Chronic exposure to tire rubber-derived contaminant 6PPD-quinone impairs sperm quality and induces the damage of reproductive capacity in male mice.

It has been reported that N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q), a derivative of the tire antioxidant, N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), exhibits acute toxicity towards organisms. However, the possible reproductive toxicity of 6PPD-Q in mammals has rarely been reported. In this study, the effects of 6PPD-Q on the reproductive toxicity of C57Bl/6 male mice were assessed after exposure to 6PPD-Q for 40 days at 4 mg/kg body weight (bw). Exposure to 6PPD-Q not only led to a decrease in testosterone levels but also adversely affected semen quality and in vitro fertilization (IVF) outcomes, thereby indicating impaired male fertility resulting from 6PPD-Q exposure. Additionally, transcriptomic and metabolomic analyses revealed that 6PPD-Q elicited differential expression of genes and metabolites primarily enriched in spermatogenesis, apoptosis, arginine biosynthesis, and sphingolipid metabolism in the testes of mice. In conclusion, our study reveals the toxicity of 6PPD-Q on the reproductive capacity concerning baseline endocrine disorders, sperm quality, germ cell apoptosis, and the sphingolipid signaling pathway in mice. These findings contribute to an enhanced understanding of the health hazards posed by 6PPD-Q to mammals, thereby facilitating the development of more robust safety regulations governing the utilization and disposal of rubber products.

Gene expression analysis of Chironomus riparius in response to acute exposure to tire rubber microparticles and leachates.

Tire rubber microparticles (TRPs) entering aquatic ecosystems through stormwater runoffs is a significant challenge. TRPs are formed by the abrasion of tires with the road surface and include chemical additives that are an additional cause for concern. Currently, information on the molecular effects of TRPs, or especially its additives, in freshwater organisms is scarce. To address this problem, an array covering different cellular processes has been designed for the freshwater midge Chironomus riparius. Fourth-instar larvae were exposed to two concentrations of TRPs (1 mg L-1, 10 mg L-1) and tire rubber leachates (TRLs) (0,0125 %, 5 %) to evaluate the transcriptional activity by Real-Time PCR. To assess acute toxicity, larvae were exposed for 24 h and genes related to the endocrine system, stress response, DNA repair mechanisms, immune system, oxidative stress, and detoxification mechanisms were evaluated. The activity of the enzymes: glutathione S-transferase (GST) and catalase was also examined. The main pathway affected was the stress response showing overexpression of HSPs (HSC70.3, HSC70.4, HSC70.5, HSP60). Moreover, there was a reduction of the GSTd3 and catalase disrupting the antioxidant system. The upregulation of InR indicates a potential disturbance in the insulin pathway and ABCB6 activation only in TRPs exposure suggests its potential implication in their transport. However, most of these alterations are caused by TRLs, showing higher toxicity than TRPs. The results obtained in this work provide the first approach at the molecular and cellular levels to elucidate the impact of TRLs in freshwater organisms. To perform a realistic evaluation of the TR effects, additional research is required to assess the TR's long-term effects at the molecular level.

Non-targeted analysis based on quantitative prediction and toxicity assessment for emerging contaminants in tire particle leachates.

Non-targeted analysis (NTA) has great potential to screen emerging contaminants in the environment, and some studies have conducted in-depth investigation on environmental samples. Here, we used a NTA workflow to identify emerging contaminants in used tire particle (TP) leachates, followed by quantitative prediction and toxicity assessment based on hazard scores. Tire particles were obtained from four different types of automobiles, representing the most common tires during daily transportation. With the instrumental analysis of TP leachates, a total of 244 positive and 104 negative molecular features were extracted from the mass data. After filtering by a specialized emerging contaminants list and matching by spectral databases, a total of 51 molecular features were tentatively identified as contaminants, including benzothiazole, hexaethylene glycol, 2-hydroxybenzaldehyde, etc. Given that these contaminants have different mass spectral responses in the mass spectrometry, models for predicting the response of contaminants were constructed based on machine learning algorithms, in this case random forest and artificial neural networks. After five-fold cross-validation, the random forest algorithm model had better prediction performance (MAECV = 0.12, Q2 = 0.90), and thus it was chosen to predict the contaminant concentrations. The prediction results showed that the contaminant at the highest concentration was benzothiazole, with 4,875 µg/L in the winter tire sample. In addition, the joint toxicity assessment of four types of tires was conducted in this study. According to different hazard levels, hazard scores increasing by a factor 10 were developed, and hazard scores of all the contaminants identified in each TP leachate were summed to obtain the total hazard score. All four tires were calculated to have relatively high risks, with winter tires having the highest total hazard score of 40,751. This study extended the application of NTA research and led to the direction of subsequent targeting studies on highly concentrated and toxic contaminants.

