# Distinct toxicity profiles of conventional and biodegradable fishing nets' leachates after artificial aging

Edgar Dusacre, Coralie Le Picard, Valerian Hausard, Camille Rigolet, Faith Ekoja, Morgane Jean, Sandrine Villette, Fabienne Lagarde, Sophie Lecomte, Bénédicte Morin, et al.

► To cite this version:

Edgar Dusacre, Coralie Le Picard, Valerian Hausard, Camille Rigolet, Faith Ekoja, et al. Distinct toxicity profiles of conventional and biodegradable fishing nets' leachates after artificial aging. Journal of Hazardous Materials, 2025, 489, pp.137609. 10.1016/j.jhazmat.2025.137609. hal-05365754

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Submitted on 15 Nov 2025

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Distributed under a Creative Commons Attribution 4.0 International License
# Distinct toxicity profiles of conventional and biodegradable fishing nets' leachates after artificial aging

Edgar Dusacre<sup>a,b,c</sup>, Coralie Le Picard<sup>a</sup>, Valerian Hausard<sup>a,b</sup>, Camille Rigolet<sup>a</sup>, Faith Ekoja<sup>a,d</sup>, Morgane Jean<sup>d</sup>, Christelle Clérandeau<sup>a</sup>, Sandrine Villette<sup>e,d</sup>, Fabienne Lagarde<sup>d</sup>, Sophie Lecomte<sup>e</sup>, Bénédicte Morin<sup>a</sup>, Miren P. Cajaraville<sup>b,c,d</sup>, Jérôme Cachot<sup>a,*d</sup>

<figure><img src="image_2.png" /></figure>

## HIGHLIGHTS

* Fishing nets under extreme aging conditions produced microplastics and nanoplastics.

* Fishing nets released soluble organic compounds that were toxic to aquatic species.

* Leachate toxicity varied among fishing nets and increased with aging time.

* Fishing net leachates significantly disrupted behaviour of fish larvae.

* Toxicity of biobased and biodegradable nets was similar to that of conventional nets.

## GRAPHICAL ABSTRACT

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

### Keywords:

Fishing gear

Micro and nanoplastics

Additives

Artificial degradation

Leachate ecotoxicity

## ABSTRACT

Fishing nets (FNs) represent a significant source of plastic waste, but their contribution to pollution by micro- and nanoplastics (MNPs) and associated additives is poorly understood. We studied the degradation of a high-performance-polyethylene-polypropylene (HPPE-PP) trawl net and two trammel nets made of polyamide 6 (PA6) or biodegradable polybutylene-succinate-polybutyrate-adipate-terephthalate (PBS-PBAT). Accelerated artificial ageing (AA) was performed using UV irradiation under environmental or extreme conditions followed by abrasion in water with glass microbeads. FN degradation and organic compound release were studied as well as the toxicity of leachates on the marine bacteria *Allivibrio fischeri* and larvae of the fish *Oryzias latipes*. AA of FNs under environmental conditions caused slight polymer degradation and did not produce significant MNPs. However, under extreme conditions, PA6 and PBS-PBAT FNs produced 9.1 × 10⁴ MP/mL and 2.0 × 10⁴ MP/mL, respectively. FNs released a total of 27 organic compounds in the leachates from which 7 were quantified at concentrations between 0.35 µg/L (Phthalimide) to 200 µg/L (Succinic-acid 2-methylallyl-undecyl-ester). Only the PBS-PBAT FN leachates induced significant toxicity on bacteria, bioluminescence inhibition ranging from

<footnote><sup>a</sup> Univ. Bordeaux, CNRS, Bordeaux INP, EPOC, UMR 5805, Pessac F-33600, France</footnote>

<footnote><sup>b</sup> Faculty of Science and Technology and Research Center for Experimental Marine Biology and Biotechnology PiE, University of the Basque Country UPV/EHU, Basque Country, Spain</footnote>

<footnote><sup>c</sup> CBET Research Group, Dept. Zoology and Animal Cell Biology, University of the Basque Country UPV/EHU, Basque Country, Spain</footnote>

<footnote><sup>d</sup> IMMM UMR 6283, CNRS-Le Mans University, Le Mans 72085, France</footnote>

<footnote><sup>e</sup> Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, Pessac F-33600, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: jerome.cachot@u-bordeaux.fr (J. Cachot).</footnote>
26 % to 56 %. Exposure of fish larvae to leachates of AA FNs disrupted their behavior. PBS-PBAT FN leachates caused the highest behavior stress indicator at day 12 (8.5), followed by PA6 at day 25 (8) and HPPE-PP at day 12 (7). We concluded that the toxicity of FN leachates was related more to the release of organic compounds than to the release of MPs. The toxicity of bio-based and biodegradable FNs should be further evaluated before their wider implementation in the fishing sector.

# 1. Introduction

The boom in plastics production starting in the 1950s brought about a profound transformation in all sectors, including the fishing industry [31,65]. Cheaper, lighter, more resistant to traction, and offering higher fishing yields, plastic became the primary material used in fishing gear (FG) in the second half of the 20th century [25,65]. It is currently estimated that between 1.5 Mt and 2.09 Mt of plastic FG are used yearly worldwide [50]. The annual consumption of plastic FG in the European Union is estimated at 53 Kt [21]. Generally, bottom trawl nets are made of polyethylene, high-performance polyethylene (HPPE), polypropylene (PP), or a mixture of HPPE-PP called polysteel [71]. Longlines, gillnets, and trammel nets are often made of monofilament polyamide (PA) 6 or 6.6 [7]. The lifespan of FG can vary from days to decades, depending on the quality of FG, the fishing fleet, the target fish species and the practices [40]. At their end-of-life, plastic FG are often stored, buried, incinerated or dumped into the environment, contributing to global plastic pollution [7,90]. Moreover, during the fishing activity FG can be abandoned, lost or otherwise discarded (ALDFG) [65]. Schwarz et al. [80] reported that ALDFG represented 6 % of annual global ocean macroplastic pollution in 2017, approximately 522 Kt. More conservatively, Kuczenski et al., [50] estimated a loss of 48.4–100 Kt (2.3–4.7 %) of the global FG used yearly, while Richardson et al. [79] estimated a loss of 2.0 % annually. More precisely, longline branchlines (3.6 %), trawl nets (3.6 %) and seines are the three most lost FG. On the European scale, it is reported that the average ALDFG was 20 % (10 Kt) of the FG used yearly and 3–20 % in the number of waste items collected on beaches of the Atlantic and North Sea [1,21,52,90]. Therefore, FG contribute significantly to the pollution of aquatic environments and are a major threat to flora, fauna and marine habitats due to the well-known phenomenon of ghost fishing [32,64,70].

FG and fishing nets (FNs) may degrade and therefore contribute to secondary micro- and nanoplastic (MNP) pollution. Plastic degradation is becoming better understood, and it is recognized that the main driver is photooxidation, followed by thermodegradation, abrasion, and biodegradation [14,36,5,66,81,83]. However, limited information is available on the degradation of FNs. Indeed, to our knowledge, only two papers focused on FNs [11,55] but they did not study their additive composition, their potential release of MNPs and their potential desorption of chemical compounds. The current trend is rather to solve the problem of ghost fishing and end-of-life FNs by developing biodegradable FNs [11,13,34,35,47]. A growing number of publications highlighted adverse lethal and sublethal effects of plastic degradation products [3,49,58,93]. Indeed, it has been demonstrated for a wide variety of plastics that toxic effects can be induced by MNPs and/or the release of additives, non-intentionally added substances, and adsorbed pollutants, or a combination of them [19,20,45,46,75,9,91]. However, to date, only one work has been published on the ecotoxicological effects of beach-sampled FG [88], but this study did not focus on FNs commonly used by fishermen and did not address the ecotoxicity of their specific degradation products. Concerning the novel biodegradable FNs, no study has been found on their potential effects on aquatic organisms. Thus, this work aimed to investigate the degradation kinetics of artificially aged new FNs through accelerated aging and to study the effects of their degradation products on two aquatic organism models. We hypothesized that the production of MNPs and the composition of additives vary with the FNs. Furthermore, the degradation products of FNs were expected to be toxic, with toxicity increasing with degradation

time and the release of MNPs and additives. To test these hypotheses, polysteel, polyamide 6 and polybutylene succinate-polybutyrate adipate terephthalate FNs were artificially aged using UV irradiation and then leachates were produced in deionized water using glass microbeads to simulate abrasion. The remaining FN mesoparticles were studied using scanning electron microscopy (SEM), attenuated total-reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, and polarized Raman scattering. The MPs produced were quantified and characterized using a laser direct infrared chemical imaging system (LDIR). The released compounds were analyzed by gas chromatography coupled with mass spectrometry (GC/MS). Finally, the toxicity of the FN leachates was assessed using the bioluminescence inhibition assay on the marine bacteria *Aliivibrio fischeri* (Microtox®) and the behavioral assay on larvae of the Japanese medaka, *Oryzias latipes*.

