Crawling towards complex interactions: the impact of 6PPD-quinone and increased temperatures on the freshwater snail Ampullaceana balthica

Crawling towards complex interactions: the impact of 6PPD-quinone and increased temperatures on the freshwater snail Ampullaceana balthica | 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 Crawling towards complex interactions: the impact of 6PPD-quinone and increased temperatures on the freshwater snail Ampullaceana balthica Núria Castro-Català, Catalina Lizama, Jordi Serra, Mira Čelić, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8114676/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Apr, 2026 Read the published version in Ecotoxicology → Version 1 posted 11 You are reading this latest preprint version Abstract N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone (6PPD-quinone or 6PPDQ) is an oxidation product of 6PPD, an antioxidant used in tyres to prevent rubber degradation, that has been associated with high mortality in juvenile coho salmon at concentrations as low as 95 ng/L. While research has focused primarily on fish, the effects of 6PPDQ on freshwater invertebrates remain limited. In this study, we assessed the toxicological impact of this contaminant on the freshwater snail Ampullaceana balthica over a 10-day experiment under two different temperature conditions. A. balthica was chosen because it is widely distributed in temperate and Mediterranean regions and is commonly used as a model organism in environmental toxicology studies. Although 6PPDQ had a limited impact on embryonic development, adult snails experienced significant effects on reproduction, growth, and motility, with more pronounced impacts at higher temperatures. Specifically, 6PPDQ reduced clutch and egg production, particularly during the first days of exposure. Elevated temperature increased reproduction, but its interaction with 6PPDQ lowered the overall reproductive output. The combined stressors also impaired growth and motility. Development was mainly affected by temperature, with reduced hatching and increased embryo arrest at 20°C. These sublethal effects may lead to population declines and cascading impacts on freshwater community structure and ecosystem functioning, particularly under climate change scenarios. This highlights the urgent need for comprehensive risk assessments of emerging contaminants such as 6PPDQ to better understand their ecological impacts. Tyre wear contaminant Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlights First evidence of sublethal 6PPDQ effects on molluscs at environmental levels. Repeated 6PPDQ exposure impaired reproduction, growth, and motility in Ampullaceana balthica . High temperatures increased egg production, but 6PPDQ reduced reproductive output. Growth rates declined with combined exposure to high temperatures and 6PPDQ. Introduction Tire wear particles (TWP) are a growing source of pollution in urban freshwater ecosystems, released through tire abrasion and transported via stormwater runoff. These particles contain complex chemical mixtures that can persist in aquatic environments and disrupt biodiversity and ecosystem functioning by altering species composition, impairing reproduction, and destabilizing trophic interactions (Liu et al., 2024 ; Song et al., 2025 ). Among the most concerning components of TWP is 6PPD (N-(1,3-dimethylbutyl)-N′-phenyl-1,4-phenylenediamine), a widely used rubber antioxidant that accounted for over 50% of global antioxidant consumption in 2017 (Li et al., 2023 ). During tire wear, 6PPD is released into the environment and undergoes oxidative transformation, particularly in the presence of ozone and UV radiation, forming 6PPD-quinone (6PPDQ), a derivative that has been detected in stormwater runoff at concentrations up to 19 µg/L and in surface waters during storm events at levels up to 3.5 µg/L (Di et al., 2022 ; Johannessen et al., 2021 ; Seiwert et al., 2022 ; Tian et al., 2022 ). At the same time, climate change is introducing additional stressors that may exacerbate these impacts such as frequent extreme weather events and increased thermal stress, particularly in semiarid regions where water availability is limited (IPCC, 2023; Terrado et al., 2014 ). Rising water temperatures, driven by global warming, can reduce the dilution capacity of aquatic systems by enhancing evaporation and lowering river discharge (Bolan et al., 2024 ). Warmer conditions also increase the solubility and bioavailability of pollutants such as 6PPDQ, intensifying exposure for aquatic organisms (Holmstrup et al., 2010 ). These changes can lead to increased physiological stress, disrupting biological processes such as reproduction and embryo development (Hooper et al., 2013a ; Seeland et al., 2012 ), and ultimately reducing the fitness and resilience of natural populations (Martínez-De León & Thakur, 2024 ; Weiskopf et al., 2020 ). Moreover, chemical stressors may interact synergistically with increased temperatures associated with climate change, amplifying ecological impacts beyond those caused by each stressor individually (He et al., 2025 ; Zitoun et al., 2024 ). Notably, Holmstrup et al. ( 2010 ) found that in more than half of the studies reviewed, heat stress significantly increased pollutant toxicity. Understanding these combined effects is essential for assessing the risks posed by TWP-derived contaminants under future climate scenarios. Recent studies have identified 6PPDQ as the chemical responsible for unexplained mortality in coho salmon ( Oncorhynchus kisutch ) in the Pacific Northwest, with lethal concentrations (LC50) as low as 95 ng/L for juveniles (Tian et al., 2022 ). This has spurred research into its toxicity across other salmonid species. White spotted char ( Salvelinus leucomaenis) is another sensitive species, with an LC50 of 0.51 µg/L, whereas rainbow trout ( Oncorhynchus mykiss ) and brook trout ( Salvelinus fontinalis ) show moderate sensitivity, with LC50 values between 0.59 and 1.96 µg/L. Alevins of lake trout (Salvelinus namaycush) exhibit a 45 day median lethal dose (LC50) of 0.39 µg/L (Roberts et al., 2025 ). In contrast, the Arctic char ( Salvelinus alpinus ) and the nonsalmonid white sturgeon ( Acipenser transmontanus ) show greater tolerance, with LC50 values above 14.2 and 12.7 µg/L, respectively (Brinkmann et al., 2022 ; Hiki & Yamamoto, 2022 ). Zebrafish ( Danio rerio ) and Japanese medaka ( Oryzias latipes ) show even greater tolerance, with LC50 values exceeding 40 µg/L (Mayer et al., 2024 ). However, sublethal effects have been observed in zebrafish at lower concentrations. For instance, (Varshney et al., 2022 ) reported neurotoxic effects, including altered motor behaviour and bradycardia, after prolonged exposure to 10–20 µg/L of 6PPDQ. Similarly, Ricarte et al., ( 2023 ) demonstrated that short-term exposure to 2 µg/L in zebrafish larvae leads to significant disruptions in essential behaviours, neurotransmitter profiles, circadian rhythms, and heart rates, highlighting the physiological disruptions that can occur at low concentrations even in the absence of mortality. Prosser et al. ( 2023 ) have found that 6PPDQ does not cause significant mortality in four invertebrate species, the mayfly Hexagenia spp., the cladoceran Daphnia magna , the gastropod Planorbella pilsbryi , and the bivalve Megalonaias nervosa , at relatively low concentrations. However, the NOECs that they have reported, particularly for the gastropod P. pilsbryi (11.7 µg/L), do not eliminate the possibility of sublethal, long-term effects at concentrations that are still environmentally relevant. Despite the critical ecological roles of aquatic invertebrates, data on the sublethal toxicity of 6PPDQ in these species remain limited. Recent studies have shown that prolonged exposure to 6PPDQ at concentrations ranging from 1 to 10 µg/L inhibits lifespan and induces multisystem toxic responses in Caenorhabditis elegans (Hua et al., 2024 ) and that Daphnia pulex experiences significant growth inhibition at 10 µg/L (Shi et al., 2024). In primary producers, such as the green algae Chlorella vulgaris , 6PPDQ caused growth stimulation at concentrations ranging from 50 to 200 µg/L but inhibited growth at higher concentrations (400 µg/L). Additionally, C. vulgaris experienced increased oxidative stress, affecting cell permeability and mitochondrial membrane potential stability (Liu et al., 2024 ). These findings also suggest that the sublethal effects of 6PPDQ could have broader ecological implications at low environmentally relevant concentrations. Freshwater snails, which are important primary consumers in freshwater ecosystems, are particularly vulnerable to pollution because of their low motility. Unlike more mobile species such as fish, snails cannot escape from contaminated environments, increasing their susceptibility to prolonged exposure to pollutants (Baroudi et al., 2020 ). Freshwater snails feed primarily on biofilm that grow on submerged surfaces like stones or cobbles (Hladyz et al., 2011 ). This feeding behaviour exposes them to sunlight and, consequently, to increases in temperature, making them particularly vulnerable under warming conditions. Our study aimed to assess the impact of 6PPDQ on the gastropod Ampullaceana balthica (Linnaeus, 1758) in this context. We hypothesized that chronic exposure to environmentally relevant concentrations of 6PPDQ would lead to sublethal effects on the snails, with these impacts becoming more pronounced and potentially lethal at higher temperatures. To assess these toxicological impacts, we exposed the snails and their offspring to environmentally relevant levels of 6PPDQ at temperatures of 15°C and 20°C. With this research, we would like to contribute to a more accurate assessment of the risks that 6PPDQ poses to freshwater invertebrates under future warming scenarios. Materials and Methods Experiment setup A. balthica snails were collected in late spring from the headwaters of the Ter River (42° 15' 36'', 2° 21' 55''). They were acclimated in 4 aquariums filled with 7L of dechlorinated water at 15°C and provided with constant aeration for 4 days prior to the experiment. After acclimation, they were exposed for 10 days in 5-L microcosms to 6PPDQ at two different temperatures: 15°C and 20°C. The lower temperature of 15°C falls within the natural range of water temperatures at the collection site, whereas 20°C is above this range. All microcosms were first prepared at 15°C, and then gradually adjusted by distributing them into two climate-controlled chambers, one maintained at 15°C and the other increased to 20°C. The microcosms were maintained with calcium carbonate stones, constant aeration, and a 12h:12h light-dark cycle under controlled conditions. Each treatment group consisted of three replicates (microcosms), with 13 snails per replicate. The experiment lasted 10 days, and the analysed endpoints included mortality, growth, mobility, reproduction, embryonic development, and the CN ratio of the soft bodies (Fig. 1 ). Snails were exposed to a nominal 6PPDQ concentration of 10 µg/L, chosen to reflect environmentally relevant levels detected in surface waters during storm events (Cadena-Aizaga et al., 2025 ; Johannessen et al., 2021 ; Tian et al., 2022 ). The stock solution was added to dechlorinated water at the appropriate concentration and thoroughly mixed before adult snails or embryos were introduced. A media renewal protocol was implemented, in which dechlorinated water (with or without the contaminant) was replaced every three days. This approach reflects real-world environmental conditions, particularly repetitive rainfall events that drive the continuous input of contaminants into freshwater ecosystems. At the beginning of the experiment and after each renewal, 1 mg/snail of fish food (Tetramin®) was provided, following the recommendations of (Zimmer et al., 2012 ). The physical and chemical characteristics of the water were held constant throughout the experiment. Snail mortality was checked daily by gently touching each snail with the tip of a plastic pipette. If a snail showed no movement or response, such as retracting into its shell, it was considered dead. The number of dead snails was recorded each day, and any dead snails were promptly removed from the microcosms after being confirmed to be unresponsive. This method ensures accurate mortality tracking while preventing contamination from decomposing individuals. Reproduction and development follow-up Clutches of A. balthica snails were systematically counted and collected every 3–4 days (on days 2, 4, 7, and 10), prior to water renewal. The collected clutches were placed in 6-well plates, where the eggs were counted, and their development was monitored until all surviving embryos hatched, following the same exposure pattern and media renewal protocol as in the first-generation experiment. The endpoints included the total number of clutches produced, the number of eggs per clutch, the number of embryos that successfully hatched, and the number of nondeveloped embryos, defined as those where development had stopped at the morula or gastrula stage. Growth and CN Ratio Analysis At the end of the experiment, all snails were dried at 60°C, weighed (dry weight), and their shells were measured to calculate growth rates. The carbon and nitrogen contents of the dried soft body samples were analysed via a Carlo Erba CN 1500 Analyzer (CCiT of the Universitat the Barcelona). Motility assessment On Day 10, three snails from control and each treatment group were individually placed in Petri dishes to assess their movement. Each snail was video recorded for 15 minutes to capture their behaviour and mobility patterns. The footage from minute 2 (after acclimation) to minute 8 of each video was selected for analysis using the AnimApp application (Rao et al., 2019 ). Statistical analyses We used linear models (LM) to examine the relationships between our response variables and the effect predictor variables (6PPDQ treatment, temperature, and time). We compared the models using Akaike Information Criterion (AIC) corrected for small sample size (AICc). The selected model was refined by iteratively removing the least significant terms until an optimal model was reached. The best model had the lowest AICc, with significant differences identified when the ΔAICc exceeded two units (Hobbs & Hilborn, 2006 ; Johnson & Omland, 2004 ).The data was log-transformed when necessary to meet the normality and homoscedasticity assumptions. For the developmental data (hatching success rates and percentage of nondeveloped embryos), GLMs were applied. Overdispersion was checked by examining the ratio of residual variance to degrees of freedom (Bolker et al., 2009 ). Due to high overdispersion, a quasi-Poisson distribution was used. The detailed R code and results for regression model selection are provided in Text S1. All the statistical analyses were conducted in R v4.3.2 (R Core Team, 2023), using the packages MASS, car, lme4, MuMIn, AICcmodavg, lmtest and ggplot2. Statistical significance was determined using a conventional α = 0.05 threshold. Chemical analysis of water samples Compound preparation and sample handling The compound 2-((4-methylpentan-2-yl)amino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione (6PPDQ; purity 97.1%, lot 1341848) was obtained from Dr. Ehrenstorfer (LGC). Stock solutions (6 mg/L) were prepared by dissolving 6PPQ in absolute ethanol (final solvent 0.01% v/v), stored at 4°C in the dark for up to 7 days, and subsequently kept at − 20°C until analysis. Exposure water samples were collected immediately before media renewal and stored at − 20°C, following recommended guidelines. For direct analysis, stock solutions were diluted to 200 µg/L to prevent precipitation or column overloading, and all samples were filtered through 0.45 µm PVDF membranes prior to LC analysis. Sample preparation, extraction, and degradation experiment Water samples (100 mL) were extracted in triplicate following the methodology describe in detail elsewhere (Gago-Ferrero et al., 2015 ). Just briefly, samples were sequentially filtered through glass fiber (0.7 µm) and PVDF (0.45 µm) membranes to remove particulates and were adjusted to pH 6.5–7.0 using 1.5 M ammonia or formic acid and spiked with 50 µL internal standard solution (0.5 µg/mL). For recovery assessment one blank sample was spiked with 25 or 50 µL of a standard mixture (1 µg mL). SPE was performed on Oasis HLB cartridges (200 mg, 6 mL) preconditioned with dichloromethane, methanol, and water. Samples were loaded at ~ 2 mL/min, rinsed with water, dried under air for 30 min, and eluted with methanol and