Differential ecotoxicity of marine litter components; identification of typologies posing higher environmental risk

Differential ecotoxicity of marine litter components; identification of typologies posing higher environmental risk | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Differential ecotoxicity of marine litter components; identification of typologies posing higher environmental risk Melissa Calviño, Alejandro Vilas, Mirco Haseler, Ricardo Beiras This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9498033/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 2 You are reading this latest preprint version Abstract Marine litter was sampled in beaches from the Baltic (Germany), NE Atlantic (Galicia) and Mediterranean (Morocco, Tunisia, Egypt, Italy) coasts according to standard procedures, and occasionally from the NE Atlantic seafloor by trawling. Collected items were associated to OSPAR and JRC typologies. The ecotoxicity of specific types of marine litter was assessed by measuring the inhibition of Paracentrotus lividus embryo-larval development caused by their leachates. Cigarette butts exhibited the highest toxicity consistently across all sampling sites, followed in decreasing order by foam, fisheries related items, and strings. The food-related items plastic caps, metal caps, and PET water bottles showed the least ecotoxicity. Ecotoxicology Marine litter Cigarette butts Sea urchin larvae Plastics Figures Figure 1 Figure 2 Figure 3 Highlights • Ecotoxicity of marine litter assessed using sea urchin larvae. • Cigarette butts showed highest toxicity from both tobacco and filter components. • Plastic foam had the second highest toxicity, linked to additives rather than polymer type. • Common food packaging showed non relevant toxic effects. 1. Introduction The Marine Strategy Framework Directive (MSFD) characterizes marine litter as “any persistent, manufactured or processed solid material discarded, disposed of or abandoned in the marine and coastal environment” (UNEP, 2005) and it is one of the eleven descriptors considered in the EU to assess the good environmental status (GES) of marine ecosystems. Progress has been made to standardize marine litter sampling protocols (GESAMP, 2019; NOAA, 2013; OSPAR Commission, 2010, 2020) but the methods to assess the impact of litter on representative marine organisms remain comparatively overlooked. This is partly due to the broad spectrum of potential deleterious effects of litter in the marine environment. Impacts of marine litter range from deterring the aesthetical values of landscape (Galgani et al., 2019), to entangle macro and megafauna with eventually lethal effects (Derraik, 2002; Kühn et al., 2015). Recently, increasing concern has been raised on the ecotoxicological effects of plastics due to their unknown composition in chemical additives (Gunaalan et al., 2020). Apart from the generally non-reactive polymeric matrix, every plastic item contains on average around 20 chemical additives (van Oers et al., 2012), mostly not covalently bound to the polymer chains and thus easily leaching into the surrounding aquatic moiety (Beiras et al., 2019). The United Nations has quantified over 13,000 chemicals associated with plastics, of which over 3,200 were of potential concern due to their hazardous properties (UNEP, 2023). Due to littering and inadequate waste disposal, an estimated 11% of global plastic production ends up in aquatic ecosystems (Borrelle et al., 2020). However, with few exceptions (Pannetier et al, 2019; Gambardella et al. 2024), most ecotoxicological studies targeting microplastics used engineered materials not representative of actual secondary microplastics originated from marine litter (reviewed by Beiras & Schönemann, 2020). Seven of the ten most common marine litter items found in coastal areas are made of plastic, including food and beverage packaging, bottle caps and plastic bags (UNEP, 2021). Over 80% of seafloor anthropogenic litter also consists of plastic (Maes et al., 2018). Even though items found in coastal areas are often similar to the ones found on the seafloor, composition and abundance differ, therefore some potentially important or common items are likely to be missed if efforts focus solely on land or the seafloor and studies should focus on a multiple compartment approach (Roman et al., 2020). The European Commission estimates that the ten most found single-use plastic items make up 43% by count of all marine litter on European beaches (Halleux, 2019). Fishing gear containing plastics accounts for another 27% (Halleux, 2019). Marine litter, a major threat to marine and coastal biodiversity, also has significant socioeconomic impacts, with costs for the EU economy estimated between €259 million and €695 million per year (Halleux, 2019). Cigarette butts (CBs) are among the most common(Araújo & Costa, 2019; UNEP, 2015) ranking first in countries such as Denmark, Germany, Poland, and Finland, and accounting for the second-highest proportion overall (15.3%) across several surveyed areas, having been found in 150 out of 197 surveys (76%) (Haseler et al., 2020). Leachate studies have shown that CBs release trace metals, aliphatic and polycyclic aromatic hydrocarbons, nicotine, and cotinine into artificial seawater within 24 hours. These compounds significantly inhibited bacterial bioluminescence, oyster embryo development, and algal growth (Lucia et al., 2023). The discarded CBs consist of unsmoked tobacco remnants, paper wrap, and a filter composed primarily of nonbiodegradable cellulose acetate, which contributes to their persistence and potential ecotoxicity in marine environments (Novotny and Slaughter 2014). Exposure studies on rotifers revealed significantly reduced population growth and density in groups treated with Unsmoked Cigarette Filters (UCFs) compared to controls by the eighth day. UCF leachates also had a pronounced inhibitory effect on fecundity, particularly through maternal exposure (Lian et al., 2024). Given these findings, it appears essential to determine whether the primary source of toxicity arises from residual tobacco compounds or from the filter material itself in order to design environmentally safer cigarette components. The aim of this study was to assess the ecotoxicity of frequently encountered types of marine litter from both the coastline and seafloor from Europe and Africa, to identify those posing the highest ecotoxicological risk to marine organisms. UCF were also included to evaluate the baseline toxicity of the filter material in the absence of tobacco combustion products, allowing comparison with smoked filters. This analysis supports the development of prevention strategies and evidence-based policies to reduce ecological harm, contributing not only to the achievement of Good Environmental Status (GES) within the European Union but also to the broader objectives of the United Nations Sustainable Development Goals, particularly Goal 14, which calls for the conservation and sustainable use of the oceans and marine resources (United Nations Department of Economic and Social Affairs., 2018) 2. Material and methods 2.1. Study sites: The marine litter items tested are listed in Suppl Mat., Table 1 . Seafloor litter was collected in 2019 by trawling fishing boats from the Ports of Marín and Vigo (Galicia, NW Iberian Peninsula) as part of the Repescaplas Project ( Fundación Biodiversidad ). Intertidal litter was gathered from 100m macro-litter and meso-litter surveys conducted according to JRC-approved standard methods (MSFD Technical Subgroup on Marine Litter (MSFD TSG ML), 2023) in beaches in Europe and North Africa through multiple initiatives, including the Spanish marine litter monitoring program in Galicia (NW Iberian Peninsula), the IOW marine debris surveys on German and Lithuanian Baltic coasts (2017–2019), the JPI Oceans RESPONSE Project (2022), the H2020 LABPLAS Project (2022–2023), and African beach assessments (TouMali Project). Further details on the field surveys are described by Haseler et al. (2025). The most common items found were tentatively identified (Suppl Mat. Figure A), and processed as below described. 2.2. Tested materials All marine litter items were collected, classified based on typologies outlined in the OSPAR Agreement (2020), the Joint Research Centre list (Hanke et al., 2021) and monitoring guidelines provided by Ferreira (2013), and then stored in the dark at room temperature. Unsmoked cigarette filters (Suppl Mat. Figure B) from seven different commercial brands were also collected and pooled together to represent the variability of cigarette butts typically found on beaches. When possible, the polymer identity was ascertained by using Fourier- transform infrared spectroscopy (FTIR) with a Thermo Scientific Nicolet 6700. 