# Are alternatives to polyethylene less toxic than conventional polyethylene? A case study of MP toxicity on Scrobicularia plana using an Integrative Biomarker Response index (IBR-T)

Clémentine Labbé, Isabelle Métais, Hanane Perrein-Ettajani, Mohammed Mouloud, Antoine Le Guernic, Oihana Latchere, Nicolas Manier, Amélie Châtel

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Clémentine Labbé, Isabelle Métais, Hanane Perrein-Ettajani, Mohammed Mouloud, Antoine Le Guernic, et al.. Are alternatives to polyethylene less toxic than conventional polyethylene? A case study of MP toxicity on Scrobicularia plana using an Integrative Biomarker Response index (IBR-T). Aquatic Toxicology, 2026, 291, pp.107680. 10.1016/j.aquatox.2025.107680. hal-05426780

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# Are alternatives to polyethylene less toxic than conventional polyethylene?
## A case study of MP toxicity on *Scrobicularia plana* using an Integrative Biomarker Response index (IBR-T)

Clémentine Labbé<sup>a,∗</sup> <sup> ID </sup>, Isabelle Métais<sup>a</sup>, Hanane Perrein-Ettajani<sup>a</sup>, Mohammed Mouloud<sup>a</sup>, Antoine Le Guernic<sup>a</sup>, Oihana Latchere<sup>a</sup>, Nicolas Manier<sup>b</sup>, Amélie Châtel<sup>a</sup>

ARTICLE INFO

Keywords:

Polylactic acid
Oxo-polyethylene
Bivalve
Biomarkers
Burrowing
Comet assay

ABSTRACT

Microplastics (MP) are now widely contaminating multiple ecosystems. To mitigate this pollution, the development of plastic alternatives is being promoted. However, scientific data on the absence of toxicity of these alternatives is still lacking. The present study assesses the impact of petroleum-based fragmentable MP, oxo-polyethylene (oxoPE), and biobased biodegradable MP, polylactic acid (PLA), on the bivalve *Scrobicularia plana*. Clams were exposed for 21 days to environmental concentrations (0.008, 10 and 100 µg L⁻¹) of both PLA and oxoPE MP and impacts were evaluated from individual to molecular levels. The effects of alternative MP were then compared with polyethylene MP ones, resulted from a previous study. An Integrative Biomarker Response - Threshold index (IBR-T) was calculated to assess the differences in toxicity between alternative and conventional plastics. An impact of PLA and oxoPE MP was shown on energy reserves, with a reduction in glycogen concentration in organisms exposed to the highest concentration (100 µg L⁻¹). A decrease in burrowing was also reported in *S. plana* exposed to 0.008 and 100 µg L⁻¹ of PLA MP. At cellular and molecular levels, catalase (CAT) and glutathione-S-transferases (GST) activities decreased after exposure to PLA MP (10 and 100 µg L⁻¹ respectively) suggesting a disruption of antioxidant and detoxication systems, while no impact was noted on DNA damage, whatever the biodegradable MP. Analysis using IBR-T revealed greater effects of PLA (100 µg L⁻¹) on *S. plana* physiology compared to the different types of PE MP tested, while oxoPE MP showed limited toxicity. These findings support the importance of assessing the toxic effects of biodegradable and fragmentable MP to evaluate whether they can really be considered as a reliable alternative to conventional plastics.

1. Introduction

Plastic manufacturing and accumulation of plastic waste in the environment is continuing to increase worldwide. Global plastics production was estimated at 413,8Mt in 2023 (Plastics Europe, 2024) and is expected to reach 736Mt in 2040 (OECD, 2024). The most widely produced plastics in the world are so-called “conventional” plastics, such as polypropylene (PP), polyethylene (PE), polyvinyl chloride (PVC) and polystyrene (PS) (Plastics Europe, 2024). Most of these plastics are derived from fossil fuels and none of them can be degraded rapidly in natural ecosystems. In 2019, around 20Mt of plastic waste were released into the environment (OECD, 2022) resulting in pollution and toxicity problems for aquatic and terrestrial ecosystems (MacLeod et al., 2021).

Thus, to decrease both pollution and toxicity, alternatives to conventional plastics were developed such as bioplastics. They include bio-based or “bio sourced” (e.g. starch blends, cellulose films, polylactic acid) or petroleum-based (e.g. PBAT) biodegradable plastics but also biobased plastics that are not biodegradable (Paul-Pont et al., 2023). The biodegradability of a material is defined as its capacity to be completely transformed by the action of microorganisms into natural substances such as water, carbon dioxide or mineral salts. This degradation is subject to abiotic and biotic environmental conditions, i.e. temperature, humidity, the presence of oxygen or microorganisms (ISO 472, 2013). In 2024, the global bioplastics production was estimated at 2.47Mt. Biodegradable plastics accounted for 56 % of this production, with a predominance of polylactic acid (PLA)

<footnote><sup>a</sup> Biology of Organisms Stress Health Environment (BIOSSE), Université Catholique de l'Ouest, Angers, France</footnote>

<footnote><sup>b</sup> Institut National de l'Environnement Industriel et des Risques (INERIS), Parc Technologique ALATA, 60550, Verneuil-en-Halatte, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: clabbe@uco.fr (C. Labbé).</footnote>
(EUBP_Market_Data_Report_2024.pdf s. d). PLA combines the double advantage of being bio-sourced and biodegradable. PLA is produced from plants in the form of fibers, films, sheets, coatings intended to produce objects for the medical and agricultural as well as packaging, textile and even electronic sectors. Its strength properties and reduced production costs are drawing increasing attention to this material as an alternative to conventional plastics (Schneiderman and Hillmyer, 2017). Other types of alternatives were also developed, in particular the oxo-degradable plastics (e.g. petroleum based oxo-polyethylene (oxoPE)) containing an oxo-degradation catalyst such as metal complexes as a pro-oxidant additive enhancing its supposed entire degradation under first external factors such as heat, oxygen or UV light and then under the presence of microorganisms as fungi or bacteria (Markowicz and Szymańska-Pulikowska, 2019). Their use as an environmental alternative to standardized plastics is, however, controversial as their degradation can contribute to total plastic contamination (European Commission, 2018). Although they have been banned in the European Union since 2019, they can still be marketed in other countries, and it is essential not to exclude them from studies on plastics toxicity.

