PET bottles and and their affect on water

1. Effect of temperature on the release of intentionally and non-intentionally
added substances from polyethylene terephthalate (PET) bottles into water:
Chemical analysis and potential toxicity
Cristina Bach a,c,⇑
, Xavier Dauchy a
, Isabelle Severin b
, Jean-François Munoz a
, Serge Etienne c
,
Marie-Christine Chagnon b
a
ANSES, Nancy Laboratory for Hydrology, Water Chemistry Department, 40 rue Lionnois, 54000 Nancy, France
b
Derttech ‘‘Packtox’’, Nutox team, Inserm U866, AgroSupDijon Nord, 1 Esplanade Erasme, 21000 Dijon, France
c
Institute Jean Lamour, UMR 7198, Department SI2M, Ecole des Mines de Nancy, University of Lorraine, Parc de Saurupt, CS 14234, 54042 Nancy, France
a r t i c l e i n f o
Article history:
Received 21 August 2012
Received in revised form 30 November 2012
Accepted 15 January 2013
Available online 29 January 2013
Keywords:
PET-bottled water
By-products
Chemical mixtures
Cyto-genotoxicity
Endocrine disruption
a b s t r a c t
The purpose of this study was to investigate the impact of temperature on the release of PET-bottle con-
stituents into water and to assess the potential health hazard using in vitro bioassays with bacteria and
human cell lines. Aldehydes, trace metals and other compounds found in plastic packaging were analysed
in PET-bottled water stored at different temperatures: 40, 50, and 60 °C. In this study, temperature and
the presence of CO2 increased the release of formaldehyde, acetaldehyde and antimony (Sb). In parallel,
genotoxicity assays (Ames and micronucleus assays) and transcriptional-reporter gene assays for estro-
genic and anti-androgenic activity were performed on bottled water extracts at relevant consumer expo-
sure levels. As expected, and in accordance with the chemical formulations specified for PET bottles,
neither phthalates nor UV stabilisers were present in the water extracts. However, 2,4-di-tert-butylphe-
nol, a degradation compound of phenolic antioxidants, was detected. In addition, an intermediary mono-
mer, bis(2-hydroxyethyl)terephthalate, was found but only in PET-bottled waters. None of the
compounds are on the positive list of EU Regulation No. 10/2011. However, the PET-bottled water
extracts did not induce any cytotoxic, genotoxic or endocrine-disruption activity in the bioassays after
exposure.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Today, the most common polymer used for the bottling of
drinking water is polyethylene terephthalate (PET). Since migra-
tion can occur between packaging and foodstuffs, consumers
may be exposed to the potentially harmful chemicals (additives,
un-reacted monomers, and processing aids) used in manufacturing
the packaging. These intentionally-added substances (IAS)
are listed and controlled by European Regulation No. 10/2011
and do not pose any risk to humans. However, over 50% of
compounds migrating from food contact materials are non-
intentionally added substances (NIAS) (Grob, Biedermann,
Scherbaum, Roth, & Rieger, 2006). Indeed, Mittag and Simat
(2007) reported that 98% of the toxicity evidenced by several epoxy
coating migrates was due to NIAS and/or reaction products. Euro-
pean Regulation No. 10/2011 concerning plastics and multilayers
recently became more strict, stating that ‘‘the risk assessment of
a substance should cover the substance itself, relevant impurities
and foreseeable reaction and degradation products in the intended
use’’ (EU, 2011).
PET is characterised by a limited range of additives and low dif-
fusion of potential migrants in the polymer matrix (EFSA, 2011b).
However, in PET-bottled waters non-polymer origins of NIAS also
exist, namely the water itself, the bottling process, disinfection
agents and environmental pollutions.
Furthermore, PET can be degraded due to several exposure
factors under normal conditions of use (heat and UV light). In addi-
tion, certain physicochemical properties of bottled water, such as
inorganic composition, carbonation or bacterial presence, influence
0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.01.046
Abbreviations: BHT, butylated hydroxytoluene; BHET, bis(2-hydroxyethyl)
phthalate; DBP, dibutyl phthalate; DEHA, di-2-ethylhexyl adipate; DEHP, di-2-
ethylhexyl phthalate; DEP, diethyl phthalate; DHT, dihydrotestoterone; DiBP, di-
isobutyl phthalate; DMP, dimethyl phthalate; DMSO, dimethyl sulfoxide; 2,4-dtBP,
2,4-di-tert-butylphenol; ECCVAM, European Center for the Validation of Alternative
Methods; EFSA, European Food Safety Authority; IAS, intentionally added sub-
stances; LOQ, limit of quantification; NIAS, non-intentionally added substances;
SML, specific migration limit.
⇑ Corresponding author at: ANSES, Nancy Laboratory for Hydrology, Water
Chemistry Department, 40 rue Lionnois, 54000 Nancy, France. Tel.: +33 (0)3 83 38
87 29; fax: +33 (0)3 83 38 87 21.
E-mail address: cristina.bach@anses.fr (C. Bach).
Food Chemistry 139 (2013) 672–680
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

2. the leaching of constituents from PET bottles into water. It has also
been established that the migration of several compounds (formal-
dehyde, acetaldehyde and Sb) from PET packaging to water is a
thermally activated process (see review in Bach, Dauchy, Chagnon,
and Etienne (2012)). However, little or nothing is known about the
release of other NIAS (Grob et al., 2006) from the PET bottles into
water and the final effect in terms of toxicity of all the migrated
substances.
Over the last few years, certain studies have reported finding
chemical mixtures with cytogenotoxic effects and endocrine dis-
ruption activity in PET-bottled water (see review and comments
in Bach et al. (2012)). Toxic effects, and especially endocrine dis-
ruption, could be attributed to a ‘‘cocktail effect’’ due to compound
mixtures (Muncke, 2009). However, migration studies of PET-bot-
tled water rarely combine chemical analysis with toxicological
assessments. Therefore when bioassays demonstrate positive re-
sponses, analytical data to identify the responsible compounds
are always lacking and conclusions are difficult to draw.
