Science of the Total Environment 838 (2022) 155933

Contents lists available at ScienceDirect

Science of the Total Environment

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

Review

Effects of microplastics on physiological performance of marine bivalves,

potential impacts, and enlightening the future based on a comparative study
Robert Mkuye a, Shunlian Gong a, Liqiang Zhao a, Fortunatus Masanja a, Charles Ndandala a, Edna Bubelwa b,
⁎
Chuangye Yang a,c,d,e, Yuewen Deng a,c,d,e,
a
Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China
b
Nyang'hwale District, 352 Geita, Tanzania
c
Guangdong Science and Innovation Center for Pearl Culture, Zhanjiang 524088, China
d
Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Zhanjiang, China
e
Pearl Breeding and Processing Engineering Technology Research Centre of Guangdong Province, Zhanjiang 524088, China

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

• Microplastics have the potential to cause

respiratory inefficiencies.
• SOD from bivalves have the potential to
be used for human medicinal purposes.
• Contaminated bivalves have the potential
to cause biomagnification and bioaccumu-
lation upon ingestion.
• Microplastics affect the rate of respiration
of marine bivalves

A R T I C L E I N F O A B S T R A C T

Editor: Julian Blasco This review aims to explore the effects of microplastics and their corresponding additives on the physiological perfor-
mances of marine bivalves together with their related genes. We identified gaps based on studies that were conducted
Keywords: on other organisms, and we conducted a comparative study on similar and relevant aspects for exploring future poten-
Microplastics tial areas of study and interest. Microplastics are widely dispersed in all forms of media (solid, liquid, and gas). Expo-
Lipid peroxidation
sure to an organism (including humans) is inevitable. However, impacts depend on the concentration of exposure,
Bioacumulation
Physiological parameters
location of a biomarker being observed, and treatment involved. Different shapes, colors, and polymer types are re-
ported and the transfer of microplastics along the food chain are recorded. The impacts of microplastics intensify
when coupled with other chemicals or additives (referred to as xenobiotics) in a treated group. Thus, the degree of in-
hibition or enhancement of a physiological response magnifies when a coexposure of microplastic and a xenobiotic oc-
curs. Microplastics have been observed to reduce immune system functionality by reducing hemocytes count,
distorting oxidative system, respiration, and increasing energy consumption in bivalves due to physiological modula-
tions that result from ingestion of microplastics or their additives. We found knowledge gaps and suggested future re-
search directions to fully understand the impact of microplastics and their additives on marine bivalves.

⁎ Corresponding author at: Guangdong Ocean University, Zhanjiang 524088, China.

E-mail address: [email protected] (Y. Deng).

http://dx.doi.org/10.1016/j.scitotenv.2022.155933
Received 7 February 2022; Received in revised form 17 April 2022; Accepted 10 May 2022
Available online 14 May 2022
0048-9697/© 2022 Elsevier B.V. All rights reserved.
R. Mkuye et al. Science of the Total Environment 838 (2022) 155933

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Literature collection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.1. Transfer, bioaccumulation, and biomagnification of microplastic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.2. MPs composition reported in bivalve species. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.3. Impacts of MPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.3.1. Total haemocytes, composition, and phagocytosis rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.3.2. Reactive oxygen species (ROS), lipid peroxidation (LPO), and deoxyribonucleic acid (DNA) damage − . . . . . . . . . . . . . . . . . 6
3.3.3. LPO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.4. Gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.5. Enzymatic modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.5.1. Glutathione S transferase (GST) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.5.2. Superoxide dismultase (SOD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.5.3. Catalase (CAT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.5.4. Glutathione (GSH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.5.5. Glutathione peroxidase (GPX) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
4. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
CRediT authorship contribution statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Declaration of competing interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1. Introduction However, 65,238,500 MPs are found every day in the final effluent or 23
billion MPs annually.
Plastic production and use have become ubiquitous and widespread in The volume of effluent discharged from WWTPs as reported by studies
everyday human life with an estimated global production of 359 million suggests a high number of MPs discharges daily. For instance, 17 different
tons per year of in 2018 (Facciol et al., 2021; Guzzetti et al., 2018). Plastic WWTPs in the United States (US) have been reported to release 4 × 106
debris are discarded into the environment by general littering, illegal MPs per day per WWTP.The total emission from US WWTPs into aquatic
dumping, and losses of waste from landfill sites, recycling facilities, plastic habitats has been estimated to be 8 trillion microbeads per day, and the
mulching in agriculture, and tire and textile industry (Rebelein et al., United Kingdom (UK) has been estimated to release 65 × 106 MPs every
2021). The plastic debris (macroplastics) acted upon agents of weathering day (Lise et al., 2018; Mason et al., 2016; Murphy et al., 2016; Rochman
such as ultraviolet (UV) radiation, fluctuating temperatures, abrasion on et al., 2015).
beaches, wind, salinity, and physical stress such as wave action; MPs can cause damage chemically, physically, and biologically to or-
(Martinelli et al., 2021; Rebelein et al., 2021) disintegrate to form smaller ganisms that ingest them directly and indirectly through the consumption
mesoplastic (5–25 mm) or microplastics (MPs <5 mm) (Andrady, 2011). of contaminated prey. Estimations show that more than 800 species of ma-
MPs can also be manufactured and they are known as primary MPs. Pri- rine organisms including bivalves, polychaetes, crustaceans, and fish are af-
mary MPs include abrasive micro-beads in face scrubber cosmetics and fected by marine debris. This condition is mostly observed through
toothpaste, synthetic fibers, and pre-production resin pellets, and second- entanglement and ingestion which might cause potential health risks to
ary MPs are generated in situ by the mechanical and oxidative breakdown humans (Cauwenberghe and Janssen, 2014; Cho et al., 2019; Jahani
of larger plastics for specific reasons and by different weathering agents et al., 2019; Savoca et al., 2019; Setälä et al., 2014).
(Abbasi et al., 2018; Qin et al., 2019). Given their physical and chemical Studies have documented the ingestions and translocation of MPs by the
properties, MPs can be transported in water, soil, and air with the occur- mussels (Magni et al., 2018) that are cultured, captured in the wild, or that
rence of exchange between environmental compartments, adsorption of are being exposed to the lab. In the study that involved the exposure of
toxic compounds, and being carried a long distance, with the potential to zebra mussels to polyester, microbeads under lab conditions revealed the
causing physical and chemical harm to a multitude of species including ingestion and translocation of 10 and 1 μm MPs. Larger MPs concentrated
humans (Birch et al., 2020). more in the gut lumen and can pass through the biological barriers, moving
MPs can be ingested by organisms due to their small sizes and enter food to the basal lamina of the gut epithelium and reaching the tissues of the di-
chains that ultimately reach human beings (Savoca et al., 2019). Aquatic gestive gland and into the hemolymph. The study observed the presence of
animals can ingest MPs directly and indirectly through trophic interactions; 10 μm and 1 μm MPs in the mussel hemolymph (Magni et al., 2018) upon
thus, MPs can move from one trophic level to another (Ward et al., 2019; exposure but fewer 1 μm MPs were observed due to instruments. Therefore,
Woods et al., 2018). Facilitated by their sizes, MPs can enter the circulatory more advancements are needed to improve and facilitate the localization of
system by passing through the intestine cavity due to their sizes (Browne beads in mussel tissues. Smaller MPs (≤1 μm) are difficult to detect when
et al., 2008; Shi et al., 2020), and they can also act as a surface for the bac- compared with the larger beads (≥10 μm) in mussels (Magni et al., 2018).
terial community thereby increasing the risk of transmission of an MPs have been reported to transfer from one organism to another, in-
antibiotic-resistant gene in a bacteria community (Arias-Andres et al., cluding humans across the gut lumen toward other tissues, such as the lym-
2018). In humans, exposure routes are thought to be through inhalation, in- phatic system (Farrell and Nelson, 2013; Gouin, 2020; Li et al., 2015; Setälä
gestion, and dermal absorption, with evidence confirming the inhalation et al., 2014). Magni et al. (2018) reported that MPs are present in the circu-
and ingestion with MPs being detected in human wastes; urine, feces and latory system of marine bivalves. This condition confirms that these MPs
blood (Leslie et al., 2022; Zarus et al., 2020). can pass through the gut epithelium interface via the microfold cells (M
Terrestrial ecosystems are the main sources of MPs. Up to 80% of MPs cells) in the Peyer's patches of gut epithelium translocated in the soft tissues
enter the marine ecosystems through industrial and domestic sewage and in the hemolymph. With such an ability, MPs have the potential to
water including wash water and laundry effluent. Lise et al. (2018) reported cause mechanical effects on the gut structure and less effect in terms of tox-
that toothpaste contains MPs. Murphy et al. (2016) indicated that wastewa- icity due to their fast elimination (Cauwenberghe and Janssen, 2014;
ter treatment plants (WWTPs) can remove more than 98% of MPs. Magni et al., 2018).

