**1. Introduction**

The advent of plastics is unclear but could be matched between the discovery of natural rubbers by Charles Marie de La Condamine (1736) and the subsequent introduction of vulcanization by Thomas Hancock and Charles Goodyear (1843–1844), which confers to rubbers resistance for a wide plethora of uses [1]. In this context, there is not a welldefined position by the scientific community regarding the inclusion of some types of elastomers in the classification of plastics. For example, rubbers are not plastics according to the definition of the International Organization for Standardization (ISO) [2]. However, some of these materials, including tire rubbers, contain from 40 to 60% synthetic polymers (i.e., styrene-butadiene and polybutadiene rubbers) and are characterized by physical proprieties such as solid state and insolubility in water; therefore, some agencies and environmental researchers classify them among plastics [3–6]. Tires, in addition to synthetic polymers, contain silica, oil, carbon black, sulfur compounds and zinc oxide [7]. Zinc (Zn) represents 1–2% of the total weight of tires, and the leaching of this element in

**Citation:** Magni, S.; Tediosi, E.; Maggioni, D.; Sbarberi, R.; Noé, F.; Rossetti, F.; Fornai, D.; Persici, V.; Neri, M.C. Ecological Impact of End-of-Life-Tire (ELT)-Derived Rubbers: Acute and Chronic Effects at Organism and Population Levels. *Toxics* **2022**, *10*, 201. https://doi.org/ 10.3390/toxics10050201

Academic Editor: Susanne M. Brander

Received: 21 March 2022 Accepted: 17 April 2022 Published: 19 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

water represents an important environmental concern [8]. In turn, tires are considered by some authors a considerable source of plastic debris due to, e.g., their mechanical abrasion during the activity of transport [9–13]. This aspect originates tire-wear particles (TWPs) and tire-road-wear particles (TRWPs) when TWPs are aggregated with other road debris/pollutants [6,14]. The contribution of tires to the total amount of released plastic debris in the environment ranges from 30 to 50% in Germany, Denmark and Norway [3,9,10,15–18]. In this context, a study by Knight et al. [19] reported a concentration of these contaminants ranging from 0.6 ± 0.33 to 65 ± 7.36 particles/5 mL of analyzed material from a natural area close to a road. However, relative to studies related to the monitoring of conventional (micro)plastics, few have reported the presence of tire particles in the environment [17,20]. Indeed, the main detected plastics, e.g., in aqueous matrices, are constituted by polymers, such as polystyrene (PS), polyethylene (PE), polypropylene (PP), polyester (PEST), polyacrylate (PAK) and polyamide [21–24]. In this context, it is important to consider that tire particles are difficult to detect with the identification methodologies of conventional (micro)plastics, such as the Fourier transform infrared spectroscopy (FT-IR), due to the lack of appropriate reference standards, as well as the presence of carbon black in tires, which absorbs in the infrared region [25]. Therefore, tire particle monitoring is hampered by analytical methods, currently represented by chromatography–mass spectrometry (GC/MS) and pyrolysis GC/MS [26]. The small amounts of TWPs and TRWPs identified in monitoring studies do not correlate with the massive use of tires around the world; in Europe alone every year, about 3.4 million tons of these products reach the end of life [27]. So-called end-of-life tires (ELTs) are normally recycled into ELT-derived rubber granules (ELT-dg) or ELT-derived rubber powder (ELT-dp) and transformed into other products, such as performance infills for artificial turfs and playground safety floorings [28–30]. However, regarding the potential toxicity of these substances, few studies (some of which are very old) have been conducted, and many research gaps currently affect the available knowledge, such as the lack of data on long-term toxicity; the differences in the results expression; and terminological inconsistency about TWP, TRWP and ELT [10,14]. For instance, a study on the crustacean *Hyallela azteca* revealed acute and chronic effects of TWPs after an exposure for 21 days to 500–2000 particles/mL as significant impacts on mortality, reproduction and growth [31], identifying 50% lethal concentration (LC50) at 3426 ± 172 particles/mL. Another study revealed the absence of effects of ELTs at 10% sediment dry weight after 28 days of exposure in the freshwater crustaceans *Gammarus pulex* and *Asellus aquaticus* and in the Oligochaeta worms *Tubifex* spp. [32]. On the other hand, a larger number of studies have reported the toxicity of tire particle leachates [10,14]. A study on the rainbow trout *Oncorhynchus mykiss*, the crustacean *Daphnia magna* and the fathead minnow *Pimephales promelas* exposed to a leachate of worn, pristine and breakwater tires revealed no effects on *D. magna* and *P. promelas* and a 96 h LC50 for *O. mykiss* at 11.8–19.3% *v/v* for old tire leachate, which is more toxic than leachate from new tires [33]. On *O. mykiss*, an increase in ethoxyresorufin-O-deethylase (EROD) activity after exposure to water with whole tires was also observed [34]. Other studies highlighted a wide range of 50% effect concentration (EC50) on *D. magna* after 48 h exposure to TWP leachates, ranging from 0.13 to 10 g/L [35,36], and 0.55 to 5 g/L for *Ceriodaphnia dubia* [37]. On the contrary, no significant effects were obtained in *H. azteca*, *P. promelas*, *C. dubia* and in the Diptera *Chironomus dilutus* exposed for 42 days to TRWP leachate (10 g/kg sediment) [38]. The effects of TRWPs on *Pseudokirchneriella subcapita*, *D. magna* and *P. promelas* were also evaluated using sediment elutriate and under standard test temperatures, reporting an EC50 greater than 10,000 mg/L [39]. Lastly, in another study, the toxicity of micro- (1–20 μm) and nano- (<1 <sup>μ</sup>m) tire particles (from 0 to 3.0 × 109 particles/mL), as well as their leachate (from 0 to 100%), was assessed on *Danio rerio* and *D. magna*. This study, which combined the assessment of both particles and leachate toxicity, reported mortality and malformations on exposed specimens, especially those treated with nanoparticles [40].

Based on these complicated and heterogeneous pieces of evidence and considering the sparse information about the potential effects related to ELTs in particular, we evaluated the acute and chronic effects induced by aqueous suspensions of ELT-dg (size ranging from 0.8 to 2.5 mm) and ELT-dp (size < 0.8 mm) on freshwater organisms. The material used in the ecotoxicity tests were derived from the mechanical shredding of whole tires containing all compounds used in tire production (inner liners, treads, etc.), as well as the substances normally detected in TRWPs, such as bituminous residues and agglomerates of exhaust fumes. We tested different ELT-dg and ELT-dp concentrations, from 0.12 to 100.0 mg/L, using a battery of classical ecotoxicological tests on freshwater species, such as the unicellular green alga *P. subcapitata*, the crustacean *D. magna* and the teleost *D. rerio* (zebrafish), which constitute a simple aquatic trophic chain.
