VPM)], and their select metabolites, were analyzed in wastewater influent, primary effluent, final effluent, SPM, and treated sludge from

two WWTPs in the Albany area of New York. In addition to psychoactive pharmaceuticals, one antiplatelet [clopidogrel (CPG)], one antihistamine [diphenhydramine (DPH)], and their metabolites were analyzed. This is the first study to describe the fate of psychoactive pharmaceuticals including influx assessment as well as the fraction sorbed to the sewage sludge and SPM in WWTPs in the USA. The fraction of each of the target pharmaceuticals sorbed to SPM was calculated and utilized in the estimation of mass loadings and emissions of these chemicals from WWTPs. The removal efficiencies of pharmaceuticals through wastewater treatment processes also were calculated. 2. Material and methods 2.1. Reagents and chemicals Standard stock solutions (100 or 1000 μg/mL) of individual pharmaceuticals and their corresponding isotopically-labeled standards were purchased from commercial vendors, as described elsewhere (Subedi et al., 2013). Purity of all of the standards was ≥ 95%. Formic acid (98.2%) was from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water was prepared with the milli-Q ultrapure system (Barnstead International, Dubuque, IA, USA). All standard stock solutions were stored at − 20 °C. 2.2. Sample collection and preparation Information on the collection of wastewater samples and sludge has been described earlier (Subedi and Kannan, 2014). Briefly, 24-h composite wastewater samples, including raw wastewater (influent), primary treated wastewater (primary effluent), secondary treated wastewater (effluent or final effluent), and sludge were collected over a seven-day period, from July 12 to 18, 2013, consecutively from two WWTPs in the Albany area of New York. The two plants are denoted as WWTPA (population served ~ 15,000) and WWTPB (population served ~100,000), with a treatment capacity of 2.5 and 35 MGD, respectively. Both WWTPs used activated biological treatment. The activated sludge samples (3.9% solid, determined gravimetrically) were collected for seven days in WWTPA; however, activated sludge and dewatered sludge samples were collected from WWTPB for four consecutive days within the sampling week. The activated sludge samples from both WWTPs were the combined sludge produced after primary and secondary treatments (dewatered and thickened). More detailed information on WWTPs is provided elsewhere (Sinclair and Kannan, 2006) and Table S1. All samples were collected in certified pre-cleaned amber glass jars with Teflon-faced caps, shipped to the laboratory, and stored in a refrigerator at 4 °C until extraction. The detailed procedure for the extraction of wastewater, SPM, and sludge has been described elsewhere (Subedi and Kannan, 2014). Briefly, wastewater samples (100 mL) were centrifuged at 5000 ×g for 10 min, and the supernatant was filtered through a glass fiber filter (37 mm, pore size, 1 μm; GE Osmonics Inc., Minnetonka, MN) to separate SPM. The filtered wastewater was spiked with a mixture of labeled internal standards (25 ng), mixed well, allowed to equilibrate for ~ 30 min at room temperature, and extracted by passage through Oasis® HLB 6 cm3 (200 mg; Waters, Milford, MA) solid phase extraction (SPE) cartridges. Prior to use, the cartridges were conditioned with 5 mL of methanol and 5 mL of milli-Q water, and wastewater samples were loaded at ~ 1 mL/min. Cartridges were allowed to dry for ~ 30 min under vacuum and then eluted with 6 mL of methanol followed by 3 mL of a mixture of acetone, methanol, and ethyl acetate (2:2:1 v/v/v). Cartridges were also eluted with 3 mL of methanol containing 5% ammonia. The eluents were combined and concentrated to ~100 μL under a gentle stream of nitrogen at 35 °C using a TurboVap® Evaporator (Zymark, Inc., Hopkinton, MA). The final volume of the extract was adjusted to one milliliter with methanol in an amber glass vial, and 10 μL of the extract was injected into HPLC –MS/MS.

B. Subedi, K. Kannan / Science of the Total Environment 514 (2015) 273–280

Freeze-dried sludge (0.1 g) was spiked with a mixture of internal standards (25 ng) prior to extraction. The entire contents of SPM obtained by centrifugation and filtration of 100 mL of wastewater were transferred into a clean, pre-weighed polypropylene tube along with a preweighed glass-fiber filter, freeze-dried, and analyzed. Spiked sludge samples were vortex-mixed for 1 min and extracted with 6 mL of methanol:water mixture (5:3 v/v) using an ultrasonic bath (Branson® Ultrasonics 3510R-DTH; Danbury, CT) for 30 min. Extracts were centrifuged at 4500 ×g for 5 min (Eppendorf Centrifuge 5804, Hamburg, Germany), the supernatant was collected in a polypropylene tube, and the extraction was repeated with 6 mL of methanol. The extracts were combined and concentrated to ~1 mL under a gentle stream of nitrogen. The concentrated extract was diluted with milli-Q water to ~12 mL and purified by passage through Oasis® HLB (6 cm3, 200 mg) cartridges, as described above for wastewater samples. The final volume of the extract was one milliliter, and 10 μL of the extract were injected into HPLC –MS/ MS for analysis.

