toxics

Article
Prevalence of Microplastics in the Eastern Oyster Crassostrea
virginica in the Chesapeake Bay: The Impact of Different
Digestion Methods on Microplastic Properties
Thet Aung 1,2 , Inayat Batish 1,2 and Reza Ovissipour 1,2,3, *

1 Department of Food Science and Technology, Virginia Tech, Blacksburg, VA 24061, USA; [email protected] (T.A.);
[email protected] (I.B.)
2 FutureFoods Lab and Cellular Agriculture Initiative, Seafood Agricultural Research and Extension Center,
Hampton, VA 23669, USA
3 Center for Coastal Studies, Virginia Tech, Blacksburg, VA 24060, USA
* Correspondence: [email protected]; Tel.: +1-757-727-4861

Abstract: This study aimed to determine the microplastic prevalence in eastern oysters (C. virginica)
in three sites in the Chesapeake Bay in Virginia and optimize the digestion methods. The digestion
results illustrate that the lowest recovery rate and digestion recovery were related to enzymatic,
enzymatic + hydrogen peroxide (H2 O2 ), and HCl 5% treatments, while the highest digestion recovery
and recovery rate were observed in H2 O2 and basic (KOH) treatments. Nitric acid digestion resulted



in satisfying digestion recovery (100%), while no blue polyethylene microplastics were observed due

 to the poor recovery rate. In addition, nitric acid altered the color, changed the Raman spectrum
Citation: Aung, T.; Batish, I.; intensity, and melted polypropylene (PP) and polyethylene terephthalate (PET). In order to determine
Ovissipour, R. Prevalence of
the number of microplastics, 144 oysters with an approximately similar size and weight from three
Microplastics in the Eastern Oyster
sites, including the James River, York River, and Eastern Shore, were evaluated. Fragments were the
Crassostrea virginica in the
most abundant microplastics among the different microplastics, followed by fibers and beads, in the
Chesapeake Bay: The Impact of
three sites. A significantly higher number of fragments were found in the James River, probably due
Different Digestion Methods on
Microplastic Properties. Toxics 2022,
to the greater amount of human activities. The number of microplastics per gram of oyster tissue was
10, 29. https://doi.org/10.3390/ higher in the James River, with 7 MPs/g tissue, than in the York River and Eastern Shore, with 6.7
toxics10010029 and 5.6 MPs/g tissue.

Academic Editors: Monia Renzi,

Keywords: microplastics; bivalves; isolation; Chesapeake Bay; Raman spectroscopy
Cristiana Guerranti, Manuela
Piccardo, Francesca Provenza and
Andrea Broccoli

Received: 14 December 2021 1. Introduction

Accepted: 6 January 2022
Global plastic production has increased in recent decades from 1.9 tons in 1950 to
Published: 10 January 2022
368 million tons in 2019 [1]. Approximately 269,000 tons of plastic is floating on the
Publisher’s Note: MDPI stays neutral ocean surface, equivalent to 5.25 trillion plastic particles [2], with an abundance of 103 to
with regard to jurisdictional claims in 105 particles per m3 [3] or 0.001 to 0.1 particles per mL [4]. The current estimates of total
published maps and institutional affil- plastic in the world’s oceans may be underestimated since 50% of the plastics are negatively
iations. buoyant and, as a result, may sink to the bottom of the ocean [2].
A wide range of plastic polymers, with different shapes (e.g., spheres, fiber, film,
irregular) and different densities, have been detected in the ocean and marine organ-
isms, comprising polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET),
Copyright: © 2022 by the authors.
polyvinylchloride (PVC), polyester, polystyrene, and polyamide [5–10]. Different densities
Licensee MDPI, Basel, Switzerland.
make microplastics (MPs) across the water column, from the surface to the bottom of the
This article is an open access article
ocean, impact MP availability in marine organisms. Every marine organism tested to date
distributed under the terms and
has been shown to ingest MPs [11], and in many cases, translocation of MPs from the
conditions of the Creative Commons
Attribution (CC BY) license (https://
digestive tract to other organs has been reported [11].
creativecommons.org/licenses/by/
When plastics enter the ocean, the degradation is dependent on the polymer composi-
4.0/).
tion, shape, and density of the plastics in combination with the environmental conditions

Toxics 2022, 10, 29. https://doi.org/10.3390/toxics10010029 https://www.mdpi.com/journal/toxics

Toxics 2022, 10, 29 2 of 11

such as weathering, temperature, irradiation, and pH [12]. Plastic particles contaminating

