Estrogenic, Genotoxic, and Antibacterial Effects of Chemicals from Cryogenically Milled Tire Tread.

Tire and road wear particles (TRWP) contain complex mixtures of chemicals and release them to the environment, and potential toxic effects of these chemicals still need to be characterized. We used a standardized surrogate for TRWP, cryogenically milled tire tread (CMTT), to isolate and evaluate effects of tire-associated chemicals. We examined organic chemical mixtures extracted and leached from CMTT for the toxicity endpoints genotoxicity, estrogenicity, and inhibition of bacterial luminescence. The bioassays were performed after chromatographic separation on high-performance thin-layer chromatography (HPTLC) plates. Extracts of CMTT were active in all three HPTLC bioassays with two estrogenic zones, two genotoxic zones, and two zones inhibiting bacterial luminescence. Extracts of CMTT artificially aged with thermooxidation were equally bioactive in each HPTLC bioassay. Two types of aqueous leachates of unaged CMTT, simulating either digestion by fish or contact with sediment and water, contained estrogenic chemicals and inhibitors of bacterial luminescence with similar profiles to those of CMTT extracts. Of 11 tested tire-associated chemicals, two were estrogenic, three were genotoxic, and several inhibited bacterial luminescence. 1,3-Diphenylguanidine, transformation products of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine, and benzothiazoles were especially implicated through comparison to HPTLC retention factors in the CMTT samples. Other bioactive bands in CMTT samples did not correspond to any target chemicals. Tire particles clearly contain and can leach complex mixtures of toxic chemicals to the environment. Although some known chemicals contribute to estrogenic, genotoxic, and antibacterial hazards, unidentified toxic chemicals are still present and deserve further investigation. Overall, our study expands the understanding of potential adverse effects from tire particles and helps improve the link between those effects and the responsible chemicals. Environ Toxicol Chem 2024;00:1-11. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Evaluation of tire tread particle toxicity to fish using rainbow trout cell lines.

Tire and road wear particles (TRWP) resulting from tire abrasion while driving raise concerns due to their potential contribution to aquatic toxicity. Our study aimed to assess cryogenically milled tire tread (CMTT) particle toxicity, used as a proxy for TRWP, and associated chemicals to fish using two Rainbow Trout (Oncorhynchus mykiss) cell lines representing the gill (RTgill-W1) and the intestinal (RTgutGC) epithelium. CMTT toxicity was evaluated through several exposure pathways, including direct contact, leaching, and digestion, while also assessing the impact of particle aging. Following OECD TG249, cell viability was assessed after 24 h acute exposure using a multiple-endpoint assay indicative of cell metabolic activity, membrane integrity and lysosome integrity. In vitro EC50 values for the fish cell lines exceeded river TRWP concentrations (2.02 g/L and 4.65 g/L for RTgill-W1 and RTgutGC cell lines, respectively), and were similar to in vivo LC50 values estimated at 6 g/L. Although toxicity was mainly driven by the leaching of tire-associated chemicals, the presence of the particles contributed to the overall toxicity by inducing a continuous leaching, highlighting the importance of considering combined exposure scenarios. Aging and digestion conditions were also found to mediate CMTT toxicity. Thermooxidation resulted in a decreased chemical leaching and toxicity, while in vitro digestion under mimicked gastrointestinal conditions increased leaching and toxicity. Specific chemicals, especially Zn, 2-mercaptobenzothiazole, 1,3-diphenylguanidine, and N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) were identified as contributors to the overall toxicity. Although 6PPD-quinone was detected in CMTT digestate, cytotoxicity assays with RTgill-W1 and RTgutGC cell lines showed no toxicity up to 6 mg/L, supporting the notion of a specific mode of action of this chemical. This study provides insights into the toxicological mechanisms induced by tire particles and their associated chemicals and can help in the evaluation of potential risks to aquatic life associated with TRWP.

Environmental DNA metabarcoding for benthic monitoring: A review of sediment sampling and DNA extraction methods.

Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring.

Chromium bioavailability in aquatic systems impacted by tannery wastewaters. Part 1: Understanding chromium accumulation by indigenous chironomids.

The tanning industry uses large quantities of Cr whose contribution to the contaminant burden of aquatic organisms is not yet fully understood. The present study investigated Cr bioaccumulation by indigenous chironomids in a freshwater ecosystem impacted by tannery effluents. Total Cr content in sediments and in chironomids was determined on several occasions. Chromium distribution among sediments and pore waters, and Cr speciation in overlying and pore waters were studied in detail to understand possible factors controlling Cr bioavailability to chironomids. Total chromium concentration ranged from 69 to over 3000 µg g-1 dry weight in sediments and from negligible to over 300 µg g-1 dry weight in chironomids (values corrected for sediment gut content). Filterable (<0.45 µm) Cr concentration in overlying waters and pore waters from the surface sediment layers (upper 2 cm) ranged from 3 to 120 µg L-1, with Cr(VI) representing 0.5-28% of the total filterable Cr. Chromium profiles in pore waters as determined by diffusive equilibration in thin films (DET) and diffusive gradient in thin films (DGT) were comparable. DGT-labile Cr accounted for <2% of the total Cr measured by DET. Although Cr concentrations in sedimentary and aqueous matrices were not directly proportional to Cr levels measured in chironomids, the available findings suggested that Cr inputs from tanneries were bioavailable to resident chironomids. These observations are of particular importance considering that Cr(III), putatively of limited bioavailability and ecotoxicological concern, is the predominant redox form of Cr in bed sediments impacted by tannery discharges. The companion paper provides further insight into Cr bioavailability and effects in tannery impacted ecosystems using a combination of in situ and laboratory approaches.

Chromium bioavailability in aquatic systems impacted by tannery wastewaters. Part 2: New insights from laboratory and in situ testing with Chironomus riparius Meigen (Diptera, Chironomidae).

Chromium is widely used as a tanning agent and can become a contaminant of concern in aquatic ecosystems receiving discharges from industrial or artisanal tanning activities. In a companion study, we showed that Cr discharged by tanneries was bioavailable to indigenous chironomids with accumulation via sediment ingestion likely to represent the predominant exposure route. However, Cr accumulation by chironomids did not directly reflect the degree of sediment contamination and the potential adverse effects of Cr accumulation on chironomids were not evaluated. In the present study, chironomids were exposed to homogenised, field-collected sediments in the laboratory and to intact sediments in situ using a customized caging system. Chromium concentrations were assessed in sediments, exposed larvae of laboratory-reared Chironomus riparius and overlying waters of in situ cages. Experimental results of Cr bioaccumulation were compared with expected Cr body burden in chironomids calculated using biodynamic modelling. Our data provided strong support to the hypothesis that Cr bioaccumulation in the field is specifically controlled by the deposition of contaminated suspended particulate matter (SPM) containing a pool of Cr readily bioavailable to surface deposit feeders. Considering freshly deposited SPM as an additional route of exposure for surface deposit feeders leads to a good agreement between the modelling and experimental results. Additionally, a Cr body burden of about 77 µg g-1 d.w. was identified as a tentative threshold above which effects on the growth of C. riparius may appear. While both laboratory and in situ experiments provide evidence for the availability of Cr in aquatic system impacted by tannery wastewaters, standard laboratory exposure conditions may miss additional exposure routes in the field and underestimate possible adverse effects on benthic organisms.