dichloromethane at 1 mL/min, followed by 1 min high-vacuum drying. Eluates were evaporated under nitrogen and reconstituted in 0.25 mL methanol:water (1:1, v/v; ×400 enrichment) and filtered through 0.2 µm PVDF membranes when necessary. For the degradation assessment, 1 L microcosms were spiked with 6PPDQ (10 mg/L stock prepared as above) and incubated at 15°C and 20°C for 48 h (n = 2). Samples were collected at 30 min and 48 h and immediately analyzed alongside exposure water using UHPLC coupled to Orbitrap high-resolution mass spectrometry (UHPLC-Orbitrap-HRMS). Instrumental analysis All samples were analyzed using an ultra-high-performance liquid chromatography (UHPLC) system coupled to a high-resolution Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific). UHPLC separation was achieved on a Cortecs C18 + column (2.1 × 100 mm, 2.7 µm) with a VanGuard cartridge (2.1 × 5 mm, 2.7 µm) using water (0.1% formic acid) and methanol (0.1% formic acid) as mobile phases. The Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific) operated in positive ESI mode, with data acquired using all-ion fragmentation (AIF), following the method described by Gago-Ferrero et al. ( 2020 ). Quality control and method validation Instrument stability and analytical performance were assessed using calibration curves prepared over nine concentration levels, injected at the beginning and end of each sequence. Method limits of detection (LOD) and quantification (LOQ) were determined following standard procedures. Recoveries were evaluated by spiking exposure water blanks at two concentration levels, and all samples were analyzed in triplicate to ensure reproducibility. Routine quality control checks were performed to verify measurement reliability. Results Stock solution stability and instrumental performance The 6PPDQ stock solutions were stable when stored at 4°C for up to 7 days and at − 20°C for extended storage. UHPLC-Orbitrap-HRMS analysis demonstrated consistent retention times (± 0.05 min) and accurate mass measurements (mass error < 2 ppm). Instrumental reproducibility was confirmed through repeated injections of the internal standard, showing relative standard deviations (RSD) below 5%. Calibration curves spanning 5–500 µg/L exhibited excellent linearity (R² > 0.99) across all analytical sequences. Method validation and recovery Recovery studies conducted on spiked exposure water blanks (100 and 200 µg /L) yielded recoveries of 85–95%, with RSD values below 6%, confirming method precision. Limits of detection and quantification were determined as 0.5 µg/L and 5 µg/L, respectively. No significant matrix effects were observed, indicating that the SPE procedure and sample filtration effectively removed interfering substances from water microcosmos. Exposure water sample analysis 6PPDQ concentrations in exposure water samples were consistently measurable using both direct analysis and SPE-prepared extracts. We observed that freeze–thawing can lead to partial precipitation of the compound. This behavior may result in reduced solubility and uneven distribution in the exposure medium, and is most likely attributable to the low solubility of solid 6PPDQ and potential crystallization (Hu et al., 2023 a). To minimize this effect, all frozen samples were thawed gently and homogenized prior to analysis. Samples filtered through 0.45 µm PVDF membranes prior to UHPLC analysis showed sharp, symmetric peaks with high signal-to-noise ratios (Fig. S1 ). The preconcentration factor achieved through SPE (×400) allowed detection of low concentrations with high reproducibility. The measured concentration of the stock solution (nominal concentration of 6 mg/L) was 2.09 mg/L. The samples collected on Day 0 and Day 2, under 20°C conditions, presented concentrations of 1.9 µg/L and 0.8 µg/L, respectively. The samples collected prior to water renewal on Days 3 and 7 presented concentrations below the detection limit of 0.5 µg/L. This decline over time may be attributed to photodegradation of 6PPDQ, as the compound is known to degrade upon exposure to sunlight or UV radiation (Redman et al., 2023 ). Degradation kinetics of 6PPDQ under different temperature conditions In 48h degradation experiment, 6PPDQ exhibited temperature-dependent degradation. At 15°C, the compound decreased by 57.5% ± 3.1% over 48 h, whereas at 20°C, degradation was faster, with a reduction of 82.8% ± 7.9% over the same period (Fig. S2). These results demonstrate that temperature significantly influences 6PPDQ stability in aqueous environments. Analytical reliability Overall, the UHPLC-Orbitrap-HRMS method provided sensitive, accurate, and reproducible quantification of 6PPDQ in both direct and SPE-prepared water samples. Quality control measures, including triplicate analysis and internal standard monitoring, confirmed the robustness and reliability of the analytical workflow. Mortality The mean control mortality did not exceed 20% at the end of the experiment. Two-way ANOVA selected revealed a near-significant effect of treatment (F 1,43 =4.47, p = 0.08) and a significant effect of time (F 1,43 = 96.92, p = 0.043) in the daily mortality rate (Fig. 2 ). Reproduction For the number of clutches laid per snail alive during the experiment, the final model selected was a three-way ANOVA without interaction (Text S1). The model revealed that there was a statistically significant negative effect of 6PPDQ on the number of clutches laid per snail (F 1,1= 4.19, p = 0.048), but no significant effects of temperature (F 1,1= 1.02, p = 0.32) and time (F 1,1= 0.89, p = 0.35) (Fig. 3 A). For the number of eggs per clutch, the final selected model was a three-way ANOVA (Text S1). The model indicated that clutches exposed to the contaminant and laid from day 0 to day 7 presented a lower number of eggs per clutch compared with nonexposed clutches (F 1,49= 5.37, p = 0.025). Additionally, there was a significant interaction effect between 6PPDQ and time (F 1,49= 4.93, p = 0.031), indicating that in the 6PPDQ-exposed aquaria, the clutches laid from Days 8 to day 10 presented a greater number of eggs than those laid earlier (Fig. 3 B). No significant effects related to the temperature factor were found (F 1,49= 2.15, p = 0.149). Finally, we calculated the number of eggs laid per snail as a measure of reproductive fitness (i.e. the number of potential descendants per adult snail). The final selected model was a two-way ANOVA. The number of eggs per snail and per day increased significantly with temperature (F 1,19= 9.64, p = 0.006) but there was a negative interaction effect of 6PPDQ with temperature (F 1,19= 5.69, p = 0.028) (Fig. 4 A). This indicates that the combined exposure to the contaminant and elevated temperature (20°C) leads to a synergistic negative interaction, resulting in a marked reduction in daily egg production beyond what would be expected from the individual effects of each stressor. Development The quasi-Poisson selected models (Tables S1 and S2) revealed that the hatching success decreased in the microcosms at 20°C (Fig. 3 C) and the proportion of nondeveloped zygotes increased (Fig. 3 D). No significant effects related to the exposure to the contaminant were found. Growth The three-way ANOVA revealed that there was a significant positive effect of temperature on the growth rate of the snails at the end of the experiment (F 1,92= 11.46, p = 0.001), and, also, a significant interaction effect between temperature and 6PPDQ (F 1,92= 0.50, p = 0.002) (Fig. 4 B; Text S1). This interaction indicates a negative synergistic effect, where the combined exposure to elevated temperature and 6PPDQ led to reduced growth rates, below the control baseline. CN ratio The final model selected was a one-way ANOVA with treatment as the sole factor (Text S1). The analysis indicated a near-significant effect of 10-day exposure to 6PPDQ on the CN ratio of the snail soft tissues (F 1,91= 3.40, p = 0.069) (Fig. 5 A). CN ratio was slightly higher when snails were exposed to the toxicant. Motility Two-way ANOVA selected revealed that there were significant negative effects of both temperature (F 1,92= 5.95, p = 0.046) and 6PPDQ (F 1,92= 6.29, p = 0.036) on the velocity of the snails after the 10-days experiment (Fig. 5 B; Text S1). Discussion This study reveals the toxicological impact of 6PPDQ, an emerging contaminant linked to tyre wear, on various life history traits of snails. As hypothesized, the sublethal effects observed on reproduction, growth, and motility reveal a broad spectrum of ecological risks associated with 6PPDQ exposure at environmentally relevant concentrations, emphasizing the need for regulatory measures and safer alternatives. Our results also reveal that some of these effects becoming more pronounced at higher temperatures. The results indicate that the exposure to 10.45 µg/L of 6PPDQ has a significant negative impact on the reproductive output of the freshwater snail Ampullaceana balthica , specifically affecting both the number of clutches laid and the number of eggs per clutch. Compared with nonexposed snails, snails exposed to 6PPDQ laid fewer clutches. With respect to the number of eggs per clutch, the results revealed a more complex interaction. Initially, during the first week, 6PPDQ exposure resulted in a lower number of eggs per clutch compared to nonexposed clutches. However, a significant interaction effect between 6PPDQ and time was observed, with clutches laid later in the experiment (Days 8 to 10) in the contaminated aquaria resulting in an increase in the number of eggs per clutch. This temporal pattern may suggest a delayed compensatory response, where snails initially reduce egg production but later attempt to increase reproductive output as exposure continues. Similar responses have been observed in various organisms, where reproductive efforts are maximized following an initial period of stress to improve overall reproductive success (Minchella & Loverde, 1981 ; Rollo & Hawryluk, 1988 ). Interestingly, similar compensatory reproductive responses have been documented in other freshwater gastropods exposed to environmental stressors. For example, Bi et al. ( 2025 ) observed that Pomacea canaliculata exposed to low concentrations of arsenite significantly increased egg production. This was interpreted as a hormetic response, where mild environmental stress stimulates reproductive output through physiological or endocrine mechanisms. Similarly, Liang et al., ( 2023 ) reported enhanced spawning and estradiol levels in P. canaliculata under low-level pollution, suggesting an adaptive strategy to ensure reproductive success under suboptimal conditions. Likewise, Lymnaea stagnalis exposed to cigarette butt leachate initially reduced clutch production, but reproductive output recovered during a post-exposure phase (Olah-Kovacs et al., 2025 ). These findings suggest that freshwater snails may exhibit plastic reproductive strategies that allow partial recovery from sublethal stress. The analysis of overall reproductive fitness, measured as the number of eggs laid per snail per day, revealed a significant negative interaction between 6PPDQ and temperature suggesting that the detrimental effects of the contaminant may be exacerbated at relatively high temperatures, potentially due to increased metabolic demands (Clarke & Fraser, 2004 ; Cloyed et al., 2019 ; Scrine et al., 2017 ) or increased uptake of the contaminant (Guo et al., 2018 ; Heugens et al., 2001 ; Hooper et al., 2013b ). While the temporary increase in egg production per clutch may reflect an adaptive reproductive response to stressful conditions, the overall reduction in reproductive fitness indicates that this compensatory strategy is insufficient. The decline in reproductive fitness is likely driven by the repetitive peak exposure to 6PPDQ. Even though the contaminant levels drop below the LOD after each three-day cycle, likely result from the low solubility of solid 6PPDQ (Hu et al., 2023 ) the renewal of contaminated media introduces fresh pulses of 6PPDQ, mimicking real-world conditions that can arise under diverse climatic or microclimatic regimes, where intermittent rainfall and runoff repeatedly transport contaminants into aquatic environments. In natural situations, despite the tendency of 6PPDQ to partition onto soil and organic matter due to its hydrophobic nature (log K ow =4.3 ± 0.02) (Hu et al., 2023 ; Hua et al., 2024 ), a portion remains in the dissolved aqueous phase, particularly during storm events. These events introduce fresh pulses of the contaminant into aquatic environments, leading to repeated high-concentration exposures similar to those observed in our experimental setup. This cycle of exposure, followed by temporary recovery periods, could explain the initial reduction in reproductive output, followed by a compensatory increase in egg production later in the experiment. However, despite this compensatory effort, the overall negative impact on reproductive fitness suggests that the snails were unable to fully recover from the stress induced by repetitive peak exposures. Over time, chronic exposure to 6PPDQ reduces the reproductive capacity of snails, potentially threatening population sustainability in contaminated environments. This aligns with findings from other studies, where environmental stress or limited resources cause snails to reduce their reproductive efforts to survive. For example, in Lymnaea stagnalis reproduction was delayed under food limitation, diverting energy toward maintenance, reducing the number of eggs produced (Zonneveld & Kooijman, 1993 ). Similar trade-offs between reproduction and survival have been observed in snails exposed to contaminants such as copper, which also reduced both reproductive output and growth (Gao et al., 2017 ; Khangarot & Das, 2010 ; Real et al., 2000). Additionally, findings by (Wang & Liu, 2023 ) show that under silver nanoparticle exposure, adult gastropods allocate more resources to combat oxidative stress rather than to growth or reproduction, further emphasizing the energy trade-offs under environmental stress. In our study, growth rates were also negatively impacted by the contaminant, especially at relatively warm temperatures. Normally, higher temperatures promote growth by accelerating metabolic rates, but this positive effect was diminished by the contaminant. This suggests that 6PPDQ disrupts energy allocation, forcing snails detoxification over growth (Wang & Liu, 2023 ). Marginal changes in the CN ratio further support this finding, indicating metabolic stress that impairs the snails' ability to balance energy storage and protein synthesis, contributing to reduced growth and reproduction. Motility was also significantly reduced in snails exposed to 6PPDQ, with additional negative effects observed at relatively high temperatures. The reduction in motility could further decrease survival chances, as limited movement may impair foraging and escape from. The combined effects of 6PPDQ and temperature on motility suggest that the contaminant induces physiological stress, limiting the capacity of the snails to perform essential behaviours. Reduced motility could further compound the negative effects on growth and reproduction, as limited movement restricts access to food and mates, ultimately diminishing the overall fitness of the population. The effects of 6PPDQ at similar concentrations have also been documented in other invertebrates, with nematodes showing abnormal locomotion behaviours and neurodegeneration at 10 µg/L (Hua et al., 2023 ), and Daphnia pulex exhibiting significant reductions in growth rates at exposure levels of 0.1 and 10 µg/L (Shi et al., 2024). While 6PPDQ clearly affected reproduction, its effects on embryonic development were less pronounced. This may be due to the protective features of the egg masses and eggshells. Initially, 6PPDQ must penetrate the gelatinous matrix surrounding the egg masses, which consists of proteins and polysaccharides that can limit the entry of external contaminants (Benkendorff et al., 2001 ). The degree of protection offered by this matrix varies depending on factors such as the chemical's size, polarity, and lipophilicity (Arman, 2023 ). For example, snail eggs within the matrix showed reduced sensitivity to cadmium compared to isolated eggs (Liu et al., 2013 ), although some contaminants (e.g., nanoparticles) can agglomerate and adhere to egg masses (Musee et al., 2010 ). Once it passes through the gelatinous matrix, 6PPDQ needs to cross the egg capsule membrane itself. Studies have shown that molecules such as raffinose (504.42 g/mol) and polyethylene glycol (500–600 g/mol) can pass through this membrane (Beadle, 1969 ). Given