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.


INTRODUCTION
N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone (6PPD-quinone) is a prevalent contaminant of emerging concern resulting from oxidation of 6PPD, which is an antidegradant substance added to tires.Tire wear is the primary source of 6PPD-quinone in nature, typically constituting between 0.4% and -2% of the total weight of car tires (Babbit, 2010).
Following the initial discovery of the acute toxic effects of 6PPD-quinone in coho salmon (Oncorhynchus kisutch) by Tian et al. (2021), subsequent testing of other species revealed that toxicity of 6PPD-quinone is highly specific to only certain species.Toxicity tests conducted using either commercial standards of 6PPD-quinone, tire leachate, or untreated urban roadway runoff have so far been conducted on 17 species of fish and 9 other organisms (Table 1).Acute toxicity at environmentally relevant concentrations (<2.43 μg/L; Cao et al., 2022;Challis et al., 2021;Johannessen et al., 2022;Kryuchkov et al., 2023;Rauert et al., 2022) has been documented in five species within the Oncorhynchus and Salvelinus genera.Risk assessment of this chemical is complicated by the fact that closely related species within these genera differ widely in their sensitivity to 6PPD-quinone.For example, the median lethal concentration (LC50) for coho salmon is 0.041 μg/L (Lo et al., 2023) whereas the closely related sockeye salmon (Oncorhynchus nerka; French et al., 2022) and chum salmon (Oncorhynchus keta; McIntyre et al., 2021) lack a response to the chemical.Moreover, there are indications that the toxicity of 6PPD-quinone can change substantially throughout the ontogeny; the LC50 for coho alevins (3 weeks old) has been reported to be 0.041 μg/L (Lo et al., 2023) whereas the LC50 for parr (>1 year) is more than double, at 0.095 μg/L (Tian et al., 2022).
Because the underlying cause of the species-specific toxicity pattern of 6PPD-quinone remains elusive, toxicity testing to determine the sensitivity of a given species is necessary.We present the first results from acute toxicity testing of 6PPD-quinone on pink salmon, the only Pacific salmon that has not previously been tested.

Exposure experiment
Exposure of pink salmon to 6PPD-quinone was conducted at the Norwegian Institute for Nature Research (NINA) Aquatic Research Station located in Rogaland, western Norway (58°54′ N, 5°57′ E).The fish used in the experiment originated from a stock of wild parent fish caught in the River Kongsfjord (Finnmark, Norway; 70°39′ N, 29°15′ E).Eggs were fertilized and incubated at the Norwegian Institute of Food, Fisheries, and Aquaculture Research Tromsø Aquaculture Research Station (69°52′ N, 18°55′ E) and transported to the NINA Aquatic Research Station during the "eyed" stage.The eggs hatched between September 26 and 29, and the experiment was conducted from October 17 to 19, 2023; hence the pink salmon alevins were between 19 and 22 days old at the start of the experiment.
The experiment was performed in static water treatments for 48 h, with a total of eight different 6PPD-quinone concentrations (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8 μg/L) and two controls.One of the controls consisted of water from the research station, and the other control consisted of the same fish media as used in the preparation of the 6PPD-quinone solutions.Fish media and 6PPD-quinone solutions were prepared at the Norwegian Veterinary Institute.Fish media were prepared to match the chemical properties of the NINA research station water, which comes from a deep intake (13 m) in a nearby lake and is filtered (mesh 0.1 mm; Bernoulli filter) and UV and ozone treated before entering the station.The water quality parameters from August 2023 were as follows: pH 6.7, turbidity 0.54 FNU, conductivity 6.5 mS/m, Ca 2+ 3.6 mg/L, Mg 2+ 1.2 mg/L, total N 0.59 mg/L, and total organic carbon 4.7 mg/L.All data collected during the experiment and complete water chemistry data are openly available (see Data Availability Statement).
To minimize changes in environment and handling, the experiment was conducted in the same room, and treatments were placed in the same type of hatching chambers and kept at the same temperature as the fish had experienced prior to the experiment.Fish were gently transferred to glass containers containing 1 L of fish media or control water.Each treatment had a minimum of 10 fish (some treatments had 11).Treatments were oxygenated prior to the experiment.A subset (n = 10) of the pink salmon alevins used in the experiment was measured individually and had an average length of 25.9 mm (SD 1.37 mm) and a combined weight of 1.44 g.Throughout the experiment oxygen, pH, and temperature were monitored, and fish were observed every 3 to 6 h, using a Handy Polaris 2 device (Oxyguard); pH measurements were done on 30-mL aliquots using a PHM92 device (Radiometer).Fish that were not actively moving were gently touched with a rounded metal probe to induce movement.Water samples were collected in preprepared probes (see the Analysis of 6PPD-quinone concentrations section) after 0.5, 3, 12, 24, 30, 36, 42, and 48 h for analysis of 6PPD-quinone concentrations.

