Science of the Total Environment 618 (2018) 142–150

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Concentrations of several phthalates contaminants in Egyptian bottled

water: Effects of storage conditions and estimate of human exposure
Ghada Zaki, Tamer Shoeib ⁎
Department of Chemistry, The American University in Cairo, New Cairo 11835, Egypt

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• First time measurements of phthalates

in Egyptian bottled water
• DEHP and DBP detected in 50 and 58% of
samples respectively 2 weeks after pro-
duction
• Positive correlation obtained between
storage time and phthalate concentra-
tions.
• Estimated intake of DEHP and DBP from
bottled water were far below their TDI
values.

a r t i c l e i n f o a b s t r a c t

Article history: The occurrence and concentrations of six common phthalates were investigated for the first time in bottled water
Received 22 July 2017 locally produced in the Egyptian market. The compounds investigated were dimethyl phthalate (DMP), diethyl
Received in revised form 30 October 2017 phthalate (DEP), dibutyl phthalate (DBP), n-butyl benzyl phthalate (BBP), diethyl hexyl phthalate (DEHP), and Di-
Accepted 31 October 2017
n-octyl phthalate (D-n-OP). A set of 108 bottled water samples from six different commercial brands of water bottled
Available online 8 November 2017
in transparent polyethylene terephthalate (PET) plastic bottles with high density polyethylene (HDPE) plastic caps
Editor: Yolanda Picó were investigated. Water samples were analyzed immediately after purchasing (~2 weeks after production), after
being stored at room temperature (25 ± 5 °C), in a refrigerator (4 ± 1 °C) and outdoor under sun exposure (daylight
Keywords: temperature of 40 ± 5 °C). Samples were stored up to six months depending on the tested condition. Among the tar-
Phthalates get compounds, only DEHP and DBP were detected in the samples analyzed immediately after purchasing with a de-
Bottled water tection frequency of 50 and 58% and mean concentrations of 0.104 and 0.082 μg l−1 respectively. Significant positive
Storage conditions correlation was obtained between the storage time, temperature and the concentration of phthalate compounds de-
Migration tected in the bottled water, indicating possible migration from the PET plastic material as the source. The estimated
Estimated human exposure
contribution of bottled water consumption to the tolerable daily intake (TDI) levels of the two most abundant
phthalates observed here for adults and toddlers did not exceed 0.16 and 0.72% for DBP while these values were
0.04 and 0.16% for DEHP respectively. These estimated daily intake values from PET bottled water consumption
were far below their respective TDI values and therefore should constitute no adverse health effects.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction

⁎ Corresponding author. Packaging materials protect foods and beverages from chemical and
E-mail address: T.Shoeib@aucegypt.edu (T. Shoeib). environmental contaminations (Bhunia et al., 2013; De Fátima Pocas