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R. Mkuye et al. Science of the Total Environment 838 (2022) 155933

Bivalves are sessile marine ecosystems that are widely distributed and et al., 2021). Plastics (including MPs) originate from the terrestrial ecosys-
are important species in an ecosystem. As filter feeders, bivalves can accu- tem and they are brought into the water system either intentionally to facil-
mulate large numbers of pollutants (including MPs) and microorganisms itate human socio-economical activities such as tourism, and fishing, which
from the aquatic environment that might influence their physiological per- are due to the mismanagement in the marine ecosystems. They are also car-
formance and their overall condition (Canesi and Pruzzo, 2016; Filgueira ried unintentionally by other agents of transport such as water runoff, wind,
et al., 2013). Bivalve also form a base in ecological niches and the accumu- and landslides from source points where plastics are being used such as in
lation of MPs through ingestion affects the species and higher consumers the farm and homes, enter the river and gets carried away into the oceans
(including humans) through trophic transfer. Bivalves have an important (Bhuyan et al., 2021; Birch et al., 2020). In a recent study, rivers, and
economic and nutritional values and play important role economically lakes have been accredited for their roles in transporting plastics to other
and ecologically (Cho et al., 2019; Farrell and Nelson, 2013; Li et al., water bodies. The abundance of MPs in the seas and oceans have been
2015; Q. Wang et al., 2021; Wang et al., 2021a, 2021b). They are endowed studied and studies have shown that a substantial variation is observed
with an effective and complex innate immune system (humoral and cellular their spatiotemporal distribution with different morphological and
defenses) similar to that of vertebrates. However, they lack an adaptive im- chemical characteristics (Yu et al., 2022) as shown in Tables 1, 2, and 3.
mune mechanisms to environmental challenges (Canesi and Pruzzo, 2016). Few have managed to reveal the potentiality of bioaccumulation and
Therefore bivalves are a suitable candidate for the study due to their biomagnification by considering the sources available MPs in the ecosys-
economical, nutritional and ecological values. tem.
This review aimed to explore the effects of MPs on the physiological per- Apart from the bioavailability of MPs in water sources leading to being a
formance of marine bivalves together with their related genes and to con- potential threat for bioaccumulation (Table 2), ingestion of MPs affects the
duct a comparative study on similar and relevant aspects for exploring rate of respiration in bivalves. Few studies such as in catfish, copepods, and
future potential areas of study and interest. Reports based on quantification Manila clam (Tongo and Erhunmwunse, 2021; W. Jiang et al., 2022;
of MPs in water and bivalves (mostly found and reported in/ from China) Svetlichny et al., 2021) have considered demostrated this hypothesis.
were considered for the sake of comprehending the study. Therefore, a More studies are needed to determine necessary condition factors and de-
deep literature studywas performed from obtained scientific reports in dif- rive a potential mathematical model.
ferent scientific journals, and research enabling websites about MPs, their
corresponding additives, and observed chemicals such as bisphenol A 3.2. MPs composition reported in bivalve species
(BPA), polycyclic aromatic hydrocarbon (PAH), persistent organic pollut-
ants (POP). The obtained studies were about the effects of MPs on marine MPs with different compositions have been extracted from bivalve that
bivalves in relation to gene expression, chemical modulation, and enzy- are from the wild (Gomiero, 2019; Su et al., 2018; Webb et al., 2019), fish
matic activities. The review focused on the impacts that have been reported farms, and markets (Ding et al., 2020; Li et al., 2015) indicating the uncon-
in the immunotoxicity on bivalves upon exposure to MPs. From a compar- trollable contamination. With the use of a microscope and micro-Fourier
ison of the studies on bivalves and other animals (including humans) gaps, transformed infrared spectroscopy, MPs have been observed, verified and
potential mathematical models, and potential threats were identified and classified successfully into different physical (size, shape, and color) and
suggested for future studies. chemical (polymer type) characteristics (Li et al., 2015). On the basis of
their physical and chemical observations, studies have associated them
2. Literature collection with their potential place of origin, associated activities and forecasted
their implication for the environment, humans, and animals. Future studies
In this review, published scientific reports on MPs' abundance in water, can be conducted to formulate policies based on the scientific findings that
and bivalves, laboratory experiments on MPs exposure on bivalve, and rel- have been reported (Table 3).
evant reports from other organisms were used. These reports were collected
from Science Direct, Springer, Google scholar, and Web of Science by using 3.3. Impacts of MPs
“microplastics”, “bivalve”, and “immuotoxicity” as keywords and the pa-
pers were selected based on its contents. For the sake of comprehending On the basis of the experimental procedures and designs, MPs in the bi-
the study, studies that reported the abundance of MPs in water and bivalve valves and the infiltration in tissues and haemolymph may or may not cause
were considered with much focus given in reports from the Southern part of particular biological responses or affect the cellular functionality (Magni
China. For the sake of comparison and better understanding of the area of et al., 2018). This condition may be due to the length of the experiment.
interest, relevant and related keywords including but not limited to “reac- More studies need to be done to understand the long-term effect of MPs
tive oxygen species,” “cell death,” “superoxide dismultase,” “catalase,” on bivalves.
“glutathione S transferase,” “glutathione peroxidase,” and “hemocytes”
were used. Valid and relevant comparison, as well as the inclusion of liter- 3.3.1. Total haemocytes, composition, and phagocytosis rate
ature were made from obtained results for better understanding of the Hemocytes are cells that circulate in the hemolymph and are responsi-
study. ble for numerous physiological functions (including shell formation, tissue
repair, nutrient transportation, digestion, excretion reproduction, and de-
3. Results and discussion toxification) and internal defense (immune functioning) (Han et al., 2022;
Hyun-KiHong and Choi, 2019; Le Guernic et al., 2020). Haematic parame-
In this work, published scientific reports between the years of 1993 and ters (total haemocytes counts, THC; hemocyte composition;, and phago-
2022 were obtained and studied. Reports that were considered for physio- cytic rate) of the clams can be affected by MPs, their corresponding
logical performance were from 2017 to 2021, abundances and quantifica- additives and the co-exposure of both MPs and additives.
tion were from 2015 to 2022, and for comparison and elaboration were The THC and viability are affected by the exposure of MPs and their co-
from 1993 to 2022. From the reports, laboratory experiments were con- exposure to additives. Measurement of cell viability enables the determina-
ducted based on single MPs and a combined mixture of MPs and xenobi- tion of the degree of cytotoxicity and immunotoxicity caused by xenobi-
otics exposure on bivalves. otics, thereby, helping in understanding biocompatibility. In a study
conducted in zebra mussels at concentration mixtures of 5 × 105 of 1 μm
3.1. Transfer, bioaccumulation, and biomagnification of microplastic MPs/L and 5 × 105 of 10 μm MPs/L (hereafter referred to as MIX 1), and
2 × 106 of 1 μm MPs/L and 2 × 106 of 10 μm MPs/L (hereafter referred
Recent studies have widely reported the presence of MPs in sediments, to as MIX 2), the percentage of hemocyte viability was reduced to 89 ±
water, and organisms (Claessens et al., 2013; Dou et al., 2021; Truchet 7% and 91 ± 1% for MIX 1 andMIX 2 respectvively, when compared to

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R. Mkuye et al. Science of the Total Environment 838 (2022) 155933

Table 1
showing the spatio range and average concentration of microplastics.
Location Concentration range (items/m3) Average concentration (items/ m3) Reference

South China Sea and East Indian ocean 0–4.97 0.40 ± 0.62 (Li et al., 2022)
Zhubi reef 1400–8100 4933 ± 1369 (Huang et al., 2019)
Guangdong province 3000–19,000 8895 (Zhang et al., 2020)
Nanxun reef 1250–3200 1773 (Nie et al., 2019)
Xijin National Wetland Park 150–1067 431 ± 15 (Q. Wang et al., 2021; Wang et al., 2021a, 2021b)
Hainan island 83–2300 892 (GM), 154 (XC) (Lin et al., 2021)
Western Pacific and South China sea 0.02–0.10 0.06 ± 0.03 (Liu et al., 2021)
Qiantang river 1.5–9.4 3.9 (Dai et al., 2022)
Pearl River Estuary 0.688–8.221 2.376 ± 0.700 (Lam et al., 2020)
Beijiang River and Pearl River 400–8400, Beijiang river 3183 Beijiang river (S. Wang et al., 2020; Wang et al., 2020a)
2400–18,200 Pearl river delta 7571 Pearl river
Ningbo 300–4000 1620.16 ± 878.22 summer (Xu et al., 2021a, 2021b)
1696.08 ± 983.52 winter
Jiangsu coastal waters 0.0206 – 0.2897 0.1037 ± 0.0751 (Q. Wang et al., 2021; Wang et al., 2021a)

Note: South China Sea (SCS), Eastern Indian Ocean (EIO), Xincun bay (XC), Gangmen bay (GM).

