Wastewater-based epidemiology as a new tool for estimating

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Wastewater-based epidemiology as a new tool for estimating population exposure to phthalate plasticizers Iria Gonzalez-Marino, Rosario Rodil, Ivan Barrio, Rafael Cela, and Jose Benito Quintana Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05612 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on February 28, 2017

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Environmental Science & Technology

Wastewater-based epidemiology as a new tool for estimating

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population exposure to phthalate plasticizers

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Iria González-Mariño*, Rosario Rodil, Iván Barrio, Rafael Cela, José Benito Quintana*

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Department of Analytical Chemistry, Nutrition and Food Sciences, IIAA – Institute for

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Food Analysis and Research, University of Santiago de Compostela, Constantino

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Candeira S/N, 15782 – Santiago de Compostela, Spain.

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*Corresponding authors:

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Iria González-Mariño:

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Candeira S/N, 15782 – Santiago de Compostela, Spain

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phone: +34 881 816035

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e-mail: [email protected]

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José Benito Quintana:

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Candeira S/N, 15782 – Santiago de Compostela, Spain

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phone: +34 881 814263 and +34 881 816035

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e-mail: [email protected]

22 23

Abstract

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This study proposes the monitoring of phthalate metabolites in wastewater as a

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non-intrusive and economic alternative to urine analysis for estimating human exposure 1

ACS Paragon Plus Environment


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to phthalates. To this end, a solid-phase extraction-liquid chromatography-tandem mass

27

spectrometry method was developed, allowing for the determination of eight phthalate

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metabolites in wastewater (limits of quantification between 0.5 and 32 ng L-1). The

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analysis of samples from the NW region of Spain showed that these substances occur in

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raw wastewater up to ca.1.6 µg L-1 and in treated wastewater up to ca. 1 µg L-1.

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Concentrations in raw wastewater were converted into levels of exposure to six

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phthalate diesters. For three of them, these levels were always below the daily exposure

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thresholds recommended by the US-Environmental Protection Agency and the

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European Food Safety Authority. For the other three, however, estimates of exposure

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surpassed such threshold (especially the toddler threshold) in some cases, highlighting

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the significance of the exposure to phthalates in children. Finally, concentrations in

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wastewater were also used to estimate metabolite concentrations in urine, providing a

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reasonable concordance between our results and the data obtained in two previous

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biomonitoring studies.

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Keywords: sewage analysis; endocrine disruptors; biomonitoring; phthalic acid esters;

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human exposure to chemicals.

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INTRODUCTION

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Disubstituted phthalate esters (phthalate diesters or just phthalates) are high

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production volume chemicals that have been widely used in the manufacturing and

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processing of plastics for more than 80 years. They are added as solubilizing and/or

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stabilizing agents to a huge variety of products such as furnishing materials, detergents,

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lubricants, adhesives, pesticides, textiles, food packages, personal care products,

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pharmaceuticals, and even medical devices.1 Following this widespread utilization, and

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since they are used as additives (i.e. not chemically bound to containing materials), their

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easy migration into the environment derives in a continuous exposure to the more

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volatile and low-molecular weight phthalates through inhalation and dermal contact,

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respectively.2 However, ingestion is considered the major route of exposure to

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phthalates, either by consuming contaminated food, accidental ingestion of

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contaminated dust and soil or licking of products containing them (e.g. by babies).3 As

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known endocrine disrupting (anti-androgenic) compounds, phthalates have shown to

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speed up and delay pubertal onset in girls and boys, respectively, and to play a role in

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the manifestation of children obesity.4 Childhood exposure is also associated to asthma

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and allergic symptoms, hypertension, and learning and behavioral problems. In adults,

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they mainly affect sex hormone levels, impairing semen quality and causing infertility

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and gynecological disorders. Additionally, they have been linked to respiratory

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problems, type 2 diabetes and pregnancy-induced hypertension.4,5 Finally, and although

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the association of prenatal exposure with birth outcomes is a controversial issue, most

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studies associate prenatal exposure to phthalates with developmental toxicity (lower rate

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of weight gain, shorter birth weight and length), neurotoxicity, shorter pregnancy

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duration and preterm birth.4,6 In view of all these effects, phthalates have been included

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in the priority lists of dangerous substances in most of the industrialized countries7−9. 3



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Oral Reference Doses (RfD)10-14 and Tolerable Daily Intakes (TDI)15-17 have been

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provided for some of them by the US Environmental Protection Agency (EPA) and the

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European Food Safety Authority (EFSA), respectively.