relatively small molecular weight of 298.4 g/mol, 6PPDQ likely has the ability to permeate the egg capsule as well. Despite these barriers, the reduced number of eggs laid and overall reproductive impairment suggest that the long-term developmental success of the population could be compromised (Gao et al., 2017 ; Khangarot & Das, 2010 ). Further research is needed to explore whether embryos exposed to 6PPDQ experience persistent or delayed sublethal effects after hatching. In addition to the direct exposure from contaminated water during rainfall events, 6PPDQ can interact with organic matter, such as leaf litter and biofilms, which aquatic organisms may ingest. Tyre wear particles in the water can also be accidentally ingested, releasing 6PPDQ into the digestive systems of these organisms (Arman, 2023 ). This raises concerns about the potential for bioaccumulation of 6PPDQ when contaminated food is consumed. Therefore, alongside the direct toxicity of dissolved 6PPDQ, it is essential to consider its effects from both contaminated food and tyre wear particles. The impact of 6PPDQ and other tyre-associated chemicals on aquatic organisms through these ingestion pathways remains largely unexplored, highlighting a critical area for future research to fully understand the ecological implications of tyre-related contaminants. Conclusion In summary, repetitive exposure to 6PPDQ at environmentally relevant concentrations presents a serious threat to the freshwater snail Ampullaceana batlhica , affecting key life history traits such as reproduction, embryo development, growth, and motility. While the protective properties of the egg masses may mitigate developmental risks, their effects on adult snails are severe and can potentially lead to long-term population declines. Overall, our study underscores the need for further investigation into the sublethal effects of 6PPDQ on aquatic species and the broader ecological risks associated with this contaminant. The findings contribute to a better understanding of how 6PPDQ impacts aquatic ecosystems, particularly in the context of climate change and increased contaminant levels. These significant impacts on aquatic life highlight the need for regulatory measures and the development of safer alternatives in the tyre industry to address 6PPDQ pollution. Data Availability All raw data associated with this study are available in the CORA Data Repository at https://doi.org/10.34810/data1906 . Declarations Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper. Funding This paper was funded by the project “City runoff pollution impacts on river biodiversity under extreme climatic events”, CityPoll (TED2021-129966B-C31 and TED2021-129966B-C33) funded by MCIN/AEI/ 10.13039/501100011033 and by the European Union NextGeneration EU/PRTR, and by the project Rivstress PID2020-115708RB-C21 funded by MCIN/AEI/ 10.13039/501100011033 . We would also like to acknowledge the CCiTUB (Centres Científics i Tecnològics de la Universitat de Barcelona) of the University of Barcelona for their services. Author Contribution C.L. (Catalina Lizama): Conceptualization, Data curation, Formal analysis.J.S. (Jordi Serra): Data curation, Formal analysis.M.Č. (Mira Čelić): Data curation, Writing – Review & Editing.I.C. (Isabel Cadena): Data curation, Writing – Review & Editing.M.P. (Mira Petrovic): Investigation, Writing – Review & Editing, Funding acquisition.I.M. (Isabel Muñoz): Investigation, Writing – Review & Editing, Funding acquisition.N.D.C.C. (Núria De Castro Català): Conceptualization, Methodology, Investigation, Writing – Original Draft, Writing – Review & Editing, Supervision.All authors reviewed and approved the final manuscript. Data Availability All raw data associated with this study are available in the CORA Data Repository at [https://doi.org/10.34810/data1906](https:/doi.org/10.34810/data1906) . Author contributions CRediT: Núria de Castro-Català: Conceptualization, Methodology, Data Curation, Formal analysis, Visualization, Writing - Original Draft, Writing - Review & Editing Catalina Lizama: Conceptualization, Data Curation, Formal analysis Jordi Serra: Data Curation, Formal analysis Mira Čelić: Data Curation, Writing - Review & Editing Isabel Cadena: Data Curation, Writing - Review & Editing Mira Petrovic: Investigation, Writing - Review & Editing, Funding acquisition Isabel Muñoz: Investigation, Writing - Review & Editing, Funding acquisition References Arman S (2023) Effects of acute and chronic bendiocarb exposure during early life stages of the pond snail (Lymnaea stagnalis). Chem Ecol 39(1):44–58. https://doi.org/10.1080/02757540.2022.2134351 Baroudi F, Alam A, Fajloun J, Z., Millet M (2020) Snail as sentinel organism for monitoring the environmental pollution; a review. Ecol Ind 113:106240. https://doi.org/10.1016/J.ECOLIND.2020.106240 Beadle LC (1969) Salt and water regulation in the embryos of freshwater pulmonate molluscs. I. The embryonic environment of Biomphalaria sudanica and Lymnaea stagnalis. J Exp Biol 50(2):473–479. https://doi.org/10.1242/JEB.50.2.473 Benkendorff K, Davis AR, Bremner JB (2001) Chemical Defense in the Egg Masses of Benthic Invertebrates: An Assessment of Antibacterial Activity in 39 Mollusks and 4 Polychaetes. J Invertebr Pathol 78(2):109–118. https://doi.org/10.1006/JIPA.2001.5047 Bi X, Shi Z, Li D, Zhan W, Wang Z, Li H, Zhao B, Chen G (2025) Arsenic accumulation and reproductive toxicity in freshwater snail (Pomacea canaliculata). Ecotoxicol Environ Saf 289:117688. https://doi.org/10.1016/J.ECOENV.2025.117688 Bolan S, Padhye LP, Jasemizad T, Govarthanan M, Karmegam N, Wijesekara H, Amarasiri D, Hou D, Zhou P, Biswal BK, Balasubramanian R, Wang H, Siddique KHM, Rinklebe J, Kirkham MB, Bolan N (2024) Impacts of climate change on the fate of contaminants through extreme weather events. Sci Total Environ 909:168388. https://doi.org/10.1016/J.SCITOTENV.2023.168388 Bolker BM, Brooks ME, Clark CJ, Geange SW, Poulsen JR, Stevens MHH, White JSS (2009) Generalized linear mixed models: a practical guide for ecology and evolution. Trends Ecol Evol 24(3):127–135. https://doi.org/10.1016/J.TREE.2008.10.008/ATTACHMENT/BC246FFE-E4DF-4091-BACA-87ABB8F552E6/MMC3.ZIP Brinkmann M, Montgomery D, Selinger S, Miller JGP, Stock E, Alcaraz AJ, Challis JK, Weber L, Janz D, Hecker M, Wiseman S (2022) Acute Toxicity of the Tire Rubber-Derived Chemical 6PPD-quinone to Four Fishes of Commercial, Cultural, and Ecological Importance. Environ Sci Technol Lett 9(4):333–338. https://doi.org/10.1021/ACS.ESTLETT.2C00050/SUPPL_FILE/EZ2C00050_SI_003.MOV Cadena-Aizaga MI, García-Gómez E, Gago-Ferrero P, Sabater S, Petrovic M (2025) Comprehensive characterization of microcontaminants in urban runoff: Unveiling chemical signatures using wide-scope target screening by LC-HRMS. Environ Chem Ecotoxicol 7:2133–2141. https://doi.org/10.1016/J.ENCECO.2025.09.009 Clarke A, Fraser KPP (2004) Why does metabolism scale with temperature? Funct Ecol 18(2):243–251. https://doi.org/10.1111/J.0269-8463.2004.00841.X Cloyed CS, Dell AI, Hayes T, Kordas RL, O’Gorman EJ (2019) Long-term exposure to higher temperature increases the thermal sensitivity of grazer metabolism and movement. J Anim Ecol 88(6):833–844. https://doi.org/10.1111/1365-2656.12976 Di S, Liu Z, Zhao H, Li Y, Qi P, Wang Z, Xu H, Jin Y, Wang X (2022) Chiral perspective evaluations: Enantioselective hydrolysis of 6PPD and 6PPD-quinone in water and enantioselective toxicity to Gobiocypris rarus and Oncorhynchus mykiss. Environ Int 166:107374. https://doi.org/10.1016/J.ENVINT.2022.107374 Gago-Ferrero P, Bletsou AA, Damalas DE, Aalizadeh R, Alygizakis NA, Singer HP, Hollender J, Thomaidis NS (2020) Wide-scope target screening of > 2000 emerging contaminants in wastewater samples with UPLC-Q-ToF-HRMS/MS and smart evaluation of its performance through the validation of 195 selected representative analytes. J Hazard Mater 387:121712. https://doi.org/10.1016/J.JHAZMAT.2019.121712 Gago-Ferrero P, Schymanski EL, Bletsou AA, Aalizadeh R, Hollender J, Thomaidis NS (2015) Extended Suspect and Non-Target Strategies to Characterize Emerging Polar Organic Contaminants in Raw Wastewater with LC-HRMS/MS. Environ Sci Technol 49(20):12333–12341. https://doi.org/10.1021/ACS.EST.5B03454/SUPPL_FILE/ES5B03454_SI_001.PDF Gao L, Doan H, Nidumolu B, Kumar A, Gonzago D (2017) Effects of copper on the survival, hatching, and reproduction of a pulmonate snail (Physa acuta). Chemosphere 185:1208–1216. https://doi.org/10.1016/J.CHEMOSPHERE.2017.07.101 Guo SN, Zheng JL, Yuan SS, Zhu QL (2018) Effects of heat and cadmium exposure on stress-related responses in the liver of female zebrafish: Heat increases cadmium toxicity. Sci Total Environ 618:1363–1370. https://doi.org/10.1016/J.SCITOTENV.2017.09.264 He W, Chen C, Yang L, Chen Z, Zhu L, Zhu L, Feng J (2025) Chemical Ecological Risk Assessment for Aquatic Life Under Climate Change: A Review from Occurrence, Bioaccumulation, and Toxicity Perspective. Rev Environ Contam Toxicol 2025 263:1(1):1–27. https://doi.org/10.1007/S44169-025-00085-5 . 263 Heugens EHW, Hendriks AJ, Dekker T, Van Straalen NM, Admiraal W (2001) A Review of the Effects of Multiple Stressors on Aquatic Organisms and Analysis of Uncertainty Factors for Use in Risk Assessment. Crit Rev Toxicol 31(3):247–284. https://doi.org/10.1080/20014091111695 Hiki K, Yamamoto H (2022) The Tire-Derived Chemical 6PPD-quinone Is Lethally Toxic to the White-Spotted Char Salvelinus leucomaenis pluvius but Not to Two Other Salmonid Species. Environ Sci Technol Lett 9(12):1050–1055. https://doi.org/10.1021/ACS.ESTLETT.2C00683/SUPPL_FILE/EZ2C00683_SI_001.PDF Hladyz S, Cook RA, Petrie R, Nielsen DL (2011) Influence of substratum on the variability of benthic biofilm stable isotope signatures: Implications for energy flow to a primary consumer. Hydrobiologia 664(1):135–146. https://doi.org/10.1007/S10750-010-0593-0/TABLES/2 Hobbs NT, Hilborn R (2006) Alternatives To Statistical Hypothesis Testing In Ecology: A Guide To Self Teaching. Ecol Appl 16(1):5–19. https://doi.org/10.1890/04-0645 Holmstrup M, Bindesbøl AM, Oostingh GJ, Duschl A, Scheil V, Köhler HR, Loureiro S, Soares AMVM, Ferreira ALG, Kienle C, Gerhardt A, Laskowski R, Kramarz PE, Bayley M, Svendsen C, Spurgeon DJ (2010) Interactions between effects of environmental chemicals and natural stressors: A review. Sci Total Environ 408(18):3746–3762. https://doi.org/10.1016/J.SCITOTENV.2009.10.067 Hooper MJ, Ankley GT, Cristol DA, Maryoung LA, Noyes PD, Pinkerton KE (2013a) Interactions between chemical and climate stressors: A role for mechanistic toxicology in assessing climate change risks. Environ Toxicol Chem 32(1):32–48. https://doi.org/10.1002/ETC.2043 Hooper MJ, Ankley GT, Cristol DA, Maryoung LA, Noyes PD, Pinkerton KE (2013b) Interactions between chemical and climate stressors: A role for mechanistic toxicology in assessing climate change risks. Environ Toxicol Chem 32(1):32–48. https://doi.org/10.1002/ETC.2043 Hu X, Zhao H, Tian Z, Peter KT, Dodd MC, Kolodziej EP (2023) Chemical characteristics, leaching, and stability of the ubiquitous tire rubber-derived toxicant 6PPD-quinone. Environ Science: Processes Impacts 25(5):901–911. https://doi.org/10.1039/D3EM00047H Hua X, Feng X, Liang G, Chao J, Wang D (2023) Exposure to 6-PPD Quinone at Environmentally Relevant Concentrations Causes Abnormal Locomotion Behaviors and Neurodegeneration in Caenorhabditis elegans. Environ Sci Technol 57(12):4940–4950. https://doi.org/10.1021/ACS.EST.2C08644/ . ASSET/IMAGES/LARGE/ES2C08644_0005.JPEG Hua X, Liang G, Chao J, Wang D (2024) Exposure to 6-PPD quinone causes damage on mitochondrial complex I/II associated with lifespan reduction in Caenorhabditis elegans. J Hazard Mater 472:134598. https://doi.org/10.1016/J.JHAZMAT.2024.134598 Hua X, Wang D (2023) Tire-rubber related pollutant 6-PPD quinone: A review of its transformation, environmental distribution, bioavailability, and toxicity. J Hazard Mater 459:132265. https://doi.org/10.1016/J.JHAZMAT.2023.132265 Johannessen C, Helm P, Metcalfe CD (2021) Detection of selected tire wear compounds in urban receiving waters. Environ Pollut 287:117659. https://doi.org/10.1016/J.ENVPOL.2021.117659 Johnson JB, Omland KS (2004) Model selection in ecology and evolution. Trends Ecol Evol 19(2):101–108. https://doi.org/10.1016/J.TREE.2003.10.013 Khangarot BS, Das S (2010) Effects of copper on the egg development and hatching of a freshwater pulmonate snail Lymnaea luteola L. J Hazard Mater 179(1–3):665–675. https://doi.org/10.1016/J.JHAZMAT.2010.03.054 Lee H, Romero J (2023) (n.d.). Climate Change 2023 Synthesis Report IPCC , : Sections. In: Climate Change 2023: Synthesis Report. Contribution of Working Groups I, II and III to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change [Core Writing Team. 35–115. https://doi.org/10.59327/IPCC/AR6-9789291691647 Li C, Zhang Y, Yin S, Wang Q, Li Y, Liu Q, Liu L, Luo X, Chen L, Zheng H, Li F (2023) First insights into 6PPD-quinone formation from 6PPD photodegradation in water environment. J Hazard Mater 459:132127. https://doi.org/10.1016/J.JHAZMAT.2023.132127 Liang D, Li Y, Li S, Meng D, Li F, Huang S, Gong M, Qin J, Li H (2023) Dose-dependent inhibitory effects of glyphosate on invasive Pomacea canaliculata reproductive and developmental growth under oxidative deposition. Ecotoxicol Environ Saf 253:114659. https://doi.org/10.1016/J.ECOENV.2023.114659 Liu J, Yu M, Shi R, Ge Y, Li J, Zeb A, Cheng Z, Liu W (2024) Comparative toxic effect of tire wear particle-derived compounds 6PPD and 6PPD-quinone to Chlorella vulgaris. Sci Total Environ 951:175592. https://doi.org/10.1016/J.SCITOTENV.2024.175592 Liu T, Koene JM, Dong X, Fu R (2013) Sensitivity of isolated eggs of pond snails: A new method for toxicity assays and risk assessment. Environ Monit Assess 185(5):4183–4190. https://doi.org/10.1007/S10661-012-2860-1/METRICS Liu Z, Yang H, Zhang Y, Shao Y, Hu S, Zhang H, Shen G (2024) Tire-wear particle leachate at environmentally relevant concentrations exert a hepatotoxic impact on the black-spotted frog by disrupting the gut–liver axis. Environ Chem Ecotoxicol 6:380–389. https://doi.org/10.1016/J.ENCECO.2024.08.004 Martínez-De León G, Thakur MP (2024) Ecological debts induced by heat extremes. Trends Ecol Evol. https://doi.org/10.1016/J.TREE.2024.07.002 Mayer PM, Moran KD, Miller EL, Brander SM, Harper S, Garcia-Jaramillo M, Carrasco-Navarro V, Ho KT, Burgess RM, Hampton T, Granek LM, McCauley EF, McIntyre M, Kolodziej JK, Hu EP, Williams X, Beckingham AJ, Jackson BA, Sanders-Smith ME, Mendez RD, M (2024) Where the rubber meets the road: Emerging environmental impacts of tire wear particles and their chemical cocktails. Sci Total Environ 927:171153. https://doi.org/10.1016/J.SCITOTENV.2024.171153 Minchella DJ, Loverde PT (1981) A Cost of Increased Early Reproductive Effort in the Snail Biomphalaria glabrata. Https://Doi.Org/10.1086/283879 , 118 (6), 876–881. https://doi.org/10.1086/283879 Musee N, Oberholster PJ, Sikhwivhilu L, Botha AM (2010) The effects of engineered nanoparticles on survival, reproduction, and behaviour of freshwater snail, Physa acuta (Draparnaud, 1805). Chemosphere 81(10):1196–1203. https://doi.org/10.1016/J.CHEMOSPHERE.2010.09.040 Olah-Kovacs B, White HJ, Tingey T, Boots B, Green DS (2025) The impacts of smoked cigarette butt leachate on a common freshwater gastropod, Lymnaea stagnalis. Environ Pollut 376:126425. https://doi.org/10.1016/J.ENVPOL.2025.126425 Prosser RS, Salole J, Hang S (2023) Toxicity of 6PPD-quinone to four freshwater invertebrate species. Environ Pollut 337:122512. https://doi.org/10.1016/J.ENVPOL.2023.122512 Rao SR, Olechnowicz SWZ, Krätschmer P, Jepson JEC, Edwards CM, Edwards JR (2019) Small Animal Video Tracking for Activity and Path Analysis Using a Novel Open-Source Multi-Platform Application (AnimApp). Scientific Reports 2019 9:1 , 9 (1), 1–9. https://doi.org/10.1038/s41598-019-48841-7 Real M, Muñ Oz I, Guasch H, Navarro E, Sabater S (2024) (n.d.). The effect of copper exposure on a simple aquatic food chain . Retrieved October 2, from Redman ZC, Begley JL, Hillestad I, DiMento BP, Stanton RS, Aguaa AR, Pirrung MC, Tomco PL (2023) Reactive Oxygen Species and Chromophoric Dissolved Organic Matter Drive the Aquatic Photochemical Pathways and Photoproducts of 6PPD-quinone under Simulated High-Latitude Conditions. Environ Sci Technol 57(49):20813–20821. https://doi.org/10.1021/ACS.EST.3C05742/SUPPL_FILE/ES3C05742_SI_001.PDF Ricarte M, Prats E, Montemurro N, Bedrossiantz J, Bellot M, Gómez-Canela C, Raldúa D (2023) Environmental concentrations of tire rubber-derived 6PPD-quinone alter CNS function in zebrafish larvae. Sci Total Environ 