Tesni Hyett, student Biomedical Scientist studying at Cardiff University and Sapienza University of Rome
Tesni Hyett is studying BioMedical Sciences at Cardiff University. She is the daughter of 3P Technik UK’s Director Glyn Hyett. During the summer break between her 2nd academic year and 3rd-year professional training year at Sapienza University of Rome, Italy, researching Oncology, Glyn asked Tesni to research 6PPD in Stormwater. Glyn had read some articles about the toxic effects of this chemical used in tyres. He wanted to understand the extent of research on the chemical in the environment and get the perspective of a young genetics-focused Biomedical Scientist who has no prior experience with stormwater The brief was deliberately kept very loose. Here is Tesni’s paper.
6PPD in the natural environment – a concern for Biomedicine and wider Scientific fields
6PPD is commonly added in rubber products, in particular tyre rubber.
Tyres, Stormwater and 6PPD, 6PPD-q
200 thousand tonnes of 6PPD (N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine) was produced in China alone in 20201. This seems like a great unimaginable amount, but when put into context of every vehicle in use today having at least two tyres, you can begin to comprehend the vast volume of 6PPD being produced and that which already exists. But what is 6PPD and where does the worn-off rubber including added 6PPD go?
What is 6PPD?
Figure 1. showing structural diagrams of 6PPD and 6PPD-quinone
6PPD is very unstable and readily undergoes a reaction with Ozone (O3) to produce a transformation product; 6PPD-quinone (6PPD-q).
6PPD and 6PPD-q has been detected in runoff water, wastewater discharges from wastewater treatment plants and river waters.
However, 6PPD-q was first reported in water samples in 2021, only 3 years ago.
It seems likely that the potential impact of 6PPD (and 6PPD-q) on the wider environment and ecosystems, including the human impact remains is critically unresearched.
Causes for concern
- 6PPD has a short hydrolysis half-life of ~54.8-64 hours, meaning a large amount of hydrolysis will occur whilst the water samples are being stored and analysed3. Therefore, it is likely current levels of 6PPD measured are a gross underestimate.
- Furthermore, there are concerns over many studies analysing 6PPD and its transformation products in their water phase. A more accurate assessment of their environmental presence would be gathered if analysed in both their water and particle phases.
- 6PPD and 6PPD-q are both hydrophobic, meaning they don’t bond well with water, and have a preference to dissolving in fats.3 Due to this preference of dissolving in fats, bioaccumulation could occur, threatening every level of the food chain.
- 6PPD-q can last longer than 6PPD in aquatic environments, increasing aquatic organisms’ exposure time to 6PPD-q.2
- Worryingly, 6PPD has now been reported in human samples (urine, serum and cerebrospinal fluid samples).6
Toxic effects of 6PPD – Fish
To date, much of the current research into the toxic effects of 6PPD focuses on fish species.
Interestingly, 6PPD-q exhibits species-specific effects. The lethal concentration 50 (LD50, i.e. the dose of 6PPD-q that causes death in half the sample of organisms tested) varied between species of fish (for more information see the table below). It has been suggested that differences in the target sites of 6PPDs and its interactions with these target sites, and the differences in absorption, distribution, metabolism and excretion of 6PPD within the organism may be responsible for these species-specific effects.3
Coho salmon is the most researched species of fish, attracting a lot of attention due to its importance in commercial and recreational fishing. 6PPD-q causes acute mortality and toxicity in Coho Salmon.3 This has been reported in the UK, and has gained some notoriety with stormwater quality scientists.
The exact mechanisms by which 6PPD and 6PPD-q exert their toxic effects are not known.
Research on Coho Salmon has shown that acute mortality may be driven by leakage from blood vessels into tissues at the blood-brain barrier, after exposure to 6PPD-q from roadway runoff water.3
In contrast, a study into the gill and liver cells of Rainbow Trout fish revealed tissue-specific disruption of mitochondrial respiration as a potential mechanism of 6PPD toxicity.3
Recent studies into Zebrafish have suggested other potential mechanisms. It has been suggested that Zebrafish growth may be stunted and inhibited due to 6PPD altering the distribution of endocrinal systems and pathways.3
Long-term exposure of 6PPD in Zebrafish leads to intestinal toxicity, through disruption of the functional state of the intestinal barrier.
Although much of the current and up to date research is mainly based on fish species, we can already see the complexity of the workings of 6PPD. It is also surely apparent what its potential implications as a water pollutant may prove to be.
Toxic effects of 6PPD – Mammals
Limited research has been conducted into 6PPD and mammals.
6PPD-q can quickly distribute itself around the mammalian body and be absorbed by the bloodstream and main organs. A study saw peak concentration of 6PPD-q within mouse models just one hour after oral administration.4
Researchers also observed 6PPD-q penetrating the blood-brain barrier in mice, only 30 minutes after administration.4 (It is worth noting that this same mechanism was observed in fish species).
Another interesting finding of this study is that biologically male mice had higher levels of circulating 6PPD-q.
A study on chronic exposure of 6PPD-q in male mice has since been conducted, revealing a consequential decrease in testosterone levels. It was found that 6PPD-q decreased all signalling pathways promoting the production of sperm, but an increase in the signalling pathways promoting cell death occurred.5 Ultimately leading to decreased sperm production, impacting sperm quality and their reproductive capacity.
