Article

Emerging Contaminants from Bioplastic Pollution in

Marine Waters
Amedeo Boldrini 1,2 , Nicola Gaggelli 1 , Francesco Falcai 1 , Alessio Polvani 1,2 , Luigi Talarico 1 ,
Luisa Galgani 1 , Riccardo Cirrone 1,3 , Xinyu Liu 1,3 and Steven Loiselle 1,4, *

1 Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2,
53100 Siena, Italy; [email protected] (A.B.); [email protected] (N.G.);
[email protected] (F.F.); [email protected] (A.P.);
[email protected] (L.T.); [email protected] (L.G.); [email protected] (R.C.);
[email protected] (X.L.)
2 Center for Colloids and Surface Science (CSGI)-Siena Research Group, University of Florence, Via della
Lastruccia 3, 50019 Firenze, Italy
3 NBFC-National Biodiversity Future Centre, 90121 Palermo, Italy
4 National Interuniversity Consortium of Materials Science and Technology (INSTM)—Siena Research Unit,
Via G. Giusti 9, 50121 Firenze, Italy
* Correspondence: [email protected]

Abstract: The increasing presence of compostable bioplastics as substitutes for conventional fossil-
based plastics necessitates a deeper understanding of their environmental impacts, particularly in
marine ecosystems, where they often accumulate. This study examines the leaching potential of differ-
ent phthalic acid esters (PAEs) from commercial biodegradable plastic bags into natural seawater over
a three-month period. Degradation experiments were conducted to investigate the release of PAEs
under direct solar radiation exposure and in shielded conditions. 1 H-NMR analysis of the seawater
confirmed the release of phthalates, with higher concentrations observed in the samples exposed
to sunlight. The leaching rate ranged from 264–342 microgram/g plastic under light exposure to
20–167 microgram/g in dark conditions. These results indicate that the accumulation of compostable
plastic waste in coastal marine environments leads to the release of phthalic acid esters, with potential
Citation: Boldrini, A.; Gaggelli, N.;
implications for marine ecosystem health and human exposure to these emerging contaminants.
Falcai, F.; Polvani, A.; Talarico, L.;
Galgani, L.; Cirrone, R.; Liu, X.; Keywords: marine pollution; emerging contaminants; phthalates; marine water; HPLC; NMR
Loiselle, S. Emerging Contaminants
from Bioplastic Pollution in Marine
Waters. Water 2024, 16, 3676.
https://doi.org/10.3390/w16243676 1. Introduction
Academic Editor: José Luis Plastics constitute a wide class of synthetic, semi-synthetic, and natural polymers,
Sánchez-Lizasoe characterized by an elevated molecular weight and long hydrocarbon chains. Fossil-based
plastics are commonly referred to as “conventional” or “traditional” and are ubiquitous
Received: 13 November 2024
in industrial and household uses [1]. One of the key features of plastic-based products is
Revised: 12 December 2024
their resistance to degradation. However, this aspect has generated a number of challenges
Accepted: 18 December 2024
for waste management [2], resulting in an estimated 5 to 13 million tons of plastic waste
Published: 20 December 2024
released yearly into the aquatic environment [3].
Inland-generated plastic litter can be transported through rivers to the marine en-
vironment, where it can accumulate, alongside local plastic pollution related to fishing
Copyright: © 2024 by the authors. activities and aquaculture [4]. The physical degradation of macroplastics to the nanoscale
Licensee MDPI, Basel, Switzerland. has a number of impacts. Fragments are ingested by aquatic fauna and can act as carriers
This article is an open access article of multiple pollutants. Their hydrophobic properties and high surface area-to-volume
distributed under the terms and ratio favor the adsorption of heavy metals, polycyclic aromatic hydrocarbons (PAHs), and
conditions of the Creative Commons polychlorinated biphenyls (PCBs) [5] that can bioaccumulate.
Attribution (CC BY) license (https:// Even without their capacity to accumulate pollutants from their surroundings, addi-
creativecommons.org/licenses/by/ tives present in nearly all plastic products can be released during degradation [6]. Additives
4.0/).

Water 2024, 16, 3676. https://doi.org/10.3390/w16243676 https://www.mdpi.com/journal/water