# 2. Materials and methods

## 2.1. Fishing nets

Three fishing nets (FNs) of different composition were studied. The first was a conventional green cod-end trawl net made of polysteel, referred to as HPPE-PP in this study (Appendix A, Figure A.1), purchased from the Spanish company Naberan (reseller reference: 3psco0704d, cul de chalut polysteel j-70 4 mm double). Polysteel is a copolymer of polypropylene and high-performance polyethylene (HPPE). The diameter of a braided string was 4.25 mm. The second FN studied was a conventional trammel net made of polyamide 6 (PA6) (Appendix A, Figure A.1), purchased from the French company Alprech Filet (reseller reference: Monofilament kit 45 ×0.33x2496x30.5 mm green - 200 ×3.5–0.60 ×416 mm white). In this work, only the two outer panels were studied. Their monofilaments had a diameter of 0.60 mm and were translucent. The third FN tested was a partially bio-based 55 %, biodegradable and industrially compostable outer panel trammel net made of polybutylene succinate-polybutyrate adipate terephthalate (PBS-PBAT) (Appendix A, Figure A.1). It also contained polyvinyl acetate at a final concentration of 0.5–10 % [67]. The monofilament was opaque white with a diameter of 0.60 mm. The Arcachon Bay Marine Natural Park (Office Français de la Biodiversité) provided this FN. The detailed composition of these three plastic FNs remains unknown, although the patent details for PBS-PBAT were available [67]. The two conventional FNs were selected because they were used by fishermen in the southeastern Bay of Biscay [74].

## 2.2. Artificial aging (AA)

Due to the ubiquitous nature of MNP pollution, strict precautions inspired by Lefebvre et al. [57] were taken to avoid sample contamination. All handling of FNs and leachates was carried out in a laminar flow hood, regularly cleaned and disinfected. All tools and containers used were made of stainless steel or glass. They were washed three times with filtered (0.22 µm) 70 % ethanol and deionized water before each use or sample change. Vinyl gloves and cotton coats were used systematically during the experiments. Blanks were performed in parallel with all steps to estimate cross-contaminations. Any transport of FNs, net pieces, or leachates was conducted only in previously cleaned and sealed containers.

For the AA of the FNs, they were cut into mesopieces of 2–2.5 cm, weighed in batches of 0.6 g (environmental conditions) or 0.2 g
(extreme conditions), placed in covered quartz pots, and exposed to UV light without water for 6, 12, 25 or 60 days. The UV irradiation process indicated below was partially based on the international guidelines ISO-14892-3:2016(E) for plastic exposure to laboratory light sources [42]. Two aging methods were used. First, 60 days of dry UV irradiation (25 % UV-B280-315nm and 75 % UV-A315-400nm at 31 mW/cm²) at 64°C was used to simulate the worst-case aging scenario (extreme condition) only for PA6 and PBS-PBAT. Second, an artificial aging protocol with conditions closer to environmental parameters (6, 12 and 25 days of dry UV irradiation with 25 % UV-B305-315nm and 75 % UV-A365-375nm, at 40 mW/cm² at 23°C) was used to study the degradation kinetics of the three FNs (environmental conditions). The 'environmental' UV A&B chamber was designed and constructed by the Aquatic Ecotoxicology team of the EPOC laboratory (University of Bordeaux, France). This UV chamber was designed to be open source, easy to build, financially accessible, energy efficient, and capable of emitting high irradiation power at low temperatures (23°C) [26]. The correspondence between artificial UV irradiation in the chamber and sunlight was calculated using the method of Gewert et al. [30]. The mean solar irradiance used is the annual average irradiance of the southeastern Bay of and is equal to 68 W/m² [39]. Therefore, 6, 12, and 25 days of UV irradiation in this chamber are equivalent to one, two, and four years of exposure to sunlight, respectively. After the photo-oxidation period, the leachates were obtained by shaking for 24 h at 190 rpm on an orbital shaker (Ika KS 501 digital, radius 15 mm) with filtered (0.22 µm) deionized water and 62 glass beads (diameter of 1.5 mm) to increase abrasion. Because there was no standardization for the speed of agitation [22,68,86,92], we used as shaking speed, the maximum tidal current velocity (0.3 m/s) on the continental margin of the Basque Country and the New-Aquitaine region [53]. Conversion from m/s to rpm was performed using the following formula:

$$N_{(rpm)} = \frac{60}{2\pi r} * v_{(m.s^{-1})} \quad (1)$$

where N is the number of rotations per minute (rpm), 60 is the number of seconds per minute, r is the radius of the orbital shaker, and v is the tidal current velocity (m.s⁻¹).

Finally, leachates were filtered using a 1 mm stainless steel filter to separate the mesopieces from the leachate. The final volume of the leachates was 60 mL (environmental conditions) and 40 mL (extreme conditions), with initial equivalent mesoplastic concentrations of 1 × 10⁴ mg/L (environmental conditions) and 5 × 10³ mg/L (extreme conditions). FN pieces were dried until a constant mass was achieved, weighed, and stored at 4°C. Leachates were divided into different aliquots for the experiments and stored at -20°C. For each condition, three replicates were made and three blanks were also made, following all the steps.

## 2.3. Structural analysis of fishing nets

Images were captured by scanning electron microscopy (SEM) to characterize the effects of AA on the surface structure of FNs. The FN pieces were randomly selected, thinly coated with platinum using a platinum coater (JEOL JFC-1300) and placed in the SEM (JEOL, JSM 6510 LV, IMMM, Le Mans, France). The images were taken at four different magnifications (x50, x100, x250, x500) with an accelerating voltage of 20 kV, a working distance of 20 mm, and a spot size of 30 nm.

Polarized Raman scattering analyses were conducted on FN filaments to calculate the dichroic ratio and to observe the evolution of polymer orientation, particularly the crystalline phase. Analyses were performed using a WITec Alpha 300RS instrument (WITec GmbH, Ulm, Germany) equipped with an EMCCD high-intensity low-noise camera and a high-precision piezoelectric scanning stage. Raman spectra were recorded from 200 to 3700 cm⁻¹ (600 g per mm grating, spectral resolution of 8 cm⁻¹) using a 532 nm excitation wavelength. The beam was

focused on the sample with a Zeiss objective (20 ×/0.4 NA), and the laser intensity in the sample was approximately 30 mW. A polarizer was used to change the laser orientation, and two spectra were acquired at 0° and 90° with a 10-second acquisition time per spectrum. This process was repeated on five different filaments for each FN. For each FN, two bands sensitive to the crystalline phase were selected to monitor polymer orientation during the AA kinetics. For HPPE-PP, the band at 1421 cm⁻¹ (assigned to the stretching mode of the crystalline (orthorhombic) C-C bond) and the band at 2847 cm⁻¹ (assigned to the δCH2 of the methylene group) were chosen [76]. For PA6, the bands at 1009 cm⁻¹ (methylene group) and 1657 cm⁻¹ (assigned to C=O of the amide group) were selected. For PBS-PBAT, the bands were chosen at 1617 cm⁻¹ (assigned to νC=C bond) and 864 cm⁻¹ (assigned to δC-C bond). All data were processed using WITec Project FIVE Plus software (WITec GmbH, Ulm, Germany). Preprocessing included cosmic-ray removal (CRR) and background subtraction using a shape function. Data analysis was focused on the 400-3600 cm⁻¹ range. For each selected band, the area under the curve was measured for the 0°, and 90° spectra and the absolute dichroic ratio (spectra 90°/spectra 0°) was calculated. The dichroic ratio values for each AA time were statistically compared for each FN.

Attenuated-total-reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy (Nicolet, Nexus 870 equipped with MIRacle diamond crystal) was used to monitor the chemical evolution of the surface structure of the FNs. Three spectra (OMNIC software V9.2.98, Thermo Fisher) were acquired for each pristine FN and for each FN at different aging times (6, 12, 25 days) with a spectral range of 400-4000 cm⁻¹, a spectral resolution of 4 cm⁻¹ and an acquisition of 100 scans. Before being superimposed, spectra of the same FN and irradiation time were averaged, and the baselines were manually corrected.

## 2.4. Microplastic quantification and nanoplastic detection in fishing nets leachates

After the AA process and after having been dried to a constant mass, remaining FN filaments (> 1 mm) were weighted using a precision balance (Ohaus Discovery DV215CD, Ohaus Corporation, USA) with a readability of 0.1 mg. Their mass after AA was subtracted from the initial mass of FN put under UV irradiation to calculate the mass lost. Regarding the liquid phase, 2 mL of leachate from each condition and time were collected and stored at -20 °C in a sterile glass vial. Samples were thawed at room temperature for MP quantification and used directly. The particles in the leachate were quantified using laser direct infrared chemical imaging system (Agilent 8700 LDIR Chemical Imaging System). The company protocol [2] was followed with a few adjustments described below. Five hundred microliters of leachate from each sample were filtered using a 0.8 µm polyethylene terephthalate glycol gold-coated filter from Sterlitech (Auburn, USA). To reduce analysis time and filter consumption, the three replicates of each artificially aged FN were combined and diluted tenfold in 96 % filtered (0.22 µm) ethanol to distribute particles homogeneously in the filter and minimize aggregation. The laser direct infrared chemical imaging analysis was performed in two steps. First, particles above 100 µm were quantified in a 180 mm² area (half of the total filter area) using infrared detection mode and scan acquisition (infrared spectrum range: 975-1800 cm⁻¹). Then, particles between 4 and 100 µm were quantified using the real image mode and scan acquisition for better size resolution (infrared spectrum range: 800-1800 cm⁻¹). The 180 mm² area was divided into 25 smaller squares of 7.2 mm², and seven squares were randomly chosen. A power statistical analysis determined that this number of squares was representative (α = 0.05, 1-β = 0.75, size effect = 0.38, k = 25). The "Microplastic 2.0" library of the "Agilent Clarity" software (version 1.5.58) was used for spectral matching, consolidated with spectra of the new and artificially aged FN samples. Three blanks were prepared for each condition, following all the steps of the AA process. The MP quantification data were corrected using blank values according to the
methods proposed by Dawson et al. [23]. The method selected only particles corresponding to the FN polymers with a 'spectral matching score' > 70 % and statistical comparisons with blanks.