275

2.4. Calculations The fraction of the total mass of analytes sorbed to SPM, removal efficiency of pharmaceuticals through the treatment processes, mass loadings in WWTPs, and emission from WWTPs were calculated using the following equations (Eqs. (1)–(4)), as reported by Subedi and Kannan (2014) and Jelic et al. (2012). 

PSPM

 CSPM  MSPM VW  ¼  100 CSPM  MSPM þ CW VW

ð1Þ

       100 100 − Ce  F  þ ðCS  TSPÞ Ci  F  100−PSPM 100−PSPM    Removal efficiency ð%Þ ¼ 100 Ci  F  100−PSPM  100

ð2Þ

2.3. Analysis  Target chemicals were analyzed using an API 2000 electrospray triple quadrupole mass spectrometer (ESI-MS/MS; Applied Biosystems, Foster City, CA, USA), interfaced with an Agilent 1100 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA). The analytes were separated using a Hypersil Gold® column (150 mm × 2.1 mm, 3 μm) (Thermo Scientific, Chelmsford, MA, USA). Methanol and water (0.1% formic acid) were used as mobile phases; a description of the mobile phase gradient flow is presented in Table S2. Target analytes were determined by multiple-reaction monitoring (MRM) in the positive ionization mode. Detailed information on the MS/MS transitions is provided in the Supporting Information (Table S3) (Subedi et al., 2013). Briefly, analyte peak identification was based on the retention time (±0.05 min) and the ratio of qualitative to quantitative transition-ion responses (±20%). The quantitation of pharmaceuticals was based on the isotope dilution method. The calibrations curves were prepared by plotting concentration-dependent response factor of each target analyte (peak area of analyte divided by peak area of internal standard) versus the response-dependent concentration factor (concentrations of analyte divided by concentration of internal standard). However, metabolites were quantified using the internal standards of the corresponding parent compound (due to the lack of labeled standards available for the metabolites), and BPP was quantified using VLF-D8. The regression coefficients (r2) for seven- to nine-point calibration standards calculated by equal weighting quadratic regression were ≥ 0.99 for all target analytes. The limits of quantitation (LOQs) and limits of detection (LODs) were determined as a minimum concentration of analytes in sample extracts that provide a signal to noise ratio ≥10 and ≥3, respectively. LODs of the target pharmaceuticals and their metabolites in wastewater, SPM, and sludge samples were in the ranges of 0.05– 10 ng/L, 0.05–5.0 ng/g, and 0.1–10 ng/g, respectively. LOQs of the target pharmaceuticals and their metabolites in wastewater, SPM, and sludge samples were ranged from 0.1–20 ng/L, 0.1–20 ng/g, and 0.5–20 ng/g, respectively. The continuing calibration verification standards injected before and after every batch (n = 21) of sample analysis showed recoveries at 100 ± 30%. A method blank was analyzed with every batch of samples. The concentrations of target pharmaceuticals and their metabolites in method blanks were below the corresponding LOQ. The concentrations of the target chemicals in SPM and sludge are reported on a dry-weight basis unless stated otherwise. One sample was selected randomly for matrix spike (MS) and matrix spike duplicate (MSD) analyses with each batch of samples analyzed. Target pharmaceuticals and their corresponding internal standards were each spiked at 10 and 25 ng, respectively, and were passed through the entire analytical procedure. The average relative recoveries of target pharmaceuticals from wastewater, sludge, and SPM were 74 ± 32%, 81 ± 26%, and 84 ± 34%, respectively (Table S2).

Mass load ¼ Ci  F 

100 100−PSPM



 

1 106



 

1000 Population



    100 þ ðCS  TSPÞ Emission=1000 people ¼ ðCE  FÞ 100−P   SPM  1000 1  Population 106