https://doi.org/10.1016/j.scitotenv.2017.10.337
0048-9697/© 2017 Elsevier B.V. All rights reserved.
 G. Zaki, T. Shoeib / Science of the Total Environment 618 (2018) 142–150
and Hogg, 2007). However, the packaging material itself may also pres-
ent a potential source of chemical contamination to the many packaged
products through the possible migration of some of their chemical con-
stituents into the packaged products. These migrating constituents may
include substances intentionally or non-intentionally added to the
packaging materials (De Fátima Pocas and Hogg, 2007; Grob et al.,
2006). Monitoring exposure of chemicals from packaging materials
into foods has thus become a fundamental part of ensuring food safety
and protecting human health (De Fátima Pocas and Hogg, 2007). Di-
esters of ortho-phthalic acid are among such chemicals which require
firm monitoring (Bhunia et al., 2013; Cao, 2010; De Fátima Pocas and
Hogg, 2007).
Di-esters of ortho-phthalic acids, generally recognized as phthalates,
are a class of synthetic organic chemical compounds that are extensively
used in the manufacturing of polymers and many commonly used com-
mercial products (Guo and Kannan, 2012; Swan, 2008; Latini, 2005).
Phthalates are used primarily as plasticizers, which are added to the
polymeric materials, especially polyvinyl chloride (PVC), to enhance
their softness, durability and ease of processing (Meeker et al., 2009;
Schettler, 2006). To date, phthalates are the most frequently used plas-
ticizers accounting for over 80% of the plasticizers used globally (Guo
and Kannan, 2012).
Phthalates are not chemically bound to the polymer matrices to
which they are added. Accordingly, they can easily leach out from the
products, during their use or after their disposal, migrating into air,
dust, soil, water and food (Cao, 2010; Wormuth et al., 2006). Exposure
to phthalates can cause serious adverse health effects on the kidney,
liver, respiratory and endocrine systems (Meeker et al., 2007;
Bornehag et al., 2004; Hoppin et al., 2004; ATSDR, 2002). Phthalates ex-
hibit serious hepatotoxic effects and the International Agency for Re-
search on Cancer classified DEHP as a class 2B probable carcinogen
(Erkekoglu et al., 2014; ATSDR, 2002). The main health concerns are re-
lated to the endocrine disrupting potency of phthalates and their me-
tabolites. Animal and in-vitro studies demonstrated that phthalates
have anti-androgenic and weak estrogenic activities that consequently
resulted in hazardous reproductive and developmental effects (Hauser
et al., 2007; Lee and Koo, 2007; Meeker et al., 2007). Human studies
are limited however, epidemiological studies seem to show correlations
between phthalates exposure and adverse reproductive outcomes, in-
cluding sexual hormones disorder, infertility, premature breast devel-
opment and preterm labor (Swan, 2008; Hauser et al., 2007; Zhang
et al., 2006; Latini et al., 2003). Studies have also shown that exposure
to phthalates is associated with altering thyroid hormones levels, in-
creased abdominal obesity, insulin resistance, asthma and respiratory
allergic symptoms (Meeker et al., 2007; Stahlhut et al., 2007; Hoppin
et al., 2004).
Recent reports suggest food and beverage to be the predominant
source of human exposure to phthalates (Heinemeyer et al., 2013;
Fierens et al., 2012; Fasano et al., 2012; Rudel et al., 2011; Cao, 2010).
Bottled water as a potential source of human exposure to phthalates
has drawn considerable attention lately due to its high and regular con-
sumption despite its higher price relative to tap water, in part due to the
perception of higher quality, purity and safety (Marcussen et al., 2013;
Diduch et al., 2013; Bach et al., 2012). In fact the Codex Alimentarius
general standard for bottled/packaged drinking waters requires compli-
ance with the World Health Organization guideline for DEHP in drinking
water not to exceed 8 μg l−1 (WHO, 2011; Codex Alimentarius, 2001)
while both the US FDA and US EPA have regulated the maximum per-
missible level of DEHP to be 6 μg l− 1 in bottled water and drinking
water respectively (FDA, 2011; EPA, 2009).
The most common polymer used for the packaging of bottled water
is polyethylene terephthalate (PET). Although PET is reported to be free
from plasticizers or phthalates, several studies have shown the presence
of phthalates in bottled water packed in PET containers (Jeddi et al.,
2015; Guart et al., 2014; Keresztes et al., 2013; Al-Saleh et al., 2011;
Amiridou and Voutsa, 2011; Montuori et al., 2008; Cao, 2008; Leivadara
143
et al., 2008; Casajuana and Lacorte, 2003). Phthalates occurrence in PET
bottled water is a controversial issue as PET polymers differ chemically
from phthalates. Phthalates are esters of ortho-phthalic acids, while PET
is polyester of para-phthalic acid. Thus, it is not chemically possible for
PET to yield the phthalates in question through degradation or any
other chemical pathway during the manufacturing or storage (Sax,
2010; Enneking, 2006). Furthermore, Phthalates are not typically used
in PET manufacturing as PET bottles are required to be rigid, in order
to attain good mechanical and gas barrier properties, while their elastic-
ity can be tailored during their production through extrusion and mold-
ing (Bhunia et al., 2013).
In Egypt, the occurrence of phthalates in PET bottled water was not
previously investigated. The aim of this study was to examine the occur-
rence and quantify the levels of phthalates in bottled water locally pro-
duced in the Egyptian market and to investigate the effects of various
storage conditions on the levels of these contaminants. As it is not un-
usual for vendors and distributors in Egypt to transport and store bot-
tled waters outdoors under direct sun exposure for extended periods,
the levels of phthalate compounds were analyzed directly after pur-
chasing (~2 weeks after production), the migration of phthalate com-
pounds during storage under common conditions in the Egyptian
market was investigated, comparisons of phthalate levels in the locally
produced PET plastic bottled water with the global levels for PET bottled
water are presented and finally an estimate of the exposure of adults
and toddlers in Cairo to phthalate compounds through bottled water
consumption and possible associated health effects are explored. To
our knowledge, there are no previous published studies that have ex-
amined the phthalate contaminants in PET bottled water produced in
Egypt.
2. Materials and methods
2.1. Samples collection and storage conditions
Bottled water samples (n = 108) were collected across six different
brands (equivalent to 2 bottles from each brand per condition analyzed)
that are locally produced and frequently consumed in the Egyptian mar-
ket. The bottled water samples were geographically randomly collected
in July 2013 from various local markets in Cairo, Egypt. All sampled bot-
tled waters were of the same size (1.5 L) and those from a given brand
were from the same batch. All the samples were packed in PET plastic
bottles with HDPE plastic caps with no cap liners. The only brand of car-
bonated bottled water locally produced in Egypt was also examined to
assess the effect of the presence of carbon dioxide gas on the phthalates
levels, while the remaining five were non carbonated bottled drinking
water.
Bottled water samples were analyzed a) directly after purchasing
(~ 2 weeks after production) (n = 12); b) analyzed after 1, 2, and
4 months of storage (n = 36) after being stored outdoors (exposed di-
rectly to sunlight during the period of July to November 2013 where the
daylight temperature was recorded to be 40 ± 5 °C); c) after 1, 2, and
4 months of storage at 4 °C (n = 36); and d) after 2 and 6 months of
storage (n = 24) at room temperature (25 ± 4 °C). Each sample was an-
alyzed in duplicates and the values for the duplicate analysis were aver-
aged for data summaries and statistical analyses.
2.2. Chemicals and reagents
A standard mixture (Z-014A) containing standard solutions of di-
methyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate
(DBP), n-butyl benzyl phthalate (BBP), diethyl hexyl phthalate
(DEHP), and Di-n-octyl phthalate (D-n-OP) dissolved in dichlorometh-
ane (each at a concentration of 2000 μg ml−1) was obtained from
AccuStandard (New Haven, USA). HPLC grade dichloromethane was
purchased from Fisher Scientific (Loughborough, UK). Anhydrous sodi-
um sulfate of analytical reagent grade was purchased from MP
144 G. Zaki, T. Shoeib / Science of the Total Environment 618 (2018) 142–150
Biomedicals, LLC (Strasbourg, France). Ultrapure water produced by a
Milli-Q Integral water purification system (Millipore, Bedford, MA,
USA) was used. The stock and working standard solutions were kept
at 4 °C until use. Calibration standard solutions with different concen-
trations were freshly prepared on the day of analysis by diluting the
working standard in dichloromethane.
2.3. Glassware and reagent control
Several precautions were undertaken, according to previously pub-
lished reports (Tienpont et al., 2005; David et al., 2003), in order to min-
imize the phthalates contamination that may occur throughout all the
steps of analysis. For samples and standard handling, glass separatory
funnels with Teflon stopcocks, glass vials with Teflon caps and GC syrin-
ges were used.
2.4. Sample preparation
Phthalate Esters (PAEs) were isolated from the water samples using
liquid-liquid extraction with dichloromethane. One liter of water sam-
ple was introduced into a separatory funnel and serially extracted
three times with 60 ml of dichloromethane each time. The combined
extracts were collected, dried over anhydrous sodium sulfate and evap-
orated to a final volume of 1 ml by rotary evaporator (IKA RV 10 control,
IKA-Werke GmbH and Co.Kg, Germany) at 43 °C and 33 rpm.
2.5. Instrumental methods
A gas chromatograph equipped with a split/splitless injector and
an auto-sampler (Varian CP-3800, Varian Inc., Middleburg, The
Netherlands) coupled with a triple quadrupole mass spectrometer
(Varian 320, Varian Inc., Walnut Creek, USA) was employed for the anal-
ysis. A 30 m VF-5MS capillary column (5% phenyl - 95% methyl
polysiloxane) with a 0.25 μm film thickness and 0.25 mm internal diam-
eter (CP8944, J&W Agilent, Folsom, California, USA) was used for com-
pound separation. Helium carrier gas at 99.999% purity was used at a
constant rate of 1 ml min−1. Injections of 1 μl were made in splitless
mode using injector temperature set at 275 °C. The oven temperature
was initially programmed at 40 °C for 3.5 min then was increased at a
rate of 5 °C min−1 until it reached 250 °C where it was held for
10 min. Finally, the temperature was increased at a rate of 2 °C min−1
to reach 280 °C and was held for 5 min.
Electron ionization at 70 eV was employed. The temperature of the
transfer line, ion source and manifold of the mass spectrometer were
set at 250, 200 and 40 °C respectively. The data were acquired and re-
corded in full scan (50–550 amu at a rate of 0.5 scans sec−1) and selec-
tive ion monitoring (SIM) modes on a workstation (Varian 6.9)
equipped with a National Institute of Standards and Technology (NIST,
US Department of Commerce) mass spectral library. The target PAEs
compounds were identified in the samples based on retention time
matching to the standard as well as the presence of four qualifier ions
with their correct ratios in accordance to the mass spectra obtained
using the standards. Table S1 in the supplementary information lists
the analyzed phthalates with their target and qualifier ions.
The quantification was mostly based on the phthalic anhydride ion
signal detected at m/z 149, except for DMP where a base peak at m/z
163 was employed. The quantification of PAEs was achieved using ex-
ternal standard calibration in which the amount of each compound
was calculated from the peak response using a multipoint calibration
curve. Six point calibration curves for each phthalate compound were
constructed by plotting known concentrations of the corresponding
standards versus their respective peak area responses. Linear curve fits
with high correlation coefficients ranged from 0.991 to 0.999 for the
six analyzed PAEs were obtained using linear squares analysis. The in-
strumental detection limits (IDLs) were calculated from the instrument
response to the lowest standard and extrapolated down to the
corresponding amount of analyte that would generate a signal to
noise ratio of 3:1.
2.6. Quality control and quality assurance
The accuracy of the method was determined by analyzing seven rep-
licates of ultrapure Milli-Q water blanks spiked with PAEs standards at
concentration level of 0.4 μg l−1. In addition, the precision of analysis
was estimated by determining the relative standard deviation of the
seven replicates for the above stated spiked ultrapure Milli-Q water.
The PAEs compounds recovery ranged from 80 to 140% and the relative
standard deviation (RSD) ranged from 7 to 21% (see Table S2).
Laboratory control samples (spiked ultrapure Milli-Q water as indi-
cated above, ultrapure Milli-Q water blank samples and dichlorometh-
ane solvent) were analyzed with each set of samples to examine the
background contamination. A full set of calibration phthalate standards
was analyzed before and after each set of samples, in addition one stan-
dard was run as a control after every 8 samples to ensure instrument
stability. Samples were blank corrected by subtracting the peak area
values from the corresponding sample peak area for each compound;
however the data was not recovery corrected.
The method detection limits (MDLs) were calculated as the mean of
the blank concentration (n = 7) plus 3 times the standard deviations.
Table S2 in the supplementary information lists the IDLs and MDLs of
the PAEs compounds which ranged from 0.004 to 0.013 μg l− 1 and
from 0.010 to 0.062 μg l−1 respectively.
2.7. Statistical analysis
Statistical analysis of the results was carried out using SPSS software
statistics version 22. For statistical treatment all phthalate values deter-
mined to be below their MDL were given values equal to their respec-
tive MDL values divided by the square root of 2. This statistical
treatment of values below MDL was established in studies of phthalate
contaminants (Serrano et al., 2014; Chen et al., 2012; Meeker et al.,
2009; Hornung and Reed, 1990). Initially, normality of concentrations
in each group was checked using the Shapiro-Wilk test. Non-
parametric tests were then used in our analysis to indicate statistical
significance. Friedman and Wilcoxon Signed Ranks tests were employed
in order to assess statistical significant changes in concentrations ob-
served at each storage time point of the same storage condition relative
to the concentration values observed without allowing for storage time.
Finally, the Mann Whitney test was used to compare changes in concen-
tration levels among various storage conditions irrespective of the stor-
age periods. In the Friedman and Mann Whitney tests P-values b 0.05
were considered significant. Statistically significant changes between
the concentrations of DBP, DEHP and DEP after outdoor, room tempera-
ture and refrigerated storage relative to their respective values obtained
directly after purchase in the former two cases and after one month
storage in case of DEP were determined as indicated in Figs. 1 and 2.
3. Results and discussion
The occurrence and concentrations of six common phthalate com-
pounds were investigated in different branded PET bottled water locally
produced in the Egyptian market. Bottled water is a highly consumed
product in the Egyptian market, therefore assessing phthalate contami-
nations – if any – is of importance. Bottled water samples were analyzed
directly after purchasing (~2 weeks from production) and after being
stored under different conditions to investigate the factors that could
potentially affect the PAEs leaching from the PET plastic bottles into
the water. Bottled water were collected across six popular commercial
brands commonly used in the Egyptian market.
 G. Zaki, T. Shoeib / Science of the Total Environment 618 (2018) 142–150 145