In the natural environment, the conditions required for optimal biodegradation of biodegradable plastics are not always met. For example, PLA degradation can occur under industrial and home composting but requires relatively high temperature (thermophilic temperature of around 55 °C) to improve it, without any prior chemical modification of the virgin polymer (Paul-Pont et al., 2023). Thus, despite the promised biodegradability of these bioplastics, their degradation is not complete and could even lead to faster production of microplastics (Cao et al., 2024; Shi et al., 2024). Indeed, once released into the environment, under the effect of either abiotic and biotic factors, macroplastics undergo aging and fragmentation leading to production of microplastics (MP, plastic particles between 1 µm and 5 mm in size). These generated MP, called secondary MP, are found dispersed throughout all ecosystems mixed with primary MP, fragments of plastic produced in sizes smaller than 5 mm (Thompson et al., 2024). Shi et al. (2024) reviewed the concentration of biodegradable MP in various aquatic environments, showing their occurrence mostly in untreated wastewater, sewage sludge and sediment samples. PLA MP were predominant in all environmental compartments with concentrations comprised between 90 and 180 µg g⁻¹ (Okoffo et al., 2022). In addition, as with conventional plastics, various additives are added during the production of bioplastics and may be released as they degrade in the environment (Cao et al., 2024). Once in the environment, MP can also adsorb other pollutants, contributing significantly to the contamination of global ecosystems. Small sizes of plastics, when consumed by organisms at each level of the food chain, contribute to altering human, plant, animal, and ultimately the whole environmental health.

The toxicity of biodegradable MP was previously studied on various organisms, but was mostly focused on PLA MP toxicity in soil environment. Thus, Parolini et al. (2024) compared the impact of PET MP and PLA MP on the earthworm *Eisenia foetida* and found a modified oxidative status on worms exposed to PLA for 28 days (1 g and 10 g kg⁻¹ of soil, 164 µm). Other studies focused on microorganism communities and reported that PLA MP could alter soil microbial functionalities or soil bacterial composition (Cao et al., 2024). Concerning aquatic environment, bivalves are particularly exposed to MP as filter feeders present in almost all aquatic ecosystems and have previously been widely studied in terms of MP ingestion, bioaccumulation and response to toxicity (Khanjani et al., 2023). Aiming to study the spatial distribution of MP accumulated in tissues of different bivalves, Ribeiro et al. (2025) found biobased polymers as PLA MP in oysters and mussels soft tissues. Concerning toxicity, previous studies found a reduced glycerophospholipids content on *Mytilus edulis* exposed to 10 and 100 µg L⁻¹ of PLA MP for 8 days (Khalid et al., 2021) and an alteration of the hemolymph proteome after a 52-day exposure to PLA MP (25 µg L⁻¹, 65.6 µm) (Green et al., 2019). Green (2016) conducted a study that exposed a marine benthic

community to MP: PE and biodegradable PLA exposure for 60 days resulted in a significant reduction in species richness and total number of organisms. Furthermore, the abundances of periwinkles (*Littorina sp.*) and isopods (*Idotea balthica*), as well as the biomass of clams (*Scrobicularia plana*), decreased at the highest tested concentrations of 80 µg L⁻¹. The same study showed elevated respiration rates on the oyster *Ostrea edulis* after exposure to PLA MP (80 µg L⁻¹, 65.6 µm) for 60 days compared to exposure to HDPE MP (80 µg L⁻¹, 102.6 µm) (Green, 2016).

The rare studies on this subject do not allow us to fully estimate whether bioplastics could serve as good alternatives to conventional plastic. In the present study, the impact of MP from a petroleum-based plastic alternative, oxoPE, and a biobased biodegradable plastic, PLA, was tested on the key estuarine bivalve *Scrobicularia plana*. The objectives of this study were (1) to evaluate the toxic effects of both alternative MP on *Scrobicularia plana* during a 21-day laboratory exposure to environmental concentrations (0.008, 10 and 100 µg L⁻¹) of MP from the molecular to the individual level; (2) to compare the effects of these alternative MP with those observed in *S. plana* during exposure under the same laboratory conditions to different types of PE MP (Labbé et al., 2024) using an integrating index (IBR-T) (Catteau et al., 2023).

## 2. Materials and methods

### 2.1. Preparation and characterization of microplastics

In this study, two types of commercially available plastic products were used to compare oxo-degradable and bio-based biodegradable plastics toxicity. PLA pellets (Ingeo™ Biopolymer 4043D, NatureWorks LLC) and oxoPE garbage bags (D₂w refuse sacks, Symphony environmental) were purchased. Both types of plastics were ground using a cryogenic mechanical miller (SPEX, 6770 Freezer-mill) filled with liquid nitrogen. Before cryogrinding, the oxoPE bags were manually cut into pieces of approximately 1 cm². The obtained plastic particle powders were then sieved for 60 min, on a stainless-steel sieve of 400 µm as in Jaouani et al. (2023). The entire fraction of particles <400 µm was retained. An additional morphometric analysis (Morphologi 4-ID, Malvern) was performed on MP powders to characterize particles size and shape (>1 µm) as described in Labbé et al. (2024). The maximum particle size (Max. Distance) was defined as the maximum distance between any two points on a particle and was given in µm. The shape of a particle was characterized by the value of the Aspect Ratio corresponding to the ratio between its width and length. This value ranged from 0 (fiber-like particles) to 1 (spherical particles).