ð3Þ

ð4Þ

where PSPM is the fraction of the total mass of analytes sorbed to SPM (%), CSPM is the concentration of analytes in SPM (ng/g), MSPM is the mass of SPM analyzed (g), VW is the volume of wastewater (L) used to obtain MSPM, CW is the concentration of analyte in wastewater (ng/L), Ci is the concentration of analyte in wastewater influent (ng/L), Ce is the concentration of analyte in wastewater effluent (ng/L), PSPMi is the fraction of the total load of analyte sorbed to SPM (%) in wastewater influent, PSPMe is the fraction of the total load of analyte sorbed to SPM (%) in wastewater effluent, mass load is the amount of individual pharmaceutical introduced into WWTP (mg/d/1000 inhabitants), F is the daily flow of wastewater influent (L/d) over a 24-h period, Cs is the concentration of analyte in sludge (ng/g wet weight), TSP is the total sludge production (g/d wet weight), population is the number of inhabitants served by the WWTP, and emission/1000 inhabitants is the quantity of pharmaceutical discharged through wastewater effluent, SPM, and sludge (mg/d/1000 inhabitants). 3. Results and discussion 3.1. Psychoactive pharmaceuticals in wastewater APPZ and QTP were found in 64% and 100%, respectively, of influent samples (n = 14; seven samples from each plant) (Table 1 and Fig. S1). The mean concentration of APPZ found in influent in our study was ~6 times lower than that reported for wastewater from a psychiatric hospital in China (Yuan et al., 2013); however, APPZ was not reported in wastewater from a centralized municipal WWTP earlier (Table 1). Similarly, the mean concentration of QTP in influent (19.9 ng/L) was 4.2 times higher than that reported from centralized municipal WWTPs in China and 176 times lower than that reported for wastewater from a psychiatric hospital (Yuan et al., 2013). APPZ and QTP were among the top anti-schizophrenics prescribed in the USA in 2008 (Gallini et al., 2013). QTP undergoes extensive metabolization in the human body and is excreted predominantly as metabolites (92% in urine and feces) (Sheehan et al., 2010). The mean concentration of nor-quetiapine (NQTP), one of the active metabolites of QTP, was 3.4 times higher than the concentration of QTP in influents. This is the first study to report the concentrations of NQTP in wastewater.

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LZP, APZ, and DZP were found in all influent samples (df: 100%) at mean concentrations of 18.2, 6.06, and 3.58 ng/L, respectively; the average consumption of these three drugs was 2420, 2650, and 7060 kg,

respectively, in the USA in 2010–2012 (INCB, 2013). Benzodiazepines are metabolized extensively in the human body and form pharmacologically inactive glucuronides, which are excreted through urine (Calisto

Table 1 Concentrations of select psychoactive pharmaceuticals and their metabolites in wastewater (ng/L) and sludge (ng/g dry wt) from two centralized wastewater treatment plants in Albany area, New York, USA, in July 2013. WWTPB

Analytes WWTPA Primary effluent

Effluent

*Sludge (n = 7)

Influent

Primary effluent

Effluent

*Sludge (n = 4)

Influent

Effluent

Sludge

Mean/df (%) Range

Mean/df (%) Range

Mean/df (%) Range

Mean/df (%) Range

Mean/df (%) Range

Mean/df (%) Range

Mean/df (%) Range

Mean/df (%) Range

Mean/df (%) Range

Mean/df (%) Range

Mean/df (%) Range

3.21/100 1.27–5.70 ND

10.3/86 ND-20.9 ND

115/100 21.0–267 1.49/100 0.59–2.40 17.8/100 7.41–25.3 133/100 69.3–260

5.43/71 ND-14.1 ND

1.53/41 ND-5.28 ND

1.69/57 ND-5.93 ND

NDo

NDo

–

–

15.5/100 8.29–20.9 66.1/100 34.0–131

12.5/100 0.65–18.7 60.3/86 ND-102

4.60/41 ND-29.3 82.3/100 42.9–144

16.8/100 13.0–21.1 2.93/100 0.65–6.26 21.1/100 19.4–22.1 196/100 99.1–267

6.68/94r bLOQ-23.9r 1.87/75r bLOQ-3.81r 5.41/94r bLOQ-17.3r 11.9/75r bLOQ-31.6r

27.1/86 ND-45.9 4.53/100 3.01–6.37 12.4/71 ND-28.2 3.01/86 bLOQ-8.21 7.28/100 2.77–10.4 3.82/100 2.37–6.12 238/100 112–501

78.4/100 42.8–114 4.59/100 3.10–6.20 12.7/86 ND-46.6 2.58/100 1.28–4.00 7.72/100 4.38–10.8 3.69/100 3.29–4.08 268/100 91.3–631

ND 0.28/50 ND-0.62 1.43/50 ND-4.39 0.48/50 ND-1.43 1.60/50 ND-3.81 1.08/50 ND-2.71 118/100 57.3–191

Antischizophrenics APPZ 5.58/57 ND-22.4 DAPPZ ND QTP NQTP

Literature

Influent

24.4/100 11.0–43.6 71.0/100 39.5–159

17.5/100 6.94–40.0 69.4/100 50.9–94.8

0.98/57 ND-3.43 74.3/100 25.0–131

Sedatives–hypnotics–anxiolytics LZP 20.3/43 16.2/100 9.14–33.0 4.86–28.7 APZ 6.24/100 4.85/86 3.09–12.6 ND-8.84 AHA 21.4/100 5.56/43 5.86–28.5 ND-21.3 DZP 3.38/100 1.62/57 2.29–4.57 bLOQ-4.70 OxZP 8.43/100 6.76/100 2.30–16.9 3.56–10.1 NDZP 5.30/100 4.29/100 1.68–9.73 2.56–7.23 CBZ 145/100 301/86 61–347 ND-564

