Occurrence of Organophosphorus Flame Retardants and Plasticizers

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Occurrence of organophosphorus flame retardants and plasticizers (PFRs) in Belgian foodstuff and estimation of the dietary exposure of the adult population Giulia Poma, Carlos Sales, Bram Bruyland, Christina Christia, Severine Goscinny, Joris Van Loco, and Adrian Covaci Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06395 • Publication Date (Web): 27 Jan 2018 Downloaded from http://pubs.acs.org on January 29, 2018

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Environmental Science & Technology

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Occurrence of organophosphorus flame retardants

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and plasticizers (PFRs) in Belgian foodstuff and

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estimation of the dietary exposure of the adult

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population

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Giulia Poma1*, Carlos Sales2, Bram Bruyland1, Christina Christia1, Séverine Goscinny3, Joris

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Van Loco3, Adrian Covaci1*

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1

Toxicological Center, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium

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2

Research Institute for Pesticides and Water, University Jaume I, E-12071 Castellón, Spain

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3

Food, Medicines and Consumer Safety, The Scientific Institute of Public Health, Juliette

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Wytsmanstraat 14, 1050 Brussels, Belgium

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*

corresponding author: [email protected]

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ACS Paragon Plus Environment

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KEYWORDS

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Organophosphorus flame retardants and plasticizers; food analysis; human dietary exposure; GC-

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MS/MS; processed food

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ABSTRACT

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The occurrence of 14 organophosphorus flame retardants and plasticizers (PFRs) was

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investigated in 165 composite food samples purchased from the Belgian market and divided in

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14 food categories, including fish, crustaceans, mussels, meat, milk, cheese, dessert, food for

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infants, fats and oils, grains, eggs, potatoes and derived products, other food (stocks) and

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vegetables. Seven PFRs [namely, tri-n-butyl phosphate (TnBP), tris (2-chloroethyl) phosphate

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(TCEP), tris (1-chloro-2-propyl) phosphate (TCIPP), tris(1,3-dichloro-2-propyl) phosphate

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(TDCIPP), triphenyl phosphate (TPHP), 2-ethylhexyl diphenyl phosphate (EHDPHP) and tris (2-

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ethylhexyl) phosphate (TEHP)] were detected at concentrations above quantification limits. Fats

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and oils were the most contaminated category, with a total PFR concentration of 84.4 ng/g ww,

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followed by grains (36.9 ng/g ww) and cheese (20.1 ng/g ww). Our results support the

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hypothesis that PFR contamination may occur during industrial processing and manipulation of

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food products (e.g. packaging, canning, drying, etc.). Considering the daily average intake of

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food for the modal adult Belgian (15-64 y), the dietary exposure to sum PFRs was estimated up

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to 7,500 ± 1,550 ng/day (103 ± 21 ng/kg bw/day). For individual PFRs, TPHP contributed on

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average with 3,400 ng/day (46.6 ng/kg bw/day), TCIPP with 1,350 ng/day (18.5 ng/kg bw/day),

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and EHDPHP with 1,090 ng/day (15 ng/kg bw/day), lower than their corresponding health-based

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reference doses. The mean dietary exposure mainly originated from grains (39 %), followed by

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fats and oils (21 %) and dairy products (20 %). No significant differences between the intakes of

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adult men and women were observed.


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1. Introduction

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The phasing out of brominated flame retardants (BFRs) due to toxicity concerns1 and the

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enlistment on the POP list of polybrominated diphenyl ethers (PBDEs) in 2009 and

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hexabromocyclododecane (HBCDD) in 2013 has led to a surge in the usage of alternative flame

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retardants2. Organophosphorus flame retardants and plasticizers (PFRs) have been implemented

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as the foremost substitute of BFRs and have known a substantial growth in production volumes

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over the last 15 years3. They are widely used in standard and engineering plastics, polyurethane

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foams (PUF), thermoset coatings, textiles and polymer mixtures4. PFRs can be found inside the

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metal and plastic walls of refrigerators and freezers, and are used as thermal insulation panels in

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the construction sector or as casing for electronic equipment2, floor coverings, foam seating and

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bedding products, and as food-wrapping film5.