2.3. Toxicity tests Marine aquatic toxicity of leachates was assessed using the Paracentrotus lividus sea-urchin embryo test (SET), a rapid and sensitive method following the standardized protocol described by Beiras et al . (2019). Samples from the most common typologies and UCFs were micronized using either a ZM200 ultracentrifuge mill, a CryoMill (both Retsch, Verder Scientific), or a stainless-steel file, depending on their mechanical properties. Sea urchins were supplied on the day of testing by the Marine Culture Unit of ECIMAT (CIM–University of Vigo), which maintains a stock of sexed mature P. lividus collected from the outer Ría de Vigo. In-vitro fertilization was performed according to Beiras et al. 2012. Leachates were prepared following the protocol by Almeda et al . (2023), using for all materials the < 250 µm fraction. The sieved fraction was mixed with chemically-defined artificial seawater (ASW) (Lorenzo et al., 2002) at a proportion of 10 g L-1 in glass bottles with no head space, which were incubated for 24 h at 20 ◦C in darkness using an overhead rotator (GFL 3040) at 1 rpm. Leachates were obtained by filtration through Whatman® GF/F filters and tested via serial dilutions in ASW according to standard SET procedures adapted to microplastics (e.g. Uribe-Echeverría and Beiras, 2022), using 4 mL glass vials per quadruplicate. Images of the formalin-fixed vials were captured using a Leica DMI 4000B inverted microscope, and the length increase of 35 individuals per vial, defined as the maximum dimension minus the average egg diameter, was automatically recorded using in-house Artificial Intelligence algorithms developed in collaboration with the VARPA Research Group (University of A Coruña). 2.4. Statistical analysis and assessment criteria Toxicity parameters (EC 10 , EC 50 ) and their 95% confidence intervals were calculated by fitting the data to a probit dose-response model using SPSS v24 statistical software. Toxic Units (TU) were calculated as TU = 1/EC 50 , where the EC50 is the dilution (in parts per one) of the 10 g/L leachate reducing by 50% the larval growth. Materials were classified following the assessment criteria shown in Table 1 , modified from Alonso-López et al., (2021). 3. Results 3.1. Marine litter items toxicity tests The ecotoxicity of plastic items belonging to specific types of marine litter was assessed by measuring the inhibition of Paracentrotus lividus embryo-larval development caused by their leachates. For each item class, effective concentrations (EC 10 and EC 50 ) and Toxic Units (TU) were determined to quantify toxicity, with 95% confidence intervals reported where applicable. Results are presented by litter type and geographical area in Table 2 . Cigarette butts Leachates from cigarette butts have shown consistently medium toxicity across all samples with EC50 values ranged from 1,030 to 1,859 mg/L, and Toxic Units (TU) between 5.38 and 9.70. Highest toxicity was observed in the Baltic Sea, sample 136E (TU = 9.70; 95% CI: 8.07–11.70), while the lowest was in Egypt, sample 160D (TU = 5.38; 95% CI: 4.50–6.43). The samples 160D (Egypt) and 157D (Morocco), were also tested using the Microtox assay (measurement of Vibrio fischeri bioluminescence inhibition). After 30 minutes of exposure, results showed mean EC50 values of 29.35 mg/L (95% CI: 17.49–49.24) and 37.96 mg/L (95% CI: 24.11–59.77), and Toxicity Units (TU) values of 0.03 and 0.02, respectively. These results are consistent with the toxicity levels observed in the SET, supporting the overall assessment of their ecotoxic potential. Two additional tests were conducted with a pool of unsmoked cigarette filters (UCFs) from different brands and with Unsmoked Biodegradable Filters (UBFs), which are marketed under the promise of being organic, natural, biodegradable, and unbleached. The UCFs showed ecotoxicity but to a lower extent than all the cigarette butt samples (Suppl Mat. Figure C). The EC10 was calculated at 764 mg/L, and the EC50 at 3,257 mg/L, corresponding to 3.1 (95% confidence interval: 2.54–3.66) TU (yellow ecotoxicity class). While the UBFs showed no toxicity at all, with noncalculable EC10 and EC50 values, indicating they were non-toxic. Foam Leachates from plastic foam items generally ranged from non-toxic (< 1 TU) to low (2.9 TU). Most samples exhibited high EC50 values or were non-calculable (NC), with Toxic Unit (TU) values below 1, indicating minimal toxic effects. Sample 136D showed the highest toxicity, with an EC50 of 3,448 mg/L and a TU of 2.90 (95% CI: 2.22–3.66). Sample 160C showed low toxicity, with an EC50 of 7,874 mg/L and a TU of 1.26 (95% CI: 0.88–1.64) and the NE Atlantic sample, a piece of insulating cork (REP-05), also showed low toxicity with a TU of 1.01 (95% CI: 0.76–1.46). Strings and cords Among the tested marine litter types, the leachates from string and cord samples revealed generally low toxicity, with values ranging from 10,000 mg/L and TU values < 1, indicating negligible effects. In contrast, the Galician coast, sample 153E, which consisted of strings and cords thicker than those from other origins identifiable as rests of derelict fishing nets, exhibited slight but measurable toxicity, with an EC50 of 6,803 mg/L and a TU of 1.50 (95% CI: 1.10–1.90). Aquaculture Leachates from aquaculture-related plastic litter showed from negligible to low ecotoxicity. The beach samples collected in Galicia (082), which consisted of polyethylene (PE) plastic sticks used in mussel rafts, and Italy (ID091-RSP), which consisted of plastic nets used for mussel culture (66% polypropylene, 32% PE), both had EC50 values above 10,000 mg/L and TU values below 1. Two of the fisheries related samples collected from the seafloor: a buoy (REP-04) and an octopus trap (REP-03) also lack toxicity, but the third one, a piece of rope (REP-02), did exhibit inhibitory effects at the levels tested (EC50 = 3,546 mg/L). Plastic caps Leachates from plastic caps generally showed low toxicity. Most of the samples presented EC50 values greater than 10,000 mg/L and TU values below 1, indicating negligible toxic effects. Sample from Egypt (160A) was an exception, with an EC50 of 9,009 mg/L and a TU of 1.11 (95% CI: 1.00–1.22), suggesting low toxicity. The remaining samples had non-calculable (NC) values or very high EC50s, reinforcing the overall negligible toxicity of plastic cap leachates. Metal bottle caps, lids and pull tabs Leachates from metal bottle caps, lids, and pull tabs showed negligible toxicity. Both the Baltic Sea (136G) and Galician (153D) samples had EC50 values exceeding 10,000 mg/L and TU values below 1, indicating minimal inhibitory effects. Plastic bottles (PET) Leachates from PET plastic bottles, either coming from the beach or the seafloor, exhibited no toxicity. The NE Atlantic sample (REP-01) showed an EC10 of 5,555 mg/L, while the Italian sample (ID094-RSP) had no calculable EC10. Both PET samples had EC50 values above 10,000 mg/L and TU values below 1, indicating minimal inhibitory effects. 4. Discussion Cigarette butts showed higher toxicity compared to other major types of marine litter, all of which fell within the “medium” toxicity category. In contrast, unsmoked cigarette filters (UCFs) also demonstrated toxicity but were classified as “low,” indicating a smaller but still measurable level of ecotoxicity. This suggests that the toxic effects observed in cigarette butts are not only due to tobacco combustion byproducts, but also partially attributable to the materials that make up the filter itself. These results align with the findings of Novotny and Slaughter (2014), who noted that cigarettes and their waste, particularly discarded filters with remnant tobacco, contain a wide range of environmentally harmful chemicals. These include residues from agricultural treatments, contaminants absorbed from soil, manufacturing additives and combustion products produced during smoking. Furthermore, our findings are consistent with those of Lucia et al. (2023), who reported that naturally smoked cigarette butts (not yet degraded in the marine environment) reduced bioluminescence in A. fischeri , caused abnormal embryo development in M. gigas , and consistently inhibited algal growth across three species ( P. tricornutum , S. costatum and D. tertiolecta ). Together, these results point to a clear dose–response relationship and reinforce the conclusion that cigarette butts are ecotoxic in multiple forms, whether freshly smoked, degraded in the marine environment, or even as unsmoked filters, supporting that the observed ecotoxicity is partially due to original filter components, and not only to those acquired upon combustion and ulterior weathering in marine conditions. Notably, all tested samples exhibited toxicity regardless of their country of origin, suggesting that the ecotoxic potential of cigarette butts is consistent across brands and regions, and not significantly mitigated by national differences in tobacco production or regulations. In addition, it is clear that the withdrawal of cigarette butts remains a major problem for both manual and mechanical (Zielinski et al., 2019) underscoring the ongoing challenges in effectively managing this persistent form of marine litter. In comparison