64.2/100 37.3–85.5 6.20/100 5.01–8.21 9.2/100 7.25–12.4 1.73/71 bLOQ-3.45 9.87/100 7.09–14.4 4.53/100 3.05–6.56 310/100 150–731

0.26/14 ND-1.80 0.61/71 ND-1.08 0.37/14 ND-2.06 ND 0.86/43 ND-1.91 0.96–57 ND-2.29 83.1/100 28.6–189

16.2/100 6.11–34.3 5.89/100 3.47–8.77 17.4/100 5.93–26.0 3.79/100 2.29–9.78 6.52/100 4.06–9.30 4.04/100 2.63–5.25 241/100 109–588

Antidepressants VLF 415/100 169–609 BPP 110/100 17.1–231 STL 80.8/100 46.7–114 NSTL 71.1/100 23.7–137 CTP 133/100 77.7–170 DCTP 55.4/71 ND-126

471/100 309–702 75.0/100 12.8–182 58.5/100 33.4–73.0 56.8/100 19.7–119 221/100 180–271 68.0/100 32.5–135

480/100 389–553 67.4/100 18.2–264 62.8/100 27.9–88.3 54.4/86 ND-78.6 280/100 205–414 118/100 35.9–310

129/100 76.5–162 23.7/100 8.23–46.2 1490/100 976–1993 688/100 269–1192 283/100 170–429 222/100 36.8–434

336/100 194–407 147/100 25.2–378 43.1/100 31.6–52.5 65.3/100 11.0–183 59.4/100 35.1–146 12.8/29 ND-55.3

359/100 245–451 73.8/100 39.3–108 47/100 23.5–70.4 33.3/100 11.7–50.6 127/100 47.6–211 35.6/71 ND-105

339/100 209–431 34.1/100 7.31–89.2 24.5/100 15.7–49.8 16.4/41 ND-83.6 150/100 104–215 79.3/86 bLOQ-183

84.2/100 66.6–106 12.5/100 7.07–19.9 862/100 788–961 394/100 175–716 170/100 131–230 130/100 33.2–223

1040/100 537–1390 46.5/100 23.2–82.9 NA

23.0/100 9.33–40.8 83.3/100 35.0–137 61.9/100 38.2–134 179/100 23.2–503 218/100 112–380 385/100 246–509

606/100 377–904 24.1/100 2.99–97.5 168/100 75.0–354 473/100 109–1840 7.30/100 1.79–17.4 8.88/100 1.11–24.1

568/100 363–722 64.5/100 12.1–125 NA

426/100 299–852 74.2/100 28.2–153 NA

361/100 188–613 21.1/100 6.76–33.5 19.3/100 9.36–42.5

594/100 382–754 82.2/100 29.8–225 194/100 179–218 294/100 163–368 49.2/100 28.8–63.2 20.3/100 2.63–39.9

295/100 118–618 23.5/100 8.82–42.9 15.2/100 2.53–28.1

Antiplatelet CPG 35.5/100 7.39–92.8 CPGC 160/100 43.5–278

16.3/100 14.0–23.0 129/100 75.0–193

21.8/100 13.9–30.1 194/100 166–249

32.4/100 19.1–66.4 3.24/100 1.59–5.19

31.4/100 22.9–38.3 124/100 67.8–166

Antihistamine DPH 462/100 286–615 DPMA 3.7/86 bLOQ-5.52

609/100 421–764 3.65/29 ND-9.55

194/86 bLOQ-704 4.5/29 bLOQ-12.9

444/100 351–621 ND

227/100 105–390 3.79/29 ND-9.45

Antihypertensions ANL 1220/100 377–2000 PPN 24.5/100 3.04–53.8 DTZ 105/100 15.3–215 DAD 483/100 125–1090 VPM 18.5/100 5.34–42.8 NVP 14.8/100 4.22–33.5

o

4–6

NDo

–

–

74e 51–82e NDf,o

140/100f 30–160f,j NDf,o

–

– e

11.6r 10.6/38r bLOQ-14.1r –

17 13–19e 480/100h up to 860h –

1.4/100m ND-6.2g,m 320/100h 18.9–630g,h,j –

757/100i 6–1032b,d,e,i

713/100i 6–961b,d,i

1343/100i 40–1769f,i,k 191k

1087/100i 8.94/94r 60–2190f,i,k,n 0.97–499i,r 104–500k,n –

20/100i 7.6–34i,k 20/100i 15–30i,k 236/100i 144–326i,k 133k

12/100i 5.7–70i,k,n 15/100i 12–50i,n 173i 86–500i,k,n 111k

56.7/100r 11.3–2117c,i,l,r 117/100r 24.3–279i,r 26.8/94r 10.7–1381i,r 41.5r

7801b 916–11239b,e 183e 72–309e 6–19d

2772b 118–5910b,j 93/100a 16–284a,j 6–13d

NDc

327/100 138–1000 38.5/100 25.3–58.6 8.29/100 1.87–18.3

27.6/100 17.7–46.4 49.7/100 13.9–115 48.5/100 35.9–68.7 84.9/100 69.7–104 170/100 145–219 175/100 92.4–249