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PFRs are used as additive substances and are not chemically bound to the polymers they are

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applied to4. This allows them to leak into the environment, mainly through volatilization and

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abrasion5. Because of the structural differences among PFRs, leading to a variety of

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physicochemical properties within this class of compounds, they have been already found in all

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environmental compartments (like air6, water7, sediments and soil8,9, dust10,11, biota4,12,13) and

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humans11,14. However, data on the environmental persistence and toxicity of PFRs are scarce and

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few reports on adverse effects have been published2. For instance, tris (1-chloro-2-propyl)

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phosphate (TCIPP) and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) are known carcinogens

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in rats15; TDCIPP has also shown endocrine disrupting properties, altering the concentrations of

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thyroid hormone thyroxine (T4) and triiodothyronine (T3) in zebrafish3 and decreasing human

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semen quality16 and mobility17. Tris (2-chloroethyl) phosphate (TCEP), tri-n-butyl phosphate

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(TnBP) and triphenyl phosphate (TPHP) have neurotoxic aspects2, while 2-ethylhexyl-diphenyl


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phosphate (EHDPHP) is considered highly toxic to fish and aquatic plants and has potential to

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bioaccumulate13. Because of the frequent occurrence of PFRs in the environment, and their

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possible accumulation in biota samples, humans can be exposed to PFRs via different exposure

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routes2: mainly ingestion (and inhalation) of indoor dust18 and ingestion of contaminated food19–

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21

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the presence of PFRs in food is due to migration from food packaging, bioaccumulation, uptake

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from the agricultural environment, contamination during industrial processing or some other

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sources is still not clear19,20. This leads to uncertain and insufficient information to estimate the

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risk of human exposure to PFRs through diet.

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In the present study, the concentrations of 14 PFRs (namely, TCIPP, TDCIPP, TCEP, TnBP,

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TPHP, EHDPHP, tri-n-propyl phosphate (TnPP), tris(2-ethylhexyl) phosphate (TEHP), tritolyl

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phosphates (TOTP, TMTP, TPTP), tris(2-isopropylphenyl) phosphate (T2IPPP), tris(3,5-

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dimethylphenyl) phosphate (T35DMPP), tris(p-t-butylphenyl) phosphate (TBPP)) were

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investigated in 165 food samples belonging to 14 different food groups (namely, fish,

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crustaceans, mussels, meat, milk, cheese, dessert, food for infants, fats and oils, grains, eggs,

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potatoes and derived products, other food (stocks) and vegetables), all purchased from the

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Belgian food market. The dietary exposure to PFRs of the Belgian adult population was then

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estimated based on consumption data from the most recent Belgian National Food Consumption

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Survey22.

. However, there are few studies on the occurrence of PFRs in food and information on whether

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2. Experimental section

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2.1 Sample collection


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The food samples were available as previously collected and prepared in the framework of the

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FLAREFOOD project on the monitoring of the presence of BFRs in foodstuff23,24. In brief,

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individual food items were purchased from supermarkets, discount retailers and specialized meat

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and fish stores in main Belgian cities (Brussels and Antwerp) and in one village (Dessel) in

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April-December 2015 and May-November 2016. The individual samples were ground and

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homogenized, and equal amounts (±75 g) of the food items belonging to the same typology were

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pooled to create a unique composite sample. The composite samples were then either freeze-

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dried, analyzed fresh (samples with high fat content, e.g. included in “fats and oils” and “other

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food” categories), or directly aliquoted for analysis (such as grains, potatoes, and milk powder

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formulas for infants). A total of 165 samples, belonging to 14 food categories (overview in

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Supporting Information, Table S1), were prepared for analysis. Pending analysis, the samples

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were stored at -20°C in Falcon tubes (50 mL) previously tested to be free from the targeted

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PFRs.

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2.2 Analytical procedures

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A schematic overview of the extraction procedure is given in Figure S1. About 300 mg of freeze-

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dried sample was weighed in a 15 mL polypropylene (PP) tube with 100 mg NaCl. For high-fatty

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matrices (>20 % lip), a lower amount (variable from 50-150 mg) of sample was taken to reduce

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possible matrix effects. The lipid content of each sample was gravimetrically determined as

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previously reported21 and is shown in Table S1. The sample was added with 5 mL of acetonitrile

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(ACN) and 50 µL of internal standard (IS) mixture (containing TPHP-d15, TDCIPP-d15, TCEP-

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d12 and TAP), vortexed 1 min, and centrifuged at 3500 rpm for 5 min. The supernatant was then

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transferred to a pre-cleaned glass tube and evaporated to 2 mL under gentle nitrogen stream. A


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dispersive-SPE (d-SPE) was performed by adding to the sample 100 mg C18 and 50 mg of

