# Contamination of Pyrenees mountain streams by agricultural synthetic fibre ropes and a first assessment of their potential effect on aquatic and terrestrial gastropods

Laurent Seuront

► To cite this version:

Laurent Seuront. Contamination of Pyrenees mountain streams by agricultural synthetic fibre ropes and a first assessment of their potential effect on aquatic and terrestrial gastropods. Jean-Yves Reynaud; Eric Armynot du Châtelet. Short Papers from the 19th French Congress of Sedimentology, Lille, 84, Association des Sédimentologistes Français, pp.87-95, 2025, 2-907205-83-8. 10.70665/HXMQ9979. hal-05005448

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Submitted on 1 Apr 2025

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# CONTAMINATION OF PYRENEES MOUNTAIN STREAMS BY AGRICULTURAL SYNTHETIC FIBRE ROPES AND A FIRST ASSESSMENT OF THEIR POTENTIAL EFFECT ON AQUATIC AND TERRESTRIAL GASTROPODS

Laurent SEURONT<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

laurent.seuront@cnrs.fr

**Abstract:** Despite the ever-growing amount of work devoted to plastic pollution on a global scale, we are still lacking information on the extent and effects of microplastic pollution in mountain terrains and foothills ecosystems worldwide. The present study was specifically triggered by the nearly ubiquitous presence of synthetic fibre ropes in two otherwise pristine valleys of the foothills of the central Pyrenees. In this context, the present work (i) assessed the presence of fibres in the riverbank sediments of two streams of the Pyrenees foothills, and (ii) inferred the potential effects on the local aquatic and terrestrial gastropods fauna on behavioural assays conducted on representative species of Pyrenees springs and streams, i.e. *Ancyclus fluviatilis* and *Radix balthica*, and two terrestrial gastropods, the endemic snail *Obscurella nouleti* and the widespread grove snail *Cepaea nemoralis*. Fibres were present in all samples considered but notably at relatively low concentrations. Other common microplastic items such as fragments, film and foam were remarkably consistently absent from all the samples considered in the present work. These results indicate that the Nistos and Ourse streams are only marginally impacted by plastic pollution in general and by plastic pollution related to agricultural activities in particular. However, the polyethylene fibres studied contained three low molecular weight phthalates which when incorporated into an inert gel, induced systematic behavioural avoidance in all species tested. To avoid underestimating the role of Nistos and Ourse streams as potential microplastic sinks, further work is needed to assess the nature and amount of microplastic items (including fibres) that may have been trapped in foothill soil and in stream sediment surface depending on their shape, density and polymer and/or advected downstream. Similarly, the observed consistent avoidance of rope leachates questions the potential long-term effects of the chemical compounds contained in the synthetic ropes that are extensively used in the agricultural sector and found nearly everywhere in the foothill landscapes.

**Keywords:** Plastic pollution, synthetic fibres, phthalate, riverbank sediment, behavioural assay.

## 1 INTRODUCTION

In an era of ever-growing anthropogenic pressure on both terrestrial and aquatic ecosystems, plastic pollution is increasingly acknowledged as a pervasive environmental problem due to its persistent behaviour and increased resistance to biodegradation; see e.g. MacLeod et al. (2021), Li et al. (2021) and Richard et al. (2024) for recent reviews. Specifically, due to plastic abuse and the worldwide struggle to cope with the increasing influx of plastic waste, this material has become one of the most ubiquitous sources of both contamination and pollution of the Anthropocene (Geyer et al., 2017). Plastic wastes are accumulating on landfills and beaches worldwide as well as in the most remote terrestrial and oceanic locations (Napper et al., 2020; Bergmann et al., 2022; De-la-Torre et al., 2024), threatening both terrestrial and aquatic environments, the economy and human well-being on a global scale (Amelia et al. 2021).