–

–

–

–

28.4/100 14.7–40.7 166/100 64.8–168

23.7/100 18.2–39.9 116/100 49.5–170

29.0/100 19.0–34.8 2.26/100 1.33–3.25

124e 106–133e –

–

287/100 14.4–575 11.4/57 ND-32.7

85.7/100 5.63–426 4.14/14 ND-16.5

293/100 225–339 ND

160–600p

586q

–

–

3100

h

510h

–

3.3r 2.03–23l,r 4.62–13.1r 3.82r 1.27–8.71r 23.3/100r 17–245i,r

53.7/100r 22.7–849c,l,r 2.59/44r bLOQ-3.91r 27.5/100r 1.70–159r 3.66/94r bLOQ-551c,l,r 7.43/94r bLOQ-458c,r 8.51/81r bLOQ-20.8r 3.74/100r 1.19–12.6r 87.2/100r 87.2–5957l,r –

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Notes to Table 1: Italics refer to metabolites. df: detection frequency (%), *sludge corresponds to the combined sludge produced after primary and secondary treatments; ND: non-detect; LOQ: limit of quantitation; aripiprazole (APPZ); dehydro-aripiprazole (DAPPZ); quetiapine (QTP); norquetiapine (NQTP); lorazepam (LZP); alprazolam (APZ); α-hydroxy alprazolam (AHA); diazepam (DZP); oxazepam (OxZP); nordiazepam (NDZP); carbamazepine (CBZ); venlafaxine (VLF); bupropion (BPP); sertraline (STL); norsertraline (NSTL); citalopram (CTP); N-desmethyl citalopram (DCTP); atenolol (ANL); propranolol (PPN); diltiazem (DTZ); desacetyl diltiazem (DAD); verapamil (VPM); norverapamil (NVP); clopidogrel (CPG); clopidogrel carboxylic acid (CPGC); diphenhydramine (DPH); 2-(diphenylmethoxy) acetic acid (DPMA). a In effluent from five WWTPs in the UK (Ashton et al., 2004). b In effluent from five WWTPs in South Korea (Behera et al., 2011). c In composite biosolid samples from 96 WWTPs in the USA (Chari and Halden, 2012). d In wastewater from 4 WWTPs in Korea (Choi et al., 2008). e In a pilot scale WWTP in Spain (Dolar et al., 2012). f In 3 WWTPs in Spain (Gracia-Lor et al., 2012). g In 6 WWTPs in Berlin, Germany (Hass et al., 2012). h In 11 WWTPs in Germany (Hummel et al., 2006). i In 5 WWTPs in Canada (Lajeunesse et al., 2012). j In a WWTP in Spain (Lopez-Serna et al., 2010). k In a WWTP in Canada (Metcalfe et al., 2010). l In seven sewage sludge samples in France (Peysson and Vulliet, 2013). m In eleven WWTPs in Korea (Ryu et al., 2011). n In a WWTP in the USA (Schultz et al., 2010). o In WWTPs in China (Yuan et al., 2013). p In municipal wastewater in the USA (Du et al., 2014). q In a WWTP in the USA (Bartelt-Hunt et al., 2009). r In 16 WWTPs in Korea (Subedi et al., 2013).