Water 2024, 16, 3676 2 of 13

are used as antioxidants, stabilizers, plasticizers, and flame retardants, with common exam-
ples including bisphenol A, nonylphenol, and polybrominated diphenyl ethers [7]. These
compounds are incorporated into the surface or physically dispersed into the polymer
structure; the absence of a chemical bond allows their leaching in both controlled recycling
processes [8] as well as during uncontrolled degradation in the environment [9]. The degree
and rate of leaching will depend on a wide range of factors, including temperature, local
chemical conditions [10], and irradiance [11]. Upon release, additives or their byproducts
can accumulate, representing a potentially important ecotoxicological risk.
Biodegradable plastics represent a promising replacement for traditional synthetic
polymers, providing new waste disposal opportunities such as composting. According
to the IUPAC, biodegradable plastics are polymeric compounds capable of being decom-
posed by enzymatic processes into carbon dioxide, water, methane, inorganic compounds,
and biomass. Bioplastics refer to materials fully or partially made of renewable biomass
sources and include poly(lactic acid), polyhydroxyalkanoates, and bio-based poly(butylene
succinate), as well as plastics based on starch, cellulose, proteins, and lignin [12,13]. Not all
bioplastics are necessarily biodegradable, depending on their polymer structure [14].
The degradation of biodegradable materials is not solely influenced by the intrinsic
properties of the polymer (length of the chain, degree of crystallinity, and molecular
complexity) or the additives incorporated during their production, but it is also determined
by the local conditions [15]. Compostable plastics must degrade within a defined timeframe
and under specific conditions [16].
Polylactic acid (PLA) is a compostable bioplastic, the main degradation product of
which, lactic acid, is an approved food additive. Its release or that of its derivatives poses
no harm to the environment [17]. Polybutylene adipate-co-terephthalate (PBAT) also shows
good compostability due to its aliphatic chains. An increase in PBAT crystallinity during
biodegradation suggests that the amorphous domain tends to decompose faster than the
rigid aromatic segment [18]. PBAT is certified as compostable according to the ASTM
D6400 [19] criteria of biodegradation, disintegration, and compost quality [20]. PLA/PBAT
mixtures have found a growing use as sheets and films in packaging applications.
To evaluate the real opportunities of a widespread use of bioplastics, a more complete
understanding of the additives used during production stages, their presence in the final
products, and their potential release into the environment is required. The most common
additives used in bioplastics are plasticizers, such as phthalate acid esters (PAEs), applied
to improve softness and flexibility as well as to facilitate handling during production stages.
Despite their usefulness, the release of PAEs into the environment can have significant
consequences. PAEs released from the polymer may occur during any phase of the product
life cycle (e.g., production, practical use, weathering, recycling). Being primarily lipophilic
and highly persistent, they can be easily absorbed and bioaccumulate [21].
Phthalic acid esters, or phthalates, are a family of dialkyl and alkyl aryl esters of
phthalic acid. They are colorless, odorless, and liquid at room temperature, characterized by
low volatility and solubility in water [22]. The most common phthalates used in consumer
products are dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP),
benzyl butyl phthalate (BzBP), dicyclohexyl phthalate (DCHP), di-2-ethylhexyl phthalate
(DEHP), diisobutyl phthalate (DiBP), diisononyl phthalate (DiNP), diisodecyl phthalate
(DiDP), di-n-hexyl phthalate (DnHP), and di-n-octyl phthalate (DnOP). When leached
into the environment, phthalates undergo different reaction pathways depending on local
conditions. In aquatic ecosystems, photolysis, hydrolysis, and microbial degradation are
the main causes of their transformation. In general, phthalic acid diesters are first degraded
to the corresponding monoesters and then to phthalic acid [23,24].
Phthalates are known to impact the environment as well as human health, as expo-
sure has been associated with impaired reproductive capacity and carcinogenesis [25–27].
Leaching from conventional plastics related to food, pharmaceuticals, and construction
materials has led to their presence in the environment [28]. Recent regulations (REACH) by
the European Chemicals Agency contain 14 phthalates categorized as toxic for reproduction
Water 2024, 16, 3676 3 of 13

and endocrine disruptors, due to their capacity to mimic estrogens and impact hormonal
functions and pathways [27,29]. DMP, DEP, DBP, BzBP, DEHP, and DnOP have been listed
as priority pollutants by the United States Environmental Protection Agency (US EPA) [30].
Biodegradability represents a possibility to limit the presence and impact of plastic
pollution. However, this requires specific conditions to be met, most importantly that the
biodegradable plastic reaches appropriate waste-treatment plants [31]. If these conditions
are not met, the increased use of bioplastic bags, combined with non-optimal waste manage-
ment, will lead to their further accumulation in both aquatic and terrestrial environments.
There have been numerous studies on the impacts of conventional plastic pollution on
the aquatic environment. However, there is limited information on the potential impacts
regarding the growing use of biodegradable plastics. The present study focused on the
release of phthalic acid esters from widely used commercial biodegradable plastic bags to
determine the leaching potential of these materials in seawater under conditions of sunlight
and in darkness.

2. Materials and Methods

2.1. Experimental Design
Marine water (20 L) was sampled from the coastal waters near Livorno, Italy (olig-
otrophic waters of the Tyrrhenian Sea) (pH = 8.18 ± 0.01, salinity 38 PSU). Vacuum filtration
was performed using a 0.45 µm Millipore filter (47 mm) to reduce the concentrations of
phytoplankton and particulate matter.
Nine different shopping bags, declared compostable and biodegradable (biobags, BB),
were purchased from local stores, and three of them (BB4, BB8, and BB9) were randomly
selected. Three strips from each bag were cut (approximately 2 cm × 4 cm) from different
parts of the bag to have more homogeneity and representativeness of the composite sample.
All strips had similar total surface areas and a similar total weight for each bag (Table 1).

Table 1. Total weight of biobag strips at the beginning of the experiment and microcosm types
(500 mL).

