Microchemical Journal 155 (2020) 104719

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Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

Phthalate acid esters (PAEs) in highly acidic juice packaged in polyethylene T

terephthalate (PET) container: Occurrence, migration and estrogenic
activity-associated risk assessment
Leila Arfaeiniaa, Sina Dobaradaranb,c, Farzaneh Nasrzadehb, Shiva Shamsib, Yousef Poureshghd,
Hossein Arfaeiniab,c,
⁎

a
Department of Natural Resources, Faculty of Engineering, Islamic Azad University-Bushehr Branch, Bushehr, Iran
b
Systems Environmental Health and Energy Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr,
Iran
c
Department of Environmental Health Engineering, Faculty of Health and Nutrition, Bushehr University of Medical Sciences, Bushehr, Iran
d
Department of Environmental Health Engineering, School of Health, Ardabil University of Medical Sciences, Ardabil, Iran

ARTICLE INFO ABSTRACT

Keywords: The present study aimed to investigate the concentration of PAEs, their migration from the walls of PET con-
Phthalate esters tainers into highly acidic juices (such as lemon juice, vinegar, and verjuice) and the risk assessment based on the
Highly acidic juice estrogenic potential of these widespread pollutants. We analyzed and quantified six priority compounds of PAEs,
Pet container including dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n‑butyl phthalate (DnBP), butyl benzyl
Estrogenic activity
phthalate (BBP), bis (2-ethylhexyl) phthalate (DEHP), and di-n-octyl phthalate (DnOP) in 40 samples of afore-
Health risk assessment
mentioned juices packed in PET containers. The results showed that DEHP and DnBP were the major compounds
with the median value of 8.1 and 6.8 µg/L, 10.5 and 7.2 µg/L, and 9.8 and 6.7 µg/ L, in lemon juice, vinegar, and
verjuice, respectively. The results also showed that ∑PAEs concentration was higher in juices packed in PET
containers than in glass containers (two sample t-test, pvalue < 0.05). A significant and positive correlation was
observed between the concentration of PAEs and the storage temperature, storage time, and sun exposure of
containers of juices (Pearson correlation test, pvalue <0.05). However, there was a significant and negative
correlation between pH and the concentration of these pollutants (pvalue < 0.05). The human daily intake-based
risk assessment showed that the safety factor (SF) calculated for all PAEs was much higher than 1. This indicates
that the consumption of these acidic juices would not put consumers at the risk of these compounds. However,
the risk assessment based on the estrogenic potential of PAEs compounds showed that the average of estrogen
equivalence (EEQ) calculated for the ∑PAEs was higher than 1 ng E2/L and could have long-term adverse effects
on human health.

1. Introduction widespread application of PAEs and their ubiquitous presence, unin-

Phthalates are a group of chemicals that are widely used as plasti-
cizers in various products such as toys, blood bags, shopping and gar-
bage bags, food packaging, water bottles, wall coverings, wire, and
cable jacketing [1-3]. Due to the high global demand for phthalate-type
plasticizers, the production rate of these types of plasticizers will be
increased by 1.3% from 2017 to 2022 [4].
Phthalate esters (PAEs) are a group of such substances that are
mainly used to enhance the flexibility and softness of industrial poly-
mers; their most important applications are in the synthesis of polyvinyl
chloride (PVC), rubber, and styrene materials [1,5,6]. Because of these
tentional contamination of PET containers with these contaminations is
happened easily [7]. PAEs in plastic products such as PET containers
are not chemically bonded; therefore, there are many concerns and
questions regarding the migration of these compounds from the wall of
the plastic containers into the water and food products packaged
therein. In fact, these compounds form a reversible bond with the
polymer chains; and they may also be separated from the polymer
structure under various environmental conditions and stresses (at high
temperatures, acidic pH, direct sunlight, freezing, long shelf life, etc.)
and migrated to the contents of this container [8,9].
PAEs include DMP, DEP, DnBP, BBP, DEHP, and DNOP (their

⁎
Corresponding author: Department of Environmental Health Engineering, Faculty of Health and Nutrition, Bushehr University of Medical Sciences, Bushehr, Iran.
E-mail address: arfaeiniah@bpums.ac.ir (H. Arfaeinia).