and Esteves, 2009). The glucuronide conjugates are transformed to the corresponding parent compounds by β-glucuronidase enzyme produced by fecal bacteria, e.g., Escherichia coli. OxZP and NDZP were found in all influent samples at mean concentrations of 7.47 and 4.67 ng/L, respectively. The measured concentrations of OxZP and NDZP in wastewater represent both the parent molecule and the metabolite of benzodiazepines (Hass et al., 2012). α-hydroxy alprazolam (AHA), a metabolite of APZ, was found for the first time in wastewater influents at mean concentrations of 19.4 ng/L (df: 100%). CBZ is one of the co-prescribed pharmaceuticals, along with antischizophrenics and anxiolytics (Kim et al., 2007), and is found at the highest concentration (194 ng/L; df: 100%) in wastewater among several schizophrenics and sedative–hypnotics–anxiolytics analyzed in this study (Table 1). VLF, a selective serotonin and norepinephrine re-uptake inhibitor (SSNRI), was found at the highest mean concentration (376 ng/L) in all influents, followed by BPP (a dopamine and norepinephrine inhibitor) and selective serotonin re-uptake inhibitors (SSRI: CTP and STL) (Table 1). Metcalfe et al. (2010) reported the occurrence of VLF at the highest concentrations among all antidepressants analyzed (N500 ng/L) in wastewater effluents from two WWTPs employing conventional activated sludge and tertiary treatment followed by UVdisinfection in Canada. The parent compounds and the metabolites of antidepressants are excreted predominantly as conjugates. The mean concentration of norsertraline (NSTL), a pharmacologically active metabolite of STL, was similar to that of STL in influents. However, the mean concentration of CTP was ~ 3 times higher than its metabolite, N-desmethyl citalopram (DCTP: 34.1 ng/L; df: 50%) in influents. ANL, an antihypertensive, was found at the highest concentration (913 ng/L, df: 100%) among all pharmaceuticals analyzed in influents; this concentration was 2 and 9 times lower than those reported in Spain (Dolar et al., 2012) and Korea (Behera et al., 2011), respectively (Table 1). However, Dolar et al. studied the removal of pharmaceuticals from municipal wastewater through a membrane bioreactor coupled to reverse osmosis in Spain (Dolar et al., 2012) whereas Behera et al. monitored pharmaceuticals in Korean WWTPs employing conventional treatment process including primary treatment and activated sludge process (Behera et al., 2011). The mean concentrations of antiplatelet (CPG) and antihistamine (DPH) analyzed in influents were 3.8 times lower than those reported in Spain (Dolar et al., 2012) but similar to those reported in an earlier study in the USA (Table 1) (Du et al., 2014). Clopidogrel carboxylic acid (CPGC, a metabolite of CPG) was not reported in wastewater before; however, in this study, this metabolite was found at concentrations 4.2 times higher than those of CPG

in influents (df: 100%). Similarly, 2-(diphenylmethoxy) acetic acid (DPMA: metabolite of DPH) was found for the first time in influents at 3.76 ng/L (df: 57%) in our study. 3.2. Psychoactive pharmaceuticals in sludge APPZ and DAPPZ were consistently detected in all sludge samples (Table 1). APPZ is excreted from human bodies mainly through feces, as a metabolite (37%) as well as an unchanged parent molecule (~18%) (Sheehan et al., 2010). It can be presumed that pharmaceuticals found in feces often partition to solid particles, such as SPM, rather than to the aqueous phase (Metcalfe et al., 2010). Similarly, QTP is excreted predominantly as a metabolite (72.5%) in urine and (19.5%) through feces (Sheehan et al., 2010). NQTP was detected in all effluent and sludge samples at concentrations of 28 and 8.2 times higher, respectively, than the concentrations of QTP. In dewatered sludge, the mean concentrations of ANL, DTZ, BPP, CBZ, and NDZP were 3.3 times lower than those found in activated sludge (Table S5). The sorption coefficients (Kd) of psychoactive pharmaceuticals were determined based on the assumption that the analytes are in equilibrium between the aqueous phase and the particulate phase as reported by Radjenovic et al. (2009). The concentrations measured in influent (ng/L) and SPM (ng/kg dw) were applied in the calculation of Kd (L/kg) (Table S5). The highest Kd values were found for VPM (12000 L/kg) and its metabolite norverapamil, NVP (14300 L/kg) and some antidepressants (STL: 3460 L/kg and NSTL: 5680 L/kg). The Kd values of APPZ and QTP were 1930 and 430 L/kg, respectively. The reported Kd values for STL, NSTL, and VLF, based on the measured concentrations in sludge and effluent (Lajeunesse et al., 2012) were similar to those found in our study. The sorption coefficients of PPN and CBZ were ~ 3.5 times higher than those reported in a study from Spain (Radjenovic et al., 2009). Removal by sorption is considered negligible for substances having log Kd values below two and considered significant for substances having log Kd values above four (Deegan et al., 2011). The sorption of antischizophrenics (APPZ and QTP), antidepressants (STL and NSTL), antihypertensive metabolite (NVPM), and antihistamine (DPH) was significant (Kd N 4) (Table S5); however, the sorption of LZP, ANL, and CPGC (log Kd = 1.21–1.73) was low. 3.3. Sorption of psychoactive drugs on SPM Based on the measured concentrations of psychoactive pharmaceuticals in SPM and the aqueous phase (Eq. (1)), the fraction of drugs found

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in SPM ranged from 0.68% (ANL) to 80.2% (VPM) (Table 2). Many studies that examined the fate of psychoactive pharmaceuticals in WWTPs based their measurements on filtered wastewater (Lajeunesse et al., 2012; Metcalfe et al., 2010; Radjenovic et al., 2009). We found, however, that the fraction of APPZ, NQTP, STL, NSTL, CTP, DCTP, PPN, VPM, and NVP sorbed to SPM was N 50% of the total mass. This suggests that studies that determine mass loadings of psychoactive pharmaceuticals in WWTPs based only on aqueous phase concentrations can underestimate the total loadings by up to 80%. Nevertheless, the fraction of select sedative–hypnotics–anxiolytics (APZ, DZP, NDZP, and CBZ), antidepressants (VLF and BPP), and antihypertensives (ANL and DTZ) sorbed to SPM was b 11%, which suggests that these pharmaceuticals are present predominantly in the aqueous fractions.

