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A mini review on 6PPD quinone: A new threat to aquaculture and fisheries ☆
Abstract
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.
... Several of the detected compounds, including benzothiazole, 2-(methylthio)benzothiazole (MTBT), hexamethoxy methyl melamine (HMMM), 4-tert-octylphenol, phenanthrene, 4 H-cyclopenta[def]phenanthren-4-one, and fluoranthene and pyrene, have been identified as micropollutants and PAHs originating from tires [91]. Notably, 1,4-Benzenediamine, N-(1,3-dimethylbutyl)-N'-phenyl-, also known as 6PPD, was detected and serves as a precursor of 6PPD-quinone, a significant marker indicating TWP contamination across ecosystems, particularly affecting fish toxicity [10]. ...
... Benzothiazoles and arylamines have been identified as potential causative agents in the developmental impairments induced by TWP leachate in fathead minnows [18]. The 6-PPD detected in the leachate and its derivative, the 6-PPD-Q, also has been demonstrated to impact zebrafish embryo development and induce myopia [10]. Additionally, three PAHs (fluorene, pyrene, and phenanthrene) have been linked to reduced eye pigmentation in zebrafish embryos and a subsequent decrease in swimming activity [25]. ...
... Several of the detected compounds, including benzothiazole, 2-(methylthio)benzothiazole (MTBT), hexamethoxy methyl melamine (HMMM), 4-tert-octylphenol, phenanthrene, 4 H-cyclopenta[def]phenanthren-4-one, and fluoranthene and pyrene, have been identified as micropollutants and PAHs originating from tires [91]. Notably, 1,4-Benzenediamine, N-(1,3-dimethylbutyl)-N'-phenyl-, also known as 6PPD, was detected and serves as a precursor of 6PPD-quinone, a significant marker indicating TWP contamination across ecosystems, particularly affecting fish toxicity [10]. ...
... Benzothiazoles and arylamines have been identified as potential causative agents in the developmental impairments induced by TWP leachate in fathead minnows [18]. The 6-PPD detected in the leachate and its derivative, the 6-PPD-Q, also has been demonstrated to impact zebrafish embryo development and induce myopia [10]. Additionally, three PAHs (fluorene, pyrene, and phenanthrene) have been linked to reduced eye pigmentation in zebrafish embryos and a subsequent decrease in swimming activity [25]. ...
... While 6PPD-Q acute toxicity to coho salmon has been estimated, toxicity to other salmonids, other fish, and aquatic biota remains under investigation (Tian et al., 2021;Tian et al., 2022a;Brinkmann et al., 2022). A 6PPD-Q review by Bohara et al. is revealing to how ubiquitous this CEC is within nature and points out that the CEC is not only toxic to coho salmon but also other aquatic species (Bohara et al., 2023). ...
Coho salmon (Oncorhynchus kisutch) are highly sensitive to 6PPD-Quinone (6PPD-Q). Details of the hydrological and biogeochemical processes controlling spatial and temporal dynamics of 6PPD-Q fate and transport from points of deposition to receiving waters (e.g., streams, estuaries) are poorly understood. To understand the fate and transport of 6PPD and mechanisms leading to salmon mortality Visualizing Ecosystem Land Management Assessments (VELMA), an ecohydrological model developed by US Environmental Protection Agency (EPA), was enhanced to better understand and inform stormwater management planning by municipal, state, and federal partners seeking to reduce stormwater contaminant loads in urban streams draining to the Puget Sound National Estuary. This work focuses on the 5.5 km2 Longfellow Creek upper watershed (Seattle, Washington, United States), which has long exhibited high rates of acute urban runoff mortality syndrome in coho salmon. We present VELMA model results to elucidate these processes for the Longfellow Creek watershed across multiple scales–from 5-m grid cells to the entire watershed. Our results highlight hydrological and biogeochemical controls on 6PPD-Q flow paths, and hotspots within the watershed and its stormwater infrastructure, that ultimately impact contaminant transport to Longfellow Creek and Puget Sound. Simulated daily average 6PPD-Q and available observed 6PPD-Q peak in-stream grab sample concentrations (ng/L) corresponds within plus or minus 10 ng/L. Most importantly, VELMA’s high-resolution spatial and temporal analysis of 6PPD-Q hotspots provides a tool for prioritizing the locations, amounts, and types of green infrastructure that can most effectively reduce 6PPD-Q stream concentrations to levels protective of coho salmon and other aquatic species.
... ated by friction between vehicles and the road surface, have recently attracted attention for their impact on the environment.Tyre wear particles are rubber-based product similar to micro plastics that can be transported and dispersed into the environment, including air, water and soil, by various mechanisms such as saltation, abrasion of larger particles, the sheer force of moving air and water(Figure 7)[16][17]. ...
... Fang et al. [18] reported that 6PPD exposure induced hepatotoxicity (increased liver weight) and altered glycolipid metabolism and glutathione metabolism in mice. In addition, some metabolites of PPDs (e.g., PPDs-derived quinones) in the human body may be more toxic than their parent PPDs [4,19]. For instance, the oxidized product of 6PPD (LC 50 , 250 µg/L), known as 6PPD-quinone (0.82 µg/L), has been observed to induce acute mortality in coho salmon [20]. ...
Human exposure to p-phenylenediamine derivatives (PPDs) may induce hepatotoxicity and altered glycolipid metabolism. Recent studies have demonstrated the wide presence of PPDs in environmental matrixes. However, until now, the occurrence of PPDs in tap water has not been well known. This study analyzed nine PPDs in tap water collected from Hangzhou and Taizhou, China. The results showed that seven PPDs were detected in tap water samples from Hangzhou (n = 131), with the concentration of total detected PPDs ranging from 0.29 to 7.9 ng/L (mean: 1.6 ng/L). N-(1, 3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD; mean: 0.79 ng/L, <LOD−5.7 ng/L) was the predominant PPD in tap water from Hangzhou, followed by N, N′-di-2-butyl-p-phenylenediamine (44PD; 0.39 ng/L, <LOD−2.2 ng/L) and N-isopropyl-N′-phenyl-1, 4-phenylenediamine (IPPD; 0.31 ng/L, <LOD−1.4 ng/L). Five PPDs were detected in tap water collected from Taizhou (n = 30). N-phenyl-N′-cyclohexyl-p-phenylenediamine (CPPD; mean: 1.0 ng/L, <LOD−4.2 ng/L) was the predominant PPD in tap water from Taizhou, followed by 6PPD (0.93 ng/L, <LOD−2.6 ng/L) and 44PD (0.78 ng/L, <LOD−1.8 ng/L). The mean daily intake (DI) of PPDs for adults and children in Hangzhou was estimated to be 4.9–24 and 6.4–32 pg/kg bw/day, respectively. Meanwhile, the mean DI of PPDs for adults and children living in Taizhou was 11–31 and 14–40 pg/kg bw/day, respectively. To our knowledge, this study provides the first data on the occurrence of PPDs in tap water, which is vital for human exposure risk assessment.
... 6PPD is an extensively used additive to protect tyres from wear and tear. When 6PPD enters the aquatic environment with stormwater or urban runoff, it reacts with atmospheric ozone, forming a highly toxic compound known as 6PPD-quinone (6PPDq) (Bohara et al., 2023;Li et al., 2023;Tian et al., 2021). Tian et al. (2021) found 6PPDq to be the causative toxicant for the mysterious mortality of coho salmon (Oncorhynchus kisutch) over many years in stormwater-affected streams in the Western United States. ...