Detection of nanoplastics (particles < 1 µm) in the leachates using scanning electron microscopy (SEM) was performed in the leachates of FNs artificially aged for 0, 25, or 60 days. After thawing, the leachates were stirred for 10 seconds and 10 µL of each leachate was deposited on a thin copper foil fixed on a support. The support was placed in a closed glass container and incubated at 35°C for 15 hours. The support surface was coated with a thin layer of platinum and placed in the SEM (Hitachi S-4800 SEM, General Service SGiker, UPV/EHU, Leioa, Basque Country, Spain). The images were taken at two magnifications (x5000, x10000) with an accelerating voltage of 5 kV and a working distance of 4.2 mm.

## 2.5. Chemical analysis of organic chemicals in fishing net leachates

A preliminary non-target analysis was performed using artificially aged FN leachates for 25 days. The leachates (200 mL, equivalent mesoplastic concentration: $1 \times 10^4$ mg/L) were frozen on dry ice and sent to LABOCEA (Plouzané, France). First, a purification step was performed using a Florisil® column. Then three extractions were carried out using the following methods: 1) 25 mL of dichloromethane (Pestipur Carlo Erba, Italy); 2) 10 mL of dichloromethane, 15 mL of ethyl acetate (Pestipur Carlo Erba, Italy), 500 µL of acetic acid and 250 µL sulfuric acid; and 3) 5 mL of dichloromethane. The extracts were purified and concentrated for analysis by gas chromatography coupled with a mass spectrometer (GC/MS SCAN) (column HP 5MS 30, 0.25 mm, 0.25 µM Agilent, USA). The obtained mass spectra were compared with the National Institute of Standards and Technology (NIST) (USA) library (version 2.2, June 10, 2014) for compound identification.

In the second phase, using the results of the non-target analysis (Appendix A, Table A.1), ten compounds were selected for quantitative analysis: $\epsilon$-Caprolactam, Tetradecanoic acid, 1,8-Diazacyclo-tetradecane-2,9-dione, Succinic acid 2-methylallyl undecyl ester, N-butyl Benzenesulfonamide, 2-Ethyl-1-hexanol, 2,4-Di-tert-butylphenol, Diethyl phthalate, Acetyl-tributyl-citrate, Phthalimide (Appendix A, Table A.2). New artificially aged FN leachates (0, 6, 12, 25 days) (80 mL, equivalent mesoplastic concentration $1 \times 10^4$ mg/L) were produced, frozen in dry ice, and sent to LABOCEA (Plouzané, France) for quantification of the ten selected organic compounds. Unfortunately, the leachate of PA6 FNs artificially aged for 6 days was lost during the analysis process. Standards for target analysis were purchased from Toronto Research Chemicals (Toronto, Canada) or HPC Standards GmbH (Borsdorf, Germany) for the following compounds: $\epsilon$-Caprolactam (CAS: 105-60-2), tetradecanoic acid (CAS: 544-63-8), 1,8-Diaza-2,9-diketocyclotetradecane (CAS: 56403-09-9), N-Butyl benzenesulfonamide (CAS: 3622-84-2), 2-Ethyl-1-hexanol (CAS: 104-76-7), 2,4-Di-tert-butylphenol (CAS: 96-76-4), Diethyl phthalate (CAS: 84-66-2), Tributyl O-acetylcitrate (CAS: 77-90-7), and Phthalimide (CAS: 85-41-6). The standard for Succinic acid 2-methylallyl undecyl ester was unavailable, so tetradecanoic acid (CAS: 544-63-8) was used to estimate its concentration. Liquid-liquid extraction was used for analysis. Briefly, an aliquot of leachate was diluted in ultrapure water. Then, extraction with a mixture of methyl chloride (Carlo Erba) and ethyl acetate (Carlo Erba) was performed under acidic conditions. The extract was purified in Florisil® and concentrated at 250 µL. The analyses were performed on a GC/MS Agilent (USA) with a VF 5 MS, 30 m column (Agilent). Helium was used as the carrier gas. Each molecule was identified by three ions and the specific retention time was used to ensure reasonable specificity. The quantification limits of the ten organic compounds are described in Appendix A (Table A.3).

## 2.6. Microtox® bioassay

The acute toxicity of leachates to the marine bacteria *Aliivibrio fischeri* was assessed using the Microtox® bioassay and ISO 11348-3

guidelines [41]. Marine bacteria cultures were exposed for 30 min to 100 % leachates with NaCl at $2 \times 10^4$ mg/L. The pH of the controls and FN leachates was adjusted to $7.3 \pm 0.2$ (the optimal pH range for *Aliivibrio fischeri* is 6.5-8.5). Data were normalized to the seawater control containing bacteria in seawater and bioluminescence inhibition was calculated for each sample. Finally, data were statistically compared to blanks.

## 2.7. Fish larvae bioassay

### 2.7.1. Exposure conditions, survival, malformations and heart rate

Japanese medaka (*Oryzias latipes*) embryos (Carbio strain) were purchased and received 24 hours after fertilization from the experimental facility PGP (Physiology Genomique Poissons, INRAE, Rennes, France). The reception and development of the embryos were carried out as described by Bedrossiantz et al. [8]. The temperature of the climate chamber (Snidjers Scientific, Tilburg, The Netherlands) was maintained at $24 \pm 0.5^\circ C$. The embryo development medium was renewed daily (1/2 v/v), and dead embryos were counted and removed. Immediately after hatching (10-day post fertilization), 18-20 larvae per replicate were exposed for 48 hours to 100 % artificially aged FN leachates. The exposure medium was renewed daily (1/2 v/v). Only FN leachates produced under environmental conditions were used. Triplicates were performed for each condition. A binocular magnifier was used to assess mortality, swimming ability, and abnormal body curvature at 24 and 48 hours after exposure. The effects on swimming ability and abnormal body curvature were considered according to what was described in the works of Horie et al. [38] and Yamagishi et al. [96]. Dead fish were counted and eliminated at 24 hours and at 48 hours the cumulated mortality was calculated, while swimming ability and abnormal body curvature data were recorded only at the end of the 48 hour exposure period.

To measure their heart rate, larvae were prepared as described by Bedrossiantz et al. [8] at a room temperature of $24 \pm 0.5^\circ C$. Sixteen individuals (12-day post fertilization) per blank or FN leachate sample were analyzed. Only alive and healthy organisms were selected. A near-infrared camera (Basler acA1300-60gm) mounted on a stereomicroscope (Olympus SZX2-TR30), connected to the open-source software Open Broadcaster Software Studio, was used to capture the videos (30 fps, 1280 × 720 pixels, 60 seconds). The videos were analyzed using DanioScope™ software Version 1.2 (Noldus, Wageningen, The Netherlands). Three 20-second measurements were recorded and averaged for each individual. Heart rates of fish in blanks were normalized to 100, and data of fish exposed to FN leachates were normalized relative to their respective blanks.

### 2.7.2. Fish behavior

The analysis of fish behavior was carried out using the DanioVision System (Noldus, Wageningen, The Netherlands) composed of a DanioVision observation chamber equipped with a near-infrared camera, a panel of LED for light simulation, a vibrational stimulus mechanism (tapping), a Temperature Control Unit to maintain the chamber at $24 \pm 0.5^\circ C$, and a computer running the analysis software EthoVision XT (version 17.0.1630). The analysis followed methodologies reported by Bedrossiantz et al. [8].

Larvae (12-day post fertilization) were placed in 48-well plates and subjected to a 51 min routine which enabled the determination of six behavioral endpoints:

* **Basal Locomotor Activity (BLA):** Distance travelled (cm) by the larvae in the dark during the period from 10 to 20 min (LOFF1 period).

* **Vibrational Startle Response (VSR):** Escape reaction to vibrational stimuli (51 stimuli, intensity 8) in the dark, measured as the distance swum (cm) during the second after the first vibrational stimulus.
* **Habituation:** Area under the curve of the distance travelled during the tapping period from minute 20–21 in the dark, indicating the non-associative learning process of ignoring irrelevant stimuli [10, 18].

* **Visual Motor Response (VMR):** Hyperactivity of the larvae in reaction to a sudden reduction of light intensity, estimated by subtracting the distance travelled during the first 2 minutes of the dark period from minute 41–51 (LOFF3) and the last 2 minutes of the light period (intensity 100 %) from minute 31–41 (LON).

* **Acclimatization to Sudden Darkness (ASD):** Ability of the larvae to recover their BLA over an extended period after the light shut off suddenly. Measured by the area under the curve of the swum distance (cm) during the LOFF3 period.