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Once in the human body, phthalates are hydrolyzed to their corresponding

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monoesters and excreted in such form or further oxidized and excreted afterward. Both

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types of metabolites can also be conjugated into glucuronide complexes prior to

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excretion, but these forms are rapidly hydrolyzed in wastewater by the action of β-

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glucuronidase enzymes produced by fecal bacteria.18 Since 2000,19 several studies have

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been conducted worldwide to assess daily intake levels of phthalates by measuring the

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concentrations of their metabolites in urine.20-23 Although useful, this approach is

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restricted to a limited set of samples and, therefore, to a limited number of people.

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Instead, and assuming that raw wastewater is a highly diluted urine sample of an entire

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community, levels of a substance in it can be correlated with the consumption/exposure

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of/to the aforementioned substance in the investigated community.24 This methodology,

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called wastewater-based epidemiology (WBE), has already provided successful results

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for comparing the consumption of illicit drugs in different countries.25,26 Additionally, it

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has been extended to other stimulant and psychoactive substances such as caffeine,27

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nicotine,27,

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consciously consumed substances, some studies have recently expanded the WBE

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approach to estimate the exposure and the effects of exposure to undesired chemicals,

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such as pesticides32 or an oxidative stress biomarker used as indicator of overall

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population health status.33

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alcohol29,30 and pharmaceuticals.31 Besides these applications to self-

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Following this trend, this study aims to develop and apply a WBE-method to

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estimate the exposure to six of the most frequently used phthalate diesters (dimethyl

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phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DnBP), di-iso-butyl 4


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phthalate (DiBP), benzyl butyl phthalate (BzBP) and di-(2-ethylhexyl) phthalate

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(DEHP) by the analysis of their hydrolyzed metabolites in wastewater.34-36 To the best

99

of authors’ knowledge, this is the first time that the presence of phthalate monoesters in

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wastewater is used to estimate exposure to phthalate plasticizers. In fact, only three

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studies so far have considered the determination of phthalate metabolites in the aqueous

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environment,37,38 or in wastewater,39 but without involving any kind of WBE

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application.

104 105

EXPERIMENTAL

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Chemicals and reagents

112

Phthalate diesters and monoesters considered in the study are displayed in Table

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1. DMP, DEP, DnBP, BzBP and DEHP were purchased from Sigma-Aldrich

114

(Steinheim, Germany). The metabolites monomethyl phthalate (MMP), monoethyl

115

phthalate (MEP), mono-n-butyl phthalate (MnBP) and monobenzyl phthalate (MBzP)

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were purchased from AccuStandard (New Haven, CT, USA). Mono-i-butyl phthalate

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(MiBP), mono-(2-ethylhexyl) phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl)

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phthalate (MEHHP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono-(2-ethyl-5-

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carboxypentyl) phthalate (MECPP) and the deuterated species monomethyl phthalate-

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D4 (MMP-D4), mono-n-butyl phthalate-D4 (MnBP-D4) and mono-(2-ethyl-5-

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hydroxyhexyl) phthalate-D4 (MEHHP-D4) were purchased from TRC (Toronto, ON,

122

Canada). Mixed stock solutions containing 10 µg mL-1 of all the analytes or all the

123

deuterated analogs were prepared in methanol (MeOH) and stored in the dark at -20°C.