896:165240. https://doi.org/10.1016/J.SCITOTENV.2023.165240 Roberts C, Lin J, Kohlman E, Jain N, Amekor M, Alcaraz AJ, Hogan N, Hecker M, Brinkmann M (2025) Acute and Subchronic Toxicity of 6PPD-Quinone to Early Life Stage Lake Trout (Salvelinus namaycush). Environ Sci Technol 59(1):791–797. https://doi.org/10.1021/ACS.EST.4C09090/SUPPL_FILE/ES4C09090_SI_001.PDF Rollo CD, Hawryluk MD (1988) Compensatory Scope and Resource Allocation in Two Species of Aquatic Snails. Ecology 69(1):146–156. https://doi.org/10.2307/1943169 Scrine J, Jochum M, Ólafsson JS, O’Gorman EJ (2017) Interactive effects of temperature and habitat complexity on freshwater communities. Ecol Evol 7(22):9333. https://doi.org/10.1002/ECE3.3412 Seeland A, Oehlmann J, Müller R (2012) Aquatic ecotoxicity of the fungicide pyrimethanil: Effect profile under optimal and thermal stress conditions. Environ Pollut 168:161–169. https://doi.org/10.1016/j.envpol.2012.04.020 Seiwert B, Nihemaiti M, Troussier M, Weyrauch S, Reemtsma T (2022) Abiotic oxidative transformation of 6-PPD and 6-PPD quinone from tires and occurrence of their products in snow from urban roads and in municipal wastewater. Water Res 212:118122. https://doi.org/10.1016/J.WATRES.2022.118122 Shi C, Wu F, Zhao Z, Ye T, Luo X, Wu Y, Liu Z, Zhang H (2024a) Effects of environmental concentrations of 6PPD and its quinone metabolite on the growth and reproduction of freshwater cladoceran. Sci Total Environ 948:175018. https://doi.org/10.1016/J.SCITOTENV.2024.175018 Shi C, Wu F, Zhao Z, Ye T, Luo X, Wu Y, Liu Z, Zhang H (2024b) Effects of environmental concentrations of 6PPD and its quinone metabolite on the growth and reproduction of freshwater cladoceran. Sci Total Environ 948:175018. https://doi.org/10.1016/J.SCITOTENV.2024.175018 Song W, Lin L, Oh S, Grossart HP, Yang Y (2025) Tire wear particles in aquatic environments: From biota to ecosystem impacts. J Environ Manage 388:126059. https://doi.org/10.1016/J.JENVMAN.2025.126059 Terrado M, Acuña V, Ennaanay D, Tallis H, Sabater S (2014) Impact of climate extremes on hydrological ecosystem services in a heavily humanized Mediterranean basin. Ecol Ind 37(PART A):199–209. https://doi.org/10.1016/J.ECOLIND.2013.01.016 Tian Z, Gonzalez M, Rideout CA, Zhao HN, Hu X, Wetzel J, Mudrock E, James CA, McIntyre JK, Kolodziej EP (2022) 6PPD-Quinone: Revised Toxicity Assessment and Quantification with a Commercial Standard. Environ Sci Technol Lett 9(2):140–146. https://doi.org/10.1021/ACS.ESTLETT.1C00910/SUPPL_FILE/EZ1C00910_SI_001.PDF Varshney S, Gora AH, Siriyappagouder P, Kiron V, Olsvik PA (2022) Toxicological effects of 6PPD and 6PPD quinone in zebrafish larvae. J Hazard Mater 424:127623. https://doi.org/10.1016/J.JHAZMAT.2021.127623 Wang T, Liu W (2023) Chronic and transgenerational effects of silver nanoparticles in freshwater gastropod Lymnaea stagnalis. Chemosphere 313:137386. https://doi.org/10.1016/J.CHEMOSPHERE.2022.137386 Weiskopf SR, Rubenstein MA, Crozier LG, Gaichas S, Griffis R, Halofsky JE, Hyde KJW, Morelli TL, Morisette JT, Muñoz RC, Pershing AJ, Peterson DL, Poudel R, Staudinger MD, Sutton-Grier AE, Thompson L, Vose J, Weltzin JF, Whyte KP (2020) Climate change effects on biodiversity, ecosystems, ecosystem services, and natural resource management in the United States. Sci Total Environ 733:137782. https://doi.org/10.1016/J.SCITOTENV.2020.137782 Zimmer EI, Jager T, Ducrot V, Lagadic L, Kooijman SALM (2012) Juvenile food limitation in standardized tests: A warning to ecotoxicologists. Ecotoxicology 21(8):2195–2204. https://doi.org/10.1007/S10646-012-0973-5/TABLES/1 Zitoun R, Marcinek S, Hatje V, Sander SG, Völker C, Sarin M, Omanović D (2024) Climate change driven effects on transport, fate and biogeochemistry of trace element contaminants in coastal marine ecosystems. Commun Earth Environ 2024 5:1(1):1–17. https://doi.org/10.1038/s43247-024-01679-y . 5 Zonneveld C, Kooijman SALM (1993) Comparative kinetics of embryo development. Bull Math Biol 55(3):609–635. https://doi.org/10.1016/S0092-8240(05)80242-3 Additional Declarations No competing interests reported. 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11:50:05","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":185666,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8114676/v1/a649f6ed4bd37873fce90db2.html"},{"id":100370763,"identity":"49485081-1a94-478d-8234-a3a464b99423","added_by":"auto","created_at":"2026-01-16 08:08:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":66898,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperiment design and endpoints analysed.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8114676/v1/c77bace34c4161b6b890afb7.png"},{"id":100232095,"identity":"533f32c5-fa56-478c-a2f6-00fff0dd44eb","added_by":"auto","created_at":"2026-01-14 11:50:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":48225,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDaily mortality rates plotted by 6PPDQ treatment throughout the experiment. Data are shown as mean ± s.e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8114676/v1/c5bc6ccc65ff82db3466abe1.png"},{"id":100370762,"identity":"2b11aa51-619d-424d-99b0-a183a4a98fe3","added_by":"auto","created_at":"2026-01-16 08:08:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":38835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReproductive and developmental endpoints plotted according to the factor identified as statistically significant in the final model (either 6PPDQ treatment or temperature). Data are shown as mean ± s.e. A) Number of clutches laid per snail alive per day. B) Number of eggs per clutch. C) Hatching success. D) Percentage of non-developed embryos.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8114676/v1/650210b60f468969b32a80db.png"},{"id":100370909,"identity":"a1f6af3e-4ba4-4461-bb5d-068125d6f78d","added_by":"auto","created_at":"2026-01-16 08:08:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":29038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA) Effect size of temperature and 6PPDQ on the total number of eggs laid per snail alive per day; B) Effect size of temperature and 6PPDQ on the daily growth rate (measured as the change in dry mass) of the snails. Data are shown as mean ± s.e. The dashed line represents the expected additive effect under the Concentration Addition (CA) model. In both panels, the observed combined effects fall below this expectation, indicating synergistic interactions between temperature and 6PPDQ.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8114676/v1/6a6b3bc63f5ffd34fd094525.png"},{"id":100232104,"identity":"d98dcb7f-496b-41a9-9b9f-707c4b7330ec","added_by":"auto","created_at":"2026-01-14 11:50:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":18788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA) CN ratio of the snail soft bodies plotted by the 6PPDQ treatment and temperature; B) Velocity of the snails plotted by the 6PPDQ treatment and temperature. Data are shown as mean ± s.e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8114676/v1/6ff33ca9447118dfc4a8e4cf.png"},{"id":107928457,"identity":"31ff78a6-47e6-4581-92cc-9fbe36fb744a","added_by":"auto","created_at":"2026-04-27 16:10:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":512766,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8114676/v1/75e76c82-bf10-44bd-aedc-8e4d89ad718c.pdf"},{"id":100370928,"identity":"490f11d5-060c-4b72-aaba-9f72556d7248","added_by":"auto","created_at":"2026-01-16 08:09:00","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":435931,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8114676/v1/6e01992abc17a020e3a47636.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Crawling towards complex interactions: the impact of 6PPD-quinone and increased temperatures on the freshwater snail Ampullaceana balthica","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eFirst evidence of sublethal 6PPDQ effects on molluscs at environmental levels.\u003c/li\u003e\n \u003cli\u003eRepeated 6PPDQ exposure impaired reproduction, growth, and motility in \u003cem\u003eAmpullaceana balthica\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eHigh temperatures increased egg production, but 6PPDQ reduced reproductive output.\u003c/li\u003e\n \u003cli\u003eGrowth rates declined with combined exposure to high temperatures and 6PPDQ.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eTire wear particles (TWP) are a growing source of pollution in urban freshwater ecosystems, released through tire abrasion and transported via stormwater runoff. These particles contain complex chemical mixtures that can persist in aquatic environments and disrupt biodiversity and ecosystem functioning by altering species composition, impairing reproduction, and destabilizing trophic interactions (Liu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Among the most concerning components of TWP is 6PPD (N-(1,3-dimethylbutyl)-N\u0026prime;-phenyl-1,4-phenylenediamine), a widely used rubber antioxidant that accounted for over 50% of global antioxidant consumption in 2017 (Li et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). During tire wear, 6PPD is released into the environment and undergoes oxidative transformation, particularly in the presence of ozone and UV radiation, forming 6PPD-quinone (6PPDQ), a derivative that has been detected in stormwater runoff at concentrations up to 19 \u0026micro;g/L and in surface waters during storm events at levels up to 3.5 \u0026micro;g/L (Di et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Johannessen et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Seiwert et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Tian et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt the same time, climate change is introducing additional stressors that may exacerbate these impacts such as frequent extreme weather events and increased thermal stress, particularly in semiarid regions where water availability is limited (IPCC, 2023; Terrado et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Rising water temperatures, driven by global warming, can reduce the dilution capacity of aquatic systems by enhancing evaporation and lowering river discharge (Bolan et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Warmer conditions also increase the solubility and bioavailability of pollutants such as 6PPDQ, intensifying exposure for aquatic organisms (Holmstrup et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). These changes can lead to increased physiological stress, disrupting biological processes such as reproduction and embryo development (Hooper et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013a\u003c/span\u003e; Seeland et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and ultimately reducing the fitness and resilience of natural populations (Mart\u0026iacute;nez-De Le\u0026oacute;n \u0026amp; Thakur, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Weiskopf et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, chemical stressors may interact synergistically with increased temperatures associated with climate change, amplifying ecological impacts beyond those caused by each stressor individually (He et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zitoun et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Notably, Holmstrup et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) found that in more than half of the studies reviewed, heat stress significantly increased pollutant toxicity. Understanding these combined effects is essential for assessing the risks posed by TWP-derived contaminants under future climate scenarios.\u003c/p\u003e \u003cp\u003eRecent studies have identified 6PPDQ as the chemical responsible for unexplained mortality in coho salmon (\u003cem\u003eOncorhynchus kisutch\u003c/em\u003e) in the Pacific Northwest, with lethal concentrations (LC50) as low as 95 ng/L for juveniles (Tian et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This has spurred research into its toxicity across other salmonid species. White spotted char (\u003cem\u003eSalvelinus leucomaenis)\u003c/em\u003e is another sensitive species, with an LC50 of 0.51 \u0026micro;g/L, whereas rainbow trout (\u003cem\u003eOncorhynchus mykiss\u003c/em\u003e) and brook trout (\u003cem\u003eSalvelinus fontinalis\u003c/em\u003e) show moderate sensitivity, with LC50 values between 0.59 and 1.96 \u0026micro;g/L. Alevins of lake trout (Salvelinus namaycush) exhibit a 45 day median lethal dose (LC50) of 0.39 \u0026micro;g/L (Roberts et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In contrast, the Arctic char (\u003cem\u003eSalvelinus alpinus\u003c/em\u003e) and the nonsalmonid white sturgeon (\u003cem\u003eAcipenser transmontanus\u003c/em\u003e) show greater tolerance, with LC50 values above 14.2 and 12.7 \u0026micro;g/L, respectively (Brinkmann et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hiki \u0026amp; Yamamoto, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) and Japanese medaka (\u003cem\u003eOryzias latipes\u003c/em\u003e) show even greater tolerance, with LC50 values exceeding 40 \u0026micro;g/L (Mayer et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, sublethal effects have been observed in zebrafish at lower concentrations. For instance, (Varshney et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported neurotoxic effects, including altered motor behaviour and bradycardia, after prolonged exposure to 10\u0026ndash;20 \u0026micro;g/L of 6PPDQ. Similarly, Ricarte et al., (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) demonstrated that short-term exposure to 2 \u0026micro;g/L in zebrafish larvae leads to significant disruptions in essential behaviours, neurotransmitter profiles, circadian rhythms, and heart rates, highlighting the physiological disruptions that can occur at low concentrations even in the absence of mortality.\u003c/p\u003e \u003cp\u003eProsser et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) have found that 6PPDQ does not cause significant mortality in four invertebrate species, the mayfly \u003cem\u003eHexagenia\u003c/em\u003e spp., the cladoceran \u003cem\u003eDaphnia magna\u003c/em\u003e, the gastropod \u003cem\u003ePlanorbella pilsbryi\u003c/em\u003e, and the bivalve \u003cem\u003eMegalonaias nervosa\u003c/em\u003e, at relatively low concentrations. However, the NOECs that they have reported, particularly for the gastropod \u003cem\u003eP. pilsbryi\u003c/em\u003e (11.7 \u0026micro;g/L), do not eliminate the possibility of sublethal, long-term effects at concentrations that are still environmentally relevant. Despite the critical ecological roles of aquatic invertebrates, data on the sublethal toxicity of 6PPDQ in these species remain limited. Recent studies have shown that prolonged exposure to 6PPDQ at concentrations ranging from 1 to 10 \u0026micro;g/L inhibits lifespan and induces multisystem toxic responses in \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e (Hua et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and that \u003cem\u003eDaphnia pulex\u003c/em\u003e experiences significant growth inhibition at 10 \u0026micro;g/L (Shi et al., 2024).\u003c/p\u003e \u003cp\u003eIn primary producers, such as the green algae \u003cem\u003eChlorella vulgaris\u003c/em\u003e, 6PPDQ caused growth stimulation at concentrations ranging from 50 to 200 \u0026micro;g/L but inhibited growth at higher concentrations (400 \u0026micro;g/L). Additionally, \u003cem\u003eC. vulgaris\u003c/em\u003e experienced increased oxidative stress, affecting cell permeability and mitochondrial membrane potential stability (Liu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These findings also suggest that the sublethal effects of 6PPDQ could have broader ecological implications at low environmentally relevant concentrations.\u003c/p\u003e \u003cp\u003eFreshwater snails, which are important primary consumers in freshwater ecosystems, are particularly vulnerable to pollution because of their low motility. Unlike more mobile species such as fish, snails cannot escape from contaminated environments, increasing their susceptibility to prolonged exposure to pollutants (Baroudi et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Freshwater snails feed primarily on biofilm that grow on submerged surfaces like stones or cobbles (Hladyz et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). This feeding behaviour exposes them to sunlight and, consequently, to increases in temperature, making them particularly vulnerable under warming conditions. Our study aimed to assess the impact of 6PPDQ on the gastropod \u003cem\u003eAmpullaceana balthica\u003c/em\u003e (Linnaeus, 1758) in this context. We hypothesized that chronic exposure to environmentally relevant concentrations of 6PPDQ would lead to sublethal effects on the snails, with these impacts becoming more pronounced and potentially lethal at higher temperatures.