https://doi.org/10.1016/j.microc.2020.104719
Received 9 November 2019; Received in revised form 7 February 2020; Accepted 8 February 2020
Available online 10 February 2020
0026-265X/ © 2020 Elsevier B.V. All rights reserved.
L. Arfaeinia, et al.
Table 1
Basic physicochemical properties of the six target phthalates.
PAEs Abbreviations CAS Number Estrogenic potential
Dimethyl phthalate DMP 131–11–3 – 1100
Diethyl phthalate DEP 84–66–2 5 × 10−7
di-2-(ethylhexyl) phthalate DEHP 117–81–7 3 × 10−7
Benzyl butyl phthalate BBP 85–68–7 2 × 10−4
Di-n‑butyl phthalate DnBP 84–74–2 4.1 × 10−5
di-N-octyl phthalate DnOP 117–84–0 – 0.022
physicochemical properties are given in Table 1 [10-13]). These com-
pounds are able to enter the human body in various ways such as di-
gestion, inhalation or skin contact. Among the mentioned pathways,
most human intake occurs through the digestion of food packaged in
plastic containers [14,15]. PAEs have been suggested as endocrine
disruption compounds. In fact, through binding to hormone receptors in
the body, PAEs lead to anti-androgenic activity, change in the function
and structure of sertoli cells of a testis, reduction in the number and
quality of spermatozoon motility in the male, alteration in female
steroid hormones, early maturity, preterm delivery, and fetal death
[16,17]. Furthermore, chronic exposure to these compounds causes a
variety of cancers, including breast cancer in women and prostate
cancer in men [18,19], hyperactivity in children, sterility, the effect on
the thyroid gland, teratogenic and mutagenic activities [20], obesity,
liver damage, increased zoom proxy, and allergies [21,22].
Acidic juices are among the most common food items in the world,
including Iran, which are used in cases such as cooking, medicine, and
treatment [23]. Lemon juice, verjuice, and vinegar are highly acidic
juices that are widely used in cooking, diet, and even medicine. In Iran,
these products are often manufactured in traditional or industrial ways,
packaged in PET plastic containers, and stored for a long time. It is
noteworthy that sometimes, due to lack of knowledge, these foods are
exposed to direct sunlight in pickling workshops, homes, or shops. Due
to the acidic properties of these products, the probability of PAEs mi-
grating from the wall of these containers into the food matrix increases
if food is stored in plastic containers for a long time and exposed to the
sun [8,24]. In addition, PET raw materials are manufactured in dif-
ferent factories with varying qualities; in certain cases, they are pre-
pared from recycled materials, which has doubled the need to in-
vestigate the migration of PAEs from the structure of PET bottles.
However, there is no study on the measurement of the amount of PAEs
in highly acidic juices stored in plastic containers. Therefore, the ob-
jectives of the present study are to: 1) determine the levels of different
PAEs in highly acidic juices (lemon juice, verjuice, and vinegar) stored
in PET containers, 2) determine the effect of various factors, including,
pH, storage time, and container size of storage on the concentration of
PAEs in these products, and 3) determine the potential value of the
estrogenic activity of PAEs introduced into the body of consumers by
consuming these acidic juices and the assessment of their effects.
2. Materials and Method
2.1. Chemicals and reagents
A mixed standard solution PAEs (six PAEs containing DMP, DEP,
DnBP, BBP, DEHP and DnOP) prepared with 2 mg mL−1 of n-hexane
was purchased from Sigma-Aldrich (St. Louis MO, USA). Analytical-
grade diisophenyl phthalate, di-n-phenyl phthalate and di-n-benzyl
phthalate were purchased from Sigma-Aldrich (St. Louis MO, USA).