showed a negative removal efficiency in WWTPs. Microbial transformation of conjugated forms of drugs during the wastewater treatment processes can increase the residue levels of parent drugs in waste streams (Calisto and Esteves, 2009). Gracia-Lor et al. (2012) reported a negative removal for LZP in WWTPs. Negative removal efficiency for CBZ was attributed to cleavage of hydroxylated carbamazepine metabolite to CBZ by microbial activity in WWTPs (Miao et al., 2005). APPZ, QTP, and NQTP were removed in WWTPs at 68%, 87%, and 31%, respectively. The average removal efficiencies of antidepressants and their metabolites in WWTPs (27%) were similar to those reported in two Canadian WWTPs (Metcalfe et al., 2010). The secondary biological treatment using activated sludge enhanced the removal of QTP (49%), STL (16%), NVP (26%), and DPH (50%) (Fig. 1).

3.4. Removal of pharmaceuticals from WWTPs 3.5. Mass loading and emission of psychoactive drugs through WWTPs Mass loadings (mg/d/1000 inhabitants) of psychoactive pharmaceuticals were calculated using the concentration of psychoactive pharmaceuticals determined in wastewater influent, including the fraction sorbed to SPM (Eq. (3)) (Table 2). The mass loadings of psychoactive pharmaceuticals ranged from 0.91 (DZP) to 347 mg/d/1000 inhabitants (ANL). The mass load of psychoactive pharmaceuticals in the WWTPA was 3.5 (PPN) to 16 (STL) times higher than in the WWTPB. The average mass loadings of antidepressants and antihypertensives were similar; however, the average mass loading of sedative–hypnotic–anxiolytic

Table 2 The fraction of pharmaceutical sorbed, average mass load, and emission of pharmaceuticals and their metabolites in two centralized wastewater treatment plants in Albany area, New York, USA. Analytes

PSPM (%)a

Mass load (mg/d/1000 inhabitants)

Environmental emission (mg/d/1000 inhabitants)

Antischizophrenics Aripiprazole (APPZ) Dehydro-aripiprazole (DAPPZ) Quetiapine (QTP) Norquetiapine (NQTP)

68.3 NA 22.9 50.4

7.41 36.4

3.82 0.01 2.27 59.5

Sedatives–hypnotics–anxiolytics Lorazepam (LZP) Alprazolam (APZ) α-Hydroxy alprazolam (AHA) Diazepam (DZP) Oxazepam (OxZP) Nordazepam (NDZP) Carbamazepine (CBZ)

34.7 NA NA NA 48.2 NA 4.13

7.74 1.66 5.60 0.92 4.84 1.38 43.8

47.7 3.36 7.44 1.48 6.56 2.60 186

Antidepressants Venlafaxine (VLF) Bupropion (BPP) Sertraline (STL) Norsertraline (NSTL) Citalopram (CTP) Desmethyl citalopram (DCTP)

10.8 1.03 71.5 68.5 60.9 69.0

122 31.0 78.9 76.6 72.2 30.7

255 30.8 38.0 71.7 131 61.6

Antihypertensions Atenolol (ANL) Propranolol (PPN) Diltiazem (DTZ) Desacetyl diltiazem (DAD) Verapamil (VPM) Norverapamil (NVP)

0.68 56.1 8.93 13.5 80.2 71.5

347 12.6 34.5 143 18.8 10.6

316 50.7 45.8 217 30.0 11.2

Antiplatelet Clopidogrel (CPG) Clopidogrel carboxylic acid (CPGC)

19.5 0.72

12.3 41.8

15.1 94.3

Antihistamine Diphenhydramine (DPH) 2(Diphenylmethoxy)acetic acid (DPMA)

42.4 NA

184 1.00

116 2.77

a

6.46

Average PSPM in influents of two WWTPs; metabolites are italicized.

Total Treatments

Primary Treatment AAPZ QTP NQTP LZP APZ AHA DZP OxZP NDZP CBZ VLF BPP STL NSTL CLP DCTP ANL PPN DTZ DAD VPM NVP CPG CPGC DPH DPMA

Antischizophrenic

Antisedative-hypnotic-anxiolytic

Antidepressant

Antihypertensive

Antiplatalet Antihistamine

-100

-75

-50

-25

0

25

50

75

Analytes

The removal efficiencies of psychoactive pharmaceuticals through wastewater treatment were calculated based on PSPM corrected mean concentrations in influent and effluent (Eq. (2)) (Fig. 1). The average removal efficiencies of psychoactive pharmaceuticals and their metabolites in two WWTPs ranged from 0% (VLF) to 71% (APPZ) through the primary treatment and from 0.3% (DCTP) to 87% (QTP) through the final (primary and secondary) treatment. Select pharmaceuticals and their metabolites, including LZP, CBZ, NSTL, PPN, CPGC, and DPMA,