Uptake and Biotransformation of the Tire Rubber-derived Contaminants 6-PPD and 6-PPD Quinone in the Zebrafish Embryo (Danio rerio).

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6-PPD) is a widely used antioxidant in tire rubber known to enter the aquatic environment via road runoff. The associated transformation product (TP) 6-PPD quinone (6-PPDQ) causes extreme acute toxicity in some fish species (e.g., coho salmon). To interpret the species-specific toxicity, information about biotransformation products of 6-PPDQ would be relevant. This study investigated toxicokinetics of 6-PPD and 6-PPDQ in the zebrafish embryo (ZFE) model. Over 96 h of exposure, 6-PPD and 6-PPDQ accumulated in the ZFE with concentration factors ranging from 140 to 2500 for 6-PPD and 70 to 220 for 6-PPDQ. A total of 22 TPs of 6-PPD and 12 TPs of 6-PPDQ were tentatively identified using liquid chromatography coupled to high-resolution mass spectrometry. After 96 h of exposure to 6-PPD, the TPs of 6-PPD comprised 47% of the total peak area (TPA), with 4-hydroxydiphenylamine being the most prominent in the ZFE. Upon 6-PPDQ exposure, >95% of 6-PPDQ taken up in the ZFE was biotransformed, with 6-PPDQ + O + glucuronide dominating (>80% of the TPA). Among other TPs of 6-PPD, a reactive N-phenyl-p-benzoquinone imine was found. The knowledge of TPs of 6-PPD and 6-PPDQ from this study may support biotransformation studies in other organisms.

The toxicity effects of the individual and combined exposure of methyl tert-butyl ether (MTBE) and tire rubber powder (RP) on Nile tilapia fish (Oreochromis niloticus).

Methyl tert-butyl ether (MTBE) is soluble in water and can contaminate water sources when it spills during transportation or leaks from underground storage tanks. Incomplete combustion releases MTBE as exhaust fumes that can be deposited on urban surfaces. Meanwhile, car tires erosion emits of large amounts of rubber dust (RP), easily transported to water bodies. Therefore, this study has the objective of assessing the toxicity of varying concentrations of MTBE (0, 2.5, 5.0 µL L-1) and RP (0, 5.0, 10.0 mg L-1 RP), both individually and in combination, over a period of 28 days on Nile tilapia (Oreochromis niloticus). MTBE and PR decreased fish growth performance. Blood biochemical analytes indicated that MTBE and RP led to increasing Aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and creatinine phosphokinase (CPK), alkaline phosphatase and gamma-glutamyl transferase (GGT) activities. Alterations related to glucose, triglycerides, cholesterol, and creatinine, plasma contents, were also observed. Increased antioxidant biomarkers, including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), glutathione reductase (GR), and malondialdehyde (MDA), was observed. Exposure fish to MTBE and PR changed metabolic profile of muscle tissue. Moreover, results showed that MTBE, its metabolites, and PR could accumulate in the muscle tissue of fish. Results suggest that MTBE and RP can impact fish health, both individually and when combined. The presence of MTBE enhances the toxicity of RP, indicating a synergistic effect. Nevertheless, further studies are needed to understand the impact of toxic compounds on aquatic environments and organisms' health.

Tire-rubber related pollutant 6-PPD quinone: A review of its transformation, environmental distribution, bioavailability, and toxicity.