### 2.2. Suspensions of microplastics

For each type of microplastic particles (PLA and oxoPE), stock suspensions at concentrations of 1 and 0.1 g L⁻¹ were prepared in ultrapure (Milli-Q) water, pH 7. Working solutions of 0.1 mg L⁻¹ were then obtained by three ten-fold serial dilutions of the 0.1 g L⁻¹ stock suspension in ultrapure water (Milli-Q), pH 7 (Revel et al., 2019). A specific volume of the stock suspensions (1 g L⁻¹ or 0.1 g L⁻¹) or of the working solution (0.1 mg L⁻¹) was distributed into the aquaria to obtain the final concentrations of 0.008, 10, and 100 µg L⁻¹. These concentrations were chosen according with Labbé et al. (2024). No surfactants were used during the MP preparation to avoid any additional effects. Each solution was hand mixed before adding to the aquaria. All MP solutions were added in the aquaria every 3 days, immediately after the water change, to maintain the same concentration throughout the exposure.

A Transmission electron microscopy (TEM) analysis was performed on 1 g L⁻¹ stock solutions to detect the presence of particles smaller than 1 µm (Labbé et al., 2024). Briefly, solutions were filtered on 5 µm filter units (Sartorius Minisart®) to eliminate the larger particles. Three microliters of each solution were deposited on Formvar Carbon coated 200 mesh copper grids (Agar Scientific AGS162) pre-treated by glow discharge and then let to evaporate for 2 h at room temperature.
Samples were imaged with a JEOL JEM-1400 PLUS transmission electron microscope at 120 kV (JEOL) and recorded with a JEOL CCD Camera Ruby (8 M pixel) using JEM1400 PLUS software.

## 2.3. Organism sampling and laboratory exposure assay

Individuals of *S. plana* were hand-collected at low tide in sediment of a bay of the French Atlantic coast (47°26′36″ N, 2°27′4″ W; Pont-Mahé) in April 2023. Clams were brought back to the laboratory in metal buckets filled with water and sediment from the sampling site and introduced into aquaria (30 L) containing 27 L of aerated artificial seawater (Tropic Marine) at 25 psu in a temperature-controlled room at 17 ± 1 °C for an acclimatization period of 7 days. A photoperiod of 12:12 h was set up. A total of 427 clams were distributed in seven glass aquaria (61 clams per aquaria). The artificial seawater was fully renewed every 3 days before adding the MP solutions. Salinity and temperature were monitored daily throughout the exposure period and did not show any significant variations in any of the aquariums. The clams were fed once a week with the marine microalgae *Tetraselmis suecica* (Teramer) (70,000 cells/clam/L).

Clams were then exposed for 21 days to two MP conditions: oxo-polyethylene microplastics (oxoPE MP, <400 µm) and polylactic acid microplastics (PLA MP, <400 µm) at three different concentrations: 0.008 µg L⁻¹, 10 µg L⁻¹, and 100 µg L⁻¹; and to a negative control without any added plastic. The three tested concentrations were chosen according to environmentally relevant MP concentrations used in Revel et al. (2020), representative of coastal regions and gyres.

## 2.4. Ecophysiology measurement

The physiological status of exposed clams was monitored through several parameters including the condition index (CI), the clearance rate (CR) and energetic reserves status.

### 2.4.1. Condition index

At the end of the acclimatization period, the physiological status of the clams at T0 was determined on 10 sampled individuals. After 21 days exposure (T21), 10 clams per condition were sampled. The total and flesh weights of clams were determined individually. The condition index (CI) was calculated as follows:

$$CI=[(\text{wet weight of soft tissues}/\text{total wet weight})*100] \ (\text{AFNOR, 1985})$$

Although dry weight is generally used to calculate the condition index, the wet weight condition index was chosen in our study to limit the number of individuals required, as it allows measurements of IC and energy reserves on the same specimens. A positive correlation between dry weight and wet weight was reported in *S. plana* allowing the integration of the wet weight in the calculation of the condition index (Mouneyrac et al. 2008).

### 2.4.2. Clearance rate

Clearance rate (CR), defined as the rate at which clams can remove particles from solution (Salerno et al., 2018), was estimated for clams previously exposed for 21 days to PLA, oxoPE MP (0.008, 10, and 100 µg L⁻¹) or to artificial seawater without added MP (control condition) using a system adapted from Revel et al. (2019). Clams (n = 3) were placed individually on metal grids immersed in 600 mL beakers filled with 400 mL of water from the aquaria for 1 h before starting measurements. Clams were then fed with *Tetraselmis suecica* (Teramer) at an initial concentration of 300,000 cells mL⁻¹. A fine bubble aeration was installed on the beaker wall to promote mixing without resuspending feces. The clearance rate was measured by assessing the concentration of the algal solution every 15 min for 45 min according to the classical algal cell counting technique on Malassez counting chamber (Utermöhl, 1958) as previously described in Vidal et al. (2023). The clearance rate was expressed as the number of cells of *T. suecica* per clam per hour.

### 2.4.3. Energy reserves

The status of energy reserves was evaluated by measuring glycogen, lipid and protein concentrations in the total homogenate of 10 clams at the beginning of experiment and after a 21-day exposure to PLA MP, oxoPE MP or no MP (control) as in Vidal et al. (2023) with a minor modification of the grinding buffer (Citrate buffer (0.1 M citric acid monohydrate, 0.1 M sodium citrate tribasic dihydrate)). Lipid, glycogen and protein concentrations were expressed in mg g⁻¹ of wet weight (ww).

## 2.5. Burrowing test

Burrowing tests were performed at T21 as described in Métais et al. (2023). The burial behavior was evaluated by placing 10 individuals per condition on the sediment surface in the dark and observing how many of them burrowed at frequent intervals: every 5 min for the first hour, 10 min for the second hour, and 20 min for the third to the sixth hours. The percentages of unburied individuals over time were determined for each condition.