93 ± 6% for the control group (Magni et al., 2018). The results are similar phagocytosis. Similar to the use of hemocytes and their viability as an indi-
to other studies by Sun et al. (2020, 2021) who reported a reduction in THC cator of toxicity, future studies can focus on finding the exiating relation-
and viability upon exposure to MPs and their additives. The haemocytes in ship between hemocytes and bivalve's lifespan, growth rates, cell division,
the control group of the study by S. Wang et al. (2020) and Wang et al. and other body parameters.
(2020b) were compared to the haemocytes in treatment groups of PAH Therefore, the cellular toxicity of hemocytes increases when exposed to
mixture (containing 64 μg/L standard mix of PAHs equivalent to 4 μg/L MPs and the effect magnifies when a co-exposure such as using MPs and
for each PAH), MPs-PAH co-exposure group (30 μm containing 0.26 any other associated contaminant, occurs. MPs affect hemocytes in such a
mg/L polystyrene (PS) MPs and 64 μg/L standard mix of PAHs) and the manner that increase in MPs concentration results in a decline in cell viabil-
MPs (30 μm at 0.26 mg/L PS MPs containing approximately 1.76 × 104 ity (Magni et al., 2018; Sun et al., 2021) as shown in Table 4.
particles/L), the THC values of clams exposed to the MPs, PAH mixture The composition of hemocytes may vary considerably among species.
and MPs-PAHs co-exposure groups were remarkably reduced to approxi- Three main types of hemocytes, namely, agranulocytes (absence of cyto-
mately 85.47%%, 705.21%, and 47.86%, respectively. A decline in the plasmic granule with exception of few), hyalinocytes (large with small
THC indicates a lower number of available hemocytes. On the bases of nuclear-cytoplasmic ratio, NCR but have few or lack granules), and
the studies, a great reduction in THC and viability is observed when mussels granulocytes (possessing cytoplasmic granules), are found in bivalve. In
are being exposed to two xenobiotics at the same time. However, more most cases, granulocytes constitute the dominant subpopulation although
studies that link MPs and other uncontrollable parameters such as temper- some bivalves such as a pacific oyster (Crassostrea gigas) have been shown
ature rise, should be conducted. Therefore, it will be of great interest to ex- to have a prevalence of subpopulations of hyalinocytes (Anisimova et al.,
amine the immune response of bivalves under uncontrollable parameters 2017; Dudognon et al., 2014). Morphologically, agranulocytes are the
(MPs influx and global warming) that cause a general temperature rise alto- smallest haemocyte type, with a high nucleus to cytoplasm ratio (NCR),
gether. Future research can focus on understanding mussel response to granulocytes are the largest ameboid-shaped haemocyte type with low
combined effects of temperature and MPs. Similarly Sun et al. (2021) re- NCR, and hyalinocytes are smaller than granulocytes with an intermediate
ported a decline in hemocytes viability upon PS MPs, PAH, and MP-PAH NCR compared with agranulocytes and granulocytes (Andreyeva et al.,
coexposure to 85.78%, 70.21%, and 49.27% respectively. 2021; Hine, 1999). Among the many studies that have been conducted on
The overall reduction in THC and haemocytes viability is due to the cel- the effects of MPs on bivalves, a few studies have considered the composi-
lular stress that is caused by the xenobiotics, resulting in a degree of DNA tion of hemocytes. Future studies may focus on the impact of MPs and their
damage. DNA integrity is essential to maintaining the normal function of additives and other parameters on the physiology and functions of bivalve
the cell (Han et al., 2019). Therefore, aggravated DNA damage induced hemocytes and their composition in a range of species to fill the knowledge
by the tested pollutants may result in reduced cell viability and inhibited gap. Few studies have focused on the effects of MPs and their xenobiotics

Table 2
showing the concentration of microplastics in water and bivalves.
species Location Concentration in water Concentration in bivalve (items/ individual)/(items/g of Reference
(itemes/m3) wet tissue)

Pinctada margaritifera French Polynesia 3.30 surface water 263.88 particles/ individual (11,083 particles/ 42 (Gardon
N/A in vertical water individuals) et al., 2021)
Amarilladesma mactroides and Claromecó beach, Argentina 10,330 ± 4700 at the 0.33 ± 0.1 items g-1 wet weight (w.w.) for A. mactroides, (Truchet
Brachidontes. rodriguezii estuary, 0.17 ± 0.07 item g-1 w.w for B. rodriguezii et al., 2021)
10,000 ± 1417 at the
beach
9670 ± 7027 at the
Lighthouse
Crassostrea gigas, Mytilus edulis, South Korean coasts 1400 ± 560 0.33 ± 0.23 n/g and 1.21 ± 0.68 n/individual in C. gigas/ (Cho et al.,
Ruditapes philippinarum M. edulis (composite sample) 2021)
0.43 ± 0.32 n/g and 2.19 ± 1.20 n/individual in R.
philippinarum
Perna viridis Meretrix lyrata Tapi-Phumduang River and its 1770 ± 280 1–13 items/individual (0.16–1.85 items/g) in small P. (Chinfak
corresponding branched rivers Tapi-Phumduang River Virdis et al., 2021)
330 ± 140 branched 0–6 items/individual (0–0.49 items/g) in large P. virdis
rivers 0–3 items/individual (0–1.06 items/g) in small M. lyrata
0–2 items/individual (0–0.20 items/g) in large M. lyrata

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Table 3
showing microplastic polymers, sizes, quantity and forms.
Species observed Polymer type Sizes color Quantity Forms Reference