124

HPLC-grade MeOH, acetonitrile (ACN), acetic acid (100%), hydrochloric acid

125

(37%) and ammonium acetate (≥ 98%) were purchased from Merck (Darmstadt,

126

Germany). Formic acid (95-97%) was purchased from Sigma-Aldrich (San Luis, Mi, 5



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USA). Ultrapure water was obtained in the laboratory by purifying demineralized water

128

in a Milli-Q Gradient A-10 system (Merck-Millipore, Bedford, MA, USA).

129 130

Wastewater sampling and sample treatment

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Wastewater samples were collected at six wastewater treatment plants (WWTPs)

132

from the coastal NW region of Spain; all of them treat mostly domestic wastewater and

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serve a population between 12,000 and 136,000 inhabitants. In the plant with the

134

greatest capacity (136,000), grab samples of both treated and raw wastewater were

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taken in March and April 2015. Samples of 24 h composite raw wastewater were

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collected for seven consecutive days in April 2016, from 10.00 a.m. to 10.00 a.m. of the

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following day. In the remaining plants, 24 h composite samples of treated and raw

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wastewater were taken for one day in June 2016. In all cases, samples were collected by

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automatic samplers operating in time proportional mode. Exact date, location,

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population and particular characteristics of the sampling mode are displayed in the

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Supporting Information, Table S1. In all cases, samples were transferred to the

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laboratory and extracted within 8 h after the end of the sampling.

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Under final working conditions, 100 mL of water were subsequently filtered

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through 1.6 µm glass microfiber filters GF/A (Whatman, Kent, U.K.) and 0.45 µm

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mixed cellulose acetate & cellulose nitrate filters (Millipore, Bedford, MA, USA)

146

acidified to pH 2.0 with 37% HCl and spiked with 50 ng of IS. Then, they were solid-

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phase extracted (SPE) on Oasis HLB-60 mg cartridges (Waters Corp., Milford, MA,

148

USA), previously rinsed with 3 mL of MeOH and 3 mL of pH 2.0 ultrapure water.

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Sorbents were dried under nitrogen for 30 min and analytes eluted with 5 mL of MeOH.

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Eluates were concentrated down to ~0.5 mL under nitrogen (99.999%) in a Turbovap II

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concentrator (Zymark, Hopkinton, MA USA) and diluted to a final volume of 1 mL 6


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(MeOH) for their liquid chromatography-tandem mass spectrometry (LC-MS/MS)

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analysis. Every sample was processed and analyzed in triplicate.

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Liquid chromatography-tandem mass spectrometry

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Ten µL of the extracts (or the standards) were injected into a Varian HPLC system

157

(Walnut Creek, CA, USA) equipped with a vacuum degasser, two high-pressure mixing

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pumps Varian ProStar-210, an automatic sampler Varian ProStar-410 and a

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thermostated LC column compartment kept at 40°C. Analyte separation was carried out

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on a Luna Phenyl-Hexyl column (150×2 mm I.D., particle size 3 µm) from Phenomenex

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(Torrance, CA, USA) using a dual eluent system of (A) 0.1% acetic acid in ultrapure

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water and (B) 0.1% acetic acid in MeOH. The flow rate was set at 200 µL min-1 and the

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elution gradient was as follows: 0 min (60% B), 10 min (80% B), 10.5 min (100% B),

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15.5 min (100% B), 15.6 min (60% B), 20 min (60% B).

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The HPLC system was coupled to a triple quadruple mass spectrometer Varian

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320-MS equipped with an Electrospray Ion Source (ESI) operating in negative mode.

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Nitrogen, used as drying and nebulizing gas, was provided by a nitrogen generator (Erre

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Due srl, Livorno, Italy). Argon, for the collision induced dissociation, was purchased

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from Praxair (Madrid, Spain). Working parameters were set at: 4000 V (Needle

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Voltage), 50°C (Source Temperature), 200°C (Drying Gas Temperature), 18 psi (Drying

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Gas Pressure), 55 psi (Nebulizing Gas Pressure) and 2.0 mTorr (Collision Gas

172

Pressure). The MS system was operated in Selected Reaction Monitoring (SRM) mode

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recording one (IS) or two (analytes) precursor/product ion transitions per compound.