the environment can negatively impact the ecosystem by influencing the food web, from
microorganisms to marine mammals, easily bioaccumulated and transferred via food to
humans [13]. Large plastic particles are often found in the digestive tracts of marine ver-
tebrates or birds, while smaller particles such as MPs can be translocated to the circular
system or into surrounding tissue [11]. Ingested MPs by more than 140 different species of
marine organisms showed negative impacts, including DNA damage in clams [14], delayed
larvae development in oysters [15], valve closure in mussels [16], transcriptional alterna-
tion in mussels [17], and reduced energy budget in crabs [18]. Among marine organisms,
filter-feeding bivalves are exposed to MPs to a greater extent since they are sessile and
non-selectively filter water, resulting in bioaccumulation of MPs, having a negative impact
on their performance, reducing production yield in aquaculture and fisheries, negatively
impacting rural and coastal communities, and possibly serving as a vehicle for the transfer
of MPs to humans.
Aquaculture and fisheries are rapidly growing industries, and their production has
almost doubled in the last ten years, with 179 million tons of seafood produced in 2018 and
additional increases in production anticipated in the future [19]. In the U.S., the annual per
capita consumption of seafood products is around 19.2 kg and is projected to approach
22.5 kg by 2030 [19]. Oysters are the top marine aquaculture product, which justifies a thor-
ough investigation of how MPs can negatively impact the oyster industry. Recent research
showed that oysters could ingest MPs, and after a two-month exposure to polystyrene
pellets, the oysters exhibited a significant decrease in sperm velocity and quantity, size of
oocytes, and larvae development [15]. This study demonstrates the potential impact of MPs
on oyster production and the importance of assessing MP prevalence in oysters. Multiple
studies have examined the type and abundance of MPs in field-collected organisms [20–23].
However, to the best of our knowledge, there is only one study on the prevalence of MPs
in oysters in Florida [23] and one study on oysters in Georgia [24]. In the Chesapeake
Bay, there are only a few studies on MPs in the water [25,26]. Yonkos et al. [25] studied
the abundance of MPs in water samples in four estuarine rivers in the Chesapeake Bay in
Maryland, and Yanez et al. [26] evaluated the number of MPs in the water in the James
River and York River. However, there is no report on the prevalence of MPs in oysters
in the Chesapeake Bay. Considering that MPs might influence the oyster industry, more
research is required for understanding the abundance of MPs.
Quantifying MPs in seafood requires an efficient isolation step to remove all the organic
materials and soft tissues without affecting the polymer integrity [27]. Four different diges-
tion methods have been used for eliminating organic materials including acids [21,28–30],
bases [30–33], oxidative agents [30,34,35], and enzymes [36,37]. Most of these methods
are corrosive, affecting the MP structure, expensive, and time consuming. For example,
many researchers found that nitric acid can damage the polymers and, in many cases,
melt them, resulting in underestimating MPs in marine organisms. Thus, this study aimed
to evaluate the influence of different digestion methods on standard MP recovery and
chemical structures. In addition, in this study, we determined the prevalence of MPs in
oysters from three sites in the Chesapeake Bay in Virginia for the first time.

2. Materials and Methods

2.1. Experiment 1: Optimizing the Digestion Method
2.1.1. Selecting the Digestion Method
During the entire experiment, to prevent any cross-contact, all the solvents were
filtered using 0.45 um filter paper, and all the glassware was washed with 1 M nitric
acid and rinsed three times with deionized water, then washed with 70% ethanol and
dried in an oven, and then covered with aluminum foil. Six digestion approaches were
used for removing the organic tissue from 10 oysters for each approach to collect the blue
polyethylene microplastics (300–355 µm) to determine the digestion efficiency and recovery
rate. Approach 1 was selected based on enzymatic hydrolysis, using 3% Alcalase at 60 ◦ C
Toxics 2022, 10, 29 3 of 11

for 48 h. Approach 2 was based on Approach 1, followed by hydrogen peroxide 30% and
incubation for 24 h at 60 ◦ C. Approach 3 was based on using 30% hydrogen peroxide at
the ratio of 40:1 and digesting the samples for 24 h at 65 ◦ C, continued by 48 h digestion at
room temperature, and adding 30 ppt sodium chloride and maintaining samples at room
temperature for another 24 h. Approach 4 was selected based on the conventional digestion
method using 69% nitric acid and incubation at 60 ◦ C for 24 h. Approach 5 was based
on using HCl 5% at 60 ◦ C for 24 h. Approach 6 was selected based on using KOH 10%
and incubation at 60 ◦ C for 24 h. All the samples were filtered after digestion, using 0.45
filter paper via a vacuum filter. Filters were dried at room temperature under the hood
for 3 h. Digestion efficiency (%) was calculated using the following equation according to
Karami et al. [27]:

Digestion efficiency (%) = (Wi − (Wa − Wb))/Wi × 100

where Wi is the initial weight of the biological materials, Wa is the weight of the dry filter
membrane after filtration, and Wb is the weight of the dry filter membrane before filtration.
Karami et al. [27] set more than 95% digestion efficiency as the acceptable threshold to
reduce the optical examination of MPs by organic materials left from the digestion.
To determine the proper digestion method with the highest recovery rate, 5 g of oyster
soft tissue was mixed with blue polyethylene microplastics (300–355 µm, Cospheric LLC.,
Goleta, CA, USA) at a rate of 20 microplastics per gram of oyster tissue (total of 100 MPs
per sample) in triplicates (n = 3). Oysters were obtained from local stores for experiment 1.
The MP recovery rate was calculated based on the number of microplastics added to the
raw materials and the number of MPs recovered after digestion and filtration.

2.1.2. The Impact of Digestion Methods on Plastic Chemical Structure

Raman spectra of the intact plastics (control), including polypropylene (PP), polyethy-
lene terephthalate (PET), and polystyrene (PS), before and after exposure to different
digestion methods (Approaches 1–6), were collected in the range of 2500–500 cm cm−1
using a DXR2 microscopy Raman spectrometer (Thermo Fisher Scientific Inc., Waltham,
MA, USA) equipped with a 785 nm diode laser source.

2.2. Experiment 2: Microplastic Prevalence in Oysters from the Chesapeake Bay

2.2.1. Sampling Sites
Samples for this study were collected from the Chesapeake Bay area in Virginia from
three different locations (Figure 1) with a salinity range of 10 to 25 ppt during the summer
of 2020. We selected the Chesapeake Bay watershed due to the degraded stormwater
runoff, rainfall, groundwater, and water from canals from nearby urban and suburban
areas, as well as the sea level rise and high-tide flooding, which has critically polluted the
watershed [38,39]. After collecting the samples from the sites, oysters were placed into
coolers with ice and transferred to the lab within 2 h.
Toxics 2022,10,
Toxics2022, 9, 29
x FOR PEER REVIEW 44 of 11
11

Figure1.1.Sampling
Figure Samplingsites
sitesin
inthe
theChesapeake
ChesapeakeBay
Bayin
inVirginia.
Virginia.Site
Site1:1: Eastern
Eastern Shore;
Shore;Site
Site2:
2: York
YorkRiver;
River; Site 3: James River.
Site 3: James River.