Scapharca subcrenata Tegillarca PE, PET, PA 5 mm to 5 mm black, red, blue, 2.1 to 10.5 items/g Fibers, (Li et al., 2015)
granosa Mytilus galloprovincialis white, and (wet weight) and from 4.3 to 57.2 fragments
Patinopecten yessoensis Alectryonella transparent items/individual and pellets
plicatula
Sinonovacula constricta Ruditapes
philippinarum,
Meretrix lusoria, Cyclina sinensis
Mytilus galloprovincialis, Eighteen polymers Qingdao - 10 to 4377 Nine colors of Qingdao - 1.2 to 4.1 items/individual Fibers, (Ding et al.,
Perna viridis, Ruditapes philippinarum, predominantly μm, microplastics in the (i.e., 0.8 to 4.4 items/g, wet weight, fragments, 2020)
Crassostrea gigas, Sinonovacula Rayon, PET, CPE, Xiamen - 58 to 5000 shellfish Xiamen - 1.3 to 6.0 items/individual granules,
constricta, Scapharca subcrenata, PVC in Qingdao, μm from Qingdao and (i.e., 2.1 to 4.0 items/g) and film
Meretrix lusoria, Busycon canaliculatu Ten (not listed) eight colors Xia-
polymers men
predominantly but predominant
rayon, PVDF, CPE, colors were
PVC, and PET. predominantly
white, black, and
transparent
Perna canaliculus PE, PA 50 to 700 μm Blue, red 0 to 1.5 per musse Fragments, (Webb et al.,
0 to 0.48 particles g tissue −1 four beads, 2019)
and two
fibre
Corbicula flumine PET, PP, PE 0.021–4.02 mm Blue, yellow, green, 0.3–4.9 items/g Fibre, (Su et al., 2018)
black, white 0.4–5.0 items/individual pellet, fil,
transparent fragment
Mytilus edulis LDPE, HDPE, PS 20–90 μm in tissue N/A 0.2 ± 0.3 particles/ g tissue N/A (Cauwenberghe
et al., 2015)
Mytilus galloprovincialis PE, PP, PET PVC, PS, N/A N/A Fragment (Coastal 1.06–1.33 items/g Fragments, (Gomiero,
and PLY for particle WW pellet 2019)
shape Offshore 0.65–0.66 items/g WW)
PE, PP, PET, PS, and Fibers (of0.62–0.63 items/g coatal and
PA for fibers 0.24–0.35 items/gWW offshore)
Villorita sp CPVC, PE, PP, and 0.012–2.85 mm in Black, blue, red, 23 ± 20 nos./ind Fibre, (Cho et al.,
PES digestive tissues and transparent, green fragment, 2019)
0.013–4.35 mm in and yellow, film,
non-digestive tissues
Ruditapes decussatus N/A N/A Red, blue, green, M. galloprovincialis Fragment (Yozukmaz,
Mytilus galloprovincialis yellow, white, and Site 1 film and 2021)
other 0.76 ± 1.10 particles/ individual for fibre
fragemnt, 1.73 ± 1.60 particles/
individual for film, 14.76 ± 8.24
particles/ individual fibre
R. decussatus
0.60 ± 1.00 particles/ individual for
fragment, 0.66 ± 1.30 particles/
individual for film, 11.30 ± 7.21
particles/ individual for fibre
Site 2
M. galloprovincialis
0.43 ± 0.94 particles/ individual for
fragment, 0.93 ± 1.62 particles/
individual for film, 11.10 ± 5.60
particles/ individual for fibre
R. decussatus 1.13 ± 71.70 particles/
individual for fragment, 0.90 ± 0.99
particles/ individual for film, 11.73 ±
4.73 particles/ individual for fibers
Cerastoderma edule, PE, PP, PS, ABS, 15–50 mm, 50–100 Blue, black, Mussel Fibers, MP (Hermabessiere
Mytilus edulis SBR, and PET mm, 100–500 mm transparent, pink, 0.76 ± 0.40 MP/individual, 0.25 ± 0.16 et al., 2019)
and > 500 mm brown and green MP/ g of tissue, in Le Portel (LP)
0.78 ± 0.30 MP/ individual, 0.15 ±
0.06 MP/ g of tissue in Baie d’Authie
(BA).
Cockle
2.46 ± 1.16 MP/ individual, b 0.74 ±
0.35 MP/ g of tiisue in BA
0.56 ± 0.22 MP/ individual 0.19 ± 0.08
MP/ g of tissue in in BdV

Note: polyethylene (PE), polyamide (PA), poly(vinyl chloride (PVC), polyvinylidene fluoride (PVDF), polyethylene chlorinated (CPE), polyethylene terephthalate (PET), low
density polyethylene (LDPE), high density polyethylene (HDPE), polystyrene (PS), polyvinyl chloride chlorinated (CPVC), poly(ether sulfone) (PES), polypropylene (PP), Ac-
rylonitrile-Butadiene-Styrene (ABS), poly(styrene-co-butadiene), styrene butadiene rubber (SBR).

5
R. Mkuye et al. Science of the Total Environment 838 (2022) 155933

Table 4
showing the effects of microplastic exposure treatments and concentrations in the percentage of haemocytes viability across different mussel species.
Mussel used Treatment Compounds/ Concentration and size Viability (%) Reference
chemicals

Blood clam PS PS MP and 1.76 × 104 particles/L of 30 μm 85.47 (S. Wang et al., 2020;
PAH PAH 4 μg/L for each PAH 75.21 Wang et al., 2020b)
PS 1.76 × 104 particles/L and 4 μg/Lfor each PAH 47.86
Zebra mussel MIX 1 PS 5 × 105 PM/L of 1 μm size 89 ± 7 (Magni et al., 2018)
5 × 105 PMs/L of 10 μm size
MIX 2 PS 2 × 106 PM/L of 1 μm 91 ± 1
2 × 106 PM/L of 10 μm PMs/L.
Zebra mussel PVC PVC and 16,875 PVCMDs/L of 56 ± 35 μm 96.1 ± 2.7 (t = 6) and (Magni et al., 2020)
Mater – Bi® 95.5 ± 2.2 (t = 14)
Mater – Bi® group 18,750 Mater-Bi® MDs/L of 41 ± 36 μm 96.7 ± 1.8 (t = 6) and
group 93.7 ± 6.8 (t = 14)
Mediterranean 4.5 μm MPs PS, BPA 0.058 mg/L, corresponding to 1000 particles/mL for 4.5 μm MPs with Benzo Reduced (González-soto et al.,
mussel alone (LMP), [a]pyrene (Bap) adsorption capacity of from144.99 to 242.89 μg/g 2019)
0.5 μm MPs 7.44 × 105 particles/ mL for 0.5 μm MPs Reduced
alone (SMP),
4.5 μm MPs 0.058 mg/L, corresponding to 1000 particles/mL for 4.5 μm MPs Reduced
with BaP
(LMPB)
0.5 μm MPs 7.44 × 105 particles/ mL for 0.5 μm MPs Benzo[a]pyrene (Bap) adsorption Reduced
with BaP capacity of 30.5 to 67.65 μg/g
(SMPB).

Note: microplastics (MPs), polystylene (PS), polyaromatic hydrocarbon (PAH) mixture containg acenaphthene, acenaphthylene, anthracene, benzo[a]anthracene, benzo[b]
fluoranthene, benzo[k]fluo- ranthene, benzo[g,h,i]perylene, benzo[a]pyrene, chrysene, dibenz[a,h] anthracene, fluoranthene, fluorene, indeno[1,2,3-c,d]pyrene, naphtha-
lene, phenanthrene, and pyrene, Mixture 1 (MIX 1), Polyvinyl chloride (PVC), 4.5 μm microplastics (LMP), 0.5 μm microplastics (SMP), 4.5 μm sorbed with benzo[a]pyrene
(LMPB), benzo[a]pyrene (BaP), 0.5 μm microplasticssorbed with benzo[a]pyrene SMPB).

