CONTROLLING TOXIC CHEMICAL POLLUTION OF URBAN AQUATIC ECOSYSTEMS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: MCINTIRE-STENNIS
• Reporting Frequency: Annual
• Accession No.: 1015288
• Project Start Date: Feb 27, 2018
• Project End Date: Jan 31, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001

Performing Department
School of the Environment

Non Technical Summary
Due in part to the success of the Clean Water Act controlling large point-sources of pollution, toxic chemicals in the U.S. enter urban water systems primarily through stormwater runoff. From a limited number of investigations, we know that urban road runoff is acutely toxic to a variety of fish and aquatic invertebrates (McIntyre et al. 2015, Kayhanian et al. 2008, Skinner et al. 1999, Bay et al. 2003), including adult coho salmon (Spromberg et al. 2016), as well as juveniles (McIntyre et al. 2015), and post-hatch embryos (McIntyre in prep). Overall, we have a very poor understanding of the vulnerability of aquatic wildlife to urban road runoff (e.g., Puget Sound; (Mackenzie & McIntyre 2017) in forested watersheds.Green stormwater infrastructure (GSI) encompasses an evolving set of technologies to mitigate the impacts that urban stormwater runoff has on physical and chemical habitats of aquatic ecosystems. Prominent among GSIs is the approach of 'bioretention' - methods for encouraging stormwater to infiltrate into the ground. By infiltrating rather than running off into waterways, bioretention has the potential to mimic the hydrology of undeveloped basins (DeBusk et al. 2011) and to filter contaminants present in urban stormwater (Ahiablame et al. 2012). Only recently has the biological effectiveness of GSI been explored. Bioretention can prevent acute mortality in adult coho salmon (Spromberg et al. 2016), in juvenile coho salmon and their prey (McIntyre et al. 2015), and can prevent sublethal toxicity in developing fish embryos (McIntyre et al. 2014), but much more work is needed to determine the most effective GSI implementation for protecting aquatic species and thus the forests that depend on them.In addition to a narrow understanding of the species that are sensitive to urban runoff in the Pacific Northwest (or elsewhere), there is little to no mechanistic understanding of the toxicity of urban runoff to aquatic animals. As a result, our toolbox for evaluating the biological effectiveness of GSI is extremely limited. A popular experimental model for exploring mechanisms of toxicity is the zebrafish (Danio rerio). Initial work into the mechanistic toxicology of urban runoff with zebrafish embryos has shown cardiovascular damage (McIntyre et al. 2014) related to exposure to aromatic hydrocarbons (McIntyre et al. 2016). Concomitant with visible cardiotoxicity, we identified a small set of cardiac-specific genes that were responsive to runoff exposure. Cardiotoxicity may be an important underlying pathophysiology in fishes exposed to urban stormwater runoff, but more studies are needed to elucidate the molecular initiating events that lead to toxicity in aquatic species exposed to urban runoff so that we can sustain forest ecosystems.In symptomatic coho salmon spawners, RNA sequencing has identified a number of candidate genes that are upregulated. Primary among these are egr1 (early growth factor-1) in gill and heart, and c-jun and c-fos in gill, heart, and liver. All three genes are important to cell signaling pathways and are activated by conditions of inflammatory, oxidative, and other kinds of cellular stress. Much more research is needed to understand the role of these genes in the acute mortality syndrome of coho salmon exposed to urban runoff.In order to develop and implement GSI that will effectively protect sensitive species like coho salmon from extirpation in areas undergoing development, we need tools to assess GSI that are inexpensive to use, sensitive to urban runoff, relevant for the protection of at-risk species, and reliable in terms of reproducibility.

Animal Health Component

50%

Research Effort Categories

Basic

30%

Applied

50%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
314	0812	1150	100%

Knowledge Area
314 - Toxic Chemicals, Poisonous Plants, Naturally Occurring Toxins, and Other Hazards Affecting Animals;

Subject Of Investigation
0812 - Fish habitats;

Field Of Science
1150 - Toxicology;