with the “biodegradable” filters, which showed no toxicity, it can be assumed that cigarette butt toxicity occurs at two levels: primarily due to tobacco and combustion products, and secondarily due to the plastic material in conventional filters. Considering the availability of less toxic alternatives, it may be interesting to promote their use. This is not a definitive solution, since the main source of toxicity comes from the tobacco itself, but it could contribute to reducing the overall environmental impact. The “foam” typology includes the expanded polystyrene (EPS) commonly used in shock-sensitive packaging and insulation, and polyurethane. Three of the five plastic foam samples tested showed EC10 values < 2,000 mg/L, supporting a remarkably higher toxicity than caps, bottles, strings and cords, but consistently lower than cigarette butts. The heterogeneous EC10 values obtained for the five samples tested suggest that the observed effects are not inherent to the polymer type itself, but either to the specific chemical composition of each item, or to the hydrophobic organic chemicals (HOCs) sorbed from the seawater in each area. Some types of EPS commonly found in marine debris carry brominated flame retardants and other potentially toxic additives (Jang et al., 2016). This supports the conclusions of Beiras et al. (2021), who emphasized the importance of reducing the environmental impact of plastic materials through careful selection of polymer additives. However, alternative explanations of heterogeneous results for common typologies that cannot be ruled out include differential sorption of HOCs from the surrounding water in each sampling area. Plastics can accumulate HOCs such as PCBs and pesticides at concentrations many orders of magnitude higher than in the surrounding water (Mato et al., 2001), and PS shows higher sorption coefficients than other common polymers such as HDPE (Uber et al., 2019) Strings and cords, as well as plastic bottle caps, generally exhibited non-toxic behaviour in our tests, with only one sample from each category falling into the low toxicity range. These discordances may be attributed to the variability in chemical additives, particularly colorants, as these items were visually diverse (Suppl Mat. Figure A) and illustrate the need to expand this kind of study to larger marine litter collections. Metal marine litter items, primarily bottle caps, lids, and pull tabs, showed no toxic effects on P. lividus larval growth. However, during the 24-hour leachate preparation period, a notable drop in dissolved oxygen levels was observed, from 7.14 mg/L to 4.54 mg/L, probably due to metal oxidation. To prevent confounding effects from hypoxia, all leachates underwent a 10-minute aeration before being used in the bioassay, ensuring that any observed effects were due to the material’s toxicity rather than reduced oxygen availability. Although no relevant toxicity was detected, these findings highlight that metal litter in confined environments may still contribute to decrease key water quality parameters, such as oxygen levels, which could indirectly affect marine organisms. Regarding plastic bottles (PET), results showed no significant toxicity from leachates, consistent with the findings of Gambardella et al., (2024), who also found no adverse effects in marine invertebrates and vertebrates exposed to PET. In contrast, Piccardo et al. (2020) reported significant reductions in larval growth of Paracentrotus lividus when exposed to PET leachates derived from particles of varying sizes. Although all studies used the same test species and bioassay techniques, the contrasting results point to differences likely driven by the specific chemical additives or residues present in the plastics. Piccardo et al . (2020) also reported morphological deformities in larvae exposed to PET leachates, which is consistent with our observations, although in our case no significant reduction in larval size was observed compared to the control. These findings suggest that even within a single litter category like PET bottles, ecotoxicity can vary significantly depending on the product’s formulation, highlighting the importance of chemical composition in assessing environmental impact. Leachates from most aquaculture derived products, like mussel raft sticks and mussel nets, showed no detectable toxicity in our study. Although neither leachate toxicity nor ingestion-based effects were evident, these materials remain an important source of marine litter due to their persistence, requiring further research on potential long-term effects. Wu et al. (2020), similarly observed that aquaculture operations contribute to microplastic contamination in sediments and seawater, and that microplastic accumulation occurs in commercial species. However, the levels in edible tissues were found to be negligible. Moreover, one of the items, a piece of blue rope, did inhibit embryo development at the concentrations tested. Further research on the chemical content of this specific item would be necessary in order to explain its ecotoxicity. Unlike other typologies, plastic pieces associated to fishing and aquaculture do not derive from littering or inadequate solid waste disposal but from the accidental loss of pieces of fishing gear (e.g. dolly ropes), fishing traps, and extensive aquaculture devices (e.g. plastic sticks used in mussel ropes). Fishing activities are the source of more than two-thirds of plastic litter in the Mediterranean coasts of Spain (García-Rivera et al., 2017) and in the North Atlantic it accounts for 83% of marine litter, being the most abundant typology (Gil Gamundi & Martínez-Gil Pardo de Vera, 2020). For some specific plastic pieces of fisheries equipment prone to get lost upon use and not suitable for recovery and reuse, replacement of conventional by biodegradable plastic materials may greatly contribute to reduce their long-term environmental impact. Ecological impacts of marine litter are complex. Physical impacts beyond the scope of the present study include entanglement of wildlife and ghost fishing, and blockage of digestive system upon ingestion (reviewed by Kühn et al., 2015). Ecotoxicological impacts, targeted here, stem from the potential toxicity of leaching chemicals originally present in the plastic as functional additives (Beiras et al., 2021) or sorbed during weathering in the sea (Ferrari et al., 2024). We have found that leachates from different plastic litter items remarkably differ in ecotoxicity. This will reflect in very different environmental risk posed by each typology. Following the conventional risk assessment methodology, risk is estimated from the ratio of environmental concentrations vs the toxicity thresholds, which can be estimated by the EC10 values. In this first approach we have found that average EC10 for different litter typologies can vary over 10-fold (Fig. 3 ). Therefore, future research should further address these differences by using additional test species and exploiting more comprehensive marine litter collections in order to identify the typologies posing the highest risk. This will help prioritize remediation actions and will guide more ecologically relevant plastic pollution prevention policies. 5. Conclusions These results stress the need to advance beyond total numbers of marine litter items in marine monitoring and consider the different impacts of various typologies to produce a more ecologically meaningful assessment of environmental status within the context of the MSFD. Research should prioritize the most toxic litter categories to identify the specific additives responsible for their effects, especially in items like plastic foam or cigarette butts, which pose a remarkably higher ecotoxicological risk than other plastic litter components mostly originating from food packaging. Importantly, cigarette butts are often overlooked during professional beach cleanups, whether mechanical or manual, and consequently remain embedded in the sediment where they may continue to leach harmful substances into the environment. This highlights the urgent need for effective mitigation strategies against littering and inadequate solid waste disposal in order to prevent cigarette butts, foams, and other litter types from reaching the marine environment in the first place. Declarations Acknowledgements We thank Patricia Rubio, Sara López-Ibáñez, Pedro Campoy-López and Leticia Vidal-Liñán for their helpful technical assistance, and Olalla Alonso, Aaron Beck and Ciara Gambardella for providing samples from Galicia, Baltic Sea and Adriatic, respectively. CRediT authorship contribution statement Melissa Calviño : Conceptualization, Methodology, Formal analysis, Writing -Original draft, Writing -Review & editing, Investigation, Visualization. Alejandro Vilas : Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data Curation, Project administration. Mirco Haseler : Investigation, Resources, Writing -Review & editing, Supervision, Funding acquisition. Ricardo Beiras : Conceptualization, Methodology, Software, Validation, Formal analysis, Resources, Writing - review & editing, Supervision, Funding acquisition. Funding This research was partially funded by the Ministry of Science and Innovation—Spanish Agency of Research (AEI) through the Project: Safe Additives for the Plastic INdustry (SAPIN) (PID2022-138421OB- C22), by the Ministry of ecological Transition (MITECO) through the REPESCAPLAS Project (Fundación Biodiversidad), by the EC through the H2020 LABPLAS Project (H2020 EU3.5.4 Ref 101003954), by the BMU/ZUG project TouMaLi (Beitrag der nachhaltigen Abfallwirtschaft im Tourismus zum Schutz der Meeresökosysteme), grant number 65MM0001 (litter collection in Africa), and by the JPI Oceans RESPONSE Project (litter collection in the Adriatic). Program of Consolidation and Structuring of Units of Competitive Investigation of the University System of Galicia (Xunta de Galicia) potentially co-financed by ERDF (ED431C 2021/56) is also acknowledged. 