Though much attention has focused on the impacts of plastic waste on the world's oceans, remote and mountainous systems are increasingly acknowledged as areas that are significantly affected by plastic waste. Microplastics (MPs, i.e. plastic items in the 5 mm to 1 µm size range) are introduced into remote aquatic ecosystems, especially mountain terrains and foothills, including high-altitude ecosystems, via the degradation, fragmentation and subsequent transport of plastic products that accumulate in various mountainous regions — including Mount Everest (SkyNews, 2019) — due to the lack of proper waste disposal and recycling facilities (Free et al., 2014; Zhang et al., 2016). Rain and snow are, however, considered to be the primary drivers of microplastics (MPs) deposition in high altitude ecosystems, together with the wide range of synthetic fibres released by the clothing and equipment of hikers, mountaineers and other tourist activities, which are transferred by the wind into the main water body; see Padha et al. (2022) for a review. Synthetic fibre ropes (typically made of polyethylene and polypropylene) are also used in the agricultural sector, notably for binding bundles to be picked up by automatic balers and binders or similar

<footnote n="1">¹CNRS, Univ. Lille, Univ. Littoral Côte d'Opale, IRD, UMR 8187 - LOG - Laboratoire d'Océanologie et de Géosciences, Station Marine de Wimereux, F-59000 Lille, France</footnote>

<footnote n="2">²Department of Marine Resources and Energy, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo, 108-8477, Japan</footnote>

<footnote n="3">³Department of Zoology and Entomology, Rhodes University, Grahamstown 6140, South Africa</footnote>
machines. A common practice in rural areas of the Central Pyrenees foothills is to use these ropes to demarcate agricultural plots, hiking path and private properties, and are subsequently observed in various state of degradation in a range of landscapes (Fig. 1).

Research on remotely located mountainous MP-polluted ecosystems is, however, still in its early stage, very fragmented and MP abundance reported in the literature should be considered with caution given the diversity of potential sources, as well as both the sampling and extraction methods considered (Padha et al., 2022) for a review. Despite a drastic increase in the number of publications assessing microplastic pollutions in rivers over the last decade (see D'Avignon et al. (2022), their Fig. 2), there is still a critical lack of information on (i) MP abundances in mountain terrains and foothills ecosystems worldwide, (ii) the links between anthropogenic activities and the type of MPs, and (iii) the potential ecotoxicological consequences toward specific aquatic organisms. In this context, this preliminary study aims at assessing (i) the presence of plastic fibres in the riverbank sediment of two streams of the Pyrenees foothills where synthetic fibre ropes are extensively used, and (ii) the potential effects that these fibres may have on the local aquatic and terrestrial gastropods were investigated based on behavioural assays on representative species of Pyrenees springs and streams, i.e. *Ancylus fluviatilis* and *Radix balthica*, and two terrestrial gastropods, the endemic snail *Obscurella nouleti* and the widespread grove snail *Cepaea nemoralis*. Gastropods have been specifically targeted because the Pyrenees are a biodiversity hotspot for land snails and freshwater molluscs, with many endemic, specialised and relict species which reflect the region's uniquely varied climate, geology and range of microhabitats. In addition, freshwater gastropods play critical ecological roles through nutrient cycling, biofilm control, energy transfer, and habitat modification, influencing both biotic and abiotic components of their environments and their terrestrial counterparts serve as decomposers (hence contribute to nutrient cycling, soil health, and plant regulation), herbivores, prey species, and bioindicators, influencing ecosystem stability and biodiversity.

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

Figure 1. Illustration of the nearly ubiquitous presence of synthetic fibre ropes in the rural areas of the Central Pyrenees foothills (in the present case the Nistos valley, but similar observations hold for our second study site, the Barousse valley) where they are used in (A) in the vicinity of agricultural facilities to restrict access, to demarcate (B) hiking paths and (C) grazing plots, and are subsequently found in various state of degradations (E,F), even in remote and pristine areas (D). These ropes were consistently blue, though some colour variations do exist (F, inset) likely due to differences in manufacturers and/or the duration of exposure to environmental conditions.

