# "Groundbreaking study: Combined effect of marine heatwaves and polyethylene microplastics on Pacific oysters, Crassostrea gigas"

Leila Parizadeh, Camille Saint-Picq, Pierrick Barbier, Arno Bringer, Valérie Huet, Emmanuel Dubillot, Hélène Thomas

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## To cite this version:

Leila Parizadeh, Camille Saint-Picq, Pierrick Barbier, Arno Bringer, Valérie Huet, et al. "Groundbreaking study: Combined effect of marine heatwaves and polyethylene microplastics on Pacific oysters, Crassostrea gigas". Environmental Pollution, 2025, 364, pp.125164. 10.1016/j.envpol.2024.125164. hal-04957810

HAL Id: hal-04957810

https://univ-rochelle.hal.science/hal-04957810v1

Submitted on 4 Apr 2025

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"Groundbreaking study: Combined effect of marine heatwaves and polyethylene microplastics on Pacific oysters, *Crassostrea gigas*"<sup>a,*</sup>

Leila Parizadeh<sup>a,*</sup>, Camille Saint-Picq<sup>a</sup>, Pierrick Barbier<sup>b</sup>, Arno Bringer<sup>c</sup>, Valérie Huet<sup>a</sup>, Emmanuel Dubillot<sup>a</sup>, Hélène Thomas<sup>a</sup>

ARTICLE INFO

Keywords:

Temperature

Microplastics

Combined environmental factors

Defence markers

Crassostrea gigas health

A B S T R A C T

Microplastics (MPs) and rising marine seawater temperatures are one of the major environmental problems threatening the survival of marine organisms and biodiversity. However, interactions between such multiple stressors are virtually unexplored. This study aimed to assess the combined effect of two temperatures and polyethylene MPs on the Pacific oyster *Crassostrea gigas*, one of the most globalized mollusc species for aquaculture. Our work highlights the potential ecological risk posed by these two factors on marine bivalve molluscs. The experimental design was carried out following a 14-day exposure of oysters to environmental concentrations of polyethylene MPs (0.01 mg.L⁻¹), and to two temperatures (15 °C and 22 °C). Sampling was performed on days 0, 7, and 14. The µ-FTIR analysis was applied to quantify MPs of interest and to check a potential environmental contamination. Tissue samples of digestive glands were collected from the oysters to evaluate the activity of biomarkers including superoxide dismutase, glutathione-s-transferase, malondialdehyde and laccase through protein levels. We note that the combination of MPs and high water temperature (HWT, 22 °C) had a significant impact both on the survival of animals and on stress markers, by modifying lipid peroxidation and immune responses. This original study gave the first innovative results on this topic and provides us with knowledge of the combined effects of MPs pollution and HWT (simulating marine heatwaves situation) on *C. gigas*. There remains a lack of information on the toxicity and the potential environmental hazard of plastics in the marine environment.

# 1. Introduction

In France, shellfish farming is the second most widespread form of aquaculture after fish farming, and the most widely produced species is *Crassostrea gigas*, representing almost 83% of European production (European Commission, 2022; Botta et al., 2020). However, following the continuous expansion of aquaculture, unusual mortality events have frequently been observed during the summer season worldwide. Because of the intense urbanisation and industrialisation around coastal zones, coastal waters are exposed to a growing number of harmful substances that threaten marine and coastal life (Landrigan et al., 2020; Gogoi et al., 2018; Taheran et al., 2018). Human activities have contributed to the occurrence of extreme weather events, such as marine heat waves (MHWs), an event in which water temperature rises above

seasonal normal for a duration exceeding 5 days (Intergovernmental Panel On Climate Change, 2023; Darmaraki et al., 2019a; Darmaraki et al., 2019b; Oliver et al., 2018; Hobday et al., 2016). Over the past 20 decades, MHWs have doubled in overall frequency, becoming longer, more severe, and more widespread (Collins et al., 2022). Rising frequency of MHWs appears to be associated with a worldwide surge in mass mortality events (Garrabou et al., 2022). Rising global temperature and extreme events like MHWs may have a significant impact on the metabolic engineering, reproducibility, physiological responses of aquatic animals, on marine biodiversity and on the disease development in invertebrates (Sharma et al., 2024; Li et al., 2023; Peruzza et al., 2023; Rolton et al., 2022). In the ark clam *Scapharca broughtonii*, high temperatures significantly enhanced the pathogenicity of the OsHV-1 virus (Xin et al., 2020). High temperatures, significantly reduced the survival

<footnote><sup>a</sup> Littoral Environnement et Sociétés (LIENSs, UMRi, 7266, CNRS, La Rochelle Université, 2 rue Olympé de Gouges, 17000, La Rochelle, France</footnote>