Fig. 1. Mean concentrations of DBP, DEHP and DEP in μg l−1 observed in PET water samples analyzed at various time points under different storage conditions. Panel A: room temperature
storage (25 ± 5 °C, data collected at the 2 and 6 months time points); panel B: outdoor storage with sun exposure (daylight temperature 40 ± 5 °C, data collected at the 1, 2 and 4 months
time points); panel C: refrigerated Storage (4 ± 1 °C, data collected at the 1, 2 and 4 months time points). The zero month storage time point refers to analysis ~2 weeks after production.
The P values for statistical significance are indicated as * for P ≤ 0.05, ** for P ≤ 0.01 relative to values at zero month storage for DBP and DEHP and values at 1 month storage for DEP.

3.1. Phthalate compounds in PAE bottled water detected directly after pur-
chase (no storage)
Table 1 shows the results of the bottled water samples (n = 12) an-
alyzed directly after purchasing (~2 weeks from production). Statistical
details are also included in Table S3 in the supplementary material. DBP
and DEHP were detected at levels above their respective MDL values,
DEP was found to be below its MDL of b 0.012 μg l−1 while DMP, BBP
and D-n-OP were not detected in any of the samples. DEHP was the
most abundant phthalate compounds detected in 6 out of the 12

Fig. 2. Box and whisker plots of DBP, DEHP and DEP in μg l−1 observed in PET water samples analyzed at various time points under different storage conditions. Panel A: room temperature
storage (25 ± 5 °C); panel B: outdoor storage with sun exposure (40 ± 5 °C); panel C: refrigerated storage (4 ± 1 °C). Middle, top, and bottom lines of the boxes are the mean, 75th and
25th percentile values respectively. Upper and lower whiskers are maximum and minimum values respectively. The zero month storage time point refers to analysis ~2 weeks after
production. The P values for statistical significance are indicated as * for P ≤ 0.05, ** for P ≤ 0.01 relative to values at zero month storage for DBP and DEHP and values at 1 month
storage for DEP.
146 G. Zaki, T. Shoeib / Science of the Total Environment 618 (2018) 142–150

Table 1
Concentration levels of phthalates in μg l−1 reported from PET bottled waters samples analyzed directly after purchasing or production. Top values are for range, middle values are for
mean and bottom values are for median. Dashed lines indicate data is not available and ND is listed when compounds were not detected.

Reference Country Number of samples Phthalate analyzed

DEP DBP DEHP DMP BBP DnOP

This study Egypt 12 ND ND-0.171 ND-0.298 ND ND ND

ND 0.082 0.104 ND ND ND
ND 0.06 0.067 ND ND ND
Jeddi et al., 2015 Iran 12 – – – – – –
– 0.135 0.217 – b0.01 –
– – – – – –
Santana et al., 2014 Portugal 6 – 0.06–2.94 0.07–0.18 – – –
– 0.92 0.11 – – –
– – – – – –
Cao, 2008 Canada 7 0.054–0.100 0.075–0.317 0.052–0.338 ND ND ND
0.080 0.138 0.118 ND ND ND
– – – ND ND ND
Amiridou and Voutsa, 2011 Greece 5 – – – ND ND ND
– – – ND ND ND
0.033 0.044 0.350 ND ND ND
Serodio and Nogueira, 2006 Portugal 1 0.04a 0.350a 0.170a b0.008a 0.020a 0.010a
Psillakis and Kalogerakis, 2003 Greece 2 0.07–0.12 0.08–0.14 0.36–0.46 ND ND ND
0.095 0.11 0.41 ND ND ND
– – – ND ND ND
Guart et al., 2014 Spain 224 1.02–20.5 ND ND-1.520 ND-0.022 0.619–1.28 –
– ND – – – –
– ND – – – –
c
Keresztes et al., 2013 Hungary ND b0.007–0.8 b0.016–1.7 ND b0.006–0.1 –
ND – – ND – –
ND – – ND – –
Leivadara et al., 2008 Greece 8 – – ND-0.2 – – –
– – – – – –
– – – – – –
Casajuana and Lacorte, 2003 Spain 5 b0.002 b0.003–0.059 NQb b0.002 b0.004 –
– – – – – –
– – – – – –
Montuori et al., 2008 Italy 71d 0.140–0.350 0.170–0.520 b 0.02 b 0.02–0.10 – –
– – – – – –
0.22 0.23 b 0.02 0.060 – –
Dévier et al., 2013 France 2 b 0.030 b 0.020 b0.010 b0.030 ND ND
– – – – ND ND
– – – – ND ND
a
Data from only one sample were reported.
b
Not quantified due to contamination problems.
c
Number of samples not specified.
d
Data reported are for the 25th and 75th percentile values.