Plastic pollution, including micro- and nanoplastics, is a growing concern. Tyre-wear particles (TWPs) are the second largest source of microplastics in the ocean following abrasion of synthetic fibres. In addition to the particles themselves, TWPs contain many harmful chemicals, including 6PPD. This chemical reacts with atmospheric ozone and forms the toxic compound 6PPD-quinone (6PPDq), which poses a danger to aquatic life. There is a knowledge gap in understanding risks associated with the combined toxicity of nanoplastics (NPs) and 6PPDq. The present study aimed to investigate the toxicity of NPs and 6PPDq on adult zebrafish using phenotypic (behaviour, histology) and transcriptomic endpoints. Zebrafish were exposed to four treatments: control (contaminant-free), 50 μg/L 6PPDq, 3 mg/L polystyrene (PS)-NPs, and a combination of 50 μg/L 6PPDq and 3 mg/L PS-NPs. We did not observe locomotory dysregulation in zebrafish exposed to NPs. However, we found significant hyperlocomotion in zebrafish exposed to 6PPDq and this effect was even more substantial after co-exposure with PS-NPs. This study explores the molecular mechanisms behind these effects, identifying genes associated with neurotransmitters and fatty acid metabolism that were dysregulated by the co-exposure. Transcriptomic analysis further showed that both 6PPDq and PS-NPs impacted cellular processes associated with sterol biosynthesis, cholesterol metabolism, and muscle tissue development. The effects on these mechanisms were stronger in co-exposed zebrafish, indicating a heightened risk to cellular integrity and mitochondrial dysfunction. These results highlight the significance of mixture toxicity when studying the effects of NPs and associated chemicals like 6PPDq.
... 6PPD is an extensively used additive to protect tyres from wear and tear. When 6PPD enters the aquatic environment with stormwater or urban runoff, it reacts with atmospheric ozone, forming a highly toxic compound known as 6PPD-quinone (6PPDq) (Bohara et al., 2023;Li et al., 2023;Tian et al., 2021). Tian et al. (2021) found 6PPDq to be the causative toxicant for the mysterious mortality of coho salmon (Oncorhynchus kisutch) over many years in stormwater-affected streams in the Western United States. ...
Sediments containing diatom microfossils extensively deposit in lacustrine and marine environments. The silty and porous diatoms, unlike common rock-forming minerals, are of strong water adsorption, whose introduction makes the soil an intractable problem. This study discussed the transmutation of clayey soils with diatom inclusions from physical and microstructural perspectives.
Mineralogy and microstructure of kaolin and diatomite were first analyzed, and their mixture was used to simulate the natural sediments. The consistency limit, specific surface, and activity of the mixtures with different diatom proportions were achieved by laboratory experiments, and those from literatures were also collected. Thereinafter, the evolution of physical property and characterization by diatom incorporation was analyzed to reveal the diatom inclusion effect.
With enrichment of diatom microfossils within the silt fraction, the consistency limits, soil activity, and specific surface area significantly diverge from the traditional cognition of clay mineral and particle size-dependent behavior. Microstructure reveals that the physicochemical and structural differences between diatoms and clay minerals underlie the paradoxical performance. The plasticity chart would be updated to classify the fine-grained soils with diatom inclusions by excluding the water stored by diatoms. A modified multi-phase and porosity-division model was proposed to identify the water retention of diatom in volume and mass to characterize the plasticity nature of this sediment.
The evolution of soil properties after diatom intervention was clarified. New porosity division and “real plasticity” identification system helps to establish an accurate understanding of diatomaceous soils.
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.
To date, previous studies have reported the adverse effects of microplastics (MPs) and nanoplastics (NPs) on both freshwater and marine organisms. However, the information on MPs' and NPs' effects on shrimp species is scarce. In addition, the factors influencing the distribution of these particles in aquatic systems have been explained, yet the mechanisms behind MPs and NPs distribution and consumption, specifically to crustaceans and shrimp, have not been elucidated in detail. The effects of MPs and NPs as well as plastic-carried contaminants and pathogens on shrimp are critical to shrimp production and subsequent human consumption. Recent findings are required to review and discuss to open up new avenues for emerging Shrimp and crustacean research for sustainability. This review summarizes the distribution and fate of MPs and NPs along with contaminants and pathogens and identifies potential risks to shrimp health. The transport of MPs and NPs is influenced by their plastic properties, hydrodynamics, and water properties. Additionally, the fate of these particles on a plastic surface (plastisphere) is regulated by contaminant properties. Pathogens thriving on plastic surfaces and contaminants adsorbed can reach aquatic organisms directly with plastic particles or indirectly after release to an aquatic environment. MPs and NPs can be absorbed by shrimp through their gills and mouth and accumulate in their internal organs. Innate immunity influenced the degree of survival rate, tissue damage, alteration of gut microbiota, and increased oxidative stress caused by MPs and NPs accumulation. The studies on the effects of MPs and NPs are still not sufficient to understand how these particles are absorbed from various parts of the shrimp body and the fate of these plastics inside the body.
The chemical 6PPD-quinone is highly toxic to some fish species of the Oncorhynchus and Salvelinus genera and is the oxidation product of the common car tire additive 6PPD. We present a new sample preparation method that involves liquid-liquid extraction of water samples followed by silica-based solid phase extraction prior to LC-MS/MS analysis. The new sample preparation method showed good analyte recovery from spiked water samples (78%-91%) and a low ion suppression effect, surpassing previously published methods. This new method was successfully validated, achieving a limit of quantification of 5 ng/L and estimated expanded measurement uncertainty of 18.6%. In a proof-of-concept study, the method was applied to several water samples from various sources in Southern Norway. These were runoff samples from tunnel washing, from a tunnel runoff treatment plant and downstream of the plant drain. In addition, two water samples from puddles were included: one was runoff from an artificial soccer turf field and one from a puddle on a country road. The results of the analyses revealed that the concentration of 6PPD-quinone was above the LC50 reported for coho salmon (Oncorhynchus kisutch) in all samples except the samples from and downstream of the treatment plant. The highest measured concentration was 258 ng/L, which is the 2.7-fold of the reported LC50 in coho salmon (95 ng/L). Our initial data emphasize the need for more comprehensive environmental monitoring of 6PPD-quinone as well as toxicological studies in aquatic organisms.
The discovery that the commercial rubber antidegradant 6PPD reacts with ozone (O3) to produce a highly toxic quinone (6PPDQ) spurred a significant research effort into nontoxic alternatives. This work has been hampered by lack of a detailed understanding of the mechanism of protection that 6PPD affords rubber compounds against ozone. Herein, we report high-level density functional theory studies into early steps of rubber and PPD (p-phenylenediamine) ozonation, identifying key steps that contribute to the antiozonant activity of PPDs. In this, we establish that our density functional theory approach can achieve chemical accuracy for many ozonation reactions, which are notoriously difficult to model. Using adiabatic energy decomposition analysis, we examine and dispel the notion that one-electron charge transfer initiates ozonation in these systems, as is sometimes argued. Instead, we find direct interaction between O3 and the PPD aromatic ring is kinetically accessible and that this motif is more significant than interactions with PPD nitrogens. The former pathway results in a hydroxylated PPD intermediate, which reacts further with O3 to afford 6PPD hydroquinone and, ultimately, 6PPDQ. This mechanism directly links the toxicity of 6PPDQ to the antiozonant function of 6PPD. These results have significant implications for development of alternative antiozonants, which are discussed.
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.
Tire wear particles (TWP) are assumed to be one of the major sources of microplastic pollution to the environment. However, many of the previously published studies are based on theoretical estimations rather than field measurements. To increase the knowledge regarding actual environmental concentrations, samples were collected and analyzed from different matrices in a rural highway environment to characterize and quantify TWP and other traffic-derived non-exhaust particles. The sampled matrices included road dust (from kerb and in-between wheeltracks), runoff (water and sediment), and air. In addition, airborne deposition was determined in a transect with increasing distance from the road. Two sieved size fractions (2–20 µm and 20–125 µm) were analyzed by automated Scanning Electron Microscopy/Energy Dispersive X-ray spectroscopy (SEM/EDX) single particle analysis and classified with a machine learning algorithm into the following subclasses: TWP, bitumen wear particles (BiWP), road markings, reflecting glass beads, metals, minerals, and biogenic/organic particles. The relative particle number concentrations (%) showed that the runoff contained the highest proportion of TWP (up to 38 %). The share of TWP in kerb samples tended to be higher than BiWP. However, a seasonal increase of BiWP was observed in coarse (20–125 µm) kerb samples during winter, most likely reflecting studded tire use. The concentration of the particle subclasses within airborne PM80-1 decreases with increasing distance from the road, evidencing road traffic as the main emission source. The results confirm that road dust and the surrounding environment contain traffic-derived microplastics in both size fractions. The finer fraction (2–20 µm) dominated (by mass, volume, and number) in all sample matrices. These particles have a high potential to be transported in water and air far away from the source and can contribute to the inhalable particle fraction (PM10) in air. This highlights the importance of including also finer particle fractions in future investigations.