As with some fish species, 6PPD toxicologically affects mammalian intestines. Studies have found that when 6PPD-q is administered orally, the intestines are affected in a dose-dependent manner. Again, as seen in fish species, the intestinal barrier was disrupted.7
Another study observed increased liver weight and liver enzyme levels at high doses of 6PPD-q.8
Whilst a wide range of impacts have been researched, it is surely reasonable to conclude that much more is yet to be unveiled.
Similarities between fish and mice have been found. However, fish showed significant species-specific effects, and most mammalian studies have only been conducted in mice models.
There may yet be very many potentially significant questions on the effects on other mammalian species.
On a perhaps more hopeful note, a potential pathway of intervening the mechanism of 6PPD-q within mammalian intestines has been discovered in a recent study. Key inflammation molecules have been specified as where site specific impairment takes place.7
Toxic effects of 6PPD – Humans
6PPD-q has been identified in human samples, as already mentioned.
A small study based on 200 samples (from 50 biological males and 50 biological females) reported 6PPD and 6PPD-q in plasma and urine samples. Data analysis of these 200 samples revealed a significant difference between 6PPD-q measured in urine samples between the male sex and female sex. No other demographic features showed significant correlations. The female sex tends to experience higher daily exposure to 6PPD and 6PPD-q.9
This difference between the two sexes can potentially be explained by differences in daily excretions and liver immune functions. Females were seen to typically experience higher excretion.
Of course, further research needs to be carried out, and larger populations and samples must be analysed.
Concluding remarks
Having reviewed the current research released on 6PPD and its potential impacts it has shocked me how little analysis of mammals, and in particular of humans has been done.
As a Biomedical Scientist, when I was asked to research and write this article I originally ignorantly thought:
What does this have to do with me and the science I am interested in? Why should I look into tyre rubber pollutants in the aquatic environment and in stormwater runoff?
It is much more obvious to me now, science is one global subject, much like an ecosystem, whether that be a microbiome or a tropical rainforest. Every component and area influences the others. Science is an interlinking field, we must utilise it to look after our ever-changing planet.
Further Information
The table below is extracted from a recent review article (Hua and Wang 20231) to show the range of studies carried out and their conclusions in terms of toxic effect after long-term 6PPD exposure and their indicative LD50s.
The current range of 6PPD studies
References:
- Hua, X., Wang, D. 2023. Tire-rubber related pollutant 6-PPD quinone: A review of its transformation, environmental distribution, bioavailability, and toxicity. Journal of Hazardous Materials 459(132265). https://doi.org/10.1016/j.jhazmat.2023.132265
- Tian, Z. et al. 2022. A ubiquitous tire rubber–derived chemical induces acute mortality in coho salmon. Science 375(6582) pp. 185-189. https://doi.org/10.1126/science.abd6951
- Chen, X., He, T., Yang, X., Gan, Y., Qing, X., Wang, J., Huang, Y. 2023. Analysis, environmental occurrence, fate and potential toxicity of tire wear compounds 6PPD and 6PPD-quinone. Journal of Hazardous Materials 452(131245) https://doi.org/10.1016/j.jhazmat.2023.131245
- Zhang, J. 2024. Stable isotope-assisted mass spectrometry reveals in vivo distribution, metabolism, and excretion of tire rubber-derived 6PPD-quinone in mice. Science of The Total Environment 912(169291) https://doi.org/10.1016/j.scitotenv.2023.169291
- Yao, K., Kang, Q., Liu, W., Chen, D., Wang, L., Li, S. 2024. Chronic exposure to tire rubber-derived contaminant 6PPD-quinone impairs sperm quality and induces the damage of reproductive capacity in male mice. Journal of Hazardous Materials 470(134165) https://doi.org/10.1016/j.jhazmat.2024.134165
- Jiang, Y., Wang, C., Ma, L., Gao, T., Wāng, Y. 2024. Environmental profiles, hazard identification, and toxicological hallmarks of emerging tire rubber-related contaminants 6PPD and 6PPD-quinone. Environmental International 187(108677) https://doi.org/10.1016/j.envint.2024.108677
- Yang, Y. et al. 2024. Environmentally realistic dose of tire-derived metabolite 6PPD-Q exposure causes intestinal jejunum and ileum damage in mice via cannabinoid receptor-activated inflammation. Science of The Total Environment 918(170679) https://doi.org/10.1016/j.scitotenv.2024.170679
- Fang, L., Fang, C., Di, S., Yu, Y., Wang, C., Wang, X., Jin, Y. 2023. Oral exposure to tire rubber-derived contaminant 6PPD and 6PPD-quinone induce hepatotoxicity in mice. Science of The Total Environment 869(161836) https://doi.org/10.1016/j.scitotenv.2023.161836
- Liu, C. et al. 2024. Emerging N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) and 6PPD quinone in paired human plasma and urine from Tianjin, China: Preliminary assessment with demographic factors. Journal of Hazardous Materials 476(134818) https://doi.org/10.1016/j.jhazmat.2024.134818