Neutral silica gel (100- to 200-mesh) and anhydrous sodium sulfate
were obtained from Sinopharm Co. Ltd. (Shanghai, China). HPLC-grade
acetone and n-hexane were purchased from Tedia Company Inc., USA.
All chemicals and reagents were > 99% pure and used without further
purification. In this experiment, ultrapure water (18.2 MΩ cm−1) from
a Synergy UV ultrapure water system equipped with a Millipak-40 filter
2
Microchemical Journal 155 (2020) 104719
Water solubility (mg/L) Saturated vapor pressure (25 ºC,Pa)
0.22
4200 0.22
0.003 8.6 × 10−4
2.7 1.1 × 10−3
11.2 9.7 × 10−3
26.65
unit (Merck KGaA, Germany) was used to make the solutions.
2.2. Sample collection
In this study, at the first step, shops and stores selling lemon juice,
verjuice and vinegar in Bushehr city were listed. Afterwards, 20 stores
(out of the 43 stores identified in the city) were randomly selected. For
random selection, the stores first were coded from 1 to 43. Next, 20
cases were randomly selected by the formulation in Excel using the
following randomization function (Eq. (1)).
0 43 × Rand() 43. (1)
After this step, from each selected store, we chose one sample from
the products (vinegar, verjuice, and lemon juice) stored in PET con-
tainers. In total, 20 samples of these acidic juices stored in PET con-
tainers were chosen. Moreover, 20 samples kept in glass containers
(control samples) were selected from each acidic juice. The specifica-
tions of each sample, including the storage time, sun exposure, and
container size of storage were further recorded. After collecting the
samples, they were placed in amber glass containers and transferred to
the laboratory along a box cooled with ice. In the laboratory, the pH of
the samples was determined and they were stored at −20 °C to perform
the chemical analysis.
2.3. Preparation of samples
The extraction process of the PAEs from juice samples has been
described elsewhere [25]. For this experiment, 5 mL of each sample was
selected and placed in glass centrifuge tubes. Then, 20 mL of acetone
and n-hexane with 1: 1 volumetric ratio (v/v) was added to the glass
centrifuge tubes. After that, the surrogate standard mixture (diiso-
phenyl phthalate, di-n-phenyl phthalate and di-n-benzyl phthalate) was
added to each sample at a final concentration of 1 μg/L. Control sam-
ples were also prepared by a similar process without a food matrix
sample. A stock solution 100 μg mL−1 of phthalates was prepared.
Standard samples used for quality control were prepared by adding
standard solution to acetone / n-hexane with 1: 1 vol-to-volume ratio
(acetone / n-hexane (v / v) 1: 1). In addition, spiked samples were
prepared through adding a certain amount (based on the levels of re-
quired working standard) of standard solution to the samples. The
samples were then left for one night and then ultrasonicated for 30 min.
The volume of the extraction solution was reduced to 1–2 mL by rotary
evaporation in a water bath at 40 °C; then, 5 mL of hexane was added to
the residual solvent and rotary evaporation method was once again
performed to obtain a volume of less than 1 mL. The concentrated
samples were purified and then loaded onto a glass chromatography
column (25 cm length and 1 cm internal diameter), which consisted of
6 cm alumina with 12 cm silica gel and 2 cm anhydrous sodium sulfate.
2.4. GC–MS analysis
To determine the concentration of PAEs, the prepared samples were
injected into the gas chromatography-mass spectrometry with Agilent
7890 GC-5975MSD model equipped with capillary columns operated in
electron impact and selective ion monitoring mode. High purity helium
L. Arfaeinia, et al. Microchemical Journal 155 (2020) 104719