Growing natural calamities as a consequence of global warming are one of the most pondering subjects today. The exponential growth of environmental pollution due to unscientific human exploitation of natural resources is considered the prime reason for the harsh responses of nature. Researchers from various fields of industry and academia are working hard to develop and implement products/technologies that are environmentally friendly or less harmful to the ecosystem. Material researchers, specifically those working in the automobile sector are also not behind in search of green products from eco-friendly raw materials and production methods. The automobile industry is collectively responsible for around 40% of global pollution in terms of greenhouse gas emissions. Out of which around 20–30% is originating from tyre production and its end-use. In this view, tyre production from eco-friendly raw materials and technologies that have minimum hazardousness to the environment is a hot research topic today. A few products in the market with “green” tags and many are in the pipeline for the recent future. This review summarises a detailed discussion of the emerging green technologies for tyre production and depicted comprehensive data from the available literature. The paper has been drafted from a well-balanced academic and industrial point of view since the researchers from both sectors are working in harmony for a better future for green tyre technology.
We compared the sensitivity of closely related Pacific salmon and steelhead (Oncorhynchus spp.) to untreated urban stormwater runoff across three storm events. Juvenile coho, sockeye, steelhead, and Chinook were exposed for 24 h to untreated urban runoff and then transferred to clean water for 48 h. As anticipated from previous studies, coho were highly susceptible to runoff toxicity, with cumulative mortality rates ranging from 92%-100% across the three storms. By contrast, juvenile sockeye were unaffected (100% survival), and cumulative mortality rates were intermediate for steelhead (4%-42%) and Chinook (0%-13%). Furthermore, coho died rapidly following the onset of stormwater exposure (generally <4 h), whereas mortality in Chinook and steelhead was delayed by 1-2 days. Similar to previous findings for coho, steelhead and Chinook did not recover when transferred to clean water. Lastly, significant mortality occurred in coho even when roadway runoff was diluted by 95% in clean water. Our findings extend the urban runoff mortality syndrome in salmonids and point to a near-term need for sublethal studies in steelhead and Chinook to more precisely understand stormwater risks to threatened species recovery efforts in the western United States.
Aquatic pollution may continue to deepen following the emergence of new class of toxicants. The present study investigated the effect of water-soluble fraction of burnt tire-ash on Clarias gariepinus. The fish were exposed to sublethal doses; 0.00 g/L, 2.24 g/L, 1.12 g/L and 0.56 g/L of tire-ash solution, representing 1/5, 1/10 and 1/20 of 11.2 g/L median lethal concentration (96 LC50), for 28 days, followed by 14 days recovery trial. Biological sampling was done on exposure day 1, 14 and 28, and on day14
recovery period for biochemical analysis such as the liver and gill Naþ/Kþ and Ca2þ-ATPase, serum immunoglobulin (IgM) and brain acetylcholinesterase (AChE) of the experimental fish. Also, body biomass and behavior were evaluated. The behavioral responses exhibited by the fish to BTA exposure include reduced feeding, hypoactivity, air gulping and skin discoloration, which was observed to be concentration dependent. The body weight of 2.24 g/L and 1.12 g/L BTA-exposed fish decreased significantly
than 0.56 g/L exposed fish and the control. Furthermore, findings revealed evident induction of Naþ/Kþ and Ca2 þ-ATPase activities in both tissues, elevation of serum immunoglobulin content and inhibition of AChE activity in the brain of the exposed fish relative to the control. However, it was also observed that the biochemical parameters normalized after the recovery period. In conclusion, watersoluble fraction of burnt tire-ash produced toxicological effects in the experimental model, hence the present study provides the ecotoxicological insight of tire ash.
p-Phenylenediamines (PPDs) have been extensively used in the rubber industry and found to be pervasive in various environmental compartments for decades, while their transformation products and associated ecological and human health risks remain largely unknown. Herein, we developed and implemented a mass spectrometry-based platform combined with self-synthesized standards for the investigation of rubber-derived quinones formed from PPD antioxidants. Our results demonstrated that five quinones are ubiquitously present in urban runoff, roadside soils, and air particles. All of the identified sources are closely related to mankind's activities. Among the identified quinones, N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone has been recently found to be highly toxic, causing acute mortality of coho salmon in the Pacific Northwest. Ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry was then applied for quantification of the five quinones and their corresponding PPD antioxidants. The results revealed interesting distinct distribution and concentration patterns of PPD-derived quinones in different environmental matrices. Daily intake rates of these quinones in a compact city of Hong Kong were estimated to be varied from 1.08 ng/(kg·day) for adults to 7.30 ng/(kg·day) for children, which were higher than the exposure levels of their parent compounds. Considering the prevalence of the use of rubber products, the outcome of this study strongly suggests for additional toxicological studies to investigate potential ecological and human health risks of the newly discovered quinones.
N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) is the most widely used antioxidant in automobile tyres and many rubber products. We investigated the impact of 6PPD and 6PPD quinone on acute toxicity, morphology, swimming behaviour, heart rate, and oxygen consumption in zebrafish larvae. Zebrafish embryos were exposed to 6PPD and 6PPD quinone at concentrations of 1, 10, and 25 μg/L during the development period of 1 to 96 hpf. In the present study, 6PPD quinone was found to be toxic to zebrafish larvae with a 24 h LC50 of 308.67 μg/L. No significant mortality was observed at any of the tested concentrations. A dose-dependent reduction in swimming performance was observed in the exposed larvae at 116 hpf for both toxicants. Overall, our study shows that exposure of zebrafish embryos to 6PPD and 6PPD quinone at environmentally relevant concentrations (1 μg/L) does not affect its behaviour. However, exposure to higher but still sublethal concentrations of 6PPD and 6PPD quinone (10 and 25 μg/L) can affect behavioural endpoints. These findings reveal the toxicity of 6PPD and 6PPD quinone to early life stages of fish.
Recent findings that 2-anilo-5-[(4-methylpentan-2-yl)amino]cyclohexa-2,5-diene-1,4-dione (6PPD-quinone), the transformation product of a common tire rubber antioxidant, is acutely toxic in stormwater-impacted streams has highlighted the need for a better understanding of contaminants in urban runoff. This study represents one of the first reports of 6PPD-quinone and other tire rubber-derived compounds in stormwater and snowmelt of a cold-climate Canadian city (Saskatoon, 2019–2020). Semiquantification of the five target compounds, N,N′-diphenylguanidine (DPG), N,N-dicyclohexylmethylamine (DCA), N,N′-dicyclohexylurea (DCU), 1-cyclohexyl-3-phenylurea (CPU), and 6PPD-quinone, revealed DPG was most abundant, with average concentrations of 60 μg L–1 in stormwater and 1 μg L–1 in snowmelt. Maximum observed concentrations of DPG were greater than 300 μg L–1, equivalent to loadings of 15 kg from a single rain event. These concentrations of DPG represent some of the highest reported in urban runoff globally. 6PPD-Quinone was detected in 57% (12/21) of stormwater samples with a mean concentration of approximately 600 ng L–1 (2019) and greater than 80% (28/31) of snowmelt samples with mean concentrations of 80–370 ng L–1 (2019 and 2020). Concentrations of 6PPD-quinone exceeded the acute LC50 for coho salmon (0.8–1.2 μg L–1) in greater than 20% of stormwater samples. Mass loadings of all target chemicals correlated well with roads and residential land-use area.
