CHARACTERIZATION OF THE EFFECTS OF 6PPD-QUINONE EXPOSURE ON EARLY-LIFE STAGE ONCORHYNCHUS MYKISS AND SALVELINUS NAMAYCUSH

Urban Runoff Mortality Syndrome (URMS) refers to a phenomenon in which fish, particularly coho salmon (Oncorhynchus kisutch), suffer acute mortality after exposure to stormwater runoff in urban environments, with pre-mortem symptoms of gasping, loss of coordination, and surface swimming. First identified in 2011, the cause of URMS has been a focus of ongoing research. Recently, N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q), a derivative of the rubber tire antiozonant N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), was identified as the causative agent. 6PPD-Q enters surface waters through roadway runoff, as tire particles are swept into aquatic systems, resulting in pulses in concentration. 6PPD-Q toxicity has been shown to be highly variable across fish species, particularly within salmonids. While adult rainbow trout (Oncorhynchus mykiss) are known to be a sensitive species, there is limited research as to the vulnerability of their early-life stages. In addition, much of the research conducted thus far has focused on acute exposure scenarios, and little is known regarding sub-chronic 6PPD-Q toxicity. In addition, no data is currently published on the potential sensitivity or tolerance of lake trout (Salvelinus namaycush), another important Canadian salmonid. Given that environmental concentrations of 6PPD-Q can reach or exceed toxic thresholds for sensitive fish, the potential for significant ecological impacts on freshwater ecosystems is substantial. Thus, the overall objective of this thesis was to assess the acute and sub-chronic effects of 6PPD-Q on two salmonid species, rainbow trout and lake trout. The first study (Chapter 2) focused on determining life-stage specific toxicity of 6PPD-Q to early-life stage Oncorhynchus mykiss (rainbow trout), a species in which adults had previously been described as sensitive. A 28-day sub-chronic study was conducted on rainbow trout alevins, beginning at hatching and continuing through the exogenously feeding stage. The alevins were exposed to time-weighted average concentrations of 6PPD-Q ranging from 0.06 to 2.35 µg/L This first study revealed concentration- and time-dependent increases in mortality with a 28-day median lethal concentration (LC50) of 0.56 µg/L. Over the exposure period, increasing incidence of morphological deformities were observed, including spinal curvature, yolk sac edema, and blood pooling in the caudal fin. In a subsequent acute study, exogenously feeding rainbow trout fry exhibited a 96-hour LC50 of 0.47 µg/L. These findings indicate that early-life stages of rainbow trout are more sensitive to 6PPD-Q than previously studied sub-adults, with exogenously feeding fry being the most sensitive life stage of rainbow trout identified to date. Additionally, sub-chronic exposure to 6PPD-Q was associated with developmental abnormalities. This research underscores the importance of early-life stage studies in identifying sensitive benchmark concentrations and assessing sub-lethal effects. The second study (Chapter 3) focused on generating toxicity data for early-life stage Salvelinus namaycush (lake trout), a species for which no toxicity data for 6PPD-Q had been published previously. In the sub-chronic study, lake trout alevins were exposed starting at hatch, to time-weighted incremental average concentrations of 6PPD-Q between 0.22 and 13.5 µg/L for 45 days. This determined a 45-day LC50 of 0.39 µg/L, and growth deformities were noted in exposed alevins, including spinal curvature, yolk sac edema, and distinctive pooling of blood in the caudal fin and eye. Subsequently, a 96-hr acute study was conducted on juvenile lake trout (8 weeks post-hatch) to determine acute toxicity to the exogenously feeding life stage. The 96-hour LC50 of acutely exposed exogenously feeding lake trout fry was slightly greater than the 45-day LC50 at 0.50 µg/L. These findings reveal that both lake trout alevins and exogenously feeding fry are sensitive to 6PPD-Q, highlighting the significance of this compound for inland freshwater ecosystems. This thesis as a whole examined the acute and sub-chronic toxicity of 6PPD-Q on early-life stages of rainbow trout and lake trout. Acute exposure revealed that both species, and particularly the juvenile life stage, are highly sensitive to 6PPD-Q. Sub-chronic exposure led to significant mortality and sub-lethal effects like yolk sac edema and spinal curvature, which could impair fitness and survival in the wild. While the sensitivity of rainbow trout to contaminants underscores its value as a model organism for ecotoxicity testing, this thesis research also highlighted gaps in understanding, such as exposure pathways in spawning habitats and effects on unstudied species. Current regulatory frameworks may inadequately assess the risks of chemicals like 6PPD-Q, emphasizing the need for early-life stage testing and alternatives development. The findings support enhanced chemical regulation, targeted research on sensitive fish species, and mitigation strategies to address the broader ecological impacts of roadway runoff.

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