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Reducing marine litter from plastics . https://www.europarl.europa.eu/RegData/etudes/ATAG/2019/635580/EPRS_ATA(2019)635580_EN.pdf Hanke, T., Msfd, G., & Hanke, G. (2021). A Joint List of Litter Categories for Marine Macrolitter Monitoring Manual for the application of the classification system Title: A Joint List of Litter Categories for Marine Macrolitter Monitoring . https://doi.org/10.2760/127473 Haseler, M., Balciunas, A., Hauk, R., Sabaliauskaite, V., Chubarenko, I., Ershova, A., & Schernewski, G. (2020). Marine Litter Pollution in Baltic Sea Beaches – Application of the Sand Rake Method. Frontiers in Environmental Science , 8 , 599978. https://doi.org/10.3389/FENVS.2020.599978/BIBTEX Haseler, M., Ben Abdallah, L., El Fels, L., El Hayany, B., Hassan, G., Escobar-Sánchez, G., Robbe, E., von Thenen, M., Loukili, A., Abd El-Raouf, M., Mhiri, F., El-Bary, A. A., Schernewski, G., & Nassour, A. (2025). Assessment of beach litter pollution in Egypt, Tunisia, and Morocco: a study of macro and meso-litter on Mediterranean beaches. Environmental Monitoring and Assessment , 197 (1). https://doi.org/10.1007/s10661-024-13517-x Jang, M., Shim, W. J., Han, G. M., Rani, M., Song, Y. K., & Hong, S. H. (2016). Styrofoam Debris as a Source of Hazardous Additives for Marine Organisms. Environmental Science and Technology , 50 (10), 4951–4960. https://doi.org/10.1021/acs.est.5b05485 Kühn, S., Bravo Rebolledo, E., & van Franeker, J. A. (2015). Deleterious Effects of Litter on Marine Life. In Anthropogenic marine litter (pp. 75–116). Springer. Lian, H., Zhu, L., Li, M., Feng, S., Gao, F., Zhang, X., Zhang, F., Xi, Y., & Xiang, X. (2024). Emerging threat of marine microplastics: Cigarette butt contamination on Yellow Sea beaches and the potential toxicity risks to rotifer growth and reproduction. Journal of Hazardous Materials , 478 . https://doi.org/10.1016/j.jhazmat.2024.135534 Lorenzo, J. I., Nieto, O., & Beiras, R. (2002). Effect of humic acids on speciation and toxicity of copper to Paracentrotus li6idus larvae in seawater. In Aquatic Toxicology (Vol. 58). www.elsevier.com/locate/aquatox Lucia, G., Giuliani, M. E., d’Errico, G., Booms, E., Benedetti, M., Di Carlo, M., Fattorini, D., Gorbi, S., & Regoli, F. (2023). Toxicological effects of cigarette butts for marine organisms. Environment International , 171 , 107733. https://doi.org/10.1016/J.ENVINT.2023.107733 Maes, T., Barry, J., Leslie, H. A., Vethaak, A. D., Nicolaus, E. E. M., Law, R. J., Lyons, B. P., Martinez, R., Harley, B., & Thain, J. E. (2018). Below the surface: Twenty-five years of seafloor litter monitoring in coastal seas of North West Europe (1992–2017). Science of the Total Environment , 630 , 790–798. https://doi.org/10.1016/j.scitotenv.2018.02.245 Mato, Y., Isobe, T., Takada, H., Kanehiro, H., Ohtake, C., & Kaminuma, T. (2001). Plastic resin pellets as a transport medium for toxic chemicals in the marine environment. Environmental Science and Technology , 35 (2), 318–324. https://doi.org/10.1021/es0010498 MSFD Technical Subgroup on Marine Litter (MSFD TSG ML). (2023). Guidance on the monitoring of marine litter in European seas: An update to improve the harmonised monitoring of marine litter under the Marine Strategy Framework Directive . https://doi.org/10.2760/59137 NOAA. (2013). Marine debris monitoring and assessment: Recommendations for monitoring debris trends in the marine environment . Novotny, T. E., & Slaughter, E. (2014). Tobacco Product Waste: An Environmental Approach to Reduce Tobacco Consumption. In Current environmental health reports (Vol. 1, Issue 3, pp. 208–216). Springer. https://doi.org/10.1007/s40572-014-0016-x OSPAR Commission. (2010). Guidelines for Monitoring Marine Litter on the Beaches . OSPAR Commission. (2020). CEMP Guidelines for Beach Litter Monitoring . https://repository.oceanbestpractices.org/bitstream/handle/11329/1886/20-02e_cemp_guideline_beach_litter.pdf?sequence=1&isAllowed=y Pannetier, P., Morin, B., Clérandeau, C., Laurent, J., Chapelle, C., Cachot, J., 2019. Toxicity assessment of pollutants sorbed on environmental microplastics collected on beaches: Part II-adverse effects on Japanese medaka early life stages. Environ. Pollut. 248, 1098–1107. https://doi.org/10.1016/j.envpol.2018.10.129 Piccardo, M., Provenza, F., Grazioli, E., Cavallo, A., Terlizzi, A., & Renzi, M. (2020). PET microplastics toxicity on marine key species is influenced by pH, particle size and food variations. Science of The Total Environment , 715 , 136947. https://doi.org/10.1016/J.SCITOTENV.2020.136947 Roman, L., Hardesty, B. D., Leonard, G. H., Pragnell-Raasch, H., Mallos, N., Campbell, I., & Wilcox, C. (2020). A global assessment of the relationship between anthropogenic debris on land and the seafloor. Environmental Pollution , 264 . https://doi.org/10.1016/j.envpol.2020.114663 Uber, T. H., Hüffer, T., Planitz, S., & Schmidt, T. C. (2019). Sorption of non-ionic organic compounds by polystyrene in water. Science of the Total Environment , 682 , 348–355. https://doi.org/10.1016/j.scitotenv.2019.05.040 UNEP. (2015). Marine litter assessment in the Mediterranean. United Nations Environment Programme Mediterranean Action Plan (UNEP/MAP) . UNEP. (2021). Drowning in Plastics – Marine Litter and Plastic Waste Vital Graphics . United Nations Department of Economic and Social Affairs. (2018). The Sustainable Development Goals Report 2018. In UN DESA, New York, NY, USA . https://unstats.un.org/sdgs/files/report/2018/TheSustainableDevelopmentGoalsReport2018-EN.pdf Uribe-Echeverría, T., & Beiras, R. (2022). Acute toxicity of bioplastic leachates to Paracentrotus lividus sea urchin larvae. Marine Environmental Research , 176 . https://doi.org/10.1016/j.marenvres.2022.105605 van Oers, L., van der Voet, E., & Grundmann, V. (2012). Additives in the Plastics Industry. Handbook of Environmental Chemistry , 18 (August 2011), 133–149. https://doi.org/10.1007/698_2011_112 Wu, F., Wang, Y., Leung, J. Y. S., Huang, W., Zeng, J., Tang, Y., Chen, J., Shi, A., Yu, X., Xu, X., Zhang, H., & Cao, L. (2020). Accumulation of microplastics in typical commercial aquatic species: A case study at a productive aquaculture site in China. Science of The Total Environment , 708 , 135432. https://doi.org/10.1016/J.SCITOTENV.2019.135432 Zielinski, S., Botero, C. M., & Yanes, A. (2019). To clean or not to clean? A critical review of beach cleaning methods and impacts. In Marine Pollution Bulletin (Vol. 139, pp. 390–401). Elsevier Ltd. https://doi.org/10.1016/j.marpolbul.2018.12.027 Tables Tables 1 & 2 are available in the Supplementary Files section. Supplementary Files MarineLitterEcotoxicityTable1.docx MarineLitterEcotoxicityTable2.docx MarineLitterEcotoxicitySupplementaryMaterial.docx MarineLitterEcotoxicityGraphicalAbstract.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 24 Apr, 2026 First submitted to journal 22 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9498033","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":628962919,"identity":"f996a46c-a350-4420-b36d-06634dcf6282","order_by":0,"name":"Melissa Calviño","email":"data:image/png;base64,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","orcid":"https://orcid.org/0009-0000-6660-4390","institution":"Universidade de Vigo","correspondingAuthor":true,"prefix":"","firstName":"Melissa","middleName":"","lastName":"Calviño","suffix":""},{"id":628962920,"identity":"a4cfb14d-5864-4420-9240-8adc68d94714","order_by":1,"name":"Alejandro Vilas","email":"","orcid":"","institution":"University of Vigo: Universidade de Vigo","correspondingAuthor":false,"prefix":"","firstName":"Alejandro","middleName":"","lastName":"Vilas","suffix":""},{"id":628962921,"identity":"d163cd3d-4375-4724-b2cb-8192a46d04f6","order_by":2,"name":"Mirco Haseler","email":"","orcid":"","institution":"Leibniz-Institut fur Meereswissenschaften: Helmholtz-Zentrum fur Ozeanforschung Kiel","correspondingAuthor":false,"prefix":"","firstName":"Mirco","middleName":"","lastName":"Haseler","suffix":""},{"id":628962922,"identity":"86fceab2-c7e1-4876-81f7-cd0c271f1bb0","order_by":3,"name":"Ricardo Beiras","email":"","orcid":"","institution":"University of Vigo: Universidade de Vigo","correspondingAuthor":false,"prefix":"","firstName":"Ricardo","middleName":"","lastName":"Beiras","suffix":""}],"badges":[],"createdAt":"2026-04-22 15:06:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9498033/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9498033/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109067810,"identity":"fd4fa90b-3d75-4d80-b3fa-ab3e47fdf592","added_by":"auto","created_at":"2026-05-12 10:01:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106933,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMost frequently collected items during the 2018 International Coastal Cleanup (Ocean Conservancy, 2019), with corresponding OSPAR reference codes.