# 2 MATERIALS AND METHODS

## 2.1 Study sites

The Nistos stream and the Ourse River are located in the foothills of the Central Pyrénées (Fig. 2). The 18.5 km long Nistos stream is a sub-tributary of the Garonne River via the Neste River, and the 25.4 km long
Ourse river is sensu stricto formed at the confluence of the 'Ourse de Ferrère' and the 'Ourse of Sost' streams at Mauléon-Barousse and is a tributary of the Garonne River (Fig. 2C). Five sampling locations were considered, and were consistently chosen for the lack of vegetation between the shoreline and the water's edge as the presence of vegetation typically acts as natural buffer that increases retention and accumulation of microplastics within sediments compared to bare or sparsely vegetated areas (Tasseron et al., 2024), hence could lead to confounding results. Given the acknowledged role of flow velocity in microplastic deposition and accumulation on riverbanks and within sediments (Owowenu et al., 2023; Munz et al., 2024) two stations were selected along the Nistos stream, based on a visual assessment of the nature of their flow: the first one was characterized by a steep river slope with lots of rock and boulders creating highly turbulent flows (station N₁, Fig. 2C) and the second one by a nearly flat bottom with gently flowing water over a soft sediment bottom (station N₂, Fig. 2C). The four stations considered along the River Ourse (O₃ and O₄) and along each of its two tributaries (O₁ and O₂; Fig. 2C) were consistently characterized by water flowing over a gently sloping rocky bottom. All samples were taken in August 2022.

## 2.2 Sediment sampling

The sediment samples were consistently collected in an area between the shoreline and the water's edge in August 2023. At each site, five sediment samples were randomly selected at a depth of 2 cm, using a metal frame with dimensions of 15 x 15 cm and a height of 2 cm and a stainless-steel shovel. Each sample was gently mixed with a metal rod within a glass beaker and subsequently placed in an aluminum foil, frozen and transported to the laboratory for further analyses.

<figure><img src="image_3.png" /><figcaption>Figure 2. Localization of (A, B) the study area in the Central Pyrénées, and (C) the sampling stations along Nistos stream and the Ourse river and its two tributaries, the 'Ourse de Ferrère' and 'Ourse de Sost' streams.</figcaption></figure>

## 2.3 Samples treatment

Each sediment sample was dried at 50 °C for 72 h and 30 g of each sample was investigated and subsequently stored in a clean polyethylene bag (Sigma Aldrich Z162965). MPs were subsequently extracted by density separation with a hypersaline solution (300 g NaCl l⁻¹) prepared with filtered water (Whatman GF/C, 47 mm of diameter and 1.2 µm of pore size). The supernatant was subsequently GF/C filtered, the filters transferred to a clean glass Petri dish and sealed with Parafilm, pending microscopic examination. The number of fibres on each filter was counted, averaged over the five replicate samples for each sampling location, and expressed in fibres kg⁻¹ of dry sediment. Note that the filters were also examined for other MPs items such as fragments, film and foam.

## 2.4 Polymer assessment and additive content

Polymer type and additive content were assessed from light (n = 3) and dark (n = 3) blue fibres randomly sampled in the Nistos and Barousse valley (see inset of Fig. 1F), and polymer type was assessed from a sub-sample (n = 18) of fibres collected in the riverbanks of stations N₁ and N₂, and O₁, O₂, O₃, and O₄. Fibre
polymer types and additive content were assessed respectively using Fourier transform infrared (FTIR) spectroscopy and gas-chromatography coupled with mass spectrometry (GC-HRMS).

## 2.5 Behavioural assays

Despite the growing awareness of the ubiquity and toxicity of plastic compounds and their leachates — i.e. the cocktail of chemical compounds released by plastic items in the environment (Delaeter et al., 2022) — in aquatic (both freshwater and marine) and terrestrial systems (see e.g. Gunaalan et al. (2020), Delaeter et al. (2022), Nam et al. (2022), Omidoyin and Jho (2024) and Junior et al. (2024) for recent reviews), we are still critically lacking information on how leachates from agricultural fibre ropes may impact local invertebrates, in particular aquatic and terrestrial gastropods.