The antioxidant 6-PPD has been widely used to prevent cracking and thermal oxidative degradation and to extend the service life of tire rubber. 6-PPD quinone (6-PPDQ) is formed via the reaction of 6-PPD with O3. Due to its acute lethality in coho salmon, 6-PPDQ has become an emerging pollutant of increasing concern. In this review, we provide a critical overview of the generation, environmental distribution, bioavailability, and potential toxicity of 6-PPDQ. The transformation pathways from 6-PPD to 6-PPDQ include the N-1,3-dimethylbutyl-N-phenyl quinone diamine (QDI), intermediate phenol, and semiquinone radical pathways. 6-PPDQ has been frequently detected in water, dust, air particles, soil, and sediments, indicating its large-scale and potentially global pollution trend. 6-PPDQ is bioavailable to both aquatic animals and mammals and acute exposure to 6-PPDQ can be lethal to some organisms. Exposure to 6-PPDQ at environmentally relevant concentrations could induce several types of toxicity, including neurotoxicity, intestinal toxicity, and reproductive toxicity. This review also identifies and discusses knowledge gaps and research needs for the study of 6-PPDQ. This review facilitates a better understanding of the environmental occurrence and exposure risk of 6-PPDQ.

Environmental concentrations of tire rubber-derived 6PPD-quinone alter CNS function in zebrafish larvae.

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPD-quinone) is a degradation product of 6PPD, an antioxidant widely used in rubber tires. 6PPD-quinone enters aquatic ecosystems through urban stormwater runoff and has been identified as the chemical behind the urban runoff mortality syndrome in coho salmon. However, the available data suggest that the acute effects of 6PPD-quinone are restricted to a few salmonid species and that the environmental levels of this chemical should be safe for most fish. In this study, larvae of a "tolerant" fish species, Danio rerio, were exposed to three environmental concentrations of 6PPD-quinone for only 24 h, and the effects on exploratory behavior, escape response, nonassociative learning (habituation), neurotransmitter profile, wake/sleep cycle, circadian rhythm, heart rate and oxygen consumption rate were analyzed. Exposure to the two lowest concentrations of 6PPD-quinone resulted in altered exploratory behavior and habituation, an effect consistent with some of the observed changes in the neurotransmitter profile, including increased levels of acetylcholine, norepinephrine, epinephrine and serotonin. Moreover, exposure to the highest concentration tested altered the wake/sleep cycle and the expression of per1a, per3 and cry3a, circadian clock genes involved in the negative feedback loop. Finally, a positive chronotropic effect of 6PPD-quinone was observed in the hearts of the exposed fish. The results of this study emphasize the need for further studies analyzing the effects of 6PPD-quinone in "tolerant" fish species.

Toxicological effects of tire rubber-derived 6PPD-quinone, a species-specific toxicant, and dithiobisbenzanilide (DTBBA) in the marine rotifer Brachionus koreanus.

The ingredients of tire-rubber products include a complex range of chemicals additives, most of which are leached into surrounding water as unmeasured toxicants with unexplored ecotoxicological impacts. The present study summarizes the reported species-specific acute toxicity of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q), the ozonation product of anti-oxidant 6PPD used in tire rubber. Also, chronic toxicity and oxidative response of 6PPD-Q and another tire-rubber derivative, 2',2'''-dithiobisbenzanilide (DTBBA), in rotifer Brachionus koreanus were investigated. Although 6PPD-Q has been reported to be highly toxic to several species of salmonids, only moderate chronic toxicity was observed in B. koreanus. In contrast, DTBBA significantly retarded the population growth and fecundity. The varying toxicity of 6PPD-Q and DTBBA was linked to the level of reactive oxygen species in which DTBBA exposure caused a significant concentration-dependent increase. Our results imply unanticipated risks to aquatic species posed by chemical additives in tire-rubber which may be considered emerging contaminants of toxicological concern.

Characterization of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD)-induced cardiotoxicity in larval zebrafish (Danio rerio).