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9498033/v1/82e389f7896c5389be56f872.png"},{"id":108837723,"identity":"2e9475c9-fff2-4f60-b704-e97f8b87cc7d","added_by":"auto","created_at":"2026-05-09 00:16:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":224899,"visible":true,"origin":"","legend":"\u003cp\u003eSampling sites where field surveys were conducted are marked with red dots. Countries are indicated using ISO 3-letter codes.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9498033/v1/b4d2e550c18f80f483777786.png"},{"id":108977148,"identity":"7c43034f-5758-4074-907e-5c934fe587c1","added_by":"auto","created_at":"2026-05-11 11:30:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":62690,"visible":true,"origin":"","legend":"\u003cp\u003eSET EC10 values by marine litter typology. Boxplots show median, quartiles, and range; red dots indicate mean values. Jittered points represent individual locations.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9498033/v1/500163271de6e94306f81290.png"},{"id":109081333,"identity":"5a3bd029-d41f-44c9-b29b-95b831e7d233","added_by":"auto","created_at":"2026-05-12 12:17:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":676831,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9498033/v1/8dfc80bd-6a0c-4e35-ae05-faa27f52f191.pdf"},{"id":108837722,"identity":"a0d66287-5264-4fb4-abc9-0fa52815600c","added_by":"auto","created_at":"2026-05-09 00:16:06","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15663,"visible":true,"origin":"","legend":"","description":"","filename":"MarineLitterEcotoxicityTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9498033/v1/859ba2b29b72b57ea65e64b4.docx"},{"id":108837725,"identity":"ba55cc2e-8987-4a05-85f6-f33557fd27c0","added_by":"auto","created_at":"2026-05-09 00:16:06","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":19703,"visible":true,"origin":"","legend":"","description":"","filename":"MarineLitterEcotoxicityTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-9498033/v1/57ca079a7462686ad59f70d2.docx"},{"id":108977661,"identity":"5eddc54d-b93a-4c78-b466-d2e4c2662710","added_by":"auto","created_at":"2026-05-11 11:32:28","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":13628029,"visible":true,"origin":"","legend":"","description":"","filename":"MarineLitterEcotoxicitySupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9498033/v1/99cf0ae6e0554107d18017fe.docx"},{"id":108837727,"identity":"09934010-ae6d-4238-93ac-f12c020ac52d","added_by":"auto","created_at":"2026-05-09 00:16:06","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":1613409,"visible":true,"origin":"","legend":"","description":"","filename":"MarineLitterEcotoxicityGraphicalAbstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9498033/v1/0811ccb7fa6af990469ff2fc.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eDifferential ecotoxicity of marine litter components; identification of typologies posing higher environmental risk\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cp\u003e\u0026bull; Ecotoxicity of marine litter assessed using sea urchin larvae.\u003c/p\u003e\u003cp\u003e\u0026bull; Cigarette butts showed highest toxicity from both tobacco and filter components.\u003c/p\u003e\u003cp\u003e\u0026bull; Plastic foam had the second highest toxicity, linked to additives rather than polymer type.\u003c/p\u003e\u003cp\u003e\u0026bull; Common food packaging showed non relevant toxic effects.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eThe Marine Strategy Framework Directive (MSFD) characterizes marine litter as \u0026ldquo;any persistent, manufactured or processed solid material discarded, disposed of or abandoned in the marine and coastal environment\u0026rdquo; (UNEP, 2005) and it is one of the eleven descriptors considered in the EU to assess the good environmental status (GES) of marine ecosystems. Progress has been made to standardize marine litter sampling protocols (GESAMP, 2019; NOAA, 2013; OSPAR Commission, 2010, 2020) but the methods to assess the impact of litter on representative marine organisms remain comparatively overlooked. This is partly due to the broad spectrum of potential deleterious effects of litter in the marine environment. Impacts of marine litter range from deterring the aesthetical values of landscape (Galgani et al., 2019), to entangle macro and megafauna with eventually lethal effects (Derraik, 2002; K\u0026uuml;hn et al., 2015).\u003c/p\u003e \u003cp\u003eRecently, increasing concern has been raised on the ecotoxicological effects of plastics due to their unknown composition in chemical additives (Gunaalan et al., 2020). Apart from the generally non-reactive polymeric matrix, every plastic item contains on average around 20 chemical additives (van Oers et al., 2012), mostly not covalently bound to the polymer chains and thus easily leaching into the surrounding aquatic moiety (Beiras et al., 2019). The United Nations has quantified over 13,000 chemicals associated with plastics, of which over 3,200 were of potential concern due to their hazardous properties (UNEP, 2023). Due to littering and inadequate waste disposal, an estimated 11% of global plastic production ends up in aquatic ecosystems (Borrelle et al., 2020). However, with few exceptions (Pannetier et al, 2019; Gambardella et al. 2024), most ecotoxicological studies targeting microplastics used engineered materials not representative of actual secondary microplastics originated from marine litter (reviewed by Beiras \u0026amp; Sch\u0026ouml;nemann, 2020).\u003c/p\u003e \u003cp\u003eSeven of the ten most common marine litter items found in coastal areas are made of plastic, including food and beverage packaging, bottle caps and plastic bags (UNEP, 2021). Over 80% of seafloor anthropogenic litter also consists of plastic (Maes et al., 2018). Even though items found in coastal areas are often similar to the ones found on the seafloor, composition and abundance differ, therefore some potentially important or common items are likely to be missed if efforts focus solely on land or the seafloor and studies should focus on a multiple compartment approach (Roman et al., 2020).\u003c/p\u003e \u003cp\u003eThe European Commission estimates that the ten most found single-use plastic items make up 43% by count of all marine litter on European beaches (Halleux, 2019). Fishing gear containing plastics accounts for another 27% (Halleux, 2019). Marine litter, a major threat to marine and coastal biodiversity, also has significant socioeconomic impacts, with costs for the EU economy estimated between \u0026euro;259\u0026nbsp;million and \u0026euro;695\u0026nbsp;million per year (Halleux, 2019).\u003c/p\u003e \u003cp\u003eCigarette butts (CBs) are among the most common(Ara\u0026uacute;jo \u0026amp; Costa, 2019; UNEP, 2015) ranking first in countries such as Denmark, Germany, Poland, and Finland, and accounting for the second-highest proportion overall (15.3%) across several surveyed areas, having been found in 150 out of 197 surveys (76%) (Haseler et al., 2020).\u003c/p\u003e \u003cp\u003eLeachate studies have shown that CBs release trace metals, aliphatic and polycyclic aromatic hydrocarbons, nicotine, and cotinine into artificial seawater within 24 hours. These compounds significantly inhibited bacterial bioluminescence, oyster embryo development, and algal growth (Lucia et al., 2023).\u003c/p\u003e \u003cp\u003eThe discarded CBs consist of unsmoked tobacco remnants, paper wrap, and a filter composed primarily of nonbiodegradable cellulose acetate, which contributes to their persistence and potential ecotoxicity in marine environments (Novotny and Slaughter 2014). Exposure studies on rotifers revealed significantly reduced population growth and density in groups treated with Unsmoked Cigarette Filters (UCFs) compared to controls by the eighth day. UCF leachates also had a pronounced inhibitory effect on fecundity, particularly through maternal exposure (Lian et al., 2024). Given these findings, it appears essential to determine whether the primary source of toxicity arises from residual tobacco compounds or from the filter material itself in order to design environmentally safer cigarette components.