Light Exposure Biobag Total Sample Weight (g) Container

BB4 2.150
BB8 1.296
Light Quartz flasks
BB9 1.921
BLANK -

BB4 2.256
BB8 1.015
Dark Dark glass bottles
BB9 1.988
BLANK -

Eight 500 mL microcosms were prepared to simulate two different environmental

conditions. Four quartz microcosms, nearly transparent to solar radiation in the UV and
visible wavelengths, were used to simulate conditions of full exposure to solar radiation.
Acid-cleaned dark glass microcosms of the same volume, with an attenuation of 99% of
the solar UV wavelengths and 90% of visible light, were used to simulate dark conditions.
Biobag strips from BB4 were placed in one quartz microcosm (BB4-LIGHT) with a similar
set of strips placed into one dark glass microcosm (BB4-DARK). The same procedure was
applied to the other two biobag samples, resulting in three LIGHT conditions, one for each
selected biobag strip, and the corresponding three DARK conditions containing individual
biobag strips. The two remaining microcosms (one quartz and one dark glass) were filled
with seawater and used as the control (BLANK). All microcosms were sealed with glass
stoppers to prevent evaporation.
Water 2024, 16, 3676 4 of 13

The eight microcosms were set on a rooftop in a fixed position, with the stoppered side
below to avoid shadow or additional reflectance. All microcosms were placed vertically in
the same position to ensure a similar solar irradiance (LIGHT, CONTROL) and temperature
(LIGHT, DARK, CONTROL). They were exposed to local environmental conditions for
120 days. The temperature was continuously monitored by a digital thermometer data
logger placed between the microcosms. An average temperature of 30.2 ± 0.1 ◦ C was
recorded during the entire period. It should be noted that differences in daily temperature
dynamics between the digital thermometer, dark glass, and quartz microcosms were likely
to have occurred (lower daytime temperature, higher nighttime temperature), with the
average temperature being slightly higher in the DARK conditions. UV-Visible solar irradi-
ance was measured continuously near the surface of the quartz and dark-glass microcosms
with a four-channel radiometer (10-nm waveband and center wavelengths of 380, 440,
590, and 670, Skye Instruments, Llandrindod Wells, UK). The total exposure of the plastic
samples in the quartz flasks over the treatment period was 7413 and 371 kJ/m2 of plastic
for UVA (380 nm) and UVB (315 nm) radiation, accounting for the spectral attenuation of
the seawater and the plastic at half the depth of the microcosm. The average values of the
daily recorded UVA and UVB exposure were 1464 and 74.4 kJ/m2 per day, respectively.
After 120 days, biobag strips were removed from seawater, washed with ultrapure
water, dried, weighed, and stored at room temperature. Seawater samples were transferred
to polyethylene containers and stored in the dark at 4 ◦ C.

2.2. Chemicals and Selected Phthalates

A standard mixture of six phthalate esters (DMP, DEP, DBP, BzBP, DEHP, and DnOP),
TraceCERT® , certified reference material, was purchased from Merck KGaA (Milan, Italy).
For dilution and mobile phase preparation, ultrapure (UP) water (18.2 MΩ cm, Direct-
Pure, RephiLe Bioscience, Boston, MA, USA, filtered with a 0.2 µm PES filter), acetonitrile
(ACN) (gradient grade, ≥99.9%), and methanol (ACS reagent, ≥99.8%) were purchased
from Merck KGaA (Milan, Italy). Chloroform (ACS, 99.8% purity) was purchased from
Carlo Erba.
NMR solvents were purchased from Merck KGaA (Milan, Italy): acetone-d6 (99.9 atom
% D), chloroform-d (CDCl3 ) (99.8 atom % D), and deuterium oxide (D2 O) (99.9% atom D,
contains 0.05 wt. % TSP-d4 , 3-(trimethylsilyl) propionic-2,2,3,3-d4 sodium salt).
Solid-phase extraction (SPE) columns were purchased from Phenomenex (Torrance,
CA, USA): Strata-X-AW 33 µm Polymeric Weak Anion, 100 mg/3 mL; Strata-X 33 µm
Polymeric Reversed Phase, 200 mg/3 mL.

2.3. FTIR and 1 H-NMR Spectroscopy

Fourier-transform infrared spectroscopy (FTIR) and 1 H-NMR were used to investigate
the chemical composition, patent protected, of the selected biobags before the experiment,
using a Bruker Alpha II FTIR spectrometer in attenuated total reflectance (ATR) mode [32].
Three scans were performed at three different points on the surface, over a range of
4000–400 cm−1 , with 24 scans per measurement and a resolution of 4 cm−1 .
1 H-NMR measurements were carried out on both the biobags and on the seawater

before and after biobag leaching using a Bruker 600 MHz spectrometer. One-dimensional
(1D) spectra were acquired at T = 298 ± 0.1 K by a common pulse sequence without solvent
suppression, with an FID of 32,768 points, 512 scans, a spectral width of 6000 Hz, a 90◦
pulse of 8.0 µs, and a relaxation delay of 1.0 s. Two-dimensional (2D) COSY sequences, with
1024 points in the F2 dimension, 256 increments with 16 transients each, and a relaxation
delay of 3.0 s, were employed to support the identification of spin systems over a spectral
width of 6000 Hz.
For NMR structural characterization, 0.1 g of each biobag was solubilized in 5 mL
of chloroform following a solubility test with different solvents [33], evaporated under a
nitrogen stream, and re-dissolved in CDCl3 . To assess the presence of phthalates within
the biobags, ≃0.075 g of each sample was solubilized in deuterated acetone. 1D and 2D
Water 2024, 16, 3676 5 of 13