2.6. Risk assessment

2.6.1. Daily intake-associated risk assessment

The daily intake of phthalates by humans through these highly
acidic juices (lemon juice, verjuice and vinegar) was calculated using
Equation 2:
C×V
DI =
BW (2)
In this equation, DI is the daily intake of phthalates by the consumer
(µg/kg-bw/day), C is PAEs concentration in the pickle matrix (µg/L), V
is daily consumption of acidic juices (L/d) and BW is the human body
weight (kg). According to the data collected from a questionnaire of
300 people, the average consumption of lemon juice, verjuice and vi-
negar by adults and children was 0.34 and 5.23 mL/day, respectively.
The weight of the adults and children was 70 and 20 kg, respectively
[27].
For comparison, the safety factor (SF) was calculated based on
Equation 3:
RfD
SF =
DI (3)
where RfD is the value of a reference dose suggested by Regulations and
Fig. 1. Occurrence of six priority PAEs in the highly acidic juice samples packed
guidelines. SF value of less than 1 indicates a safety concern [28].
in PET containers.

2.6.2. Estrogenic activity-associated risk assessment

(99.9999%) with a constant flow rate of 1.2 mL / min was employed as As can be seen in Table 1, some of the studied phthalates are es-
carrier gas. The temperature program was as follows: the temperature trogenic; for instance, DBP, DEP, BBP, and DEHP compounds have
of the column oven was set to 50℃ for 1 min, then increased to 200 ℃ weak estrogenic properties. In this work, the concept of EEQ was ap-
with the heating ramp of 15℃ per min and kept at this temperature for plied for a better comparison [29,30].
1 min, then it increased to 280℃ with the heating ramp of 8 ℃ / min
and kept at this temperature for 3 min. Samples were injected into the EEQ = EPi × Ci (4)
system in a non-pulse and split-less mode at an injector temperature of
where EP and C are the estrogenic potential and the concentration of
250 °C. The transfer line temperature was set to 280 ℃ and the post-run
each PAE compound, respectively. EP of estrogenic compounds are
temperature was set to 280 ℃ for 2 min. Finally, the concentration of
determined through in vitro bioassay studies. Estradiol (E2) hormone is
PAEs in acidic juice samples was calculated in a unit of mass of PAEs
a combination of the highest estrogenic activity among natural estro-
(µg) to volume (L) of each juice
gens and is selected as the standard estrogen compound and its EP value
was considered as 1. When a compound is more estrogenic than E2, its
EP is above 1, otherwise it is lower than 1. Therefore, the EP unit is
2.5. Quality control and quality assurance
normally ng E2 / L.
The devices were calibrated daily by calibration standards. A
3. Results and discussion
method blank, a spiked blank (with spiking level ranging from 1 ng/L to
100 µg/L), a matrix spike (with spiking level ranging from 1 ng/L to
3.1. Occurrence of the PAEs in highly acidic juice
100 µg/L), and repeat samples were performed for each batch of sam-
ples. Additionally, the samples to which the surrogate standards were
Occurrence of six priority PAEs in the samples with highly acidic
added, were analyzed to evaluate the effects of the matrix. The recovery
juice packaged in PET and glass containers are shown in Fig. 1 and
rates of diisophenyl phthalate standard, di-n-phenyl phthalate, and di-
Fig. 2, respectively. As can be seen, median of ∑PAEs concentration in
n-benzyl phthalate were 82.3 ± 7.9%, 76.3 ± 3.4%, and
the samples kept in PET containers was 24.2 µg/L in lemon juice
104.6 ± 9.8%, respectively. The recovery rate obtained for phthalates
samples, 28.4 µg/L in verjuice samples and 25.1 µg/L in vinegar sam-
in spiked blanks samples ranged from 73.4 to 110% with a standard
ples (Fig. 1). The values observed for PAEs in highly acidic juices were
deviation of less than 10%; in the matrix spike samples, the recovery
higher than those reported by Jeddi et al. [31] for non-acidic juices.
rate was in the range of 72.0–111% with a standard deviation of about
Table 2 shows the PAEs concentrations obtained in this study and
10.4%. Method validation was carried out in accordance with the
others [8,15,25,31-34]. As observed, except for the DEHP compound in
currently accepted U.S. Food and Drug Administration Guideline for
wine samples packed in bottles with plastic-cap whose concentration
Industry [26]. Six target PAEs (DMP, DEP, DnBP, BBP, DEHP, and
was higher than the values observed in this study for highly acidic
DNOP) were prepared at concentration levels ranging 1 ng/L to 100 µg/
juices, the other concentrations were lower than this study. Therefore,
L. Under optimized conditions, the coefficient of correlation in the
it can be interpreted that the storage of juices in plastic containers
gained calibration curves was fine, ranging from 0.9944 to 0.9995. The
under acidic conditions is an important factor in increasing the occur-
limit of detection (LODs) and limit of quantification (LOQs) for the
rence of PAEs in the texture of these foods [35]. The detection fre-
target analytes ranged from 0.0011 to 0.0023 and 0.0039–0.0081 ng/L,
quency (D.F) among the studied phthalates varied from 55 to 100%.
respectively (Table S1). In the method blank samples, very trace values
DEHP, DnBP and BBP had the highest detection frequency and were
(ranging from <LOQ to 0.0047 µg/L.) were observed for some PAEs;
found in all samples. DEP and DnOP were found in 90% of the samples,
these values were appropriately subtracted from those read in the
and DMP was observed in 55% of the samples. DEHP and DnBP were
samples.
the major compounds with the median value of 8.1 and 6.8 µg/L, 10.5
and 7.2 µg/L and 9.8 and 6.7 µg/L in lemon juice, vinegar, and verjuice,