## 2.6. Biomarkers

### 2.6.1. Enzymatic activities

Enzyme activities (catalase (CAT), glutathione-S-transferases (GST), acetylcholinesterase (AChE) and acid phosphatase (AcP)) were quantified in gills and digestive gland tissues of 9 clams per condition according to the protocols used in Métais et al. (2023a). The same individual homogenate was used to evaluate all enzyme activities. All specific activities were expressed either in µmol min⁻¹ mg protein⁻¹ (CAT) or in nmol min⁻¹ mg protein⁻¹ (GST, AChE, AcP).

### 2.6.2. Genotoxicity (comet assay)

The comet assay was carried out as previously described by Métais et al. (2023a) only on gills of three clams per condition. One hundred nuclei were analyzed per slide and observed using an optical fluorescence microscope (Olympus BX60, × 40) equipped with a CDD camera (Lucas-S, Andor Technology) and a Komet 6 image analysis system (Kinetic Imaging Ltd). DNA damage was expressed as percentage of the DNA present in the comet tail (% Tail DNA) for each nucleus.

## 2.7. Quality control

To prevent cross-contamination, all equipment used in the laboratory was previously rinsed with milliQ water and 70 % ethanol, dried at room temperature in a hood and stored under aluminum foil to avoid contact with ambient air. During the experiments, cotton laboratory coats and nitrile gloves were worn.

## 2.8. Integrated Biomarker Response - Threshold (IBR-T) analysis

To visualize significant changes in selected biomarkers and to compare conditions, an IBR-T (“Integrated Biomarker Response – Threshold”) was calculated as described by Catteau et al. (2023), with some modifications. Instead of using threshold values as preconized by Catteau et al. (2023), first, only deviation indices of biomarkers statistically significant from exposed conditions compared to the control one were kept for the IBR-T calculation. Secondly, as threshold values were not assessed, variations in biomarkers of the exposed conditions compared to the control condition were considered, whether they were induction or inhibition. IBR-T was calculated on the mean of deviation indices using 16 biomarkers grouped in four categories: i) physiological biomarkers (condition index, clearance rate at 30 and 45 min and energy reserves), ii) behavior marker (burrowing behavior), iii) enzymatic biomarkers (CAT, GST, AChE and ACP activities in gills and digestive gland) and iv) genotoxicity biomarker (comet assay in gills).

In addition, for data comparison, an IBR-T calculation was
performed on data obtained from a previous study (Labbé et al., 2024) carried out under the same conditions (MP concentrations, exposure duration, species) as the present study, but with different types of MP: virgin LDPE (vPE), artificially aged LDPE (aPE) and PE from the field (fPE).

## 2.9. Statistical analysis

Statistical analysis was carried out using XLSTAT 2019 (version 21.4.63762) and R software (version 4.0.3). Normality of data distribution and variances homogeneity among groups were tested using the Shapiro-Wilk test and Bartlett test, respectively. If the data followed the conditions for applying parametric tests, differences between the treatments were tested using an ANOVA. A significative ANOVA was followed by a Tukey post hoc test to determine which means were significantly different. If the data did not comply with the parametric tests' conditions, Kruskal-Wallis test was used to test differences between the treatments. If the overall test was significant, a Dunn procedure was performed to determine which means were significantly different. For the burrowing test, the percentages of unburied individuals were first ln-transformed to linearize the data and then the regression coefficients of the least-square regression lines were compared using a covariance analysis (ANCOVA). In all cases, p-values ≤ 0.05 were considered statistically significant.

# 3. Results

## 3.1. Characterization of microplastics powders and exposure solutions

Morphometric analysis of the MP powders showed a range of particle sizes between 1.3 and 487 µm for PLA MP (mean size, 14 µm) and between 1.3 and 547 µm for oxoPE MP (mean size, 31 µm). Median particle sizes were 3.7 µm and 5.2 µm, for PLA and oxoPE powders, respectively (Table S1). A diversity of particle shapes was recorded in both MP powders with a mean aspect ratio of 0.7235 for PLA MP and 0.5865 for oxoPE MP. Regarding nanoparticle analysis, the presence of nano-plastics (<1 µm) was revealed by TEM imaging in both tested solutions (Fig. 1). Calibrated spherical nanoparticles were also detected in oxoPE

solution (Fig. 1–F).

## 3.2. Physiological status

No significant differences in the mean of CI were observed between clams at the beginning of the exposure (T0) (19.73 ± 4.03) and clams of the control condition after 21 days of exposure (17.72 ± 2.87) and between clams of the control condition at 21 days and those of all other MP conditions at 21 days. The mean CI of the clams was comprised between 17.72 ± 2.87 and 20.51 ± 2.57 (Table S2).

Concerning the clearance rate, a 21-day exposure to MP did not significantly impact the CR of *S. plana* regardless of MP types, concentrations and measurement times (Fig. S1).

Regarding the status of energy reserves, a 21-day exposure to the control condition (CTL) and to PLA (PLA) or to oxoPE at 0.008, 10, and 100 µg L⁻¹ did not modify the content of protein, lipid and glycogen in *S. plana* compared to the beginning of exposure (T0) (Fig. 2). However, a significant decrease in glycogen concentration was observed in *S. plana* exposed for 21 days to 100 µg L⁻¹ of PLA and oxoPE, compared with unexposed organisms (CTL) (Fig. 2–C).

No significant difference was noted in the protein and lipid content of clams after a 21-day exposure to PLA and oxoPE compared to the control condition, regardless of the exposure concentration (Fig. 2–A and B).