COSY spectra were acquired [34]. A 1D spectrum of a 10 mg/L solution of the standard
phthalate esters mix in methanol was acquired in D2 O, using the same sequence with
water suppression.
The NMR analysis of seawater samples was performed on 540 µL of the solution from
each flask, including the blank, and by adding 60 µL of D2 O containing 0.05% m/m of
TSP-d4 as a chemical shift reference and concentration standard. For each seawater sample,
1 H-NMR experiments were performed using the noesypr1d pulse sequence [35], with a

free induction decay (FID) of 32,768 points, over a spectral width of 6000 Hz, 256 scans, and
a relaxation delay of 2.0 sec. To estimate the phthalate concentration in seawater samples,
we used peak integrals at 7.94 and 8.10 ppm, calibrating our results to the integral of the
TSP-d4 , which was arbitrarily assigned a unit value. Equation (1) was then applied to
convert integrals into concentrations expressed in mg/L.

mx = (Ax /Aref )×[mref /(MWref /no.Href )]×(MWx /no.Hx ) (1)

where mref is the concentration (mg/L) of the internal standard; Ax and Aref are the peak
areas of the analyzed species and internal standard, respectively; MWx and MWref are the
molecular weights; no.Hx and no.Href are the number of hydrogen atoms corresponding to
the peaks of compound x and the internal standard, respectively.

2.4. HPLC-DAD Analysis

A Dionex Ultimate-3000 high-performance liquid chromatography (HPLC) system,
equipped with a diode array detector (DAD), was used to perform quantification of phtha-
late esters in biobags and in seawater samples. Analyses were carried out following [36]
with a Kinetex C18 EVO 100 Å column (150 × 2.1 mm, 2.6 µm, Phenomenex Torrance
California) under isothermal conditions at T = 298 K. The mobile phase was ultrapure water
(line A) and acetonitrile (line B) with a constant flow rate of 0.400 mL/min and a sample
injection volume of 5 µL. The detector was set to 220 nm. The total run time was 40 min
(Supplementary Materials).
For HPLC analysis, a sample of each biobag was weighed and then solubilized in
3.5 mL of chloroform [33], evaporated under a nitrogen stream, re-dissolved in 2 mL of
methanol, and filtered with a 0.22 µm PTFE filter, previously tested to ensure no contami-
nation and sample loss.
For the eight seawater samples, a preliminary solid-phase extraction (SPE) was per-
formed before HPLC and 1 H-NMR analysis.

2.5. Statistical Analysis

Datasets showed near-normal distributions (skewness < 1.5), allowing for the use of a
mean-based approach (t-test) for direct comparison between conditions, with α set to 0.05.
Bonferroni correction for multiple comparisons.

3. Results and Discussion

3.1. Biobag Composition Characterization
The FTIR spectra of the biobags (Figure 1) were compared to spectra from previous
studies on biodegradable plastic materials [37,38]. The results showed the presence of
poly(lactic acid) (PLA)/poly(butylene adipate-co-terephthalate) (PBAT) blends. BB4 and
BB9 spectra shared a high degree of similarity, suggesting a similar blending ratio of the
two polymers. A likely low interfacial interaction of the two biodegradable polymers
allowed the identification of the respective functional groups, the peak positions of which
did not result in variations with respect to the spectra of pure compounds. The infrared
spectrum of BB9 was similar to BB4, except for a slight shift of 1–2 cm−1 , suggesting an
analogous chemical composition.
 two polymers. A likely low interfacial interaction of the two biodegradable polymers
allowed the identification of the respective functional groups, the peak positions of which
did not result in variations with respect to the spectra of pure compounds. The infrared
Water 2024, 16, 3676 6 of 13
spectrum of BB9 was similar to BB4, except for a slight shift of 1–2 cm−1, suggesting an
analogous chemical composition.

FTIR spectra
Figure 1. FTIR spectra of
of BB4,
BB4, BB8, −1 .
BB8, and BB9 plastic samples over the range 4000–400 cm−1
Figure .