The safety of aquatic ecosystems has been compromised by numerous anthropogenic activities, especially leachates from non-point source toxicants, leaching into aquatic systems. This study evaluated the toxicity of the water-soluble fractions (WSFs) of burnt tire ash (BTA) on Clarias gariepinus via a battery of integrated biomarkers. Juvenile C. gariepinus were exposed to sublethal (0.56, 1.12, and 2.24 g/L) concentrations of BTA, derived from 11.2 g/L median lethal concentration (96 LC50), at duration intervals of 1, 14, and 28 days, followed by a recovery trial that lasted for 14 days. Serum biochemical parameters, antioxidant enzyme activities of the gill and liver, lysozymes activity and erythron profile were assessed. The findings of the present study revealed that BTA-WSF induced prominent alterations on biochemical parameters, lysozymes activity and antioxidant enzymes activities in the exposed fish. Furthermore, toxicant exposure promoted oxidative stress, cellular damage and genotoxicity (erythrocytic nuclear and cellular abnormalities) in the exposed fish. In general, a post-exposure trial showed partial recovery from the exposure effects of the toxicant, following the evident modifications of serum enzymes and erythron pathopathology in the experimental model. Biomonitoring of BTA, using sentinel aquatic species such as C. gariepinus, provides insights into the ecotoxicological potency of this toxicant.
Prompted by a recent report that 6PPD-quinone (6PPD-q), a by-product of a common tire manufacturing additive that is present in road runoff, is toxic to coho salmon (Oncorhynchus kisutch), extracts of water samples collected from an urban river were re-analyzed to determine if this compound was present in stormwater-influenced flows. In addition, extracts were analyzed for 1,3-diphenylguanidine (DPG), which is also used in tire manufacturing. Samples were originally collected in the fall of 2019 and winter of 2020 in the Greater Toronto Area of Canada from the Don River, a highly urbanized watershed in close proximity to several major multi-lane highways. These target compounds were analyzed using ultra-high pressure liquid chromatography with high resolution mass spectrometric detection with parallel reaction monitoring. Both 6PPD-q and DPG were detected above limits of quantification (i.e., 0.0098 µg/L) in all extracts. Maximum concentrations for 6PPD-quinone of 2.30 ± 0.05 µg/L observed in the river during storm events exceeded the LC50 for this compound for coho salmon (i.e., > 0.8 µg/L). In composite samples collected at intervals throughout one rain event, both compounds reached peak concentrations a few hours after initiation of the event (i.e., 0.52 µg/L for DPG and 2.85 µg/L for 6PPD-q), but the concentrations of 6PPD-q remained elevated above 2 µg/L for over 10-h in the middle of the event. Estimates of cumulative loads of these compounds in composite samples indicated that kg amounts of these compounds entered the Don River during each hydrological event, and the loads were proportional to the amounts of precipitation. This study contributes to the growing literature indicating that potentially toxic tire-wear compounds are present at elevated levels and are transported via road runoff into urban surface waters during rain events.
The photochemical degradation pathways of 6PPD-quinone (6PPDQ, 6PPD-Q), a toxic transformation product of the tire antiozonant 6PPD, were determined under simulated sunlight conditions typical of high-latitude surface waters. Direct photo-chemical degradation resulted in 6PPDQ half-lives ranging from 17.5 h at 20°C to no observable degradation over 48 h at 4°C. Sensitization of excited triplet-state pathways using Cs + and Ar purging demonstrated that 6PPDQ does not decompose significantly from a triplet state relative to a singlet state. However, assessment of processes involving reactive oxygen species (ROS) quenchers and sensitizers indicated that singlet oxygen and hydroxyl radical do significantly contribute to the degradation of 6PPDQ. Investigation of these processes in natural lake waters indicated no difference in attenuation rates for direct photochemical processes at 20°C. This suggests that direct photochemical degradation will dominate in warm waters, while indirect photochemical pathways will dominate in cold waters, involving ROS mediated by chromophoric dissolved organic matter (CDOM). Overall, the aquatic photodegradation rate of 6PPDQ will be strongly influenced by the compounding effects of environmental factors such as light screening and temperature on both direct and indirect photochemical processes. Transformation products were identified via UHPLC-Orbitrap mass spectrometry, revealing four major processes: (1) oxidation and cleavage of the quinone ring in the presence of ROS, (2) dealkylation, (3) rearrangement, and (4) deamination. These data indicate that 6PPDQ can photodegrade in cool, sunlit waters under the appropriate conditions: t 1/2 = 17.4 h to no observable decrease (direct); t 1/2 = 5.2−11.2 h (indirect, CDOM).
Quinones are one of the most important redox-reactive organic compounds in natural environments, such as soil, water, and sediment, playing an important role in regulating the environmental processes and biogeochemical cycles of critical elements under climate change, including the influences of extreme events such as wildfires. However, to date, no existing methods can quantify quinones in complex environmental media. To overcome this challenge, a quantification method was developed by coupling chemical tagging of quinones by cysteine-containing nonaromatic peptides (Cpep) through a Michael addition reaction with size exclusionary chromatography (SEC) separation and ultraviolet (UV) analysis─leveraging on the characteristic absorbance of aromatic rings at 254 nm and molecular size of peptide. The method was demonstrated using model quinones, including 1,4-benzoquinone (BQ), 1,4-naphthoquinone (NQ), and 1,4-anthraquinone (AQ), with a detection limit of 3.3, 0.7, and 0.2 μM, respectively. Concentrations of quinones in water extractions of biochars, soils, and wildfire-derived ashes were determined to range from 0.8 to 14 μM and were positively correlated with their redox reactivity determined by a chemical assay. This method provides a novel rapid quantification of quinones in complex environmental media as well as a quick assessment for redox reactivity and opens up new avenues for studying environmental transformation and remediation of contaminants.
N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-quinone) is an emerging contaminant of concern that is generated through the environmental oxidation of the rubber tire anti-degradant 6PPD. Since the initial report of 6PPD-quinone being the cause of urban runoff mortality syndrome of Coho salmon, numerous species have been identified as either sensitive or insensitive to acute lethality caused by 6PPD-quinone. In sensitive species, acute lethality might be caused by uncoupling of mitochondrial respiration in gills. However, little is known about effects of 6PPD-quinone on insensitive species. Here we demonstrate that embryos of fathead minnows (Pimephales promelas) are insensitive to exposure to concentrations as great as 39.97 μg/L for 168 h, and adult fathead minnows are insensitive to exposure to concentrations as great as 9.4 μg/L for 96 h. A multi-omics approach using a targeted transcriptomics array, (EcoToxChips), and proton nuclear magnetic resonance (1H NMR) was used to assess responses of the transcriptomes and metabolomes of gills and livers from adult fathead minnows exposed to 6PPD-quinone for 96 h to begin to identify sublethal effects of 6PPD-quinone. There was little agreement between results of the EcoToxChip and metabolomics analyses, likely because genes present on the EcoToxChip were not representative of pathways suggested to be perturbed by metabolomic analysis. Changes in abundances of transcripts and metabolites in livers and gills suggest that disruption of one‑carbon metabolism and induction of oxidative stress might be occurring in gills and livers, but that tissues differ in their sensitivity or responsiveness to 6PPD-quinone. Overall, several pathways impacted by 6PPD-quinone were identified as candidates for future studies of potential sublethal effects of this chemical.