## 3.3. Burrowing test

The burrowing kinetics decreased in *S. plana* exposed to 0.008 µg L⁻¹ and to 100 µg L⁻¹ of PLA (slopes = -0.0012 and -0.0014, respectively) compared with controls (slopes = -0.0047). No significant effect was observed in *S. plana* exposed to oxoPE as compared to controls (Fig. 3). Regarding the effect of concentration inside a condition, the burrowing kinetics was impaired in *S. plana* exposed to 0.008 µg L⁻¹ and to 100 µg L⁻¹ of PLA compared to 10 µg L⁻¹ of PLA (slope = -0.0039). A decrease was also reported in *S. plana* exposed to 0.008 µg L⁻¹ of oxoPE (slope = -0.0027) compared to 10 µg L⁻¹ of oxoPE (-0.004) (Table 1). Comparison between exposure to PLA and oxoPE at the same concentration showed no difference at 10 µg L⁻¹. However, exposure to PLA at 0.008 µg L⁻¹ and 100 µg L⁻¹ (slopes = -0.0012 and -0.0014, respectively)

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** TEM images of different particles observed in the stock solutions (1 g L⁻¹, 5 µm filtered) of PLA MP (A, B and C) and oxoPE MP (D, E and F).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Proteins (A), lipids (B) and glycogen (C) content in whole organism of S. plana at the beginning of exposure (T0) and after a 21-day exposure to the control condition (CTL) and to PLA (PLA) or oxoPE (oPE) at 0.008, 10, and 100 µg L⁻¹ (mean ± SD, letters a, b, c for significant differences between conditions *p* < 0.05 and the red star for significant differences between conditions and control *p* < 0.05).</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Percentage of unburied clams S. plana (n = 10) at different times (min). Animals were previously exposed for 21 days to the control condition and to PLA (PLA) or oxoPE (oPE) at 0.008, 10, and 100 µg L⁻¹ (red star for significant differences between conditions and control *p* < 0.05).</figcaption></figure>

**Table 1**

Slopes of burrowing tests for S. plana in the control condition and after a 21-day exposure to PLA (PLA) or oxoPE (oPE) microplastics at 0.008, 10, and 100 µg L⁻¹ (N = 10).

<table><thead><tr><th></th><th>Concentrations (µg L<sup>-1</sup>)</th><th>Slopes</th></tr></thead><tbody><tr><td>Control</td><td>-</td><td>- 0.0047 <sup>ac</sup></td></tr><tr><td rowspan="3">PLA</td><td>0.008</td><td>- 0.0012 <sup>b</sup></td></tr><tr><td>10</td><td>- 0.0039 <sup>ac</sup></td></tr><tr><td>100</td><td>- 0.0014 <sup>b</sup></td></tr><tr><td rowspan="3">oPE</td><td>0.008</td><td>- 0.0027 <sup>a</sup></td></tr><tr><td>10</td><td>- 0.004 <sup>c</sup></td></tr><tr><td>100</td><td>- 0.0035 <sup>ac</sup></td></tr></tbody></table>

Superscript letters a, b, c for significant differences between conditions and in bold for significant differences between control and conditions *p* < 0.05.

impaired burial rate compared to exposure to oxoPE at these concentrations (slopes = - 0.0027 and - 0.0035, respectively) (Table 1).

### 3.4. Enzymatic biomarkers

The detoxication system and oxidative stress in clams were measured through analysis of GST and CAT activities, respectively. In gills of *S. plana*, a 21-day exposure to 100 µg L⁻¹ of PLA significantly impaired GST activity compared to control organisms and to clams exposed to 0.008 µg L⁻¹ of PLA. In addition, a decrease in CAT activity was observed in gills of clams exposed to 10 µg L⁻¹ of PLA for 21 days compared to control group and to organisms exposed to 0.008 µg L⁻¹ of PLA (Fig. 4- A and C). Regarding the neurotoxicity and the immunity function, no effect of the tested MP was observed for AchE and AcP activity respectively in gills of exposed clams (Fig. 5- A and C).

In digestive mass, no significant difference was observed between tested conditions regardless of the analyzed biomarkers (Fig. 4 and
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Glutathione-S-transferase and catalase activity in gills (A, C respectively) and digestive glands (B, D respectively) of S. plana after a 21-day exposure to the control condition (CTL) and to PLA (PLA) or oxoPE (oPE) at 0.008, 10, and 100 µg L⁻¹ (mean ± SD, letters a, b, c for significant differences between conditions *p* < 0.05 and the red star for significant differences between conditions and control *p* < 0.05).</figcaption></figure>

Fig. 5- B and D).

**3.5. Genotoxicity**

In gills, after 21 days of exposure, no significant difference in DNA damage was observed in clams exposed to PLA or oxoPE compared to control organisms, regardless of the exposure concentration. However, DNA damage was significantly increased in S. plana exposed to 100 µg L⁻¹ of PLA as compared to organisms exposed to 0.008 and 10 µg L⁻¹ (Fig. 6).

**3.6. Integrated Biomarker Response - Threshold (IBR-T) analysis**

The IBR-T scores for biodegradable MP (PLA, oxoPE) and conventional PE MP (vPE, aPE, fPE) are shown in Table S3. After 21 days of exposure to selected biodegradable MP, the highest IBR-T values were found in the PLA condition at 100 µg L⁻¹ (IBR-T = 0.42). IBR-T score for oxoPE condition at 100 µg L⁻¹ was similar to those of PLA condition at 0.008 and 10 µg L⁻¹ (0.17, 0.16 and 0.14, respectively). Concerning oxoPE conditions at 0.008 and 10 µg L⁻¹, the IBR-T score was zero, meaning that there was no significant difference with control condition regarding the studied biomarkers. IBR-T was lower after 21 days of exposure to conventional PE than to PLA 100 µg L⁻¹, regardless of the type of tested PE. IBR-T was comprised between 0.36 for fPE 10 µg L⁻¹ and 0 for aPE 10 µg L⁻¹. fPE IBR-T, regardless of the concentration, was the highest calculated value in comparison with other PE conditions.

The IBR-T score is detailed according to the biomarker categories (Fig. 7) and the major mechanisms affected (Fig. 8). An impact on physiological, behavior and enzymatic biomarkers was observed in the case of exposure to biodegradable MP and to various types of conventional PE, depending on the exposure concentration. No impact on genotoxicity was observed when exposed to biodegradable MP, whereas this biomarker was affected by exposure to field-aged PE (fPE) and unaged PE (vPE). Energy reserves, burrowing and oxidative stress were the functions the most affected when considering all tested MP while detoxication system was only impacted after an exposure to PLA at 100 µg L⁻¹.