The current European Regulatory framework states that an indi-
vidual toxicological evaluation for substances used in the manufac-
turing of food contact materials is required. However, potential
interactions (dose additivity, synergism, supra-additivity, etc.) be-
tween compounds may also occur at very low doses. These two
points (low doses and interactions) are not often taken into ac-
count and represent new paradigms in toxicology. Furthermore,
another current challenge is the development of analytical meth-
ods able to detect a wide range of analytes present in bottled water
at very low levels (see review in Diduch, Polkowska, and Namies´-
nik (2011)).
The aim of this study was to determine the chemical composi-
tion of various bottled waters and, in parallel, to perform in vitro
bioassays to check the potential toxicity of these waters when ex-
posed to high temperatures. The effect of temperature on the
migration of aldehydes, trace metals and several other potential
migrants present in plastic packaging was monitored in PET-bot-
tled waters. The migration tests were performed under realistic
conditions of human exposure according to the EU Regulation
migration criteria (1 kg of water in contact with 6 dm2
of packag-
ing material). The toxicological evaluation of bottled water extracts
was carried out using toxicological endpoints of concern at low
concentrations. The bioassays retained in this study were the Ames
test (using prokaryotes) and the micronucleus assay using P53
competent human cells (HepG2 cell lines) to assess genotoxicity.
Gene reporter assays were also performed for endocrine disruption
activities (estrogenic and anti-androgenic) using human HepG2
and MBA-MB453-kb2 cell lines. All the assays were chosen for
their performance and feasibility and in accordance with EFSA
and/or ICCVAM recommendations (EFSA, 2011a; ICCVAM, 2003).
2. Materials and methods
2.1. Bottled water samples and storage conditions
Two French brands of non-carbonated water (brand A) and car-
bonated water (brand B) bottled in PET and in glass directly pur-
chased from a local store were analysed. Water samples were
bottled at the same time and were from identical batches. Three
samples were derived from each brand by replacing the commer-
cial water with ultrapure water: non-carbonated water in PET
and glass (brand A), ultrapure water in PET (brand A), carbonated
water in PET and in glass (brand B), ultrapure water in PET (brand
B). Water samples were analysed (i) before the experiments (after
10 days at 20 °C), and (ii) after 10 days of storage at three different
temperatures: 40, 50 and 60 °C.
2.2. Solid phase extraction (SPE)
Fourteen compounds, presented in Table 1, were extracted
using Oasis HLB glass cartridges (6 cc/200 mg, Waters, Milford,
USA). These compounds were previously identified in PET-bottled
waters using a preliminary GC–MS screening method (see Addi-
tional Data section). Prior to SPE extraction, three internal stan-
dards were added to water samples as surrogates (Table 1),
namely 2,6-di-tert-butyl-d9-4-methylphenol-3,5-d2, benzophe-
none-d5 and,di-2-ethylhexyl-phthalate-3,4,5,6-d4 (CDN isotopes,
Pointe-Claire, Quebec, Canada) at a concentration of 0.4 lg/L.
Carbonated water was degassed by ultrasonication. Cartridges
were conditioned with 5 mL of ethyl acetate, methanol and UPLC
grade water (Biosolve, Valkenswaard, the Netherlands), and
Table 1
Analytical parameters for the 14 compounds related to plastic packaging. Ions monitored limits of quantification (LOQ) and average recoveries and standard deviations (SD) are
indicated.
Compound Ionsb
LOQ % Recovery (SD)
(m/z) lg/L 0.1 lg/L (0.3 lg/Lc
) 0.5 lg/L (1.6 lg/Ld
)
Dimethyl phthalate (DMP) 163, 77 0.1 107 (12) 100 (7)
2,6-Di-tert-butyl-p-benzoquinone 177, 220, 135 0.1 76 (17) 65 (11)
2,6-Di-tert-butyl-d9–4-methylphenol-3,5-d2a
222, 240 – 57 (6) 66 (4)
Butylated hydroxytoluene (BHT) 205, 220, 177 0.1 63 (8) 73 (12)
2,4-Di-tert-butylphenol (2,4-dtBP) 191 0.3 85 (10) 81 (4)
Ethyl-4-ethoxybenzoate 194, 121, 166 0.1 102 (15) 97 (10)
Diethyl phthalate (DEP) 149 0.1 114 (13) 95 (15)
Benzophenone-d5a
187, 110, 82 – 106 (13) 103 (9)
Benzophenone 182, 105, 77 0.1 99 (10) 92 (10)
4-Nonylphenol (NP) 135, 121, 107 0.1 85 (16) 79 (11)
3,5-Di-tert-butyl-4-hydroxybenzaldehyde (BHT-CHO) 219, 191 0.1 56 (10) 65 (13)
Di-iso-butyl phthalate (DiBP) 149 0.1 93 (11) 87 (11)
Dibutyl phthalate (DBP) 149 0.1 100 (13) 87 (11)
2-Ethylhexyl-p-methoxycinnamate 178, 161 0.3 44 (11) 45 (6)
Di-2-ethylhexyl adipate (DEHA) 129, 111 0.1 46 (8) 41 (5)
Di-2-ethylhexyl-phthalate-3,4,5,6-d4a
153, 171 – 46 (5) 48 (7)
Di-2-ethylhexyl phthalate (DEHP) 149, 167 0.1 60 (5) 60 (4)
a
Internal standard.
b
In bold: quantification ions.
c
For 2,4-di-tert-butylphenol and 2-ethylhexylmethoxycinnamate, average recovery was calculated for a spiked level of 0.3 lg/L.
d
For 2,4-di-tert-butylphenol and 2-ethylhexylmethoxycinnamate, average recovery was calculated for a spiked level of 1.6 lg/L.
C. Bach et al. / Food Chemistry 139 (2013) 672–680 673

3. sample loading of 1L and elution with 2 mL of ethyl acetate was
carried out. Extracts were analysed by GC–MS.