* **Time in the Peripheral Zone (TPZ):** Time spent by larvae in the border area of the well (ring area: ½ of the well area (diameter of 14 mm)), calculated during the LOFF1, tapping, and LOFF3 periods.

Data were normalized to values in their respective blanks, set at 1. To quantify the overall effects of leachates on larvae, a behavioral stress index was determined based on the work of Villa et al. [89]. For each endpoint, a score was assigned based on the deviation from the blank when it was significantly different: deviation $\le 0.2 = 0.5$; $[0.2-0.4] = 1$; $[0.4-0.6] = 2$; $[0.6-0.8] = 3$; $[0.8-1] = 4$; $> 1 = 5$. The scores were then summed for each FN and AA time. A higher score indicated a greater significant behavioral effect on the Japanese medaka larvae.

## 2.8. Data treatment and statistical analysis

Data treatment and statistical analysis were performed using RStudio (R version 4.3.1, RStudio version 2024.04.0 +735). Conditions of application of statistical tests were carefully checked for each test (density plots, Shapiro-Wilk tests, and QQ-plots, Bartlett's test, Levene's test). When requirements were met, Student's t test was used for two groups comparisons. If not, non-parametric Wilcoxon-Mann-Whitney's test was used. A one-way ANOVA was performed to identify significant differences between more than two groups. If necessary, a Tukey's HSD post-hoc test was performed. When the validity criteria for the ANOVA were not met, a Kruskal-Wallis test was used, and if necessary, a Dunn's multiple comparison test with Holm's correction was carried out. For MP quantification after applying the correction method, a Napierian logarithmic transformation was used to meet the conditions for parametric statistical tests. Data were then represented as back-transformed values using the delta method [73]. A principal component analyses based on centered and scaled data and a correlation matrix (Spreaman's method) were conducted to explore the relationships between degradation variables (MP quantification, organic degradation products) and the resulting toxicity. Variables used included MP and organic compound concentrations (both with blank values subtracted), *A. fischeri* bioluminescence inhibition, and the behavioral stress index for *O. latipes*. To reduce redundancy in the principal component analysis, endpoints related to the time spent by the larvae in the peripheral zone of the well (TPZ LOFF1, TPZ LOFF3, TPZ Tapping) were summed.

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** SEM images of the pristine and artificially aged (25 and 60 days) mesopieces of fishing nets (FNs). A.0 and A.25 are the filament of the HPPE-PP codend trawl net. A.0 is the filament before artificial aging (AA), and A.25 after 25 days of AA. B.0 and C.0 show the new PA6 and PBS-PBAT FNs, respectively. B.25 and C.25 show the filaments after 25 days of AA. B.60 and C.60 show the filaments artificially aged for 60 days under extreme conditions. In B.60 and C.60, white arrows point to the fragmentation and ablation degradation phenomena. The black arrows highlight longitudinal and transversal cracks. Scale bars are 100 µm.</figcaption></figure>
## 3. Results

### 3.1. Surface structural modifications of artificially aged fishing nets

No visible differences were observed by SEM between the pristine FNs and the artificially aged ones under environmental conditions (6, 12, 25 days) for the three FNs (Fig. 1 shows only the pristine mesopieces and those aged for 25 days). However, after 60 days of aging under extreme conditions, substantial degradation was observed in PA6 and PBS-PBAT FNs (Fig. 1). PA6 FNs exhibited fragmentation through ablation and delamination of the plastic surface layers, resulting in small, fine fragments. Longitudinal cracks that extended deeper into the bulk of the plastic were also visible. PBS-PBAT FNs showed surface layer fragmentation, characterized by numerous transverse and smaller

longitudinal cracks.

As to polarized Raman spectroscopy, neither HPPE-PP nor PA6 FNs showed significant differences in crystallinity after 0, 25, or 60 days of artificial aging (df = 3, p-value > 0.05). On the contrary, for PBS-PBAT FNs artificially aged for 12 or 25 days a significant increase in the crystalline phase of the polymer was observed for both wavelengths studied (864 cm⁻¹ and 1617 cm⁻¹). Significant changes in the crystal-line phase occurred between 6 and 12 days of AA with an increase of 0.14 points of the dichroic ratio for the bands at 864 cm⁻¹ and 0.13 points of the dichroic ratio for the bands at 1617 cm⁻¹ (Appendix A, Figure A.2). PBS-PBAT FNs irradiated for 60 days were too degraded and emitted too much fluorescence to obtain reliable results using polarized Raman spectroscopy.

The surface analysis of the FNs with ATR-FTIR revealed no

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** A) Microplastic concentration in leachates of artificially aged FNs for 0, 6, 12, 25, or 60 days. The size range is 4–100 µm. The top of the bar represents the back-transformed median and the black line is the back-transformed standard error (SE). Both are calculated with the delta method. Brackets and asterisks indicate significant differences in MP concentration between the FN net and its respective blank for each FN and each time independently (Student's *t*-test). Sample size by fishing net and AA time: n = 7. B) HPPE-PP, PA6, and PBS-PBAT FNs mass loss caused by the AA. The top of the bars represents the mean percentage, and the black line is the standard deviation (SD). Sample size by FN and AA time: n = 3. In both plots, letters indicate significant differences between AA times for each FN (independent one-way ANOVA, followed by Tukey's HSD post-hoc test).</figcaption></figure>
significant modification of the chemical environment of the HPPE-PP FN between pristine FNs and FNs artificially aged for 25 days (Appendix A, Figure A.3 and Table A.4). For PBS-PBAT and PA6 FNs, differences between pristine FNs and those artificially aged for 25 days were very slight. However, under extreme AA conditions, there was an overall decrease in PA6 FN absorption intensity, accompanied by a widening of the amine group. While for the PBS-PBAT FN, there was a photooxidation of the methyl and methylene groups, a widening of the carbonyl band, and the disappearance of the bands at 1270 cm⁻¹ and 732 cm⁻¹, indicating a strong impact of AA on this trammel net (Appendix A, Figure A.3 and Table A.4). Therefore, these modifications indicated a change in the chemical environment at the surface of PA6 and PBS-PBAT FNs aged under extreme conditions.

## 3.2. Plastic particle analysis in artificially aged FN leachates

Microplastic quantification by laser direct infrared chemical imaging system revealed a non-negligible amount of MPs in the blanks (Fig. 2A), likely resulting from contamination during the AA steps or/and potential artifacts detected by the laser direct infrared chemical imaging system. Increasing the system's sensitivity to detect the smallest particles also increased the detection of artifacts, especially when there were very few real particles in the filter. After correcting the data with a "spectral matching score" > 70 %, the number of MPs in the leachates (MPs/mL) (Fig. 2A) and the concentration of plastic particles in the leachates (mg/L) (Fig. 2B) were significantly correlated for PBS-PBAT FNs (cor.coeff = 0.99, p-value = 0.002), but not for PA6 FNs or HPPE-PP FNs, primarily due to values being below the mass loss quantification limit for the latter.

AA under environmental conditions did not significantly produce MPs across AA time for HPPE-PP FN leachates (Fig. 2A). The mass loss was below the quantification limit (1 mg) and thus under 16.7 mg/L, regardless of the AA time. This corresponds to a mass loss lower than 0.1 % (Fig. 2B). For PA6 FN leachates, the estimated concentration of plastic particles in the leachates ranged from 167 ± 17 mg/L (1.7 % mass loss) to 367 ± 95 mg/L (7.3 % mass loss) after 0 and 60 days under

extreme AA conditions, respectively (Fig. 2B), with a significant difference only between 0 and 60 days. Similarly, for laser direct infrared chemical imaging MP quantification, only the worst-case scenario caused a significant difference compared to blank leachates (t-value = 10, df = 9, p-value = 6 × 10⁻⁶) or with the other AA times (F-value = 5, df = 4, p-value = 2.7 × 10⁻³), at a release median of 1.1 × 10⁵ MPs/mL (IC95 % [1.6 × 10⁴ - 1.6 × 10⁵]). Blank mean value for PA6 spectral matching was 2.3 × 10⁴ MPs/mL (IC95 % [1.5 × 10⁴ - 4.0 × 10⁴]), leading to a corrected value of 9.1 × 10⁴ MPs/mL. Most of the particles were fragments (size ratio L/W = 1.6). The median particle dimensions were 25 µm in length and 15 µm in width. The size range extended from a minimum of 5 µm for both length and width to maximum dimensions of 455 µm and 415 µm for length and width, respectively (Fig. 3). For PBS-PBAT FN leachates, the plastic mass loss and MP quantification did not vary significantly between 0 and 25 days of AA, with a maximum estimated plastic concentration of 133 ± 73 mg/L (1.3 % mass loss) and no significant difference in MP concentration between FN and blank. However, under extreme AA conditions, the degradation of PBS-PBAT led to a significant increase in particle concentration in leachates (1250 ± 651 mg/L, i.e., 25 % mass loss) (Fig. 2B) and a significant production of 2.2 × 10⁴ MP/mL (IC95 % [9.9 × 10³ - 3.8 × 10⁴]) (t-value = 6, df = 10, p-value = 2.2 × 10⁻⁴) (Fig. 2A). The blank leachate value was 2.4 × 10³ MPs/mL (IC95 % [8.7 × 10² - 6.4 × 10³]), resulting in a corrected value of 2.0 × 10⁴ MP/mL. Most of the particles were fragments (size ratio L/W = 1.5). The median particle dimensions were 16 µm in length and 11 µm in width. The size range extended from a minimum of 5 µm for both length and width to maximum dimensions of 43 µm and 33 µm for length and width, respectively (Fig. 3). For leachates of PA6 and PBS-PBAT FNs, most quantified MPs were between 8 and 12 µm (15 % for PA6 FN, 24 % for PBS-PBAT FN). The smallest size range (4–8 µm) represented only 2 % for both FN leachates, mainly because the laser direct infrared chemical imaging system focused on larger particles, preventing the detection of the smallest particles.