To assess the potential biological effect of synthetic fibres, the above-mentioned light and dark blue ropes were used in a behavioural assay specifically designed to assess if the additives contained in these fibres were susceptible to induce a behavioural response in the 4 species considered in this study. The small (i.e. 5-8 mm) freshwater, air-breathing limpet *A. fluviatilis* typically occurs in oxygen-rich fast-running waters, while the 10 to 20 mm pulmonate snail *R. balthica* is characteristic of slow-moving waters with abundant vegetation such as lakes, ponds and the numerous water troughs scattered in the Nistos and Barousse valley. The conical shell (12-15 mm in length) *O. nouleti* is often found in natural or artificial shaded limestone substrates where it is found on boulders, cliffs and screes, but it is also a forest species which is very present on the trunks of smooth-barked trees, in particular beech (*Fagus sylvatica*). In contrast, the rounded shell *C. nemoralis* (15-25 mm in shell diameter) is nearly ubiquitous in temperate Europe where it is typically found in woodland edges, hedgerows, and grasslands with shaded, humid conditions.

An avoidance assay was designed in a purpose-built arena made of Lego® tiles, with a central section separated from the outer section of the arena by a 3.2-mm deep and 7.8-mm wide trench (Fig. 3). This trench was filled by a control Ficoll gel made from GF/C filtered natural Nistos stream water and Ficoll gels made from leachate solutions of light and dark blue ropes. The behavioural assay consisted of placing one organism in the central section of the experimental arena and assess its ability to cross the gel-filled trench. Leachate solutions were prepared by immersing 0.3 g (ca. 10 cm) of either light or dark blue agricultural fibre ropes in one litre of aerated control stream water for 24 h. The light and dark blue ropes used to prepare the rope leachate solutions were respectively the light blue rope #1 and dark blue rope #1 described in Table 1. Control and leachate gels were prepared by the addition of a high-molecular weight inert polymer (Ficoll 400® PM400, Sigma Aldrich) at a concentration of 20% Ficoll (w/v). Note that the dimensions of the experimental arenas were tuned to the size of the test organisms, i.e. the surface of the central part of the experimental was consistently 4.5 to 5 times greater than the surface of the locomotory foot of the organisms (Fig. 3A).

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Schematic representation of the three types of Lego® tiles experimental arenas specifically designed to run behavioural assays on *Ancylus fluviatilis* (A), *Radix balthica* and *Obscurella nouleti* (B) and *Cepaea nemoralis* (C). Scale bar = 15.6 mm.</figcaption></figure>

For each assay, the behavioural responses of 20 individual of each species were assessed in response to control Ficoll gel, and Ficoll gels obtained from leachate solutions of light and dark blue ropes. No significant differences existed between the size of the organisms used in the control and the leachate experiments, i.e. *A. fluviatilis* (5.5 ± 0.1 mm; mean ± SD), *R. balthica* (10.2 ± 0.2 mm), *O. nouleti* (12.1 ± 0.3 mm) and *C. nemoralis* (14.2 ± 0.2 mm; mean ± SD). Each behavioural assay was run with one single test organism that was placed in the centre of the experimental arena, and its behaviour was recorded for 30 minutes (or eventually stopped if the organism left the experimental arena in less than 30 minutes) from the top with a GoPro camera placed 40 cm above the experimental arenas at a frame rate of 1 image per minute for the slow-moving *A. fluviatilis*, and 1 image every 5 seconds for *R. balthica*, *O. nouleti* and *C. nemoralis*. Three experimental arenas were considered simultaneously, consistently with a control Ficoll gel and Ficoll gels
obtained from leachate solutions of light and dark blue ropes. Between each behavioural assay, the
experimental arenas were first carefully rinsed with 95% (v/v) acetone, and twice with GF/C filtered natural
Nistos stream water. All behavioural assays were conducted in June 2024, and all individuals were returned
to their natural environment after the behavioural experiments took place.