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Transitions, together with chemical formulae, retention times, Collision Energies (CE)

175

and Capillary Voltages (CV) are displayed in Table S2.

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Method validation

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Analytes were quantified using deuterated compounds as surrogate or internal

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standards (IS). Only three deuterated analogs (MMP-D4, MnBP-D4 and MEHHP-D4)

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were used among the eight phthalate monoesters included in the study. Therefore, for

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the remaining ones, the closest structural or retention time-related deuterated substance

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was selected as IS (Table S2). Calibration curves were prepared in MeOH and linearity

183

was evaluated between 1 and 500 µg L-1 (IS level: 50 µg L-1). Instrumental

184

quantification limits (IQL) were calculated from the lowest calibration standards as the

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concentrations providing a signal-to-noise ratio (S/N) of 10. Limits of quantification of

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the whole method (MQL) were estimated in the same way by analyzing extracts of

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wastewater samples containing low concentrations of all the analytes and 50 µg L-1 of

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IS. Instrumental repeatability was assessed as the relative standard deviation (%RSD) of

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five repeated injections of a 50 µg L-1 standard (IS level: 50 µg L-1). Instrumental

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reproducibility was assessed as the %RSD of 50 µg L-1 standards (IS level: 50 µg L-1)

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analyzed throughout nine months.

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The evaluation of the trueness and precision was performed by recovery studies

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performed in ultrapure and raw wastewater. Samples of 100 mL were spiked with 0.5

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µg L-1 of IS and 0.1 or 1 µg L-1 of the analytes (ultrapure water) or 2 µg L-1 of the

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analytes (wastewater) and processed in triplicate. Ionization matrix effects were

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assessed by comparing the response of a raw wastewater extract spiked with 100 µg L-1

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of analytes and 50 µg L-1 of IS to that of a standard of the same concentration in MeOH.

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IS only-spiked aliquots were analyzed simultaneously in order to correct for the

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background levels of the analytes in wastewater. In all cases, analyses were conducted

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in triplicate.

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Blank evaluation and quality control

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Potential contamination issues were assessed both at the sample preparation stage

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and during the LC-MS analysis by i) processing Milli-Q aliquots spiked with IS and

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analyzing them normally (procedural blank); and ii) analyzing standards containing only

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IS (analysis blank). A procedural blank and an analysis blank were run simultaneously

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together with every set of samples. In most cases, a high and very variable signal was

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found for MEHP (Figure S1), what leaded us to exclude it from the final method. For

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the remaining analytes, when concentrations in any of the blanks were > IQL or >MQL,

210

these values were subtracted from the levels found in samples.

211 212

Stability tests

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Metabolite stability was tested through triplicate experiments designed to assess

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(i) their potential formation in wastewater from the also occurring parent phthalate

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diesters; (ii) their potential degradation in this matrix; and (iii) possible losses during

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filtration.

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In the first case, 10 mL aliquots of raw wastewater were spiked with 0.5 mg L-1 of

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DMP, DEP, DnBP, BzDP and DEHP and stored in amber glass vials for 24 h under

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different conditions: room temperature (22 ± 2ºC) and 4°C, natural pH and pH 2.0 (n =

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3 in every case). Aliquots of 1 mL were taken at the beginning of every experiment

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(time 0) and after 1.5, 3, 5, 8 and 24 h, filtered through 0.45 µm mixed cellulose acetate

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& cellulose nitrate syringe filters and injected (10 µL) into the LC-MS system.

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Metabolite degradation was assessed in a similar way by spiking 10 mL of

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wastewater samples (acidified to pH 2) with 0.5 mg L-1 of the eight phthalate

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monoesters and keeping them at room temperature for 48 h (n = 3).

