In-vitro digestion of Tire and Road Wear Particles: Bioavailability of metals and Polycyclic Aromatic Hydrocarbons

The potential impact of Tire and Road Wear Particles (TRWP) on aquatic organisms has recently gained attention since the occurrence of TRWP in the aquatic environment has been observed in surface water and sediments of numerous regions. Moreover, the ingestion of TRWP by aquatic species, including fish, has recently been demonstrated. However, data regarding the bioavailability and toxicity of contaminants associated with these particles are still lacking. This study aimed (i) to characterize the solubilization potential of metals and PAHs from tire particles and TRWP in Simulated Gastric Fluids (SFG) and Simulated Intestinal Fluids (SIF) designed to mimic Oncorhynchus mykiss (Rainbow Trout) gut conditions and (ii) to assess the impact of influencing factors such as co-ingestion of natural food organic matter on the compounds solubilization. Our results showed that solubilization of all metals and PAHs were enhanced by up to 10-fold in simulated gut fluids compared to solubilization in the control (phosphate buffer saline solution). Zn was highly solubilized after an in-vitro digestion of 24-h with a final concentration in SIF of 3,2 µg.L-1 corresponding to 23 % of the total Zn concentration in TRWP. Five PAHs (Fluoranthene, Phenanthrene, Pyrene, Benzo (e) Pyrene and Benzo (g,h,i) perylene) were quantified in SIF after an incubation time of 24-h and the concentration of individual PAHs in SIF ranged from 42 to 3092 ng.L-1 which represented 0,4 – 1,5 % of their total concentration measured in tire particles. Metals were highly solubilized by the acidic SGF (pH = 2) but only a weak solubilization was observed in neutral SIF (pH = 7,4). Contrastingly, PAHs were not solubilized in SGF and highly solubilized in SIF which was proved to contain high quantities of proteins and surfactants forming micelles. This emphasise the importance of pH in the solubilization of metals and of bile surfactants and organic matter in the solubilization of PAHs from TRWP and brings new insights on the bioavailability of metals and organic pollutants from TRWP. Overall, our results demonstrate the importance of accounting for the ingestion of TRWP as an important route of exposure to metals and PAHs for aquatic organisms. As the solubilization of potential toxic compounds was greatly enhanced by the gut fluids and leads to bioavailability of these compounds, further studies investigating the uptake by the epithelial cells and transfer throughout the whole organism will be performed.

Potential bioavailability of inorganic compounds from Tire and Road Wear Particles to fish in simulated gastric and intestinal fluids

Florian Frédéric Vincent Breider, Benoît Jean Dominique Ferrari, Thibault Béranger Masset

The potential impact of Tire and Road Wear Particles (TRWP) on aquatic organisms has recently gained attention since the occurrence of TRWP in the aquatic environment has been demonstrated in surface water and sediments of numerous regions. However, data regarding the bioavailability and toxicity of contaminants associated with these particles are still lacking. Several metals enter in the composition of tires including Zinc, used as a catalyst during the vulcanization process of rubber. This study aimed (i) to characterize the elemental composition of Cryogenically Milled Tire Tread (CMTT) and TRWP and (ii) to assess the solubilization potential of inorganic contaminants from CMTT and TRWP in Simulated Gastric Fluids (SFG) and Simulated Intestinal Fluids (SIF) designed to mimic Oncorhynchus mykiss (Rainbow Trout) gut conditions. Our results show an important difference in the elemental composition of CMTT and TRWP due to the input of elements such as Ti, Cr, Mn, Sr, Ba and Pb from the road constituent to the TRWP. In vitro digestion experiments of both CMTT and TRWP showed that solubilization of all metals were enhanced by up to 10-fold in simulated gut fluids compared to solubilization in phosphate buffer solution. Solubilization kinetics to the SGF and SIF were biphasic with a higher solubilization rate of elements within the first hour and a slower solubilization rate up to 26h. For most metals, solubilization rates were higher for TRWP than for CMTT. After digestion time of 26h, from 1% (Co) to 10% (Zn) of total metal in CMTT were solubilized in the gut fluids and the concentration of metals were about 1 – 100 µg/L except for Zn with 7000 µg/L. Our results emphasise the importance of pH, bile surfactants and organic matter in the solubilization of metals from TRWP and brings new insights on the bioavailability of inorganic pollutants from TRWP.