**2.6 Statistical analyses**

Multiple comparisons between sampling sites were conducted using the non-parametric Kruskal-Wallis test (KW test hereafter), which is more robust than a (parametric) analysis of variance given the small sample size (n = 5) considered in the present work. A subsequent multiple comparison procedure based on the Dunn test was then used to identify distinct groups of measurements.

**3 RESULTS AND DISCUSSION**

**3.1 Synthetic fibres are consistently present in stream sediment, but at relatively low concentrations**

Synthetic fibres have been found in all the samples, were consistently blue and made of polyethylene. These fibres ranged from to 0.12 to 2 mm in length, and their concentrations ranged from 2.35 to 16.46 fibres per kg of dry sediment (Fig. 4). No significant differences in fibre lengths were found between sites (KW test, *p* > 0.05). In contrast, fibre concentrations C significantly differ between sites (KW test, *p* < 0.05), with C(N₁) = C(O₁) = C(O₂) = C(O₃) = C(O₄) < C(N₂) (Dunn test, *p* < 0.05). Their concentrations were notably ca. 3-fold higher in station O₂, which — in contrast with the other 4 sampling stations — was characterised by a nearly flat bottom with gently flowing water. The observed increase in fibre concentration in riverbank sediment of station N₂ is consistent with the acknowledged role of flow velocity in microplastic deposition and accumulation on riverbanks and within sediments (Owowenu et al., 2023; Munz et al., 2024). It is also stressed that the concentrations of fibres observed in the present work are drastically (i.e. up to several orders of magnitude) lower than microplastic concentrations reported in the literature even from remote Himalayan and Andean streams; see e.g. Yang et al. (2021) and Canchari and Iannacone (2024). In this context, it is stressed that in sharp contrast with other studies, plastic items such as fragments, film and foam have never been found in the riverbank sediments of the 6 stations investigated in the present work. These results indicate that the Nistos and Ourse streams are only marginally impacted by plastic pollution in general and by plastic pollution related to agricultural activities in particular. Beyond the relatively limited extent of the agricultural activities held in these valleys, these results may also lie in the relative isolation of the Nistos and Barousse valleys which are both dead ends, their distance from ski resorts and the related reduced tourist activities.

<figure><img src="image_5.png" /><figcaption>Figure 4. Synthetic fibre concentration C (fibres per kilogram of dry sediment) in the riverbank sediments of the Nistos (stations N₁ and N₂) and Ourse (stations O₁, O₂, O₃ and O₄) streams.</figcaption></figure>

**3.2 Polymer type and additive content**

Both light and dark blue ropes were consistently made of polyethylene, contained only plasticizers (i.e. the low molecular weight phthalates Diethyl phthalate (DEP), Diisobutyl phthalate (DIBP) and Dimethyl phthalate (DMP)), are all acknowledged as endocrine disruptors, and for their reproductive and developmental toxicity, neurotoxicity, hepatotoxicity and nephrotoxicity and carcinogenicity; see e.g. Praveena et al. (2018) and Dutta et al. (2020) for reviews. The relative contents of these compounds differed between light and dark blue ropes (Tab. 1). Specifically, dark blue ropes (n = 3) only contained DIBP and DMP. The former was 33-, 198- and 276-fold more abundant than the latter. In contrast, light blue ropes contained either (n = 2) DEP and DIBP or (n = 1) DEP, DIBP and DMP. In any case, the proportion of these additives were highly variable as stressed for dark blue ropes. Though the resolution of this specific issue lies well beyond the scope of the
present work, further work is needed to decipher the origin of the observed variability in plasticizers content, in particular relation to the origin of their manufacturer (i.e. different manufacturers may have different additive recipes) and the impact of weathering processes (e.g. UV, wind and rain) on additive content. It is finally notable that no evidence of UV stabilisers nor antioxidants, which respectively protect the ropes against degradation to UV exposure and prevent polymer oxidation and brittleness over time.