3
L. Arfaeinia, et al. Microchemical Journal 155 (2020) 104719

significant if pvalue < 0.05. As shown in Table 3, a significant and po-

sitive correlation was observed between the storage temperature and
the concentration of PAEs in highly acidic juices (pvalue < 0.05). These
findings are in agreement with previous studies reporting that the mi-
gration of PAEs from the wall of plastic containers to its contents was
accelerated at high storage temperatures [35,38]. There was also a
significant and positive correlation between the storage time and the
migration rate of PAEs (pvalue < 0.05), which is consistent with pre-
vious studies [39]. The findings also showed that the exposure to sun-
light of acidic juices increased the migration of PAEs, thereby in-
creasing the concentration of these pollutants in the food matrix. A
significant and negative correlation was found between pH and con-
centration of these pollutants in highly acidic juices (pvalue < 0.05). In
other words, the migration rate of PAEs occurred more and faster under
acidic conditions [40].
Martin et al. [41] also observed that the acidic conditions of the
plastic container contents increased the diffusion and migration of
PAEs. However, the observations of the present study are in conflict
with the findings of studies reporting that high pH was a favorable
condition for the migration of PAEs from the wall of containers into
food [42,43]. In addition, no significant relationship was seen between
Fig. 2. Occurrence of six priority PAEs in the highly acidic juice samples packed
in glass containers.
the size of storage containers (surface or volume) and the amount of
phthalates in highly acidic juices (pvalue > 0.05).

respectively. In the control samples (samples kept in glass containers),

median of ∑PAEs concentrations were 0.05, 0.06 and 0.06 µg/L in
lemon juice, verjuice, and vinegar samples, respectively (Fig. 2). The
significance of the difference of the PAEs concentration in the acidic
juices packed in PET containers and the glass containers (control group)
was also investigated by two sample t-test. Based on results of this
statistical test, ∑PAEs concentration was higher in juices packed in PET
containers than in glass containers (pvalue < 0.05). The high amount of
PAEs observed in pickles packed in PET containers compared to glass
containers indicate that the main source of these pollutants is migrating
from the wall of the plastic containers into the pickle texture [36].
However, pollutant of the PAEs may also occur during the production,
collection and transfer of samples [37].
3.2. Migration of PAEs from the wall of pet containers into highly acidic
juice
Several factors such as storage conditions (temperature, sunlight,
and time duration), pH, and size of PET containers may affect the mi-
gration of PAEs from the PET wall into their contents [32]. The sig-
nificance of the relationship between PAEs concentration and other
measured parameters of highly acidic juice was also tested by Pearson
correlation analysis. Differences in mean values were accepted as being
3.3. Health risk assessment of PAEs
3.3.1. Human daily intake and safety factor
The maximum and average daily intakes by highly acidic juices
packed in PET containers were calculated based on the maximum and
average of concentrations of the studied phthalates. Next, the risk as-
sessment of these pollutants was calculated and the results are pre-
sented in Table 4 [44-47]. As can be seen, the maximum daily intakes of
DMP, DEP, DnBP, BBP, DEHP, and DnOP were 0.0092, 0.0058, 0.014,
0.0065, 0.012, and 0.0087 µg/kg bw/d in adults and 0.033, 0.02,
0.047, 0.023, 0.041, and 0.041 µg/kg bw/d in children. Based on the
recommended reference dose, the safety factor was calculated for each
compound. As observed in Table 4, the SF value calculated for all PAEs
was much higher than 1, indicating that the consumption of these acidic
juices does not put the consumers at the risk of these contaminants. It is
noteworthy that the SF value could not be calculated due to the lack of
RfD for DMP and DnOP compounds.
The survey of the concentration of PAEs in highly acidic juice
samples was short-lived and much shorter than the quality assurance
period of at least one year. Therefore, to more precisely investigate the
long-term monitoring challenges, the kinetic models of PAEs migration
from the wall of PET containers into foods were prepared using the

Table 2
Comparison of the findings of current work on the migration of target PAEs with other studies (ng/L).
Matrix Statistics DMP DEP DEHP BBP DnBP DnOP Reference

Highly acidic juice packed in PET containers (µg/L) Mean 5.2 2.1 9.8 4.0 7.9 4.9 Current study
Min-max 0.6–39.1 0.16–24.6 1.3–48.8 0.2–27.5 1.1–57.2 0.5–37.7
Med 3.2 1.0 9.4 2.5 6.9 4.2
D.F* (%) 55 90 100 100 100 90
Highly acidic juice packed in glass containers (control samples) Mean 0.01 0.005 0.02 0.008 0.016 0.01 Current study
(µg/L) Min-max 0.001–0.065 0.001–0.01 0.02 0.002–0.04 0.004–0.08 0.001–0.06
Med 0.006 0.004 0.003–0.12 0.007 0.014 0.008
D.F*(%) 35 60 65 55 60 65
Doogh Mean – – 2582.72 10.37 45.07 227.67 (32)
Bottled Water Mean 120 660 430 70 110 – (15)
Water Mean – – 411 20 116 – (31)
Carbonated soft drink Mean – – 2737.87 – 49.52 228.96 (33)
Wine samples packed in bottles with the plastic-cap Mean 155–375 – 10,999–17,694 151–355 1389–1948 – (25)
Drinking Bottled Water Mean – 70 580 – – – (8)
water Mean – 375 100 – – – (34)

⁎
Detection frequency.