samples at concentrations ranging from b 0.062 to 0.298 μg l−1, while
DBP was the second abundant detected in 7 out of the 12 analyzed sam-
ples at concentrations ranging from b0.043 to 0.071 μg l−1. The pres-
ence of DEHP, DBP in 50% and 58% of the tested bottles respectively
may be due to different production facilities used by the different
brands tested (Guart et al., 2014; Al-Saleh et al., 2011). The mean con-
centrations of DBP and DEHP with the standard error as determined
~2 weeks after production are presented at the zero time points in Fig.
1 while Fig. 2 shows the maximum, mean, minimum, 75 and 25 percen-
tile values obtained.
Our results are consistent with previously published data summa-
rized in Table 1 (A review of phthalate occurrence in drinking water is
presented in Erythropel et al., 2014). The concentrations detected in
this study were similar to the levels reported from Iran, Portugal,
Canada, Greece, Spain, Hungary and Italy that examined the levels of
phthalates in bottled water either directly after purchase or production
(Jeddi et al., 2015; Guart et al., 2014; Santana et al., 2014; Keresztes
et al., 2013; Amiridou and Voutsa, 2011; Cao, 2008; Leivadara et al.,
2008; Serodio and Nogueira, 2006; Psillakis and Kalogerakis, 2003;
Casajuana and Lacorte, 2003). DBP and DEHP seem to be common con-
taminants reported in many bottled water samples. DEHP reported
values are generally around 0.1 μg l−1 but was shown to reach 1.5 and
1.7 μg l− 1 in Spain and Hungary respectively (Guart et al., 2014;
Keresztes et al., 2013). The mean concentration of DBP detected in the
Egyptian PET bottled water samples being 0.082 μg l− 1 is in general
agreement with values reported in other studies but considerably
lower than the values of 0.92 and 0.35 μg l− 1 reported in Portugal
(Santana et al., 2014; Serodio and Nogueira, 2006). The maximum
level of DBP of 0.172 μg l−1 observed in this study, is among the lowest
reported maximum levels for this contaminant worldwide, being for ex-
ample 5 and 17 times lower than those reported in Hungary (Keresztes
et al., 2013) and Portugal (Santana et al., 2014).
In contrast to the above studies, reports from Greece and Italy indi-
cate the presence of DEP in samples analyzed directly after their pur-
chase or production as listed in Table 1 (Amiridou and Voutsa, 2011;
Montuori et al., 2008). The highest concentration reported for DEP
was 20.5 μg l−1 from a Spanish study where 224 samples were analyzed
(Guart et al., 2014). Additionally, the occurrence of DMP and BBP were
also reported in Spain (Guart et al., 2014), Hungry (Keresztes et al.,
2013), Italy (Montuori et al., 2008) and Portugal (Serodio and
Nogueira, 2006).
A recent French study, which claimed extremely low method detec-
tion limits that ranged from 0.0004 to 0.0016 μg l−1, reported the ab-
sence of phthalates in French bottled water samples examined directly
after production (Dévier et al., 2013). In that study, the presence of var-
ious phthalates and other organic contaminants in two brands of PET
bottled water were examined by using SPME and GC–MS. In this report,
some phthalates were detected at concentration levels similar to or
slightly higher than those of the blanks employed. The source of the de-
tected phthalates was investigated further by performing additional
 G. Zaki, T. Shoeib / Science of the Total Environment 618 (2018) 142–150
analysis of samples at the bottling factory which lead to the reported
conclusion that laboratory background contamination was likely the
source of the detected phthalates and that the PET bottled water sam-
ples were free from phthalates traces (Dévier et al., 2013).
The variation in the detected types and concentrations of phthalates
between our study and other reports might originate from regional dif-
ferences in the occurrence of contaminants as well as different industri-
al production processes and uses of phthalates among the countries
where the studies were conducted (Erythropel et al., 2014). In Egypt,
bottled water is predominantly natural water derived from deep
wells; however, it was reported that phthalate contaminants are able
to percolate down through underlying soils and reach deep aquifers
(Dévier et al., 2013; Bono-Blay et al., 2012). Several reports point to con-
taminations during production and bottling processes (Jeddi et al.,
2015; Guart et al., 2014; Amiridou and Voutsa, 2011; Cao, 2008;
Leivadara et al., 2008; Montuori et al., 2008); the use of virgin and
recycled PET (Keresztes et al., 2013; Bach et al., 2012; EFSA, 2011; Sax,
2010) as well as municipal and industrial activities such as unsatisfacto-
ry disposal of wastes leading to landfill leachate (Huang et al., 2013;
Bono-Blay et al., 2012; ATSDR, 1995) as possible contributing factors.
3.2. Effect of storage on phthalates concentration
3.2.1. Storage at room temperature
After storing samples (n = 24) at room temperature (25 ± 5 °C) for
2 and 6 months, an increase in some phthalates concentration was ob-
served as shown in Fig. 1 Panel A. The mean concentrations of DBP
after 2 and 6 months storage was 0.136 and 0.227 μg l−1 respectively
corresponding to an increase of 1.6 and 2.8 times respectively relative
to their mean values obtained directly after purchasing. DEHP mean
concentrations after 2 and 6 months storage was 0.274 and 0.396
μg l−1 respectively, indicating increases of approximately 2.6 and 4
times relative to their initial mean values. Table S4 of the supplementary
material lists all the data which are summarized in Panels A of Figs. 1