<table><caption>Table 1. Plasticizer content of light and dark blue rope samples, expressed by the surface area (arbitrary units, Al) of the chromatogram peaks of each additive normalized by the sample size (µm), i.e. Al/µm. DEP: Diethyl phthalate, DIBP: Diisobutyl phthalate, and DMP: Dimethyl phthalate.</caption><thead><tr><th>Rope type</th><th>Sample #</th><th>DEP</th><th>DIBP</th><th>DMP</th></tr></thead><tbody><tr><td rowspan="3">Dark blue</td><td>1</td><td>-</td><td>746</td><td>3</td></tr><tr><td>2</td><td>-</td><td>769</td><td>4</td></tr><tr><td>3</td><td>-</td><td>408</td><td>13</td></tr><tr><td rowspan="3">Light blue</td><td>1</td><td>317</td><td>649</td><td>-</td></tr><tr><td>2</td><td>406</td><td>82</td><td>6</td></tr><tr><td>3</td><td>111</td><td>124</td><td>-</td></tr></tbody></table>

### 3.3 Behavioural assays

All the individuals of each species of gastropod considered in the present work (*Radix balthica*, *Obscurella nouleti* and *Cepaea nemoralis*) consistently started moving within the first minute after having been placed in the centre of the experimental arena. In contrast, only 14 *Ancylus fluviatilis* moved during the behavioural assays conducted with Ficoll gel obtained from GF/C filtered stream water. The proportion of motile *A. fluviatilis* was slightly higher in the behavioural assays conducted with Ficoll gels from leachate solutions of light and dark blue ropes, where 16 and 17 individuals moved.

<figure><img src="image_6.png" /><figcaption>Figure 5. Behavioural assay conducted on *Ancylus fluviatilis* on grey Lego® tiles with a 3.2-mm deep trench gell-free (white area; A), or filled with a 20% (w/v) Ficoll gel made with (B) GF/C filtered stream water (green stippled area), (C) light blue rope leachate solution (orange stippled area) and (D) dark blue rope leachate solution (red stippled area). The characteristic behaviour exhibited by *A. fluviatilis* in each experimental conditions is illustrated by the trajectories of five distinct individuals. Scale bar = 15.6 mm.</figcaption></figure>
R. balthica, O. nouleti and C. nemoralis did not exhibit any specific behaviour upon the encounter of the 3.2-mm deep trench which they consistently crossed and subsequently resumed their basal crawling activity (Fig. 5A). It is nevertheless stressed that a relatively small proportion of them (i.e. respectively 20%, 15% and 10% in R. balthica, O. nouleti and C. nemoralis) exhibited a positive thigmotactic behaviour, i.e. a tendency to follow the topographic discontinuity related to the surface-to-drop-off transition or the surface-to-wall transition respectively when entering and exiting the trench). This observation is in sharp contrast with the behaviour exhibited by A. fluviatilis which consistently exhibited a positive thigmotactic behaviour when entering and exiting the trench (Fig. 6A). Though the resolution of the differences in the observed thigmotactic responses in A. fluviatilis (Fig. 5A) and R. balthica, O. nouleti and C. nemoralis (Fig. 6A) lies well beyond the scope of the present work, they may be related to the size of the so-called topographic discontinuity relative to the size of the organisms considered. A 5 to 6 mm long A. fluviatilis as those considered in the present work is typically 2 to 2.5 mm high, which makes the 3.2 mm topographic discontinuity related to a surface-to-drop-off transition or a surface-to-wall transition of the trench a much stronger stimulus than for the other three species. This hypothesis is consistent with the relatively small proportion of R. balthica, O. nouleti and C. nemoralis (i.e. 20%, 15% and 10% in R. balthica, O. nouleti and C. nemoralis) exhibiting a thigmotactic behaviour when compared to the 100% observed in A. fluviatilis.