SEM analysis of the leachates revealed the presence of nanoplastics below 1 µm (Fig. 4). The leachates of FNs artificially aged under extreme

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** MP size distribution quantified in the leachates of PA6 and PBS-PBAT FNs artificially aged for 60 days. Size range of 4–100 µm with size intervals of 4 µm. The black dotted line is the median length of the quantified MPs.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** SEM images of the leachates of artificially aged FNs for 0, 25 or 60 days (only PA6 and PBS-PBAT). A.0 and A.25 are leachates of HPPE-PP FNs artificially aged 0 or 25 days. B.0, B.25, and B.60 are the leachates of PA6 FNs artificially aged 0, 25 or 60 days. C.0, C.25, and C.60 are the leachates of PBS-PBAT FNs artificially aged 0, 25 or 60 days. White arrows indicate nanoparticles inferior to 1 µm. Scale bars are 1 µm.</figcaption></figure>

conditions showed more nanoplastics than those aged under environmental conditions, although a higher abundance of MNPs caused a higher particle aggregation (Fig. 4).

### 3.3. Organic chemicals in FN leachates

Non-target GC/MS analysis identified 27 compounds in the FN leachates (Appendix A, Table A.1 and Figure A.4). Among these, ten compounds were of particular concern to the aquatic environment, including ε-Caprolactam, Tetradecanoic acid, 1,8-Diazacyclo-tetradecane-2,9-dione, Succinic acid 2-methylallyl undecyl ester, N-butyl Benzensulfonamide, 2-ethyl-1-hexanol, 2,4-Di-tert-butylphenol, Diethyl phthalate, Acetyl-tributyl-citrate, Phthalamide (Appendix A, Table A.2). The rest of compounds were of lower or unknown hazard. The composition of mineral oil, although unknown, could potentially contain hazardous compounds such as PAHs, alkanes, and isoparaffins.

Quantitative analysis of these ten compounds revealed that seven of them (ε-Caprolactam, 1,8-Diazacyclo-tetradecane-2,9-dione, Succinic acid-2-dimethylallyl undecyl ester, Benzenesulfonamide, Diethyl phthalate, Acetyl-tributyl-citrate and Phthalimide) were present at concentrations exceeding the limit of quantification and blank values (Fig. 5). In particular, Acetyl-tributyl-citrate was the only compound found in HPPE-PP FN leachates, with its concentration steadily increasing with artificial AA (0 day = 122 µg/L, 6 days = 162 µg/L, 12 days = 182 µg/L, 25 days = 221 µg/L). In PA6 FN leachates, 1,8-Diazacyclo-tetradecane-2,9-dione and ε-Caprolactam were present at

concentrations > 195 µg/L and > 4200 µg/L, respectively, at each time of AA, while Acetyl-tributyl-citrate was quantified above the blank value (69 µg/L) only in leachates of FNs aged 25 days (199 µg/L). The PBS-PBAT FN leachates contained the seven compounds, with 1,8-Diazacyclo-tetradecane-2,9-dione slightly exceeding the blank value (28 µg/L), especially in the leachates of FNs aged 12 days (37 µg/L). A similar pattern was observed for Acetyl-tributyl-citrate, with concentrations exceeding the blank value (69 µg/L) in the leachates of FNs aged 12 days (85 µg/L) and 25 days (73 µg/L). Benzenesulfonamide was quantified at all AA times with concentrations > 6.5 µg/L, with a maximum concentration in leachates of FNs aged 12 days (7.1 µg/L). Diethyl phthalate and Phthalimide increased until 12 days of AA, with maximum concentrations of 3.3 µg/L and 0.55 µg/L, respectively, before decreasing at 25 days. Finally, the concentration of Succinic acid-2-dimethylallyl undecyl ester decreased from 350 µg/L in leachates of FNs that were not irradiated to 200 µg/L in leachates of FNs aged 12 days and increased slightly to 210 µg/L in leachates of the FNs aged 25 days (Fig. 5).

### 3.4. Microtox® bioassay

After 30 minutes of exposure to 100% leachates, HPPE-PP FN leachates did not cause significant bioluminescence inhibition compared to the blank leachates (Fig. 6). Additionally, no significant difference in bioluminescence inhibition was observed between the different AA times. Leachates of PA6 FNs aged 0, 12, 25 and 60 days caused
<figure><img src="image_8.png" /><figcaption>Fig. 5. Organic compounds leached during the AA of FNs. The red dotted line represents the higher value between the quantification limit and the concentrations observed in leachates of artificially aged blank samples. The values for Succinic acid, 2-methylallyl undecyl-ester, are approximated because the standard of tetradecanoic acid was used for its measurement. Sample size n = 1.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Fig. 6. Bioluminescence inhibition of the marine bacteria *Aliivibrio fischeri* exposed to FN leachates after various artificial aging times. Data normalized to seawater controls. The central black line of the boxplot represents the median, and the upper and lower borders are the 25th and 75th percentiles. The whiskers indicate 1.5 times the interquartile range (IQR). The red points represent the means, and the black points are the outliers. Letters indicate significant differences among AA times (One-way ANOVA for each FN, p-value < 0.05, Tukey's HSD post-hoc test with Holm's correction). Brackets and asterisks indicate significant differences between the blank and FN leachates (Student's t test for each FN and AA time. \* = p-value < 0.05, \*\* = p-value < 0.01, \*\*\* = p-value < 0.001, \*\*\*\* = p-value < 0.0001). Sample size by FN and AA time: n = 6.</figcaption></figure>
significant bioluminescence inhibition compared to the blanks. However, bioluminescence inhibition of leachates produced under environmental AA conditions was less than 20 %. No significant differences were observed between the different AA times. On the contrary, for PBS-PBAT FN leachates, the bioluminescence inhibition increased with AA time. The mean inhibition was greater than 20 % for all leachates: 25.7 ± 5.7 % for leachates of FNs not irradiated, and 26.7 ± 6.5 %, 29.5 ± 5.2 %, 39 ± 7.3 % and 56.3 ± 3 % for the leachates of FNs aged 6, 12, 25 and 60 days, respectively. A significant increase in inhibition was observed between the leachates of FNs aged 25 days and all other AA times [One-way ANOVA, F-value = 17, df = 4, p-value < 0.0001], as well as between the leachates of FNs aged 60 days and all the other AA times [One-way ANOVA, F-value = 17, df = 4, p-value < 0.0001] (Fig. 6). In conclusion, PA6 FN leachates slightly affected *A. fischeri*, while PBS-PBAT FN leachates were the only ones to cause a significant increase in bioluminescence inhibition with aging time and to exceed 20 % bioluminescence inhibition for all AA times.

## 3.5. Fish bioassays

### 3.5.1. Survival, malformations and heart rate

FN leachates did not significantly affect *Oryzias latipes* larvae survival, swimming ability, or body curvature after 48 hours of exposure compared to the blank leachates (Appendix A, Figure A.5). FN leachates

had little or no effects on heart rate compared to blank leachates (Appendix A, Figure A.6). Only the heart rate of fish exposed to leachates of HPPE-PP FNs aged 6 days decreased significantly compared to leachates of blanks aged 6 days (Kruskal-Wallis, Chi2 = 12, df = 3, p-value < 0.01; Dunn's test: p-value < 0.01). The leachate of the HPPE-PP FN aged 6 days was also significantly different from all other HPPE-PP FN leachates (Kruskal-Wallis, Chi2 = 21, df = 3, p-value < 0.001; Dunn's test: p-value < 0.01). PA6 and PBS-PBAT leachates did not significantly affect the heart rate ratio of the medaka larvae (Appendix A, Figure A.6).