BB8 exhibited
BB8 exhibited additional
additional bands
bands that
that were
were associated
associated with
with aa higher
higher percentage
percentage ofof
PLA. The absorption at 1759 cm −1 can be attributed to the C-O stretching vibration of
PLA. The absorption at 1759 cm can be attributed to the C-O stretching vibration of
−1
carbonyl, which
carbonyl, which isis found
found at
at higher
higher frequencies
frequenciescompared
comparedtotoPBAT,
PBAT, where
wherethe ester
the group
ester is
group
conjugated with the aromatic ring. The peaks at 1453 and 1365 cm −1 represent, respectively,
is conjugated with the aromatic ring. The peaks at 1453 and 1365 cm−1 represent,
the C-H asymmetric
respectively, the C-Hand symmetricand
asymmetric bending vibrations
symmetric of the
bending CH3 group;
vibrations the CH
of the asymmetric
3 group;
vibration is likely overlapped with the -CH2 -bending of PBAT, which is expected at slightly
the asymmetric vibration is likely overlapped with the−-CH 2-bending of PBAT, which is
higher frequencies (≃1465 cm−1 ). The band at 1390 cm 1 can be associated with the C-H
expected at slightly higher frequencies (≃1465 cm −1). The band at 1390 cm−1 can be
deformation of the α-carbon. The peak at 1080 cm−1 represents an exception, assigned to
associated with the C-H deformation of the α-carbon. The peak at 1080 cm−1 represents an
the O-C-C stretching of PBAT-saturated ester linkage, which was absent in the spectra of
exception, assigned to the O-C-C stretching of PBAT-saturated ester linkage, which was
BB4 and BB9.
absent in the spectra of BB4 and BB9.
1 The
1The
H-NMR spectroscopy of all three biobags showed similar spectra (Supplementary
H-NMR spectroscopy of all three biobags showed similar spectra
Materials); peak assignment confirmed the presence of a mixture of PLA and PBAT in
(Supplementary Materials); peak assignment confirmed the presence of a mixture of PLA
varying concentrations.
and PBAT in varying concentrations.
3.2. Detection and Quantification of Phthalate Esters in Biobag Samples
3.2. Detection and Quantification of Phthalate Esters in Biobag Samples
The investigation of phthalate presence on all biobags was initially conducted by
The investigation
1 H-NMR. of phthalate
Sample spectrums presence
(Figure on alltwo
2) exhibit biobags was signals
distinct initiallywithin
conductedthe by 1H-
region
NMR. Sample
7.80–7.60 ppmspectrums
that can be(Figure 2) exhibit
attributed two distinct
to phthalate signals within
compounds, the region
according 7.80–7.60
to comparison
ppm thatspectrum
with the can be attributed to phthalate
of the standard compounds,
phthalates according to comparison
mix (Supplementary with the
Materials). Phthalate
spectrum
esters have oftwo
the pairs
standard phthalatesequivalent
of chemically mix (Supplementary Materials).
aromatic protons Phthalate
coupling esters
differently tohave
each
two
otherpairs
and of chemically
producing twoequivalent
symmetricaromatic
complexprotons coupling
multiplets. differently
A slight variationtoof
each other
chemical
and
shift,producing two symmetric
approximately complex
0.07 ppm, was multiplets.
observed A slight
due to the variation
different of used.
solvents chemical shift,
approximately 0.07 ppm, was observed due to the different solvents used.
Water 2024, 17, x FOR PEER REVIEW 7 of 13
Water 2024, 16, 3676 7 of 13

Figure 2. 1 H-NMR spectra of BB4 (red), BB8 (green), and BB9 (blue) plastic samples in deuterated
Figure 2. 1H-NMR spectra of BB4 (red), BB8 (green), and BB9 (blue) plastic samples in deuterated
acetone, showing characteristic signals of aromatic protons of phthalate esters.
acetone, showing characteristic signals of aromatic protons of phthalate esters.
Quantification using HPLC was performed using a nine-point calibration curve for
each Quantification
phthalate under using HPLC
study. was performed
Concentration usingfrom
ranged a nine-point
0.05 to 10 calibration curve
mg/L, with for
each
each phthalate
compound under
showing goodstudy.
linearity (R2 >0.99) ranged
Concentration from 0.05Materials).
(Supplementary to 10 mg/L, with each
Phthalate con-
compound in
centrations showing goodshowed
the biobags linearity (R2 >0.99)
relative (Supplementary
differences Materials).
between biobags (TablePhthalate
2). DMP,
concentrations
DBP, and DEHPinwere the present
biobagsinshowed
all threerelative
samplesdifferences between
(Figure 3), with biobags (Table
concentrations 2).
ranging
DMP, DBP, and DEHP were present in all three samples (Figure 3), with
between 17.2–95.5, 332.0–436.1, and 95.3–770.5 µg/g, respectively. DEP was not present in concentrations
ranging
the betweenwhile
BB9 sample, 17.2–95.5,
BB4 and 332.0–436.1, and a95.3–770.5
BB8 exhibited µg/g, respectively.
similar concentration DEP
(25.5 and 24.9was not
µg/g);
present
in in the
contrast, DnOPBB9was
sample,
onlywhile
foundBB4 and with
in BB9, BB8 exhibited a similar
a concentration concentration
of 56.2 µg/g. Our(25.5 and
findings
24.9 µg/g);
were similarintocontrast, DnOP
the results found wasbyonly
Xu etfound inin
al. [39] BB9, withexpress
plastic a concentration
packagingofbags.
56.2 µg/g.
Our findings were similar to the results found by Xu et al. [39] in plastic express packaging
bags. 2. Phthalate esters identified in biobag samples (BB4, BB8, BB9) from HPLC-DAD analysis and
Table
their corresponding concentrations expressed as mg/L and µg per gram of biobag.
Table 2. Phthalate esters identified in biobag samples (BB4, BB8, BB9) from HPLC-DAD analysis
Sample Phthalate
and their corresponding concentrations expressed as RT
mg/L and µg per
(min) gram
Conc. of biobag.
(mg/L) Conc. (µg/g)
Weight (mg) Ester
Phthalate
DMP 2.28 0.41
Sample Weight (mg) RT (min) Conc. (mg/L) Conc.95.58
(μg/g)
Ester
DEP 4.63 0.11 25.58
BB4 8.6 DMP 2.28 0.41 95.58
DBP 11.77 1.67 389.07
DEP 4.63 0.11 25.58
BB4