<footnote><sup>b</sup> Centre pour l'Aquaculture, la Pêche et l'Environnement de Nouvelle-Aquitaine (CAPENA), Prise de Terdoux, 17480, Le Château d'Oléron, France</footnote>

<footnote><sup>c</sup> Qualyse, 5 allée de l'Océan, 17000, La Rochelle, France</footnote>

<footnote>* This paper has been recommended for acceptance by Maria Cristina Fossi.</footnote>

<footnote>* Corresponding author.
E-mail address: leila.parizadeh@univ-lr.fr (L. Parizadeh).</footnote>
rate of *C. gigas*, infected with *Vibrio alginolyticus*, probably by disrupting
the gut microbiota and favouring the proliferation of the pathogen (*Li
et al.*, 2023). In clam *Cyclina sinensis*, high temperatures promote shell
growth but reduce soft tissue growth, negatively affecting the animal's
development and its physiological and biochemical responses (*Chen
et al.*, 2024). The sensitivity of species of coastal marine bivalves may
vary with the synergic effect of various anthropic activities, such as
MHWs and constantly increasing pollution (*Carneiro et al.*, 2021; *Ewere
et al.*, 2021; *Veiga et al.*, 2020). Moreover, raised temperatures com-
bined with pollutants can affect the capability of the marine invertebrate
population to respond to thermal stress in many estuaries and coastal
lagoons (*Barbosa et al.*, 2023; *Carneiro et al.*, 2021; *Marigómez et al.*,
2017). Mussels in mercury-contaminated seawater exhibited greater
biochemical alterations at higher temperatures (21 °C vs 17 °C) sug-
gesting a potential compounding effect between climate change and
marine pollution. Higher temperatures may increase marine organisms'
sensitivity to heavy metal pollutants or enhance the toxicity of these
pollutants in marine environments (*Coppola et al.*, 2021). In *Mytilus
galloprovincialis* mussels exposed to lead (Pb), temperature significantly
influenced antioxidant enzyme defence against reactive oxygen species
(ROS) with a more pronounced effect at 22 °C than at 17 °C, through
endogenous protein formation and superoxide dismutase (SOD) induc-
tion (*Pirone et al.*, 2019).

In addition, plastics are one of the most critical coastal pollutants,
and annually at least 14 million tons of plastic debris end up in aquatic
ecosystems, where they remain for years as a result of their high dura-
bility (Sharma et al., 2023; Choi et al., 2022; Dos Santos et al., 2021).
Plastics can undergo physiochemical degradation to form microplastics
(MPs), which are particles <5 mm in diameter (Hingant et al., 2023;
Choi et al., 2022; Bringer et al., 2021a). These pollutants are readily
absorbed by aquatic organisms, including fish and shellfish, and nega-
tively affect their growth, survival, reproduction and gut microbiota
(Vazirzadeh et al., 2025; Bringer et al., 2022; Hoseini et al., 2022;
Bringer et al., 2021a; Teng et al., 2021; Nobre et al., 2020), while posing
a risk of transfer of MPs to humans through consumption (N, S.M., A, K.
V., 2024; Li et al., 2022; Liu et al., 2022; Revel et al., 2020). MPs act as
vectors for persistent organic pollutants (POPs), heavy metal and drugs,
at concentrations far higher than those found in surrounding waters
(Tumwesigye et al., 2023). MPs provide a habitat for diverse microbial
communities, including antibiotic-resistant and pathogenic bacteria,
while also adsorbing heavy metals that can contribute to co-resistance
and the spread of antimicrobial resistance genes (ARGs), threatening
both marine and human health (Wu et al., 2024; Milani et al., 2024; Sun
et al., 2023; Caruso, 2019). By transporting chemicals, MPs also promote
the formation of biofilms, which could increase the transfer of hydro-
phobic organic pollutants to organisms that ingest them
(Zambrano-Pinto et al., 2024; Afianti et al., 2022; Pham et al., 2021).

Among the most common MPs polymers, polyethylene (PE) consti-
tutes 42% of the marine surface MPs samples (Erni-Cassola et al., 2019)
and is discarded in the environment due to its widespread use in pack-
aging, agriculture and cosmetics products (Dąbrowska et al., 2022;
El-Sherif et al., 2022; Ainali et al., 2021; James et al., 2020; Abidli et al.,
2019; Erni-Cassola et al., 2019; Koelmans et al., 2019; Abidli et al.,
2021; Carr et al., 2016). The high prevalence of polyethylene micro-
plastics (PE-MPs) in estuaries, other aquatic and terrestrial environ-
ments poses a threat to ecosystem health as a critical source of
environmental pollutants (El-Sherif et al., 2022; Jones et al., 2020;
Rochman, 2016).

