Effects of environmental concentrations of 6PPD and its quinone metabolite on the growth and reproduction of freshwater cladoceran.

The widespread occurrence and accumulation of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its quinone metabolite, 6PPD quinone (6PPD-Q), have been globally recognized as a critical environmental issue. However, knowledge on the adverse effects of 6PPD and 6PPD-Q on freshwater invertebrates is limited. This study investigated the effects of 6PPD and its oxidative byproduct, 6PPD-Q, on the growth and reproduction of Daphnia pulex. Through 21-day exposure experiments, we measured the uptake of 0.1, 1, and 10 µg/L 6PPD and 6PPD-Q by D. pulex and assessed the effects on growth and fecundity of D. pulex. While 6PPD and 6PPD-Q did not affect the mortality rate of D. pulex, 6PPD-Q exposure inhibited the growth of D. pulex, indicating potential ecological risks. In particular, the reproductive capacity of D. pulex remained unaffected across the tested concentrations of 6PPD and 6PPD-Q, suggesting specific toxicological pathways that warrant further investigation. This study underscored the importance of evaluating the sublethal effects of emerging contaminants such as 6PPD and 6PPD-Q on aquatic invertebrates, and highlighted the need for comprehensive risk assessments to better understand their environmental impacts.

P-phenylenediamine antioxidants and their quinone derivatives: A review of their environmental occurrence, accessibility, potential toxicity, and human exposure.

Substituted p-phenylenediamines (PPDs), a class of antioxidants, have been widely used to extend the lifespan of rubber products, such as tires and pipes. During use, PPDs will generate their quinone derivatives (PPD-Qs). In recent years, PPDs and PPD-Qs have been detected in the global environment. Among them, N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPD-Q), the oxidation product of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), has been identified as highly toxic to coho salmon, with the lethal concentration of 50 % (LC50) being 95 ng/L, highlighting it as an emerging pollutant of great concern. This review summarizes the physicochemical properties, global environmental distribution, bioaccessibility, potential toxicity, human exposure risk, and green measures of PPDs and PPD-Qs. These chemicals exhibit lipophilicity, bioaccumulation potential, and poor aqueous stability. They have been found in water, air, dust, soil, and sediment worldwide, indicating their significance as emerging pollutants. Notably, current studies have identified electronic waste (e-waste), such as discarded wires and cables, as a non-negligible source of PPDs and PPD-Qs, in addition to tire wear. PPDs and PPD-Qs exhibit strong bioaccumulation in aquatic organisms and mammals, with a tendency for biomagnification within the food web, posing health threats to humans. Available toxicity data indicate that PPDs and PPD-Qs have negative effects on aquatic organisms, mammals, and invertebrates. Acute exposure leads to death and acute damage, and long-term exposure can cause a series of adverse effects, including growth and development toxicity, reproductive toxicity, neurotoxicity, intestinal toxicity, and multi-organ damage. This paper discusses current research gaps and offers recommendations to understand better the occurrence, behavior, toxicity, and environmental exposure risks of PPDs and PPD-Qs.

Ferrostatin-1 mitigates cellular damage in a ferroptosis-like environment in Caenorhabditis elegans.

Although iron (Fe) is the most biologically abundant transition metal, it is highly toxic when it accumulates as Fe2+, forming a labile Fe pool and favoring the Fenton reaction. This oxidative scenario leads to a type of caspase-independent programmed cell death, referred to as ferroptosis, where following processes take place: (i) Fe2+ overload, (ii) glutathione peroxidase 4 inactivation, (iii) lipid peroxidation, and (iv) glutathione depletion. The present study sought to evaluate the consequences of Fe2+ administration on ferroptosis induction in Caenorhabditis elegans. We demonstrated higher mortality, increased lipid peroxidation, reduced glutathione peroxidase activity, and morphological damage in dopaminergic neurons upon Fe2+ overload. Pharmacological intervention at the level of lipid peroxidation with ferrostatin-1 (250 µM) mitigated the damage and returned the biochemical parameters to basal levels, revealing the potential of this therapeutical approach. Finally, to assess the relationship between ferroptosis and dopamine in a Parkinsonian background, we evaluated the UA44 worm strain which overexpresses the alpha-synuclein protein in cherry-labeled dopaminergic neurons. We demonstrated that Fe2+ administration reduced lethality associated with similar alterations in biochemical and dopaminergic morphological parameters in wild-type animals. These experiments provide mechanistic-based evidence on the efficacy of a pharmacological approach to mitigate the physiological, biochemical, and morphological consequences of Fe2+ overload. At the same time, they encourage further research on the impact of the combined effects resulting from the genetic background and dopamine signaling in a Parkinsonian phenotype.