### 3.5.2. Fish behavior

Sublethal effects on larvae behavior caused by exposure to FN leachates were summarized in a heatmap (Fig. 7). Various endpoints, including Basal Locomotor Activity (BLA), Vibrational Startle Response (VSR), Tapping habituation, Visual Motor Response (VMR), Acclimatization to Sudden Darkness (ASD), and Time in the Peripheral Zone (TPZ) during LOFF1, tapping, and LOFF3 periods were analyzed. Leachates of HPPE-PP and PBS-PBAT FNs aged 12 days significantly affected the BLA [H = 13.37, df = 3, p < 0.0001]. For the same period, the TPZ LOFF1 was affected more homogeneously among the different FN leachates. We observed a significant increase in the time spent in the peripheral zone for larvae exposed to leachates of HPPE-PP, PA6 and PBS-PBAT FNs aged 12 days [H = 30.61, df= 3, p < 0.0001], while for leachates of FNs aged 25 days, only HPPE-PP and PA6 FNs caused significant behavioral

<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Effect of leachates of artificially aged FNs on the swimming behavior of *Oryzias latipes* larvae. The abbreviations of the different endpoints are BLA = Basal Locomotor Activity, TPZ = Time in Peripheral Zone, VSR = Vibrational Startle Response, VMR = Vibrational Motor Response, ASD = Acclimatization to Sudden Darkness, and LOFF1 and LOFF3 = Light Off during the first and third periods. The numbers after each endpoint name (0, 6, 12, 25) are the AA times in days. Values in the cells are the normalized (around 1) deviation from their respective blank values for each endpoint. The scale color on the right represents the deviation from the blank. White cells (value = 1) indicate a low deviation from the blank, red cells illustrate a decrease in the endpoint value according to the blank, and blue cells express an increase in the endpoint values. Asterisks indicate significant differences between larvae exposed to blank leachates and larvae exposed to FN leachates at each AA time for each endpoint (Independent Dunn's test: \* = p-value < 0.05, \*\* = p-value < 0.01, \*\*\* = p-value < 0.001, \*\*\*\* = p-value < 0.0001). Sample size by FN and AA time: n = 48.</figcaption></figure>
effects [H = 57.99, df = 3, p < 0.0001]). Exposure to FN leachates did not affect the VSR. However, the two other endpoints calculated for the same stimulus period (TPZ tapping, Habituation) showed a significant response. Indeed, the TPZ tapping increased significantly for the leachates of HPPE-PP and PBS-PBAT FNs aged 12 days [H = 13.12, df = 3, p < 0.001], and for the leachates of the three FNs aged 25 days [H = 62.45, df = 3, p < 0.0001]. The habituation was less affected, with only one significant difference for larvae exposed to leachates of HPPE-PP FNs aged 25 days [H = 11.88 df= 3, p < 0.001]. Concerning the VMR, a significant decrease occurred for larvae exposed to leachates of the HPPE-PP and PA6 FNs aged 6 days [H = 32.97, df = 3, p < 0.0001]. The ASD was significantly affected by leachates of HPPE-PP FNs aged 6 days [H = 19.11, df = 3, p < 0.001]. Furthermore, the leachates of PA6 FNs aged 25 days also impacted the ASD, with a significant and sharp decrease [H = 18.7, df = 3, p < 0.0001]. For PA6 and PBS-PBAT FN leachates, the ASD increased from 0 to 12 days of AA. Finally, for the TPZ during the LOFF3 period, the three FN leachates caused a similar effect. Larvae stayed significantly longer in the peripheral area when exposed to leachates of FNs aged 12 days [H = 159.24, df= 3, p < 0.0001] or 25 days [H = 302.07, df= 3, p < 0.0001]. Interestingly, the effects on the TPZ were very similar, regardless of the period studied, with a significant impact from 12 days of AA.

Overall, leachates of FNs irradiated for 12 and 25 days caused more behavioral effects on *Oryzias latipes* larvae than shorter AA (Fig. 7 and Fig. 8). However, the behavioral stress index indicated different response patterns among the FNs (Fig. 8). The highest adverse impacts on fish behavior of HPPE-PP and PBS-PBAT FN leachates occurred after 12 days of AA. The BLA was the most strongly impacted behavior endpoint since it represented 57 % of the HPPE-PP FN leachates behavioral stress index and 41 % of the PBS-PBAT FN leachates behavioral stress index. PBS-PBAT FN leachates had the highest behavioral effect with a behavioral stress index of 8.5, mainly due to an increased effect on the TPZ during the LOFF1 period. HPPE-PP FN leachates were the only ones to cause an impact after six days (ASD was significantly affected). PA6 leachates yielded very different outcomes. Indeed, leachates of PA6 FNs began to disrupt larvae behavior after 12 days of AA, as indicated by a behavioral stress index of 1.5. Subsequently, a substantial disruption was observed following exposure to leachates from PA6 FNs aged 25 days (behavioral stress index = 8), with significant impacts on both VMR and ASD, accounting for 86 % of the behavioral stress index.

### 3.6. Principal component analysis

The principal component analysis aimed to elucidate the relationship between FN degradation products occurring in the leachates and their toxicity. Analysis revealed that three dimensions were necessary to explain most of the variance of data (76.3 %) (Fig. 9). Organic compounds contributed the most to the first two dimensions, followed by the bioluminescence inhibition of *A. fischeri* and three of the six *O. latipes* behavior endpoints (ASD, VMR, and habituation). Four organic compounds (Benzenesulfonamide, Diethyl-phthalate, Phthalimide and Succinic acid-2-dimethylallyl undecyl ester) were loaded in the same dimension and direction as bioluminescence inhibition, indicating their influence on this endpoint. The correlation matrix (Appendix A, Figure A.7) also showed that the influence of these organic compounds was significantly correlated to the bioluminescence inhibition of *A. fischeri* ($r \ge 0.77$, p-value < 0.01). On the other hand, $\epsilon$-Caprolactam, 1,8-Diazacyclo-tetradecane-2,9-dione, and the behavior endpoints VMR and ASD were loaded identically and in the same direction, suggesting a relationship between these variables (Fig. 9). Additionally, all PA6 FN leachates were grouped in the same area, particularly, leachates of PA6 FNs aged 25 days, indicating their toxicity for Japanese medaka larvae. The principal component analysis also highlighted a relationship between the leachates of HPPE-PP FNs aged 12 and 25 days, the Acetyl-tributyl-citrate and the disruption of the habituation behavior in response to vibrational stimuli. This suggests that Acetyl-tributyl-citrate released by the HPPE-PP FNs during the AA could had an effect on the behavior of the fish larvae.

The third dimension provided additional information, particularly on MP concentration and behavior endpoints TPZ and BLA, which had not contributed significantly to the first two dimensions (Fig. 9). This third dimension strengthened the relationship between the release of phthalimide and the inhibition of bioluminescence in *A. fischeri*, especially for leachates of PBS-PBAT FNs aged 25 days. Furthermore, MP and Acetyl-tributyl-citrate concentrations were loaded identically and in the same direction as VMR and ASD, suggesting their role in behavioral effects of leachates. The presence of 25-day PA6 FN leachates and 12-day HPPE-PP FN leachates in the same sector emphasized their effects on *O. latipes* larvae. On the other hand, the toxicity of PBS-PBAT FN leachates to *O. latipes* larvae was not explained by explanatory variables such MP concentration and quantified organic compounds.

<figure><img src="image_11.png" /><figcaption>**Fig. 8.** *Oryzias latipes'* larvae behavioral stress index. The stacked bars illustrate the cumulative behavioral stress indexes across different AA times (0, 6, 12, and 25 days) for three types of fishing nets (HPPE-PP, PA6, and PBS-PBAT). Each colored segment within the bars represents the relative contribution of distinct behavioral endpoints, including Basal Locomotor Activity (BLA), Time in Peripheral Zone (TPZ) during period with light off (LOFF1), Vibrational Startle Response (VSR), TPZ Tapping, Habituation, Vibrational Motor Response (VMR), Acclimatization to Sudden Darkness (ASD), and TPZ LOFF3.</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>**Fig. 9.** Principal component analysis of toxicity endpoints, MP concentrations, and organic compound concentrations. (A) The first and second dimensions explain 58.3 % of the variance. (B) The first and third dimensions explain 56.4 % of the variance. The pink arrow represents bioluminescence inhibition; the red arrows are the larvae behavior endpoints; the sky blue arrow represents MP concentration, and the dark blue arrows are the seven concentrations of organic compounds. Numbers near the FN points indicate the AA times under 'environmental conditions' (0, 6, 12 or 25 days).</figcaption></figure>
# 4. Discussion

## 4.1. Fishing nets degradation

### 4.1.1. Structural modifications and production of microplastics and nanoplastics

Fishing activity plays a vital role in the global economy and as a primary food source for humans [32,65]. However, fishing gear, including fishing nets (FNs), contributes significantly to global plastic pollution. Despite the substantial environmental impact of abandoned, lost, or discarded fishing gear (ALDFG), research on its role as a source of micro- and nanoplastics (MNPs) and dissolved chemical compounds remains limited. Understanding the degradation kinetics and potential toxicity of ALDFG is essential for effective mitigation strategies and better management of end-of-life FG.