co-exposure on the composition of the THC upon exposure. Studies re- are chemically reactive oxygen-containing species produced during
ported that the exposure of clams to MPs, PAH, and PAH-MPs co- mitochondrial respiration from molecular oxygen (O2− ) during vital
exposure led to alterations in the composition of hemocytes. The proportion processes occurring in humans and other living organisms (Kwon
of red granulocytes and hyalinocytes decreased more upon exposure to et al., 2021). Several factors such as MPs and their corresponding addi-
MPs, PAHs, and their corresponding MPs-PAH,; such as MPs and petroleum tives and compounds have the ability to change the levels of ROS and
hydrocarbon and MPs-petroleum hydrocarbon co-exposure unlike basophil LPO when an organism is exposed to them. Sun et al. (2021) observed
that increased with exposure (Sun et al., 2020, 2021). With the understand- that the intracellular content of ROS increased by 17% and 22% for
ing of the change in hemocytes' composition, the changes that may occur in
phagocytotic activities can be realized, as shown in Table 5.
Phagocytosis is a key element of an organism's immunity. Therefore, the Table 5
ability of hemocytes to undertake phagocytosis demonstrates well the im- showing the effects of microplastic exposure treatments on the haemocytes compo-
mune response. However, inhibition of the hemocyte adhesion, aggrega- sition and its corresponding effect on phagocytosis.
tion, and phagocytic behavior may render the bivalve's immune system Treatment Percentage (%) Reference
and compromise the condition in the bivalve by making it vulnerable to Red Basophil Hylinocytes Phagocytotic
other environmental challenges that include parasitism (Chakraborty granulocytes granulocytes
et al., 2021; Le Guernic et al., 2020). Sun et al. (2020) reported that the de- MP 70.0 ± 0.9 28.7 ± 0.8 1.3 ± 0.2 26.7 ± 0.5 (Sun et al.,
crease in phagocytosis in mussels is caused by a reduction of the hemocytes PAHs 64.3 ± 0.9 34.9 ± 0.9 1.0 ± 0.2 24.2 ± 0.5 2021)
specifically. The proportion of red granulocytes proportion the most effi- MP – 54.9 ± 0.9 44.5 ± 1.0 0.9 ± 0.1 18.2 ± 0.5
cient than the rest of the hemocytes and cell viability. Phagocytosis is sup- PAHs
MPs 70.0 ± 0.9 29.0 ± 1.0 75.3 ± 0.2 Reduced (Sun et al.,
pressed upon exposure to MPs, PAH and MPs-PAH co-exposure. Sun et al.
PL 68.4 ± 0.4 31.0 ± 0.4 75.3 ± 0.2 Reduced 2020)
(2021) reported that after two weeks of exposure to MPs or PAHs, the PL _ MP 66.5 ± 0.3 32.9 ± 0.5 75.3 ± 0.4 Reduced
phagocytic rate declined to approximately 87.83% and 79.61% whereas PH 59.0 ± 0.3 40.2 ± 0.5 75.3 ± 0.5 Reduced
the co-exposed to MPs and PAHs resulted to lower rates than those from sin- PH + 50.2 ± 0.6 49.5 ± 0.7 75.3 ± 0.2 Reduced
gle exposure (Sun et al., 2021). S. Wang et al. (2020) and Wang et al. MPs
M 82.1 ± 0.2 16.7 ± 0.3 1.3 ± 0.2 47.1 ± 0.2 (Tang et al.,
(2020b) observed that the lysosome activity is substentially affected by N 77.9 ± 0.1 20.5 ± 0.1 1.6 ± 0.1 32.4 ± 0.8 2020)
pH and MPs on days 7 and 14 of the study. During the exposure period, E2 79.2 ± 0.7 18.9 ± 0.6 1.9 ± 0.1 45.7 ± 0.8
at pH 8.1, a significantly increased lysosome activity occurred in the high MEL 82.5 ± 0.4 16.1 ± 0.4 1.4 ± 0.2 48.2 ± 0.7
concentration micro-PS (106 particles/L) group on day 14. With the magni- NEL 75.0 ± 0.5 22.9 ± 0.3 2.2 ± 0.1 27.5 ± 0.8
EH 78.7 ± 0.4 20.1 ± 0.4 1.2 ± 0.3 43.7 ± 0.6
fied intensity of xenobiotics and other parameters under natural and culti-
MEH 80.6 ± 0.6 17.8 ± 0.4 1.7 ± 0.2 47.4 ± 0.5
vated conditions, a significant change in phagocytosis might have NEH 73.0 ± 0.2 25.0 ± 0.2 2.0 ± 0.1 26.2 ± 0.8
potential health effects on other organisms that might ingest bivalves. Stud-
Note: microplastics (MPs),polycyclic aromatic hydrocarbons (PAHs), degree of alka-
ies should focus on the potentiality of immune distortion with the ingestion
linity/ acidity (pH), Petrollium hydrocarbon at high concentration (pH), petroleum
of contaminated bivalves across trophic levels.
hydrocarbon at low concentration (PL), microplastics with size 30 μm (M),
microplastics with size 500 nm (N), 17β-estradiol at 0.1 μg/L concentration (E2),
3.3.2. Reactive oxygen species (ROS), lipid peroxidation (LPO), and microplastics with size 30 μm and 17β-estradiol at concentration of 0.1 μg (MEL),
deoxyribonucleic acid (DNA) damage − microplastics with size 500 nm and concentration of 0.1 mg (NEL), 17β-estradiol
Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), at concentration of 1 μg/L (EH), microplastics at size of 30 μm and 17β-estradiol
hypochlorous acid/ hypochlorite (HOCl/ClO−), the hydroxyl radical at 1 μg concentration (MEH), microplastics with size 500 nm and 17β-estradiol at
(-OH−), the superoxide anion radical (O−
2 ), and singlet oxygen (O
2−
) 1 μg concentration (NEH).

6
R. Mkuye et al. Science of the Total Environment 838 (2022) 155933

clams treated with MPs and PAHs, further increased when co-exposed by countervailing biological mechanisms and pathological conditions. LPO
with MPs-PAHs, and rincreased up to 25% when co-exposed to petro- has been documented not to be readily affected by MPs but they can be
leum hydrocarbon and MPs (Sun et al., 2020). Overproduction of affected by either corresponding additives or co-exposure of additive and
ROS can directly cause the aggravation in LPO and DNA damage. A MPs; however, the level of LPO increases with a prolonged exposure to
similar trend of increase in ROS contents was observed by (Paul-Pont MPs. Sun et al. (2021) observed that the LPO level in hemocytes is unaffected
et al., 2016) on exposure to fluoranthene (FLU), microbeads (micro- by MPs exposure alone but changes with PAH exposure and MPs-PAH co-
PS) xenobiotics. exposure to approximately 4.23 and 5.38 times of the control respectively
Xenobiotics may cause oxidative stress that generates ROS, resulting in and a great change is observed upon co-exposure. Similarly, the LPO levels
DNA damage (depending on the radical of ROS) to a certain degree. For in- increases upon exposure to polyethylene (PE) MPs, and co-exposure to
stance, PAH ingested by humans or bivalves is activated into highly reactive other additives until day 14 (Donovan et al., 2018; Islam et al., 2021).
and toxic metabolites such as dihydrodiols, quinones, and diol epoxides by Given that a similar effect might occur in other organisms including humans,
the intracellular xenobiotic-metabolizing enzymes such as cytochrome future studies should focus on the manner of reducing or eliminating the
P450 monooxygenases (CYP) and microsomal epoxide hydrolase (EPHX1) changes in ROS and LPO that are caused by MPs and their corresponding
(Luckert et al., 2013; Martins et al., 2013; Palackal et al., 2001). However, additives. The use of vitamin B12 has been reported to have antioxidant
MPs have been reported not to cause irreversible DNA damage when a mus- abilities on reducing the oxidative stress and LPO in buffalo bull spermatozoa
sel is exposed to it alone. However, when exposed to certain contaminants as- (Ahmed et al., 2021) during an appropriate time before harvest for human
sociated with MPs such as PAHS or when MPs is coupled with other consumption so as to reduce health risks.
compounds such as PAHs, the degree of damage increases. In a study by The LPO level has been reported to be affected by the treatment and
Magni et al., 2018, and Zhou et al., 2021, no DNA damages were observed time duration of exposure but no interaction is found between the treat-
upon exposure to MPs alone but there was a degree of DNA damage in hemo- ment and time in mussels when using PS. No interaction has been docu-
cytes on exposure to PAH, and more effect was observed upon co-exposure to mented when polyvinyl chloride (PVC) and mater - Bi were used (Magni
MPs-PAH, which were approximately 3.46 and 4.58 times of the control (Sun et al., 2018, 2020). As reported by Magni et al. (2018), the LPO level in
et al., 2020, 2021). In humans, DNA damage might cause mutagenic or car- MIX 1 and MIX 2 increased in dayes 3 and 6, whereas the level decreased
cinogenic effects. Therefore, the high chances of bioavailability and the bio- for MIX 1 and 2 on days 6 and 3, similar to a study conducted by Paul-
accumulation in MPs and its corresponding additives, are a potential threat Pont et al. (2016) upon exposure to MPs-PS and FLU. Magni et al. (2020)
to the well-being of the human population. did not observe significant effects of time, treatment, and time/treatment.
In a review article by Liu et al. (2020) and a study by Sussarellu et al.
(2016), exposure to MPs enhanced the ingestion and absorption efficiency, 3.4. Gene expression
and energy flows shifted toward organism maintenance and structural
growth at the expense of reproduction. The study further reported that a re- Gene expression is the process in which the information encoded in a
duction in sperm velocity that may, lower their ability to fertilize oocytes in gene (gene sequences) is transcribed into a functional gene product such
exposed oysters. Severin (which was found to be upregulated) and arginine as proteins, and RNA (rRNA, tRNA, and so on). Gene modulation indicates
kinase (which was found to be down-regulated) are responsible to regulate the state of the body systems at that time. When a gene is expressed or reg-
the completion of cell division and adenosine triphosphate (ATP) metabo- ulated, it allows the cell to adapt to different conditions, and, they are the
lism. Therefore, they can cumulatively reflect the effects of micro-PS on ac- basis of cell development and differentiation (Arasappan et al., 2010;
olyte maturation, fertilization, and subsequent embryo development Doug Chung and Le Roch, 2013; Preziosi, 2007). MPs and their correspond-
(Gardon et al., 2018; Liu et al., 2020; Sussarellu et al., 2016). However, ing additives have the ability to modulate genes (up regulate or down reg-
studies in humans have found that in addition to severin and arginine ki- ulate) in the bivalves depending on the concentration or treatments (single
nase modulations, ROS has an effect on male reproduction. Similar to the exposure or co-exposure), as shown in Table 6.
hemocytes, spermatozoa can generate ROS via the sperm plasma mem- Immune-related and detoxification related genes (nuclear factor NF-
brane (ROS may be produced by the nicotinamide adenine dinucleotide kappa-B p105 subunit (NFκB), inhibitor of nuclear factor kappa-B kinase
phosphate (NADPH) oxidase system) and at the mitochondrial level. ROS subunit alpha (IKKα), mitogen-activated protein kinase (MAPK),
are generated via the nicotinamide adenine dinucleotide (NAD)-dependent glutathione-S-transferase (GST), and cytochrome P450 protein family 1
redox reaction as reviewed by Dutta et al. (2019). Thus, an increase in ROS subfamily A member 2 (CYP1A2) have been found to be downregulated
is a well-documented response by bivalves upon ingestion of xenobiotics. by MPs alone and upon co-exposure with other xenobiotics (Zhou et al.,
Similar to human beings, bivalves might undergo a series of metabolic path- 2021), with exception of Tumor necrosis factor receptor-associated factor
ways that will ultimately cause sperm malfunctioning. In human beings', 6 (TRAF6) has been reported by (Sun et al., 2021) not to be affected by a
the prime ROS in human spermatozoa is superoxide (O− 2 ), which reacts single exposure to MPs. The observed responses in the genes (except
with itself through dismutation reactions to yield H2O2. For transition TRAF6) can be endorsed by the accumulation of oxytetracycline and
metals, such as iron and copper, H2O2 and O− 2 can generate the most de- florfenicol (FLO) in blood clams that is promoted by the presence of MPs,
structive and extremely reactive hydroxyl radical (OH−) via the Haber– probably through the Trojan horse effect and the inhibition of detoxifica-
Weiss reaction, which can initiate LPO cascade disrupting membrane fluid- tion. TRAF6 is less sensitive to MPs because it serves as a molecular bridge
ity and impairing sperm function (Dutta et al., 2019). Future studies should with the downstream NFκB and MAPK signaling pathways. Although stud-
further explore the above hypothesis above in bivalves. Currently, few stud- ies have been conducted on MAPK, a knowledge gap is found on the types
ies, including (Sussarellu et al., 2016), have been done to link ROS and the of MAPK (extracellular regulated protein kinase [ERK], c-Jun- NH2 – termi-
overall effects on reproduction. nal kinase [JNK], or p38 – MAPK) which are been reported to be affected by
environmental pollutants (including MPs). The extent of the effect that is
3.3.3. LPO being shown by each MAPK is demonstrated by Châtel et al. (2010). How-
LPO is induced by excess ROS production (mainly hydroxyl radical [• HO] ever, the activation of NFκB plays essential roles such as apoptosis, prolifer-
and hydroperoxyl [HO2•]) that can inflict direct damage to lipids, and less ation, and phagocytosis via activating Bcl-2 (B-cell lymphoma-2), which
effective antioxidant defense which may cause cell death through apoptosis, subsequently inhibits the activation of apoptosis executor, the caspases
autophagy, and ferroptosis (iron-dependent cell death).Thus, LPO can induce (Sun et al., 2021).
membrane rupture and increase membrane permeability, leading to cell The expression of genes might induce production and protection against
death and is a means of determining oxidative stress (Jiang et al., 2021; ROS. For instance, the aryl hydrocarbon receptor (AHR) can induce intra-
Jianga et al., 2021; Xu et al., 2021a, 2021b). These peroxidation reactions cellular production of ROS by combining with aromatic compounds such
are toxic to cells and cell membranes; however, they are normally controlled as PAH. In a study by Sun et al. (2021), it was observed that activation of