Manganese overexposure results in ferroptosis through the HIF-1α/p53/SLC7A11 pathway in ICR mouse brain and PC12 cells.

Manganese (Mn) overexposure has been associated with the development of neurological damage reminiscent of Parkinson's disease, while the underlying mechanisms have yet to be fully characterized. This study aimed to investigate the mechanisms leading to injury in dopaminergic neurons induced by Mn and identify novel treatment approaches. In the in vivo and in vitro models, ICR mice and dopaminergic neuron-like PC12 cells were exposed to Mn, respectively. We treated them with anti-ferroptotic agents ferrostatin-1 (Fer-1), deferoxamine (DFO), HIF-1α activator dimethyloxalylglycine (DMOG) and inhibitor LW6. We also used p53-siRNA to verify the mechanism underlying Mn-induced neurotoxicity. Fe and Mn concentrations increased in ICR mice brains overexposed to Mn. Additionally, Mn-exposed mice exhibited movement impairment and encephalic pathological changes, with decreased HIF-1α, SLC7A11, and GPX4 proteins and increased p53 protein levels. Fer-1 exhibited protective effects against Mn-induced both behavioral and biochemical changes. Consistently, in vitro, Mn exposure caused ferroptosis-related changes and decreased HIF-1α levels, all ameliorated by Fer-1. Upregulation of HIF-1α by DMOG alleviated the Mn-associated ferroptosis, while LW6 exacerbated Mn-induced neurotoxicity through downregulating HIF-1α. p53 knock-down also rescued Mn-induced ferroptosis without altering HIF-1α protein expression. Mn overexposure resulted in ferroptosis in dopaminergic neurons, mediated through the HIF-1α/p53/SLC7A11 pathway.

The lethal and sublethal impacts of two tire rubber-derived chemicals on brook trout (Salvelinus fontinalis) fry and fingerlings.

Recent toxicity studies of stormwater runoff implicated N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-quinone) as the contaminant responsible for the mass mortality of coho salmon (Oncorhynchus kisutch). In the wake of this discovery, 6PPD-quinone has been measured in waterways around urban centers, along with other tire wear leachates like hexamethoxymethylmelamine (HMMM). The limited data available for 6PPD-quinone have shown toxicity can vary depending on the species. In this study we compared the acute toxicity of 6PPD-quinone and HMMM to Brook trout (Salvelinus fontinalis) fry and fingerlings. Our results show that fry are ∼3 times more sensitive to 6PPD-quinone than fingerlings. Exposure to HMMM ≤6.6 mg/L had no impact on fry survival. These results highlight the importance of conducting toxicity tests on multiple life stages of fish species, and that relying on fingerling life stages for species-based risk assessment may underestimate the impacts of exposure. 6PPD-quinone also had many sublethal effects on Brook trout fingerlings, such as increased interlamellar cell mass (ILCM) size, hematocrit, blood glucose, total CO2, and decreased blood sodium and chloride concentrations. Linear relationships between ILCM size and select blood parameters support the conclusion that 6PPD-quinone toxicity is an outcome of osmorespiratory challenges imposed by gill impairment.

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.