2020

In vitro acute toxicity assessment of tire tread particles using rainbow trout cell lines of gill and intestine

Florian Frédéric Vincent Breider, Benoît Jean Dominique Ferrari, Thibault Béranger Masset, Kristin Schirmer

Tire and road wear particles (TRWP) are generated by the abrasion of tires while driving, and recent questions were raised in regards to their potential contribution to microplastics released into the aquatic environment and their potential toxicological impacts. Our study aimed to determine the toxicity of TRWP and associated chemicals to fish using two rainbow trout (Oncorhynchus mykiss) cell lines representing the gill (RTgill-W1) and the intestinal (RTgut-GC) epithelium. Acute exposure to cryogenically milled tire tread (CMTT) particles and CMTT digestate following in vitro digestion was investigated. To obtain the effective concentrations (EC50) causing a 50% cell viability loss, tests were performed in accordance with OECD TG249: cell viability was assessed after 24 hours exposure using a multiple-endpoint assay indicative of cell metabolic activity, membrane integrity and lysosome integrity. Finally, chemical composition of the exposure medium was analyzed to assess which chemicals could be responsible for the observed acute effects. Our results show that metabolic activity was the most sensitive indicator of CMTT and CMTT digestate toxicity. EC50 values after direct exposure to RTgill-W1 and RTgutGC cells were estimated at 2.01 g/L and 3.80 g/L, respectively. 2-Mercaptobenzothiazole was found at the highest concentration among organic chemicals leaching out from the particles. It could be a major contributor to the loss in metabolic activity based on its toxicity to rainbow trout in vivo, although additional particle or mixture effects cannot be ruled out at this point. For digestate toxicity, EC50 was determined at 12.15 g of tire particle/L of digestive fluid. The observed toxicity could be due to high amounts of Zn, N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), 1,3-Diphenylguanidine (DPG) or 2-Mercaptobenzothiazole leaching out during in vitro digestion. Traces of 6PPD-quinone were also detected in the exposure medium, but additional cytotoxicity assays using the RTgill-W1 cell line showed no significant toxicity of this compound up to 3 mg/L. Overall, our in vitro toxicity data agree with reports of acute fish (in vivo) toxicity and show that acutely toxic concentrations of CMTT are well above concentrations measured in river water (0.3 to 4 mg/L). However, we determined the highest no-observed-effect concentration at 10 mg/L, while up to 100 mg/L were measured in road runoffs.

2021

Interaction of silver nanoparticles with algae and fish cells: a side by side comparison

Renata Behra, Xiaomei Li, Kristin Schirmer, Laura Sigg, Yang Yue

Background: Silver nanoparticles (AgNP) are widely applied and can, upon use, be released into the aquatic environment. This raises concerns about potential impacts of AgNP on aquatic organisms. We here present a side by side comparison of the interaction of AgNP with two contrasting cell types: algal cells, using the algae Euglena gracilis as model, and fish cells, a cell line originating from rainbow trout (Oncorhynchus mykiss) gill (RTgill-W1). The comparison is based on the AgNP behavior in exposure media, toxicity, uptake and interaction with proteins. Results: (1) The composition of exposure media affected AgNP behavior and toxicity to algae and fish cells. (2) The toxicity of AgNP to algae was mediated by dissolved silver while nanoparticle specific effects in addition to dissolved silver contributed to the toxicity of AgNP to fish cells. (3) AgNP did not enter into algal cells; they only adsorbed onto the cell surface. In contrast, AgNP were taken up by fish cells via endocytic pathways. (4) AgNP can bind to both extracellular and intracellular proteins and inhibit enzyme activity. Conclusion: Our results showed that fish cells take up AgNP in contrast to algal cells, where AgNP sorbed onto the cell surface, which indicates that the cell wall of algae is a barrier to particle uptake. This particle behaviour results in different responses to AgNP exposure in algae and fish cells. Yet, proteins from both cell types can be affected by AgNP exposure: for algae, extracellular proteins secreted from cells for, e. g., nutrient acquisition. For fish cells, intracellular and/or membrane-bound proteins, such as the Na+/K+-ATPase, are susceptible to AgNP binding and functional impairment.