Marine species such as oysters are especially vulnerable to MPs
exposure, as they are filter feeders that often occupy sediment layers, an
aquatic environment that is conducive to contaminant accumulation
(Baroja et al., 2021). Significant concentrations of MP particles could
accumulate in the soft tissues of various Molluscs (N, S.M., A, K.V., 2024;
Chinfak et al., 2024; Zhao et al., 2024), which act as sentinel species
involved in the trophic transfer of MPs (Ribeiro et al., 2024; D'Costa.,
2022; Bom and Sá, 2021). Indeed, exposure to MPs can affect the

growth, survival, reproduction rate, and defence biomarker responses,
as well as changing the intestinal microbiota of various marine organ-
isms (Bringer et al., 2022; Hoseini et al., 2022; Bringer et al., 2021a;
Teng et al., 2021; Nobre et al., 2020).

Compared to found concentrations in the environment, most studies
on MPs are carried out at much higher concentrations (50 to 10³ par-
ticles.ml⁻¹ (Wenclawiaket al. 2024; Capolupo et al., 2018) versus 10 or
100 particles.L⁻¹ (Alam et al., 2019; Zhu et al., 2019). For bivalve
models such as *C. gigas*, experimental studies have often tested con-
centrations of MPs, frequently finding very high levels (between 23 µg.
L⁻¹ and 2.5 g.L⁻¹) (Baroja et al., 2021; Nobre et al., 2020; Le Bihanic
et al., 2020) which are not representative of levels of MPs found in the
environment (Rani-Borges et al., 2022; Bringer et al., 2021a; Revel et al.,
2020). Due to technical challenges, most environmental studies focus on
particles > 300 µm, whereas MPs concentrations in surface waters
worldwide range from 0.002 to 0.5 mg.L⁻¹ (particles >10 µm), which
may introduce a bias leading to an underestimation of the ecological
impact of these anthropogenic pollutants. (Rani-Borges et al., 2022;
Bringer et al., 2021b; Fok et al., 2020; Enders et al., 2015; Eriksen et al.,
2013).

Our work aims to assess the impact of climate change and realistic PE-MPs concentrations on the health status of *C. gigas* and to enable early diagnosis of ecosystem health disturbances. Oxidative stress biomarkers provide a useful tool for early detection of environmental stress effects on bivalves (Milinkovitch et al., 2015; Revel et al., 2019; Bringer et al., 2022). So, we evaluate the biological response of *C. gigas* by studying the activity of SOD, glutathione-s-transferase (GST), malondialdehyde (MDA), and laccase (LAC), through protein levels and by monitoring the animal's survival rate, after an exposure to an environmentally relevant concentration of PE-MPs (0.01 mg.L⁻¹) (Labbé et al., 2024; Abidli et al., 2021; Revel et al., 2020) at two temperatures (15 °C and 22 °C) over 14 days. These experimental laboratory conditions were used to simulate a specific environmental situation in coastal areas around La Rochelle (France), along the French Atlantic coast in the Pertuis Charentais region. This is the first time that the joint effect of PE-MPs and HWT has been assessed in the Pacific oyster model, providing a better understanding of the physiological response of bivalves to environmental change.

2. Material and methods

2.1. Environmental temperature monitoring

Seawater temperature data were recorded using a WiSens CTD Autonomous data logger (NKE Instrumentation, Hennebont, France) placed in plastic bags (9 mm mesh) alongside the containers housing the oysters reared at the “La Mortane” site. The data logger was securely deployed on a cultivation table positioned approximately 70 cm above the sea bottom. Temperature measurements were acquired at 15-min intervals from January 1, 2020, to December 31, 2022. Data were cleaned, according to the depth parameter, to exclude records of air temperature when the probe was outside water. Due to instrument maintenance, data were not acquired on October 3, 13 and 14, 2020, November 23, 2021, or on December 4, 2022 (Fig. 1).

## 2.2. Animal collection

The study was carried out in La Rochelle (France, experimental site of LIENSs), in April 2023 with juvenile *C. gigas* (6.3 ± 0.89 cm long; 3.4 ± 0.52 cm wide; 1.9 ± 0.35 cm thick; 22.7 ± 7.89 g mass) originating from “La Mortane” oyster farming area in Marennes-Oléron bay (45°53,502' N; 001°10,060' W). These oysters originated from wild spat that settled on collectors during the summer of 2021 in the “Longées” oyster farming area (Fouras, France) and were subsequently reared for 18 months in plastic bags at “La Mortane”.
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Daily mean temperature (±sd) of the water column between January 2020 and December 2022 at "La Mortane" (Pertuis Charentais, France). Mean, minimum and maximum daily temperatures are represented by solid, dotted and dashed lines, respectively.</figcaption></figure>