Amino accelerators and antioxidants (AAL/Os), as well as their degradation derivatives, are industrial additives of emerging concern due to their massive production and use (particularly in rubber tires), pervasiveness in the environment, and documented adverse effects. This study delineated their inter-regional variations in road dust collected from urban/suburb, agricultural, and forest areas, and screened for less-studied AAL/O analogues with high-resolution mass spectrometry. 1,3-Diphenylguanidine (DPG; median concentration: 121 ng/g) and N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPD-Q; 9.75 ng/g) are the most abundant congeners, constituting 69.7% and 41.4% of the total concentrations of AAL/Os (192 ng/g) and those of AAO transformation products (22.3 ng/g), respectively. The spatial distribution across the studied sites suggests evident human impacts, reflected by the pronounced urban signature and vehicle-originated pollution. Our nontargeted analysis of the most-contaminated road dust identified 16 AAL/O-related chemicals, many of which have received little investigation. Particularly, environmental and toxicological information remains extremely scarce for five out of the 10 most concerning compounds prioritized in terms of their dusty residues and toxicity including 1,2-diphenyl-3-cyclohexylguanidine (DPCG), N,N''-bis[2-(propan-2-yl)phenyl]guanidine (BPPG), and N-(4-anilinophenyl)formamide (PPD-CHO). Additionally, dicyclohexylamine (DChA), broadly applied as an antioxidant in automobile products, had an even greater median level than DPG. Therefore, future research on their health risks and (eco)toxic potential is of high importance.
N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ) is the ozonation product of tire antioxidant 6-PPD. 6-PPDQ can be detected in different environments, such as roadway runoff and dust. Although 6-PPDQ toxicity has been frequently assessed in aquatic organisms, the possible toxic effects of 6-PPDQ on mammals remain largely unclear. We here aimed to perform systematic assessment to evaluate 6-PPDQ toxicity on multiple organs in mice. Male BALB/c mice were intraperitoneally injected with 6-PPDQ for two exposure modes, single intraperitoneal injection and repeated intraperitoneal injection every four days for 28 days. Serum, liver, kidney, lung, spleen, testis, brain, and heart were collected for injury evaluation by organ index, histopathology analysis and biochemical parameters. In 0.4 and 4 mg/kg 6-PPDQ single injected mice, no significant changes in organ indexes and biochemical parameters were detected, and only moderate pathological changes were observed in organs of liver, kidney, lung, and brain. Very different from this, in 0.4 and 4 mg/kg 6-PPDQ repeated injected mice, we observed the obvious increase in organ indexes of liver, kidney, lung, testis, and brain, and the decrease in spleen index. Meanwhile, the significant pathological changes were formed in liver, kidney, lung, spleen, testis, and brain in 0.4 and 4 mg/kg 6-PPDQ repeated injected mice. Biochemical parameters of liver (alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP)) and kidney (urea and creatinine) were all significantly upregulated by repeated injection with 0.4 and 4 mg/kg 6-PPDQ. After repeated exposure, most of 6-PPDQ was accumulated in liver and lung of mice. Therefore, our results suggested the risk of repeated exposure to 6-PPDQ in inducing toxicity on multiple organs in mice.
Although it has been shown that exposure to 6-PPDQ can cause toxicity on environmental organisms, its possible effects on metabolic state remain largely unclear. We here determined the effect of 6-PPDQ exposure on lipid accumulation in Caenorhabditis elegans. We observed increase in triglyceride content, enhancement in lipid accumulation, and increase in size of lipid droplets in 6-PPDQ (1-10 μg/L) exposed nematodes. This detected lipid accumulation was associated with both increase in fatty acid synthesis reflected by increased expressions of fasn-1 and pod-2 and inhibition in mitochondrial and peroxisomal fatty acid β-oxidation indicated by decreased expressions of acs-2, ech-2, acs-1, and ech-3. The observed lipid accumulation in 6-PPDQ (1-10 μg/L) exposed nematodes was also related to the increase in synthesis of monounsaturated fatty acylCoAs reflected by altered expressions of fat-5, fat-6, and fat-7. Exposure to 6-PPDQ (1-10 μg/L) further increased expressions of sbp-1 and mdt-15 encoding two metabolic sensors to initiate the lipid accumulation and to regulate the lipid metabolism. Moreover, the observed increase in triglyceride content, enhancement in lipid accumulation, and alterations in fasn-1, pod-2, acs-2, and fat-5 expressions in 6-PPDQ exposed nematodes were obviously inhibited by sbp-1 and mdt-15 RNAi. Our observations demonstrated the risk of 6-PPDQ at environmentally relevant concentration in affecting lipid metabolic state in organisms.
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.
The automobile tire antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its quinone metabolite 6PPDQ have recently received much attention for their acute aquatic toxicity. The present study investigated the mechanistic developmental toxicity of 6PPD and 6PPDQ in embryonic zebrafish. Neither compound induced significant mortality but significantly decreased spontaneous embryo movement and heart rate. Both compounds induced malformations with different phenotypes; the 6PPD-exposed larvae manifested a myopia-like phenotype with a convex eyeball and fusion vessels, while the 6PPDQ-exposed embryonic zebrafish manifested enlarged intestine and blood-coagulated gut, activated neutrophils, and overexpressed enteric neurons. mRNA-Seq and quantitative real-time PCR assays showed that 6PPD- and 6PPDQ-induced distinct differential gene expression aligned with their toxic phenotype. 6PPD activated the retinoic acid metabolic gene cyp26a, but 6PPDQ activated adaptive cellular response to xenobiotics gene cyp1a. 6PPD suppressed the gene expression of the eye involved in retinoic acid metabolism, phototransduction, photoreceptor function and visual perception. In contrast, 6PPDQ perturbed genes involved in inward rectifier K+ and voltage-gated ion channels activities, K+ import across the plasma membrane, iron ion binding, and intestinal immune network for IgA production. The current study advances the present understanding the reason of why many fish species are so adversely impacted by 6PPD and 6PPDQ.
Recently, 6-PPD quinone (6-PPDQ), a derivative of tire antioxidant 6-PPD, was reported to have acute toxicity for organisms. However, the possible reproductive toxicity of 6-PPDQ is still largely unclear. In this study, the reproductive toxicity of 6-PPDQ after long-term exposure was further investigated in Caenorhabditis elegans. Exposure to 1 and 10 μg/L 6-PPDQ reduced the reproductive capacity. Meanwhile, exposure to 1 and 10 μg/L 6-PPDQ enhanced the germline apoptosis, which was accompanied by upregulation of ced-3, ced-4, and egl-1 expressions and downregulation of ced-9 expression. The observed increase in germline apoptosis in 1 and 10 μg/L 6-PPDQ exposed nematodes was associated with the enhancement in DNA damage and increase in expressions of related genes of cep-1, clk-2, hus-1, and mrt-2. The detected enhancement in germline apoptosis in 1 and 10 μg/L 6-PPDQ exposed nematodes was further associated with the increase in expressions of ced-1 and ced-6 governing the cell corpse engulfment process. Molecular docking analysis indicated the binding potentials of 6-PPDQ with three DNA damage checkpoints (CLK-2, HUS-1, and MRT-2) and corpse-recognizing phagocytic receptor CED-1. Therefore, our data suggested the toxicity on reproductive capacity by 6-PPDQ at environmentally relevant concentrations by enhancing DNA damage- and cell corpse engulfment-induced germline apoptosis in organisms.
6PPD, a tire rubber antioxidant, poses substantial ecological risks because it can form a highly toxic quinone transformation product (TP), 6PPD-quinone (6PPDQ), during exposure to gas-phase ozone. Important data gaps exist regarding the structures, reaction mechanisms, and environmental occurrence of TPs from 6PPD ozonation. To address these data gaps, gas-phase ozonation of 6PPD was conducted over 24-168 h and ozonation TPs were characterized using high-resolution mass spectrometry. The probable structures were proposed for 23 TPs with 5 subsequently standard-verified. Consistent with prior findings, 6PPDQ (C18H22N2O2) was one of the major TPs in 6PPD ozonation (∼1 to 19% yield). Notably, 6PPDQ was not observed during ozonation of 6QDI (N-(1,3-dimethylbutyl)-N'-phenyl-p-quinonediimine), indicating that 6PPDQ formation does not proceed through 6QDI or associated 6QDI TPs. Other major 6PPD TPs included multiple C18H22N2O and C18H22N2O2 isomers, with presumptive N-oxide, N,N'-dioxide, and orthoquinone structures. Standard-verified TPs were quantified in roadway-impacted environmental samples, with total concentrations of 130 ± 3.2 μg/g in methanol extracts of tire tread wear particles (TWPs), 34 ± 4 μg/g-TWP in aqueous TWP leachates, 2700 ± 1500 ng/L in roadway runoff, and 1900 ± 1200 ng/L in roadway-impacted creeks. These data demonstrate that 6PPD TPs are likely an important and ubiquitous class of contaminants in roadway-impacted environments.