Environmental profiles, hazard identification, and toxicological hallmarks of emerging tire rubber-related contaminants 6PPD and 6PPD-quinone.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) is commonly used in rubber compounds as antioxidants to protect against degradation from heat, oxygen, and ozone exposure. This practice extends the lifespan of rubber products, including tires, by preventing cracking, aging, and deterioration. However, the environmental consequences of waste generated during rubber product use, particularly the formation of 6PPD-quinone (6PPD-Q) through the reaction of 6PPD with ozone, have raised significant concerns due to their detrimental effects on ecosystems. Extensive research has revealed the widespread occurrence of 6PPD and its derivate 6PPD-Q in various environmental compartments, including air, water, and soil. The emerging substance of 6PPD-Q has been shown to pose acute mortality and long-term hazards to aquatic and terrestrial organisms at concentrations below environmentally relevant levels. Studies have demonstrated toxic effects of 6PPD-Q on a range of organisms, including zebrafish, nematodes, and mammals. These effects include neurobehavioral changes, reproductive dysfunction, and digestive damage through various exposure pathways. Mechanistic insights suggest that mitochondrial stress, DNA adduct formation, and disruption of lipid metabolism contribute to the toxicity induced by 6PPD-Q. Recent findings of 6PPD-Q in human samples, such as blood, urine, and cerebrospinal fluid, underscore the importance of further research on the public health and toxicological implications of these compounds. The distribution, fate, biological effects, and underlying mechanisms of 6PPD-Q in the environment highlight the urgent need for additional research to understand and address the environmental and health impacts of these compounds.

Exposure to 6-PPD quinone causes ferroptosis activation associated with induction of reproductive toxicity in Caenorhabditis elegans.

Exposure to N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ) caused toxicity on Caenorhabditis elegans, including reproductive toxicity. However, the underlying mechanisms for this induced reproductive toxicity by 6-PPDQ remain largely unclear. We examined possible association of ferroptosis activation with reproductive toxicity of 6-PPDQ. In 1-100 µg/L 6-PPDQ exposed nematodes, Fe2+ content was increased, which was accompanied with enhanced lipid peroxidation, increased malonydialdehyde (MDA) content, and decreased L-glutathione (GSH) content. Exposure to 1-100 µg/L 6-PPDQ decreased expressions of ftn-1 encoding ferritin, ads-1 encoding AGPS, and gpx-6 encoding GPX4 and increased expression of bli-3 encoding dual oxidase. After 6-PPDQ exposure, RNAi of ftn-1 decreased ads-1 and gpx-6 expressions and increased bli-3 expression. RNAi of ftn-1, ads-1, and gpx-6 strengthened alterations in ferroptosis related indicators, and RNAi of bli-3 suppressed changes of ferroptosis related indicators in 6-PPDQ exposed nematodes. Meanwhile, RNAi of ftn-1, ads-1, and gpx-6 induced susceptibility, and RNAi of bli-3 caused resistance to 6-PPDQ reproductive toxicity. Moreover, expressions of DNA damage checkpoint genes (clk-2, mrt-2, and hus-1) could be increased by RNAi of ftn-1, ads-1, and gpx-6 in 6-PPDQ exposed nematodes. Therefore, our results demonstrated activation of ferroptosis in nematodes exposed to 6-PPDQ at environmentally relevant concentrations, and this ferroptosis activation was related to reproductive toxicity of 6-PPDQ.

6PPDQ induces cardiomyocyte senescence via AhR/ROS-mediated autophagic flux blockage.

Recently, attention has been drawn to the adverse outcomes of N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPDQ) on human health, but its cardiac toxicity has been relatively understudied. This work aims to investigate the effects of 6PPDQ on differentiated H9c2 cardiomyocytes. Our findings demonstrated that exposure to 6PPDQ altered cellular morphology and disrupted the expression of cardiac-specific markers. Significantly, 6PPDQ exposure led to cardiomyocyte senescence, characterized by elevated ß-Galactosidase activity, upregulation of cell cycle inhibitor, induction of DNA double-strand breaks, and remodeling of Lamin B1. Furthermore, 6PPDQ hindered autophagy flux by promoting the formation of autophagosomes while inhibiting the degradation of autolysosomes. Remarkably, restoration of autophagic flux using rapamycin counteracted 6PPDQ-induced cardiomyocyte senescence. Additionally, our study revealed that 6PPDQ significantly increased the ROS production. However, ROS scavenger effectively reduced the blockage of autophagic flux and cardiomyocyte senescence caused by 6PPDQ. Furthermore, we discovered that 6PPDQ activated the Aryl hydrocarbon receptor (AhR) signaling pathway. AhR antagonist was found to reverse the blockage of autophagy and alleviate cardiac senescence, while also reducing ROS levels in 6PPDQ-treated group. In conclusion, our research unveils that exposure to 6PPDQ induces ROS overproduction through AhR activation, leading to disruption of autophagy flux and ultimately contributing to cardiomyocyte senescence.