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) is a type of p-phenylenediamine (PPD), which is widely used in the manufacture of rubber tires owing to its excellent antiozonant properties. In this study, the developmental cardiotoxicity of 6PPD was evaluated in zebrafish larvae, and the LC50 was approximately 737 µg/L for the larvae at 96 h post fertilization (hpf). In the 6PPD treatment of 100 µg/L, the accumulation concentrations of 6PPD were up to 2658 ng/g in zebrafish larvae, and 6PPD induced significant oxidative stress and cell apoptosis in the early developmental stages of zebrafish. Transcriptome analysis showed that 6PPD exposure could potentially cause cardiotoxicity in larval zebrafish by affecting the transcription of the genes related to the calcium signal pathway and cardiac muscle contraction. The genes related to calcium signaling pathway (slc8a2b, cacna1ab, cacna1da, and pln) were verified by qRT-PCR, which were significantly downregulated in larval zebrafish after exposing to 100 µg/L of 6PPD. Simultaneously, the mRNA levels of the genes related to cardiac functions (myl7, sox9, bmp10, and myh71) also respond accordingly. H&E staining and heart morphology investigation indicated that cardiac malformation occurred in zebrafish larvae exposed to 100 µg/L of 6PPD. Furthermore, the phenotypic observation of transgenic Tg (myl7: EGFP) zebrafish also confirmed that 100 µg/L of 6PPD exposure could change the distance of atria and ventricles of the heart and inhibit some key genes (cacnb3a, ATP2a1l, ryr1b) related to cardiac function in larval zebrafish. These results revealed the toxic effects of 6PPD on the cardiac system of zebrafish larvae.

Analysis, environmental occurrence, fate and potential toxicity of tire wear compounds 6PPD and 6PPD-quinone.

Tire wear compounds N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its derivative 6PPD-quinone have been considered as emerging pollutants and attracted much attention recently. As an antioxidant and antiozonant widely used, 6PPD would be released during the production or use of rubber-related products. Because of the mass production and wide use of rubber-related products, 6PPD and 6PPD-quinone have been identified to be ubiquitous in the environment. In this study, we firstly reviewed the current available literature on the analytical procedures, concentrations and distribution of 6PPD and 6PPD-quinone, and then investigated the potential toxic effects of these two compounds on aquatic organisms. Current studies have been mainly focused on the occurrence of 6PPD and 6PPD-quinone in dust and water, while available information on atmosphere, soil, sediments and organisms is limited. The fate and distribution of 6PPD and 6PPD-quinone would be influenced by environmental factors such as temperature, illumination, and storm events, etc. Although 6PPD and 6PPD-quinone have potential adverse effects on aquatic organisms, and 6PPD-quinone has species-specific toxicity, toxicological mechanisms of these compounds are still unclear. Based on the review and analysis of current studies, some suggestions for future research of 6PPD and 6PPD-quinone are given.

Oral exposure to tire rubber-derived contaminant 6PPD and 6PPD-quinone induce hepatotoxicity in mice.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) is a widely used additive for protecting various rubber products, and its product of oxidation N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPDQ) has attracted extensive attention in aquatic toxicity. However, the toxicity of 6PPD and 6PPDQ in mammals has not been reported yet. In this study, the effects of 6PPD and 6PPDQ on the liver of C57BL/6 mice were assessed by orally administering different doses of 6PPD and 6PPDQ (10, 30, and 100 mg/kg) in mice for 6 weeks. 6PPD and 6PPDQ were found to bioaccumulate in the liver in a dose-dependent manner. Moreover, a high dose of 6PPD and 6PPDQ exposure increased not only the liver weights but also liver triglyceride levels, indicating that 6PPD and 6PPDQ exposure induced hepatotoxicity in mice. Furthermore, transcriptomic analysis revealed that 6PPD and 6PPDQ induced differential expression of genes mainly enriched in glycolipid metabolism, immune-related, and glutathione metabolism pathways. Therefore, 6PPD and 6PPDQ altered hepatic metabolism in mice. Furthermore, 6PPDQ could induce an immune response by upregulating the transcription of immune-related genes and promoting macrophage infiltration in the liver. In conclusion, our study revealed the toxic effects of 6PPD and 6PPDQ exposure on multi-endpoints in the liver of mice and improve our understanding of the health risks of 6PPD and 6PPDQ to mammals. The findings of our study may help formulate better safety regulations for the use and disposal of rubber products.

Long-term exposure to tire-derived 6-PPD quinone causes intestinal toxicity by affecting functional state of intestinal barrier in Caenorhabditis elegans.