Keywords

urban stormwater runoff

aquatic toxicology

molecular biology

coho salmon

zebrafish

herring

biomarkers

molecular initiating event

green stormwater infrastructure

bioretention

Goals / Objectives
Develop molecular biomarkers for exposure to urban stormwater runoff that are sensitive, relevant, and reliableUse molecular biomarkers to help determine the molecular initiative events that cause observed acute toxicity from urban runoff in relevant fishesTest the ability of green stormwater infrastructure technologies to prevent biomarker activation leading to acute toxicityGiven the potential deleterious effects of stormwater on important Puget Sound fishes, we propose developing tools to accurately assess the impacts of stormwater, and the ability of GSI to mitigate those effects. Specifically, we propose exploring the molecular initiating events that lead to the most sensitive and relevant impacts of urban runoff in coho salmon and zebrafish. Coho salmon are very sensitive to urban road runoff, dying after just a few hours of exposure in both the lab and the field. Zebrafish is a model fish species with rapid developmental timing that is useful for high throughput toxicology screening. Zebrafish can also be a useful surrogate for studying toxicity mechanisms in wild species-of-concern.Ideally, this work will result in reliable biomarkers of runoff exposure that are mechanistically linked to sensitive, relevant impacts in the field. Examples of questions that will be addressed include: Are multiple organ systems involved in acute toxicity from urban runoff? What are the molecular initiating events (MIE) that lead to observed impacts on affected organ systems? Are MIEs species-specific?Biomarkers are biochemical measurements used to assess an organism's response to acute or chronic exposure to contaminants. For example, induction of cyp1a transcription can be used to identify exposure of fish to PAHs (Whyte et al. 2000, Incardona et al. 2009, Incardona et al. 2015), or vitellogenin expression is used to identify male and immature fish exposed to estrogenic chemicals (Tyler et al. 1999). Ideally robust biomarkers of exposure should be specific to the contaminant of concern and indicative of the level and duration of exposure. However, the molecular markers identified in response to stormwater exposure so far (e.g. cyp1a, c-jun, c-fos) are genes commonly induced in response to stress. Moreover, with a complex mixture of contaminants such as stormwater, a suite of biomarkers may be necessary to form a "molecular fingerprint" of organisms' response to exposure.In order to identify a "fingerprint" of stormwater exposure, we propose to conduct high-throughput sequencing followed by RNA-Seq and pathway analyses. RNA-Seq analysis enables comparison of all the genes expressed in a given tissue without a priori knowledge of the molecular pathways involved. In this way, RNA-Seq is a useful tool for identifying novel biomarkers of stormwater exposure. In addition, pathway analyses can be used to identify signaling pathways and upstream molecular events involved in stormwater toxicity. These methods can improve our understanding of molecular mechanism of toxicity and prespawn mortality in salmon.