2.2.2. Sample
2.2.2. Sample Preparation
Preparation
Uponarrival
Upon arrivalininthe
the lab,
lab, whole
whole livelive oysters
oysters werewere
placedplaced
in zipinlockzipbags
lockand
bagswereandfrozen
were
frozen
for 24 hfor 24 hprocessing.
before before processing.
Oysters Oysters
were thawed were atthawed
room at room temperature
temperature for furtherforstudies,
further
studies,
shell lengthshell
andlength
widthandwere width were measured,
measured, and afterthe
and after removing removing
soft tissue,thethe
softweight
tissue,was the
weight was
recorded recorded
before before the
the digestion digestion
process. Before process. Before the
the digestion digestion
process, 500 mLprocess, 500 mL
Erlenmeyer
flasks were washed
Erlenmeyer flasks werethree timesthree
washed withtimes
deionized water, previously
with deionized filtered with
water, previously filtered0.2 with
µm
nitrocellulose membrane filter paper using vacuum filtration. Each
0.2 µ m nitrocellulose membrane filter paper using vacuum filtration. Each oyster soft tis- oyster soft tissue was
cut
sueinto
wassmall
cut intopieces
small and thenand
pieces placed
theninto
placedthe into
500 the
mL 500
flask.
mLSamples were digested
flask. Samples were digestedusing
the chemical
using digestion
the chemical method method
digestion according to Li et al.to[40]
according and
Li et al.the
[40]NOAA
and thestandard
NOAAprocedure
standard
for marine debris
procedure [41]. debris
for marine First, 200
[41].µL of 0.1%
First, 200 Tween 20 and
µ L of 0.1% 30%20
Tween hydrogen
and 30%peroxide
hydrogen at per-
the
ratio
oxideofat40:1
the(200
ratiomL:5 g) (200
of 40:1 was added.
mL:5 g)Then, flasks were
was added. Then,incubated
flasks were in incubated
a shaking incubator
in a shaking at
65 ◦ C for 24 h, followed by maintaining them at room temperature for another 48 h. Then,
incubator at 65 °C for 24 h, followed by maintaining them at room temperature for another
30
48gh.sodium
Then, 30 chloride
g sodiumwaschloride
added towas each flask,to
added and flasks
each were
flask, andkept at room
flasks temperature
were kept at room
for another 24 h. The top solution was filtered using a 0.45 µm
temperature for another 24 h. The top solution was filtered using a 0.45 µ m filter filter and vacuum filtration.
and
vacuumIn total, 144 live oyster samples were collected from three sites with no significant
filtration.
differences
In total,in 144
soft live
tissue, shellsamples
oyster length, were
and shell widthfrom
collected (p <three
0.05).sites
Mean shell
with nolengths
significantfor
sites
differences in soft tissue, shell length, and shell width (p < 0.05). Mean shell lengths for
1, 2, and 3 were 8.9, 9.2, and 9.3 cm, respectively. The mean weight of soft tissue for
sites
sites1,1,2,2,and
and33waswere 18.6,
8.9,19.4, and 9.3
9.2, and 20 g,
cm,respectively
respectively.(Table
The1).mean weight of soft tissue for
sites 1, 2, and 3 was 18.6, 19.4, and 20 g, respectively (Table 1).
Table 1. Number of oysters collected from each site, and biometric information.
Table 1. Number of oysters collected from each site, and biometric information.
Mean Weight of Soft
Site Number of Oysters Mean Shell Length (cm)
Tissue
Mean(g)Weight of Soft Mean Shell Length
Site Number of Oysters a a
1 58 18.6 ± 2.1Tissue (g) 8.9 ± 1.6
(cm)
2 1 47 58 a ± 2.1 a
19.4 ± 2.118.6 9.2 ±8.91.2±a1.6 a
3 2 39 47 20 ± 3.419.4
a ± 2.1 a 9.3 ±9.21.18± a1.2 a
3 ± sd. Values in the same39
Values are mean 20 ±are
column with different letter 3.4significantly different
a 9.3(p±<1.18
0.05).a
Values are mean ± sd. Values in the same column with different letter are significantly different (p
< 0.05).
2.3. Data Analysis
The results are presented as the mean of the replicates ± standard deviation. The
2.3. Data Analysis
significance of the differences was determined with one-way analysis of variance (p < 0.05).
Raman The resultswere
spectra are presented as theby
pre-processed mean of thebaseline
applying replicates ± standardnormalization,
correction, deviation. Theand
sig-
nificance of the differences was determined with ®
one-way analysis of variance
smoothing to reduce the noise using Unscrambler X software (version 10.5) (CAMO (p < 0.05).
Raman spectra were pre-processed by applying baseline correction, normalization, and
Toxics 2022, 10, 29 5 of 11

Software, Oslo, Norway). Microplastic color and size were measured using a Nikon Eclipse
Ci microscope (Melville, NY, USA), and ImageJ software.

3. Results
3.1. Experiment I: Optimizing the Digestion Methods
This experiment was conducted to evaluate the impact of six digestion approaches
on the digestion efficiency and recovery rate of standard blue polyethylene (300–355 µm),
and the chemical structure of three plastics, including PP, PET, and PS (Table 2, Figure 2).
The results illustrate that treatment with H2 O2 , KOH, and HNO3 had the highest digestion
efficiency, meaning that the organic tissues were digested completely (p < 0.05). Meanwhile,
enzymatic hydrolysis, enzymatic-H2 O2 , and HCl 5% treatments showed a low digestion
efficiency. The recovery rate for the blue PE microplastics was satisfying for H2 O2 and
KOH, while for HNO3 , due to its strong acidity, the blue PE microplastics were melted
completely. Strong acidic and basic solutions can digest organic tissue by degrading
proteins, carbohydrates, and fats [35]. However, lower concentrated acids such as 5%
HCl are not suitable for digesting a high amount of organic materials. Neulle et al. [34]
and Karami et al. [27] also reported poor digestibility of tissues using 5% and 20% HCl
solutions, respectively. Previous studies also reported that digesting bivalves in 30% H2 O2
at 60–65 ◦ C for 24 h followed by incubation at room temperature for 48 h resulted in
complete digestion of soft tissues [27,40,42]. Von Friesena et al. [37] developed an efficient
enzymatic digestion method with a high recovery rate (87%) and digestion efficiency (97%)
for bivalve tissue, which is in contrast to our findings. This could be explained by the
fact that we used the Alcalase enzyme, a proteinase enzyme, while the other researchers
used pancreatic enzymes consisting of amylase, lipase, and proteinase, which can digest
the whole tissue [37]. We also observed that the enzyme, enzyme-H2 O2 , and 5% HCl
treatments, which partially digested the oysters, resulted in foam formation and clogged
the filters, reducing the microplastics’ recovery and characterization. We selected Alcalase
based on the fact that oysters have a higher protein content, and low carbohydrate content.
However, Alcalase alone did not digest the oyster tissue completely.