and 2. The detection frequency of DBP and DEHP increased from 50%
and 58% prior to storage to 100% for both compounds at the 2 and
6 months storage time points at room temperature. DEP which was
not detected in samples analyzed before storage is observed at low con-
centrations after storage at room temperature. At the two month time
point, DEP was detected in 67% of the samples with a mean concentra-
tion of 0.017 μg l−1 which is just above the method detection limit (see
Table S2) and a concentration range of b 0.012 to 0.036 μg l− 1 while
after 6 months, its detection frequency increased to 75% with a maxi-
mum concentration of 0.062 μg l−1. DMP, BBP and D-n-OP were not de-
tected in any of the samples analyzed at the two time points of this
storage condition.
The changes in phthalate concentrations after each period of storage
as compared to their respective initial levels recorded before storage
were statistically analyzed using Friedman and Wilcoxon Signed
Ranks tests in order to assess their significance. Statistically significant
changes in the concentrations of DBP and DEHP were obtained after
6 months storage at room temperature relative to their initial values ob-
tained after purchasing. These observations agree with previous reports
showing an increasing trend in the concentrations of phthalates as a
function of storage time (see Table S5 of the Supporting material). For
example, the concentration of DEHP was shown to increase after
being stored at room temperature for 3 months (Leivadara et al.,
2008). In another study the detection of DEHP was reported after
9 months of storage at room temperature, while no phthalates were de-
tected throughout the first 8 months of storage (Biscardi et al., 2003). A
more recent study reported a sharp increase in the concentrations of
DEHP and a modest increase in the DBP and BBP after 44 days of storage
at room temperature (Keresztes et al., 2013), while a 3 fold increase in
the detection frequency of phthalates in PET water samples after
being stored for one-year at room temperature relative to samples
that are were not stored was reported (Guart et al., 2014). Mineral
147
water contained in plastic bottles was shown to contain significant con-
centrations of PAEs after 30 day of storage at 22 °C (Bošnir et al., 2007).
The mean concentrations reported in that study of DBP and DEHP of
11.33 and 8.78 μg l−1 respectively are in fact the highest reported con-
centrations for these contaminates at this storage condition and are ap-
proximately 80 and 30 times higher respectively than our reported
values.
3.2.2. Outdoor storage
The levels of phthalates were examined in samples (n = 36) after
outdoor storage with direct sun exposure for 1, 2 and 4 months,
where the average daylight temperature was 40 ± 5 °C and are listed
in Table S6. Panels B in Figs. 1 and 2 show a trend of increasing phthalate
concentrations upon outdoors storage. The mean concentration of DEHP
was recorded to be 0.190, 0.306 and 0.432 μg l−1 with detection fre-
quencies of 92, 100 and 100% after outdoor storage for 1, 2 and
4 months respectively. This represented concentration increases of 1.8,
2.9 and 4.2 fold, respectively relative to the initial mean concentration
determined in the water samples before storage. DBP mean concentra-
tions of 0.124, 0.167 and 0.229 μg l−1 with detection frequencies of 83,
83 and 100% were obtained after outdoor storage for 1, 2 and 4 months
respectively. These values corresponded to 1.5, 2.0 and 2.8 fold in-
creases respectively relative to the initial mean concentration deter-
mined in the water samples before storage. DEP was detected in 42%
of the samples after one month of storage with a concentration range
of b0.012 to 0.042 μg l−1. The detection frequency for DEP increased
to 67 and 75% after 2 and 4 months storage under this condition respec-
tively reaching a maximum concentration of 0.057 μg l−1. It is also inter-
esting to note that for all time points considered under this storage
condition, as in the case of room temperature storage, DMP, BBP and
D-n-OP were not detected in any of the samples analyzed.
Analysis of the data indicated that the increases for DBP and DEHP
were statistically significant after 1, 2 and 4 months of storage relative
to their respective concentrations before storage. Increases for DEP on
the other hand were shown to be only statistically significant at the
4 months storage period. These results suggest that increased storage
time under the conditions employed here may increase the migration
level of phthalates from the PET bottles.
This finding is consistent with other studies reporting an increase in
phthalates concentrations after outdoor exposure for different periods.
For instance an increase of 20 fold in phthalate concentrations was ob-
served in water packed in plastic bottles after 10 weeks of outdoor stor-
age in temperatures up to 30 °C (Casajuana and Lacorte, 2003). In
another report an increase in phthalates levels in PET bottled water
was observed after being stored at different temperature conditions
with the highest migration rate detected for samples stored at 40 °C
for 45 days (Jeddi et al., 2015). An increase in phthalate concentration
was also observed after storage for only 24 h at 60 °C (Keresztes et al.,
2013).
On the other hand, several reports suggest no significant changes in
phthalate concentrations in PET bottled water samples after their stor-
age at different time points and at different temperatures (Bach et al.,
2013; Amiridou and Voutsa, 2011; Guart et al., 2011; Ceretti et al.,
2010), while one study reported a decrease in DEHP concentrations in