Under artificial aging conditions closer to the environment (UV irradiation at 23°C for 25 days and shaking at 190 rpm), the structural modifications caused on the three FNs was different, with slight degradation only observed for the crystalline/amorphous phase ratio of the bio-based/biodegradable PBS-PBAT trammel net. Because to the best of our knowledge, no studies have been conducted on the controlled degradation of polysteel, we could not compare the results obtained for the HPPE-PP trawl net. However, a study on polyethylene and polypropylene pellets indicated no degradation after two months of a weak UV irradiation, first cracks appearing after six months, and significant degradation occurring after 12 months for polyethylene pellets [83]. For polypropylene, they observed first cracks after two months and pronounced signs of degradation after six months. On the contrary, in our study, even 25 days of AA (approximately equivalent to 4 years) induced minimal or no structural modifications of the HPPE-PP FN surfaces, as observed through SEM, polarized Raman, or ATR-FTIR analyses. Furthermore, unlike what is often observed for polyethylene or polypropylene weathering [4,44,66,83], the AA treatment of HPPE-PP did not provoke photooxidation of the carbonyl band at 1730 cm⁻¹. This minimal degradation of HPPE-PP FN was further corroborated by the low mass loss of HPPE-PP FN after AA and the insignificant release of MPs by HPPE-PP FN over the UV irradiation period. These differences compared to the literature, can be partially explained by the composition of polyethylene or polypropylene pellets of the raw material that differs from commercial HPPE-PP trawl nets, the latter containing more additives, at least partly to withstand physical and chemical degradation processes [83]. Second, the temperature of the experiment made by Song et al. [83] was approximately twice as high as in our study. It has been reported that elevated temperature, combined with UV irradiation, activates photooxidation and significantly accelerates plastic degradation processes [12,33,66,87]. Moreover, the aging and fragmentation of plastics in the environment are highly dependent on several intrinsic parameters, such as the types and contents of their additives, their initial crystallinity, and their shaping and processing, which can explain differences in degradation patterns and kinetic for the same type of plastic.

Concerning the PA6 trammel net, previous studies have highlighted the appearance of holes in PA fibers and filaments when weathered in seawater at room temperature (24–25°C) [56,84]. In these works, degradation was hypothesized to be due mainly to hydrolysis, salts, and photooxidation. The low degradation observed in the present work under realistic environmental conditions does not agree with the results of Sørensen et al. [84]. However, the PA used was not the same (PA 6,6 vs. PA 6) and the textile microfibers used in Sørensen et al. [84] were thinner than the FN filaments studied in this work. Nevertheless, in another experiment, exposure of PA6 to natural weathering for six months caused few visual modifications of the plastic surface [6]. In our case, the low visual degradation was consistent with the non-significant production of microplastics compared to the control. For PBS-PBAT FNs, the results of our study were aligned with the conclusions of Le Gué et al. [55] who showed few changes in the surface and characteristics of filaments weathered for 18 months in deionized water and seawater at 4 or

15 °C. Regarding the chemical structure, there was a slight photooxidation for both trammel nets, which is consistent with several studies [34,4,56,6,84] even though the modifications were less marked in the present study. The significant changes observed by polarized Raman only for PBS-PBAT FNs indicated that this FN was more sensitive to AA than the other oil-based fishing nets. The modification of the orientation of the polymer chain can be explained by the photooxidation of the amorphous phase [24,43,66]. When the amorphous phase decreased due to oxygen attack, it may have led to chemo-crystallization of the polymer [66]. Other studies have also reported modifications of polymer properties without visual degradation [34,55,6]. In summary, while remaining conservative and not ignoring the limitations of the laser direct infrared chemical imaging system and the AA protocol, the new HPPE-PP cod-end trawl net and the new 0.60 mm diameter outer panel trammel net of PA6 or PBS-PBAT did not appear to produce any significant quantity of microplastics, after 25 days of AA (equivalent to 4 years in the environment).

However, studying the degradation of the FNs in the worst-case scenario enabled us to understand the evolution of FNs when they become ALDFG and remain in the natural environment exposed to sun light for extended periods. In this context, Grimaldo et al. [34] and Le Gué et al. [55] weathered fishing nets under UV irradiation for 41 days at 38°C and in seawater for 18 months at 40°C and 60°C, respectively. Both studies showed greater degradation of the filament (longitudinal embrittlement and cracks) under extreme conditions than under environmentally relevant conditions. These observations were consistent with the results of our work, in which extreme conditions led to drastic changes on the surface of the PA6 and PBS-PBAT FNs (ablation, delamination, splitting), as well as to significant modifications of the chemical structure, high mass loss, significant microplastic production, and nanoplastic release (Figs. 1, 2, 3, 4). The PBS-PBAT FN produced fewer MPs than the PA6 FNs despite experiencing a higher mass loss during the AA process. This could be explained by the biodegradability of the PBS-PBAT FN, which may result in the production of smaller MPs and nanoplastics, as observed through SEM or even in degradation into oligomers, monomers, or other degradation by-products. However, since the complete degradation of PBS-PBAT FNs could not be reached experimentally, it can also be hypothesized that the lower amount of MPs in the PBS-PBAT leachates could come from the impossibility to accurately quantify MPs smaller than 5 µm by laser direct infrared chemical imaging. Indeed, the laser direct infrared chemical imaging system struggled with particles < 10 µm, and robust detection was impossible for particles < 5 µm [59]. Moreover, the wavelength range of 975 cm⁻¹ to 1800 cm⁻¹ of the system was inadequate for identifying polymers with bands above 1800 cm⁻¹. Nevertheless, the laser direct infrared chemical imaging system and SEM enabled us to study the shape of the released MNPs and revealed that 100 % of them were fragments and not fibers, according to the definition of Liu et al. [62,60]. This finding was in line with the results of Napper et al. [71], who reported that degradation of maritime ropes produced fragments and not fibers. Therefore, our results suggested that the outer panels of both oil-based or bio-based trammel nets weathered in the environment for very long periods may produce microplastics and nanoplastics with the shape of fragments.

### 4.1.2. Release of organic chemicals in the leachates

To better understand the potential impact of FNs over time, it was essential to analyze the release of soluble organic compounds in the leachates. Results underlined that even under conditions that caused only slight degradation of the FNs, 27 organic compounds were released in the leachates. Among them, ten were known or suspected to be hazardous to the aquatic environment (ε-Caprolactam, Tetradecanoic acid, 1,8-Diazacyclo-tetradecane-2,9-dione, Succinic acid 2-methylallyl undecyl ester, N-butyl Benzensulfonamide, 2-Ethyl-1-hexanol, 2,4-Ditert-butylphenol, Diethyl phthalate, Acetyl-tributyl-citrate, Phthalamide) (Chen et al., 2022; [38,91]). Three of these hazardous organic
compounds were quantified in all FN leachates (ε-Caprolactam, 1,8-Diazacyclo-tetradecane-2,9-dione and Acetyl-tributyl-citrate), including non-irradiated ones. Two studies also emphasized the release of chemical compounds from fishing gear. Vilke et al. [88] reported the leaching of 41 organic compounds and 6 heavy metals from various ALDFG collected on beaches in southern Portugal. Among the 41 organic compounds, only Acetyl-tributyl-citrate and Diethyl phthalate, were to the same as those identified and quantified in our work. Chen et al. [16] quantified 26 additives (mainly antioxidants and plasticizers) in fishing net leachates commonly used in the Yangtze River and the East China Sea. However, none of the 26 organic compounds they identified were those we detected in this work. Using three different plastic weathering methods, they demonstrated that photooxidation and constant abrasion by waves contributed to the degradation of plastics and promoted additive leaching. In this present work, we demonstrated that leaching of dissolved organic compounds was not always linearly and positively correlated with the duration of AA. This non-linear relationship between the concentration of organic compounds in leachates and AA time can be explained by the volatility of specific compounds or their photo-oxidation during UV irradiation [61,72]. Lastly, it is important to acknowledge that only a fraction of the organic compounds leached by the FNs during AA were identified and quantified. Therefore, further non-targeted analyses methods, such as high-performance liquid chromatography, are required to obtain a more comprehensive list of organic compounds in the FN leachates.

## 4.2. Toxicity of fishing net leachates

### 4.2.1. Effects on the bioluminescence of *A. fischeri*

After characterizing and quantifying some of the degradation products of artificially aged FNs, we were able to study the toxicity of leachates containing the mixture of MNPs and chemical degradation products. The Microtox® assay revealed varying levels of toxicity depending on the composition of FNs and AA times. For HPPE-PP FN leachates, the low toxicity for *A. fischeri* can be attributed to the slow degradation rate of this net and the release of compounds below the toxicity threshold of this bacteria (ε-Caprolactam and 1,8-Diazacyclo-tetradecane-2,9-dione). On the contrary, PA6 and PBS-PBAT FN leachates exhibited significant acute toxicity, but PBS-PBAT FN leachates, which caused bioluminescence inhibition greater than 20 % at all AA times, were the most toxic. The organic compounds released (N-butyl benzenesulfonamide, Diethyl phthalate, Phthalimide, and Succinic acid 2-methylallyl undecyl ester) were strongly linked to this inhibition of bioluminescence and accounted for most of this toxicity. These organic compounds have been reported as endocrine disruptors, cytotoxic agents, and inducers of oxidative stress [15,85,91,95,97]. Comparing low density polyethylene, bio-polybutylene succinate, and polybutylene adipate terephthalate pellets, Klein et al. [48] observed similar results with *A. fischeri*, showing no toxicity after exposure to low density polyethylene leachate but high toxicity with bio-polybutylene succinate and polybutylene adipate terephthalate leachates. They hypothesized that the higher toxicity of bio-based and biodegradable plastics was attributed to the release of active chemicals. Lastly, the highly significant toxicity of PA6 and PBS-PBAT FNs artificially aged under extreme conditions raised concerns, as it reproduced the worst-case scenario that ALDFG can suffer.