8.6 DEHP 24.25 3.31 770.47

DBP 11.77 1.67 389.07
DMP
DEHP 24.252.26 3.310.13 35.49
770.47
DEP
DMP 2.264.69 0.088
0.13 24.79
35.49
BB8 7.1
DBP
DEP 4.6911.77 0.0881.55 436.06
24.79
BB8

7.1
DBP
DEHP 11.7724.25 1.550.51 436.06
143.94
DEHP 24.25 0.51 143.94
DMP 2.18 0.052 17.33
DMP 2.18 0.052 17.33
DBP 11.76 0.99 332.00
BB9 6.0 DBP 11.76 0.99 332.00
BB9

6.0 DEHP 24.25 0.27 95.33

DEHP 24.25 0.27 95.33
DnOP 25.39 0.168 56.00
DnOP 25.39 0.168 56.00
 17, 3676
Water 2024, 16, x FOR PEER REVIEW 8 8of
of 13
13

Figure 3. Chromatograms of each biobag compared

compared to
to aa 3 mg/L standard solution
mg/L standard solution of
of phthalate
phthalate ester
mix, with
mix, with relative
relative peak
peak assignment.
assignment.

3.3. Detection of Phthalates in Seawater Samples After Degradation Experiments

3.3. Detection of Phthalates in Seawater Samples After Degradation Experiments
Microcosm seawater samples from light and dark conditions were analyzed to assess
Microcosm
the leaching seawater
potential samplescompounds
of phthalic from light and
afterdark conditions
exposure wereenvironmental
to natural analyzed to assess
con-
the leaching
ditions for 120potential of phthalic
days, comparing the compounds
degradation after exposure
processes to natural
that included environmental
photodegradation
conditions
(LIGHT) andfor 120 days,
excluded comparing (DARK).
photodegradation the degradation processes that included
photodegradation (LIGHT) and excluded photodegradation
Phthalate leaching into seawater was determined by 1 H-NMR (DARK).
spectra. In all seawater
Phthalate leaching into seawater was determined by 1H-NMR spectra. In all seawater
samples containing biobags, resonances associated with the aromatic moiety of phthalate
samples containing
esters were observed,biobags, resonances
corresponding associated
to the presence with
of twothe aromaticat
multiplets moiety of phthalate
8.10 and 7.94 ppm,
esters
assignedwere observed,
to H3/H4 andcorresponding
H2/H5 of the to the presence
aromatic of two multiplets
ring, respectively. at 8.10
The control and 7.94
microcosm
ppm,
seawater (BLANK) did not show the presence of phthalate in either LIGHT or control
assigned to H3/H4 and H2/H5 of the aromatic ring, respectively. The DARK
microcosm seawater
conditions after (BLANK)
120 days did not(Figure
of treatment show the4).presence of phthalate in either LIGHT or
DARK conditions after 120 days of treatment (Figure 4).
The effect of different environmental conditions was evidenced by the intensity of
the 1H-NMR signals for seawater samples (Figure 4). All seawater samples under LIGHT
conditions showed higher intensity peaks compared to their DARK condition
counterparts. Furthermore, the peaks of the BB4 samples were more intense than the
others. All NMR spectra were acquired using the same conditions and parameters. A
Water 2024, 17, x FOR PEER REVIEW 9 of 13

comparison with a spectrum of monomethyl phthalate [40] confirmed that these peaks
Water 2024, 16, 3676 were attributable to phthalate derivatives, as the signal was comparable in terms
9 of of
13
chemical shift and multiplicity.