## 2.3. MPs exposure and thermal challenge

The PE-MPs were obtained as a dry powder from Sigma-Aldrich Chemical (PE 34–50 µm 434272-100G, Micropowders Inc. USA). The MPs were added to the challenge tanks at 15 °C and 22 °C at a concentration of 0.01 mg.L⁻¹ in the 100 L buffer tank. Approximately every two days, 95% of the tank water was changed to ensure the well-being of the oysters. Brewers were used to homogenize the MPs in the tank water. The temperatures were selected to reflect the temperature of their natural environment on tidal area, as obtained by environmental temperature monitoring in "La Mortane" (Fig. 1). We choose the average seasonal temperature at the time of experimentation (15 °C) as our lowest temperature, and 22 °C as the highest temperature recorded on the foreshore. Based on data from the ECOSCOPA network with a normal seasonal seawater temperature of 20.7 °C on the Atlantic coast (Petton et al., 2024) and, those from CAPENA (Centre pour l'Aquaculture, la Pêche et l'Environnement de Nouvelle-Aquitaine, l'Ile d'Oléron, France) with threshold exceeding 23 times in 2020, 15 times in 2021 and 28 times in 2022, often for periods of 5–9 consecutive days (Fig. 1), 22 °C appears to be the right temperature to stimulate the MHWs.

## 2.4. Experimental design

A realistic experiment was carried out using tanks in a closed system containing 30 L of natural seawater from the Pertuis Charentais (South-West France) directly connected to the experimental site (La Rochelle, LIENS experimental site) and filtered with 50, 10 and 1 µm membranes (Diproclean). Oysters were distributed (N = 26 oysters/tank/condition) in 12 glass tanks (0.5 m in length, 0.25 m in width and 0.3 in height) placed under oxygen in a larger buffer tank (1 m in length, 0.3 m in width and 0.42 in height). We designed four experimental conditions (in triplicate) considering two temperatures (15 °C and 22 °C) and two combined conditions (15 °C + MPs and 22 °C + MPs) (Fig. 2).

The MPs concentration used was based on environmental concentration of 0.01 mg.L⁻¹ (Abidli et al., 2021; Bringer et al., 2021a; Revel et al., 2020; Le Bihanic et al., 2020). Oysters were acclimatised for 7 days, before being exposed to MPs for 14 days. A total of 18 oysters were collected on D0 (no MPs) and 36 oysters on D07 and D14 (all conditions). Physicochemical parameters such as temperature, salinity, oxygen, and pH were measured twice weekly using a HANNA Probe (HI9829 multiparameter), and a natural photoperiod was used. Oysters were fed phytoplankton naturally present in the marsh water.

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Experimental design of juvenile Pacific oysters exposed to two temperatures (15 °C and 22 °C) and two combined conditions (15 °C + PE-MPs and 22 °C + PE-MPs) over 14 days. A total of 18 oysters were collected on D0 (conditions without MPs) and 36 oysters on D07 and D14 (all conditions).</figcaption></figure>
## 2.5. Seawater's MPs analysis

The identification and quantification of all types of MPs from all collected seawater samples at the beginning and end of the study (D0 and D14 respectively) were performed by laboratory QUALYSE. All preparations and manipulations were carried out under a laminar flow hood to avoid external contamination. Glassware and utensils were cleaned with ultrapure water and filtered ethanol (0.2 µm). A blank was carried out to diagnose ambient MPs contamination. Volumes of 1000 mL were filtered on the Tulipe system. The organic content of the samples was quite low, so no pre-treatments (digestion, separation) were carried out. For filtration, 5 µm gold-coated polyester membranes were used (i3 TrackPor P, Germany), and a stainless-steel mesh with a porosity of 25 µm (The Mesh Company, UK). Filters were kept in a labelled Petri dish until being examined under a micro-FTIR (micro-FTIR, PerkinElmer Spotlight 400, USA). The system was operated by SPECTRUM IMAGE. Each filter was scanned one time for 1 h in the wave number range of 4000–690 cm⁻¹. Background spectrum imaging was collected from the gold filter at 25 µm pixels. To identify MPs structures in this study, a freeware, siMPle, developed by Aalborg University, Denmark, and Alfred Wegener Institute, Germany (https://simple-plastics.eu/), was adopted, and the target infrared spectrum was compared with the standard spectral library (PerkinElmer Library).

## 2.6. Animal sample processing

For each sample at D0, D07 and D14, biometric measurements were taken on oysters (n = 3/replicate). The dissected digestive glands (DG) were immediately submerged in liquid nitrogen to preserve the biochemical activities of interest. DG were ground in phosphate-buffered saline (PBS; pH = 7,2; 1100 mOSm) followed by 2 centrifugations (Sorvall Legend Micro 17r centrifuge, Thermo Scientific) at 4 °C, 12500 g for 15 min. Tissues were quick-frozen in liquid nitrogen until biomarker assays.