2-((4-Methylpentan-2-yl)amino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione (6-PPDQ) is the ozonation product of 6-PPD, a commonly used tire preservative. Although the 6-PPDQ has been frequently detected in different environmental ecosystems, its long-term effects on organisms remain still largely unknown. We here used Caenorhabditis elegans as an experimental animal to investigate the toxic effect of prolonged exposure to 6-PPDQ (0.1-100 µg/L). After the exposure, we found that 100 µg/L 6-PPDQ caused the lethality. We further selected concentrations of 0.1-10 µg/L to examine the possible intestinal toxicity induced by 6-PPDQ. Although 0.1-10 µg/L 6-PPDQ could not influence intestinal morphology, the intestinal permeability was significantly enhanced by 1-10 µg/L 6-PPDQ as indicated by erioglaucine disodium staining. In addition, the expression of intestinal fatty acid transporter ACS-22 governing functional state of intestinal barrier was decreased by exposure to 1-10 µg/L 6-PPDQ. Meanwhile, intestinal reactive oxygen species (ROS) production was induced by 0.1-10 µg/L 6-PPDQ and lipofuscin accumulation reflected by intestinal autofluorescence was activated by 1-10 µg/L 6-PPDQ. Accompanied with activation of intestinal oxidative stress, expressions of some anti-oxidation related genes (ctl-2, sod-2, sod-3, and sod-4) were significantly increased by 0.1-10 µg/L 6-PPDQ. Moreover, intestinal RNAi of acs-22 strengthened the susceptibility of nematodes to intestinal toxicity of 6-PPDQ. Therefore, considering that the environmentally relevant concentrations of 6-PPDQ were ≤10 µg/L, our data suggested that long-term exposure to 6-PPDQ at environmentally relevant concentrations potentially results in intestinal toxicity by disrupting functional state of intestinal barrier in organisms.

Rubber Antioxidants and Their Transformation Products: Environmental Occurrence and Potential Impact.

Antioxidants are prevalently used during rubber production to improve rubber performance, delay aging, and extend service life. However, recent studies have revealed that their transformation products (TPs) could adversely affect environmental organisms and even lead to environmental events, which led to great public concern about environmental occurrence and potential impacts of rubber antioxidants and their TPs. In this review, we first summarize the category and application of rubber antioxidants in the world, and then demonstrate the formation mechanism of their TPs in the environment, emphasizing their influence on the ozone oxidative degradation. The potential toxic effects of antioxidants and their TPs are further reviewed to improve understanding of their biological health impact and environmental risks. Finally, the environmental occurrences of antioxidants and their TPs are summarized and their environmental impacts are demonstrated based on the recent studies. Due to the currently limited understanding on the toxic and biological effects of these compounds, further studies are required in order to better assess various TPs of these antioxidants and their environmental impact. To our knowledge, this is the first review on antioxidants and their TPs in the environment, which may elevate the environmental risk awareness of rubber products and their TPs in the near future.

Protective role of Nrf2 in zinc oxide nanoparticles-induced lung inflammation in female mice and sexual dimorphism in susceptibility.

BACKGROUND: Zinc oxide nanoparticles (ZnO-NPs) are currently employed in various products such as rubber, paint, and cosmetics. Our group reported recently that Nrf2 protein provides protection against pulmonary inflammation induced by ZnO-NPs in male mice. The current study investigated the effect of Nrf2 deletion on the lung inflammatory response in female mice exposed to ZnO-NPs. METHODS: An equal number of female Nrf2-/- mice and female Nrf2+/+ mice (24 each) were allocated into three equal groups, and each was exposed to ZnO-NPs at either 0, 10 or 30 µg ZnO-NPs/mouse through pharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) and lungs were examined 14 days later to determine the number of inflammatory cells, the protein level, and for scoring inflammation histopathologically. The mRNA levels of Nrf2-dependent antioxidant enzymes and proinflammatory cytokine in lung tissue were also measured. RESULTS: Exposure to ZnO-NPs increased all types of BALF cells and lung inflammation scores in both of female Nrf2-null (Nrf2-/-) and wild-type (Nrf2+/+) mice, and Nrf2 deletion enhanced ZnO-NPs-induced increase in the number of eosinophils in BALF. Exposure to ZnO-NPs dose-dependently increased the level of oxidized glutathione (GSSG), and mRNA levels of proinflammatory cytokines/chemokines; KC, MIP-2, IL-6, IL-1ß and MCP-1 only in wild-type mice. Nrf2 deletion decreased total glutathione levels and basal mRNA levels of SOD1 and NQO1, and increased the basal mRNA level of above proinflammatory cytokines/chemokines. Nrf2 deletion enhanced ZnO-NPs-induced downregulation of GcLc, GR and TGF-ß and upregulation of HO-1 and TNF-α. Taken together with our previous results in male mice, our results showed a lower susceptibility of females to lung tissue inflammation, relative to males, irrespective of Nrf2 deletion, and that enhancement of ZnO-NPs-induced upregulation of HO-1 and TNF-α and downregulation of GcLc, GR and TGF-ß by deletion of Nrf2 is specific to female mice. CONCLUSION: We conclude that Nrf2 provides protection in female mice against increase in BALF eosinophils, probably through down-regulation of proinflammatory cytokines/chemokines and upregulation of oxidative stress-related genes. The study also suggests lower susceptibility to lung tissue inflammation in female mice relative to their male counterparts and the synergistic effects of Nrf2 and exposure to ZnO-NPs on mRNA expression of GcLc, GR, HO-1, TGF-ß or TNF-α in female mice.