Acute Toxicity Testing of Pink Salmon (Oncorhynchus gorbuscha) with the Tire Rubber-Derived Chemical 6PPD-Quinone.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-quinone) is a widespread contaminant of emerging concern resulting from oxidation of 6PPD, which is an antidegradant substance added to tires. The recent identification of 6PPD-quinone as the cause of acute mortality in coho salmon has quickly motivated studies on 6PPD-quinone toxicity to other species. Subsequent findings have shown that 6PPD-quinone toxicity is highly species specific. Closely related species can differ widely in response to 6PPD-quinone from extremely sensitive to tolerant. Hence toxicity testing is currently the only way to establish whether a species exhibits 6PPD-quinone toxicity. We investigated the acute toxicity of 6PPD-quinone in pink salmon alevins (sac fry). This species has is the only Pacific salmon that so far has not been tested for 6PPD-quinone sensitivity. Fish were exposed in static water in eight treatments with initial concentrations ranging from 0.1 to 12.8 µg/L. Fish were observed for 48 h, and changes in concentrations of 6PPD-quinone were monitored throughout the experiment. No mortalities or substantial changes in behavior were recorded. Environ Toxicol Chem 2024;43:1332-1338. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Comparison of intestinal toxicity in enhancing intestinal permeability and in causing ROS production of six PPD quinones in Caenorhabditis elegans.

As the derivatives of p-phenylenediamines (PPDs), PPD quinones (PPDQs) have received increasing attention due to their possible exposure risk. We compared the intestinal toxicity of six PPDQs (6-PPDQ, 77PDQ, CPPDQ, DPPDQ, DTPDQ and IPPDQ) in Caenorhabditis elegans. In the range of 0.01-10 µg/L, only 77PDQ (10 µg/L) moderately induced the lethality. All the examined PPDQs at 0.01-10 µg/L did not affect intestinal morphology. Different from this, exposure to 6-PPDQ (1-10 µg/L), 77PDQ (0.1-10 µg/L), CPPDQ (1-10 µg/L), DPPDQ (1-10 µg/L), DTPDQ (1-10 µg/L), and IPPDQ (10 µg/L) enhanced intestinal permeability to different degrees. Meanwhile, exposure to 6-PPDQ (0.1-10 µg/L), 77PDQ (0.01-10 µg/L), CPPDQ (0.1-10 µg/L), DPPDQ (0.1-10 µg/L), DTPDQ (1-10 µg/L), and IPPDQ (1-10 µg/L) resulted in intestinal reactive oxygen species (ROS) production and activation of both SOD-3::GFP and GST-4::GFP. In 6-PPDQ, 77PDQ, CPPDQ, DPPDQ, DTPDQ, and/or IPPDQ exposed nematodes, the ROS production was strengthened by RNAi of genes (acs-22, erm-1, hmp-2, and pkc-3) governing functional state of intestinal barrier. Additionally, expressions of acs-22, erm-1, hmp-2, and pkc-3 were negatively correlated with intestinal ROS production in nematodes exposed to 6-PPDQ, 77PDQ, CPPDQ, DPPDQ, DTPDQ, and/or IPPDQ. Therefore, exposure to different PPDQs differentially induced the intestinal toxicity on nematodes. Our data highlighted potential exposure risk of PPDQs at low concentrations to organisms by inducing intestinal toxicity.

Toxicity of substituted p-phenylenediamine antioxidants and their derived novel quinones on aquatic bacterium: Acute effects and mechanistic insights.