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primary-secondary amine (PSA) sorbent powder to remove fats, sugars, fatty acids and most

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polar pigments from the extract. After 1 min vortex and 5 min centrifugation at 3500 rpm, the

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supernatant was transferred to a clean glass tube and evaporated to dryness. The sample was then

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reconstituted in 0.5 mL hexane (n-Hex) and further cleaned-up by elution onto Florisil® cartridge

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pre-conditioned with 6 mL ethyl acetate (ETAC) and 6 mL n-Hex. Fractionation was achieved

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with 12 mL n-Hex:DCM (4:1, v/v) (F1, discarded) and 10 mL ETAC (F2, containing the target

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compounds). F2 was evaporated to near-dryness, reconstituted in 0.5 mL n-Hex and further

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cleaned-up by elution onto aminopropyl silica (APS) cartridge pre-conditioned with 6 mL n-

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Hex:DCM (1:1, v/v) and 6 mL n-Hex. A second fractionation was achieved with 10 mL n-Hex

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(F3, discarded) and 12 mL n-Hex:DCM (1:1, v/v) (F4, containing the target compounds). F4 was

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evaporated, resolubilized in 50 µL iso-octane:ETAC (4:1, v/v) and 50 µL of recovery standard

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(RS, CB-207), transferred to injection vials for GC-MS/MS analysis. Full information on the

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chemicals and materials used in the present study is reported in Supporting Information, Section

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S1.

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The chromatographic analysis was performed using an Agilent 7890B gas chromatograph,

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equipped with an Agilent 7693A autosampler with multimode inlet (MMI), coupled to a triple

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quadrupole mass spectrometer, 7000C (Agilent Technologies Inc., Palo Alto, CA, USA), with a

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EI source working in electron impact mode. The GC separation was performed using a Zebron

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Semivolatile column (5% phenyl-arylene-95% dimethyl-polysiloxane) with a length of 20 m ×

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0.18 mm ID and a film thickness of 0.18 µm (Phenomenex, Torrance, CA, USA) working at a

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flow of 1.2 mL/min of helium (99.999%; Air Liquide, Liège, Belgium). The oven program was

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set as follows: 90 °C (1.25 min); 10 °C/min to 220 °C; then 5 °C/min to 300 °C; then 40 °C/min


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to 325 °C. The injection of 1 µL of sample extracts was performed in splitless mode at a

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temperature of 110 °C. The pulse pressure was set to 50 psi, with a split purge flow of 50

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mL/min and purge time of 1 min. TAP (IS) corrected for TnPP, TnBP and TEHP; TCEP-d12

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corrected for TCEP and TCIPP; TDCIPP-d15 corrected for TDCIPP and TDBPP; TPHP-d15

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corrected TPHP, EHDPHP, TOTP, TMTP, TPTP, T2IPPP and T35DMPP. The full list of

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targeted PFRs and MS/MS quantitation parameters for their analysis is reported in Table S2.

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2.3 Quality control

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The quality control check was performed using the menhaden fish oil used in the first worldwide

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interlaboratory study (ILS) on PFRs25. The measured values were within the range of the

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reference concentration (± 15%), while the mean IS recoveries ranged between 74 and 130%,

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with a relative standard deviation (RSD) below 15%. As blank contamination is the biggest

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challenge in the analysis of PFRs, due to their ubiquitous presence in dust25, three procedural

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blanks were run in parallel with every batch of samples (n = 21) to control any potential

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background contamination. To limit the levels of PFRs in the blank samples, the working space

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and all laboratory equipment was repeatedly cleaned with acetone; SPE and evaporation was

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performed in a pre-cleaned fume hood; glass tubes were washed, pre-cleaned with n-hexane and

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left in oven (at 300°C) for 2.5 hours to remove contaminant traces. Even following these

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precautionary steps along the whole sample procedure, mean PFR values in the blank were found

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at 1.3 ± 2.7 ng/g (range 1.6 pg/g to 21.2 ng/g). Average blank levels per batch were then

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subtracted from the sample results and a value equal to 3*SD of the blank measurement was used

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as limits of quantification (LOQs) before reporting results. For compounds not present in the


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blanks, LOQs were based on a signal/noise ratio of 10. Due to the different weighed amount of

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the analyzed samples, LOQs differed per matrix and per compounds and are listed in Table S3.