In parallel, bottled water samples were extracted for the bioas-
says following the protocol described above but without internal
standards. GC–MS analysis and toxicological tests were then per-
formed on aliquots of ethyl acetate extracts after concentration
(factor 500) in order to reproduce realistic consumer exposure
in vitro. Preliminary cytotoxicity tests on ethyl acetate alone were
performed on human cell lines (HepG2 and MDA-MB453-kb2
cells). Ethyl acetate was not cytotoxic at the final concentration
of 1% in the culture medium (data not shown).
2.3. GC–MS analysis
Instrumental analyses were performed using gas chromatogra-
phy coupled with ion trap mass spectrometry (GC–MS) (Varian GC
4400-MS 240). Separation was achieved with an Rxi-5MS column
(30 m  0.25 mm ID; 0.25 lm) connected with a 5 m  0.53 mm
deactivated pre-column (Restek, Bellefonte, USA) with the follow-
ing oven programme: 40 °C (hold 1 min) to 280 °C at 8 °C/min and
280 °C (hold 15 min). Helium flow rate was 1 mL/min. Large vol-
ume injections (4 lL) in the split mode (1:25) were carried out.
The inlet temperature programme was: 40 °C (hold 1 min) to
300 °C at 100 °C/min and 300 °C (hold 15 min). Acquisitions were
in Full Scan mode with ion extraction at specific m/z (Table 1).
Calibration was performed in the 10–1000 lg/L concentration
range, depending on the target compound. Table 1 shows the limits
of quantification (LOQ) determined on the basis of a signal-to-noise
ratio of 10. However, LOQ for DiBP, DBP and DEHP were calculated
in such a way as to never reach 1/3 LOQ of the blanks values.
Blanks were prepared with UPLC water (1L) spiked with the three
deutered internal standards (Table 1) at 0.4 lg/L following the
same protocol used for the samples. The background contributions
of laboratory glassware, TeflonÒ
connexions, solvents and UPLC
water were monitored. For each sample batch, several samples
were fortified with target compounds in order to detect a matrix
effect due to the mineralisation and/or carbon dioxide in the water.
In addition, UPLC water blanks were also prepared for each sample
batch. The recovery experiments were determined in UPLC grade
water. The recoveries obtained and spiked levels for each com-
pound are presented in Table 1. Mean recoveries ranged from
44% to 114% for the lower spiked level (0.1 and 0.3 lg/L depending
on the compound), from 40% to 103% for the higher spiked level
(0.5 and 1.6 lg/L depending on the compound). Due to the lower
extraction performance, around 40% for 2-ethyl-p-methoxycinna-
mate, the DEHA and DEHP concentration values obtained in bottled
water were corrected using the recovery value obtained through
fortification of the corresponding samples.
2.4. Aldehyde analysis in PET-bottled waters
Aldehyde analysis (formaldehyde, acetaldehyde, propanal, but-
anal, crotonaldehyde, butanal, pentanal, hexanal, heptanal, octanal,
nonanal, decanal) in water was based on derivatisation with 2,4-
dinitrophenylhydrazine (2,4-DNPH) prior to SPE and liquid chro-
matography/diode array detector (HPLC/DAD) analysis. A 40 mg
2,4-DNPH in 20 mL of acetonitrile (AcCN) reagent solution was
purified to reduce background formaldehyde contamination in
accordance with Zwiener, Glauner, and Frimmel (2002). Derivati-
sation reaction in water samples (550 mL) was carried out follow-
ing the EPA 554 standard method (USEPA, 1992) with some
modifications: 500lL of 2,4-DNPH reagent were added to samples,
and reaction conditions were set up at 60 °C for 4 h. After derivat-
isation, carbonated water samples were degassed by ultrasonica-
tion for 1 h. Aldehydes-DNPH were extracted and concentrated
with Oasis HLB cartridges (200 mg adsorbent, 6 cc; Waters,
Milford, MA, USA) using a Gilson’s GX-274 ASPEC™ instrument
(Middleton, USA). The cartridges were conditioned successively
with 10 mL AcCN (2 Â 5 mL) and, 10 mL of citrate buffer 1 M
(2 Â 5 mL). Water samples were sucked through the adsorbent at
a flow rate of about 5 mL/min. The elution consisted of 6 mL AcCN
(2 Â 3 mL). Extracts were adjusted to 7 mL with ultrapure water.
Chromatographic separation was performed using an Agilent
1200 HPLC system equipped with a diode array detector (DAD).
A SunFire™ C18 column (250 Â 4.6 mm I.D.; particle size, 5 lm)
was used for the separation of DNPH derivates with a flow rate
of 1.2 mL/min and detection was performed at a wavelength of
360 nm. The mobile phase was a binary mixture of AcCN (A) and
water (B). The gradient program was as follows: isocratic at 60%
A for 20 min, 60% A to 90% A for 15 min, isocratic at 90% A for
15 min. Calibration was performed in the 1–10 lg/L concentration
range. Quantification limits (LOQ) were defined as the tenfold va-
lue of the results obtained with ultrapure water blanks. The LOQ
was 3.5 lg/L for formaldehyde, 2 lg/L for acetaldehyde and oct-
anal, 3 lg/L for nonanal and decanal and 1.5 lg/L for the other
aldehydes.
2.5. Analysis of trace metals
Bottled water samples were analysed using a Series XII
induc-
tively coupled plasma mass spectrometer (ICP-MS) (Thermo, Ger-
many) following the ISO 17294-2 standard method (ISO, 2003).
The operation conditions were as follows: RF power was 1318 W,
the carrier, the auxiliary and the nebuliser argon gas flow were
13.0, 0.88, and 0.69 dm3
/min respectively. Rhodium in a concentra-
tion of 1 lg/L was used as the internal standard. The LOQ for trace
metals was 1 lg/L, except for Sb (0.2 lg/L), Pb (0.1 lg/L) and V
(0.5 lg/L).
2.6. Human cells
Routine monitoring has shown the cells to be mycoplasma-free
(Mycoalert kit from Cambrex, Verviers, France). Stocks of cells
were routinely frozen and stored in liquid N2. All experiments were
performed using the cell lines on 10 passages after thawing.