## 2.7. Condition index and animal survival rate

Oysters (n = 9/condition/day) were measured (length, width, and thickness) using a digital caliper, and weighed (total weight, wet meat, and empty shell). A condition index (CI) was calculated to compare the growth of exposed oysters with that of control oysters. CI is defined as the ratio of wet meat to total weight minus empty shell (Bodoy et al., 1986). The difference between the beginning and end of the exposure period was used to determine the delta CI. Oyster survival was assessed daily by removing and counting dead specimens in each treatment group.

## 2.8. Biomarker assays

Biochemical assays (SOD, GST, MDA and LAC) were measured at a specific absorbance using a spectrophotometer SpectroStar Nano. Biomarker's data are expressed as IU.mg protein, except for MDA (expressed as µM.mg protein). Total protein concentrations were determined using a single-point spectrophotometer at an absorbance of 562 nm and at 37 °C. All assays were determined according to previous publications (Barbarin et al., 2023; Barbarin et al., 2022; Breitwieser et al., 2017; Luna-Acosta et al., 2015; Luna-Acosta et al., 2011).

## 2.9. Data processing and statistical analyses

All statistical analyses were performed using R software (RStudio, version 4.1.3). The homogeneity and normality of variances was evaluated using the Levene and Shapiro-Wilk tests respectively. Survival rates and biomarkers data were analysed using Student's t-test for MPs and temperature separately. Survival rate results are presented in the form of a histogram, considering standard deviations on the mean of live

animals for each time. Biomarker results are presented as whisker boxes, considering means and medians. Combined effects of temperature and MPs were analysed with a two-factor ANOVA test for survival rate, and biomarkers studied. Significance difference was accepted when p-value <0.05. Principal component analysis (PCA) was carried out following the method of Barbarin et al. (2023) to assess the variability between conditions in relation to biomarkers.

# 3. Results

## 3.1. Environmental temperature monitoring

Daily mean temperature ranged between 6.01 °C and 22.08 °C during the monitoring. Overall, temperature increased from February to June, and stabilised at its warmest during summer before decreasing to its minimum in November (Fig. 1). Moreover, temperature oscillations between neap tide and spring tide were clearly visible in the water column, with higher variations during the day. During the warm periods (i.e. July to September) of the 3 years of monitoring, the mean temperature was 20.24 °C (±1.08). At the daily scale, the limit of 22 °C was exceeded for 23, 15 and 28 days in 2020, 2021, and 2022, respectively. Temperatures are consistent with those obtained from environmental monitoring at the "La Mortane" site (Fig. 1).

## 3.2. Identification of MP particles in sea water using µ-FTIR

The µ-FTIR analyses was applied to 1) assess the background value of all types of MPs in the natural seawater (NSW) used for the experiment (control condition at D0), 2) detect the MPs of interest (PE-MPs in exposure conditions) and 3) check a potential environmental contamination. Analyses showed the presence of PE-MPs in all combined conditions. A total of 80 MPs.L⁻¹ and 110 MPs.L⁻¹ were detected in the 15 °C + MPs and 22 °C + MPs conditions, respectively. PE-MPs was detected as an abundant polymer in each combined condition (57 MPs. L⁻¹ in 15 °C + MPs condition and 60 MPs.L⁻¹ 22 °C + MPs). In NSW samples (D0) and control condition at 15 °C (D14), PE-MPs was not detected but traces of polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS) and polyvinyl chloride (PVC) were detected. In the control condition at 22 °C (D14) traces of PE were detected. The control samples were contaminated with MPs, probably from plastic laboratory experimental equipment and atmospheric MP particles (Supplementary data (Table S2)).

## 3.3. Condition index and animal survival rate

The condition index (CI) a key indicator of shellfish meat quality was assessed at D0, D07 and end D14 of the experiment. No significant effects were observed between the four experimental conditions: 15 °C, 22 °C, 15 °C + MPs, and 22 °C + MPs. However, an increase in temperature (22 °C) in the presence of MPs (0.01 mg.L⁻¹) led to a significant reduction in oyster survival from D07 (88.89% survival rate on D07 and 81.94% on D14; t-test: P < 0.05) (Fig. 3).

## 3.4. Biomarker responses

Fig. 4 shows the results obtained for the defence markers in the digestive gland of juvenile oysters for all experimental conditions:15 °C, 22 °C, 15 °C + MPs, and 22 °C + MPs (Fig. 4A–D; Table S1).