**4. Discussion**

As a potential alternative to conventional plastics, biodegradable and oxo-fragmentable plastics have emerged as early as 1990s. However, once in the environment, the conditions for the biodegradability of these plastics are rarely fulfilled (Paul-Pont et al., 2023). They even undergo faster MP fragmentation processes (Shi et al., 2024) than conventional plastics and little is known about their toxicity. Most studies concerning substitutes to conventional plastics have focused on the toxicity of PLA, a biodegradable biosourced polymer (Cao et al., 2024). Thus, the aim of this study was to provide new data on the toxicity of alternatives polymers by considering the impact of PLA MP but also MP from a fragmentable plastic, oxoPE, on a key organism of the estuarine compartment. In addition, the originality of this work was to compare
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Acetylcholinesterase and acid phosphatase activity in gills (A, C respectively) and digestive glands (B, D respectively) of S. plana after a 21-day exposure to the control condition (CTL) and to PLA (PLA) or oxoPE (oPE) at 0.008, 10, and 100 µg L⁻¹ (mean ± SD).</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 6.** DNA integrity in gills of S. plana after a 21-day exposure to the control condition (CTL) and to PLA (PLA) or oxoPE (oPE) at 0.008, 10, and 100 µg L⁻¹ (mean ± SD, red star for significant differences between conditions and control *p* < 0.05).</figcaption></figure>

the results obtained on the effects of alternatives MP with those obtained from a previous study on PE MP, using an IBR-T integrating index (Catteau et al., 2023).

## 4.1. Toxicity of alternative (OxoPE and PLA) MP

### 4.1.1. Organism level impact

Numerous studies were conducted on the impact of MP on the physiological status of bivalves. In our study, none of the tested plastic conditions had an impact on the clearance rate of *S. plana*, while Green et al. (2017) showed that PLA exposure (25 µg L⁻¹, 52 days, 65.6 µm (0.6–263 µm)) reduced filtration rates compared to controls in *Mytilus edulis*. Joyce & Falkenberg (2022) exposed for 4 weeks the marine green mussel *Perna viridis* to non-biodegradable and degradable MP (200 µm diameter) of PET and PLA at 100 µg L⁻¹ and 1000 µg L⁻¹ (~17–20 particles L⁻¹ and ~ 135–140 particles L⁻¹) and showed small to no direct effects on bivalve whole organism functioning (mortality, oxygen consumption rate, clearance rate or condition index). The various impacts on the filtration capacity of organisms can be explained by differences in the types of microparticles used between studies. In our study, the presence of very small MP (median size of 3.7 µm and 5.2 µm, for PLA and oxoPE respectively) and NP differs from the previously cited studies (65.6 µm PLA particles in Green et al., 2017; 200 µm PLA particles in Joyce and Falkenberg, 2022). Even if the mechanisms involved are not yet fully understood, it has been shown that the characteristics of particles such as size, shape or surface properties, can influence the ingestion and egestion capacities of bivalves, particularly through pseudofeces production. Bivalves would then be able to maintain their organic matter filtration functions under certain conditions by selecting the particles they ingest (Ward et al., 2019).

Feeding behavior is linked to the energy reserves of organisms
<figure><img src="image_9.png" /><figcaption>**Fig. 7.** IBR-T scores in S. plana after 21 days of exposure to biodegradable MP (PLA and oxoPE (oPE)) (A) and various types of conventional PE MP (aged PE (aPE), virgin PE (vPE) or field PE (fPE)) (B) at 0.008, 10, and 100 µg L⁻¹ according to biomarker categories in exposed organisms.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**Fig. 8.** Star plots of IBR-T scores in S. plana after 21 days of exposure to biodegradable MP (PLA and oxoPE (oPE)) (A) and various types of conventional PE MP (aged PE (aPE), virgin PE (vPE) or field PE (fPE)) (B) at 0.008, 10, and 100 µg L⁻¹ according to impacted biomarkers in exposed organisms.</figcaption></figure>

exposed to contamination. Measuring these reserves provides a better understanding of the physiological state of the organisms. Among energy reserves, glycogen is the first molecule preferentially mobilized to counteract the effect of stress (Shang et al., 2021). A 26 and 25 % depletion in glycogen was observed in our study after exposure to both PLA and oxoPE MP respectively, at the highest concentration (100 µg L⁻¹) compared to control group, showing that the energy balance was affected in those exposed clams compared to control ones. As only changes in glycogen content were observed, it appeared that energy requirement to face the presence of MP requires only glycogen, at least initially. Shang et al. (2021) described that a 14-day exposure of mussels (*Mytilus coruscus*) to polystyrene MP (2 µm, 10⁴ or 10⁶ particles L⁻¹) led to a reduction in carbohydrate reserves after 7 days, while it took 14 days to observe a decrease in lipid and protein content. Glycogen reserves are necessary to ensure the energy balance of bivalves to cope with stress and to maintain numerous functions such as growth, reproduction and burrowing behavior (Mouneyrac et al., 2008; Smolders et al., 2004). In our study, despite the decrease in glycogen reserves, no impact on the condition index was observed in clams. This lack of effect on the condition index was already noted in previous studies, demonstrating the condition index as an insensitive marker in response to MP exposure in bivalves (Bour et al., 2018). This also suggests that the growth function may have been maintained at the cost of other functions such as reproduction, as previously shown in oysters exposed to PS MP (0.023 mg L⁻¹, 2 and 6 µm, 2 months) (Sussarellu et al., 2016).

