Case Study on Screening Emerging Pollutants in Urine and Nails

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Case study on screening emerging pollutants in urine and nails Andreia Alves, Georgios Giovanoulis, Ulrika Lena Nilsson, Claudio Erratico, Luisa Lucattini, Line Småstuen Haug, Griet Jacobs, Cynthia A. de Wit, Pim E.G. Leonards, Adrian Covaci, Jorgen Magnér, and Stefan Voorspoels Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05661 • Publication Date (Web): 15 Mar 2017 Downloaded from http://pubs.acs.org on March 15, 2017

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Environmental Science & Technology

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ACS Paragon Plus Environment


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Case study on screening emerging pollutants in

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urine and nails

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Andreia Alvesa,b,1, Georgios Giovanoulisc,d,1, Ulrika Nilssond, Claudio Erraticob, Luisa

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Lucattinie, Line S. Haugf, Griet Jacobsa, Cynthia A. de Witd, Pim E.G. Leonardse, Adrian

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Covacib, Jörgen Magnerc, Stefan Voorspoelsa*

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a

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Health, Lovisenberggata 8, 0456 Oslo, Norway

Flemish Institute for Technological Research (VITO NV), Boeretang 200, 2400 Mol, Belgium b

Toxicological Centre, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium c

IVL Swedish Environmental Research Institute, SE-100 31, Stockholm, Sweden

d

Department of Environmental Science and Analytical Chemistry (ACES), Stockholm University, SE-106 91, Stockholm, Sweden e

Institute for Environmental Studies, VU University Amsterdam, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands f

Domain of Infection Control and Environmental Health, Norwegian Institute of Public

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-equal contribution from first authorship between A. Alves and G. Giovanoulis

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*corresponding author: Stefan Voorspoels (VITO NV)

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Tel.+32 14 33 50 21, Fax.+ 32 14 31 94 72

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E-mail address: [email protected]

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Abstract

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Alternative plasticizers and flame retardants (FRs) have been introduced as replacements

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for banned or restricted chemicals, but much is still unknown about their metabolism and

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occurrence in humans. We identified the metabolites formed in vitro for four alternative

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plasticizers (acetyltributyl citrate (ATBC), bis(2-propylheptyl) phthalate (DPHP), bis(2-

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ethylhexyl) terephthalate (DEHTP), bis(2-ethylhexyl) adipate (DEHA)) and one FR (2,2-bis

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(chloromethyl)-propane-1,3-diyltetrakis(2-chloroethyl) bisphosphate (V6)). Further, these

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compounds and their metabolites were investigated by LC/ESI-Orbitrap-MS in urine and

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finger nails collected from a Norwegian cohort.

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Primary and secondary ATBC metabolites had detection frequencies (% DF) in finger

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nails ranging from 46 to 95%. V6 was identified for the first time in finger nails, suggesting

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that this matrix may also indicate past exposure to FRs as well as alternative plasticizers.

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Two isomeric forms of DEHTP primary metabolite were highly detected in urine (97% DF)

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and identified in finger nails, while no DPHP metabolites were detected in vivo. Primary

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and secondary DEHA metabolites were identified in both matrices, and the relative

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proportion of the secondary metabolites was higher in urine than in finger nails; the opposite

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was observed for the primary metabolites. As many of the metabolites present in in vitro

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extracts were further identified in vivo in urine and finger nail samples, this suggests that in

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vitro assays can reliably mimic the in vivo processes. Finger nails may be a useful non-

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invasive matrix for human biomonitoring of specific organic contaminants, but further

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validation is needed.

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Keywords: Finger nails, Urine, LC/ESI-Orbitrap-MS, in vitro metabolism, alternative

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plasticizers, V6

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1. Introduction

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Recently, new alternative plasticizers have been introduced on the market to replace

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bis(2-ethylhexyl) phthalate (DEHP), one of the major toxic phthalates used worldwide.

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DEHP is listed under REACH legislation as a ‘substance of very high concern’ (SVHC),

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with demonstrated carcinogenic (Group 2B), reproductive toxicity and endocrine disrupting

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effects on humans.1

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Alternative plasticizers have been found to migrate out of polymeric products to a lesser

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extent.2 Bui et al.2 categorized more than ten different chemical families of alternative

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plasticizers (i.e. adipates, citrates, terephthalates, among other). Many of these alternative

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substances are used in similar applications as phthalates, e.g. in toys, vinyl flooring, coated

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fabrics, gloves, plastic tubing, artificial leather, shoes, sealants, carpet, cosmetic products,

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medical devices, pharmaceutical tablet coatings, food packaging and beverage closures.2,3