7
R. Mkuye et al. Science of the Total Environment 838 (2022) 155933

Table 6 from destruction and help in the recovery of damaged proteins. Gu et al.
showing the effects of microplastic exposure and its corresponding treatments in (2020) explored the effect of MPs and reported that the expression levels
gene expression (0 representing no effect, + representing upregulation, and – rep- of HSP70 and HSP90 decreased in thick shell mussels Mytilus coruscus
resenting downregulation). upon exposure to MPs at a high concentration of polybrominated biphenyl
Gene Treatment and effect Reference ether (PBDE) but no changes are observed upon single exposure to MPs in
MP PAH MP-PAH the expression levels of Hsp70 and 90.
NFκB1 − − − (Sun et al., 2021) Cell death is a vital event in multicellular organisms and occurs in apo-
TRAF6 0 − − ptosis, necrosis, and aponeurosis/apocrosis. Apoptosis is the form of genet-
BAX 0 + +
ically regulated cell death induced for the overall benefit of the organism
Caspase-3 + + +
AHR 0 + + (programmed death); necrosis is the form of cell death that is not planned
ARNT 0 + + nor programmed. Necrosis affects groups of contiguous cells, and an inflam-
CYP1A1 0 + + matory reaction usually develops in the adjacent viable tissue in response to
MP OTC FLO MP + (Zhou et al., 2021) the released cellular debris. Aponeurosis/apocrosis is the form of cell death
FLO
that have the features of apoptotic and necrotic (Brown, 2014; Caballero-
IKKα − − 0 −
NFκB − − − − Díaz and Valcárcel, 2014; Ray and Mehendale, 2005). MPs and their corre-
MAPK − − − − sponding additives are known to causing cell deaths and they can induce or
Caspase-3 0 + + + inhibit the expression levels of cell death-related genes. MPs can have an ef-
GST − − − −
fect on cell death-related genes depending on the locality of the gene and
CYP1A2 − − − −
50mp/ml 500mp/ml change the gene expression in accordance with the treatment of the expo-
LYS + + (Capolupo et al., 2018; Détrée sure to the bivalve. In a study by Teng et al. (2021), exposure of oysters
MYTC + + and Gallardo-Escárate, 2017; to MPs led to an increase in the expression of caspase-3 (an important pro-
MYTLB + + Sendra et al., 2021) tein for the apoptotic enzyme cascade and regulates neuron apoptosis) sim-
HEX − −
ilar to (Sun et al., 2021; Zhou et al., 2021) and tumor necrosis factor
GUSB − −
CTSL − + receptor superfamily member 6 associated death domain (FADD) in the di-
5-HT1 − + gestive glands. In the gills no change was observed in FADD and the expres-
MeER1 0 0 sion of caspase-3 was stimulated only in the 1000 μg L−1 concentration of
MeER2 + −
MPs treatment groups. FADD and caspase – 3 are involved in cell death with
Hemolymph Gills Digestive Mantle
gland
the FADD being an initiator of the apoptosis program through interactions
PK − − + + with the death domain of Fas, and caspase-3 is an integral part of the execu-
ID 0 0 − + tion phase of cell apoptosis and they are expressed because of the produc-
SD − − + + tion of ROS (Teng et al., 2021). The Fas binding of FADD activates FADD-
TLR − 0 + +
procaspase-8 interaction that activates the downstream “executioner
PGRP + + − 0
myticin A 0 + + + caspases,” such as caspase-3, caspase-6, and caspase-7, which perform the
mytilin B 0 0 − + apoptotic process (Chen et al., 2020). A growing knowledge of understand-
p53, − 0 + + ing is found on the genes that are responsible for apoptosis. However, few
caspase 0 + + +
studies have managed to investigate the expression levels of apoptotic
3/7
inhibiting genes on exposure to MPs. Teng et al. (2021) reported no
Note: microplastics (MPs), glutathione-S-transferase (GST), cytochrome P450 1A1 changes in the expression of B-cell lymphoma-2 (Bcl-2) in neither gills
(CYP1A1), cytochrome P450 protein family 1 subfamily A member 2 (CYTP1A2), nor digestive glands in C. gigas regardless of the treatments thereby giving
nuclear factor kappa B subunit 1 (NFκB1), tumor necrosis factor receptor associated
an insight into the interaction of genes toward apoptosis. However, more
factor (TRAF6), Bcl-2 Associated X-protein (BAX), Polycyclic aromatic hydrocarbon
genes such as Fas apoptotic inhibiting molecule (FAIM) should be consid-
(PAH), hexosaminidase (HEX), β-glucorinidase (GUSB), cathepsin-L (CTSL),
ered in future studies. FAIM was studied in Manila clam (Ruditapes
myticin C (MYTC), mytilin B (MYTB),Lysis (LYS),5-hydroxytryptamine; serotonin
(5 -HT1), estrogen-related receptor 1 (MeER1), estrogen-related receptor 2 philippinarum) and was highly expressed in hemocytes upon exposure to
(MeER2), pyruvate kinase (PK), isocitrate dehydrogenase (ID), succinate dehydro- Vibrio anguillarum and Micrococcus luteus pathogens (Chen et al., 2020).
genase (SD), toll-like receptor (TLR), peptidoglycan recognition protein (PGRP), The alteration of BAX affects the balance between the genes promoting
tumor suppressor (p53), oxytetracycline (OTC), florfenicol (FLO), inhibitor of nu- and those inhibiting apoptosis, inducing or accelerating neuron apoptosis
clear factor kappa-B kinase subunit alpha (IKKα), Mitogen-activated protein kinase such as activation of caspase-3. High BAX expression levels and the forma-
(MAPK), cysteinyl aspartate specific proteinase-3 (caspase – 3), arylhydrocarbon re- tion of homo- or heterodimers with Bcl-2 (Bax-Bcl-2 heterodimer) inhibit
ceptor (AHR), arylhydrocarbon nuclear translocator (ARNT). Bcl-2 and initiates cell apoptosis in rats; l reperfusion injury enhances the
caspase-3 activity by inducing its expression, thereby promoting neuron ap-
the aryl hydrocarbon receptor - cytochrome P450 1A1 (AHR-CYP) signal- optosis in rats (Liu et al., 2013; Vince et al., 2018). Sun et al. (2021) re-
ing pathway indicated by the upregulation of AHR, ARNT nuclear ported that the expression levels of Bcl-2- associated X (BAX) are not
translocator, and cytochrome P450 protein family 1 subfamily A member affected by exposure to MPs. Investigating the antiapoptotic gene affected
1 (CYP1A1) leads to the overproduction of ROS upon exposure to PAH by MPs will provide a comprehensive immune response for bivalves.
and MPs-PAH. A single exposure of the bivalve to MPs can cause the pro-
duction of ROS in bivalves. However, few studies have investigated this 3.5. Enzymatic modulation
phenomenon. Thus, the pathways and the genes involved in the production
of ROS by the MPs are unclear. For instance, a positive relationship has 3.5.1. Glutathione S transferase (GST)
been reported between glutathione (GSH) and ROS production. However, GST expression is induced by the presence of varying types and concen-
more genes such as heat shock protein 32 (HSP32), and metallothionein trations of xenobiotics. GST is important in detoxification because it com-
(MT), must be studied considering the number of genes that are involved bines with contaminants and generates compounds that are more easily
with ROS. HSP32 and MT are involved in cellular protection against ROS excreted (Ribeiro et al., 2020). They are the metabolizing enzymes that
such as in the razor clam (Sinonovacula constricta) upon exposure to xenobi- can protect tissues against oxidative damage caused by the excessive pro-
otic (in particular cadmium) (Wang et al., 2016). Future studies might ex- duction of ROS under pollutants and exposure to other xenobiotics com-
plore the possibility of MPs inducing the expression of the gene upon pounds and they prevent y cells from carcinogenic factors. (Tsuchida,
exposure to MPs. HSPs are a family of proteins that protect organisms 1997; Zhou et al., 2022). Similar to the observations made by Li et al.