Project Methods
Runoff collectionUrban stormwater runoff is collected during rain events in stainless steel totes (240 gallon and/or 450 gallons) from an elevated urban highway in Seattle, WA at the facility of our collaborators at NOAA-NMFS Northwest Fisheries Science Center. Runoff can then be transported to Puyallup or other locations for use in toxicity testing within 72 h of collection. Runoff may alsobe frozen for later toxicity testing. We previously showed that frozen runoff does not cause a loss of toxicity in zebrafish embryo exposures (McIntyre et al. 2014).Green stormwater infrastructure treatment of runoffIn some experiments, runoff will be treated by GSI and co-exposed to test organisms with untreated runoff. The GSI used will be those for research projects funded with other grants and may include bioretention, bioswales, and green roofs.FacilitiesExperiments will take place at a combination of the freshwater Fish Toxicology Laboratory at the WSU Research & Extension Center in Puyallup, the freshwater facilities at the NWFSC in Seattle, and the freshwater Suquamish Tribe Grovers Creek Salmon Hatchery in Indianola.The water quality and rearing capacity at the Fish Toxicology Lab need to be upgraded. The dechlorinated municipal city water used for rearing and as control water in all experiments contains dissolved zinc at concentrations that exceed limits recommended for the health of aquatic animals. Other water on the WSU campus does not contain these elevated zinc concentrations, therefore the problem is local to the fish lab and likely a result of old pipes that locally leach zinc. We will replace the existing water quality system with a reverse osmosis system that will continuously produce Type III purity water. The pure water will then be conditioned with salts to maintain a constant pH and concentration of micro- and macronutrients for healthy fish populations. This new water source will feed both the salmon rearing and zebrafish rearing facilities at the Fish Toxicology Lab.The rearing facilities for salmon will expanded to enable simultaneous rearing of more fish of different ages as well as the capacity to hold species other than coho salmon. Finally, the filtration system will be upgraded to one that needs less regular cleaning.Animals and ExposuresCoho salmon are reared at the Fish Toxicology Laboratory at the WSU Research & Extension Center in Puyallup. They can be used in toxicity testing beginning in the spring of their first year, through the spring of their third year. Exposures take place in glass or fiberglass tanks for up to 96 h following standard acute toxicity testing protocols (U.S. EPA 2002), including 3 replicates of 10 fish each maintained at 13 °C. Euthanized fish will be sampled for blood for hematology and other tissues for use in histology as well as transcriptomics. The Suquamish Tribe generously allows us use of their Grovers Creek facilities for experiments with adult coho salmon that have recently returned from the Pacific Ocean to spawn. Exposures with adult salmon take place in 800-L HDPE tanks for up to 24 h. Blood and tissues are similarly sampled from euthanized adult fish.Zebrafish are reared at both the Fish Toxicology Lab and the NWFSC. Embryos are exposed at 2-4 hours post fertilization (hpf) for up to 96 h. Four replicates of 15 embryos each are exposed in glass petri dishes containing 10 mL of rearing water (control) or runoff incubated at 28°C. At test termination, individuals are dechorionated (if necessary), embedded in 2% methylcellulose, and imaged live with a Nikon SMZ stereomicroscope. Digital photographs and are taken and morphometrics assessed with image analysis software, including embryo length, eye size, and pericardial area. From digital video, heart rates are counted and embryos are scored for presence/absence of cardiovascular defects such as arrhythmia, cardiac looping defects, and internal hemorrhaging. Groups of embryos are snap frozen in liquid nitrogen and later homogenized for molecular assays such as transcriptional changes in important genes such as biomarkers of contaminant exposure and cardiac injury.ExperimentsStudies will be conducted with each fish species to further explore the mechanistic toxicology of urban runoff. In concert with pathophysiological investigations supported by other funding sources, tissues will be sampled from individuals at various dilutions of runoff, exposure durations, and symptomatic stages. Total RNA will be isolated from whole embryos (zebrafish) or tissues of interest (salmon) from control and stormwater exposed fish and submitted for Illumina paired-end sequencing. Sequences will be aligned to existing reference genomes (zebrafish or salmon) or aligned de novo using the De novo RNA-Seq Assembly Pipeline (DRAP) and Trinity transcriptome assembler. For the de novo assembly, contiguous sequences (contigs) will be annotated using BLASTX (Altschul et al, 1990) againstthe NCBI non-redundant protein database (www.ncbi.nlm.nih.gov). Sequences with an e-value ≤ E-05 will be retained in the backbone.To examine differential transcript levels between control and exposed fish, RNA-Seq will be performed using RSEM (Li & Dewey 2011). Count estimates generated by RSEM will be analyzed for differentially expressed genes using DESeq (Anders & Huber 2010). Transcripts with an adjusted p-value ≤ 0.05 will be considered significantly altered. Clustering analysis will be performed on differentially expressed transcripts using the cluster::agnes package in R with the Spearman method (Maechler 2015). Pathway and network analyses will be conducted using Ingenuity Pathway Analysis (IPA) software. Significantly altered pathways and biological functions will be determined using the Fisher Exact Test (p ≤ 0.05). Following RNA-Seq and pathway analyses, targeted gene expression analysis will be conducted to confirm the utility of putative biomarkers. Confirmed biomarkers of stormwater exposure can then be used to determine the effectiveness of GSI to mitigate the toxicity of stormwater runoff.