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For instance, the high molecular weight phthalate bis(2-propylheptyl) phthalate (DPHP) is

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currently used as a major DEHP substitute.4

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Limited information is available on the toxicological aspects of the alternative

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plasticizers, but there are indications of lower toxicity than for DEHP (e.g. no acute toxicity

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or genotoxicity). However, some recent studies have shown endocrine disrupting potential,

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developmental and neurological effects and liver carcinogenicity in rats for some of these

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new chemicals (i.e. for acetyltributyl citrate (ATBC) and bis(2-ethyhexyl) adipate

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(DEHA)).2,5,6 In contrast, other studies did not show any adverse systemic toxicity7 or

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reproductive toxicity in rats8 for some other alternatives (e.g. bis(2-ethylhexyl) terephthalate

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(DEHTP)).

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As described in Bui et al.2, humans are exposed to these alternative plasticizers. The

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estimated risk ratio between the daily intake and the threshold limit for DPHP for infants

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was 3.4, raising some concern about the use of this plasticizer for example in childcare

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products and toys.9

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Another group of chemicals of high concern in regard to human exposure is flame

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retardants (FRs). Similar to phthalate esters, FRs have been used in a wide range of

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consumer products (e.g. furniture, textiles, electronic devices, plastics, etc.).10 As a

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consequence of their toxicity, polybrominated diphenyl ether (PBDEs) (e.g. penta-, octa-

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and deca-BDE) were banned or restricted from the market11,12 leading to demand for new

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alternative FRs, such as 2,2-bis(chloromethyl)-propane-1,3-diyltetrakis(2-chloroethyl)

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bisphosphate (V6). According to an EU risk assessment report13, V6 is primarily used in


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flexible polyurethane foam and is highly suitable for automotive and furniture applications.

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Although in the EU report, no acute toxicity to fish, algae and other invertebrates has been

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observed, possible neurotoxicity and reproductive toxicity were associated with V6

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exposure.13,14

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phosphate (TCEP)15,16, which is present as an impurity in the commercial V6 mixture at

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levels of 4.5-7.5%.17-19 Little is known about V6 toxicity in humans, but studies in dust and

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air indicate increasing concentrations indoors.17 Very high concentrations were found in

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foam used to produce baby products (> 24 mg/g) raising concern about human exposure17.

In fact, V6 is structurally related to the phased-out tris(2-chloroethyl)

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Recent biomonitoring studies identified the presence of a few alternative plasticizers,

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such as DPHP, DEHTP and DEHA through their oxidative metabolites in urine.4, 20-23 To

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our knowledge, the presence of other alternative plasticizers and FRs, such as V6, in urine

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and/or in other non-invasive matrices such as finger nails has not yet been explored. For

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urine the exposure is almost immediate depending on the metabolic rate and consequent

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excretion, while for nails the time window is approximately six months, which is the time it

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takes for nails to grow out of the nail bed. Therefore, nails have the advantage to indicate

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long-term exposure. In addition, due to sampling advantages (e.g. cost reduction, less

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storage and sample stability), finger nails were considered as an alternative matrix for

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human biomonitoring (HBM).24-27

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This study is the first where: 1) the metabolites of ATBC, DEHTP, DEHA, DPHP, and

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V6 identified in in vitro assays were screened in vivo in two non-invasive matrices (finger

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nails and urine); 2) the elucidation of the new metabolites of alternative plasticizers and FRs

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(and their fragments) was performed by LC/ESI-Orbitrap-MS; 3) the validity of using urine

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and finger nails as a non-invasive method to assess exposure to new pollutants was tested in

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a human cohort.

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2. Materials and Methods

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2.1 Reagents and Chemicals

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Neat standards for ATBC (98%), DEHA (99%), DEHTP (≥96%) were obtained from

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Sigma-Aldrich (Schenelldorf, Germany). Standards for V6 and DPHP were purchased from

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AccuStandard (Hattersheim, Germany) and Toronto Research Chemicals (Toronto,

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Canada), respectively. All standard stock solutions (1 mM) were prepared in methanol.



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LC/MS-grade methanol and acetonitrile were purchased from Fisher Scientific

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(Loughborough, UK). The ultrapure water was obtained from a Milli-Q ultrapure water

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system (Advantage A10 system, Overijse, Belgium). Trichloroethylene and formic acid (99

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%) were supplied by Merck (Darmstadt, Germany). Trifluoroacetic acid (TFA, 99 %) was

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supplied by Sigma-Aldrich (Steinheim, Germany). Ammonium acetate buffer (NH4Ac) was

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prepared by dissolving 1.93 g of ammonium acetate (99.99% purity, Sigma-Aldrich,

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Diegem, Belgium) in 100 mL ultra-pure water and acidifying the solution dropwise to pH

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6.5 with glacial acetic acid. The enzyme β-glucuronidase (E. coli K12) was supplied by

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Roche Applied Sciences (Mannheim, Germany).