2.6.1. HepG2 cell line
The HepG2 cell line was obtained from the ECACC (European
Collection of Cell Cultures, UK). The cells were grown in monolayer
culture in MEM supplemented with 2 mM L-glutamine, 1% non-
essential amino acids and 10% FBS in a humidified atmosphere of
5% CO2 and at 37 °C. Continuous cultures were maintained by sub-
culturing flasks every 7 days at 2.2 Â 106
cells/75 cm2
flask by
trypsination (trypsin (0.05%)–EDTA (0.02%)).
2.6.2. MDA-MB453-kb2 cell line
This stable transfected human mammary cancer cell line was
obtained from the ATCC (LGC Promochem, Molsheim, France).
The cells were grown in monolayer culture in Leibovitz medium
(L15) supplemented with 10% FBS in a humidified atmosphere at
37 °C. Continuous cultures were maintained by subculturing flasks
every 7 days at 4.0 Â 106
cells/75 cm2
flask by trypsination (trypsin
(0.05%)–EDTA (0.02%)).
2.6.3. Cells exposure to extracts
Bioassays were performed with concentrated bottled water ex-
tracts after 10 days at 60 °C. Extracts were tested in bioassays un-
der realistic consumer exposure conditions (1 kg of foodstuff/
6 dm2
of packaging material) in accordance with EU Regulation
(EU, 2011).
Cells sensitivity differs depending on the origins and protocols
performed: transfected cells are more sensitive to vehicle. Due to
674 C. Bach et al. / Food Chemistry 139 (2013) 672–680

4. this, for the Ames test and micronucleus assay, the final concentra-
tion of bottled water extract was five times more concentrated (1%
of ethyl acetate) than for endocrine disruption assays (0.2% of ethyl
acetate).
2.7. Genotoxicity assays
2.7.1. Ames test
The Ames test was carried out using the plate incorporation
method with or without metabolic activation, with two histidine-
dependent auxotrophic mutants of Salmonella typhimurium strains,
TA 98, TA 100, essentially as described by Maron and Ames (1983).
The S. typhimurium strains were provided by B. Ames (University of
California, Berkeley). The S9 mix was purchased from Trinova Bio-
chem (Giessen, Germany).The test strains were cultured in the li-
quid broth medium for 10 h at 37 °C under agitation. After
incubation, 0.5 mL of 0.1 M sodium phosphate buffer (pH 7.4) (ab-
sence of metabolic activation) or 0.5 mL of S9 mix (presence of
metabolic activation), 0.1 ml of bacterial culture and 10, 25 or
50 lL of ethyl acetate extracts were placed in a test tube. Two
mL of semi-liquid superficial agar was added to the mixture and
poured onto a minimal glucose agar plate.
The top agar was supplemented with 10 mL of 0.5 mM histi-
dine/biotin solution per 100 mL agar, and mutations to histidine
independence were scored on minimal glucose agar plates. The
plates were incubated for 48 h at 37 °C and then revertant colonies
were counted. All experiments were carried out in triplicate using
the three extract concentrations. Mutagenic activity was expressed
as an induction factor, i.e. as multiples of the background levels.
2.7.2. Micronucleus assay
This assay was performed following the protocol of Séverin, Jon-
deau, Dahbi, and Chagnon (2005). HepG2 cells were seeded at
2.5 Â 105
cells/well. After 24 h, cells were treated with 1% of the
ethyl acetate extract and cytochalasin B (4.5 lg/mL) for 44 h. Cells
were then washed with PBS and allowed to recover for 1.5 h in
MEM with 10% FBS. The cells were then washed with PBS, trypsin-
ised (trypsin (0.05%)-EDTA (0.02%) solution from Invitrogen labo-
ratories (Cergy-Pontoise, France)), fixed in two steps with acid
acetic/methanol (1/3) (v/v), spotted on a glass slide and stained
with acridine orange (0.1%) diluted in Sorensen buffer (1/15, v/v)
just before reading. Micronuclei were counted visually in 1000
binucleated cells (BNC)/slide using a fluorescence microscope
(Olympus CK40) and two slides/concentration were counted. To
identify micronuclei, the criteria of Kirsch-Volders et al. (2000)
were applied: the diameter of micronuclei should be under one-
third that of the main nucleus. They should be clearly distinguish-
able from the main nucleus and should have the same staining as
the main nucleus.
2.8. In vitro endocrine disruptor potential
2.8.1. Estrogenic activity: Transcriptional activation assay with HepG2
cell line
2.8.1.1. Seeding. The estrogenic potential of extracts was deter-
mined using HepG2 cells transiently transfected with hERa and
with an ER-responsive luminescent reporter gene. HepG2 cells
were seeded at a density of 1.2 Â 105 cells per well in 24-well tis-
sue culture plates (Dutscher, France) and maintained in MEM med-
ium without phenol red, supplemented with 10% dextran-coated
charcoal foetal calf serum (DCC-FCS), 1% L-glutamin and 1% non-
essential amino acids. The microplates were then incubated at
37 °C in a humidified atmosphere of 5% CO2 for 24 h.
2.8.1.2. Transient transfection. Plasmids ERE-TK-Luc and pRST7-hERa
were kindly provided by Dr. D. McDonnell (Ligand Pharmaceutical,
San Diego, USA). Plasmids pCMVb-Gal and pSG5 were kindly pro-
vided by Pr M. Cherkaoui-Malki (LBMC, Dijon, France). Each plas-
mid was first diluted in 0.15 M NaCl (sterile) to a final
concentration of 100 ng/lL. HepG2 cells were transiently transfec-
ted using the Exgen500 procedure (Euromedex) with the following
plasmid mix: 100 ng ERE-TK-Luc and 100 ng hERa, 100 ng of
pCMVb-Gal and pSG5 to a final concentration of 0.5 lg DNA. Then,
2 lL of Exgen500 diluted in NaCl 0.15 M was added to the DNA.