Progress 02/27/18 to 01/31/21

Outputs
Target Audience:Managers, regulators, researchers, and concerned cititizens of water resources, aquatic wildlife, and fisheries. Changes/Problems:The impacts of stormwater on coho salmon are very acute; progressing from mild changes in behavior to death within hours. This does not leave much time for the fish to mount a response that will be moderated bychanges in gene transcription. As such, the proposal to search for molecular pathways in coho affected by stormwater that can be used to develop tools for evaluating stormwater treatment effectiveness is premature. Our focus for the project therefore shifted to elucidating the underlying physiological changes that occur in coho leading from health to death within hours of exposure to stormwater as a first step towards identifying the target tissue(s), which is necessary for ultimately establishing the molecular initiating events. What opportunities for training and professional development has the project provided?The project offered training opportunities for 2 postdocs, 1 PhD student, and 2 MS students. Two MS thesis were completed, three first-author papers for the graduate students, and co-authorship on an additional 4 papers. How have the results been disseminated to communities of interest?Results have been shared with communities of interest through regional and national presentations and meetings where the PIs, postdocs, and graduate students give invited or submitted talks, as well as through published peer-reviewed research papers, and interviews. 13 presentations at scientific conferences given by the PI 19 interviews published on radio, TV, or news paper/online 28 invited presentations given by the PI 34 poster or oral presentations at scientific conferences given by graduate students/postdocs What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Key outcomes for the project were changes in knowledge generated by 7 experiments/datasets: 1) Characterized the acute mortality syndrome in coho spawners Adult coho returning to freshwater to spawn were exposed to stormwater collected from a busy roadway in Seattle. After 4-6 h behavior was scored and blood physiology assessed compared with time-matched controls exposed to well water. Coho in stormwater displayed a spectrum of altered behaviors ranging from lethargy to immobility, with the most common being extreme lethargy. Controls were overwhelmingly asymptomatic. Blood of exposed fish had reduced pH and plasma ions, with significantly elevated hematocrit (thicker blood), compared with controls. Co-exposed adult chum showed no changes in behavior or blood parameters relative to controls. (McIntyre et al. 2018) 2) Confirmed the vulnerability of juvenile coho salmon, more fully characterized syndrome Juvenile coho salmon showed acute mortality during exposure to stormwater. Surface- and bottom-oriented behavior tracked during exposure showed a progression from discrete surfacing to continuous surface swimming, to loss of equilibrium, and immobility on the bottom of the tank prior to mortality. With mortality occurring on average 6 h into the exposure, the first change in behavior was evident at 45 min. Fish transitioned to clean water at the appearance of continuous surface swimming were not able to recover. They died in the same amount of time as fish that remained in runoff. In agreement with observed behaviors, blood parameters of sick fish were consistent with those observed in adult coho, confirming that juveniles can be used as a model for studying impacts on adults. (Chow et al. 2019) 3) Blood cells are not the target of contaminants in coho salmon affected by stormwater Blood from coho salmon was used to develop a robust new method for determining methemoglobin concentrations in fish. Methemoglobin (metHb) is formed when the hemoglobin in red blood cells is oxidized. This can occur when an organism is exposed to strongly oxidizing substances. MetHb cannot transport oxygen and can lead to tissue hypoxia if insufficient reducing power in the blood results in elevated concentrations of metHb. Fish hemoglobins are different than those in humans, requiring direct determination of their molar adsorptivities prior to using standard spectroscopic techniques to measure specific hemoglobin species like metHb. In determining these molar adsorptivities, we discovered that coho tend to have unstable metHb that spontaneously converts to hemichrome and precipitates from solution, resulting in overestimation of metHb. We developed a multivariate modeling technique to complement multi-component analysis in determining the molar adsorptivities of coho hemoglobin species. This model was then used to determine that coho affected by stormwater did not have significantly elevated metHb. Changes in behavioral and blood chemistry in sick coho that are consistent with cardio-respiratory distress therefore are not a result of insufficient oxygen transport capability by red blood cells. (Blair et al. 2020; Blair et al. 2021) 4) Hemoconcentration in coho affected by stormwater is related to changes in the vascular system Coho affected by stormwater show extremely elevated hematocrit (HCT) in addition to behaviors consistent with cardiorespiratory distress. The goal of this study was to explore reasons for the elevated HCT. We exposed juvenile coho salmon to collected roadway runoff previously shown to produce acute symptoms and mortality in this species. We sampled their blood and also conducted tests of vascular integrity via injection of a high molecular weight tracer followed by saline perfusion. We did not observe a significant production of metHb, confirming that oxygen transport by red blood cells was not involved in the cardiorespiratory distress. Antioxidant power of the blood plasma was also not different in stormwater-exposed coho compared with controls. Evans Blue dye was injected into fish hearts and allowed to circulate through the vasculature before being rinsed out by clean saline perfusion. Significant dye retention was observed in the brains of coho exposed to stormwater, suggesting that plasma - including high molecular weight molecules - was leaking from the cerebrovasculature. Anecdotally, dye was also observed leaking from the gills in runoff-exposed coho but not in control coho. A loss of vascular integrity appears to explain the elevated HCT in sick coho and led us to postulate that blood-brain barrier disruption underlies the symptoms of cardiorespiratory distress in sick coho exposed to stormwater. (Blair et al. 2021) 5) Coho exposed to stormwater have lost integrity of their blood brain barrier In the study by Blair et al. (2021), we wanted to confirm that the accumulation of the dye in the brain was due to extravasation rather than possibly retained blood (and dye) within the vasculature. We perfused coho with horseradish peroxidase (HRP) - an enzyme that has commonly been used to study blood brain barrier disruption. Followed by a clean saline perfusion, brain tissues were prepared for histology. Prepared tissue slices were treated with hydrogen peroxide to react with retained HRP and allow its localization as either intravascular or extravascular. Although we expected to see discrete locations with diffuse staining in the brains of coho exposed to stormwater, we instead saw massive diffuse HRP staining. This was compared with essentially no staining in controls. These results confimed plasma leakage and suggest that sick coho are suffering from a very pronounced failure of the blood brain barrier. Ongoing studies are exploring the timing and severity of the development of these symptoms. 6) Tire chemicals are sufficient to recreate the acute mortality syndrome in coho Land use analysis and water chemistry studies by our group and others suggested that tires might be the source of chemicals causing toxicity to coho salmon. We tested the response of coho to a 'tire particle leachate', made by recirculating water over particles abraded from the tread of tires. Adult coho salmon died when exposed to the leachate at concentrations that were environmentally realistic levels. Behavioral changes and blood physiology were identical to those caused by collected stormwater. Adult chum did not show any of these impacts, similar to their response to stormwater. (McIntyre et al. 2021) 7) The primary toxicant responsible for acute mortality of coho is 6PPD-quinone Tire particle leachate containing >2000 chemicals was fractionated to reduce chemical complexity and enable toxicant identification. Fractions were tested for toxicity to coho and also examined by high resolution mass spectrometry. Utilizing techniques including cation exchange, polarity-based separation, reverse phase high performance liquid chromatography (HPLC), and multi-dimensional HPLC, chemical complexity of the toxic fraction was reduced to just four chemicals. The most abundant chemical in the final fraction was a previously unknown chemical; C18H22N2O2. The parent chemical was determined to be 6PPD (N-(1,3- dimethylbutyl)-N′-phenyl-p- phenylenediamine) - the most widely used anti-ozonant in tire rubber. By subjecting commercial 6PPD (C18H24N2) to ozone, we produced C18H22N2O2, confirming by nuclear magnetic resonance that the unknown chemical was 6PPD-quinone. Purified 6PPD-quinone, isolated from tire leachate or from 6PPD subjected to ozone, was highly acutely lethal to coho salmon. The median lethal concentration (LC50) was estimated as 0.79 μg/L (0.63-0.96 μg/L 95% confidence interval). This was not different from the LC50 for tire particle leachate and roadway runoff on a 6PPD-quinone basis of 0.82 μg/L (0.56-1.10 μg/L), supporting that this was the primary causal toxicant for coho salmon exposed to roadway runoff. (Tian et al. 2021)