We here report chemical characteristics relevant to the fate and transport of the recently discovered environmental toxicant 6PPD-quinone (2-((4-methylpentan-2-yl)amino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione or "6PPDQ"). 6PPDQ is a transformation product of the tire rubber antioxidant 6PPD that is ubiquitous in roadway environments, including atmospheric particulate matter, soils, runoff, and receiving waters, after dispersal from tire rubber use and wear on roadways. The aqueous solubility and octanol-water partitioning coefficient (i.e. log KOW) for 6PPDQ were measured to be 38 ± 10 μg L-1 and 4.30 ± 0.02, respectively. Within the context of analytical measurement and laboratory processing, sorption to various laboratory materials was evaluated, indicating that glass was largely inert but loss of 6PPDQ to other materials was common. Aqueous leaching simulations from tire tread wear particles (TWPs) indicated short term release of ∼5.2 μg 6PPDQ per gram TWP over 6 h under flow-through conditions. Aqueous stability tests observed a slight-to-moderate loss of 6PPDQ over 47 days (26 ± 3% loss) for pH 5, 7 and 9. These measured physicochemical properties suggest that 6PPDQ is generally poorly soluble but fairly stable over short time periods in simple aqueous systems. 6PPDQ can also leach readily from TWPs for subsequent environmental transport, posing high potential for adverse effects in local aquatic environments.
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.
6-PPD quinone (6-PPDQ) can be transformed from 6-PPD through ozonation. Nevertheless, the potential neurotoxicity of 6-PPDQ after long-term exposure and the underlying mechanism are largely unclear. In Caenorhabditis elegans, we here observed that 0.1-10 μg/L of 6-PPDQ caused several forms of abnormal locomotion behaviors. Meanwhile, the neurodegeneration of D-type motor neurons was observed in 10 μg/L of 6-PPDQ-exposed nematodes. The observed neurodegeneration was associated with the activation of the Ca2+ channel DEG-3-mediated signaling cascade. In this signaling cascade, expressions of deg-3, unc-68, itr-1, crt-1, clp-1, and tra-3 were increased by 10 μg/L of 6-PPDQ. Moreover, among genes encoding neuronal signals required for the control of stress response, expressions of jnk-1 and dbl-1 were decreased by 0.1-10 μg/L of 6-PPDQ, and expressions of daf-7 and glb-10 were decreased by 10 μg/L of 6-PPDQ. RNAi of jnk-1, dbl-1, daf-7, and glb-10 resulted in the susceptibility to 6-PPDQ toxicity in decreasing locomotory ability and in inducing neurodegeneration, suggesting that JNK-1, DBL-1, DAF-7, and GLB-10 were also required for the induction of 6-PPDQ neurotoxicity. Molecular docking analysis further demonstrated the binding potential of 6-PPDQ to DEG-3, JNK-1, DBL-1, DAF-7, and GLB-10. Together, our data suggested the exposure risk of 6-PPDQ at environmentally relevant concentrations in causing neurotoxicity in organisms.
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.
The breakdown product of the rubber tire antioxidant N-(1,3-dimethylbutyl)-N-phenyl-p-phenylenediamine-quinone (6PPD)-6-PPD-quinone has been strongly implicated in toxic injury and death in coho salmon (Oncorhynchus kisutch) in urban waterways. While recent studies have reported a wide range of sensitivity to 6PPD-quinone in several fish species, little is known about the risks to Chinook salmon (Oncorhynchus tshawytscha), the primary prey of endangered Southern Resident killer whales (Orcinus orca) and the subject of much concern. Chinook face numerous conservation threats in Canada and the United States, with many populations assessed as either endangered or threatened. In this study, we evaluated the acute toxicity of 6PPD-quinone to newly feeding (~3 weeks post-swim-up) juvenile Chinook and coho. Juvenile Chinook and coho were exposed for 24 h under static conditions to five concentrations of 6PPD-quinone. Juvenile coho were three orders of magnitude more sensitive to 6PPD-quinone compared to juvenile Chinook, with 24 h LC50 estimates of 41.0 ng/L and >67,306.8 ng/L, respectively. The coho LC50 was 2.3-fold lower than what was previously reported for 1+ year coho (95 ng/L), highlighting the value of evaluating age-related differences in sensitivity to this toxic tire-related chemical. Both fish species exhibited typical 6PPD-quinone symptomology (gasping, increased ventilation, loss of equilibrium, erratic swimming), with fish that were symptomatic generally exhibiting mortality. The LC50 values derived from this study for coho are below concentrations that have been measured in salmon-bearing waterways, suggesting the potential for population-level consequences in urban waters. The higher relative LC50 values for Chinook compared to coho merits further investigation, including the potential for population-relevant sublethal effects. This article is protected by copyright. All rights reserved. Environ Toxicol Chem 2023;00:0-0. © 2023 SETAC.
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.
Recent identification of 6PPD‐quinone as the chemical causing acute toxicity in coho salmon has led to substantial concern regarding toxicity of this contaminant for other aquatic species. Environmental occurrence of 6PPD‐quinone is probably high as it is an oxidation product of a common tire rubber additive. Research on 6PPD‐quinone toxicity in fish has revealed a rather unusual pattern, with closely related species exhibiting responses ranging from extreme sensitivity to no effect. Of eleven previously studied fish species, 6PPD‐quinone was toxic to four. The species‐specific toxicity of 6PPD‐quinone complicates urgently needed environmental risk assessment. We investigated acute toxicity of 6PPD‐quinone in Atlantic salmon and brown trout alevins (sac fry). These species have previously not been tested for sensitivity to 6PPD‐quinone. The fish were exposed in static conditions in eight treatments with initial concentrations ranging from 0.095–12.16 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 behaviour were recorded in either Atlantic salmon or brown trout. This provides an important first step in assessing effects of 6PPD‐quinone on these highly economically and culturally important species. This article is protected by copyright. All rights reserved. Environ Toxicol Chem 2022;00:0–0.
Unprecedented urbanization-induced population migration in China severely affects the scale and geographic distribution of anthropogenic pollutant discharge. Understanding how pollutant discharge patterns respond to population migration can help guide future efforts to maintain water sustainability. Here, based on a new calculation framework with 18 dynamic parameters designed for anthropogenic discharges, we finely tracked and visualized the effects of population migration on the spatial and temporal changes in anthropogenic discharge from 1980 to 2019 in the Minjiang River basin. The results indicate that the increasing effect of population migration on anthropogenic discharges peaked in 2002 and started to contribute to pollutant reduction from 2010 onward. The direct impact of population migration only contributes to the shift of anthropogenic discharges from rural to urban areas, while the migration bonus is the key factor leading to the reduction in anthropogenic discharges. Population migration is highly beneficial for chemical oxygen demand (COD) reduction, which has contributed to a shift from COD to NH4+-N and total phosphorus (TP) as hotspot pollutants in the whole basin (NH4+-N in urban areas and TP in rural areas). Moreover, pollution reduction resulting from the demographic bonus phenomenon has remained limited only to urban areas. Since approximately 2010, the per capita amount and total amount of anthropogenic pollutant discharges in rural areas have exceeded those in urban areas; in particular, the per capita COD and TP discharges in rural areas reached four times those in urban areas. This suggests that future pollution control strategies should give more attention to rural areas and focus on the differentiation and targeting of urban and rural areas.