Table 2. The impact of digestion approaches on digestion efficiency, recovery rate, and morphological
changes.

Standard Microplastics
Morphological Changes in Plastics
Digestion (Polyethylene) (300–355 µm)
Approach Digestion
Recovery Rate PP PET PS
Efficiency
Enzyme 57 ± 4 b 38 ± 5 b - - -
Enzyme + H2 O2
62 ± 3 b 35 ± 8 b - - -
(30%)
H2 O2 (30%) 100 ± 1.23 a 92 ± 6 a - - -
HNO3 (69%) 100 ± 0 a 0 Melted Melted Altered the color
48.1 ± 0.2 c b
HCl (5%) 42 ± 11 Altered the color Altered the color Altered the color
Formed opaque Formed opaque Formed opaque
KOH (10%) 100 ± 0.4 a 96 ± 4 a
color color color
Values are mean ± sd. Values in the same column with different letters are significantly different (p < 0.05). For
Enzyme, Enzyme+H2 O2 and H2 O2 treatments (-) means no changes were observed.
 group [44]. For PS, the peak intensity around 1004 cm–1, which is related to the ring breath-
ing mode, was increased after exposure to the chemicals, which might be due to the deg-
radation of the polymer structure and rearrangement and aggregation of the polymer
chains [27]. In addition, in PS treated with HNO3, the peak at 1350 cm–1 was very sharp,
and the intensity was higher than the control and treated samples, which is due to the CH
Toxics 2022, 10, 29 6 of 11
deformation.

Figure 2.
Figure Raman spectra
2. Raman spectra of
of (a)
(a) PET,
PET, (b)
(b) PS,
PS, and
and (c)
(c) PP
PP exposed
exposed to
to different
different chemicals.
chemicals.

We also applied six digestion approaches to three plastics, including PP, PET, and PS,
3.2. Experiment II: Prevalence of MPs in Oyster Samples
and characterized them using morphological changes and chemical structure changes via
Raman Theconfocal
numbermicroscopy
of microplastics
(Tablewas notFigure
2 and significantly
2). different among the tested sites (p
> 0.05). The average number of microplastics in each oyster
The results of the Raman spectra analysis of untreated and from the Jamestreated
chemically River (Site 3)
plastic
materials illustrate that the chemicals caused changes in the spectrum peaks (Figure 2).
These changes in peak intensity and band shifting might be due to polymer molecular
alteration because of the chemical digestion [35]. Peak intensities around 810, 844, 976,
1152, 1171, 1131, 1438, and 1460 cm−1 were reduced significantly after chemical treatments
in PP. Thiele et al. [33] found that the PP Raman spectrum peak intensity was significantly
reduced after chemical treatments. The peak intensity around 1460 cm−1 , which is assigned
to asymmetric bending of the CH3 group, was significantly reduced after exposing PP to the
chemicals, particularly HNO3 . Karami et al. [27] also found that the Raman peak intensities
were reduced for PP treated with HNO3 . For PET, the chemical treatments reduced the
peak intensity for the ring C = C stretching at 1610 cm−1 and C = O stretching band around
1722 cm−1 , which is in agreement with other studies [27,42,43]. This reduction might be
related to the depolymerization of the polymer structure compared to the control group [44].
For PS, the peak intensity around 1004 cm−1 , which is related to the ring breathing mode,
was increased after exposure to the chemicals, which might be due to the degradation of
the polymer structure and rearrangement and aggregation of the polymer chains [27]. In
addition, in PS treated with HNO3 , the peak at 1350 cm−1 was very sharp, and the intensity
was higher than the control and treated samples, which is due to the CH deformation.

3.2. Experiment II: Prevalence of MPs in Oyster Samples

The number of microplastics was not significantly different among the tested sites
(p > 0.05). The average number of microplastics in each oyster from the James River (Site 3)
Toxics 2022, 10, 29 7 of 11

was 140 microplastics per oyster, followed by the York River (Site 2) with 128 microplastics
per oyster. The Eastern Shore (Site 1) had the lowest number of microplastics (104 per
oyster). The highest number of microplastics per gram of soft tissue was found for the
James River (7 MPs/g tissue), followed by the York River (6.7 MPs/g tissue) and Eastern
Shore (5.6 MPs/g tissue). In all sampling locations, the highest number of microplastics
was related to fragments (80–88%), followed by fibers (9–12%) and beads (2–6%) (p < 0.05).
The most common colors were black, white, and transparent (Table 3). MPs found in this
study had different size ranges, including 110–2300 µm for microfibers, 14–1280 µm for
fragments, and 6–282 µm for beads.

Table 3. The number of microplastic types and total MPs per gram of tissue for each site.

Site Fragment Fiber Bead

1 84 ± 18 a 13 ± 6 a 7±4a
2 108 ± 23 a 13 ± 6 a 7±5a
3 123 ± 32 a 14 ± 6 a 3±4a
Values are mean ± sd. Values in the same column with different letter are significantly different (p < 0.05).