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2.2 In vitro metabolism of ATBC, DEHTP, DEHA, DPHP and V6

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To the authors’ knowledge, no authentic standards for most of the metabolites of ATBC,

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DPHP, DEHTP, DEHA and V6 (see Table SI-1) identified in vitro, were commercially

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available at the time of this study. To bridge this key gap, each compound of interest was

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individually incubated with human liver/intestinal microsomes and/or human liver S9

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fractions (see FIG SI-1). Afterwards, the formed metabolites were extracted by liquid-liquid

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extraction. Positive and negative controls were also prepared to monitor the marker activity

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of key families of enzymes

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metabolite standards can be found in the SI. These in vitro metabolic extracts were used as

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reference solutions to create an in house fingerprint mass spectra database for target

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screening in urine and nail samples.

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More details regarding the in vitro generation of the

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2.3 Nails and urine sampling from a Norwegian cohort

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Details about the sampling are described in Papadopoulou et al.32 Finger nail (N=59) and

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urine (morning) samples (N=61) were collected from 61 volunteers living in the greater

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Oslo area (Norway). The sampling was approved by the Regional Committee for Medical

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Research Ethics (REK) in Norway (N° 2013/1269) and before starting the campaign, all

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participants gave written informed consent to participate. Until analysis all samples were

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stored at -20°C. Before the extraction, residues of nail polish and dirt were cleaned from the

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nails using acetone, as previously described.25-27,33

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2.4 Extraction of alternative plasticizers and V6 from nails and urine

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Previously optimized methods were used for extraction of the target plasticizers, V6 and

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their metabolites in nails and urine.25,26,34 For nails, 30 mg of cut pieces were weighed into

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glass vials followed by extraction of the target analytes using ultrasound-assisted dispersive

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liquid-liquid micro-extraction (US-DLLME) as described by Alves et al.25,26 Urine samples

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were extracted following a method involving deconjugation of the phthalate glucuronide,

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validated by Servaes et al.34 Since no internal standards and individual standards of the

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metabolites were commercially available, only qualitative analyses (screening) were

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possible. Similarly to Giovanoulis et al.33 in which the same urine and finger nail sample

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extracts were used for the quantification of phthalate and DINCH metabolites, field (n1=10)

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and procedural blanks (n2=5) were analyzed for each extraction method without any

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phthalate contamination problems.25,26,31

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2.5 LC/ESI-Orbitrap-MS analysis

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For analysis, an LC (ThermoFisher Scientific, Bremen, Germany) equipped with an

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Accela quaternary pump, a photodiode array detector (PDA) and a CTC PAL™ autosampler

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(CTC Analytics, Zwingen, Switzerland) was used. Chromatographic separations was done

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on a C18 column (100 × 2.1 mm, 1.7 µm) from Phenomenex Inc. (Torrance, CA, USA)

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maintained at 40°C. The mobile phases used for elution of the target analytes were water

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(A) and MeOH (B), both buffered with 0.1% formic acid, following the elution gradient of

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B: initially 10%; at 9 min 60% keeping conditions constant until 13.6 min; 13.6-15 min 80

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% keeping conditions constant until 15.5 min; 15.5-20.4 min 10%. The flow was kept at 0.3

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mL/min and the injection volume was 7 µL.

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For accurate mass measurements, an Orbitrap mass spectrometer (Q-Exactive™; Thermo

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Fisher Scientific, Bremen, Germany) equipped with an ESI source working in polarity

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switching mode was used (one analysis per mode). Operation parameters were as follows:

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source voltage, 2.5 kV; sheath gas, 53 (arbitrary units); auxiliary gas, 14 (arbitrary units);

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sweep gas, 3 (arbitrary units); and capillary temperature, 350°C.

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Both in vitro metabolite extracts and human nail and urine samples were analyzed in

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product ion scan followed by selective ion fragmentation (data dependent MS2 or ddMS2) in

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positive and negative mode (one analysis per mode) to obtain additional structural

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information. (resolving power set at 17500 FWHM, stepped collision energy 10, 30, 50 V,

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isolation window: 1 m/z).



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Data were analyzed using the XCalibur software v.2.2 (ThermoFisher Scientific). The

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accurate identification of the suspect analytes was performed using a mass deviation

Case Study on Screening Emerging Pollutants in Urine and Nails

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