N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone (6PPD-quinone), a rubber tire oxidation product found in road runoff, is highly and acutely toxic to selected salmonids including coho salmon, brook trout, and rainbow trout but not other fish species and invertebrates studied to date. Sensitive species displayed increased ventilation and gasping, suggesting a possible impact on respiration. Here, adherent cell lines RTL-W1 and RTgill-W1 were exposed to 5− 80 μg/L 6PPD-quinone, and cytotoxicity, oxygen consumption rate (OCR), and biotransformation of 6PPD-quinone were measured to assess the ability of 6PPD-quinone to uncouple mitochondrial respiration in vitro. RTL-W1 cells were not sensitive to 6PPD-quinone, and exposure did not result in significant impacts on cytotoxicity or OCR. In contrast, RTgill-W1 cells demonstrated decreased cell viability at 80 μg/L and a 2-fold increase in OCR at 20 μg/L. Effects appear to be partly driven by toxicokinetic differences where incubation of RTL-W1 cells with 6PPD-quinone led to almost quantitative conversion of 6PPD-quinone into a suspected hydroxy-metabolite, which was not observed in RTgill-W1 cells. Exposure studies with primary cultures of rainbow trout gill cells indicated that 6PPD-quinone increased OCR by uncoupling the mitochondrial electron transport chain. Together, these findings suggest that 6PPD-quinone toxicity might be driven by a tissue-specific disruption of mitochondrial respiration.
As a ubiquitous tire antidegradant, N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) is persistently released into the environment. It is highly toxic to aquatic organisms, and its transformation product 6PPD-quinone (6PPD-Q), is "very highly toxic" to Oncorhynchus kisutch at a median lethal concentration (LC50) of < 0.1 ng/mL. Notably, 6PPD and 6PPD-Q are chiral compounds. Here, enantioselective evaluations, including hydrolysis and acute toxicity were conducted after preparing the enantiomer, confirming the enantiomer absolute configuration and establishing enantioseparation methods. In the 6PPD hydrolysis experiments, the products 6PPD-Q, phenol, 4-[(1,3-dimethylbutyl)amino]- (4-DBAP) and 4-hydroxydiphenylamine (4-HDPA) were detected. In different water solutions, the hydrolysis of 4-DBAP and 4-HDPA was very fast (0.87-107 h), while the 6PPD-Q hydrolysis half-lives (12.8-16.3 d) were significantly longer than 6PPD (4.83-64.1 h). At the enantiomeric level, no enantioselective hydrolysis and conversion occurred. R-6PPD generated R-6PPD-Q, and S-6PPD generated S-6PPD-Q, and the formation rate of S-6PPD-Q was 1.77 times faster than R-6PPD-Q. In terms of the enantioselective toxicity, the 6PPD enantiomer was highly toxic to China-specific Gobiocypris rarus (LC50, 162-201 ng/mL), and it had no enantioselective difference. 6PPD-Q was "very highly toxic" (LC50, 1.66-4.31 ng/mL) to Oncorhynchus mykiss, which is of commercial importance, and the toxicities of rac-6PPD-Q and S-6PPD-Q were 1.9 and 2.6 times higher than R-6PPD-Q. Furthermore, the formation concentrations of S-6PPD-Q and R-6PPD-Q in 6PPD water solutions were higher than the LC50 values of O. kisutch and O. mykiss, and the toxicity of 6PPD-Q was highly species-specific, which should raise concern. These results provide important information for environmental risk assessments of 6PPD and 6PPD-Q, especially from the perspective of enantiomers.
The typical tire manufacturing additive 6PPD, its metabolites 6PPDQ and 4-Hydroxy should be monitored because of their ubiquitous presence in the environment and the high toxicity of 6PPDQ to coho salmon. The toxic effect of 6PPD and its metabolites have been revealed superficially, especially on behavioral characteristics. However, the behavioral indicators explored so far are relatively simple and the toxic causes are poorly understood. With this in mind, our work investigated the toxic effects of 6PPD, 6PPDQ and 4-Hydroxy on adult zebrafish penetratingly through machine vision, and the meandering, body angle, top time and 3D trajectory are used for the first time to show the abnormal behaviors induced by 6PPD and its metabolites. Moreover, neurotransmitter changes in the zebrafish brain were measured to explore the causes of abnormal behavior. The results showed that high-dose treatment of 6PPD reduced the velocity by 42.4% and decreased the time at the top of the tank by 91.0%, suggesting significant activity inhibition and anxiety. In addition, γ-aminobutyric acid and acetylcholine were significantly impacted by 6PPD, while dopamine exhibited a slight variation, which can explain the bradykinesia, unbalance and anxiety of zebrafish and presented similar symptoms as Huntingdon's disease. Our study explored new abnormal behaviors of zebrafish induced by 6PPD and its metabolites in detail, and the toxic causes were revealed for the first time by studying the changes of neurotransmitters, thus providing an important reference for further studies of the biological toxicity of 6PPD and its metabolites.
A recently identified chemical, 2-((4-Methylpentan-2-yl)amino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione (6PPD-quinone; 6PPD-Q), is a transformation product of an additive used in the manufacture of tire rubber and causes acute lethality in coho salmon (Oncorhynchus kisutch) in urban watersheds. Despite its potential presence and ecotoxicity in receiving waters worldwide, information on the occurrence and fate of 6PPD-Q is limited. Here, we investigated the concentrations of 6PPD-Q and its parent chemical, 6PPD, in road dust collected from arterial and residential roads in Tokyo, Japan from May to October 2021. 6PPD-Q concentrations were highest from May to June, when atmospheric ozone concentrations are the highest in Japan; a correlation between 6PPD-Q and photochemical oxidants, as an alternative to ozone, corroborated this finding. We also found that 6PPD-Q concentrations at photochemical oxidant concentrations ranging from 35 to 47 ppbv were higher in dust collected from roads with high traffic volumes (i.e., arterial roads; median: 8.6 μg/g-OC) than in dust collect from roads with lower traffic volumes (i.e., residential roads; median: 6.3 μg/g-OC), indicating that 6PPD-Q is generated from traffic-related sources. We also found that 6PPD-Q was leached from dust particles within a few hours, with a log partitioning coefficient between organic carbon and water (KOC) of about 3.0. The present results will help to understand the environmental occurrence, fate, and behavior of 6PPD-Q.
Palladium nanoparticles (PdNPs) play an integral role in motor vehicles as the primary vehicle exhaust catalyst (VEC) for tackling environmental pollution. Automobiles equipped with Pd-based catalytic converters were introduced in the mid-1970s and ever since the demand for Pd has steadily increased due to stringent emission standards imposed in many developed and developing countries. However, at the same time, the increasing usage of Pd in VECs has led to the release of nano-sized Pd particles in the environment, thus, emerging as a new source of environmental pollution. The present reports in the literature have shown gradual increasing levels of Pd particles in different urban environmental compartments and internalization of Pd particles in living organisms such as plants, aquatic species and animals. Occupational workers and the general population living in urban areas and near major highways are the most vulnerable as they may be chronically exposed to PdNPs. Risk assessment studies have shown acute and chronic toxicity exerted by PdNPs in both in-vitro and in-vivo models but the underlying mechanism of PdNPs toxicity is still not fully understood. The review intends to provide readers with an in-depth account on the demand and supply of Pd, global distribution of PdNPs in various environmental matrices, their migration and uptake by living species and lastly, their health risks, so as to serve as a useful reference to facilitate further research and development for safe and sustainable technology.