Figure 4. The aromatic region of 1 H-NMR spectra of seawater samples after exposure to biobags for
Figure 4. The aromatic region of 1H-NMR spectra of seawater samples after exposure to biobags for
120 days; asterisk (*) indicates the peaks assigned to H3/H4 and H2/H5 of phthalate.
120 days; asterisk (*) indicates the peaks assigned to H3/H4 and H2/H5 of phthalate.
The effect of different environmental conditions was evidenced by the intensity of
It shouldsignals
the 1 H-NMR be noted forthat polymeric
seawater chains
samples could4).have
(Figure been cleaved
All seawater during
samples the LIGHT
under biobag
degradation processes in seawater, whether thermal, photo,
conditions showed higher intensity peaks compared to their DARK condition counter- or microbial. For
quantification, we considered a generic hydrolyzed phthalate without
parts. Furthermore, the peaks of the BB4 samples were more intense than the others. All side chains
(C
NMR8H4O4) as a reference to estimate phthalate leaching using two dominant chemical shifts
spectra were acquired using the same conditions and parameters. A compari-
(ppm).
son with Concentrations
a spectrum of in monomethyl
seawater of phthalate
phthalate amount (mg/L), estimated
[40] confirmed that theseusing Equation
peaks were
(1), at both ppm, provided similar values (Table 3).
attributable to phthalate derivatives, as the signal was comparable in terms of chemical
shift and multiplicity.
TableIt3.should
Amount be of phthalate
noted (mg/L) released
that polymeric chainsfrom
coulddifferent biobag
have been samples
cleaved and microcosms
during the biobag
degradation
measured processes in seawater, whether thermal, photo, or microbial. For quantification,
in seawater.
we considered a generic hydrolyzed phthalate without side chains (C8 H4 O4 ) as a reference
BB4 LIGHT BB4 DARK BB8 LIGHT BB8 DARK BB9 LIGHT BB9 DARK
to estimate phthalate leaching using two dominant chemical shifts (ppm). Concentrations in
7.94 ppm 1.425 0.893 0.696 0.065 1.143 0.470
seawater of phthalate amount (mg/L), estimated using Equation (1), at both ppm, provided
8.10 ppm 1.523 0.617 0.709 0.020 0.893 0.624
similar values (Table 3).
Seawater hydrolyzed phthalate estimates were then compared to the weight of
biobag
Table 3.samples
Amountpresent in each
of phthalate microcosm
(mg/L) releasedtofrom
determinate its leaching
different biobag samplespotential (ug/g of
and microcosms
biobag, Table 4).
measured in seawater. The leaching results were consistent with the study of Xu et al. [39]
despite the different matrix and conditions.
Considering BB4 the total concentration
BB4 ofBB8
the six phthalates
BB8 determined
BB9 in the BB9 original
biobag samples, it was possible to estimate a percentage of phthalates leached, as
LIGHT DARK LIGHT DARK LIGHT DARK
hydrolyzed
7.94 ppm phthalate,
1.425 from the original biobag,
0.893 0.696 considering
0.065 the different
1.143 biobag 0.470
samples
and different
8.10 ppm
environmental
1.523
conditions.
0.617
Phthalate
0.709
leaching
0.020
was greater
0.893
(p = 0.03)
0.624
in the
LIGHT samples exposed to solar radiation compared to the samples that underwent
thermal and microbial degradation (DARK). Samples exposed to a light environment
Seawater
resulted hydrolyzed
in a leaching phthalate
percentage estimates
that were that
was double thenin compared
the BB4 andto the weight
BB9 of biobag
biobags, with
samples present in each microcosm to determinate its leaching
similar fractions of PLA and PBAT. For the samples with a higher percentage of potential (µg/g of biobag,
PLA
Table 4).
(BB8), Thewas
there leaching
a moreresults were
than 10× consistent
increase with the
in leaching study of Xu et al. [39] despite the
percentage.
different matrix and conditions.
Table 4. Quantification of hydrolyzed phthalates present in microcosm expressed as mg/L and µg
per gram of BB.

Average Phthalate Concentration Leachate Concentration per

Leachate %
in Microcosm (mg/L) Biobag (ug/g)
Water 2024, 16, 3676 10 of 13

Table 4. Quantification of hydrolyzed phthalates present in microcosm expressed as mg/L and µg

per gram of BB.

Average Phthalate Leachate

Concentration in Concentration per Leachate %
Microcosm (mg/L) Biobag (µg/g)

BB4 LIGHT 1.47 342.79 26.77

BB4 DARK 0.756 167.33 13.07
BB8 LIGHT 0.70 271.03 42.33
BB8 DARK 0.043 20.94 3.27
BB9 LIGHT 1.018 264.97 52.92
BB9 DARK 0.55 137.58 27.48

Considering the total concentration of the six phthalates determined in the original
biobag samples, it was possible to estimate a percentage of phthalates leached, as hy-
drolyzed phthalate, from the original biobag, considering the different biobag samples and
different environmental conditions. Phthalate leaching was greater (p = 0.03) in the LIGHT
samples exposed to solar radiation compared to the samples that underwent thermal and
microbial degradation (DARK). Samples exposed to a light environment resulted in a
leaching percentage that was double that in the BB4 and BB9 biobags, with similar fractions
of PLA and PBAT. For the samples with a higher percentage of PLA (BB8), there was a
more than 10× increase in leaching percentage.
According to the last report of Plastics Europe [41], in 2022, 400 million tons of plastic
materials were produced globally, of which 0.5% consisted of bioplastic. This translates to
an annual production of 2 million tons of bioplastic. Only a fraction of plastic produced
ends up in the environment; this percentage depends on a range of factors. This fraction
has been estimated to be approximately 0.5% for marine plastic pollution. Once present,
this plastic waste undergoes different degradation pathways based on local conditions,
such as temperature, solar irradiance, and water depth, leading to the release of additives
and their derivatives. According to our results, with an average leaching rate of 200 µg/g,
there is a potential release of several tons of phthalate esters per year, with consequent
impacts on the environment and human health. This estimate agrees with a recent study
of leaching from conventional plastics to the aquatic environment. Despite the different
polymer types, the estimated amount of released additives into the environment is within
the same range [42].
It should be noted that leaching potential was also the focus of an HPLC analysis of the
seawater samples. With respect to the total phthalate concentration estimated by 1 H-NMR
analysis, both the limit of detection (LOD) and the limit of quantification (LOQ) of the
HPLC method (Table 5) should have been sufficient to provide a second estimate of leached
phthalate. However, no peaks attributable to the six phthalate esters were detected, further
supporting the hypothesis that degradation of the polymeric chains had occurred in the
seawater. As degradation could have followed multiple pathways, including hydrolysis,
photolysis, thermal degradation, and biodegradation, phthalic acid derivatives were likely
present in the hydrolyzed form. This is supported by the corresponding multiplet in
1 H-NMR analysis and the loss of the singlet in the biobag samples, where the aromatic