After vortex shaking, the microtubes were incubated at room tem-
perature for 10 min. The Exgen500-DNA mixture was then added
to OptiMEM without phenol red medium and distributed into the
wells (300 lL/well). The microplate was then incubated at 37 °C
in a humidified atmosphere of 5% CO2 for 1 h (Dumont, 2010).
2.8.1.3. Treatment and analysis. After incubating HepG2 cells for
1 h, the OptiMEM was removed and replaced by 1 mL of treatment
medium (MEM without phenol red, without FCS, 1% L-glutamin
and 1% non-essential amino acids), containing the water extract,
or the vehicle ethyl acetate (1%, negative control), or 17b-estradiol
(10À8
M, positive control). The plate was then incubated for 24 h.
At the end of the treatment, the medium was aspirated and the
wells were rinsed with 100 lL PBS and then lysed using 100 lL Re-
porter Lysis Buffer 1X (Promega). The microplate was then frozen
at À80 °C for at least 15 min. After thawing, cells were scraped
and placed into microtubes. The cells then underwent 3 freezing/
thawing cycles in liquid nitrogen and at 37 °C in a water bath. After
centrifugation (5 min at 10,600g), luciferase and b-galactosidase
activities were determined.
For luciferase measurement, 10 lL from each well were trans-
ferred into an opaque white-walled plate (Perkin Elmer, Courtab-
oeuf, France) and mixed with 50 lL of luminol. The plate was
quickly covered with an adhesive seal and the mixture was imme-
diately analysed using a luminometer (TopCountNT, Packard).
For b-galactosidase activity, 10 lL of each well were used to
measure the chlorophenol-red b-D-galactopyranoside (CPRG)
(Roche) product with a spectrophotometer at 570 nm (MRX
Dynex). Protein absorbance was also measured using 2 lL of the ly-
sate according to the Bradford method on a spectrophotometer at
595 nm (Bradford, 1976). Luciferase induction responses for each
treatment group were normalised for b-galactosidase activity and
protein level (Luc  Prot/Gal) and the results of the different tested
extracts were compared with the response observed for 17b-estra-
diol 10À8
M (set at 100%).
2.8.2. Anti-androgenic activity: Transcriptional activation assay using
the human MDA-MB453-kb2 cell line
The MDA-MB-453 (AR+
) cell line was stably transfected with
MMTV-neo-Luc with an (anti)-AR-responsive luminescent reporter
gene (Wilson, Bobsein, Lambright, & Gray, 2002). Cells were seeded
into a 24-well plate (Dutscher, France) in 1 mL of L15 medium
without phenol red, supplemented with 5% of dextran-coated char-
coal fetal calf serum (FCS), at a density of 5 Â 104
cells/well. For
anti-androgenic activity, 24 h after seeding, the medium was re-
moved and cells were exposed to ethyl acetate extracts (0.05, 0.1
and 0.2%) in the presence of dihydrotestosterone (DHT), the andro-
genic reference (4 Â 10À10
M, prepared in ethyl acetate). Niluta-
mide (Nil) (10À6
M, prepared in ethyl acetate) was used as a
positive control for anti-androgenic activity. After 24 h treatment,
luciferase activity was measured (Stroheker, Picard, Lhuguenot,
Canivenc-Lavier, & Chagnon, 2004). Cells were washed once with
1 mL of phosphate buffered saline. Following 30 min incubation
with 200 lL/well lysis buffer at room temperature with shaking,
lysate were briefly vortexed and centrifuged at 3000g at 4 °C
(Stroheker et al., 2004). Ten microlitre from each well were trans-
ferred into an opaque white-walled plate and mixed with 40 lL of
luciferase assay reagent. The plate was quickly covered with an
C. Bach et al. / Food Chemistry 139 (2013) 672–680 675

5. adhesive seal and the mixture was immediately analysed using a
luminometer (TopCountNT, Packard). Results are expressed as a
percentage of the androgenic positive control (DHT).
3. Results
3.1. Migration of 14 compounds linked to plastic packaging
PET- and glass-bottled waters exposed to the worst-case condi-
tions in this study (10 days at 60 °C) were analysed hypothesising
that the highest temperature would promote maximal migration.
2,4-di-tert-butylphenol (2,4-dtBP) was detected in bottled waters,
while the other compounds were below the quantification limits.
As shown in Fig. 1, before experiments (storage time = 0) 2,4-dtBP
was already present (0.4 lg/L) in glass-bottled non-carbonated
water (brand A) and in PET-bottled water or glass-bottled carbon-
ated water (brand B). After 10 days of exposure at 60 °C, at least a
twofold increase of 2,4-dtBP was observed in these bottled waters
and the same increase was also found in the ultrapure water placed
in brand B bottles (0.7 lg/L). In the same conditions (10 days at
60 °C), an unexpected chromatographic peak appeared exclusively
in PET-bottled water extracts. Bis(2-hydroxyethyl) terephthalate
(BHET), an intermediate monomer in PET synthesis, was identified
with the spectral library (NIST 98 Mass Spectral Library). BHET was
not quantified because the analytical standard was not available in
the laboratory. The BHET chromatogram and mass spectrum are
shown in Fig. 1A in the Additional Data section. According to the
molecular ion of BHET (m/z 254), the most intensive peak (m/z
193) corresponds to ion M-61 (M-C2H5O2) due to a double rear-
rangement of acetate. The m/z 211 peak corresponds to ion M-43
(M-CH3CO) and m/z 149 (typical of phthalate esters) corresponds
to ion C8H5O3
+
.
3.2. Migration of aldehydes
3.2.1. Effect of temperature on formaldehyde and acetaldehyde
migration
The effect of temperature on aldehydes migration into PET-bot-
tled water was assessed with results obtained in ultrapure water
for both brands of PET bottles. With ultrapure water, formaldehyde
migration was higher at 60 °C (Fig. 2A) while the release of acetal-
dehyde (Fig. 2B) had already begun at 50 °C. Acetaldehyde migra-
tion appears to be more sensitive to temperature than
formaldehyde. At 60 °C, formaldehyde concentration was 4 times
lower than acetaldehyde in ultrapure water packaged in brand A
bottles and 13 times lower in water in brand B bottles.