Substituted para-phenylenediamines (PPDs) are synthetic chemicals used globally for rubber antioxidation, with their quinone derivatives (PPD-Qs) raising particular environmental concerns due to their severe toxicity to aquatic organisms. Emerging research has identified a variety of novel PPD-Qs ubiquitously detected in the environment, yet experimental proof for the toxicity of PPD-Qs has not been forthcoming due to the unavailability of bulk standards, leaving substantial gaps in the prioritization and mechanistic investigation of such novel pollutants. Here, we use synthesized chemical standards to study the acute toxicity and underlying mechanism of 18 PPD-Qs and PPDs to the aquatic bacterium V. fischeri. Bioluminescence inhibition EC50 of PPD-Qs ranged from 1.76-15.6 mg/L, with several emerging PPD-Qs demonstrating significantly higher toxicity than the well-studied 6PPD-Q. This finding suggests a broad toxicological threat PPD-Qs pose to the aquatic bacterium, other than 6PPD-Q. Biological response assays revealed that PPD-Qs can reduce the esterase activity, cause cell membrane damage and intracellular oxidative stress. Molecular docking unveiled multiple interactions of PPD-Qs with the luciferase in V. fischeri, suggesting their potential functional impacts on proteins through competitive binding. Our results provided crucial toxicity benchmarks for PPD-Qs, prioritized these novel pollutants and shed light on the potential toxicological mechanisms.

Toxic effects and comparison of common amino antioxidants (AAOs) in the environment on zebrafish: A comprehensive analysis based on cells, embryos, and adult fish.

The ubiquity of amino antioxidants (AAOs) in the environment has attracted increasing attention, given their potential toxicity. This investigation represents a pioneering effort, systematically scrutinizing the toxicological effects of four distinct AAOs across the developmental spectrum of zebrafish, encompassing embryonic, larvae, and adult stages. The results indicate that four types of AAO exhibit varying degrees of cell proliferation toxicity. Although environmentally relevant concentrations of AAOs exhibit a comparatively circumscribed impact on zebrafish embryo development, heightened concentrations (300 µg/L) of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD) distinctly evoke developmental toxicity. Behavioral analysis results indicate that at concentrations of 20 and 300 µg/L, the majority of AAOs significantly reduced the swimming speed and activity of larvae. Moreover, each AAO triggers the generation of reactive oxygen species (ROS) in larvae, instigating diverse levels of oxidative stress. The study delineates parallel toxicological patterns in zebrafish exposed to 300 µg/L of 6PPD and IPPD, thereby establishing a comparable toxicity profile. The comprehensive toxicity effects among the four AAOs is as follows: IPPD >6PPD > N-Phenyl-1-naphthylamine (PANA) > diphenylamine (DPA). These findings not only enrich our comprehension of the potential hazards associated with AAOs but also provide data support for structure-based toxicity prediction models.

Widespread occurrence of two typical N, N'-substituted p-phenylenediamines and their quinones in humans: Association with oxidative stress and liver damage.

While N, N'-substituted p-phenylenediamines (PPDs) and their quinone derivatives (PPDQs) have been widely detected in the environment, there is currently limited data on their occurrence in humans. In this study, we conducted the first serum analysis of two PPDs and PPDQs in the healthy and secondary nonalcoholic fatty liver disease (S-NAFLD) cohorts in South China. The concentrations of four oxidative stress biomarkers (OSBs), namely, 8-iso-prostaglandin F2α (8-PGF2α), 11ß-prostaglandin F2α (11-PGF2α), 15(R)-prostaglandin F2α (15-PGF2α), and 8-hydroxy-2'-deoxyguanosine in serum samples were also measured. Results showed that N-(1,3-dimethybutyl)-N'-phenyl-p-phenylenediamine (6PPD) quinone was the predominant target analytes both in the healthy and S-NAFLD cohorts, with the median concentrations of 0.13 and 0.20 ng/mL, respectively. Significant (p < 0.05) and positive correlations were found between 6PPD concentration and 8-PGF2α, 11-PGF2α, and 15-PGF2α in both the healthy and S-NAFLD cohorts, indicating that 6PPD may be associated with lipid oxidative damage. In addition, concentrations of 6PPD in serum were associated significantly linked with total bilirubin (ß = 0.180 µmol/L, 95%CI: 0.036-0.396) and direct bilirubin (DBIL, ß = 0.321 µmol/L, 95%CI: 0.035-0.677) related to hepatotoxicity. Furthermore, 8-PGF2α, 11-PGF2α, and 15-PGF2α mediated 17.1%, 24.5%, and 16.6% of 6PPD-associated DBIL elevations, respectively. Conclusively, this study provides novel insights into human exposure to and hepatotoxicity assessment of PPDs and PPDQs.