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2.4 Dietary exposure estimation

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Consumption data from the most recent Belgian National Food Consumption Survey (data from

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2014) were used for the exposure assessment22. The target population included all Belgian

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inhabitants from 3 to 64 year-old (divided in age categories), for a total of 3,200 participants,

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randomly selected from the National Register by a multi-stage stratified procedure. The diet of

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the participants was assessed by performing two non-consecutive 24-hour recalls (describing the

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food and drinks consumed the day before the interview) using the EPIC-Soft program26,

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developed to conduct standardized interactive 24-hour recall interviews or to enter dietary

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records in a standardized way. In the present study, it was chosen to estimate the daily intake of

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PFRs for the adult population, including adolescent (age group: 15-64 years). The individual

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intake of PFRs from the Belgian adult population (ng/day) was estimated for each compound by

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multiplying the average concentration of that compound in a particular food group (ng/g ww)

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with the average consumption rate of that particular group (g/day) by the Belgian adult

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population (Table S4). The total dietary intake of PFRs was obtained by summing the respective

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intakes from each food category. The daily dietary intake was also calculated per body weight

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(ng/kg body weight/day), based on an average body weight of 73 kg22. Since no consumption

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data for age groups younger than 3 years old were available, no calculations concerning intake of

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baby food products (mostly composed by food items for infants younger than 3 years old, such

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as milk powder and baby meals) were provided in the present study.

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2.5 Statistics

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For data treatment and elaboration, statistical software IBM SPSS Statistics 24 was used. When

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calculating the mean and median concentrations of PFRs in food composite samples, the non-

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detects were substituted with half the quantification limits ( TCIPP (24 %) > EHDPHP (20 %) > TDCIPP (14 %) > TEHP and TnBP (6 %) > TCEP

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(3 %) (Figure S2). However, the distribution of PFRs highly differed among the analyzed food

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groups when they were considered individually (Fig. 1). TCIPP was the most representative

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compound in fats and oils (FAT-OC), accounting for 46 % of the total measured concentration.

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EHDPHP was mainly found in stock/other food (OTC = 12.5 ng/g ww, 66 %), dessert (DC = 7.2

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ng/g ww, 63 %), food for infants (BC = 4.1 ng/g ww, 34 %) and potatoes (POC = 2.2 ng/g ww,

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31 %), while TPHP was mainly found in grains (GRAC = 26.1 ng/g ww, 71 %), cheese (CHC =

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10.9 ng/g ww, 54 %), mussels (MC = 6.3 ng/g ww, 80 %), and vegetables (VEC = 2.0 ng/g ww,

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92 %). The remaining food groups were characterized by a more homogeneous distribution of

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PFRs. The migration of EHDPHP from food packaging materials can be a possible source of

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EHDPHP in the analyzed foodstuff, since it was approved by the US Food and Drug

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Administration (US-FDA) for such application27. The presence of TPHP, used as flame retardant

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and plasticizer, can derive from its application in electrical industrial equipment2 and its usually

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high concentrations in indoor dust (which can reasonably deposit in industrial facilities)28–30, but

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the source of the presence of the other PFRs remains unclear.

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Considering all food categories, the most frequently contaminated groups were fats and oils

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(FAT-OC = 40 %), grains (GRAC = 17 %), cheese and stock/other food (CHC and OTC = 9 %),

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baby food (BC = 6 %) and dessert (DC = 5 %) (Figure S3). The main food groups from animal

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origin, such as fish, crustaceans, mussels, meat, and eggs, contributed only marginally to the

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overall PFR distribution (1 %, 1 %, 4 %, 2 %, and 0.3%, respectively) and this might be due to

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the rapid metabolism/excretion of PFRs17,31,32. A similar trend was observed in our previous

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study, where we suggested that the higher contamination with PFRs of certain food groups might


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derive from industrial processing19. To further explore this hypothesis, the whole sample data-set

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was divided in “non-processed food” and “processed food”. In the first group, we included all

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foodstuff that had been only minimally altered from its natural state, for example by being

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frozen, freshly cut, or directly packed (namely, vegetables, eggs, some fish, meat and milk

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samples). Food items included in the second group were instead more heavily manipulated and

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industrially altered, such as being canned, smoked, dried, fried, minced, etc. (namely, baby food,

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stock, desserts, cheese, fats and oils, grains and potatoes, and the other samples of fish, meat and

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milk). The full list of samples belonging to both categories is reported in Table S1. The total

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distribution of PFRs in the two groups showed a clear predominance of PFRs in processed food

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(89 %) vs non-processed food (11 %), with a similar and homogeneous contribution of each PFR