Preparation of fish media and test solutions
Fish media containing 6PPD-quinone were prepared using Milli-Q water.Initially, 20 L of media was made by diluting salt to match the water chemical properties of the NINA research station (CaCO 3 0.091 mM, MgCO 3 0.051 mM, NaHCO 3 0.071 mM, NaCl 0.175 mM, Na 2 SO 4 0.022 mM, and K 2 SO 4 0.013 mM).An acetone solution of approximately 200 µg of 6PPD-quinone was applied to the bottom of a 1-L flask and evaporated to dryness under a gentle nitrogen flow.The residue was then dissolved in 1 L of fish media and filtered through a 53-µm filter.The concentration of 6PPD-quinone in the resulting solution was assessed using the same liquid chromatography-tandem mass spectrometry (LC-MS/MS) method as described in the following section, Analysis of 6PPD-quinone concentrations.This stock solution was utilized to prepare dilutions (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8 μg/L), and each dilution was verified before being used in the experiment.

Analysis of 6PPD-quinone concentrations
Probes, each containing 1 ng of 13 C 6 -6PPD-quinone, were prepared prior to the experiment and utilized to sample 1 mL of fish media at time points throughout the experiment (see the previous section, Exposure experiment).After the exposure experiment was completed, all probes were stored at +5 °C and sent for analysis.An LC-MS/MS device was employed, comprising an Agilent 1100 high-performance (HP)LC system and an Agilent 6410 triple quadrupole MS/MS.Analyses were conducted on 30-µL aliquots of the samples.The samples were compared against six calibration solutions with varying levels of 6PPD-quinone (from 0.05 to 10 ng/mL) and a constant level of 13 C 6 -6PPD-quinone (1.0 ng/mL).The limit of quantification (LOQ) was determined as 10 times σ/S, where S represents the slope of the calibration curve, and σ is the standard deviation of y-intercepts.The LOQ was found to be 0.049 ng/mL.Two multiple reaction monitoring transitions were acquired for each 6PPD-quinone and its isotopically labeled internal standard spiking solution in positive ionization mode: 6PPD-quinone, m/z 299 → 187 (collision energy [CE] = 34 V) and m/z 299.0 → 241 (CE = 34 V), 13 C 6 -6PPD-quinone, m/z 305 → 193 (CE = 34 V) and m/z 305 → 247 (CE = 34 V) for quantifier and qualifier ion transitions, respectively.For further details on probe preparation and the LC-MS/MS method evaluation, see Foldvik et al. (2022).