4
L. Arfaeinia, et al. Microchemical Journal 155 (2020) 104719

Table 3
Pearson correlation test among the measured parameters of highly acidic juice (lemonjuice, verjuice and vinager) samples.
Parameters pH Sunlight exposure Storage time Storage temperature DMP DEP DEHP BBP DnBP DnOP

pH 1.00 0.139 −0.193 0.118 −0.598 −0.643 −0.712 −0.803 −0.729 −0.639
Sunlight exposure 1.00 0.101 0.162 0.592 0.748 0.669 0.598 0.717 0.737
Storage time 1.00 0.876 0.665 0.719 0.669 0.810 0.609 0.834
Storage temperature 1.00 0.577 0.498 0.710 0.565 0.499 0.623
DMP 1.00 0.915 0.876 0.891 0.765 0.816
DEP 1.00 0.678 0.762 0.733 0.777
DEHP 1.00 0.825 0.817 0.786
BBP 1.00 0.778 0.675
DnBP 1.00 0.827
DnOP 1.00

following equations Eqs. (5) to (7) and the data collected during compounds in drinking water sources, and Japan has taken into con-
sampling. sideration a strict standard for the composition of E2 in drinking water
sources [51,52]. To evaluate the estrogenic potential of the studied
Linear fit C = a + b × t (5)
compounds, the EEQ value of these compounds was calculated in highly
Exponential fit C = a × Exp (b × t ) (6) acidic juices (lemon juice, vinegar and verjuice) packed in PET con-
tainers (Table 5). As can be seen, BBP had the highest value of EEQ (8 *
Polynominal model fit C = a + b × t + c × t 2 (7) 10−1 ng E2 / L), followed by the DnBP compound with an EEQ value of
3.2 * 10−1 ng E2 / L. The EEQ averages calculated for the ∑PAEs were
where C is the PAEs concentration, t is the storage time, and a, b, and c
higher than 1 ng E2/L and were able to adversely affect human health.
are all fitting constants.
It is to be noted that the possible estrogenic effects of PAEs in food
Since the expiry date of these products is more than one year, the
samples such as highly acidic juices have been poorly investigated.
concentration of PAEs in highly acidic juices was predicted for the next
Soares et al. reported that even low exposure to EEQ (0.27 ng E2 / L)
year (based on Eqs. 5 - 7) that is depicted in Fig. 3. Our findings re-
resulted in egg mortality at the end of the gastrulation and / or early
sulting from these models show that kinetics had the best fit with ex-
organogenesis in zebrafish [53]. Overall, the results of this study
ponential model. In Fig. 3., solid line and dot line are monitored and
showed that phthalates in lemon juice, vinegar and verjuice packed in
predicted concentration of PAEs in acidic juice samples, respectively. As
PET containers did not raise serious concerns as to their possible es-
can be seen in Fig. 3, the concentrations of DMP, DEP, DnBP, BBP,
trogenic effects on human health. However, the bad news is that the
DEHP and DnOP after about 57 days were 5.2, 2.1, 7.9, 4.0, 9.8 and
EEQ level of phthalates in the pickles of this container will augment
4.9 μg/L, respectively. It was anticipated that after about 365 days, the
with the increase in storage time. Estrogenic compounds (ECs) with
concentrations of these compounds would reach approximately 42.4,
identical EEQ values may also have adverse effects on human health;
19.1, 58.7, 33.2, 67.4 and 37.4 μg/L, respectively. The concentration of
therefore, caution should be exercised in interpreting the high EEQ
DEHP after one year was further observed to reach about 8.4 and 11
values calculated for phthalates in food. Given the high global con-
times the standard limit determined by the WHO [48] and USFDA [49]
sumption of highly acidic juices (lemon juice, vinegar and verjuice), it
for drinking water [8 and 6 μg/L, respectively). Therefore, it can be
should be emphasized that the possible risks of phthalates as estrogenic
clearly stated that the increase in the storage time of foods (such as
compounds need comprehensive studies in each region in order to
pickles) in PET containers under acidic conditions results in the sig-
better understand them. In general, the available information in this
nificant accumulation of PAEs in these foods, with concentrations ex-
area is still very limited. Accordingly, it is essential to conduct extensive
ceeding the limit. Therefore, the quality of PET-packed food may
research on the food safety associated with estrogenic phthalates.
change even during the warranty period, hence the necessity of the
long-term monitoring of phthalates in food packaged in these con-
tainers. 4. Conclusion