samples stored outdoors for 3 months suggesting that temperature
and sunlight might have a role in the degradation of phthalates with
time (Leivadara et al., 2008).
3.2.3. Refrigerated storage at 4 °C
All six phthalates under study here were examined in samples (n =
36) after refrigerated storage (4 ± 1 °C) for 1, 2 and 4 months. In line
with the previous two storage conditions discussed above, DMP, BBP
and D-n-OP were not detected at any time point in any of the samples
analyzed. The data obtained are presented in Table S7 of the
Supplementary material and illustrated in Panels C of Figs. 1 and 2.
The mean concentrations of DBP were determined to be 0.107, 0.128
148 G. Zaki, T. Shoeib / Science of the Total Environment 618 (2018) 142–150
and 0.173 μg l−1 at the 1, 2 and 4 month time points respectively. These
values are 1.3, 1.6 and 2 times higher than the mean value obtained for
DBP without allowing for storage. The average mean concentrations for
DEHP after 1, 2 and 4 months storage at 4 ± 1 °C were 0.135, 0.235 and
0.307 μg l−1, respectively, which corresponded to 1.3, 2.3 and 3 fold in-
creases relative to the mean value obtained directly after purchasing.
DEP just as in the previous two storage conditions was also observed
just above its method detection limit at mean concentrations of 0.014,
0.014 and 0.020 μg l−1 at the 1, 2 and 4 month time points respectively.
While the latter two values are not statistically significant relative to the
value at 1 month as shown in Figs. 1 and 2. It is however, important to
note that under refrigerated storage, the changes in DBP and DEHP con-
centrations after 4 months as well as after 2 and 4 months respectively
relative to the results obtained after ~2 weeks of production were deter-
mined to be statistically significant.
A recent report examined the levels of phthalates in water samples
after frozen and refrigerated storage (Jeddi et al., 2015). In agreement
with our findings, this recent report highlighted a slight increase in
phthalate concentrations after low temperature storage. In contrast to
our findings, higher concentrations of DEHP, BBP and DEP were ob-
served in samples stored at 4 °C relative to samples stored outdoors
and at room temperature, while the concentration of DBP was shown
to be higher in that study in room storage relative to storage at 4 °C
(Al-Saleh et al., 2011).
The overall trend observed of increasing phthalate concentrations
due to different storage conditions tested here suggests that PET plastic
bottles may be a source for the detected phthalates. This trend is also
supported by reports of phthalates concentration increases in PET bot-
tled water stored under various conditions (Jeddi et al., 2015;
Keresztes et al., 2013; Al-Saleh et al., 2011; Casajuana and Lacorte,
2003). Phthalates are not typically used in PET manufacturing and
their presence therefore, is non-intentional. The wide use of phthalates
in various industries and their ubiquitous presences as environmental
contaminants can cause their non-intentional addition in many prod-
ucts, including PET polymer and glass bottles (Guart et al., 2014;
Dévier et al., 2013; Amiridou and Voutsa, 2011; Cao, 2010; Cao, 2008;
Leivadara et al., 2008; Wormuth et al., 2006).
3.2.4. Occurrence of phthalates in carbonated PET bottled waters
One of the six brands tested was carbonated bottled water to assess
the effect of the presence of carbon dioxide gas on phthalate levels. Re-
sults obtained here indicated no statistical difference in the concentra-
tions of phthalate observed in carbonated and non carbonated water
samples (P N 0.05). This might suggest that the presence of carbon diox-
ide gas in the water likely has no effect on phthalates leaching from the
PET bottles. However, it was noted that DEP was not detected in any car-
bonated water samples analyzed here while it was detected at trace
levels in non-carbonated samples. Our findings are in line with studies
that reported no significant changes between phthalates migration
into carbonated and non carbonated water (Cao, 2008; Leivadara
et al., 2008). While some studies report a difference in phthalate con-
centrations among carbonated and non-carbonated water samples
(Keresztes et al., 2013; Montuori et al., 2008).
3.2.5. Leaching rate of different compounds at different storage temperature
Fig. 3 shows linear correlations for each of the observed DEHP, DBP
and DEP concentrations and their storage temperatures at the
2 month storage time point. It is interesting to note that for the three
contaminates shown in Fig. 3 the rate of leaching follows the order
DEHP N DBP N DEP as indicated by slopes of the three lines, this order
also follows their observed absolute concentrations. This observation
may be fortuitous as the slope of DEHP was the only one to be deter-
mined as statistically significant. However, the statistical significance
of the DEHP slope suggests that temperature does affect the migration
speed of DEHP molecules from the polymer matrix.
3.2.6. Implication for human exposure
The European Food Safety Authority (EFSA) defines the Tolerable
Daily Intake (TDI) as an estimate of the quantity of a chemical which
can be ingested daily over a lifetime without posing a significant risk
to health (EFSA, 2005a, 2005b). The measured concentrations of phthal-
ate determined in this study were used to estimate the exposure of tod-
dlers and adults through PET bottled water consumption in Cairo, Egypt.
Toddlers are generally at a higher risk of hazardous chemicals exposure