The results of this study indicate the abundance of microplastics in oyster samples
which were collected from three sites in the Chesapeake Bay in Virginia. Our results show
that fragment microplastics were the most abundant microplastics in the oysters from the
Chesapeake Bay. This is in contrast to studies that have found microfibers are the most
abundant plastics in oyster tissue [23,24,32,45]. However, few studies have reported that
fragment microplastics are the main microplastics in oysters [40,46,47]. Different factors
could explain this, including the difference in species, filtering rate, sampling location,
filtration, and instrumentation [47]. For example, Scircle et al. [47] used fluorescence
microscopy targeting larger microplastics, and Rochman et al. [32] applied µFT-IR analysis
for microplastics in bivalves and found that the most abundant microplastics are fragments.
It has been widely shown that bead and fragment particles are most likely removed from
the digestive tract. Our results suggest that fragment microplastics can also accumulate at
a high concentration in oysters, which is in agreement with other studies [40]. In another
study, samples from three rivers (Stroubles Creek, Roanoke River, and James River) in
Virginia indicated that the most abundant microplastics are fragments (80%), followed by
beads (15%) and fibers (5%) [48].
The average number of microplastic particles detected in oysters from three sites in
the Chesapeake Bay in Virginia, with an average soft tissue weight of 18.6 to 20 g, was
between 104 to 140 particles per oyster (5.6–7 particles per gram of soft tissue), which
was higher than the microplastic particles found in other bivalves. Oysters (C. virginica)
collected from a Florida estuary had 16.5 microplastic particles per oyster [23]. Others
also reported lower numbers of microplastic particles in bivalves: 2 microplastics per
oyster (C. gigas) (0.47 particles/g) [28], 0.2–0.3 particles per gram in C. gigas [29], and
0.18 MPs per gram of oyster (C. virginica) in Georgia [24]. While most studies showed lower
numbers of microplastics in other bivalves, 10–29 particles per gram of tissue in oyster (C.
gigas) in Germany [49], 34–178 particles per blue mussel (Mytilus edulis) in Canada [50],
2.1–10.5 particles per gram of nine different bivalves from a fishery market in China [40],
and 1.5–7.2 particles per gram of tissue in oysters from the Pearl River Estuary in China [51]
have been reported, indicating higher numbers of microplastics in some regions. Oysters
in our study were also larger compared to those in other studies and had larger gills
and lip pulps, which may result in the possibility of taking up more MPs [52]. Among
marine organisms, filter-feeding bivalves are exposed to MPs to a greater extent since they
are sessile and non-selectively filter water, resulting in bioaccumulation of MPs, having
a negative impact on their performance, reducing production yield in aquaculture and
fisheries, negatively impacting rural and coastal communities, and possibly serving as a
vehicle for the transfer of MPs to humans. Recent research has shown that oysters could
Toxics 2022, 10, 29 8 of 11

ingest MPs, and after intake, microplastics can attach to the guts, gills, and tissues, reducing
energy uptake and impairing muscle function and reproduction [14,15,53]. Microplastics
may also sorb harmful contaminants that, once ingested or incorporated in tissues, are
released into the organism [54]. In some studies, environmental MP concentrations have
been directly correlated with microplastic burdens in coastal bivalves [42,50].
The knowledge about microplastic quantification and distribution in the Chesapeake
Bay is very limited to only a few studies [25,26,55]. The current study evaluated the
prevalence of microplastics in oysters in the Chesapeake Bay in Virginia, which is the third
largest oyster producer in the U.S. MP pollution has been reported in areas with a higher
urban density, more commercial fishing, higher industrial waste discharge, more sewer
overflows, more wastewater treatment plants, and more shipping ports [25,26,47]. The
abundance of MPs in the surface water of the James River was 700–9000 MPs/l, and in the
York River, it was 1400 to 15000 MPs/L, mainly due to the wastewater treatment plants
near both rivers [26]. The MP concentration is also influenced by wind, rain, and extreme
meteorological conditions such as hurricanes and floods, resulting in MP transfer from
terrestrial environments to the sea [20,25,56–59]. For example, Moore et al. [58] reported
that the amount of surface plastic debris with less than a 4.75 mm diameter in California
surface waters near the Los Angeles stormwater system was six times higher after a storm.
Heavy rains increase the amount of plastic debris entering coastal regions, and strong
winds during hurricanes result in wave action, creating vertical mixing within the water
column which can resuspend plastics [25]. Yonkos et al. [25] also observed a microplastic
peak in September after Hurricane Irene and Tropical Storm Lee in the Chesapeake Bay.
More studies on the prevalence of microplastics in the water, oysters, clams, crabs, and
sediments from different locations of the Chesapeake Bay during different seasons are
required to provide a deep understanding of the MP patterns in this region.

4. Conclusions
The presence of MPs in the water, sediments, and seafood is an emerging concern.
To evaluate the prevalence of MPs in seafood products, employing a proper digestion
method to remove organic materials without negatively influencing MP chemical and
physical properties is critical. In the current study, we evaluated the impact of six different
digestion approaches on the digestion efficiency and MP recovery rate using standard
blue PE MPs and found that H2 O2 and KOH had the highest digestion efficiency and
MP recovery. Meanwhile, HNO3 digested 100% of the organic tissue, but it melted all
the blue PE MPs. Enzymatic, enzymatic-H2 O2 , and 5% HCl treatments resulted in poor
digestion and MP recovery. In addition, our results from exposing PP, PET, and PS to these
six approaches indicate that all the treatments could alter the chemical structure of the
polymers based on Raman spectroscopy. Due to the high cost of H2 O2 , and its negative
impact on the plastic chemical structure, KOH would be a suitable option for digesting
organic tissues. In addition, our results indicate high amounts of MPs in oysters collected
from three locations in the Chesapeake Bay in Virginia. MP fragments were the most
abundant particles, followed by fibers and beads. More studies are needed to determine
the MP prevalence in the water, sediments, oysters, and other organisms in the Chesapeake
Bay. The collection of more samples in different seasons is also required due to different
water inflow patterns, hurricane seasons, and water runoff into the bay.