100

Removal Efficiency (%) Fig. 1. The average removal efficiencies (%) of psychoactive pharmaceuticals from two WWTPs: aripiprazole (APPZ); dehydro-aripiprazole (DAPPZ), quetiapine (QTP); norquetiapine (NQTP); lorazepam (LZP); alprazolam (APZ); α-hydroxy alprazolam (AHA); diazepam (DZP); oxazepam (OxZP); nordiazepam (NDZP); carbamazepine (CBZ); venlafaxine (VLF); bupropion (BPP); sertraline (STL); norsertraline (NSTL); citalopram (CTP); N-desmethyl citalopram (DCTP); atenolol (ANL); propranolol (PPN); diltiazem (DTZ); desacetyl diltiazem (DAD); verapamil (VPM); norverapamil (NVP); clopidogrel (CPG); clopidogrel carboxylic acid (CPGC); diphenhydramine (DPH); 2(diphenylmethoxy) acetic acid (DPMA). Total treatments refer to the primary treatment as well as secondary treatment.

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Primary Sludge

7850 (74,100) 491 (1450) 113,000 (109,000) 52,900 (65,800)

Primary Clarifier

Primary Effluent 1800 (34,200) 377 (1030) 34,900 (29,000) 14,800 (49,200)

Secondary Treatment

Combined Sludge

Incineration

Degradation Transformation

?

Ash

?

12.4 (115) 0.17 (0.84) 508 (232) 100 (103)

Secondary Sludge

Influent

279

Secondary Clarifier

Effluent

Discharge

383 (10,700) 382 (1060) 4200 (18,900) 4130 (1020)

River

?

Fig. 2. A schematic showing the fate of select antipsychotic pharmaceuticals through a WWTP serving a population of approximately 100,000 in Albany area, New York, USA. The total amount (mg) of select antipsychotic pharmaceuticals per day at different stages of wastewater treatments is shown in the order of quetiapine (QTP, an anti-schizophrenic), alprazolam (APZ, an anti-anxiolytic), sertraline (STL, an antidepressant), and verapamil (VPM, an antihypertensive). The values in parenthesis represent the respective metabolites: norquetiapine (NQTP, a metabolite of QTP), α-hydroxy alprazolam (AHA, a metabolite of APZ), norsertraline (NSTL, a metabolite of STL), and norverapamil (NVP, a metabolite of VPM).

and antischizophrenic was approximately 7.3 and 4.1 times lower than that found for antidepressants, respectively. The emission of psychoactive pharmaceuticals from WWTPs to the environment was calculated based on the concentration in effluents, fraction sorbed to SPM in effluent, and volume of activated sludge produced (Eq. (4)). The environmental discharge of psychoactive pharmaceuticals from WWTPs, on average, ranged from 0.01 (dehydroaripiprazole) to 316 (ANL) mg/d/1000 inhabitants (Table 2). The average emission rates of antidepressants and antihypertensives analyzed in this study were 2.6–6.8 times higher than those for antischizophrenics and sedative–hypnotic–anxiolytics. The calculated per-capita emission of CBZ, ANL, and PPN in this study were 2.1–5.1 times higher than that reported for a WWTP from Spain that serves a population of 277,000 (Radjenovic et al., 2009); however, the fraction of pharmaceuticals sorbed to SPM was not calculated in the latter study. The environmental emission of psychoactive pharmaceuticals we determined here can be a lower bound estimate because the peaks of prescription of psychoactive pharmaceuticals are reported in May to early June (Skegg et al., 1986; Tansella and Micciolo, 1992). Moreover, seasonal enhancement of removal of pharmaceuticals in WWTPs was reported in summer (Lajeunesse et al., 2012; Sui et al., 2011), that can result in decreased concentration of pharmaceuticals in effluent. QTP, STL, VPM, and their metabolites were found emitted at ≤17.3% of their mass load from WWTPB (Fig. 2). However, APZ and its metabolite AHA were discharged at 77.8% and 73.1%, respectively, of the mass load from WWTPB. The removal of some psychoactive pharmaceuticals through sorption to sludge was found minimal (b 1% of initial mass) (Fig. S2), which suggests that biodegradation and/or chemical transformation can be the dominant mechanism of removal for these drugs (Fig. 2) (Meakins et al., 1994; Radjenovic et al., 2009). Further studies are required to monitor psychoactive pharmaceuticals, pharmacoactive metabolites, and potential transformed products in receiving waters.

Acknowledgments Authors would like to thank the WWTP facilities, Mr. Anthony DeJulio, and Mr. Jingchuan Xue for assistance with the sample collection. This study was funded by a grant (1U38EH000464-01) from the Centers for Disease Control and Prevention (CDC, Atlanta, GA) to Wadsworth Center, New York State Department of Health. Its contents

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