as compared to adults, due to their higher food and drink consumption
per kg of body weight. The maximum concentrations of DBP and DEHP
obtained throughout the study were used to calculate the estimated
daily intake representing the worst case scenario of exposure. The esti-
mated daily intake of phthalates through bottled water consumption
was calculated using the following equation (Schecter et al., 2013; De
Fátima Pocas and Hogg, 2007).
EDI ¼ MC  Water consumption ð1Þ
where EDI is the estimated daily intake through drinking water in μg (kg
body weight)−1 day−1 and MC is the maximum concentration levels of
DBP, DEHP and DEP in μg l−1 determined in the PET bottled water sam-
ples under the various storage conditions examined here. We assumed
an average body weight of 70 kg for adults and 12 kg for toddlers (EFSA,
2012) and used 2 and 1.5 l day−1 respectively as the dietary reference
values for water consumption for adults and toddlers (EFSA, 2010).
The contribution of the daily intake of phthalates through bottled
water was estimated as follows:
Contribution through drinking water ¼ ðEDI=TDIÞ  100 ð2Þ
While the TDI for different phthalates are available for reference as
established by EFSA (2005a, 2005b), the corresponding values for DBP
and DEHP are 10 and 50 μg/kg/bw/day, respectively. The estimated
daily intake values of DBP and DEHP from the Egyptian PET bottled
water in adults and toddlers are presented in Table S10 of the Supple-
mentary materials. The results indicate the DBP and DEHP estimated
daily intake for both adults and toddlers due to consumption of bottled
water were far below their respective TDI values. The contribution of
bottled water consumption to phthalate daily intake for adults and tod-
dlers did not exceed 0.16 and 0.72% respectively of their TDI values for
DBP while these values were 0.04 and 0.16% respectively for DEHP.
This suggests that there should be no adverse health effects through
consumption of bottled water even at the maximum concentrations de-
tected for these chemicals as determined in this study. It is also worth
mentioning that the maximum concentration observed for DEHP in
this study is considerably below the 6 μg l−1 limits set by the US EPA
(2009) and the US FDA (2011) for DEHP in drinking water and bottled
water respectively.
4. Conclusion
Six common phthalates (dimethyl phthalate (DMP), diethyl phthal-
ate (DEP), dibutyl phthalate (DBP), n-butyl benzyl phthalate (BBP),
diethyl hexyl phthalate (DEHP), and Di-n-octyl phthalate (D-n-OP))
were investigated in 108 bottled water locally produced in the
Egyptian market for the first time. The bottles were made of transparent
polyethylene terephthalate (PET) with high density polyethylene
(HDPE) plastic caps and did not contain cap liners. Water samples
were analyzed immediately after purchasing (~2 weeks after produc-
tion) and after being stored for up to 6 months under three conditions
being; room temperature (25 ± 5 °C), in a refrigerator (4 ± 1 °C) and
outdoors under sun exposure (daylight temperature of 40 ± 5 °C).
Among the target compounds, only DEHP and DBP were detected in
the samples analyzed immediately after purchasing with a detection
frequency of 50 and 58% and mean concentrations of 0.104 and 0.082
μg l− 1 respectively. The DMP, BBP and D-n-OP were not detected at
 G. Zaki, T. Shoeib / Science of the Total Environment 618 (2018) 142–150
Fig. 3. Mean concentrations of DBP, DEHP and DEP in μg l−1 determined at 4, 25 and 40 °C after 2 months storage. The P values for statistical significance are indicated as * for P ≤ 0.05, ** for
P ≤ 0.01 relative to values at zero month storage for DBP and DEHP and values at 1 month storage for DEP. The zero month storage time point refers to analysis ~2 weeks after production.
The P values for statistical significance of the slopes of the regression lines are 0.025 for DEHP, 0.270 for DBP and 0.152 for DEP. Values of P ≤ 0.05 are considered significant for the slopes of
the regression lines.
any time point in this study while the DEP was only detected after
1 month of storage. Significant positive correlation (P b 0.05) was ob-
tained between the storage time and the concentration of DEHP and
DBP relative to their respective concentrations determined immediately
after purchasing, indicating possible migration from the PTE bottles. Sig-
nificant positive correlation obtained between temperature and DEHP
concentrations after 2 months storage is obtained indicating that tem-
perature also affects the leaching rate of DEHP from the PTE bottles.
The estimated contribution of bottled water consumption to the tolera-
ble daily intake (TDI) levels of the two most abundant phthalates ob-
served here for adults and toddlers did not exceed 0.16 and 0.72% for
DBP while these values were 0.04 and 0.16% for DEHP respectively.
These estimated daily intake values from PET bottled water consump-
tion were far below their respective TDI values and therefore should
constitute no adverse health effects.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2017.10.337.
Acknowledgements
This work was funded by a graduate student grant provided by the
American University in Cairo. The authors thank the Central Laboratory
of the Egyptian Holding Company for Water and Wastewater for hosting
Ms. Zaki and Drs. Mohamed Bakr, Mohamed Farouk, Ahmed Adel and
Tamer Dagher of the same lab as well as Dr. Zeinab Amin of the Depart-
ment of Mathematics and Actuarial Science at the American University
in Cairo for numerous fruitful discussions.
Supplementary data
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