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to the total (Figure 2a). The same elaboration was then carried out considering separately meat

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(MEC), fish (including FC, CC, MC) and milk (LC), having both processed (e.g. prepared meat

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or fish, minced meat, canned meat or fish, marinated fish, fried fish, cream, milk based drinks,

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etc.) and non-processed (fresh/frozen meat and fish, fresh milk) samples. The results of the

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distribution of PFRs in processed- and non-processed-meat, fish, and milk (Figure 2b) are very

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similar among the three food groups and concordant with the general pattern (Figure 2a). These

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outcomes support the hypothesis that PFR contamination of foodstuff might likely originate from

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industrial processing and manipulation/alteration of food products.

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3.2 Estimation of human dietary exposure to PFRs

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The estimation of dietary exposure of the Belgian adult population to PFRs was based on the

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measured MB concentrations of targeted compounds in 165 food samples belonging to food

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groups representative of the general population food habit22. The category “baby food” (BC), not


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consumed by the general adult population, was not considered in the elaborations. The intake of

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fruits, beverages (e.g. water, soft-drinks, tea/coffee, and alcoholic drinks), and candy/sweets was

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not included in this study since these food categories were not purchased in the frame of this

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project.

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Mean daily dietary intakes of PFRs were estimated for the overall Belgian adult population

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(Table 2) as well as for men and women separately (Table S5). According to the consumption

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data from the National Food Consumption Survey22, men generally consume more meat, eggs,

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fish, grains and fats than women, while the consumption of the other food categories is similar

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between the two genders (Table S4).

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For the average adult population (15-64 years), the total dietary intake of PFRs was estimated at

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7,500 ± 1,550 ng/day (103 ± 21 ng/kg bw/day) (Table 2) and no significant differences between

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the PFR intakes of men and women were observed. These results were higher than those

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estimated by Xu et al. (2017) by using a duplicate diet approach (i.e. each participant provided

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diet samples by collecting duplicate portions of their meals over a 24-hour sampling period),

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where the median dietary intake of the sum PFRs was estimated at 87 ng/kg bw/day20. However,

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it is necessary to point out that this kind of approach might not be completely representative of

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habitual diet and dilution effects arising from the typology of composite samples might reduce

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the levels of contamination in food20.

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The mean daily dietary intake of PFRs estimated in our study was similar to the one estimated in

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our previous study for the Swedish adult population (5,700 ng/day and 85 ng/kg bw/day)19,

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although the contribution of each PFR to the total intake is slightly different in the two studies.

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This can be due to differences in the composition and preparation of each food group and the

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number/typology of samples included in each category. Here, TPHP accounted for 45 % of the


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total dietary intake of PFRs, followed by TCIPP (18 %), EHDPHP (15 %), TDCIPP (9 %),

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TEHP and TnBP (5 %), and TCEP (3 %). In our previous study, the major contributors to the

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total intake were in the order: EHDPHP (57%) > TDCIPP (14%) > TPHP (11%) > TCIPP (10%)

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> TCEP (7%). On the other hand, the relative contribution of each food group to the dietary

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intake of PFRs is very similar in both studies (considering the food groups analyzed in both

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studies), and in the order: grains (39 and 36 % in this study and in Poma et al., 201719,

270

respectively), fats and oils (21 and 14 %), dairy products and desserts (20 and 23 %), meat (6 and

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7 %), vegetables (6 % in both studies), fish and potatoes (4 and 3 %). More precisely, spaghetti

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(GRAC-07) and stick bread (GRAC-04) contributed here for more than 20 % each to the PFR

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dietary intake attributed to the grain category, while fish-oil food supplement (OTC-05)

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contributed alone with more than 80% to the PFR dietary intake attributed to fats and oils.

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It is likely, that the daily intake of PFRs through fat and oil consumption by the general adult

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population might have been overestimated, due to the inclusion of the fish-oil supplement in the

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FAT-OC food group, for which a total consumption of 19 g/day was estimated22. By considering

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a more realistic daily intake for commercial fish-oil food supplement (e.g. 1 g/day)22, the dietary

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intake of total PFRs of OTC-05 was thus estimated at 225 ng/day (3 ng/kg bw/day) and of FAT-

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OC at 1200 ng/day (16 ng/kg bw/day) (not shown). In addition, the relatively high dietary intake

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of PFRs of the FAT-OC category could have also been influenced by substituting the

Occurrence of Organophosphorus Flame Retardants and Plasticizers

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