3.2.2. Effect of water type (carbonated or non-carbonated) on
formaldehyde and acetaldehyde migration
In non-carbonated water, as observed before, acetaldehyde
migration (Fig. 2B) was higher at 50 °C while formaldehyde migra-
tion (Fig. 2A) was higher only at the highest temperature (60 °C).
However, data showed a higher dispersion of concentrations espe-
cially with formaldehyde in carbonated water, probably due to the
presence of residual CO2 not eliminated during sample degasifica-
tion. In carbonated water, formaldehyde and acetaldehyde were al-
ready present at 20 °C. Their concentrations were always higher in
carbonated water than in non-carbonated water. At 60 °C, a weak
increase (1.6 times) in aldehyde concentrations in carbonated
water was observed. Formaldehyde concentrations were 4 times
lower than acetaldehyde concentrations in non-carbonated water
and 8.5 times lower in carbonated water.
3.3. Migration of trace metals
Most of the inorganic elements found in both mineral waters
(carbonated and non-carbonated) were those naturally present, ex-
cept Sb.
3.3.1. Effect of temperature on Sb migration
When the water was ultrapure, Sb was not detected in PET-bot-
tled water at 20 °C (Fig. 3). In contrast, Sb concentration gradually
increased in ultrapure water between 10 days of storage at 40 °C
and at 60 °C from 0.5 to 3.5 lg/L. Therefore, higher temperatures
induce higher levels of Sb migration.
3.3.2. Effect of the water type (carbonated or non-carbonated) on Sb
migration
Sb was already present (around 1 lg/L) in both PET-bottled
waters (carbonated and non-carbonated) at 20 °C (Fig. 3). After
10 days at 60 °C, an increase in Sb concentration of 4.8 times and
7.3 times was observed in non-carbonated and carbonated water
respectively. Sb concentration in carbonated water was twice as
high as non-carbonated water, suggesting that Sb release was
accelerated by carbon dioxide.
3.4. Genotoxicity assays
3.4.1. Ames test
Induction factors obtained for negative and positive controls
performed on the TA 98 and TA 100 strains with and without S9
mix were consistent with the laboratory’s historical data. Bottled
waters were not mutagenic (induction factor <2) for S. typhimurium
0 10
0.3
0.8
1.3
1.8
2.3
Non-carbonated water in PET (brand A)
Ultrapure water in PET (brand A)
Non-carbonated water in glass (brand A)
Carbonated water in PET (brand B)
Ultrapure water in PET (brand B)
Carbonated water in glass (brand B)
Days of storage at 60°C
2,4-dtBPconcentration
(µg/L)
Fig. 1. Average concentrations ± standard deviation (SD) of 2,4-di-tert-butylphenol (2,4-dtBP) in mineral (carbonated or non-carbonated) or ultrapure water bottled in PET
and glass before and after 10 days of exposure at 60 °C. All water analyses were carried out in quintuplicate (water from five different bottles). The method’s LOQ for 2,4-dtBP
was 0.3 lg/L.
676 C. Bach et al. / Food Chemistry 139 (2013) 672–680

6. strains under the experimental conditions used (Table A.2, Addi-
tional Data section).
3.4.2. Micronucleus assay
The validity of the test was checked with the positive responses
obtained using the reference control, showing an increase in the
number of micronuclei in binucleated cells (>twice as many as
the negative control). As shown in Fig. 4, the cytotoxicity rates cal-
culated for all water extracts were below the maximum recom-
mended value of 55% (OECD, 2010). Bottled water extracts did
not induce any chromosome aberration or genomic effect in the
HepG2 cells after exposure.
3.5. Potential endocrine-disruption activity
3.5.1. Estrogenic activity
The maximum activity (100%) corresponds to the estrogenic
activity of 10À8
M 17b-estradiol (E2). Activity of the negative con-
trol and extracts was calculated relative to E2. ERa transcriptional
activity did not increase when bottled water extracts were exposed
to the HepG2 cells in our experimental conditions (Table A.3 in the
Additional Data section).
3.5.2. Anti-androgenic activity
As expected, Nilutamide (NIL), the anti-androgenic reference,
significantly decreased the androgenic activity of dihydrotestoster-
one (DHT). In contrast, extracts of the PET-bottled water did not
modify the androgenic effect of DHT, suggesting they were not
anti-androgenic even at 0.2%, the initial concentration for bottled
water (Fig. 5A–D).
However, when cells are co-exposed to glass-bottled carbon-
ated water extracts and DHT, a weak but significant increase of
the AR transcriptional activity in the MDA-MB456-kb2 cells was
observed (Fig. 5D) when compared to the response of DHT alone.
The effect was observed at two concentrations (0.05%, 0.2%) with-
out dose-dependency.
4. Discussion
Concerning chemical analysis, formaldehyde and acetaldehyde,
well-known PET degradation products, were detected in all the
PET-bottled waters. At 60 °C, the migration of these compounds
was highly accelerated. This could be explained by the storage
Fig. 2. Formaldehyde (A) and acetaldehyde (B) mean concentrations ± standard deviation (SD) in mineral (carbonated or non-carbonated) or ultrapure water packaged in PET
bottles of brands A and B after 10 days of exposure at 40, 50 and 60 °C. Room temperature exposure was arbitrarily set at 20 °C. All water analyses were performed in
quintuplicate (water from five different bottles). The method’s LOQ for formaldehyde and acetaldehyde were 3.5 and 2 lg/L, respectively.
20°C 40°C 50°C 60°C
0
1
2
3
4
5
6
7
8
9
10 Non-carbonated water (brand A)
Ultrapure water (brand A)
Carbonated water (brand B)
Ultrapure water (brand B)
Storage temperature for 10 days
Concentration(µg/L)
Fig. 3. Sb mean concentrations ± standard deviation (SD) in mineral (carbonated or
non-carbonated) or ultrapure water packaged in PET bottles from brands A and B
after 10 days of exposure at 40 °C, 50 °C and 60 °C. Exposition at room temperature
was arbitrarily fixed at 20 °C. All analyses were performed in quintuplicate (water
from five different bottles). The method’s LOQ for Sb was 0.2 lg/L.