8
R. Mkuye et al. Science of the Total Environment 838 (2022) 155933

(2021), the GST activities in bivalves exposed to MPs are time-dependent mussels were exposed to PVC and Mater-Bi® MPs, Paul-Pont et al. (2016)
with an increase in activities that declines with prolonged exposure observed a decrease in CAT activity (T = 7) with exposures to MPs-PS,
which increases the utilization of GSH. This condition strongly infers that and MPs-fluoranthene (FLU) co-exposure. The changes observed in CAT
bivalves depend on other mechanisms and molecules such as non- are due to the type of ROS contents formed. An increase in CAT activity
enzymatic antioxidants including GSH to counteract the toxicity of MPs has been well documented to be caused by the increasing ROS production.
during prolonged exposure. MPs initiate oxidative stress and cellular dam- Thus, CAT helps to protect the cells from oxidative damage (Leite et al.,
age to bivalves upon exposure and counteract the stress by releasing antiox- 2020). Apart from MPs that can alter CAT activity, other factors such as tem-
idant enzymes (including CAT, SOD, and GPx) and GSH to maintain redox perature have been reported to affect CAT activities. In a study on C. gigas
balance. Understanding and consideration of GST are vital in understand- and Mytilus galloprovincialis bivalves, it has been reported that CAT activity
ing an animal and the seasonal environmental changes as reported by increased with temperature from 15 °C to 20 °C, and decreased from 20 °C
Ribeiro et al. (2020). Magni et al. (2018) examined the effect of PS MPs to 25 °C. For this matter, future studies can explore the changes in CAT activ-
(1 and 10 μm) in detoxifying enzyme GST.No modulation (but there was ities with varying environmental factors such as temperature, pH, and MPs
a non-significant increase of about 28%) was observed noticed after the ex- across bivalve species so as to have comprehensive information.
posure by the two PM mixtures (MIX 1 and MIX2). Magni et al. (2020) also
observed an increase in GST activity with treatments (PVC and matter – Bi) 3.5.4. Glutathione (GSH)
and with time (day 7) but a decrease in activity was found when the expo- GSH is important in anti-oxidation defense, metabolism of xenobiotics,
sure was prolonged (Day 14). Guilhermino et al. (2018) observed a similar and regulation of the cell cycle, gene expression, and enzymatic activity to
trend of increase in GST activity. However, a treatment group containing a maintain redox balance in the cell. Studies have reported that, unlike antiox-
mixture of the lowest concentration of FLO and the highest concentration of idant enzymes, the GSH level varies slightly in tissues thereby, indicating the
MPs, resulted in an increase in gill GST activity (2.1 folds), and digestive importance of GSH in cell metabolism. GSH through a series of reactions can
gland GST activity (2.6 folds). The higher activity of GST in the digestive form strong reducing radical agents (GSSG.) to overcome oxidative damages
glands can be a defensive mechanism to prevent the assimilation of MPs caused by ROS upon exposure to xenobiotics (Buettner, 1993; Istomina
and their corresponding additives to prevent oxidative damage that might et al., 2021). GSH has been observed to be utilized by cells to prevent the
lead to an immune drop. effects that are brought about by MPs and their corresponding additives.
S. Wang et al. (2020) and Wang et al. (2020a), observed that the GSH
3.5.2. Superoxide dismultase (SOD) activity in the medium concentration (104 particles/L) and high concentra-
SOD has an important role in converting O− 2 into H2O2 (Amorim et al., tion (106 particles/L) are considerably increased under pH 7.7 conditions
2020). SOD can be either affected (Magni et al., 2018) or not be affected from day 1 of the study with further increase in activity observed at a higher
(Magni et al., 2020) by MPs depending on their retention in the gut and concentration at day 7. However, the degree of effect reduced on day 14, and
the effect of exposure to MPs on the antioxidant system (S. Wang et al., a higher increase in the activity was observed at pH 8.1 in medium and
2020; Wang et al., 2020b). Magni et al. (2018) reported the influence of higher concentrations on day 14 (S. Wang et al., 2020; Wang et al.,
MPs in SOD activities, a significant effect is observed in antioxidant enzyme 2020b). However, bivalves are prone to be damaged in the antioxidant
SOD with an increase in activity with MIX 1 treatment, and no significant system due to the regulatory limits of GSH and its limited ability to regulate
effect is found with time in zebra mussel with a non-significant decrease oxidative damage under pressure and pH stress on prolonged exposure to
in activity at day 3 and 6 in MIX 2. However, S. Wang et al. (2020) and xenobiotics (S. Wang et al., 2020; Wang et al., 2020b).
Wang et al. (2020) in thick shell mussels under varying pH (81 and 7.7)
and concentration (0, 10, 104, and 106 particles/L) of 2 μm PS and 3.5.5. Glutathione peroxidase (GPX)
(Magni et al., 2020) in Zebra mussels using PVC of sizes 56 ± 35 μm at GPx is an enzyme containing seleno-cysteine in the active site. GPx cat-
16,875 PVCMDs/L and 41 ± 36 μm at 18,750 Mater-Bi® MDs/L, no signif- alyzes the reduction of various hydroperoxides (e.g., H2O2) to water (H2O)
icant effect was observed during the exposure. The rapid excretion of MPs and stable alcohol via oxidation of reduced glutathione (GSH) into its disul-
by mussels (short retention time) is influenced by the differences in sizes fide form, thereby, preventing the cell from oxidative damages (Marín-
and shapes that can be perceived as harmful by the defensive mechanisms García, 2014; Tabet and Touyz, 2007). GPx activity has been observed to
and the less sensitivity of SOD to oxidative damage that may not be caused be affected by the pH, and time duration of exposure with high stimulation
by the superoxide anion production (Browne et al., 2008; Magni et al., during the short-term (≤7) days and suppression after the long-term (≥7
2018; S. Wang et al., 2020; Wang et al., 2020b) leading to the non- days), and is unaffected by the concentration of the MPs similar to the
significant effect of SOD activity. However, SOD is induced in clams to elim- SOD, and GST (Li et al., 2021). In a study conducted by (Magni et al.,
inate the high production of ROS upon exposure to xenobiotics. Stephenie 2018) it was reported that MIX 1 caused a decrease in the GPx activity of
et al. (2020) reported that there is an advantage of supplementing SOD in mussels at t = 3 days with a recovery to the control activity at the end of
mammals, where plant microencapsulation is proven effective against var- exposure, and MIX 2 did not have a significant effect. S. Wang et al.
ious diseases after the animals and human trials. Similarly, bivalves can be (2020) and Wang et al. (2020b) observed that a pH of 7.7 showed a signif-
thought of as another source of SOD to animals and humans since they form icant effect on day 1 with an increase in GPx activity but no effect with the
the base of trophic transfer. Future studies should explore the potentiality of increase in time, and micro-PS did not affect its activity throughout the ex-
using bivalve's SOD in animals and human beings. periments for all treated groups. However, a partial similarity in trend was
observed in a study by Provenza et al. (2020), who recorded a decrease in
3.5.3. Catalase (CAT) activity at pH of 7.5. This finding is possibly due to the site of GPx activity
CAT is an anti-oxidation enzyme that converts H2O2 produced during used in the study, pH scale, and duration of exposure and measurement. At
various metabolic processes to less toxic water and oxygen molecules a pH of 7.5, GSH is highly utilized compared with a pH of 7.7, thereby re-
(Amorim et al., 2020; Samanta et al., 2014). Upon exposure to MPs, CAT ac- sulting in that observation. The two partially agreeing studies by S. Wang
tivities can either be altered or not altered with more effect occurring under et al. (2020); Wang et al. (2020b) and Provenza et al. (2020) enabled for
a higher concentration of MPs relative to the low concentration. Magni et al. more future studies to confirm the effect of MPs and pH (acidity and alka-
(2018) observed a substential increase in CAT activity in MIX 1 due to the linity) in bivalve's GPx. The studies on GPx reveal that regardless of the con-
formation of ROS and a decrease in CAT activity in MIX 2 when mussels centrations, MPs will activate the antioxidant system of the organism.
are exposed to MPs. S. Wang et al. (2020) and Wang et al. (2020b) observed However, the antioxidant system becomes distorted by the reduced defen-
that mussels exposed to a high concentration of MPs (106 particles/L) sive activities of GPX and other antioxidant enzymes with prolonged expo-
have significantly higher CAT activity than low (10 particles/L) at pH 7.7. sures. The survival and existence of bivalves and their corresponding food
However, in a study by Magni et al. (2020), no effect was found when chains are threatened.