RESULTS AND DISCUSSION
During the experiment no mortality or abnormal behavior of the pink salmon alevins were observed.When undisturbed, alevins would mostly be inactive at the bottom of the glass containers.Alevins would react to disturbances caused by sampling with a burst of swimming, and movement of one fish would induce swimming in others.Specifically, no observations of behavior previously reported in sensitive species such as circling and surfacing (Blair et al., 2021;Brinkmann et al., 2022;French et al., 2022;Hiki & Yamamoto, 2022b) or excessive mucus secretion (Hiki & Yamamoto, 2022b) were recorded.As expected, concentrations of 6PPD-quinone decreased from the target concentration throughout the duration of the experiment (Figure 1).The concentrations dropped from approximately 80% of the target concentration at the start of the experiment to approximately 40% after 48 h.The treatment with the highest initial concentration (12.8 μg/L) had concentrations of 7.6 and 5.7 μg/L after 24 and 48 h, respectively, showing that the pink salmon alevins do not exhibit mortality even above environmentally relevant concentrations.The temperature the fish experienced increased from approximately 1 °C before the experiment to between 2 and 5 °C (mean 3.82 °C, SD 0.57) during the experiment, and the pH decreased from on average pH 6.89 in the first half of the experiment to pH 6.45 in the last half.All measurements conducted during the experiment are openly available (see Data Availability Statement).
Our findings demonstrating a lack of sensitivity of pink salmon alevins to 6PPD-quinone add to the rapidly growing literature studying the effects (and lack of effects) of 6PPDquinone on fish and other organisms.Pink salmon are closely related to coho salmon, chinook salmon, and rainbow trout (Oncorhynchus mykiss), all of which exhibit a strong sensitivity to 6PPD-quinone (Table 1), and they are also closely related FIGURE 1: Boxplot of relative concentration of N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone (6PPD-quinone) in relation to initial concentration (y-axis) sampled during the experiment (hours after start of experiment, x-axis).The bottom and top of the box indicate the 25th and 75th percentiles (i.e., the boxes include the middle 50% of observations), the whiskers span to the most extreme datapoint if that is more than 1.5 times the interquartile range, and the bold horizontal line represents the median value.
to sockeye salmon and chum salmon, which, like pink salmon, are tolerant of the chemical.The two other species shown to exhibit acute toxicity to 6PPD-quinone are brook trout (Salvelinus fontinalis; Brinkmann et al., 2022) and the whitespotted char (Salvelinus leucomaenis; Hiki & Yamamoto, 2022b).The phylogenetic relationship between the tested species of the family Salmonidae shows some phylogenetic structuring of sensitivity to 6PPD-quinone (Figure 2), but sensitivity to 6PPD-quinone appears to either have evolved or disappeared several times.
Variation in sensitivity at different life stages in coho salmon (Lo et al., 2023;Tian et al., 2022), and the potential for synergistic effects with other contaminants, means that caution should be used in drawing conclusions from our study in terms of pink salmon at other life stages, under other abiotic conditions, and with longer exposure durations.
Except for the genera Oncorhynchus and Salvelinus, no species have exhibited acute toxicity to 6PPD-quinone at environmentally relevant concentrations.Recently sublethal effects of 6PPD-quinone at high concentrations have been studied and documented in both fish and mammals (He et al., 2023;Ji et al., 2022;Varshney et al., 2022;Zhang et al., 2023); these studies document changes in a wide range of traits and processes such as behavior, morphology, respiration, neurotransmitters, organ injury, and genomic expression.Sublethal effects have also been documented at environmentally relevant concentrations in the nematode Caenorhabditis elegans (Hua & Wang, 2023).
The extreme variation between closely related species in response to 6PPD-quinone prevents meaningful environmental risk assessment outside of the tested species.To further complicate the picture, acute toxicity of the parent compound 6PPD has been shown for a cyprinid fish endemic to China (Gobiocypris rarus; Di et al., 2022) that is tolerant of 6PPDquinone.Rainbow trout, which is sensitive to 6PPD-quinone, is tolerant of 6PPD (Di et al., 2022).Furthermore, sublethal effects of 6PPD have been shown in zebrafish (Danio rerio, Zhang et al., 2023), and synergetic effects of 6PPD and salt have been shown in the rotifier Brachionus calyciflorus (Klauschies & Isanta-Navarro, 2022).
With the emerging data on 6PPD-quinone prevalence in the environment, the documented lethal and sublethal effects of 6PPD-quinone, and the toxicity of other paraphenylenediamine-based rubber additives, further research in these areas will be critical to advance our understanding of the effect these chemicals have on the environment, to guide the development of eco-friendly alternatives, and ensure the longterm environmental and health sustainability of rubber-based products.
This article has earned both an Open Data and an Open Materials badge for making publicly available the digitally shareable data necessary to reproduce the reported results.The data are available at https://osf.io/92jwr/?view_only=92e8ca40d07a4d5789c31cb8 443f386c.Learn more about the Open Practices badges from the Center for Open Science: https://osf.io/tvyxz/wiki.

TABLE 1 :
List of species tested for effects of 6PPD-quinone and 6PPD