Fig. 4. Micronucleus data in the HepG2 cell line after 10 days of exposure at 60 °C
to PET- and glass-bottled water extracts. The solvent control (SC) was DMSO (0.25%
final concentration). The negative control (NC) was ethyl acetate (1% final
concentration) and the positive control (PC) was a solution of vinblastine sulphate
in DMSO (0.005 lM final concentration). ApT and AvT represent non-carbonated
mineral water bottled in brand A PET- and glass-bottles respectively. BpT and BvT
represent carbonated mineral water in brand B PET- and glass-bottles respectively.
C. Bach et al. / Food Chemistry 139 (2013) 672–680 677

7. temperature which was near the PET glass transition temperature
(around 80 °C for semi-crystalline PET), which increases the mobil-
ity of polymeric chains directly linked to the migration phenome-
non (Bach, Dauchy, David, & Etienne, 2011). We demonstrated that
carbon dioxide in bottled water contributed to the increase of
migration of both aldehydes. Indeed, formaldehyde and acetalde-
hyde were substantially present in the bottled carbonated waters
before temperature experiments were conducted (10 days at
20 °C). Nijssen, Kamperman, and Jetten (1996) reported that the
relatively acid pH of carbonated waters might be responsible for
aldehyde migration. In this study, aldehydes were not detected
in PET-ultrapure water with a pH very close to carbonated water.
This confirms that the pressure exerted on the bottle wall by car-
bon dioxide could activate aldehyde migration, as previously sug-
gested by Dabrowska, Borcz, and Nawrocki (2003). The same
observations were drawn for Sb migration into carbonated waters;
in this study Sb migration was also affected by temperature. The
presence of Sb in PET-bottled waters (carbonated or non-carbon-
ated) before the experiments were conducted (10 days at 20 °C)
suggests that the water’s natural composition may promote Sb
migration. However, poor transport conditions and storage cannot
be excluded.
With regard to EU Regulations, under the worst-case scenario of
this study (10 days at 60 °C), concentrations of formaldehyde, acet-
aldehyde and Sb in PET-bottled water were largely below the spe-
cific migration limit (SML) (EU, 2011). However, it is worth noting
that the formaldehyde level in carbonated water exceeded the
French quality standard for bottled mineral waters (5.0 lg/L)
(JORF, 2011).
In this study, and consistent with chemical formulations of PET
(Enneking, 2006; Guart, Bono-Blay, Borrell, & Lacorte, 2011), nei-
ther UV stabilisers nor phthalates were detected in bottled water
before or after experiments. This is in accordance with Ceretti
et al. (2010). In contrast, Montuori, Jover, Morgantini, Bayona,
and Triassi (2008) found higher phthalate concentrations (20 times
higher) in PET-bottled water than in glass-bottled water. Leivadara,
Nikolaou, and Lekkas (2008) detected DEHP in bottled water, but
this plasticiser was not identified in tap water stored in the same
type of PET bottles. More recently, Amiridou and Voutsa (2011)
quantified phthalates in PET-bottled water but they concluded that
plasticiser migration was not significant. Phthalate pollution in the
bottling line cannot be ruled out.
2,4-dtBP was detected in the ethyl acetate extracts of PET- and
glass-bottled water after 10 days at 60 °C. Its presence was previ-
ously reported by the German Federal Institute for Risk Assessment
(BfR, 2011). The twofold increase of 2,4-dtBP in both PET- and in
glass-bottled water suggests that its occurrence was not directly
linked to PET. Its presence could be due to the plastic material in
(A) Non-carbonated water in PET
EA
D
H
T
N
IL+
D
H
T
0.05
%
+
D
H
T0.1
%
+
D
H
T0.2
%
+
D
H
T
0
25
50
75
100
125
150
175
200
*
*
Treatment
%Transcriptionalactivity
(B) Non-carbonated water in glass
EA
D
H
T
N
IL+
D
H
T
0.05
%
+
D
H
T0.1
%
+
D
H
T0.2
%
+
D
H
T
0
25
50
75
100
125
150
175
200
*
*
Treatment
%Transcriptionalactivity
(C) Carbonated water in PET
EA
D
H
T
N
IL+
D
H
T
0.05
%
+
D
H
T0.1
%
+
D
H
T0.2
%
+
D
H
T
0
25
50
75
100
125
150
175
200
*
*
Treatment
%Transcriptionalactivity
(D) Carbonated water in glass
EA
D
H
T
N
IL+
D
H
T
0.05
%
+
D
H
T0.1
%
+
D
H
T0.2
%
+
D
H
T
0
25
50
75
100
125
150
175
200
*
*
*
*
Treatment
%Transcriptionalactivity
Fig. 5. Anti-androgenic activity in MDA-MB453-kb2 cell line exposed to bottled-water extracts (10 days at 60 °C). Figures A and B represent non-carbonated water in PET and
glass respectively (brand A). Figures C and D represent carbonated water in PET and glass respectively (brand B). MDA-MB453-kb2 cell line was treated with extract
concentrations of 0.05%, 0.1% and 0.2%. Ethyl acetate (EA) represents the negative control (0.25% final concentration). Maximum activity (100%) corresponds to the activity of
dihydrotestoterone (DHT 4 Â 10À10
M), the androgenic reference. Nilutamide (Nil) (10À6
M) is the positive control for anti-androgenic activity. The à sign indicates results
statistically different from the DHT positive control (androgenic reference) using the ANOVA statistical test and then a Dunnett’s multiple comparison method. All
experiments were performed in triplicate.