\u003c/p\u003e \u003cp\u003eThe aim of this study was to assess the ecotoxicity of frequently encountered types of marine litter from both the coastline and seafloor from Europe and Africa, to identify those posing the highest ecotoxicological risk to marine organisms. UCF were also included to evaluate the baseline toxicity of the filter material in the absence of tobacco combustion products, allowing comparison with smoked filters. This analysis supports the development of prevention strategies and evidence-based policies to reduce ecological harm, contributing not only to the achievement of Good Environmental Status (GES) within the European Union but also to the broader objectives of the United Nations Sustainable Development Goals, particularly Goal 14, which calls for the conservation and sustainable use of the oceans and marine resources (United Nations Department of Economic and Social Affairs., 2018)\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Study sites:\u003c/h2\u003e\n \u003cp\u003eThe marine litter items tested are listed in Suppl Mat., Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Seafloor litter was collected in 2019 by trawling fishing boats from the Ports of Mar\u0026iacute;n and Vigo (Galicia, NW Iberian Peninsula) as part of the Repescaplas Project (\u003cem\u003eFundaci\u0026oacute;n Biodiversidad\u003c/em\u003e). Intertidal litter was gathered from 100m macro-litter and meso-litter surveys conducted according to JRC-approved standard methods (MSFD Technical Subgroup on Marine Litter (MSFD TSG ML), 2023) in beaches in Europe and North Africa through multiple initiatives, including the Spanish marine litter monitoring program in Galicia (NW Iberian Peninsula), the IOW marine debris surveys on German and Lithuanian Baltic coasts (2017\u0026ndash;2019), the JPI Oceans RESPONSE Project (2022), the H2020 LABPLAS Project (2022\u0026ndash;2023), and African beach assessments (TouMali Project). Further details on the field surveys are described by Haseler et al. (2025). The most common items found were tentatively identified (Suppl Mat. Figure A), and processed as below described.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Tested materials\u003c/h2\u003e\n \u003cp\u003eAll marine litter items were collected, classified based on typologies outlined in the OSPAR Agreement (2020), the Joint Research Centre list (Hanke et al., 2021) and monitoring guidelines provided by Ferreira (2013), and then stored in the dark at room temperature. Unsmoked cigarette filters (Suppl Mat. Figure B) from seven different commercial brands were also collected and pooled together to represent the variability of cigarette butts typically found on beaches. When possible, the polymer identity was ascertained by using Fourier- transform infrared spectroscopy (FTIR) with a Thermo Scientific Nicolet 6700.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Toxicity tests\u003c/h2\u003e\n \u003cp\u003eMarine aquatic toxicity of leachates was assessed using the \u003cem\u003eParacentrotus lividus\u003c/em\u003e sea-urchin embryo test (SET), a rapid and sensitive method following the standardized protocol described by Beiras \u003cem\u003eet al\u003c/em\u003e. (2019). Samples from the most common typologies and UCFs were micronized using either a ZM200 ultracentrifuge mill, a CryoMill (both Retsch, Verder Scientific), or a stainless-steel file, depending on their mechanical properties.\u003c/p\u003e\n \u003cp\u003eSea urchins were supplied on the day of testing by the Marine Culture Unit of ECIMAT (CIM\u0026ndash;University of Vigo), which maintains a stock of sexed mature \u003cem\u003eP. lividus\u003c/em\u003e collected from the outer R\u0026iacute;a de Vigo. In-vitro fertilization was performed according to Beiras et al. 2012.\u003c/p\u003e\n \u003cp\u003eLeachates were prepared following the protocol by Almeda \u003cem\u003eet al\u003c/em\u003e. (2023), using for all materials the \u0026lt;\u0026thinsp;250 \u0026micro;m fraction. The sieved fraction was mixed with chemically-defined artificial seawater (ASW) (Lorenzo et al., 2002) at a proportion of 10 g L-1 in glass bottles with no head space, which were incubated for 24 h at 20 ◦C in darkness using an overhead rotator (GFL 3040) at 1 rpm. Leachates were obtained by filtration through Whatman\u0026reg; GF/F filters and tested via serial dilutions in ASW according to standard SET procedures adapted to microplastics (e.g. Uribe-Echeverr\u0026iacute;a and Beiras, 2022), using 4 mL glass vials per quadruplicate.\u003c/p\u003e\n \u003cp\u003eImages of the formalin-fixed vials were captured using a Leica DMI 4000B inverted microscope, and the length increase of 35 individuals per vial, defined as the maximum dimension minus the average egg diameter, was automatically recorded using in-house Artificial Intelligence algorithms developed in collaboration with the VARPA Research Group (University of A Coru\u0026ntilde;a).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Statistical analysis and assessment criteria\u003c/h2\u003e\n \u003cp\u003eToxicity parameters (EC\u003csub\u003e10\u003c/sub\u003e, EC\u003csub\u003e50\u003c/sub\u003e) and their 95% confidence intervals were calculated by fitting the data to a probit dose-response model using SPSS v24 statistical software. Toxic Units (TU) were calculated as TU\u0026thinsp;=\u0026thinsp;1/EC\u003csub\u003e50\u003c/sub\u003e, where the EC50 is the dilution (in parts per one) of the 10 g/L leachate reducing by 50% the larval growth.\u003c/p\u003e\n \u003cp\u003eMaterials were classified following the assessment criteria shown in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, modified from Alonso-L\u0026oacute;pez et al., (2021).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Marine litter items toxicity tests\u003c/h2\u003e\n \u003cp\u003eThe ecotoxicity of plastic items belonging to specific types of marine litter was assessed by measuring the inhibition of \u003cem\u003eParacentrotus lividus\u003c/em\u003e embryo-larval development caused by their leachates. For each item class, effective concentrations (EC\u003csub\u003e10\u003c/sub\u003e and EC\u003csub\u003e50\u003c/sub\u003e) and Toxic Units (TU) were determined to quantify toxicity, with 95% confidence intervals reported where applicable. Results are presented by litter type and geographical area in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eCigarette butts\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eLeachates from cigarette butts have shown consistently medium toxicity across all samples with EC50 values ranged from 1,030 to 1,859 mg/L, and Toxic Units (TU) between 5.38 and 9.70. Highest toxicity was observed in the Baltic Sea, sample 136E (TU\u0026thinsp;=\u0026thinsp;9.70; 95% CI: 8.07\u0026ndash;11.70), while the lowest was in Egypt, sample 160D (TU\u0026thinsp;=\u0026thinsp;5.38; 95% CI: 4.50\u0026ndash;6.43).\u003c/p\u003e\n \u003cp\u003eThe samples 160D (Egypt) and 157D (Morocco), were also tested using the Microtox assay (measurement of \u003cem\u003eVibrio fischeri\u003c/em\u003e bioluminescence inhibition). After 30 minutes of exposure, results showed mean EC50 values of 29.35 mg/L (95% CI: 17.49\u0026ndash;49.24) and 37.96 mg/L (95% CI: 24.11\u0026ndash;59.77), and Toxicity Units (TU) values of 0.03 and 0.02, respectively. These results are consistent with the toxicity levels observed in the SET, supporting the overall assessment of their ecotoxic potential.\u003c/p\u003e\n \u003cp\u003eTwo additional tests were conducted with a pool of unsmoked cigarette filters (UCFs) from different brands and with Unsmoked Biodegradable Filters (UBFs), which are marketed under the promise of being organic, natural, biodegradable, and unbleached. The UCFs showed ecotoxicity but to a lower extent than all the cigarette butt samples (Suppl Mat. Figure C). The EC10 was calculated at 764 mg/L, and the EC50 at 3,257 mg/L, corresponding to 3.1 (95% confidence interval: 2.54\u0026ndash;3.66) TU (yellow ecotoxicity class). While the UBFs showed no toxicity at all, with noncalculable EC10 and EC50 values, indicating they were non-toxic.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eFoam\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eLeachates from plastic foam items generally ranged from non-toxic (\u0026lt;\u0026thinsp;1 TU) to low (2.9 TU). Most samples exhibited high EC50 values or were non-calculable (NC), with Toxic Unit (TU) values below 1, indicating minimal toxic effects. Sample 136D showed the highest toxicity, with an EC50 of 3,448 mg/L and a TU of 2.90 (95% CI: 2.22\u0026ndash;3.66). Sample 160C showed low toxicity, with an EC50 of 7,874 mg/L and a TU of 1.26 (95% CI: 0.88\u0026ndash;1.64) and the NE Atlantic sample, a piece of insulating cork (REP-05), also showed low toxicity with a TU of 1.01 (95% CI: 0.76\u0026ndash;1.46).