In our study, a negative impact of PLA MP was observed on burial behavior. Thus, the decrease in burial rate was measured in organisms exposed to 0.008 and 100 µg L⁻¹ of PLA MP but not in bivalves exposed to oxoPE MP and therefore, it did not appear to be linked to glycogen reserve depletion. Similar effects were observed in previous studies on

the bivalve *S. plana* exposed to conventional MP at environmental concentrations (0.008 µg L⁻¹) (Labbé et al., 2024; Métais et al., 2023). However, to the best of our knowledge, our study is the first to investigate the impact of biodegradable MP on the burial function of bivalves. Parolini et al. (2024) exposed earthworm *Eisenia foetida* for 28 days to different shapes and sizes of PLA MP (0.1 % and 1 % of soil weight) and observed no effect on their digging activity. The functions linked to the behavior of organisms can be impacted by numerous biotic or abiotic factors (Boldina-Cosqueric et al., 2010). Thus, the effects of MP observed on burial behavior could be related to chemical additives (Bellas et al., 2006) rather than the polymers themselves, and the mechanisms involved have yet to be clarified. Furthermore, a disturbance in the key function of burial could have an impact on population maintenance of sediment-dwelling organisms such as *S. plana* (Bertrand et al., 2017), preventing them from protecting themselves from predation at low tide (Griffiths and Richardson, 2006).

### 4.1.2. Molecular and cellular impact

The antioxidant and detoxification systems are key mechanisms to protect organisms against stress. Among these systems, several enzymes are involved, such as catalase (CAT) or glutathione-S-transferases (GST). CAT is one of the first protective enzymes activated in the antioxidant defense system, catalyzing the dismutation of H₂O₂ to H₂O and O₂, and limiting the oxidative damage of reactive oxygen species (ROS) on biological molecules (DNA, proteins, lipids). GST is also involved in antioxidant system by conjugating the degradation products of lipid peroxides to glutathione (Prokić et al., 2019). Exposures to conventional MP and biodegradable MP have previously been demonstrated to affect the antioxidant response of bivalves (Li et al., 2022). In some studies, a dose-dependent exposure increases enzyme activities that counteract
the ROS production (Métais et al., 2023; Revel et al., 2019). For example, an increase in CAT activity was previously measured in whole soft tissues of the freshwater mussel *Dreissena bugensis* after exposure to PLA MP (9 days, $67 \pm 33$ µm, $1.24 \text{ g cm}^{-3}$) compared to control (Brehm et al., 2022). However, other studies found no variation (Khalid et al., 2021) or an inhibition of the activity of such enzymes in bivalves exposed to MP, confirming that the oxidative stress responses to MP are variable (Li et al., 2022). Similarly, in our study, CAT and GST activities were evaluated in gills and digestive gland of clams, showing a decrease in activities of both enzymes in gills of organisms exposed to $10 \text{ µg L}^{-1}$ and $100 \text{ µg L}^{-1}$ of PLA, respectively. This suggests a disruption of the antioxidant capacity of exposed organisms, with a possible increase in the production of ROS and damage to biomolecules such as DNA. In the present study, no DNA damage was observed in gills of clams exposed to PLA or oxoPE, indicating that these particles do not have any genotoxicity on the clams at the tested concentrations. Other studies have also reported no DNA damage in hemocytes of *S. plana* exposed for 14 days to $1 \text{ mg L}^{-1}$ of PS (Ribeiro et al., 2017) or LDPE (Low Density Polyethylene) MP (O'Donovan et al., 2020). More variable effects, with increased DNA damage at low concentrations of PE MP (0.008 and $10 \text{ µg L}^{-1}$) and reduced damage at higher concentration ($100 \text{ µg L}^{-1}$) have also been reported in a previous study on the same clam species, suggesting the involvement of the DNA repair system in case of genotoxicity (Labbé et al., 2024). More globally, exposure times and microplastic aging parameters are likely to alter organisms' responses, particularly with regard to the antioxidant system (Zhong, Huang, et al., 2024).

## 4.2. Comparison of the toxicity of alternative MP and conventional PE MP

The aim of our study was to compare the effects of biodegradable and oxo-fragmentable MP with those of conventional PE MP tested in a previous study, on the estuarine bivalve *S. plana* (Labbé et al., 2024). Comparing the toxicity of conventional and biodegradable/oxo-fragmentable plastics is a major challenge in determining whether these plastics represent a real alternative in terms of ecosystem health. A few reviews summarized the differential effects of biodegradable and conventional MP on ecosystems. However, those studies have mainly focused on the soil environment (Cao et al., 2024). Regarding the aquatic compartment, biodegradable MP had a comparable or higher toxicity on organisms in the aquatic environment than conventional MP, in particular on growth inhibition, mortality, reproductive impairment, and gene expression in oysters, water fleas and algae (Nik Mut et al., 2024). In fact, it has been reported that the mineralization of biodegradable MP in the water environment was less efficient than in the soil environment, increasing the formation of MP and associated toxicity (Cao et al., 2024).

### 4.2.1. Using IBR-T as a comparison tool

Reviews summarizing the effects of biodegradable and conventional polymers have a methodological limit when comparing their toxicity. Indeed, the only few studies that investigated the toxicity assessment of biodegradable MP used methodologies that are difficult to compare to the ones used for conventional MP (concentrations, size of particles, etc.). The originality of the present study is that it filled this gap considering the toxicity of biodegradable and conventional MP using similar methodologies and target organisms. To fully characterize this comparison, an integrative biomarker response index (IBR-T) was used to get a general overview of the effects and be able to fully address conclusions. An IBR is a tool that integrates the measurements obtained for a set of biomarkers to account for the biological functions affected, for example during a toxicity study. Multi-biomarker approaches are now widely employed in ecotoxicology, and the IBR-T can be used to compare several studies by considering only significant modulations of biomarkers in relation to threshold values or an internal reference condition, regardless of the number of biomarkers studied (Catteau