A mini review on 6PPD quinone: A new threat to aquaculture and fisheries.

Numerous toxic substances are directly and indirectly discharged by humans into water bodies, causing distress to the organisms living on it. 6PPD, an amino antioxidant from tires reacts with ozone to form 6PPD-Q, which has garnered global attention due to its lethal nature to various organisms. This review aims to provide an understanding of the sources, transformation, and fate of 6PPD-Q in water and the current knowledge on its effects on aquatic organisms. Furthermore, we discuss research gaps pertaining to the mechanisms by which 6PPD-Q acts within fish bodies. Previous studies have demonstrated the ubiquitous presence of 6PPD-Q in the environment, including air, water, and soil. Moreover, this compound has shown high lethality to certain fish species while not affecting others. Toxicological studies have revealed its impact on the nervous system, intestinal barrier function, cardiac function, equilibrium loss, and oxidative stress in various fish species. Additionally, exposure to 6PPD-Q has led to organ injury, lipid accumulation, and cytokine production in C. elegans and mice. Despite studies elucidating the lethal dose and effects of 6PPD-Q in fish species, the underlying mechanisms behind these symptoms remain unclear. Future studies should prioritize investigating the mechanisms underlying the lethality of 6PPD-Q in fish species to gain a better understanding of its potential effects on different organisms.

Interspecies Differences in 6PPD-Quinone Toxicity Across Seven Fish Species: Metabolite Identification and Semiquantification.

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q) is a recently identified contaminant that originates from the oxidation of the tire antidegradant 6PPD. 6PPD-Q is acutely toxic to select salmonids at environmentally relevant concentrations, while other fish species display tolerance to concentrations that surpass those measured in the environment. The reasons for these marked differences in sensitivity are presently unknown. The objective of this research was to explore potential toxicokinetic drivers of species sensitivity by characterizing biliary metabolites of 6PPD-Q in sensitive and tolerant fishes. For the first time, we identified an O-glucuronide metabolite of 6PPD-Q using high-resolution mass spectrometry. The semiquantified levels of this metabolite in tolerant species or life stages, including white sturgeon (Acipenser transmontanus), chinook salmon (Oncorhynchus tshawytscha), westslope cutthroat trout (Oncorhynchus clarkii lewisi), and nonfry life stages of Atlantic salmon (Salmo salar), were greater than those in sensitive species, including coho salmon (Oncorhynchus kisutch), brook trout (Salvelinus fontinalis), and rainbow trout (Oncorhynchus mykiss), suggesting that tolerant species might detoxify 6PPD-Q more effectively. Thus, we hypothesize that differences in species sensitivity are a result of differences in basal expression of biotransformation enzyme across various fish species. Moreover, the semiquantification of 6PPD-Q metabolites in bile extracted from wild-caught fish might be a useful biomarker of exposure to 6PPD-Q, thereby being valuable to environmental monitoring and risk assessment.

Toxic Tire Wear Compounds (6PPD-Q and 4-ADPA) Detected in Airborne Particulate Matter Along a Highway in Mississippi, USA.

Tire wear particles (TWPs) are a major category of microplastic pollution produced by friction between tires and road surfaces. This non-exhaust particulate matter (PM) containing leachable toxic compounds is transported through the air and with stormwater runoff, leading to environmental pollution and human health concerns. In the present study, we collected airborne PM at varying distances (5, 15 and 30 m) along US Highway 278 in Oxford, Mississippi, USA, for ten consecutive days using Sigma-2 passive samplers. Particles (~ 1-80 µm) were passively collected directly into small (60 mL) wide-mouth separatory funnels placed inside the samplers. Particles were subsequently subjected to solvent extraction, and extracts were analyzed for TWP compounds by high resolution orbitrap mass spectrometry. This pilot study was focused solely on qualitative analyses to determine whether TWP compounds were present in this fraction of airborne PM. The abundance of airborne TWPs increased with proximity to the road with deposition rates (TWPs cm-2 day-1) of 23, 47, and 63 at 30 m, 15 m, and 5 m from the highway, respectively. Two common TWP compounds (6PPD-Q and 4-ADPA) were detected in all samples, except the field blank, at levels above their limits of detection, estimated at 2.90 and 1.14 ng L-1, respectively. Overall, this work suggests airborne TWPs may be a potential inhalation hazard, particularly for individuals and wildlife who spend extended periods outdoors along busy roadways. Research on the bioavailability of TWP compounds from inhaled TWPs is needed to address exposure risk.