\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eStrings and cords\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eAmong the tested marine litter types, the leachates from string and cord samples revealed generally low toxicity, with values ranging from \u0026lt;\u0026thinsp;1 to 1.5 TU. Most samples (136A, 157A, 160B, and 161B) showed EC50 values\u0026thinsp;\u0026gt;\u0026thinsp;10,000 mg/L and TU values\u0026thinsp;\u0026lt;\u0026thinsp;1, indicating negligible effects. In contrast, the Galician coast, sample 153E, which consisted of strings and cords thicker than those from other origins identifiable as rests of derelict fishing nets, exhibited slight but measurable toxicity, with an EC50 of 6,803 mg/L and a TU of 1.50 (95% CI: 1.10\u0026ndash;1.90).\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eAquaculture\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eLeachates from aquaculture-related plastic litter showed from negligible to low ecotoxicity. The beach samples collected in Galicia (082), which consisted of polyethylene (PE) plastic sticks used in mussel rafts, and Italy (ID091-RSP), which consisted of plastic nets used for mussel culture (66% polypropylene, 32% PE), both had EC50 values above 10,000 mg/L and TU values below 1. Two of the fisheries related samples collected from the seafloor: a buoy (REP-04) and an octopus trap (REP-03) also lack toxicity, but the third one, a piece of rope (REP-02), did exhibit inhibitory effects at the levels tested (EC50\u0026thinsp;=\u0026thinsp;3,546 mg/L).\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ePlastic caps\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eLeachates from plastic caps generally showed low toxicity. Most of the samples presented EC50 values greater than 10,000 mg/L and TU values below 1, indicating negligible toxic effects. Sample from Egypt (160A) was an exception, with an EC50 of 9,009 mg/L and a TU of 1.11 (95% CI: 1.00\u0026ndash;1.22), suggesting low toxicity. The remaining samples had non-calculable (NC) values or very high EC50s, reinforcing the overall negligible toxicity of plastic cap leachates.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eMetal bottle caps, lids and pull tabs\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eLeachates from metal bottle caps, lids, and pull tabs showed negligible toxicity. Both the Baltic Sea (136G) and Galician (153D) samples had EC50 values exceeding 10,000 mg/L and TU values below 1, indicating minimal inhibitory effects.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ePlastic bottles (PET)\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eLeachates from PET plastic bottles, either coming from the beach or the seafloor, exhibited no toxicity. The NE Atlantic sample (REP-01) showed an EC10 of 5,555 mg/L, while the Italian sample (ID094-RSP) had no calculable EC10. Both PET samples had EC50 values above 10,000 mg/L and TU values below 1, indicating minimal inhibitory effects.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCigarette butts showed higher toxicity compared to other major types of marine litter, all of which fell within the \u0026ldquo;medium\u0026rdquo; toxicity category. In contrast, unsmoked cigarette filters (UCFs) also demonstrated toxicity but were classified as \u0026ldquo;low,\u0026rdquo; indicating a smaller but still measurable level of ecotoxicity. This suggests that the toxic effects observed in cigarette butts are not only due to tobacco combustion byproducts, but also partially attributable to the materials that make up the filter itself.\u003c/p\u003e \u003cp\u003eThese results align with the findings of Novotny and Slaughter (2014), who noted that cigarettes and their waste, particularly discarded filters with remnant tobacco, contain a wide range of environmentally harmful chemicals. These include residues from agricultural treatments, contaminants absorbed from soil, manufacturing additives and combustion products produced during smoking. Furthermore, our findings are consistent with those of Lucia et al. (2023), who reported that naturally smoked cigarette butts (not yet degraded in the marine environment) reduced bioluminescence in \u003cem\u003eA. fischeri\u003c/em\u003e, caused abnormal embryo development in \u003cem\u003eM. gigas\u003c/em\u003e, and consistently inhibited algal growth across three species (\u003cem\u003eP. tricornutum\u003c/em\u003e, \u003cem\u003eS. costatum\u003c/em\u003e and \u003cem\u003eD. tertiolecta\u003c/em\u003e). Together, these results point to a clear dose\u0026ndash;response relationship and reinforce the conclusion that cigarette butts are ecotoxic in multiple forms, whether freshly smoked, degraded in the marine environment, or even as unsmoked filters, supporting that the observed ecotoxicity is partially due to original filter components, and not only to those acquired upon combustion and ulterior weathering in marine conditions. Notably, all tested samples exhibited toxicity regardless of their country of origin, suggesting that the ecotoxic potential of cigarette butts is consistent across brands and regions, and not significantly mitigated by national differences in tobacco production or regulations.\u003c/p\u003e \u003cp\u003eIn addition, it is clear that the withdrawal of cigarette butts remains a major problem for both manual and mechanical (Zielinski et al., 2019) underscoring the ongoing challenges in effectively managing this persistent form of marine litter.\u003c/p\u003e \u003cp\u003eIn comparison with the \u0026ldquo;biodegradable\u0026rdquo; filters, which showed no toxicity, it can be assumed that cigarette butt toxicity occurs at two levels: primarily due to tobacco and combustion products, and secondarily due to the plastic material in conventional filters. Considering the availability of less toxic alternatives, it may be interesting to promote their use. This is not a definitive solution, since the main source of toxicity comes from the tobacco itself, but it could contribute to reducing the overall environmental impact.\u003c/p\u003e \u003cp\u003eThe \u0026ldquo;foam\u0026rdquo; typology includes the expanded polystyrene (EPS) commonly used in shock-sensitive packaging and insulation, and polyurethane. Three of the five plastic foam samples tested showed EC10 values\u0026thinsp;\u0026lt;\u0026thinsp;2,000 mg/L, supporting a remarkably higher toxicity than caps, bottles, strings and cords, but consistently lower than cigarette butts. The heterogeneous EC10 values obtained for the five samples tested suggest that the observed effects are not inherent to the polymer type itself, but either to the specific chemical composition of each item, or to the hydrophobic organic chemicals (HOCs) sorbed from the seawater in each area. Some types of EPS commonly found in marine debris carry brominated flame retardants and other potentially toxic additives (Jang et al., 2016). This supports the conclusions of Beiras et al. (2021), who emphasized the importance of reducing the environmental impact of plastic materials through careful selection of polymer additives. However, alternative explanations of heterogeneous results for common typologies that cannot be ruled out include differential sorption of HOCs from the surrounding water in each sampling area. Plastics can accumulate HOCs such as PCBs and pesticides at concentrations many orders of magnitude higher than in the surrounding water (Mato et al., 2001), and PS shows higher sorption coefficients than other common polymers such as HDPE (Uber et al., 2019)\u003c/p\u003e \u003cp\u003eStrings and cords, as well as plastic bottle caps, generally exhibited non-toxic behaviour in our tests, with only one sample from each category falling into the low toxicity range. These discordances may be attributed to the variability in chemical additives, particularly colorants, as these items were visually diverse (Suppl Mat. Figure A) and illustrate the need to expand this kind of study to larger marine litter collections.\u003c/p\u003e \u003cp\u003eMetal marine litter items, primarily bottle caps, lids, and pull tabs, showed no toxic effects on \u003cem\u003eP. lividus\u003c/em\u003e larval growth. However, during the 24-hour leachate preparation period, a notable drop in dissolved oxygen levels was observed, from 7.14 mg/L to 4.54 mg/L, probably due to metal oxidation. To prevent confounding effects from hypoxia, all leachates underwent a 10-minute aeration before being used in the bioassay, ensuring that any observed effects were due to the material\u0026rsquo;s toxicity rather than reduced oxygen availability. Although no relevant toxicity was detected, these findings highlight that metal litter in confined environments may still contribute to decrease key water quality parameters, such as oxygen levels, which could indirectly affect marine organisms.