### 3.4.1. SOD and GST assays

SOD activity was similar at 15 °C and 22 °C for 7 days, but increased significantly at 22 °C after 14 days of exposure (+24.11%; p-value <0.05) (Fig. 4A). In 22 °C + MPs condition, SOD activity significantly decreased at D07 and D14 (−44.33% and −44.21% respectively; p-value <0.05), compared to 22 °C condition. A significant decrease in SOD activity was also observed in 22 °C + MPs condition compared to 15 °C
<figure><img src="image_7.png" /><figcaption>Fig. 3. Histogram of percentage survival of *C. gigas* for each exposure condition reported at D0, D07, and D14. Standard deviations were calculated on the mean of live animals for each time (N = 25 oysters). Lower-case letters indicate significant differences between temperatures condition with MPs at D07 and D14 (P < 0.05). Upper-case letters indicate significant differences between MPs concentrations for 22 °C at D07 and D14 (P < 0.05).</figcaption></figure>

+ MPs at D07 and D14 (–81.66% and –75.41% respectively; p-value <0.05) (Fig. 4A). In 15 °C + MPs condition, SOD activity significantly increased at D07 and D14 (+30.30% and +37.60% respectively; p-value <0.05) compared to the 15 °C condition (Fig. 4A). GST activity decreased significantly in 22 °C + MPs condition at D07 (–63.47 %; p-

value <0.05), with a tendency to decrease at D14, compared to 15 °C + MPs condition (Fig. 4B). In 15 °C + MPs condition, GST activity increased significantly, compared to the 15 °C condition (+31.88%; p-value <0.05) (Fig. 4B).

### 3.4.2. MDA assay

Exposure to PE-MPs significantly reduced MDA levels at 15 °C, but had no effect at 22 °C (Fig. 4C). MDA levels showed non-linear modulation over time in 22 °C condition (+49.53% at D0, -24.75% at D07; p-value <0.05 and no significant changes at D14) compared to 15 °C condition (Fig. 4C). In 22 °C + MPs condition, MDA levels increased significantly since D07 (+42.22% at D07 and + 42.36% at D14; p-value <0.05) compared to 15 °C + MPs condition (Fig. 4C).

### 3.4.3. LAC assay

LAC activity increased at 22 °C compared to 15 °C, regardless of exposure to MPs. In 22 °C condition, a rising trend in LAC activity was observed at D0, with a significant increase since D07 (+56.44% at D07 and + 61.64% D14; p-value <0.05) compared to 15 °C condition. In 22 °C + MPs condition, LAC activity increased significantly since D07 (+40.41% at D07 and + 26.88% at D14; p > 0.05) compared to 15 °C + MPs condition. LAC activity showed a temperature-dependent response to MPs after 14 days: it decreased by 34.37% at 22 °C + MPs condition and increased by 29.51% at 15 °C + MPs condition compared to the respective controls (without MPs) (Fig. 4D).

### 3.5. PCA analysis

At D0, the PCA explained 26.6–53% of total variance among biomarkers analysed in the oyster’s DG (Fig. 5A). In absence of MPs the

<figure><img src="image_8.png" /><figcaption>Fig. 4. Effects of PE-MPs and temperature (alone or combined) on defence biomarker responses (SOD(A), GST(B), MDA(C), LAC(D)) in the DG of *C. gigas* juveniles over 14 days. For each biomarker, 9 oysters were analysed. Lower-case letters indicate significant differences between temperatures for one MPs concentration (P < 0.05). Upper-case letters indicate significant differences between MPs concentrations for one temperature (P < 0.05).</figcaption></figure>
ellipses are not overlapping between 15 °C and 22 °C, indicating an effect of temperature on oyster health. The biomarker activity of SOD, MDA and LAC increases under the 22 °C condition. After 7 days, the PCA represented 25.2–42.4% of total variance (Fig. 5B). No differences were observed at 22 °C with and without MPs as the ellipses overlap. The 22 °C conditions with and without MPs are driven by LAC activity and are significantly different from condition 15 °C, as the ellipses do not overlap. This condition lacks a dominant factor, whereas at 15 °C with MPs, the data are driven by SOD and GST activity. Upon reaching 14 days the PCA reported 27–35.2% of total variance (Fig. 5C). The ellipses of 15 °C and 22 °C conditions do not overlap, indicating a significant difference. The SOD activity is modulated by the presence of MPs at 15 °C + MPs condition. On the contrary, in the absence of MPs, the GST seems to drive it. The 22 °C conditions are partially different, as they are half crossing. The LAC activity and MDA response is modulated by 22 °C conditions (with and without MPs).