moiety was symmetric. The lack of detection of phthalate esters by HPLC showed that
the method developed for the present study was not appropriate for the identification
of the degradation products of PAEs. This is likely due to different retention times and
interactions between the stationary phase of the SPE cartridge and C18 columns employed,
which occurs mainly with the side chains.
Water 2024, 16, 3676 11 of 13

Table 5. Limit of detection and quantification for the five phthalates detected in the original biobag.

LOD LOQ
Compound
(µg/L) (µg/L)

DMP 3.39 ± 0.64 11.23 ± 2.13

DEP 5.21 ± 1.15 17.36 ± 3.83
DBP 4.71 ± 0.3 15.71 ± 1.01
DEHP 4.60 ± 0.71 15.32 ± 2.36
DnOP 8.62 ± 1.82 28.74 ± 6.06

4. Conclusions
Although compostable bioplastics provide several waste management benefits, this
study demonstrated that there is a clear potential for the release of additives and monomers
into the environment during degradation, whether controlled in a composting facility or in
the natural environment. Additives, including plasticizers and stabilizers, are not covalently
bound to the polymer matrix and therefore can be progressively released during use and
aging. Phthalic acid esters are among the most common plasticizers used in conventional
plastics, yet very little is known about their leaching potential in biodegradable materials.
These additives are particularly important in biodegradable polymers, which often exhibit
poorer mechanical properties.
In the present study, biodegradable plastic bags (biobags) were found to contain four
different phthalic acid esters. The release of these into seawater was confirmed by NMR
analysis during a controlled degradation experiment. Furthermore, the leaching poten-
tial was shown to increase significantly in conditions where photodegradation occurred
compared to conditions where only thermal and biological degradation processes occurred.
As endocrine disruptors, the release of phthalates during the accumulation of
biodegradable plastic bags in the marine environment poses direct risks to fish and in-
vertebrates. Larger side-chain diesters of phthalic acid, such as DEHP and DnOP, have
lower solubility and are likely to pose a greater risk in the form of microplastics. Given the
increased use of bioplastics, the impacts of high molecular weight phthalates on the marine
environment warrant further study. Furthermore, more attention on the correct use and
recycling of biodegradable plastic is fundamental to reduce exposure and related health
and environmental risks.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/w16243676/s1, Figure S1: FTIR spectra of BB4 (A), BB8 (B), BB9 (C)
plastic samples over the range 4000–400 cm−1 . Each spectrum was obtained by averaging three
acquisitions of three different points on the sample surface. Figure S2: 1 H-NMR spectrum of BB4
plastic sample in CDCl3 . Figure S3: COSY spectrum of BB4 plastic sample in CDCl3 . Figure S4: PLA
protons producing NMR shift (CH and CH3) (left). All possible arrangements of PBAT monomeric
units (T = terephthalic acid, A = adipic acid, B = 1,4-butanediol), leading to different shifting for
the central tetramethylene moiety of the 1,4-butanediol (right). Figure S5: 1 H-NMR spectrum of a
10 mg/L solution of PAEs MIX in D2 O, showing characteristic signals of aromatic protons of phthalate
esters. Figure S6: HPLC-DAD chromatogram of a 10 mg/L standard solution of phthalate ester mix.
Figure S7: HPLC-DAD chromatograms of BB4 (A), BB8 (B) and BB9 (C) plastic sample compared
to the chromatogram of a 3 mg/L standard solution of PAEs mix. Table S1: HPLC elution method.
Table S2: Report of calibration curves of six phthalate esters included in the PAEs mix, built by
plotting the corresponding peak areas to the concentration of nine standard solutions.
Author Contributions: Conceptualization, S.L., N.G. and A.B.; data curation, A.B., S.L., N.G., F.F.;
formal analysis, A.B., N.G. and F.F.; investigation, A.B., N.G., F.F. and S.L.; methodology, A.B., A.P.,
L.T. and F.F.; project administration, S.L. and N.G.; supervision, S.L., L.G. and N.G.; visualization,
A.B., N.G., X.L. and R.C.; writing—original draft, A.B., N.G. and L.T.; writing—review and editing:
S.L., A.P. and N.G. All authors have read and agreed to the published version of the manuscript.
Water 2024, 16, 3676 12 of 13

Funding: The authors acknowledge the support of the Italian Ministry of University and Research,
PNRR, Missione 4 Componente 2, “Dalla ricerca all’impresa”, Investimento 1.4, Project CN00000033
and of the Programma Operativo Nazionale (PON) “Ricerca e Innovazione” 2014–2020.
Data Availability Statement: The raw data supporting the conclusions of this article will be made
available by the authors on request.
Conflicts of Interest: The authors declare no conflicts of interest.

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