et al., 2023). In our work, the clams *S. plana* exposed to PLA MP at the highest concentration ($100 \text{ µg L}^{-1}$) were more affected (IBR-T = 0.42) than those exposed to PE MP regardless of the type of PE and exposure concentration (IBR-T comprised between 0.05 and 0.36). This is consistent with the reviews mentioned above (Cao et al., 2024; Nik Mut et al., 2024) and previous studies on aquatic organisms. Thus, a higher acute toxicity (48 h of exposure) was observed in *Daphnia magna* exposed to PLA MP compared to PE MP (0, 0.5, 1, 2.5, 5, 7.5, 10, and $20 \text{ mg L}^{-1}$) potentially due to a higher bioaccumulation of biodegradable MP in organisms (Luangrath et al., 2024). An et al. (2024) also noted a higher toxicity of PLA MP on *D. magna* compared to polyethylene terephthalate (PET) MP (1 and $5 \text{ mg L}^{-1}$; 48 h) compared to control group. Other studies found no greater impact from PLA MP, but an equivalent effect to PS MP on mussels (*Mytilus coruscus*) exposed for 14 days to $10^2$, $10^4$, and $10^6$ particles $L^{-1}$ of both types of MP, especially at lower (environmental) concentrations (Zhong, Shang, et al., 2024). Green et al. (2019) exposed the blue mussel *Mytilus edulis* in an outdoor mesocosm during 52 days to PLA MP ($65.6 \text{ µm}$) and high-density PE ($102.6 \text{ µm}$) at relatively low concentration ($25 \text{ µg L}^{-1}$) and reported an alteration of the hemolymph proteome regardless of the type of plastic. In our study, energy reserves, burial behavior, and oxidative stress mechanisms were impacted by both biodegradable and conventional MP. Similar toxicity mechanisms have also been described by Zhong et al. (2024), who reported increased oxidative stress and inhibition of energy metabolism and physiological activities in mussels exposed to PLA or PS MP (14 days). However, another study showed no impact of biodegradable (PLA) or conventional MP (PET) on physiological state of the Asian green mussel *Perna viridis* (~17–20 particles $L^{-1}$ and ~135–140 particles $L^{-1}$; 28 days). Furthermore, in our work, DNA damage was only reported in the gills of individuals exposed to certain types of PE MP but not in any organisms exposed to biodegradable MP. The intervention of DNA repair system could explain this difference in the effects observed (Parolini and Binelli, 2012). However, further work is needed to fully understand this mechanism. Thus, the toxicity mechanisms of both biodegradable and non-biodegradable MP remain to be elucidated and a point to consider regarding toxicity is the presence of chemical compounds associated with plastics whatever the type of polymer (Barrick et al., 2021). After ageing conventional PE mulches and biodegradable PLA mulches outdoors for 16 months, Xiao et al. (2025) observed that the MP generated from PLA films had adsorbed more Cd than the PE MP and that increased Cd adsorption after weathering was due to biofilms formation. Thus, considering these adsorbed compounds as well as the presence of additives in the formulation of biodegradable MP should be considered in explaining the toxicity mechanisms of these plastics.

In the present study, exposure to $100 \text{ µg L}^{-1}$ of oxoPE was similar in terms of IBR (IBR-T = 0.17) to exposure to other PLA concentrations ($0.008$ and $10 \text{ µg L}^{-1}$, IBR-T = 0.16 and 0.14 respectively) and no significant impact was observed after exposure to oxoPE at 0.008 and $10 \text{ µg L}^{-1}$. This suggests that oxoPE was less toxic to clams than PLA and conventional PE at the lowest concentrations tested. Regulatory measures were taken against oxoPE, which has been banned in the European Union since 2019, due to the increased risk of pollution associated with the degradation of these plastics. However, this type of plastic continues to be sold on the international market and since it has been banned in European Union, few studies have been carried out on oxo-degradable polymer, and to our knowledge, these studies only concerned the biodegradability mechanisms of these compounds, not their toxicity (Heimowska, 2023; Markowicz and Szymańska-Pulikowska, 2019; Napper and Thompson, 2019). Assessing the toxicity of these oxo-degradable polymers under ageing conditions could be an interesting option to explore in order to better understand the potential impact of compounds released during their fragmentation. In any case, in light of our results, it seems important not to consider PLA as a risk-free biodegradable alternative to conventional plastics and to continue comparative studies including biodegradable and
oxo-fragmentable plastics to better regulate the use of all polymers. From our perspective, tools such as IBR-T could be used as a decision-making aid, thanks to the ease with which this index can be communicated to regulatory bodies and the general public.

## 5. Conclusion

This study provides new data on the effects of oxo-degradable (oxoPE) and biodegradable (PLA) MP on the physiology of a key estuarine organism, *Scrobicularia plana*. The two types of tested MP showed impacts on several functions of exposed organisms, especially for PLA MP. In addition, this study compared the toxicity of these MP with that of PE MP and established that PLA MP could have a greater impact on the bivalve *S. plana* than conventional MP depending on the exposure concentration. This confirms the need to continue assessing the toxicity of biodegradable and oxo-degradable MP currently presented as an alternative to conventional plastics. The use of tools such as IBR-T can make it possible to precisely compare data from several studies and to present clear and synthetic results to the regulatory authorities.

### CRediT authorship contribution statement

**Clémentine Labbé:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.
**Isabelle Métais:** Writing – review & editing, Supervision, Methodology, Investigation, Formal analysis, Conceptualization.
**Hanane Perrein-Ettajani:** Methodology, Formal analysis.
**Mohammed Mouloud:** Methodology.
**Antoine Le Guernic:** Writing – review & editing, Methodology.
**Oihana Latchere:** Methodology.
**Nicolas Manier:** Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization.
**Amélie Châtel:** Writing – review & editing, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization.

### 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

This work was supported by Région Pays de la Loire and Institut National de l'Environnement Industriel et des RISques (INERIS). The authors would like to thank Fabienne Lagarde and Margaux Glais from Institut des Molécules et Matériaux du Mans (IMMM) for the access and support with the morphometric platform. They are also grateful to Farid Aït ben ahmad and Yohann Baillon from INERIS (MIV/ANAE unit) for the TEM analysis. The authors thank Mélanie Bruneau for her technical assistance as well as Lalie Supiot for the help during her internship in the BIOSSE laboratory at UCO.

### Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.aquatox.2025.107680.

### Data availability

Data will be made available on request.

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