## 4. Discussion

Our study shows for the first time that the cocktail of pollutants consisting of HWT and MPs had an impact on the survival and antioxidant system of Pacific oyster *C. gigas*. Previous research has generally examined the effects of high temperatures and MPs on the physiological

state of molluscs separately, but less often combined effect of the both factors (Grimmelpont et al., 2024; Howarth et al., 2024; Georgoulis et al., 2024; Digka et al., 2024; Liu et al., 2023; Li et al., 2022; Bringer et al., 2022; Revel et al., 2020; Thomas et al., 2020). In addition, most of the studies carried out in recent years, have exposed marine species to concentrations of MPs well above those generally found in environment. Although these models are effective in generating observable responses, they may distort our understanding of the real ecological threats posed by current environmental concentrations of these pollutants (Baroja et al., 2021; Lenz et al., 2016; Qu et al., 2018; Shang et al., 2021). The originality of our study was to assess the combined effects of HWT and realistic PE-MPs concentrations (0.01 mg.L⁻¹) on the health of *C. gigas*, using CI, survival rate, and defence marker responses, which to our knowledge has never been demonstrated before.

Our results show that exposure to 0.01 mg.L⁻¹ PE-MPs combined with elevated temperature (22 °C) did not significantly affect the CI of *C. gigas* but significantly decreased their survival rate. Revel et al. (2020) similarly showed that a 10-day exposure to environmental concentrations of MPs did not alter the CI of oysters, but resulted in a 10% mortality. In contrast, Bour et al. (2018) found no effect on survival or CI of sediment-dwelling bivalves *Ennucula tenuis* and *Abra nitida* after one-month exposure to PE-MPs (up to 25 mg.kg⁻¹ sediment).

Some studies suggest synergistic effects of combined stressors on

<figure><img src="image_9.png" /><figcaption>**Fig. 5.** PCA of defence markers in DG of oysters exposed to PE-MPs and temperature (alone or combined) over 14 days. (A) D0 with axes account of 79.6% of the data, (B) D07 with axes account of 67.6% of the data and (C) D14 with axes account of 63.1% of the data. Ellipses correspond to the confidence interval in each condition.</figcaption></figure>
marine species' survival and CI. However, in *Queen Scallop Aequipecten opercularis*, a 30-day exposure to 2 °C temperature increase and 0.2 pH decrease did not affect CI (Kovačić et al., 2024). In contrast, for mussel species (*Mytilus edulis*, *M. galloprovincialis*) and oyster species (*C. gigas*, *Ostrea edulis*), the combined effects of temperature, food concentration, and oxygen levels significantly influence both CI and survival rates (Kamermans, P. and Saurel, C., 2022). Weber et al. (2020) found that for the freshwater mussel *Dreissena polymorpha* exposed to different PS-MPs concentrations (up to $10^4$ particles.mL$^{-1}$) and temperatures (14, 23 and 27 °C) for 14 days, survival decreased significantly at 27 °C, regardless of MPs concentration. Our results suggest that thermal stress may impact marine species survival more than MPs in multi-stressor scenarios. The combined effects of multiple stressors likely depend on species, environmental abiotic or biotic factors and intensity/frequency of the stress.

We reported that under low-temperature conditions (15 °C), the activity of SOD and GST was stimulated 7 days after exposure to PE-MPs (Fig. 4A–B). In contrast, we observed a significant reduction in SOD activity at 22 °C in presence of PE-MPs, as well as a negative modulation of GST activity (Fig. 4A–B), while MDA levels had significantly increased (Fig. 4C). On the one hand, these results are consistent with the findings of Li et al. (2022) who reported that short-term exposure (<7 days) to MPs will increase SOD and GST activities, whereas long-term exposure (≥7 days) resulted in suppression of these antioxidant enzymes. On the other hand, Ribeiro et al. (2017) showed that SOD activity increased in the gills and DG of *Scrobicularia plana* after 7 and 14 days of exposure to MPs, respectively. Multiple studies have indicated that rising temperatures can directly impact the metabolism of marine bivalves, disrupting their growth and homeostasis (Liu et al., 2023; Masanja et al., 2023; Zhang et al., 2023). It has been shown that long-term heat stress induces heat shock proteins, altering animal metabolism (De Marco et al., 2023). It was demonstrated by Teng et al. (2021) that exposure to MPs at concentrations of 0.01 and 1 mg.L$^{-1}$ for 21 days significantly impacts the survival rates and physiological responses of *C. gigas*. Recent studies have also reported significant combined impacts of MPs and high temperatures on the physiological responses of the shrimp *Artemia franciscana* (Han et al., 2021) and the fish *Oreochromis niloticus* (Hasan et al., 2023).