Presence of metals and metalloids in crumb rubber used as infill of worldwide synthetic turf pitches: Exposure and risk assessment.

Crumb rubber derived from end-of-life tires (ELTs) is frequently used as infill of synthetic turf pitches, promoting circular economy. Although important to reduce the accumulation of waste, the use of recycled ELTs can be a problem to human health and the environment, both by direct contact during pitch use and by the release of these elements to the surroundings, mostly via volatilization and leaching. The present study aimed to evaluate the distribution of metals in ELT-derived crumb rubber collected in artificial turf worldwide and assess possible trends by country, pitch age and type (indoor vs. outdoor). The concentration ranges observed are very wide, especially in outdoor fields and for the most abundant metals, namely Zn (2989-5246 mg/kg), Fe (196-5194 mg/kg), Mg (188-1795 mg/kg) and Al (159-1882 mg/kg). For Zn, the levels were mostly above the safe limits set in European directives for relatable matrices (soils and toy materials), and the same happened for Pb, a much more toxic metal at lower concentrations. A multi-pathway human exposure study was also performed, and the risk assessment shows non-carcinogenic and carcinogenic risks from accidental crumb rubber ingestion (with Cr and Pb as major contributors) above the acceptable values for all the receptors except adult bystanders, with a higher significance to younger individuals. These results bring a different perspective regarding most of the studies reporting low risks related with exposure to metals in crumb rubber.

Altered gene expression in Chironomus riparius (insecta) in response to tire rubber and polystyrene microplastics.

The extent until which plastics are present in our surrounding environment completely exceeds our expectations. Plastic materials in the form of microplastics have been found in terrestrial, freshwater and marine environments and are transported through the atmosphere even to remote locations. However, we are still far from understanding the effects that they may have caused and are causing to biota. In the present study, we investigated the alterations in the expression of twelve genes in the aquatic insect Chironomus riparius after 36 h exposures to polystyrene and tire rubber microplastics at nominal concentrations of 1 and 10 mg L-1. The results indicated that several genes encoding for heat shock proteins (hsp90, Glycoprotein 93 (Gp93), hsc70, hsp60, hsp40, and the small HSP hsp17) were overexpressed respect to the control. In addition, the genes coding for manganese superoxide dismutase (SOD Mn, related to alleviation of oxidative stress) and for the FK506-binding protein of 39 kDa. (FKBP39, related to development and pupation) showed altered expression. Most of the alterations on gene expression level occurred at a concentration of 10 mg L-1 of tire rubber microplastics, although specific modifications arose at other concentrations of both rubber and polystyrene. On the contrary, one hsp gene (hsp10) and genes related to biotransformation and detoxification (Cyp9f2, Cyp12a2, and ABCB6) did not alter their expression in any of the treatments. Overall, the results of the gene expression indicated that microplastics (especially tire rubber) or their additives caused cellular stress that led to some alterations in the normal gene expression but did not cause any mortality after 36 h. These results highlight the need for more studies that describe the alterations caused by microplastics at the molecular level. Additionally, it opens questions about the effects caused to aquatic fauna in environmental realistic situations, especially in hot spots of microplastic contamination (e.g., tire rubber released in storm water runoff discharge points).