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Finally, filtration losses were evaluated by comparing the signal of 0.5 mg L-1

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standards with that of ultrapure water samples spiked at the same concentration and

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filtered afterward as detailed in the Wastewater sampling and sample treatment

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subsection (n = 3).

230 231

Estimation of human exposure to phthalate diesters and back-calculation of

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monoester metabolite concentrations in urine

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Phthalate monoester concentrations in 24 h composite influent samples were

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multiplied by wastewater daily flow rates (L day-1) and divided by WWTP served

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populations (inhabitants, inh) to get monoester excretion loads in µg day-1 inh-1. These

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values were further used to estimate:

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i)

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DEHP). To this end, monoester excretion loads were multiplied by the corresponding

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correction factors (CF, Table 1), calculated according to the following equation:

human exposure to phthalate diesters (DMP, DEP, DiBP, DnBP, BzBP and

=

⁄

.

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where MPhthalate is the molecular weight of the parent phthalate diester; MMetabolite, the

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molecular weight of the phthalate monoester; and Molar Excr. Fraction, the average

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molar fraction of the parent compound excreted as that particular metabolite. Average

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excretion fractions were obtained by weighting the excretion fractions published up to

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date in human metabolism studies by the number of participants involved in every study

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(see Supporting Information, Tables S3 and S4). Exposure to DEHP was back-

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calculated from single MEHHP, MEOHP and MECPP excretion loads (applying

267

individual CFs, Table 1) and the average exposure value was obtained. MnBP excretion

268

load was considered to come entirely from the metabolization of DnBP, and so the

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contribution of BzBP (for which it is also a minor metabolite, excreted as a 6% of the

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phthalate diester dose) was considered negligible.40

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ii)

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considering a daily urine volume of 1.57 L per person. This value was obtained by

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weighting the average daily urine volume provided in three different studies41-43by the

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number of subjects involved in every study.

average phthalate monoester concentration in urine per individual (in µg L-1),

275 276

RESULTS AND DISCUSSION

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LC-MS/MS optimization and validation

278

MS/MS conditions (precursor and product ions, CE and CV) were optimized by

279

direct infusion of individual solutions (2 mg L-1) in MeOH:ultrapure water 1:1.

280

Ionization was performed in negative mode and two SRM transitions (one precursor and

281

two product ions) were acquired per analyte (Table S2). The most common products for

282

all the analytes are the ions m/z 134, m/z 121 and m/z 77, which correspond to i) the loss

283

of the carboxylic ester group, including the alkyl chain, either completely (m/z 121) or

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retaining the first carbon (m/z 134); and ii) the additional loss of the ester group,

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retaining the benzyl anion (m/z 77). For MEHHP, MEOHP and MECPP, products

286

corresponding to the alkyl cleavage are also obtained.44

287

The effect of several mobile phase modifiers (formic acid: 0-0.5%; acetic acid: 0-

288

0.5% and ammonium acetate: 0-25 mM) was also evaluated by direct injection: higher

289

responses were achieved with acetic acid (data not shown), which was finally added to

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both the aqueous and the organic phase at a concentration of 0.1%. Also, two different

291

columns of the same dimensions (Luna C18 and Luna Phenyl-Hexyl, both from

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Phenomenex) and two mobile phase combinations (ultrapure water-MeOH and

293

ultrapure water-ACN, both with 0.1% acetic acid) were considered for the 11



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chromatographic separation. As it can be seen in the Supporting Information, Figure S2,

295

isomer separation (MiBP and MnBP) improved on the Phenyl-Hexyl column and using

296

MeOH. Thus, such conditions were selected to continue with the study.

297

LC-MS performance parameters (linearity, IQL, instrumental repeatability and

298

reproducibility) are displayed in Table S2. The representation of the ratio analyte

299

area/surrogate area versus analyte concentration (1-500 µg L-1 range) fitted a linear

300

model with determination coefficients (R2) ranging from 0.9986 to 0.9995. IQL values

301

varied between 0.01 and 0.31 µg L-1; %RSD values between 1.7 and 4.4% for five

302

repeated injections of a 50 µg L-1 standard and between 2.3 and 7.0% for five 50 µg L-1

303

standards analyzed throughout nine months.