### 4.2.2. Effects on the behavior of *O. latipes* larvae

Fish behavior endpoints are increasingly recognized as indicators of physiological disturbances at the individual level [54,8] and are recommended to be used for risk assessment [28]. Locomotor disturbances are often considered as a general marker of physiological disorders in response to low concentrations of chemical pollution [8]. Increased basal locomotor activity can lead to excessive energy consumption, potentially disrupting other energy-dependent physiological functions such as growth, reproduction, and foraging [37,69]. In the case of FNs,

exposure to HPPE-PP and PBS-PBAT 12 day leachates increased the swimming distance of larvae, potentially causing significant energy loss [82]. Additionally, the sublethal effects of these two types of FNs affected the time spent in the peripheral zone, which is described as an escape behavior or a sign of anxiety [17,20]. Anxiety behavior and locomotion perturbations have been observed in the early stages of zebrafish exposed to plastic additives such as 2,4-di-tert-butylphenol and diethyl phthalate. The latter compound, found mainly in PBS-PBAT FN leachates and used in plastic manufacturing, has been described as neurotoxic [63,82,95], which may partially explain observed behavioral changes. Anxiety could also result from disturbances in steroid biosynthesis induced by endocrine disruptors such as additives potentially originating from plastic degradation, including Diethyl phthalate, Acetyl-tributyl-citrate, and 2,4-di-tert-butylphenol, even at low concentrations [38,78,95]. In our study, Acetyl-tributyl-citrate was strongly related to the toxicity of leachates of HPPE-PP FNs aged 12 and 25 days and with disruptions in habituation endpoints. The habituation to vibrational stimuli is described as a non-associative learning process in which the organism ignores irrelevant stimuli commonly found in the natural environment. Its disruption may lead to exaggerated reactions to irrelevant stimuli and energy loss [10,18]. In summary, the leachates of HPPE-PP and PBS-PBAT FNs predominantly affected behavioral endpoints, which could be associated with a disruption of the energy budget, potentially leading to adverse impacts on the growth and reproduction of fish [20,29].

On the other hand, leachates of PA6 FNs aged 25 days caused a high level of behavioral disturbance, affecting the visual motor response (VMR) and the acclimatization to sudden darkness (ASD). Shi et al. [82] described these alterations in the reaction to light stimuli (hyper- or hypoactivity) as impacting the optic nerve, potentially disrupting the development of the visual system and reducing the size of the eyes in early life stages of *Danio rerio*. They suggested using these endpoints as indicators of neurotoxicity that affects the vision of fish. At the ecological level, VMR and ASD are critical responses that enable fish to flee from looming predators [27]. Consequently, significant decrease in VMR and ASD can, in the natural environment, adversely affect prey-predator interactions, thus provoking ecological disturbances [94]. In our case, the significant decrease of VMR and ASD were correlated with the presence of ε-Caprolactam, 1,8-Diazacyclo-tetradecane-2,9-dione, and Acetyl-tributyl-citrate in leachates. ε-Caprolactam, used extensively in plastic manufacturing, was classified on the red list (chemicals that are hazardous according to well-established criteria and that should be regulated) of the plastic additives of the PlastChem report [91] and described as genotoxic and mutagenic [51]. Regarding 1,8-Diazacyclo-tetradecane-2,9-dione, the European Chemicals Agency (REACH regulation) suspected it to be hazardous to aquatic species, but our study appeared to be the first to highlight its association with the disruption of the behavior of fish larvae. Additionally, MPs played a role in the toxicity observed for PA6 and HPPE-PP FN leachates after 25 days of AA, contributing to the disruption of some medaka larvae behavior endpoints (VMR, ASD, and habituation). However, the variability in toxicity among FN leachates appeared primarily attributed to differences in the composition of plastic additives and chemical degradation products, with MPs playing a less relevant role. As discussed extensively throughout this study and in other research papers, leached plastic additives are now recognized as the main contributors to plastic toxicity [77,78,91,97]. This article highlighted for the first time that this was also the case for additives and degradation products from both commonly used oil-based and partially bio-based/biodegradable fishing nets.

## Environmental implication

The main goal of this work was to investigate potential toxic effects for aquatic organisms of leachates of artificially aged plastic fishing nets. By studying the degradation of these materials, we have proved their contribution to plastic and organic compound pollution and their
deleterious effects on aquatic species. Our findings demonstrate that fishing nets can release micro and nanoplastics and soluble organic compounds during degradation. Importantly, we have demonstrated that plastic degradation products are toxic to aquatic species of two different trophic levels. Furthermore, all the three studied fishing nets are toxic, including the partially biobased and biodegradable one, indicating that these are not yet sufficiently sustainable to replace conventional nets. In conclusion, our research highlights that leachates of plastic fishing nets could represent a threat to aquatic organisms.

## 6. Conclusions

Our research reveals that cod-end trawl nets made from polysteel and trammel nets composed of PA6 and PBS-PBAT did not release a significant quantity of MPs after artificial aging under environmentally relevant conditions. However, PA6 and PBS-PBAT fishing nets released a significant amount of MPs under extreme conditions of aging. Further investigations are needed to understand their fragmentation into nanoplastics and to determine whether microplastics from partially bio-based and biodegradable nets will fully degrade. This study also confirmed that artificially aged conventional and partially bio-based/biodegradable fishing nets released a substantial amount of soluble organic compounds. These compounds adversely affected the marine bacteria *A. fischeri* and the behavior of *O. latipes* larvae. The leachates of the HPPE-PP trawl net and PA6 trammel net mainly caused sublethal effects on *O. latipes*, while the partially bio-based and biodegradable PBS-PBAT fishing nets induced the most severe effects on both test organisms. The disruption of fish behaviour could have significant ecological consequences through affecting prey-predator relations, growth and reproduction. Therefore, while biodegradable nets hold promises for mitigating ghost-fishing, further refinement in their composition is necessary. Finally, given these results and those of other studies, the chemical composition of FNs and plastics in general must be monitored and openly accessible, especially for public research. It is fundamental to advocate for mandatory disclosure of plastic composition to ensure a comprehensive understanding and effective management of the environmental impacts of plastics.

## Funding

This study was conducted as part of the international Plasfito research project. It was funded by the French Natural Marine Park of the Arcachon Bay (Office Français de la Biodiversité), the French Water Agency Adour-Garonne and the Nouvelle Aquitaine region, the Spanish Ministry (project FIERA PID2021-128600OB-I00, MCIN/AEI/ 10.13039/501100011033 and "ERDF A way of making Europe"), the Euskampus Fundazioa (EUSK22/03), the Basque Government (consolidated research group IT1743-22), the Laboratory for Transborder Cooperation LTC AquEus (Euskampus Fundazioa and Basque Government), University of Bordeaux (doctoral fellowship to ED) and the CNRS.

## CRediT authorship contribution statement

**Lecomte Sophie:** Writing – review & editing, Visualization, Validation, Supervision, Methodology, Investigation, Data curation.
**Villette Sandrine:** Visualization, Validation, Methodology, Investigation, Data curation.
**Lagarde Fabienne:** Writing – review & editing, Validation, Supervision, Methodology, Investigation, Data curation, Conceptualization.
**Jean Morgane:** Investigation, Formal analysis.
**Clérandeau Christelle:** Writing – review & editing, Visualization, Validation, Methodology, Investigation.
**Rigolet Camille:** Investigation, Formal analysis.
**Ekoja Faith:** Methodology, Investigation, Formal analysis.
**Cachot Jérôme:** Writing – review & editing, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization.
**Le Picard Coralie:**

Investigation, Formal analysis, Data curation.
**Hausard Valerian:** Investigation, Formal analysis, Data curation.
**Morin Bénédicte:** Writing – review & editing, Visualization, Validation, Supervision, Methodology.
**Cajaraville Miren P.:** Writing – review & editing, Visualization, Validation, Supervision, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization.
**Dusacre Edgar:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

## Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jerome Cachot reports financial support was provided by University of Bordeaux, CNRS. Jerome Cachot reports financial support was provided by Regional Water Agency Adour-Garonne. Jerome Cachot reports financial support was provided by French Agency for Biodiversity. Jerome Cachot reports financial support was provided by Nouvelle-Aquitaine Regional Council. Miren Carajaville reports financial support was provided by Euskampus Foundation. Miren Cajaraville reports financial support was provided by Spain Ministry of Science and Innovation. Miren Cajaraville reports financial support was provided by Basque Government. Edgar Dusacre reports financial support was provided by University of Bordeaux. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

The authors would like to express their sincere gratitude to the fishermen who generously provided the names and contact information of companies providing their FNs. Special thanks are also extended to the French Marine Natural Park of the Arcachon Bay (Office Français de la Biodiversité) for providing the partially bio-based and biodegradable trammel net. The authors wish to thank the team from the IMMM laboratory at Le Mans University (France), including Ph.D. students Gustave Bertier and Margaux Glaïs, and research engineer Frederic Amiard, for their invaluable assistance in conducting the degradation experiments under the worst-case scenario. They would also like to thank Christian Portier and Yannick Geerebaert from the University of Bordeaux, who designed and built the "environmental" A&B UV chamber. Lastly, we thank Emeric Grillard for his artistic assistance in designing the graphical abstract.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jhazmat.2025.137609.

## Data Availability

Data will be made available on request.

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