Exposure to 6-PPD Quinone at Environmentally Relevant Concentrations Inhibits Both Lifespan and Healthspan in C. elegans.

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6-PPD), one of the most common additives used in rubber, enters the environment due to significant emissions of tire wear particles. 6-PPD quinone (6-PPDQ) is an important derivative of 6-PPD after ozonization. With concentrations ranging from nanograms per liter to µg/L, 6-PPDQ has so far been identified in a series of water samples. Acute lethality of 6-PPDQ in coho salmon (LC50 < 1 µg/L) was lower than environmental concentrations of 6-PPDQ, highlighting the environment exposure risks of 6-PPDQ. It is becoming increasingly necessary to investigate the potential toxicity of 6-PPDQ at environmental concentrations. Here, we examined the effect of 6-PPDQ exposure on lifespan and healthspan and the underlying mechanism in Caenorhabditis elegans. Exposure to 6-PPDQ (1 and 10 µg/L) shortened the lifespan. Meanwhile, during the aging process, 6-PPDQ (0.1-10 µg/L) could decrease both pumping rate and locomotion behavior, suggesting the 6-PPDQ toxicity on healthspan. For the underlying molecular mechanism, the dysregulation in the insulin signaling pathway was linked to toxicity of 6-PPDQ on lifespan and healthspan. In the insulin signaling pathway, DAF-2 restricted the function of DAF-16 to activate downstream targets (SOD-3 and HSP-6), which in turn controlled the toxicity of 6-PPDQ on lifespan and healthspan. Additionally, in response to 6-PPDQ toxicity, insulin peptides (INS-6, INS-7, and DAF-28) could activate the corresponding receptor DAF-2. Therefore, exposure to 6-PPDQ at environmentally relevant concentrations potentially causes damage to both lifespan and healthspan by activating insulin signaling in organisms.

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.

Occurrence and Fate of Substituted p-Phenylenediamine-Derived Quinones in Hong Kong Wastewater Treatment Plants.

para-Phenylenediamine quinones (PPD-Qs) are a newly discovered class of transformation products derived from para-phenylenediamine (PPD) antioxidants. These compounds are prevalent in runoff, roadside soil, and particulate matter. One compound among these, N-1,3-dimethylbutyl-n'-phenyl-p-phenylenediamine quinone (6PPD-Q), was found to induce acute mortality of coho salmon, rainbow trout, and brook trout, with the median lethal concentrations even lower than its appearance in the surface and receiving water system. However, there was limited knowledge about the occurrence and fate of these emerging environmental contaminants in wastewater treatment plants (WWTPs), which is crucial for effective pollutant removal via municipal wastewater networks. In the current study, we performed a comprehensive investigation of a suite of PPD-Qs along with their parent compounds across the influent, effluent, and biosolids during each processing unit in four typical WWTPs in Hong Kong. The total concentrations of PPDs and PPD-Qs in the influent were determined to be 2.7-90 and 14-830 ng/L. In the effluent, their concentrations decreased to 0.59-40 and 2.8-140 ng/L, respectively. The median removal efficiency for PPD-Qs varied between 53.0 and 91.0% across the WWTPs, indicating that a considerable proportion of these contaminants may not be fully eliminated through the current processing technology. Mass flow analyses revealed that relatively higher levels of PPD-Qs were retained in the sewage sludge (20.0%) rather than in the wastewater (16.9%). In comparison to PPDs, PPD-Qs with higher half-lives exhibited higher release levels via effluent wastewater, which raises particular concerns about their environmental consequences to aquatic ecosystems.