Author Contributions: Conceptualization, R.O.; methodology, R.O., T.A., I.B.; investigation, R.O.,
T.A., I.B.; resources, R.O.; data curation, R.O., T.A., I.B.; writing—original draft, R.O., T.A., I.B.;
visualization, R.O.; supervision, R.O.; project administration, R.O.; funding acquisition, R.O. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was partially supported by the Center for Coastal Studies and the College of
Agriculture and Life Sciences at Virginia Tech.
Informed Consent Statement: Not applicable.
Toxics 2022, 10, 29 9 of 11

Data Availability Statement: The data are not publicly available due to protection of subjects’ privacy
and confidentiality. The data presented in this study are available on request from the corresponding
author.
Acknowledgments: We would like to express our sincere thanks to Steve Urick, Ethan McAlhaney,
and Lexi Duscher for their support.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. PlasticsEurope Plastics-the Facts 2020. An Analysis of European Plastics Production, Demand and Waste Data. 2020, p. 44.
Available online: http//www.Plast.org (accessed on 20 December 2021).
2. Seltenrich, N. Erratum: “New Link in the Food Chain? Marine Plastic Pollution and Seafood Safety”. Environ. Health Perspect.
2016, 124, A123. [CrossRef]
3. Andrady, A.L. The plastic in microplastics: A review. Mar. Pollut. Bull. 2017, 119, 12–22. [CrossRef]
4. Woods, M.N.; Stack, M.E.; Fields, D.M.; Shaw, S.D.; Matrai, P.A. Microplastic fiber uptake, ingestion, and egestion rates in the
blue mussel (Mytilus edulis). Mar. Pollut. Bull. 2018, 137, 638–645. [CrossRef]
5. Lo Brutto, S.; Iaciofano, D.; Lo Turco, V.; Potortì, A.G.; Rando, R.; Arizza, V.; Di Stefano, V. First assessment of plasticizers in
marine coastal litter-feeder fauna in the Mediterranean Sea. Toxics 2021, 9, 31. [CrossRef]
6. Nobre, C.R.; Santana, M.F.M.; Maluf, A.; Cortez, F.S.; Cesar, A.; Pereira, C.D.S.; Turra, A. Assessment of microplastic toxicity to
embryonic development of the sea urchin Lytechinus variegatus (Echinodermata: Echinoidea). Mar. Pollut. Bull. 2015, 92, 99–104.
[CrossRef] [PubMed]
7. Kang, J.-H.; Kwon, O.Y.; Lee, K.-W.; Song, Y.K.; Shim, W.J. Marine neustonic microplastics around the southeastern coast of Korea.
Mar. Pollut. Bull. 2015, 96, 304–312. [CrossRef]
8. Lusher, A.L.; Hernandez-Milian, G.; O’Brien, J.; Berrow, S.; O’Connor, I.; Officer, R. Microplastic and macroplastic ingestion by
a deep diving, oceanic cetacean: The True’s beaked whale Mesoplodon mirus. Environ. Pollut. 2015, 199, 185–191. [CrossRef]
[PubMed]
9. Cole, M.; Lindeque, P.; Halsband, C.; Galloway, T.S. Microplastics as contaminants in the marine environment: A review. Mar.
Pollut. Bull. 2011, 62, 2588–2597. [CrossRef] [PubMed]
10. Bikker, J.; Lawson, J.; Wilson, S.; Rochman, C.M. Microplastics and other anthropogenic particles in the surface waters of the
Chesapeake Bay. Mar. Pollut. Bull. 2020, 156, 111257. [CrossRef]
11. Smith, M.; Love, D.C.; Rochman, C.M.; Neff, R.A. Microplastics in Seafood and the Implications for Human Health. Curr. Environ.
Health Rep. 2018, 5, 375–386. [CrossRef]
12. Akbay, I.K.; Özdemir, T. Monomer migration and degradation of polycarbonate via UV-C irradiation within aquatic and
atmospheric environments. J. Macromol. Sci. Part A 2016, 53, 340–345. [CrossRef]
13. Lusher, A.; Hollman, P.; Mendoza-Hill, J. Microplastics in Fisheries and Aquaculture: Status of Knowledge on Their Occurrence and
Implications for Aquatic Organisms and Food Safety; FAO: Rome, Italy, 2017.
14. Ribeiro, F.; Garcia, A.R.; Pereira, B.P.; Fonseca, M.; Mestre, N.C.; Fonseca, T.G.; Ilharco, L.M.; Bebianno, M.J. Microplastics effects
in Scrobicularia plana. Mar. Pollut. Bull. 2017, 122, 379–391. [CrossRef] [PubMed]
15. Sussarellu, R.; Suquet, M.; Thomas, Y.; Lambert, C.; Fabioux, C.; Arsenault-Pernet, E.-J.; Le Goïc, N.; Quillien, V.; Mingant, C.;
Epelboin, Y.; et al. Oyster reproduction is affected by exposure to polystyrene microplastics. Proc. Natl. Acad. Sci. USA 2016, 113,
2430–2435. [CrossRef] [PubMed]
16. Wegner, A.; Besseling, E.; Foekema, E.M.; Kamermans, P.; Koelmans, A.A. Effects of nanopolystyrene on the feeding behavior of
the blue mussel (Mytilus edulis L.). Environ. Toxicol. Chem. 2012, 31, 2490–2497. [CrossRef]
17. Capolupo, M.; Franzellitti, S.; Valbonesi, P.; Lanzas, C.S.; Fabbri, E. Uptake and transcriptional effects of polystyrene microplastics
in larval stages of the Mediterranean mussel Mytilus galloprovincialis. Environ. Pollut. 2018, 241, 1038–1047. [CrossRef]
18. Watts, A.J.R.; Urbina, M.A.; Corr, S.; Lewis, C.; Galloway, T.S. Ingestion of Plastic Microfibers by the Crab Carcinus maenas and
Its Effect on Food Consumption and Energy Balance. Environ. Sci. Technol. 2015, 49, 14597–14604. [CrossRef]
19. FAO. The State of World Fisheries and Aquaculture 2020; Food & Agriculture Organisation (FAO): Rome, Italy, 2020; ISBN 978-92-5-
1132692-3.
20. Collignon, A.; Hecq, J.-H.; Galgani, F.; Voisin, P.; Collard, F.; Goffart, A. Neustonic microplastic and zooplankton in the North
Western Mediterranean Sea. Mar. Pollut. Bull. 2012, 64, 861–864. [CrossRef]
21. De Witte, B.; Devriese, L.; Bekaert, K.; Hoffman, S.; Vandermeersch, G.; Cooreman, K.; Robbens, J. Quality assessment of the
blue mussel (Mytilus edulis): Comparison between commercial and wild types. Mar. Pollut. Bull. 2014, 85, 146–155. [CrossRef]
[PubMed]
22. Hämer, J.; Gutow, L.; Köhler, A.; Saborowski, R. Fate of Microplastics in the Marine Isopod Idotea emarginata. Environ. Sci.
Technol. 2014, 48, 13451–13458. [CrossRef]
23. Waite, H.R.; Donnelly, M.J.; Walters, L.J. Quantity and types of microplastics in the organic tissues of the eastern oyster Crassostrea
virginica and Atlantic mud crab Panopeus herbstii from a Florida estuary. Mar. Pollut. Bull. 2018, 129, 179–185. [CrossRef]
Toxics 2022, 10, 29 10 of 11