678 C. Bach et al. / Food Chemistry 139 (2013) 672–680

8. the bottle caps, as a large number of caps are made of polyethylene
(PE) and polypropylene (PP) plastics (ILSI, 2003). Simoneau, Van
den Eede, and Valzacchi (2012) recently quantified 2,4-dtBP leach-
ing from PP, polyamide and silicone baby bottles. Indeed, 2,4-dtBP
is a by-product of tris (2,4-di-tert-butylphenyl) phosphate (Irgafos
168), used to produce PE and PP. As recycled PET is authorised in
water bottles, chemicals from non-food application containers
found in collection systems of post-consumer PET are a possible
source of PET contamination (EFSA, 2011b). This phenolic com-
pound is not on the EU’s positive list (EU, 2011). 2,4-dtBP was cyto-
toxic when exposed to tumour cells or plants models (Kadoma, Ito,
Atsumi, & Fujisawa, 2009; Malek, Shin, Wahab, & Yaacob, 2009). It
is also suspected to be an endocrine disruptor. 2,4-dtBP has been
shown to be an androgen antagonist in CHO-K1 cells and in rain-
bow trout (Satoh, 2007; Tollefsen, Eikvar, Finne, Fogelberg, & Gre-
gersen, 2008) and is a compound of very high concern in substance
assessment under the REACH Regulation (CoRAP, 2011; EU, 2006).
In this study, BHET was also identified in the extracts of PET-
bottled water, but no specific migration limit (SML) has been
established in EU Regulation No. 10/2011 for this substance. Di-
methyl terephthalate, a compound of the same chemical family,
has been shown to have estrogenic activities in animals and hu-
mans (Cheung, Lam, Shi, & Gu, 2007). No other compounds were
detected in this study, although chemical analyses are never
exhaustive since it is difficult to detect all the substances present
(Bradley et al., 2009). Therefore, other non-identified NIAS may
be present in PET-bottled water.
As this study concerns low concentrations of compounds, two
relevant toxicological endpoints, cyto/genotoxicity and endocrine
disruption potential, were checked. Bottled water extracts were
neither cytotoxic (data not shown), nor genotoxic for HepG2 cells.
The extracts were not mutagenic for the S. typhimurium strains
TA98 and TA100. This is not in agreement with Biscardi et al.
(2003), who showed an eightfold increase in micronuclei when
Tradescantia cells were exposed to PET-bottled non-carbonated
water over 2 and 3 months as compared to distilled water. A sub-
stantial increase in micronuclei was also observed using Allium
Cepa cells with carbonated water bottled in PET and glass after
10 days at 40 °C by Ceretti et al. (2010). But the genotoxic effect
of bottled water was not reproduced by the latter, when they per-
formed a Comet assay on Tradescantia cells. When using in vitro
models, it is important to control the pH, the osmolarity of the cul-
ture medium and the cellular survival during compound exposure.
Also, CO2 gas in water can create cytotoxic effects (Jondeau, 2006)
that would give false positive responses in the genotoxicity assays.
In addition, according to international guidelines, plants are not
considered as primary ‘‘screening’’ tools for extrapolation to mam-
mals. (EFSA, 2011a).
With regard to potential endocrine disruption activities, no
estrogenic activity using HepG2 cells was detected in PET- or
glass-bottled water extracts prepared with Oasis HLB cartridges
under realistic consumer exposure conditions. Our data are not
in accordance with Pinto and Reali (2009), Wagner and Oehlmann
(2009) or Wagner and Oehlmann (2011), who showed estrogenic
activity PET-bottled water extracts. Divergent storage conditions,
sample preparation methods (concentration factors, SPE, etc.), cell
models (yeasts, snails, etc.) and bioassays (E-screen, YES assay,
etc.) were used, making comparison of the results difficult. Wagner
and Oehlmann (2011) reported that the use of C18 cartridges and
the addition of dimethyl sulfoxide (DMSO) was the most efficient
sample preparation method for extracting estrogen-like com-
pounds. Unfortunately, the authors did not identify the compounds
at the origin of the positive results they observed. More recently,
Plotan, Frizzell, Robinson, Elliott, and Connolly (2012) reported
that the level of hormonal activity in bottled water, estimated via
daily intake, is not a matter of concern for consumers.
The potential anti-androgenic activity of concentrated bottled
water was also checked because compounds with weak estrogenic
activity can also have anti-androgenic activity (Sohoni & Sumpter,
1998) as observed with BPA and phthalates (Stroheker et al., 2005;
Xu et al., 2005). As mentioned before, 2,4-dtBP can also have anti-
androgenic activity (Satoh, 2007). However, in our experimental
conditions, no anti-androgenic activity was detected when MDA-
MB456-kb2 cells were exposed to PET- or glass-bottled water ex-
tracts. The data only showed a weak increase of the androgenic re-
sponse of DHT in the presence of glass-bottled water extracts
compared to DHT alone. This effect could be due to an androgenic
activity of the extract itself. As the extract when tested alone was
not androgenic (data not shown), this biological response could be
due to an interaction such as a potentiation effect of the extract on
DHT activity.
5. Conclusions
The migration of IAS and NIAS in various bottled waters ex-
posed to different temperatures was investigated. In parallel,
in vitro bioassays were performed to check the potential toxicity
of chemical mixtures in PET-bottled water. As expected, formalde-
hyde, acetaldehyde and Sb were detected in PET-bottled waters.
We demonstrated that high temperatures and carbonation in-
creased migration in the worst-case scenario used in this study
(60 °C for 10 days). 2,4-dtBP and BHET were also identified as NIAS.
These two compounds are not on the EU Regulation positive list.
However, PET-bottled water after 10 days at 60 °C did not induced
any toxic activity (cytotoxicity, genotoxicity or potential endocrine
disruption) in vitro. Chemical analysis and global approaches using
pertinent and sensitive bioassays are complementary tools to iden-
tify the potential hazard of all compounds able to migrate.
Acknowledgements
This research was supported by the French Agency for Food,
Environmental and Occupational Health & Safety (ANSES) and
the Institute Jean Lamour of the University of Lorraine. The authors
wish to thank the Water Chemistry Department of ANSES’s Nancy
Laboratory for Hydrology for their excellent technical assistance.
The authors are grateful to Coralie Dumont, Khadija Raja, Anne
Novelli, Valérie Fessard, Christian Tricard, and Eric Barthélémy
for their collaboration.
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