3.3.2. Estrogenic activity-based risk assessment of PAEs
Estrogenic compounds with environmental concentrations can have
adverse effects on fish and other animals and cause vitellogenin, in-
tersex, disruption of mating behaviors and sterility, and even death
[50]. Today, strict standards are considered for some estrogenic com-
pounds in environmental sources such as drinking water. China has
considered a strict standard for DEP, DnBP, DEHP, and bisphenol A
To the best of our knowledge, this research is the first comprehen-
sive report on the occurrence, migration, and estrogenic activity-asso-
ciated risk assessment of PAEs in highly acidic juice packaged in
polyethylene terephthalate (PET) container; however, there existed a
number of gaps that could be filled in future work. First, it is possible
that a steady state concentration be reached before one year, resulting
in the predicted concentrations being an overestimate. In other words,

Table 4
Human exposure estimation and safety factor-based risk assessment of PAEs via highly acidic juice (lemonjuice, verjuice and vinager).
Factors DMP DEP DEHP BBP DnBP DnOP

Maximum concentration (µg/L) 39.1 24.6 48.8 27.6 57.2 37.6

Mean concentration (µg/L) 5.2 2.1 9.8 4.0 7.9 4.9
Daily exposure (adults, µg/kg bw/d)* 0.0092 0.0057 0.011 0.0064 0.013 0.0087
Daily exposure (children, µg/kg bw/d)* 0.032 0.02 0.040 0.022 0.047 0.031
Tolerable daily intake (TDI, µg/kg bw) – – 50.00(45) 500.00(46) 10.00(47) –
Reference dose (RfD, µg/kg bw/d)(44) – 800.00 20.00 200.00 100.00 –
Safety factor (adults, based on maximum concentration) – 139,714.60 1759.43 31,164.58 7506.52 –
Safety factor (children, based on maximum concentration) – 6,971,492.00 44,222.84 1,387,473.00 160,993.72 –

⁎
Based on maximum concentration detected in highly acidic juice.

5
L. Arfaeinia, et al. Microchemical Journal 155 (2020) 104719

Fig. 3. Measured and predicted concentrations of PAEs in highly acidic juice from PET container. Solid line: Measured concentration; Dot line: predicted con-
centration (scale for the time axis is day).

Table 5 Declaration of Competing Interest

Estrogenic effects-based health risk assessment of PAEs compounds in highly
acidic juice (lemonjuice, verjuice and vinager). The authors declare no competing financial/non-financial interests.
PAEs Average concentration Estrogenic potential EEQ (ngE2/L)
compounds (µg/L) (EP) Acknowledgments

DMP 5.2 – –
The authors are very grateful to Bushehr University of Medical
DEP 2.1 5 × 10−7 1.07×10−3
BBP 4.0 2 × 10−4 7.91×10−1 Sciences (1080) for financially supporting this research.
DEHP 9.8 3 × 10−7 2.93×10−3
DnBP 7.9 4.1 × 10−5 3.21×10−1 Supplementary materials
DnOP 4.9 – –

Supplementary material associated with this article can be found, in

the online version, at doi:10.1016/j.microc.2020.104719.
the predicted concentrations over time appears that the increase in
concentration is so accelerating, based on the findings. So, there is a
References
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why when we have data for 57 days appearing to show a slow rate
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rage temperature at store), the concentration of PAEs in highly acidic
juices should be predicted in different temperatures. Third, in regard to
risk estimates, further studies are required to better clarify the factors
contributing most significantly to risk assessments.
Author statement
All authors listed have contributed sufficiently to the project to be
included as authors. All authors have read the manuscript and have
agreed to submit it in its current form for consideration for publication
in the Journal. The authors declare that they have no conflict of in-
terest, financial or other.
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