In the presence of environmental stressors and toxic pollutants, organisms generally produce large amounts of intracellular reactive oxygen species (ROS) with a considerable ability to alter cellular constituents and produce oxidative injury. As a protective strategy against oxidative toxicity, organisms activate their antioxidant defences to overcome this ROS overload (Scopetani et al., 2020; Yu et al., 2020; Prokić et al., 2019). As a primary antioxidant enzyme, SOD plays a key role in the first line of defence against the cytotoxic effects of ROS by catalysing the dismutation of superoxide anion radical ($O_2^-$) into oxygen ($O_2$) or hydrogen peroxide ($H_2O_2$) (Li et al., 2022; Ighodaro and Akinloye, 2018). On the other hand, GST, as a key detoxification enzyme, plays a crucial role in protecting invertebrates from ROS-induced damage by eliminating lipid peroxides and detoxifying potentially toxic substances, thereby maintaining cellular homeostasis (Bringer et al., 2022; Trestrail et al., 2020). An increase in the activity of antioxidant enzymes such as SOD and GST in organisms is a protective response that eliminates harmful free radicals (Shi et al., 2020). Here, the inhibitory response of SOD and GST in *C. gigas* following combined exposure to PE-MPs and HWT (22 °C versus 15 °C) is an indication that the organism may have suffered some degree of damage due to stress from the pollutant mixture.

It is indeed possible that the antioxidant enzymes' inhibition is correlated with the increased energy costs incurred by cells in their fight against oxidative stress (Urban-Malinga et al., 2022; Hamed et al., 2020). Increased levels of MDA activity, which reflect the presence of lipid peroxidation products generated by xenobiotic exposure, support this hypothesis. Moreover, MDA is an important marker that indicates the oxidative damages of cell membranes (Trestrail et al., 2020). We also showed that under high-temperature conditions (22 °C versus 15 °C),

the activity of LAC as a marker of environmental stress increased in DG of *C. gigas* over time in the absence of PE-MPs, while no significant change was observed in the presence of PE-MPs (Fig. 4D). Recently, similar effects were demonstrated in zebra mussel *Dreissena polymorpha*, with an increase in LAC activity associated with high temperature (25 °C versus 18 °C) (Matskiv et al., 2024).

In this study, we found that co-exposure to high temperatures and PE-MPs was more toxic to oysters than exposure to either factor alone. This result is in agreement with the PCA analysis, which showed that the alterations in detoxification capacity and oxidative stress were mainly linked to the co-exposure treatment (Fig. 5). Therefore, we proposed that at HWT (22 °C), the presence of PE-MPs (0.01 mg.L$^{-1}$) could disrupt SOD and GST activities in *C. gigas*, suggesting that the risk posed by co-exposure to these anthropic factors may be linked to the lipid peroxidation mechanism.

## 5. Conclusion

Our groundbreaking research highlights the combined effects of MPs and MHWs on the survival and biological responses of juvenile Pacific oysters, *C. gigas*, under relevant environmental conditions. Fourteen days of exposure to realistic concentrations of PE-MPs (0.01 mg.L$^{-1}$) at 22 °C seem to negatively affect the survival and immunological defence of oysters. PCA analysis revealed associations between defence biomarkers and exposure to PE-MPs + temperatures in *C. gigas*. This cocktail of anthropogenic factors significantly decreases the activity of the main antioxidant enzymes involved in defence and detoxification (like SOD and GST), but increases the oxidative damage levels (as measured by MDA) in DG of *C. gigas*.

This unique laboratory study of the short-term exposure of *C. gigas* to the dual stressors of high temperatures and PE-MPs shows that lipid peroxidation may play a crucial role in the response of oysters to high temperatures and MPs. Overall, our work has led to a better understanding of how the combination of environmental stressors, in particular PE-MPs pollution and MHWs, adversely affects the physiological well-being of the commercially important oyster *Crassostrea gigas*. In the future, it would be useful to conduct a long-term experiment to study the health status of *C. gigas* in a changing environment, focusing on behavioural and molecular responses (gene expression, energy consumption): a complementary approach to our present study.

### CRediT authorship contribution statement

**Leila Parizadeh:** Writing – original draft, Conceptualization, Investigation, Formal analysis, Visualization, Project administration, Funding acquisition.
**Camille Saint-Picq:** Investigation, Formal analysis, Writing – Review & Editing.
**Pierrick Barbier:** Formal analysis, Writing – Review & Editing.
**Arno Bringer:** µ-FTIR acquisition data, Writing – Review & Editing.
**Valerie Huet:** Methodology.
**Emmanuel Dubillot:** Methodology.
**Hélène Thomas:** Conceptualization, Investigation, Methodology, Validation, Writing – Review & Editing.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

The funding was supported by the La Rochelle University (Actions incitatives ACI Project POLYENVI 2023). The authors would like to thank the Centre pour l'Aquaculture, la Pêche et l'Environnement de Nouvelle-Aquitaine (CAPENA, Le Château d'Oléron, France) for providing the oysters. We would like to thank Mrs. Lucie Rees and Mrs. Valérie Thiery for reviewing the language of the manuscript.
# Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2024.125164.

## Data availability

Data will be made available on request.

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