N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) is used as a ubiquitous rubber antioxidant worldwide and has been shown to be potentially toxic to aquatic organisms. In this study, zebrafish embryos were exposed to 6PPD for five days starting at two hours post-fertilization at concentrations of 0, 0.0022, 0.022, and 0.22 mg/L to investigate its effects on embryonic development, the growth hormone/insulin-like growth factor (GH/IGF) axis, and the hypothalamic-pituitary-thyroid (HPT) axis. The results showed that the 96 h LC50 of 6PPD was 2.2 mg/L. 6PPD exposure decreased hatchability, lowered autonomous movement, reduced body length in zebrafish embryos and caused deformities. The hormones levels and the expression of genes related to GH/IGF and HPT axis were altered after exposure to 6PPD in zebrafish larvae. These results indicated that the GH/IGF and HPT axis was disturbed. Moreover, treatment of 6PPD produced oxidative stress in zebrafish embryos. Overall, the present study thus demonstrated that exposure to 0.22 mg/L 6PPD caused developmental toxicity and disrupted the GH/IGF and HPT axis of zebrafish, which could be responsible for developmental impairment and growth inhibition.
The antiozonant N-phenyl-N’-(1,3-dimethylbutyl)-p-phenylenediamine (6-PPD) is added to tires to increase their lifetime and is emitted with tire and road wear particles into the environment. Recently, one of its transformation products (TPs), 6-PPD quinone (6-PPDQ), has gained attention due to its toxicity towards coho salmon. In this study, the abiotic oxidative transformation of 6-PPD is investigated by a series of ozonation experiments in the lab followed by analysis of TPs using liquid chromatography-high resolution-mass spectrometry (LC-HRMS). A total of 38 TPs were detected and tentatively identified, which were formed either directly from 6-PPD or via 6-PPDQ as intermediate. A suspect screening by LC-HRMS showed 32 of these TPs to occur in snow collected from urban roads as surrogate of road-runoff, where 6-PPDQ, 4-aminodiphenylamine (4-ADPA), TP 213, and TP 249 were the most prominent besides 6-PPD. More than 90% of the total load of 6-PPD and its TPs was found in the particulate fraction of snow. Thus, retaining the particulate fraction of road runoff before its discharge into surface water would substantially reduce the emission of 6-PPD and many of its TPs. Some TPs prevailed in the water phase of the snow due to their higher polarity. A total of 13 TPs were detected by suspect screening in the dissolved phase of a wastewater treatment plant (WWTP) influent. Their total load was markedly enhanced during a day of snowmelt (approx. 1100 g/d) and rainfall (approx. 2000 g/d) compared to dry weather (approx. 190 g/d). 6-PPD and 6-PPDQ contributed to less than 1% to this total load in the water phase (estimated concentrations of max 0.1 µg/L). The elimination of the estimated total loads of 6-PPD related TPs from the water phase in WWTP ranged from 22 to 67% depending on weather conditions. Eventually TP 249, 4-ADPA and TP 259_2 dominated in WWTP effluent (estimated concentration from 0.5 up to 2 µg/L). Thus TP 249 and TP 259_2 are, likely, the most specific and stable TPs of 6-PPD to be determined in the environment.
Road runoff is an important vector for the transport of chemicals originating from tire wear into receiving waters. In this study, samples of surface water were collected in the summer of 2020 from two rivers near high-traffic corridors in the Greater Toronto Area (GTA) in Canada. These samples were analyzed for two additives used in tire production, 1,3-diphenyl guanidine (DPG) and hexamethoxymethylmelamine (HMMM), as well 26 of the transformation compounds of HMMM. In addition, samples were analyzed for 6PPD-quinone (6PPD-q), an oxidation by-product of a tire additive that was recently identified as a candidate compound responsible for mass mortalities of Coho salmon (Oncorhynchus kisutch) in spawning streams in the USA. Grab and composite samples were collected during rain events (i.e., wet events) at both locations. Grab samples were collected from the Don River upstream, downstream and at the point of discharge from a municipal wastewater treatment plant (WWTP) during a period of dry weather. Of the target analytes, 6PPD-q, DPG and HMMM, as well as 15 of the transformation compounds of HMMM, were detected at concentrations above limits of quantitation. The concentrations of 6PPD-q in the receiving waters during wet events were within the range of the LC50 for adult Coho salmon. One of the transformation products (TPs) of HMMM, dimethoxymethylmelamine was detected in a composite sample from Highland Creek at an estimated concentration greater than 10 μg/L, indicating that more research is needed to evaluate the potential hazards to the aquatic environment from this compound. Sampling in the Don River during a dry period showed that discharges of wastewater from WWTPs are also continuous sources of the TPs of HMMM. This study contributes to the growing literature showing that chemicals derived from tire wear are ubiquitous in urban watersheds and may be a significant hazard to aquatic organisms.
Tire and road wear particles (TRWPs) are a major component of non-exhaust traffic emissions, but knowledge about their physico-chemical properties is limited. Road dust of a highwy tunnel was fractionated by size and density, and fractions were analyzed for TRWPs, metals, seven tire tread indicator chemicals (benzothiazoles, 6-PPD and DPG) and effects in in vitro bioassays. TRWP content in tunnel dust was very high (11 – 12%). The peak of the TRWP mass distribution was in the size fraction 20 - 50 μm, with 31 - 36% of the total TRWP mass and a content of up to 260 mg/g. The mass of organic tire constituents peaked in the smallest analyzed size fractions (<20 μm) with 35 - 55% of their total mass. They also peaked in the density fraction 1.3 - 1.7 g/cm³, indicating a lower TRWP density and a higher contribution of TP to TRWP (approx. 75%) than expected. Video-based shape analysis and SEM showed elongated particles, likely TRWP, to be present in those size and density fractions ascribed to TRWPs by chemical analysis. But also irregular heteroagglomerates could be found. Solvent extracts of size and density fractions induced effects in bioassays indicative of the activation of the arylhydrocarbon receptor (AhR-CALUX) and the adaptive response to oxidative stress (AREc32). Similar comprehensive characterization of road dust from other sites may be needed to decide on whether TRWPs occurring in high concentrations in tunnel dust are suited as representative test materials for analytical purposes and TRWP fate studies.
The substituted p-phenylenediamines (PPDs) represent a suite of effective antioxidants broadly applied in rubber industries. However, knowledge of their environmental occurrences and fate remains extremely limited. Herein, we explored the occurrence of six major PPD antioxidants and one newly defined transformation product in dust particles from different environments, including roads, underground parking lots, vehicles, and houses. The majority of the PPDs exhibited ubiquitous occurrence in these environments. Median concentrations of total PPDs were determined to be 226 ng/g in road dust, 232 ng/g in parking lot dust, and 156 ng/g in vehicle dust, orders of magnitude greater than those in house dust (14.0 ng/g). Different composition profiles of PPDs were also found between house dust and vehicle-related dust, likely indicating the influence of vehicle tires or other rubber products. In addition, a major ozonation product of N-(1,3-dimethylbutyl)-N′-phenyl-1,4-phenylenediamine (6PPD), 6PPD-qunione, was also identified in dust with levels (median range of 32.2.80.9 ng/g) comparable to that of 6PPD except in house dust. To the best of our knowledge, this is the first systematic investigation of the occurrence of major PPD antioxidants and 6PPD-qunione in various dust matrices. Our findings would attract attention to their environmental fate and ecological and human health risks.
Tire tread particles turn streams toxic
For coho salmon in the U.S. Pacific Northwest, returning to spawn in urban and suburban streams can be deadly. Regular acute mortality events are tied, in particular, to stormwater runoff, but the identity of the causative toxicant(s) has not been known. Starting from leachate from new and aged tire tread wear particles, Tian et al. followed toxic fractions through chromatography steps, eventually isolating a single molecule that could induce acute toxicity at threshold concentrations of ∼1 microgram per liter. The compound, called 6PPD-quinone, is an oxidation product of an additive intended to prevent damage to tire rubber from ozone. Measurements from road runoff and immediate receiving waters show concentrations of 6PPD-quinone high enough to account for the acute toxicity events.
Science , this issue p. 185