9
R. Mkuye et al. Science of the Total Environment 838 (2022) 155933

4. Conclusion Arasappan, D., Fang, H., Perkins, R., Tong, W., 2010. Interpretation of toxicogenomics data.
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CRediT authorship contribution statement Capolupo, M., Franzellitti, S., Valbonesi, P., Sanz, C., Fabbri, E., 2018. Uptake and transcrip-
tional effects of polystyrene microplastics in larval stages of the Mediterranean mussel
Mytilus galloprovincialis *. Environ. Pollut. 241, 1038–1047. https://doi.org/10.1016/
Robert Mkuye: Writing – original draft, Data curation, Investigation, j.envpol.2018.06.035.
Validation. Shunlian Gong: Data curation, Writing – review & editing, Val- Cauwenberghe, L.Van, Janssen, C.R., 2014. Microplastics in bivalves cultured for human con-
sumption. Environ. Pollut. 193, 65–70. https://doi.org/10.1016/j.envpol.2014.06.010.
idation. Liqiang Zhao: Writing – review & editing, Validation, Data
Cauwenberghe, L.Van, Claessens, M., Vandegehuchte, M.B., Janssen, C.R., 2015.
curation. Fortunatus Masanja: Writing – review & editing, Validation. Microplastics are taken up by mussels (Mytilus edulis) and lugworms (Arenicola marina)
Charles Ndandala: Validation. Edna Bubelwa: Writing – review & living in natural habitats. Environ. Pollut. 199, 10–17. https://doi.org/10.1016/j.envpol.
editing. Chuangye Yang: Conceptualization, Resources. Yuewen Deng: 2015.01.008.
Chakraborty, S., Ray, M., Ray, S., 2021. Bivalve haemocyte adhesion, aggregation and phago-
Conceptualization, Writing – original draft, Project administration. cytosis: a tool to reckon arsenic induced threats to freshwater ecosystem. Fish Shellfish
Immunol. 114 (March), 229–237. https://doi.org/10.1016/j.fsi.2021.05.008.
Châtel, A., Hamer, B., Talarmin, H., Dorange, G., Schröder, H.C., Müller, W.E.G., 2010. Acti-
vation of MAP kinase signaling pathway in the mussel Mytilus galloprovincialis as bio-
marker of environmental pollution. Aquat. Toxicol. 96 (4), 247–255. https://doi.org/
Declaration of competing interest
10.1016/j.aquatox.2009.11.002.
Chen, L., Li, B., Hu, S., Qiao, R., Sun, C., Yang, D., 2020. A fas apoptotic inhibitory molecule
The authors have no conflicts of interest with the contents of this article from Ruditapes philippinarum: investigation on molecular characterization and func-
tional analysis. Fish Shellfish Immunol. 104 (January), 133–140. https://doi.org/10.
1016/j.fsi.2020.05.058.
Acknowledgements Chinfak, N., Sompongchaiyakul, P., Charoenpong, C., Shi, H., Yeemin, T., Zhang, J., 2021.
Abundance, composition, and fate of microplastics in water, sediment, and shellfish in
This work was supported by China Agriculture Research System of MOF the Tapi-Phumduang River system and Bandon. Sci. Total Environ. 781, 146700.
https://doi.org/10.1016/j.scitotenv.2021.146700.
and MARA, Science and Technology Program of Guangdong Province Cho, Y., Shim, W.J., Jang, M., Han, G.M., Hong, S.H., 2019. Abundance and characteristics of
(Grant No. 2021B0202020003, 2020A1515010691 and microplastics in market bivalves from South Korea. Environ. Pollut. 245, 1107–1116.
2021A1515011199), the National Natural Science Foundation of China https://doi.org/10.1016/j.envpol.2018.11.091.
Cho, Y., Joon, W., Jang, M., Myung, G., Hee, S., 2021. Nationwide monitoring of microplastics
(Grant No.32002369 and 32102817), Department of Education of Guang- in bivalves from the coastal environment of Korea *. Environ. Pollut. 270, 116175.
dong Province (Grant No. 2020ZDZX1045 and 2021KCXTD026), and https://doi.org/10.1016/j.envpol.2020.116175.
Guangdong Provincial Special Fund for Modern Agriculture Industry Tech- Claessens, M., Van Cauwenberghe, L., Vandegehuchte, M.B., Janssen, C.R., 2013. New
techniques for the detection of microplastics in sediments and field collected organ-
nology Innovation Teams, Department of Agriculture and Rural Affairs of isms. Mar. Pollut. Bull. 70 (1–2), 227–233. https://doi.org/10.1016/j.marpolbul.
Guangdong Province (Grant No. 2022KJ146). 2013.03.009.
Dai, L., Wang, Z., Guo, T., Hu, L., Chen, Y., Chen, C., Yu, G., Ma, L.Q., Chen, J., 2022. Pollution
characteristics and source analysis of microplastics in the Qiantang River in southeastern
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