<figure><img src="image_7.png" /><figcaption>**Figure 6.** Behavioural assay conducted on *Radix balthica* on grey Lego® tiles with the 3.2-mm deep trench gell-free (white area; A), or filled with a 20% (w/v) Ficoll gel made with (B) GF/C filtered stream water (green stippled area), (C) light blue rope leachate solution (orange stippled area) and (D) dark blue rope leachate solution (red stippled area). The characteristic behaviour exhibited by *R. balthica* in each experimental conditions is illustrated by 5 trajectories. The black arrows indicate the presence of a thigmotactic behaviour (i.e. movement towards or away from the stimulus induced by a physical contact) and the white arrows the U-turn performed following a tentacle contact with the surface of a leachate gel. Scale bar = 15.6 mm.</figcaption></figure>

In accordance with previous work conducted on the intertidal species *Littorina littorea*, *L. saxatilis*, *Phorcus lineatus* and both *Steromphala cineraria* and *S. umbilicalis* (Seuront, unpublished data), none of the four species considered in the present work exhibit any avoidance behaviour towards the control Ficoll gel, which they consistently crossed without noticeably modifying the nature of their movements (Figs. 5B and 6B). These observations drastically differ from the results obtained in response to both light and dark blue rope leachate gels, where no individual ever crossed the gel-filled trench (Figs. 5C-D and 6C-D). Instead they consistently avoided the gel, with *R. balthica*, *O. nouleti* and *C. nemoralis* typically exhibiting a prodding of tentacles in the vicinity of the gel-air interface (i.e. a repeated touching of the surface with the tips of the cephalic tentacles) — as opposed to the normal tentacular motion exhibited by in the absence of foam which is that of side-to-side sweeping — and as such remained confined within the central section of the experimental arena trench (Figs. 5C-D and 6C-D). Note that *R. balthica*, *O. nouleti* and *C. nemoralis* consistently retracted their tentacles upon contact with the surface of a leachate gel — a response that was never observed when their tentacles touched the surface of a leachatre-free gel — and subsequently
exhibited a U-turn in the opposite direction of motion. This U-turn behaviour has also been observed in A. *fluviatilis* (Fig. 5C-D), but the resolution of the observations was not high enough to assess a putative tentacular response to leachate gels.

## 4 CONCLUSION

The present work was triggered by the nearly ubiquitous presence of blue synthetic ropes in the various landscapes of two valleys of the foothills of the Central Pyrénées (see Fig. 1), and the related questions of (i) their putative (i) accumulation in riverbank sediments and (ii) toxicity towards both local aquatic and terrestrial gastropods. Fibre fragments 0.12 to 2 mm in length were consistently present in the riverbank sediments of the Nistos and Ourse streams running in these valleys, but at relatively low concentrations (i.e. in the range of 2 to 16 fibres per kg of dry sediment). These results, together with the absence of other microplastic plastic items such as fragments, film and foam in the riverbank sediments of the 6 stations investigated in the present indicate that the Nistos and Ourse streams are only marginally impacted by plastic pollution in general and by plastic pollution related to agricultural activities in particular. It cannot be excluded, however, that microplastic items (including fibres) may have been trapped in stream sediment surface depending on their shape, density and polymer and/or advected downstream, leading to an underestimation of Nistos and Ourse streams as potential microplastic sinks. Though the resolution of this specific issue lies well beyond the scope of this preliminary work, it warrants the need for further work to assess the nature and concentration of microplastic items in riverbank sediment, stream sediment surface and stream water, and more generally in the soil of these foothill valleys. In this context, the non-ambiguous avoidance of rope leachates shown by the behavioural assays conducted in the present work questions the potential long-term effects of the chemical compounds contained in these synthetic ropes that are extensively used in the agricultural sector and found nearly everywhere in the foothill landscapes.

## 5 ACKNOWLEDGMENT

Leena M.Y. Seuront is acknowledged for her help in sediment sampling, and Dr. Dorine Seuront for her patience and understanding about my nearly irrepressible inclination to carry out scientific work during what should have been *stricto sensu* a holiday. Nicolas Spilmont and Mathilde Schapira are acknowledged for their suggestions and comments on a previous version of this work.

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