\u003c/p\u003e \u003cp\u003eTo assess these toxicological impacts, we exposed the snails and their offspring to environmentally relevant levels of 6PPDQ at temperatures of 15\u0026deg;C and 20\u0026deg;C. With this research, we would like to contribute to a more accurate assessment of the risks that 6PPDQ poses to freshwater invertebrates under future warming scenarios.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eExperiment setup\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eA. balthica\u003c/em\u003e snails were collected in late spring from the headwaters of the Ter River (42\u0026deg; 15' 36'', 2\u0026deg; 21' 55''). They were acclimated in 4 aquariums filled with 7L of dechlorinated water at 15\u0026deg;C and provided with constant aeration for 4 days prior to the experiment. After acclimation, they were exposed for 10 days in 5-L microcosms to 6PPDQ at two different temperatures: 15\u0026deg;C and 20\u0026deg;C. The lower temperature of 15\u0026deg;C falls within the natural range of water temperatures at the collection site, whereas 20\u0026deg;C is above this range. All microcosms were first prepared at 15\u0026deg;C, and then gradually adjusted by distributing them into two climate-controlled chambers, one maintained at 15\u0026deg;C and the other increased to 20\u0026deg;C. The microcosms were maintained with calcium carbonate stones, constant aeration, and a 12h:12h light-dark cycle under controlled conditions. Each treatment group consisted of three replicates (microcosms), with 13 snails per replicate. The experiment lasted 10 days, and the analysed endpoints included mortality, growth, mobility, reproduction, embryonic development, and the CN ratio of the soft bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSnails were exposed to a nominal 6PPDQ concentration of 10 \u0026micro;g/L, chosen to reflect environmentally relevant levels detected in surface waters during storm events (Cadena-Aizaga et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Johannessen et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tian et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The stock solution was added to dechlorinated water at the appropriate concentration and thoroughly mixed before adult snails or embryos were introduced. A media renewal protocol was implemented, in which dechlorinated water (with or without the contaminant) was replaced every three days. This approach reflects real-world environmental conditions, particularly repetitive rainfall events that drive the continuous input of contaminants into freshwater ecosystems.\u003c/p\u003e \u003cp\u003eAt the beginning of the experiment and after each renewal, 1 mg/snail of fish food (Tetramin\u0026reg;) was provided, following the recommendations of (Zimmer et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The physical and chemical characteristics of the water were held constant throughout the experiment. Snail mortality was checked daily by gently touching each snail with the tip of a plastic pipette. If a snail showed no movement or response, such as retracting into its shell, it was considered dead. The number of dead snails was recorded each day, and any dead snails were promptly removed from the microcosms after being confirmed to be unresponsive. This method ensures accurate mortality tracking while preventing contamination from decomposing individuals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReproduction and development follow-up\u003c/h2\u003e \u003cp\u003eClutches of \u003cem\u003eA. balthica\u003c/em\u003e snails were systematically counted and collected every 3\u0026ndash;4 days (on days 2, 4, 7, and 10), prior to water renewal. The collected clutches were placed in 6-well plates, where the eggs were counted, and their development was monitored until all surviving embryos hatched, following the same exposure pattern and media renewal protocol as in the first-generation experiment. The endpoints included the total number of clutches produced, the number of eggs per clutch, the number of embryos that successfully hatched, and the number of nondeveloped embryos, defined as those where development had stopped at the morula or gastrula stage.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGrowth and CN Ratio Analysis\u003c/h3\u003e\n\u003cp\u003eAt the end of the experiment, all snails were dried at 60\u0026deg;C, weighed (dry weight), and their shells were measured to calculate growth rates. The carbon and nitrogen contents of the dried soft body samples were analysed via a Carlo Erba CN 1500 Analyzer (CCiT of the Universitat the Barcelona).\u003c/p\u003e\n\u003ch3\u003eMotility assessment\u003c/h3\u003e\n\u003cp\u003eOn Day 10, three snails from control and each treatment group were individually placed in Petri dishes to assess their movement. Each snail was video recorded for 15 minutes to capture their behaviour and mobility patterns. The footage from minute 2 (after acclimation) to minute 8 of each video was selected for analysis using the AnimApp application (Rao et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eWe used linear models (LM) to examine the relationships between our response variables and the effect predictor variables (6PPDQ treatment, temperature, and time). We compared the models using Akaike Information Criterion (AIC) corrected for small sample size (AICc). The selected model was refined by iteratively removing the least significant terms until an optimal model was reached. The best model had the lowest AICc, with significant differences identified when the ΔAICc exceeded two units (Hobbs \u0026amp; Hilborn, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Johnson \u0026amp; Omland, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).The data was log-transformed when necessary to meet the normality and homoscedasticity assumptions.\u003c/p\u003e \u003cp\u003eFor the developmental data (hatching success rates and percentage of nondeveloped embryos), GLMs were applied. Overdispersion was checked by examining the ratio of residual variance to degrees of freedom (Bolker et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Due to high overdispersion, a quasi-Poisson distribution was used. The detailed R code and results for regression model selection are provided in Text S1.\u003c/p\u003e \u003cp\u003eAll the statistical analyses were conducted in R v4.3.2 (R Core Team, 2023), using the packages MASS, car, lme4, MuMIn, AICcmodavg, lmtest and ggplot2. Statistical significance was determined using a conventional α\u0026thinsp;=\u0026thinsp;0.05 threshold.\u003c/p\u003e\n\u003ch3\u003eChemical analysis of water samples\u003c/h3\u003e\n\u003cp\u003eCompound preparation and sample handling\u003c/p\u003e \u003cp\u003eThe compound 2-((4-methylpentan-2-yl)amino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione (6PPDQ; purity 97.1%, lot 1341848) was obtained from Dr. Ehrenstorfer (LGC). Stock solutions (6 mg/L) were prepared by dissolving 6PPQ in absolute ethanol (final solvent 0.01% v/v), stored at 4\u0026deg;C in the dark for up to 7 days, and subsequently kept at \u0026minus;\u0026thinsp;20\u0026deg;C until analysis. Exposure water samples were collected immediately before media renewal and stored at \u0026minus;\u0026thinsp;20\u0026deg;C, following recommended guidelines. For direct analysis, stock solutions were diluted to 200 \u0026micro;g/L to prevent precipitation or column overloading, and all samples were filtered through 0.45 \u0026micro;m PVDF membranes prior to LC analysis.\u003c/p\u003e \u003cp\u003eSample preparation, extraction, and degradation experiment\u003c/p\u003e \u003cp\u003eWater samples (100 mL) were extracted in triplicate following the methodology describe in detail elsewhere (Gago-Ferrero et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Just briefly, samples were sequentially filtered through glass fiber (0.7 \u0026micro;m) and PVDF (0.45 \u0026micro;m) membranes to remove particulates and were adjusted to pH 6.5\u0026ndash;7.0 using 1.5 M ammonia or formic acid and spiked with 50 \u0026micro;L internal standard solution (0.5 \u0026micro;g/mL). For recovery assessment one blank sample was spiked with 25 or 50 \u0026micro;L of a standard mixture (1 \u0026micro;g mL). SPE was performed on Oasis HLB cartridges (200 mg, 6 mL) preconditioned with dichloromethane, methanol, and water. Samples were loaded at ~\u0026thinsp;2 mL/min, rinsed with water, dried under air for 30 min, and eluted with methanol and dichloromethane at 1 mL/min, followed by 1 min high-vacuum drying. Eluates were evaporated under nitrogen and reconstituted in 0.25 mL methanol:water (1:1, v/v; \u0026times;400 enrichment) and filtered through 0.2 \u0026micro;m PVDF membranes when necessary.\u003c/p\u003e \u003cp\u003eFor the degradation assessment, 1 L microcosms were spiked with 6PPDQ (10 mg/L stock prepared as above) and incubated at 15\u0026deg;C and 20\u0026deg;C for 48 h (n\u0026thinsp;=\u0026thinsp;2). Samples were collected at 30 min and 48 h and immediately analyzed alongside exposure water using UHPLC coupled to Orbitrap high-resolution mass spectrometry (UHPLC-Orbitrap-HRMS).\u003c/p\u003e \u003cp\u003eInstrumental analysis\u003c/p\u003e \u003cp\u003eAll samples were analyzed using an ultra-high-performance liquid chromatography (UHPLC) system coupled to a high-resolution Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific). UHPLC separation was achieved on a Cortecs C18\u0026thinsp;+\u0026thinsp;column (2.1 \u0026times; 100 mm, 2.7 \u0026micro;m) with a VanGuard cartridge (2.1 \u0026times; 5 mm, 2.7 \u0026micro;m) using water (0.1% formic acid) and methanol (0.1% formic acid) as mobile phases. The Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific) operated in positive ESI mode, with data acquired using all-ion fragmentation (AIF), following the method described by Gago-Ferrero et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eQuality control and method validation\u003c/p\u003e \u003cp\u003eInstrument stability and analytical performance were assessed using calibration curves prepared over nine concentration levels, injected at the beginning and end of each sequence. Method limits of detection (LOD) and quantification (LOQ) were determined following standard procedures. Recoveries were evaluated by spiking exposure water blanks at two concentration levels, and all samples were analyzed in triplicate to ensure reproducibility. Routine quality control checks were performed to verify measurement reliability.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStock solution stability and instrumental performance\u003c/h2\u003e \u003cp\u003eThe 6PPDQ stock solutions were stable when stored at 4\u0026deg;C for up to 7 days and at \u0026minus;\u0026thinsp;20\u0026deg;C for extended storage. UHPLC-Orbitrap-HRMS analysis demonstrated consistent retention times (\u0026plusmn;\u0026thinsp;0.05 min) and accurate mass measurements (mass error\u0026thinsp;\u0026lt;\u0026thinsp;2 ppm). Instrumental reproducibility was confirmed through repeated injections of the internal standard, showing relative standard deviations (RSD) below 5%. Calibration curves spanning 5\u0026ndash;500 \u0026micro;g/L exhibited excellent linearity (R\u0026sup2; \u0026gt; 0.99) across all analytical sequences.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMethod validation and recovery\u003c/h3\u003e\n\u003cp\u003eRecovery studies conducted on spiked exposure water blanks (100 and 200 \u0026micro;g /L) yielded recoveries of 85\u0026ndash;95%, with RSD values below 6%, confirming method precision. Limits of detection and quantification were determined as 0.5 \u0026micro;g/L and 5 \u0026micro;g/L, respectively. No significant matrix effects were observed, indicating that the SPE procedure and sample filtration effectively removed interfering substances from water microcosmos.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExposure water sample analysis\u003c/h2\u003e \u003cp\u003e6PPDQ concentrations in exposure water samples were consistently measurable using both direct analysis and SPE-prepared extracts. We observed that freeze\u0026ndash;thawing can lead to partial precipitation of the compound. This behavior may result in reduced solubility and uneven distribution in the exposure medium, and is most likely attributable to the low solubility of solid 6PPDQ and potential crystallization (Hu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003ea). To minimize this effect, all frozen samples were thawed gently and homogenized prior to analysis. Samples filtered through 0.45 \u0026micro;m PVDF membranes prior to UHPLC analysis showed sharp, symmetric peaks with high signal-to-noise ratios (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The preconcentration factor achieved through SPE (\u0026times;400) allowed detection of low concentrations with high reproducibility. The measured concentration of the stock solution (nominal concentration of 6 mg/L) was 2.09 mg/L. The samples collected on Day 0 and Day 2, under 20\u0026deg;C conditions, presented concentrations of 1.9 \u0026micro;g/L and 0.8 \u0026micro;g/L, respectively. The samples collected prior to water renewal on Days 3 and 7 presented concentrations below the detection limit of 0.5 \u0026micro;g/L. This decline over time may be attributed to photodegradation of 6PPDQ, as the compound is known to degrade upon exposure to sunlight or UV radiation (Redman et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDegradation kinetics of 6PPDQ under different temperature conditions\u003c/h2\u003e \u003cp\u003eIn 48h degradation experiment, 6PPDQ exhibited temperature-dependent degradation. At 15\u0026deg;C, the compound decreased by 57.5% \u0026plusmn; 3.1% over 48 h, whereas at 20\u0026deg;C, degradation was faster, with a reduction of 82.8% \u0026plusmn; 7.9% over the same period (Fig. S2). These results demonstrate that temperature significantly influences 6PPDQ stability in aqueous environments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnalytical reliability\u003c/h2\u003e \u003cp\u003eOverall, the UHPLC-Orbitrap-HRMS method provided sensitive, accurate, and reproducible quantification of 6PPDQ in both direct and SPE-prepared water samples. Quality control measures, including triplicate analysis and internal standard monitoring, confirmed the robustness and reliability of the analytical workflow.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMortality\u003c/h2\u003e \u003cp\u003eThe mean control mortality did not exceed 20% at the end of the experiment. Two-way ANOVA selected revealed a near-significant effect of treatment (F\u003csub\u003e1,43\u003c/sub\u003e=4.47, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.08) and a significant effect of time (F\u003csub\u003e1,43\u003c/sub\u003e= 96.92, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.043) in the daily mortality rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eReproduction\u003c/h2\u003e \u003cp\u003eFor the number of clutches laid per snail alive during the experiment, the final model selected was a three-way ANOVA without interaction (Text S1). The model revealed that there was a statistically significant negative effect of 6PPDQ on the number of clutches laid per snail (F\u003csub\u003e1,1=\u003c/sub\u003e4.19, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.048), but no significant effects of temperature (F\u003csub\u003e1,1=\u003c/sub\u003e1.02, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.32) and time (F\u003csub\u003e1,1=\u003c/sub\u003e0.89, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.35) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eFor the number of eggs per clutch, the final selected model was a three-way ANOVA (Text S1). The model indicated that clutches exposed to the contaminant and laid from day 0 to day 7 presented a lower number of eggs per clutch compared with nonexposed clutches (F\u003csub\u003e1,49=\u003c/sub\u003e5.37, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025). Additionally, there was a significant interaction effect between 6PPDQ and time (F\u003csub\u003e1,49=\u003c/sub\u003e4.93, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.031), indicating that in the 6PPDQ-exposed aquaria, the clutches laid from Days 8 to day 10 presented a greater number of eggs than those laid earlier (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). No significant effects related to the temperature factor were found (F\u003csub\u003e1,49=\u003c/sub\u003e2.15, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.149).