24. Keisling, C.; Harris, R.D.; Blaze, J.; Coffin, J.; Byers, J.E. Low concentrations and low spatial variability of marine microplastics in
oysters (Crassostrea virginica) in a rural Georgia estuary. Mar. Pollut. Bull. 2020, 150, 110672. [CrossRef]
25. Yonkos, L.T.; Friedel, E.A.; Perez-Reyes, A.C.; Ghosal, S.; Arthur, C.D. Microplastics in Four Estuarine Rivers in the Chesapeake
Bay, U.S.A. Environ. Sci. Technol. 2014, 48, 14195–14202. [CrossRef]
26. Yañez, C.; Fortin, S.; Song, B. Quantification and identification of microplastics in James River and York River, two tributaries of
Chesapeake Bay. In Proceedings of the Ocean Sciences Meeting 2020, AGU, San Diego, CA, USA, 16–21 February 2020.
27. Karami, A.; Golieskardi, A.; Choo, C.K.; Romano, N.; Ho, Y.B.; Salamatinia, B. A high-performance protocol for extraction of
microplastics in fish. Sci. Total Environ. 2017, 578, 485–494. [CrossRef] [PubMed]
28. Van Cauwenberghe, L.; Janssen, C.R. Microplastics in bivalves cultured for human consumption. Environ. Pollut. 2014, 193, 65–70.
[CrossRef] [PubMed]
29. Van Cauwenberghe, L.; Claessens, M.; Vandegehuchte, M.B.; Janssen, C.R. Microplastics are taken up by mussels (Mytilus edulis)
and lugworms (Arenicola marina) living in natural habitats. Environ. Pollut. 2015, 199, 10–17. [CrossRef]
30. Dehaut, A.; Cassone, A.-L.; Frère, L.; Hermabessiere, L.; Himber, C.; Rinnert, E.; Rivière, G.; Lambert, C.; Soudant, P.; Huvet, A.;
et al. Microplastics in seafood: Benchmark protocol for their extraction and characterization. Environ. Pollut. 2016, 215, 223–233.
[CrossRef] [PubMed]
31. Foekema, E.M.; De Gruijter, C.; Mergia, M.T.; Van Franeker, J.A.; Murk, A.J.; Koelmans, A.A. Plastic in North Sea Fish. Environ.
Sci. Technol. 2013, 47, 8818–8824. [CrossRef]
32. Rochman, C.M.; Tahir, A.; Williams, S.L.; Baxa, D.V.; Lam, R.; Miller, J.T.; Teh, F.-C.; Werorilangi, S.; Teh, S.J. Anthropogenic debris
in seafood: Plastic debris and fibers from textiles in fish and bivalves sold for human consumption. Sci. Rep. 2015, 5, 14340.
[CrossRef]
33. Thiele, C.J.; Hudson, M.D.; Russell, A.E. Evaluation of existing methods to extract microplastics from bivalve tissue: Adapted
KOH digestion protocol improves filtration at single-digit pore size. Mar. Pollut. Bull. 2019, 142, 384–393. [CrossRef]
34. Nuelle, M.-T.; Dekiff, J.H.; Remy, D.; Fries, E. A new analytical approach for monitoring microplastics in marine sediments.
Environ. Pollut. 2014, 184, 161–169. [CrossRef]
35. Collard, F.; Gilbert, B.; Eppe, G.; Parmentier, E.; Das, K. Detection of Anthropogenic Particles in Fish Stomachs: An Isolation
Method Adapted to Identification by Raman Spectroscopy. Arch. Environ. Contam. Toxicol. 2015, 69, 331–339. [CrossRef] [PubMed]
36. Cole, M.; Webb, H.; Lindeque, P.K.; Fileman, E.S.; Halsband, C.; Galloway, T.S. Isolation of microplastics in biota-rich seawater
samples and marine organisms. Sci. Rep. 2014, 4, 4528. [CrossRef] [PubMed]
37. Von Friesen, L.W.; Granberg, M.E.; Hassellöv, M.; Gabrielsen, G.W.; Magnusson, K. An efficient and gentle enzymatic digestion
protocol for the extraction of microplastics from bivalve tissue. Mar. Pollut. Bull. 2019, 142, 129–134. [CrossRef]
38. Kim, H.W.; Li, M.-H. Managing stormwater for urban sustainability: An evaluation of local comprehensive plans in the
Chesapeake Bay watershed region. J. Environ. Plan. Manag. 2017, 60, 1702–1725. [CrossRef]
39. Macías-Tapia, A.; Mulholland, M.R.; Selden, C.R.; Loftis, J.D.; Bernhardt, P.W. Effects of tidal flooding on estuarine biogeochem-
istry: Quantifying flood-driven nitrogen inputs in an urban, lower Chesapeake Bay sub-tributary. Water Res. 2021, 60, 1702–1725.
[CrossRef] [PubMed]
40. Li, J.; Yang, D.; Li, L.; Jabeen, K.; Shi, H. Microplastics in commercial bivalves from China. Environ. Pollut. 2015, 207, 190–195.
[CrossRef]
41. Masura, J.; Baker, J.; Foster, G.; Arthur, C. Laboratory Methods for the Analysis of Microplastics in the Marine Environment: Rec-
ommendations for Quantifying Synthetic Particles in Waters and Sediments; NOAA Marine Debris Division: Silver Spring, MD,
USA, 2015.
42. Li, M.; Ebel, B.; Courtès, F.; Guedon, E.; Marc, A. In Situ Infrared Spectroscopy as a PAT Tool of Great Promise for Real-Time Monitoring
of Animal Cell Culture Processes; Austin Publishing Group: Irving, TX, USA, 2016.
43. Veres, M.; Toth, A.; Mohai, M.; Bertóti, I.; Szépvölgyi, J.; Toth, S.; Himics, L.; Koos, M. Two-wavelength Raman study of
poly(ethylene terephthalate) surfaces modified by helium plasma-based ion implantation. Appl. Surf. Sci. 2012, 263, 423–429.
[CrossRef]
44. Al-Sabagh, A.M.; Yehia, F.Z.; Eshaq, G.; Rabie, A.M.; ElMetwally, A.E. Greener routes for recycling of polyethylene terephthalate.
Egypt. J. Pet. 2016, 25, 53–64. [CrossRef]
45. Baechler, B.R.; Granek, E.F.; Hunter, M.V.; Conn, K.E. Microplastic concentrations in two Oregon bivalve species: Spatial, temporal,
and species variability. Limnol. Oceanogr. Lett. 2020, 5, 54–65. [CrossRef]
46. Phuong, N.N.; Poirier, L.; Pham, Q.T.; Lagarde, F.; Zalouk-Vergnoux, A. Factors influencing the microplastic contamination of
bivalves from the French Atlantic coast: Location, season and/or mode of life? Mar. Pollut. Bull. 2018, 129, 664–674. [CrossRef]
47. Scircle, A.; Cizdziel, J.V.; Tisinger, L.; Anumol, T.; Robey, D. Occurrence of Microplastic Pollution at Oyster Reefs and Other
Coastal Sites in the Mississippi Sound, USA: Impacts of Freshwater Inflows from Flooding. Toxics 2020, 8, 35. [CrossRef]
48. Christensen, N.D.; Wisinger, C.E.; Maynard, L.A.; Chauhan, N.; Schubert, J.T.; Czuba, J.A.; Barone, J.R. Transport and characteri-
zation of microplastics in inland waterways. J. Water Process Eng. 2020, 38, 101640. [CrossRef]
49. Leslie, H.A.; Van Velzen, M.J.M.; Vethaak, A.D. Microplastic survey of the Dutch environment—Novel data set of micro-plastics
in North Sea sediments, treated wastewater effluents and marine biota. Inst. Environ. Stud. 2013, 1–30. Available online:
tps://science.vu.nl/en/Images/IVM_report_Microplastic_in_sediment_STP_Biota_2013_tcm296-409860.pdf (accessed on 5
January 2021).
Toxics 2022, 10, 29 11 of 11