The sub-lethal impact of plastic and tire rubber leachates on the Mediterranean mussel Mytilus galloprovincialis.

Ocean contamination by synthetic polymers can represent a risk for the fitness of marine species due to the leaching of chemical additives. This study evaluated the sub-lethal effects of plastic and rubber leachates on the mussel Mytilus galloprovincialis through a battery of biomarkers encompassing lysosomal endpoints, oxidative stress/detoxification parameters, and specific responses to metals/neurotoxicants. Mussels were exposed for 7 days to leachates from car tire rubber (CTR), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS) and polyvinyl chloride (PVC), containing organic additives and metals in the ng-µg/L range. The leachate exposure affected general stress parameters, including the neutral lipid content (all leachates), the lysosomal membrane stability (PS, PP, PVC and CTR leachates) and lysosomal volume (PP, PVC and TR leachates). An increased content of the lipid peroxidation products malondialdehyde and lipofuscin was observed in mussels exposed to PET, PS and PP leachates, and PP, PVC and CTR leachates, respectively. PET and PP leachates increased the activity of the phase-II metabolism enzyme glutathione S-transferase, while a decreased acetylcholinesterase activity was induced by PVC leachates. Data were integrated in the mussel expert system (MES), which categorizes the organisms' health status based on biomarker responses. The MES assigned healthy status to mussels exposed to PET leachates, low stress to PS leachates, and moderate stress to PP, CTR and PVC leachates. This study shows that additives leached from selected plastic/rubber polymers cause sub-lethal effects in mussels and that the magnitude of these effects may be higher for CTR, PVC and PP due to a higher content and release of metals and organic compounds.

Release of Zinc and Polycyclic Aromatic Hydrocarbons From Tire Crumb Rubber and Toxicity of Leachate to Daphnia magna: Effects of Tire Source and Photoaging.

Tire crumb rubber (TCR) has been widely used in artificial turf fields, however, the potential environmental risk of TCR and the effect of sunlight exposure are scarcely studied. Here, we evaluated leachability of Zn and polycyclic aromatic hydrocarbons (PAHs) in four types of TCRs and acute toxicity of leachates to Daphnia magna. The results showed that all types of TCRs tested released Zn (0.20-1.3 µg/g) and PAHs (9.4-17 µg/g) but only two were lethal to D. magna (mortality 73%). Notably, ultraviolet (UV) irradiation induced TCR to generate acidic leachate (pH ~ 4.8), which contained 24- and 1.2-fold higher concentrations of Zn and PAHs and therefore was more toxic to D. magna than that in the absence of UV treatment. These findings demonstrate source-dependent toxicity of TCR and highlight the need to consider the effect of photoaging when evaluating the environmental risks of TCR.

A review of potentially harmful chemicals in crumb rubber used in synthetic football pitches.

Recycling end-of-life tires (ELTs) reduces waste and provides a low-cost source of energy and materials such as crumb rubber, used as infill in artificial turf football pitches. However, some concerns were raised and remain about its safety. The potentially toxic human exposure to chemicals such as polycyclic aromatic hydrocarbons (PAHs), metals and others (volatile organic compounds (VOCs), plasticizers, antioxidants and additives) existing in ELTs (and in the resulting crumb rubber) is being studied, with no definitive conclusions. The literature existing so far suggests the possibility of their release from synthetic turf infill into the environment as water leachates and to the air surrounding the pitches, but there is the need of further research, also to assess the contribution of other materials present in synthetic turf. The database available comprised crumb rubber infill studies from pitches in 6 countries (USA, Norway, Netherlands, Portugal, Italy, Spain) and revealed a myriad of hazardous chemicals, with benzo[a]pyrene (n.d.-4.31 ± 3.95 mg/kg) and zinc (n.d.-14150 ± 1344 mg/kg) often exceeding the established limits. A dependence on indoor/outdoor conditions and the age of the source material was evaluated, often showing significative differences. From this standpoint, this review is intended to add knowledge about the presence of contaminants in this recycled material, aiming to ensure the safety of end-users and the environment.