\u003c/p\u003e \u003cp\u003eFinally, we calculated the number of eggs laid per snail as a measure of reproductive fitness (i.e. the number of potential descendants per adult snail). The final selected model was a two-way ANOVA. The number of eggs per snail and per day increased significantly with temperature (F\u003csub\u003e1,19=\u003c/sub\u003e9.64, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006) but there was a negative interaction effect of 6PPDQ with temperature (F\u003csub\u003e1,19=\u003c/sub\u003e5.69, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). This indicates that the combined exposure to the contaminant and elevated temperature (20\u0026deg;C) leads to a synergistic negative interaction, resulting in a marked reduction in daily egg production beyond what would be expected from the individual effects of each stressor.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment\u003c/h2\u003e \u003cp\u003eThe quasi-Poisson selected models (Tables S1 and S2) revealed that the hatching success decreased in the microcosms at 20\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and the proportion of nondeveloped zygotes increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). No significant effects related to the exposure to the contaminant were found.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGrowth\u003c/h2\u003e \u003cp\u003eThe three-way ANOVA revealed that there was a significant positive effect of temperature on the growth rate of the snails at the end of the experiment (F\u003csub\u003e1,92=\u003c/sub\u003e11.46, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001), and, also, a significant interaction effect between temperature and 6PPDQ (F\u003csub\u003e1,92=\u003c/sub\u003e0.50, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; Text S1). This interaction indicates a negative synergistic effect, where the combined exposure to elevated temperature and 6PPDQ led to reduced growth rates, below the control baseline.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCN ratio\u003c/h2\u003e \u003cp\u003eThe final model selected was a one-way ANOVA with treatment as the sole factor (Text S1). The analysis indicated a near-significant effect of 10-day exposure to 6PPDQ on the CN ratio of the snail soft tissues (F\u003csub\u003e1,91=\u003c/sub\u003e3.40, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.069) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). CN ratio was slightly higher when snails were exposed to the toxicant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMotility\u003c/h2\u003e \u003cp\u003eTwo-way ANOVA selected revealed that there were significant negative effects of both temperature (F\u003csub\u003e1,92=\u003c/sub\u003e5.95, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.046) and 6PPDQ (F\u003csub\u003e1,92=\u003c/sub\u003e6.29, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.036) on the velocity of the snails after the 10-days experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; Text S1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study reveals the toxicological impact of 6PPDQ, an emerging contaminant linked to tyre wear, on various life history traits of snails. As hypothesized, the sublethal effects observed on reproduction, growth, and motility reveal a broad spectrum of ecological risks associated with 6PPDQ exposure at environmentally relevant concentrations, emphasizing the need for regulatory measures and safer alternatives. Our results also reveal that some of these effects becoming more pronounced at higher temperatures.\u003c/p\u003e \u003cp\u003eThe results indicate that the exposure to 10.45 \u0026micro;g/L of 6PPDQ has a significant negative impact on the reproductive output of the freshwater snail \u003cem\u003eAmpullaceana balthica\u003c/em\u003e, specifically affecting both the number of clutches laid and the number of eggs per clutch. Compared with nonexposed snails, snails exposed to 6PPDQ laid fewer clutches. With respect to the number of eggs per clutch, the results revealed a more complex interaction. Initially, during the first week, 6PPDQ exposure resulted in a lower number of eggs per clutch compared to nonexposed clutches. However, a significant interaction effect between 6PPDQ and time was observed, with clutches laid later in the experiment (Days 8 to 10) in the contaminated aquaria resulting in an increase in the number of eggs per clutch. This temporal pattern may suggest a delayed compensatory response, where snails initially reduce egg production but later attempt to increase reproductive output as exposure continues. Similar responses have been observed in various organisms, where reproductive efforts are maximized following an initial period of stress to improve overall reproductive success (Minchella \u0026amp; Loverde, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Rollo \u0026amp; Hawryluk, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1988\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, similar compensatory reproductive responses have been documented in other freshwater gastropods exposed to environmental stressors. For example, Bi et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) observed that \u003cem\u003ePomacea canaliculata\u003c/em\u003e exposed to low concentrations of arsenite significantly increased egg production. This was interpreted as a hormetic response, where mild environmental stress stimulates reproductive output through physiological or endocrine mechanisms. Similarly, Liang et al., (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) reported enhanced spawning and estradiol levels in \u003cem\u003eP. canaliculata\u003c/em\u003e under low-level pollution, suggesting an adaptive strategy to ensure reproductive success under suboptimal conditions. Likewise, \u003cem\u003eLymnaea stagnalis\u003c/em\u003e exposed to cigarette butt leachate initially reduced clutch production, but reproductive output recovered during a post-exposure phase (Olah-Kovacs et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These findings suggest that freshwater snails may exhibit plastic reproductive strategies that allow partial recovery from sublethal stress. The analysis of overall reproductive fitness, measured as the number of eggs laid per snail per day, revealed a significant negative interaction between 6PPDQ and temperature suggesting that the detrimental effects of the contaminant may be exacerbated at relatively high temperatures, potentially due to increased metabolic demands (Clarke \u0026amp; Fraser, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Cloyed et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Scrine et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) or increased uptake of the contaminant (Guo et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Heugens et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Hooper et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013b\u003c/span\u003e). While the temporary increase in egg production per clutch may reflect an adaptive reproductive response to stressful conditions, the overall reduction in reproductive fitness indicates that this compensatory strategy is insufficient.\u003c/p\u003e \u003cp\u003eThe decline in reproductive fitness is likely driven by the repetitive peak exposure to 6PPDQ. Even though the contaminant levels drop below the LOD after each three-day cycle, likely result from the low solubility of solid 6PPDQ (Hu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) the renewal of contaminated media introduces fresh pulses of 6PPDQ, mimicking real-world conditions that can arise under diverse climatic or microclimatic regimes, where intermittent rainfall and runoff repeatedly transport contaminants into aquatic environments. In natural situations, despite the tendency of 6PPDQ to partition onto soil and organic matter due to its hydrophobic nature (log K\u003csub\u003eow\u003c/sub\u003e=4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02) (Hu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Hua et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), a portion remains in the dissolved aqueous phase, particularly during storm events. These events introduce fresh pulses of the contaminant into aquatic environments, leading to repeated high-concentration exposures similar to those observed in our experimental setup. This cycle of exposure, followed by temporary recovery periods, could explain the initial reduction in reproductive output, followed by a compensatory increase in egg production later in the experiment. However, despite this compensatory effort, the overall negative impact on reproductive fitness suggests that the snails were unable to fully recover from the stress induced by repetitive peak exposures.\u003c/p\u003e \u003cp\u003eOver time, chronic exposure to 6PPDQ reduces the reproductive capacity of snails, potentially threatening population sustainability in contaminated environments. This aligns with findings from other studies, where environmental stress or limited resources cause snails to reduce their reproductive efforts to survive. For example, in \u003cem\u003eLymnaea stagnalis\u003c/em\u003e reproduction was delayed under food limitation, diverting energy toward maintenance, reducing the number of eggs produced (Zonneveld \u0026amp; Kooijman, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Similar trade-offs between reproduction and survival have been observed in snails exposed to contaminants such as copper, which also reduced both reproductive output and growth (Gao et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Khangarot \u0026amp; Das, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Real et al., 2000). Additionally, findings by (Wang \u0026amp; Liu, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) show that under silver nanoparticle exposure, adult gastropods allocate more resources to combat oxidative stress rather than to growth or reproduction, further emphasizing the energy trade-offs under environmental stress.\u003c/p\u003e \u003cp\u003eIn our study, growth rates were also negatively impacted by the contaminant, especially at relatively warm temperatures. Normally, higher temperatures promote growth by accelerating metabolic rates, but this positive effect was diminished by the contaminant. This suggests that 6PPDQ disrupts energy allocation, forcing snails detoxification over growth (Wang \u0026amp; Liu, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Marginal changes in the CN ratio further support this finding, indicating metabolic stress that impairs the snails' ability to balance energy storage and protein synthesis, contributing to reduced growth and reproduction. Motility was also significantly reduced in snails exposed to 6PPDQ, with additional negative effects observed at relatively high temperatures. The reduction in motility could further decrease survival chances, as limited movement may impair foraging and escape from. The combined effects of 6PPDQ and temperature on motility suggest that the contaminant induces physiological stress, limiting the capacity of the snails to perform essential behaviours. Reduced motility could further compound the negative effects on growth and reproduction, as limited movement restricts access to food and mates, ultimately diminishing the overall fitness of the population. The effects of 6PPDQ at similar concentrations have also been documented in other invertebrates, with nematodes showing abnormal locomotion behaviours and neurodegeneration at 10 \u0026micro;g/L (Hua et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and \u003cem\u003eDaphnia pulex\u003c/em\u003e exhibiting significant reductions in growth rates at exposure levels of 0.1 and 10 \u0026micro;g/L (Shi et al., 2024).\u003c/p\u003e \u003cp\u003eWhile 6PPDQ clearly affected reproduction, its effects on embryonic development were less pronounced. This may be due to the protective features of the egg masses and eggshells. Initially, 6PPDQ must penetrate the gelatinous matrix surrounding the egg masses, which consists of proteins and polysaccharides that can limit the entry of external contaminants (Benkendorff et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The degree of protection offered by this matrix varies depending on factors such as the chemical's size, polarity, and lipophilicity (Arman, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For example, snail eggs within the matrix showed reduced sensitivity to cadmium compared to isolated eggs (Liu et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), although some contaminants (e.g., nanoparticles) can agglomerate and adhere to egg masses (Musee et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOnce it passes through the gelatinous matrix, 6PPDQ needs to cross the egg capsule membrane itself. Studies have shown that molecules such as raffinose (504.42 g/mol) and polyethylene glycol (500\u0026ndash;600 g/mol) can pass through this membrane (Beadle, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1969\u003c/span\u003e). Given relatively small molecular weight of 298.4 g/mol, 6PPDQ likely has the ability to permeate the egg capsule as well. Despite these barriers, the reduced number of eggs laid and overall reproductive impairment suggest that the long-term developmental success of the population could be compromised (Gao et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Khangarot \u0026amp; Das, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Further research is needed to explore whether embryos exposed to 6PPDQ experience persistent or delayed sublethal effects after hatching.\u003c/p\u003e \u003cp\u003eIn addition to the direct exposure from contaminated water during rainfall events, 6PPDQ can interact with organic matter, such as leaf litter and biofilms, which aquatic organisms may ingest. Tyre wear particles in the water can also be accidentally ingested, releasing 6PPDQ into the digestive systems of these organisms (Arman, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This raises concerns about the potential for bioaccumulation of 6PPDQ when contaminated food is consumed. Therefore, alongside the direct toxicity of dissolved 6PPDQ, it is essential to consider its effects from both contaminated food and tyre wear particles. The impact of 6PPDQ and other tyre-associated chemicals on aquatic organisms through these ingestion pathways remains largely unexplored, highlighting a critical area for future research to fully understand the ecological implications of tyre-related contaminants.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, repetitive exposure to 6PPDQ at environmentally relevant concentrations presents a serious threat to the freshwater snail \u003cem\u003eAmpullaceana batlhica\u003c/em\u003e, affecting key life history traits such as reproduction, embryo development, growth, and motility. While the protective properties of the egg masses may mitigate developmental risks, their effects on adult snails are severe and can potentially lead to long-term population declines. Overall, our study underscores the need for further investigation into the sublethal effects of 6PPDQ on aquatic species and the broader ecological risks associated with this contaminant. The findings contribute to a better understanding of how 6PPDQ impacts aquatic ecosystems, particularly in the context of climate change and increased contaminant levels. These significant impacts on aquatic life highlight the need for regulatory measures and the development of safer alternatives in the tyre industry to address 6PPDQ pollution.\u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eAll raw data associated with this study are available in the CORA Data Repository at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.34810/data1906\u003c/span\u003e\u003cspan address=\"10.34810/data1906\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis paper was funded by the project \u0026ldquo;City runoff pollution impacts on river biodiversity under extreme climatic events\u0026rdquo;, CityPoll (TED2021-129966B-C31 and TED2021-129966B-C33) funded by MCIN/AEI/\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.13039/501100011033\u003c/span\u003e\u003cspan address=\"10.13039/501100011033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e and by the European Union NextGeneration EU/PRTR, and by the project Rivstress PID2020-115708RB-C21 funded by MCIN/AEI/\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.13039/501100011033\u003c/span\u003e\u003cspan address=\"10.13039/501100011033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. We would also like to acknowledge the CCiTUB (Centres Cient\u0026iacute;fics i Tecnol\u0026ograve;gics de la Universitat de Barcelona) of the University of Barcelona for their services.