\u003c/p\u003e \u003cp\u003eRegarding plastic bottles (PET), results showed no significant toxicity from leachates, consistent with the findings of Gambardella et al., (2024), who also found no adverse effects in marine invertebrates and vertebrates exposed to PET. In contrast, Piccardo et al. (2020) reported significant reductions in larval growth of \u003cem\u003eParacentrotus lividus\u003c/em\u003e when exposed to PET leachates derived from particles of varying sizes. Although all studies used the same test species and bioassay techniques, the contrasting results point to differences likely driven by the specific chemical additives or residues present in the plastics. Piccardo \u003cem\u003eet al\u003c/em\u003e. (2020) also reported morphological deformities in larvae exposed to PET leachates, which is consistent with our observations, although in our case no significant reduction in larval size was observed compared to the control. These findings suggest that even within a single litter category like PET bottles, ecotoxicity can vary significantly depending on the product\u0026rsquo;s formulation, highlighting the importance of chemical composition in assessing environmental impact.\u003c/p\u003e \u003cp\u003eLeachates from most aquaculture derived products, like mussel raft sticks and mussel nets, showed no detectable toxicity in our study. Although neither leachate toxicity nor ingestion-based effects were evident, these materials remain an important source of marine litter due to their persistence, requiring further research on potential long-term effects. Wu et al. (2020), similarly observed that aquaculture operations contribute to microplastic contamination in sediments and seawater, and that microplastic accumulation occurs in commercial species. However, the levels in edible tissues were found to be negligible. Moreover, one of the items, a piece of blue rope, did inhibit embryo development at the concentrations tested. Further research on the chemical content of this specific item would be necessary in order to explain its ecotoxicity.\u003c/p\u003e \u003cp\u003eUnlike other typologies, plastic pieces associated to fishing and aquaculture do not derive from littering or inadequate solid waste disposal but from the accidental loss of pieces of fishing gear (e.g. dolly ropes), fishing traps, and extensive aquaculture devices (e.g. plastic sticks used in mussel ropes). Fishing activities are the source of more than two-thirds of plastic litter in the Mediterranean coasts of Spain (Garc\u0026iacute;a-Rivera et al., 2017) and in the North Atlantic it accounts for 83% of marine litter, being the most abundant typology (Gil Gamundi \u0026amp; Mart\u0026iacute;nez-Gil Pardo de Vera, 2020). For some specific plastic pieces of fisheries equipment prone to get lost upon use and not suitable for recovery and reuse, replacement of conventional by biodegradable plastic materials may greatly contribute to reduce their long-term environmental impact.\u003c/p\u003e \u003cp\u003eEcological impacts of marine litter are complex. Physical impacts beyond the scope of the present study include entanglement of wildlife and ghost fishing, and blockage of digestive system upon ingestion (reviewed by K\u0026uuml;hn et al., 2015). Ecotoxicological impacts, targeted here, stem from the potential toxicity of leaching chemicals originally present in the plastic as functional additives (Beiras et al., 2021) or sorbed during weathering in the sea (Ferrari et al., 2024). We have found that leachates from different plastic litter items remarkably differ in ecotoxicity. This will reflect in very different environmental risk posed by each typology. Following the conventional risk assessment methodology, risk is estimated from the ratio of environmental concentrations vs the toxicity thresholds, which can be estimated by the EC10 values. In this first approach we have found that average EC10 for different litter typologies can vary over 10-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Therefore, future research should further address these differences by using additional test species and exploiting more comprehensive marine litter collections in order to identify the typologies posing the highest risk. This will help prioritize remediation actions and will guide more ecologically relevant plastic pollution prevention policies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThese results stress the need to advance beyond total numbers of marine litter items in marine monitoring and consider the different impacts of various typologies to produce a more ecologically meaningful assessment of environmental status within the context of the MSFD. Research should prioritize the most toxic litter categories to identify the specific additives responsible for their effects, especially in items like plastic foam or cigarette butts, which pose a remarkably higher ecotoxicological risk than other plastic litter components mostly originating from food packaging. Importantly, cigarette butts are often overlooked during professional beach cleanups, whether mechanical or manual, and consequently remain embedded in the sediment where they may continue to leach harmful substances into the environment. This highlights the urgent need for effective mitigation strategies against littering and inadequate solid waste disposal in order to prevent cigarette butts, foams, and other litter types from reaching the marine environment in the first place.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Patricia Rubio, Sara L\u0026oacute;pez-Ib\u0026aacute;\u0026ntilde;ez, Pedro Campoy-L\u0026oacute;pez and Leticia Vidal-Li\u0026ntilde;\u0026aacute;n for their helpful technical assistance, and Olalla Alonso, Aaron Beck and Ciara Gambardella for providing samples from Galicia, Baltic Sea and Adriatic, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMelissa Calvi\u0026ntilde;o\u003c/strong\u003e: Conceptualization, Methodology, Formal analysis, Writing -Original draft, Writing -Review \u0026amp; editing, Investigation, Visualization. \u003cstrong\u003eAlejandro Vilas\u003c/strong\u003e: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data Curation, Project administration. \u003cstrong\u003eMirco\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHaseler\u003c/strong\u003e: Investigation, Resources, Writing -Review \u0026amp; editing, Supervision, Funding acquisition. \u003cstrong\u003e\u0026nbsp;Ricardo Beiras\u003c/strong\u003e: Conceptualization, Methodology, Software, Validation, Formal analysis, Resources, Writing - review \u0026amp; editing, Supervision, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was partially funded by the Ministry of Science and Innovation\u0026mdash;Spanish Agency of Research (AEI) through the Project: Safe Additives for the Plastic INdustry (SAPIN) (PID2022-138421OB- C22), by the Ministry of ecological Transition (MITECO) through the REPESCAPLAS Project (Fundaci\u0026oacute;n Biodiversidad), by the EC through the H2020 LABPLAS Project (H2020 EU3.5.4 Ref 101003954), by the BMU/ZUG project TouMaLi (Beitrag der nachhaltigen Abfallwirtschaft im Tourismus zum Schutz der Meeres\u0026ouml;kosysteme), grant number 65MM0001 (litter collection in Africa), and by the JPI Oceans RESPONSE Project (litter collection in the Adriatic). Program of Consolidation and Structuring of Units of Competitive Investigation of the University System of Galicia (Xunta de Galicia) potentially co-financed by ERDF (ED431C 2021/56) is also acknowledged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlmeda, R., Gunaalan, K., Alonso-L\u0026oacute;pez, O., Vilas, A., Cl\u0026eacute;randeau, C., Loisel, T., Nielsen, T. G., Cachot, J., \u0026amp; Beiras, R. (2023). 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Elsevier Ltd. https://doi.org/10.1016/j.marpolbul.2018.12.027\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 \u0026 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-environmental-contamination-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aect","sideBox":"Learn more about [Archives of Environmental Contamination and Toxicology](https://www.springer.com/journal/244)","snPcode":"244","submissionUrl":"https://submission.nature.com/new-submission/244/3","title":"Archives of Environmental Contamination and Toxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ecotoxicology, Marine litter, Cigarette butts, Sea urchin larvae, Plastics","lastPublishedDoi":"10.21203/rs.3.rs-9498033/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9498033/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMarine litter was sampled in beaches from the Baltic (Germany), NE Atlantic (Galicia) and Mediterranean (Morocco, Tunisia, Egypt, Italy) coasts according to standard procedures, and occasionally from the NE Atlantic seafloor by trawling. Collected items were associated to OSPAR and JRC typologies. The ecotoxicity of specific types of marine litter was assessed by measuring the inhibition of \u003cem\u003eParacentrotus lividus\u003c/em\u003e embryo-larval development caused by their leachates. Cigarette butts exhibited the highest toxicity consistently across all sampling sites, followed in decreasing order by foam, fisheries related items, and strings. 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