304 305

Analyte stability

306

Tests to evaluate the potential formation of phthalate monoesters from the parent

307

diesters in wastewater showed that MnBP and MBzP were the only ones being formed

308

> MQL, but only at natural pH and at very low percentages: up to 0.56% and 0.20% of

309

the parent molar concentration, respectively, after 24 h (Supporting Information, Figure

310

S3). Considering the lowness of these figures, MnBP and MBzP concentrations in

311

wastewater were attributed to human metabolism exclusively and no correction was

312

applied when estimating exposure to DnBP and BzBP.

313

Metabolite stability experiments indicated that six out of the eight analytes were

314

fairly stable and only MEP and MMP levels dropped significantly (up to 35% and 23%

315

of the initial concentration, respectively) after 48 h at room temperature and natural pH

316

(Figure 1). In the case of 24 h-sampling, used in most of the WBE works, the average

317

time the sample spends in the sampling container is 12 h; therefore, the decrease in

318

MMP and MEP is expected to range between 20 and 30%. In order to minimize such 12


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issues, samples were adjusted to pH 2 as soon as received and kept at 4°C until

320

extraction (performed within 8 h).

321

Finally, no losses were observed during sample filtration (data not shown).

322 323

SPE sorbent selection and SPE-LC-MS/MS method performance

324

Two different sorbents were considered for the SPE: Oasis HLB (polar reversed-

325

phase) and Oasis MAX (mixed mode: polar reversed-phase plus anion exchange).

326

Aliquots (100 mL) of ultrapure water and raw wastewater were spiked with all the

327

analytes at 1 µg L-1 and 2 µg L-1, respectively, and extracted on both sorbents following

328

two different protocols. For the extraction on Oasis HLB, sorbents were conditioned

329

with 3 mL of MeOH and 3 mL of pH 2.0 ultrapure water. Samples were adjusted to pH

330

2 in order to neutralize carboxylic groups and favor their retention by reversed-phase

331

interactions. Elution was performed with 5 mL of MeOH. Oasis MAX cartridges were

332

conditioned with 3 mL of 0.1% formic acid in MeOH followed by 3 mL of ultrapure

333

water (no pH adjustment). Samples were passed through them at their natural pH (~ 5.5

334

– 7.5) and analytes were recovered with 0.1% formic acid in MeOH. In both cases,

335

extracts were concentrated down to ca. 0.5 mL and made to a final volume of 1 mL with

336

MeOH.

337

Response comparison between these extracts and a standard (for ultrapure water)

338

or an spiked wastewater extract (for wastewater) provided us with the recovery of the

339

SPE process (n=3). Results were similar (and >80%) for seven out of the eight

340

compounds with both sorbents and both matrices. Only MEP showed a lower value in

341

influent wastewater when performing the SPE on Oasis HLB cartridges (77% versus

342

106%, Figure S4).

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Matrix effects were evaluated for both sorbents by comparing the responses of a

344

spiked-after-the-SPE extract with those of a standard in MeOH (n=3). The signal

345

suppression observed with Oasis MAX was similar or slightly higher than that observed

346

with Oasis HLB for four out of the eight analytes (Figure S4). For the other four (MMP,

347

MiBP, MnBP and, especially, MEP) this difference was greater, and so the lower signal

348

suppression got with Oasis HLB, together with the compatibility with the acidification

349

performed to preserve the samples, leaded us to select it as extraction sorbent.

350

Once extraction conditions were optimized, trueness and precision of the whole

351

SPE-LC-MS/MS procedure were evaluated through recovery and %RSD values for

352

triplicate analyses of: (i) ultrapure water (100 mL spiked with 0.1 or 1 µg L-1 of analytes

353

and 0.5 µg L-1 of IS); (ii) raw wastewater (100 mL spiked with 2 µg L-1 of analytes and

354

0.5 µg L-1 of IS). Percentages of recovery (%R) varied between 76 and 124%; %RSD

355

values between 1.2 and 15%. MQLs were between 0.5 to 32 ng L-1 (see Supporting

356

Information, Table S5). All of them were considered acceptable performance figures.