50. Mathalon, A.; Hill, P. Microplastic fibers in the intertidal ecosystem surrounding Halifax Harbor, Nova Scotia. Mar. Pollut. Bull.
2014, 81, 69–79. [CrossRef] [PubMed]
51. Li, J.; Green, C.; Reynolds, A.; Shi, H.; Rotchell, J.M. Microplastics in mussels sampled from coastal waters and supermarkets in
the United Kingdom. Environ. Pollut. 2018, 241, 35–44. [CrossRef]
52. Cognie, B.; Barillé, L.; Massé, G.; Beninger, P.G. Selection and processing of large suspended algae in the oyster Crassostrea gigas.
Mar. Ecol. Prog. Ser. 2003, 250, 145–152. [CrossRef]
53. Von Moos, N.; Burkhardt-Holm, P.; Köhler, A. Uptake and Effects of Microplastics on Cells and Tissue of the Blue Mussel Mytilus
edulis L. after an Experimental Exposure. Environ. Sci. Technol. 2012, 46, 11327–11335. [CrossRef] [PubMed]
54. Teuten, E.L.; Saquing, J.M.; Knappe, D.R.U.; Barlaz, M.A.; Jonsson, S.; Björn, A.; Rowland, S.J.; Thompson, R.C.; Galloway, T.S.;
Yamashita, R.; et al. Transport and release of chemicals from plastics to the environment and to wildlife. Philos. Trans. R. Soc. B:
Biol. Sci. 2009, 364, 2027–2045. [CrossRef]
55. Redford, D.P.; Trulli, W.R.; Trulli, H.K. Composition of Floating Debris in Harbours of the United States. Chem. Ecol. 1992, 7,
75–92. [CrossRef]
56. Browne, M.A.; Galloway, T.S.; Thompson, R.C. Spatial Patterns of Plastic Debris along Estuarine Shorelines. Environ. Sci. Technol.
2010, 44, 3404–3409. [CrossRef]
57. Kukulka, T.; Proskurowski, G.; Moretferguson, S.; Meyer, D.W.; Law, K.L. The effect of wind mixing on the vertical distribution of
buoyant plastic debris. Geophys. Res. Lett. 2012, 39. [CrossRef]
58. Moore, C.J.; Lattin, G.L.; Zellers, A.F. Quantity and type of plastic debris flowing from two urban rivers to coastal waters and
beaches of Southern California. Revista de Gestão Costeira Integrada/J. Integr. Coast. Zone Manag. 2011, 11, 65–73. [CrossRef]
59. Thompson, R.C.; Olsen, Y.; Mitchell, R.P.; Davis, A.; Rowland, S.J.; John, A.W.G.; McGonigle, D.; Russell, A.E. Lost at Sea: Where
Is All the Plastic? Science 2004, 304, 838. [CrossRef] [PubMed]