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eC.L. (Catalina Lizama): Conceptualization, Data curation, Formal analysis.J.S. (Jordi Serra): Data curation, Formal analysis.M.Č. (Mira Čelić): Data curation, Writing \u0026ndash; Review \u0026amp; Editing.I.C. (Isabel Cadena): Data curation, Writing \u0026ndash; Review \u0026amp; Editing.M.P. (Mira Petrovic): Investigation, Writing \u0026ndash; Review \u0026amp; Editing, Funding acquisition.I.M. (Isabel Mu\u0026ntilde;oz): Investigation, Writing \u0026ndash; Review \u0026amp; Editing, Funding acquisition.N.D.C.C. (N\u0026uacute;ria De Castro Catal\u0026agrave;): Conceptualization, Methodology, Investigation, Writing \u0026ndash; Original Draft, Writing \u0026ndash; Review \u0026amp; Editing, Supervision.All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll raw data associated with this study are available in the CORA Data Repository at [https://doi.org/10.34810/data1906](https:/doi.org/10.34810/data1906) .\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthor contributions CRediT:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNúria de Castro-Català: Conceptualization, Methodology, Data Curation, Formal analysis, Visualization, Writing - Original Draft, Writing - Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003eCatalina Lizama: Conceptualization, Data Curation, Formal analysis\u003c/p\u003e\n\u003cp\u003eJordi Serra: Data Curation, Formal analysis\u003c/p\u003e\n\u003cp\u003eMira Čelić: Data Curation, Writing - Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003eIsabel Cadena: Data Curation, Writing - Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003eMira Petrovic: Investigation, Writing - Review \u0026amp; Editing, Funding acquisition\u003c/p\u003e\n\u003cp\u003eIsabel Muñoz: Investigation, Writing - Review \u0026amp; Editing, Funding acquisition\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArman S (2023) Effects of acute and chronic bendiocarb exposure during early life stages of the pond snail (Lymnaea stagnalis). Chem Ecol 39(1):44\u0026ndash;58. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/02757540.2022.2134351\u003c/span\u003e\u003cspan address=\"10.1080/02757540.2022.2134351\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaroudi F, Alam A, Fajloun J, Z., Millet M (2020) Snail as sentinel organism for monitoring the environmental pollution; a review. Ecol Ind 113:106240. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.ECOLIND.2020.106240\u003c/span\u003e\u003cspan address=\"10.1016/J.ECOLIND.2020.106240\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeadle LC (1969) Salt and water regulation in the embryos of freshwater pulmonate molluscs. I. The embryonic environment of Biomphalaria sudanica and Lymnaea stagnalis. J Exp Biol 50(2):473\u0026ndash;479. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/JEB.50.2.473\u003c/span\u003e\u003cspan address=\"10.1242/JEB.50.2.473\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenkendorff K, Davis AR, Bremner JB (2001) Chemical Defense in the Egg Masses of Benthic Invertebrates: An Assessment of Antibacterial Activity in 39 Mollusks and 4 Polychaetes. J Invertebr Pathol 78(2):109\u0026ndash;118. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1006/JIPA.2001.5047\u003c/span\u003e\u003cspan address=\"10.1006/JIPA.2001.5047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBi X, Shi Z, Li D, Zhan W, Wang Z, Li H, Zhao B, Chen G (2025) Arsenic accumulation and reproductive toxicity in freshwater snail (Pomacea canaliculata). Ecotoxicol Environ Saf 289:117688. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.ECOENV.2025.117688\u003c/span\u003e\u003cspan address=\"10.1016/J.ECOENV.2025.117688\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBolan S, Padhye LP, Jasemizad T, Govarthanan M, Karmegam N, Wijesekara H, Amarasiri D, Hou D, Zhou P, Biswal BK, Balasubramanian R, Wang H, Siddique KHM, Rinklebe J, Kirkham MB, Bolan N (2024) Impacts of climate change on the fate of contaminants through extreme weather events. Sci Total Environ 909:168388. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.SCITOTENV.2023.168388\u003c/span\u003e\u003cspan address=\"10.1016/J.SCITOTENV.2023.168388\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBolker BM, Brooks ME, Clark CJ, Geange SW, Poulsen JR, Stevens MHH, White JSS (2009) Generalized linear mixed models: a practical guide for ecology and evolution. Trends Ecol Evol 24(3):127\u0026ndash;135. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.TREE.2008.10.008/ATTACHMENT/BC246FFE-E4DF-4091-BACA-87ABB8F552E6/MMC3.ZIP\u003c/span\u003e\u003cspan address=\"10.1016/J.TREE.2008.10.008/ATTACHMENT/BC246FFE-E4DF-4091-BACA-87ABB8F552E6/MMC3.ZIP\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrinkmann M, Montgomery D, Selinger S, Miller JGP, Stock E, Alcaraz AJ, Challis JK, Weber L, Janz D, Hecker M, Wiseman S (2022) Acute Toxicity of the Tire Rubber-Derived Chemical 6PPD-quinone to Four Fishes of Commercial, Cultural, and Ecological Importance. Environ Sci Technol Lett 9(4):333\u0026ndash;338. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ACS.ESTLETT.2C00050/SUPPL_FILE/EZ2C00050_SI_003.MOV\u003c/span\u003e\u003cspan address=\"10.1021/ACS.ESTLETT.2C00050/SUPPL_FILE/EZ2C00050_SI_003.MOV\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCadena-Aizaga MI, Garc\u0026iacute;a-G\u0026oacute;mez E, Gago-Ferrero P, Sabater S, Petrovic M (2025) Comprehensive characterization of microcontaminants in urban runoff: Unveiling chemical signatures using wide-scope target screening by LC-HRMS. Environ Chem Ecotoxicol 7:2133\u0026ndash;2141. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.ENCECO.2025.09.009\u003c/span\u003e\u003cspan address=\"10.1016/J.ENCECO.2025.09.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClarke A, Fraser KPP (2004) Why does metabolism scale with temperature? Funct Ecol 18(2):243\u0026ndash;251. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/J.0269-8463.2004.00841.X\u003c/span\u003e\u003cspan address=\"10.1111/J.0269-8463.2004.00841.X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCloyed CS, Dell AI, Hayes T, Kordas RL, O\u0026rsquo;Gorman EJ (2019) Long-term exposure to higher temperature increases the thermal sensitivity of grazer metabolism and movement. J Anim Ecol 88(6):833\u0026ndash;844. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1365-2656.12976\u003c/span\u003e\u003cspan address=\"10.1111/1365-2656.12976\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDi S, Liu Z, Zhao H, Li Y, Qi P, Wang Z, Xu H, Jin Y, Wang X (2022) Chiral perspective evaluations: Enantioselective hydrolysis of 6PPD and 6PPD-quinone in water and enantioselective toxicity to Gobiocypris rarus and Oncorhynchus mykiss. Environ Int 166:107374. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.ENVINT.2022.107374\u003c/span\u003e\u003cspan address=\"10.1016/J.ENVINT.2022.107374\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGago-Ferrero P, Bletsou AA, Damalas DE, Aalizadeh R, Alygizakis NA, Singer HP, Hollender J, Thomaidis NS (2020) Wide-scope target screening of \u0026gt;\u0026thinsp;2000 emerging contaminants in wastewater samples with UPLC-Q-ToF-HRMS/MS and smart evaluation of its performance through the validation of 195 selected representative analytes. J Hazard Mater 387:121712. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.JHAZMAT.2019.121712\u003c/span\u003e\u003cspan address=\"10.1016/J.JHAZMAT.2019.121712\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGago-Ferrero P, Schymanski EL, Bletsou AA, Aalizadeh R, Hollender J, Thomaidis NS (2015) Extended Suspect and Non-Target Strategies to Characterize Emerging Polar Organic Contaminants in Raw Wastewater with LC-HRMS/MS. Environ Sci Technol 49(20):12333\u0026ndash;12341. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ACS.EST.5B03454/SUPPL_FILE/ES5B03454_SI_001.PDF\u003c/span\u003e\u003cspan address=\"10.1021/ACS.EST.5B03454/SUPPL_FILE/ES5B03454_SI_001.PDF\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao L, Doan H, Nidumolu B, Kumar A, Gonzago D (2017) Effects of copper on the survival, hatching, and reproduction of a pulmonate snail (Physa acuta). Chemosphere 185:1208\u0026ndash;1216. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.CHEMOSPHERE.2017.07.101\u003c/span\u003e\u003cspan address=\"10.1016/J.CHEMOSPHERE.2017.07.101\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo SN, Zheng JL, Yuan SS, Zhu QL (2018) Effects of heat and cadmium exposure on stress-related responses in the liver of female zebrafish: Heat increases cadmium toxicity. Sci Total Environ 618:1363\u0026ndash;1370. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.SCITOTENV.2017.09.264\u003c/span\u003e\u003cspan address=\"10.1016/J.SCITOTENV.2017.09.264\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe W, Chen C, Yang L, Chen Z, Zhu L, Zhu L, Feng J (2025) Chemical Ecological Risk Assessment for Aquatic Life Under Climate Change: A Review from Occurrence, Bioaccumulation, and Toxicity Perspective. Rev Environ Contam Toxicol 2025 263:1(1):1\u0026ndash;27. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S44169-025-00085-5\u003c/span\u003e\u003cspan address=\"10.1007/S44169-025-00085-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cem\u003e263\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeugens EHW, Hendriks AJ, Dekker T, Van Straalen NM, Admiraal W (2001) A Review of the Effects of Multiple Stressors on Aquatic Organisms and Analysis of Uncertainty Factors for Use in Risk Assessment. Crit Rev Toxicol 31(3):247\u0026ndash;284. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/20014091111695\u003c/span\u003e\u003cspan address=\"10.1080/20014091111695\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHiki K, Yamamoto H (2022) The Tire-Derived Chemical 6PPD-quinone Is Lethally Toxic to the White-Spotted Char Salvelinus leucomaenis pluvius but Not to Two Other Salmonid Species. Environ Sci Technol Lett 9(12):1050\u0026ndash;1055. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ACS.ESTLETT.2C00683/SUPPL_FILE/EZ2C00683_SI_001.PDF\u003c/span\u003e\u003cspan address=\"10.1021/ACS.ESTLETT.2C00683/SUPPL_FILE/EZ2C00683_SI_001.PDF\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHladyz S, Cook RA, Petrie R, Nielsen DL (2011) Influence of substratum on the variability of benthic biofilm stable isotope signatures: Implications for energy flow to a primary consumer. Hydrobiologia 664(1):135\u0026ndash;146. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S10750-010-0593-0/TABLES/2\u003c/span\u003e\u003cspan address=\"10.1007/S10750-010-0593-0/TABLES/2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHobbs NT, Hilborn R (2006) Alternatives To Statistical Hypothesis Testing In Ecology: A Guide To Self Teaching. Ecol Appl 16(1):5\u0026ndash;19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1890/04-0645\u003c/span\u003e\u003cspan address=\"10.1890/04-0645\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolmstrup M, Bindesb\u0026oslash;l AM, Oostingh GJ, Duschl A, Scheil V, K\u0026ouml;hler HR, Loureiro S, Soares AMVM, Ferreira ALG, Kienle C, Gerhardt A, Laskowski R, Kramarz PE, Bayley M, Svendsen C, Spurgeon DJ (2010) Interactions between effects of environmental chemicals and natural stressors: A review. Sci Total Environ 408(18):3746\u0026ndash;3762. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.SCITOTENV.2009.10.067\u003c/span\u003e\u003cspan address=\"10.1016/J.SCITOTENV.2009.10.067\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHooper MJ, Ankley GT, Cristol DA, Maryoung LA, Noyes PD, Pinkerton KE (2013a) Interactions between chemical and climate stressors: A role for mechanistic toxicology in assessing climate change risks. Environ Toxicol Chem 32(1):32\u0026ndash;48. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ETC.2043\u003c/span\u003e\u003cspan address=\"10.1002/ETC.2043\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHooper MJ, Ankley GT, Cristol DA, Maryoung LA, Noyes PD, Pinkerton KE (2013b) Interactions between chemical and climate stressors: A role for mechanistic toxicology in assessing climate change risks. Environ Toxicol Chem 32(1):32\u0026ndash;48. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ETC.2043\u003c/span\u003e\u003cspan address=\"10.1002/ETC.2043\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu X, Zhao H, Tian Z, Peter KT, Dodd MC, Kolodziej EP (2023) Chemical characteristics, leaching, and stability of the ubiquitous tire rubber-derived toxicant 6PPD-quinone. Environ Science: Processes Impacts 25(5):901\u0026ndash;911. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D3EM00047H\u003c/span\u003e\u003cspan address=\"10.1039/D3EM00047H\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHua X, Feng X, Liang G, Chao J, Wang D (2023) Exposure to 6-PPD Quinone at Environmentally Relevant Concentrations Causes Abnormal Locomotion Behaviors and Neurodegeneration in Caenorhabditis elegans. Environ Sci Technol 57(12):4940\u0026ndash;4950. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ACS.EST.2C08644/\u003c/span\u003e\u003cspan address=\"10.1021/ACS.EST.2C08644/\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eASSET/IMAGES/LARGE/ES2C08644_0005.JPEG\u003c/span\u003e\u003cspan address=\"http://ASSET/IMAGES/LARGE/ES2C08644_0005.JPEG\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHua X, Liang G, Chao J, Wang D (2024) Exposure to 6-PPD quinone causes damage on mitochondrial complex I/II associated with lifespan reduction in Caenorhabditis elegans. J Hazard Mater 472:134598. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.JHAZMAT.2024.134598\u003c/span\u003e\u003cspan address=\"10.1016/J.JHAZMAT.2024.134598\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHua X, Wang D (2023) Tire-rubber related pollutant 6-PPD quinone: A review of its transformation, environmental distribution, bioavailability, and toxicity. J Hazard Mater 459:132265. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.JHAZMAT.2023.132265\u003c/span\u003e\u003cspan address=\"10.1016/J.JHAZMAT.2023.132265\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohannessen C, Helm P, Metcalfe CD (2021) Detection of selected tire wear compounds in urban receiving waters. Environ Pollut 287:117659. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.ENVPOL.2021.117659\u003c/span\u003e\u003cspan address=\"10.1016/J.ENVPOL.2021.117659\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohnson JB, Omland KS (2004) Model selection in ecology and evolution. Trends Ecol Evol 19(2):101\u0026ndash;108. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.TREE.2003.10.013\u003c/span\u003e\u003cspan address=\"10.1016/J.TREE.2003.10.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhangarot BS, Das S (2010) Effects of copper on the egg development and hatching of a freshwater pulmonate snail Lymnaea luteola L. J Hazard Mater 179(1\u0026ndash;3):665\u0026ndash;675. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.JHAZMAT.2010.03.054\u003c/span\u003e\u003cspan address=\"10.1016/J.JHAZMAT.2010.03.054\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee H, Romero J (2023) (n.d.). \u003cem\u003eClimate Change 2023 Synthesis Report IPCC\u003c/em\u003e, : Sections. In: Climate Change 2023: Synthesis Report. Contribution of Working Groups I, II and III to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change [Core Writing Team. 35\u0026ndash;115. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.59327/IPCC/AR6-9789291691647\u003c/span\u003e\u003cspan address=\"10.59327/IPCC/AR6-9789291691647\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C, Zhang Y, Yin S, Wang Q, Li Y, Liu Q, Liu L, Luo X, Chen L, Zheng H, Li F (2023) First insights into 6PPD-quinone formation from 6PPD photodegradation in water environment. J Hazard Mater 459:132127. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.JHAZMAT.2023.132127\u003c/span\u003e\u003cspan address=\"10.1016/J.JHAZMAT.2023.132127\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang D, Li Y, Li S, Meng D, Li F, Huang S, Gong M, Qin J, Li H (2023) Dose-dependent inhibitory effects of glyphosate on invasive Pomacea canaliculata reproductive and developmental growth under oxidative deposition. Ecotoxicol Environ Saf 253:114659. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.ECOENV.2023.114659\u003c/span\u003e\u003cspan address=\"10.1016/J.ECOENV.2023.114659\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Yu M, Shi R, Ge Y, Li J, Zeb A, Cheng Z, Liu W (2024) Comparative toxic effect of tire wear particle-derived compounds 6PPD and 6PPD-quinone to Chlorella vulgaris. Sci Total Environ 951:175592. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.SCITOTENV.2024.175592\u003c/span\u003e\u003cspan address=\"10.1016/J.SCITOTENV.2024.175592\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu T, Koene JM, Dong X, Fu R (2013) Sensitivity of isolated eggs of pond snails: A new method for toxicity assays and risk assessment. Environ Monit Assess 185(5):4183\u0026ndash;4190. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S10661-012-2860-1/METRICS\u003c/span\u003e\u003cspan address=\"10.1007/S10661-012-2860-1/METRICS\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Z, Yang H, Zhang Y, Shao Y, Hu S, Zhang H, Shen G (2024) Tire-wear particle leachate at environmentally relevant concentrations exert a hepatotoxic impact on the black-spotted frog by disrupting the gut\u0026ndash;liver axis. Environ Chem Ecotoxicol 6:380\u0026ndash;389. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.ENCECO.2024.08.004\u003c/span\u003e\u003cspan address=\"10.1016/J.ENCECO.2024.08.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMart\u0026iacute;nez-De Le\u0026oacute;n G, Thakur MP (2024) Ecological debts induced by heat extremes. Trends Ecol Evol. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.TREE.2024.07.002\u003c/span\u003e\u003cspan address=\"10.1016/J.TREE.2024.07.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMayer PM, Moran KD, Miller EL, Brander SM, Harper S, Garcia-Jaramillo M, Carrasco-Navarro V, Ho KT, Burgess RM, Hampton T, Granek LM, McCauley EF, McIntyre M, Kolodziej JK, Hu EP, Williams X, Beckingham AJ, Jackson BA, Sanders-Smith ME, Mendez RD, M (2024) Where the rubber meets the road: Emerging environmental impacts of tire wear particles and their chemical cocktails. Sci Total Environ 927:171153. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.SCITOTENV.2024.171153\u003c/span\u003e\u003cspan address=\"10.1016/J.SCITOTENV.2024.171153\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMinchella DJ, Loverde PT (1981) A Cost of Increased Early Reproductive Effort in the Snail Biomphalaria glabrata. \u003cem\u003eHttps://Doi.Org/10.1086/283879\u003c/em\u003e, \u003cem\u003e118\u003c/em\u003e(6), 876\u0026ndash;881. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1086/283879\u003c/span\u003e\u003cspan address=\"10.1086/283879\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMusee N, Oberholster PJ, Sikhwivhilu L, Botha AM (2010) The effects of engineered nanoparticles on survival, reproduction, and behaviour of freshwater snail, Physa acuta (Draparnaud, 1805). Chemosphere 81(10):1196\u0026ndash;1203. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.CHEMOSPHERE.2010.09.040\u003c/span\u003e\u003cspan address=\"10.1016/J.CHEMOSPHERE.2010.09.040\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOlah-Kovacs B, White HJ, Tingey T, Boots B, Green DS (2025) The impacts of smoked cigarette butt leachate on a common freshwater gastropod, Lymnaea stagnalis. Environ Pollut 376:126425. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.ENVPOL.2025.126425\u003c/span\u003e\u003cspan address=\"10.1016/J.ENVPOL.2025.126425\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eProsser RS, Salole J, Hang S (2023) Toxicity of 6PPD-quinone to four freshwater invertebrate species. Environ Pollut 337:122512. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.ENVPOL.2023.122512\u003c/span\u003e\u003cspan address=\"10.1016/J.ENVPOL.2023.122512\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRao SR, Olechnowicz SWZ, Kr\u0026auml;tschmer P, Jepson JEC, Edwards CM, Edwards JR (2019) Small Animal Video Tracking for Activity and Path Analysis Using a Novel Open-Source Multi-Platform Application (AnimApp). \u003cem\u003eScientific Reports 2019 9:1\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(1), 1\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-019-48841-7\u003c/span\u003e\u003cspan address=\"10.1038/s41598-019-48841-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReal M, Mu\u0026ntilde; Oz I, Guasch H, Navarro E, Sabater S (2024) (n.d.). \u003cem\u003eThe effect of copper exposure on a simple aquatic food chain\u003c/em\u003e. Retrieved October 2, from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003c/span\u003e\u003cspan address=\"http://www.elsevier.com/locate/aquatox\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRedman ZC, Begley JL, Hillestad I, DiMento BP, Stanton RS, Aguaa AR, Pirrung MC, Tomco PL (2023) Reactive Oxygen Species and Chromophoric Dissolved Organic Matter Drive the Aquatic Photochemical Pathways and Photoproducts of 6PPD-quinone under Simulated High-Latitude Conditions. Environ Sci Technol 57(49):20813\u0026ndash;20821. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ACS.EST.3C05742/SUPPL_FILE/ES3C05742_SI_001.PDF\u003c/span\u003e\u003cspan address=\"10.1021/ACS.EST.3C05742/SUPPL_FILE/ES3C05742_SI_001.PDF\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRicarte M, Prats E, Montemurro N, Bedrossiantz J, Bellot M, G\u0026oacute;mez-Canela C, Rald\u0026uacute;a D (2023) Environmental concentrations of tire rubber-derived 6PPD-quinone alter CNS function in zebrafish larvae. Sci Total Environ 896:165240. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.SCITOTENV.2023.165240\u003c/span\u003e\u003cspan address=\"10.1016/J.SCITOTENV.2023.165240\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoberts C, Lin J, Kohlman E, Jain N, Amekor M, Alcaraz AJ, Hogan N, Hecker M, Brinkmann M (2025) Acute and Subchronic Toxicity of 6PPD-Quinone to Early Life Stage Lake Trout (Salvelinus namaycush). Environ Sci Technol 59(1):791\u0026ndash;797. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ACS.EST.4C09090/SUPPL_FILE/ES4C09090_SI_001.PDF\u003c/span\u003e\u003cspan address=\"10.1021/ACS.EST.4C09090/SUPPL_FILE/ES4C09090_SI_001.PDF\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRollo CD, Hawryluk MD (1988) Compensatory Scope and Resource Allocation in Two Species of Aquatic Snails. Ecology 69(1):146\u0026ndash;156. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2307/1943169\u003c/span\u003e\u003cspan address=\"10.2307/1943169\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScrine J, Jochum M, \u0026Oacute;lafsson JS, O\u0026rsquo;Gorman EJ (2017) Interactive effects of temperature and habitat complexity on freshwater communities. Ecol Evol 7(22):9333. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ECE3.3412\u003c/span\u003e\u003cspan address=\"10.1002/ECE3.3412\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeeland A, Oehlmann J, M\u0026uuml;ller R (2012) Aquatic ecotoxicity of the fungicide pyrimethanil: Effect profile under optimal and thermal stress conditions. Environ Pollut 168:161\u0026ndash;169. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envpol.2012.04.020\u003c/span\u003e\u003cspan address=\"10.1016/j.envpol.2012.04.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeiwert B, Nihemaiti M, Troussier M, Weyrauch S, Reemtsma T (2022) Abiotic oxidative transformation of 6-PPD and 6-PPD quinone from tires and occurrence of their products in snow from urban roads and in municipal wastewater. Water Res 212:118122. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.WATRES.2022.118122\u003c/span\u003e\u003cspan address=\"10.1016/J.WATRES.2022.118122\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi C, Wu F, Zhao Z, Ye T, Luo X, Wu Y, Liu Z, Zhang H (2024a) Effects of environmental concentrations of 6PPD and its quinone metabolite on the growth and reproduction of freshwater cladoceran. Sci Total Environ 948:175018. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.SCITOTENV.2024.175018\u003c/span\u003e\u003cspan address=\"10.1016/J.SCITOTENV.2024.175018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi C, Wu F, Zhao Z, Ye T, Luo X, Wu Y, Liu Z, Zhang H (2024b) Effects of environmental concentrations of 6PPD and its quinone metabolite on the growth and reproduction of freshwater cladoceran. Sci Total Environ 948:175018. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.SCITOTENV.2024.175018\u003c/span\u003e\u003cspan address=\"10.1016/J.SCITOTENV.2024.175018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong W, Lin L, Oh S, Grossart HP, Yang Y (2025) Tire wear particles in aquatic environments: From biota to ecosystem impacts. J Environ Manage 388:126059. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.JENVMAN.2025.126059\u003c/span\u003e\u003cspan address=\"10.1016/J.JENVMAN.2025.126059\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTerrado M, Acu\u0026ntilde;a V, Ennaanay D, Tallis H, Sabater S (2014) Impact of climate extremes on hydrological ecosystem services in a heavily humanized Mediterranean basin. Ecol Ind 37(PART A):199\u0026ndash;209. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.ECOLIND.2013.01.016\u003c/span\u003e\u003cspan address=\"10.1016/J.ECOLIND.2013.01.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian Z, Gonzalez M, Rideout CA, Zhao HN, Hu X, Wetzel J, Mudrock E, James CA, McIntyre JK, Kolodziej EP (2022) 6PPD-Quinone: Revised Toxicity Assessment and Quantification with a Commercial Standard. Environ Sci Technol Lett 9(2):140\u0026ndash;146. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ACS.ESTLETT.1C00910/SUPPL_FILE/EZ1C00910_SI_001.PDF\u003c/span\u003e\u003cspan address=\"10.1021/ACS.ESTLETT.1C00910/SUPPL_FILE/EZ1C00910_SI_001.PDF\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVarshney S, Gora AH, Siriyappagouder P, Kiron V, Olsvik PA (2022) Toxicological effects of 6PPD and 6PPD quinone in zebrafish larvae. J Hazard Mater 424:127623. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.JHAZMAT.2021.127623\u003c/span\u003e\u003cspan address=\"10.1016/J.JHAZMAT.2021.127623\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang T, Liu W (2023) Chronic and transgenerational effects of silver nanoparticles in freshwater gastropod Lymnaea stagnalis. Chemosphere 313:137386. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.CHEMOSPHERE.2022.137386\u003c/span\u003e\u003cspan address=\"10.1016/J.CHEMOSPHERE.2022.137386\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeiskopf SR, Rubenstein MA, Crozier LG, Gaichas S, Griffis R, Halofsky JE, Hyde KJW, Morelli TL, Morisette JT, Mu\u0026ntilde;oz RC, Pershing AJ, Peterson DL, Poudel R, Staudinger MD, Sutton-Grier AE, Thompson L, Vose J, Weltzin JF, Whyte KP (2020) Climate change effects on biodiversity, ecosystems, ecosystem services, and natural resource management in the United States. Sci Total Environ 733:137782. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.SCITOTENV.2020.137782\u003c/span\u003e\u003cspan address=\"10.1016/J.SCITOTENV.2020.137782\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZimmer EI, Jager T, Ducrot V, Lagadic L, Kooijman SALM (2012) Juvenile food limitation in standardized tests: A warning to ecotoxicologists. Ecotoxicology 21(8):2195\u0026ndash;2204. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S10646-012-0973-5/TABLES/1\u003c/span\u003e\u003cspan address=\"10.1007/S10646-012-0973-5/TABLES/1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZitoun R, Marcinek S, Hatje V, Sander SG, V\u0026ouml;lker C, Sarin M, Omanović D (2024) Climate change driven effects on transport, fate and biogeochemistry of trace element contaminants in coastal marine ecosystems. Commun Earth Environ 2024 5:1(1):1\u0026ndash;17. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s43247-024-01679-y\u003c/span\u003e\u003cspan address=\"10.1038/s43247-024-01679-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cem\u003e5\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZonneveld C, Kooijman SALM (1993) Comparative kinetics of embryo development. Bull Math Biol 55(3):609\u0026ndash;635. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0092-8240(05)80242-3\u003c/span\u003e\u003cspan address=\"10.1016/S0092-8240(05)80242-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ecotoxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ectx","sideBox":"Learn more about [Ecotoxicology](https://www.springer.com/journal/10646)","snPcode":"10646","submissionUrl":"https://submission.nature.com/new-submission/10646/3","title":"Ecotoxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Tyre wear contaminant","lastPublishedDoi":"10.21203/rs.3.rs-8114676/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8114676/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eN-(1,3-Dimethylbutyl)-N\u0026prime;-phenyl-p-phenylenediamine-quinone (6PPD-quinone or 6PPDQ) is an oxidation product of 6PPD, an antioxidant used in tyres to prevent rubber degradation, that has been associated with high mortality in juvenile coho salmon at concentrations as low as 95 ng/L. While research has focused primarily on fish, the effects of 6PPDQ on freshwater invertebrates remain limited. In this study, we assessed the toxicological impact of this contaminant on the freshwater snail \u003cem\u003eAmpullaceana balthica\u003c/em\u003e over a 10-day experiment under two different temperature conditions. \u003cem\u003eA. balthica\u003c/em\u003e was chosen because it is widely distributed in temperate and Mediterranean regions and is commonly used as a model organism in environmental toxicology studies. Although 6PPDQ had a limited impact on embryonic development, adult snails experienced significant effects on reproduction, growth, and motility, with more pronounced impacts at higher temperatures. Specifically, 6PPDQ reduced clutch and egg production, particularly during the first days of exposure. Elevated temperature increased reproduction, but its interaction with 6PPDQ lowered the overall reproductive output. The combined stressors also impaired growth and motility. Development was mainly affected by temperature, with reduced hatching and increased embryo arrest at 20\u0026deg;C. These sublethal effects may lead to population declines and cascading impacts on freshwater community structure and ecosystem functioning, particularly under climate change scenarios. This highlights the urgent need for comprehensive risk assessments of emerging contaminants such as 6PPDQ to better understand their ecological impacts.\u003c/p\u003e","manuscriptTitle":"Crawling towards complex interactions: the impact of 6PPD-quinone and increased temperatures on the freshwater snail Ampullaceana balthica","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-14 11:50:00","doi":"10.21203/rs.3.rs-8114676/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-02T17:47:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-01T20:12:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-24T20:35:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-23T15:45:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"239418819758269257622886619812343341679","date":"2026-01-14T17:39:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"264267307647418606528858641494160452653","date":"2026-01-12T21:12:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"323990183172217993449069570767369881447","date":"2026-01-12T19:50:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-12T17:37:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-15T12:50:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-15T12:48:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Ecotoxicology","date":"2025-11-14T12:46:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ecotoxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ectx","sideBox":"Learn more about [Ecotoxicology](https://www.springer.com/journal/10646)","snPcode":"10646","submissionUrl":"https://submission.nature.com/new-submission/10646/3","title":"Ecotoxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"76dfde25-e2d7-493f-b98a-95e158d7dd5d","owner":[],"postedDate":"January 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:08:31+00:00","versionOfRecord":{"articleIdentity":"rs-8114676","link":"https://doi.org/10.1007/s10646-026-03080-1","journal":{"identity":"ecotoxicology","isVorOnly":false,"title":"Ecotoxicology"},"publishedOn":"2026-04-24 15:57:09","publishedOnDateReadable":"April 24th, 2026"},"versionCreatedAt":"2026-01-14 11:50:00","video":"","vorDoi":"10.1007/s10646-026-03080-1","vorDoiUrl":"https://doi.org/10.1007/s10646-026-03080-1","workflowStages":[]},"version":"v1","identity":"rs-8114676","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8114676","identity":"rs-8114676","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}