357 358

Occurrence of phthalate metabolites in wastewater

359

Several wastewater samples (both grab and 24 h composite samples, raw and

360

treated wastewater) were analyzed using the developed methodology. In general,

361

concentrations in effluents were lower than in the corresponding influents (Table 2)

362

except for MMP in Gondomar (68 versus 48 ng L-1) and for MEHHP, MEOHP and

363

MECPP (the three DEHP metabolites) in Ares and Cambados. In this latter case, the

364

concentration increase was remarkably high (~300-1000 ng L-1 versus ~30-60 ng L-1).

365

This may be attributed to the metabolization, through the activated sludge treatment, of

366

DEHP released from the WWTP. This observation is especially relevant, since some

367

phthalate monoesters have shown evidences of being toxic to different organisms.45,46 14


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Almost all the influent samples analyzed exhibited quantifiable concentrations of

369

all the analytes. The highest levels were found for MEP (300-1600 ng L-1), MMP (48-

370

265 ng L-1 except for Nigrán, where it reached 1800 ng L-1), MiBP (67-277 ng L-1) and

371

MnBP (55-274 ng L-1). Remaining compounds were below 100 ng L-1 in all cases, with

372

MBzP showing the lowest maximum concentration (19 ng L-1). Accordingly to what

373

could be expected from their mass excretion fractions (MEHHP: 0.16, MEOHP: 0.11,

374

MECPP: 0.14 of the DEHP dose), the highest concentrations among these three DEHP

375

metabolites were found for MEHHP in all samples but one (a grab influent sample

376

collected in Santiago). Similarly, the second highest values corresponded to MECPP in

377

11 out of 15 cases, and MEOHP was quantified, in general, at lower levels.

378

These results were compared with some of the existing studies where the levels of

379

phthalate diesters in Spanish urban wastewaters have been measured. In a review

380

compiled by Bergé et al.,47 the highest concentrations in wastewater from residential

381

areas were found for DMP (up to 93.3 µg L-1), followed by DEP (up to 45.9 µg L-1) and,

382

by far, by DEHP (up to 3.0 µg L-1) and DnBP (2.2 µg L-1). BzBP was quantified at

383

levels < 1 µg L-1 in all cases. In two studies performed in the same region of the present

384

work (NW region of Spain),48,49 DMP was, surprisingly, not detected in any sample.

385

DEHP was quantified at the highest levels (up to 6.2 µg L-1 and 1.9 µg L-1, respectively)

386

followed by DEP (3.0 and 0.7 µg L-1), DnBP (up to 0.9 and 0.6 µg L-1) and, finally,

387

BzBP (quantified only in the first study at 0.1 µg L-1). These values are between 5 and

388

400 times higher than the maximum concentrations measured for phthalate monoesters

389

in the present study, a fact that contrasts with the closeness of the figures stated in a

390

former work, where phthalate diesters and monoesters were quantified in samples from

391

the Tama River (Japan) at levels in the same order of magnitude (between not detected

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Page 16 of 37

392

and 1.3 µg L-1 in the case of monoesters, and between not detected and 3.6 µg L-1 in the

393

case of diesters).37

394 395

Phthalate monoester excretion loads from 24 h raw wastewater analysis.

396

Estimation of human exposure to phthalate plasticizers

397

Phthalate monoester concentrations in 24 h composite influent samples were

398

converted into monoester excretion loads in µg day-1 inh-1. These data, together with the

399

average of the seven daily loads calculated for Santiago, were used to perform a

400

pairwise correlation study between all the analytes. Regression coefficients and p-values

401

are compiled in Table S6. Significant correlations (for a 95% of confidence level, p

Wastewater-based epidemiology as a new tool for estimating

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