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Blair, S., C. Barlow & J. K. McIntyre (2021). Acute cerebrovascular effects in juvenile coho salmon exposed to roadway runoff. Canadian Journal of Fisheries and Aquatic Sciences, 78: 103.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: McIntyre, J. K., J. Prat, J. Cameron, J. Wetzel, E. Mudrock, K. T. Peter, Z. Y. Tian, C. Mackenzie, J. Lundin, J. D. Stark, K. King, J. W. Davis, E. P. Kolodziej & N. L. Scholz (2021). Treading water: Tire wear particle leachate recreates an urban runoff mortality syndrome in coho but not chum salmon. Environmental Science & Technology, 55((17)): 11767.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Tian, Z. Y., H. Zhao, K. T. Peter, M. Gonzalez, J. Wetzel, C. Wu, X. Hu, J. Prat, E. Mudrock, R. Hettinger, A. E. Cortina, R. G. Biswas, F. V. C. Kock, R. Soong, A. Jenee, B. Du, F. Hou, H. He, R. Lundeen, A. Gilbreath, R. Sutton, N. L. Scholz, J. W. David, M. C. Dodd, A. Simpson, J. K. McIntyre & E. P. Kolodziej (2021). Ubiquitous tire rubber-derived chemical induces acute mortality in coho salmon. Science, 371(6525): 185.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Chow, M. I., J. I. Lundin, C. J. Mitchell, J. W. Davis, G. Young, N. L. Scholz & J. K. McIntyre (2019). An urban stormwater runoff mortality syndrome in juvenile coho salmon. Aquatic Toxicology, 214.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: McIntyre, J. K., J. I. Lundin, J. R. Cameron, M. I. Chow, J. W. Davis, J. P. Incardona & N. L. Scholz (2018). Interspecies variation in the susceptibility of adult Pacific salmon to toxic urban stormwater runoff. Environmental Pollution, 238: 196.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Blair, S., C. Barlow, E. Martin, R. Schumaker & J. McIntyre (2020). Methemoglobin determination by multi-component analysis in coho salmon (Oncorhynchus kisutch) possessing unstable hemoglobin. MethodsX, 7: 100836.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Through presentations at national and regional conferences by the PIs, graduate students and postdocs: Stormwater managers regionally and nationally, regulators, researchers, interested citizens Changes/Problems:The impacts of storwmater on coho salmon are very acute; progressing from mild changes in behavior to death within hours. This does not leave much time for the body to mount a response that will be moderated by transcriptional responses. As such, the search for molecular pathways in coho affected by stormwater that can be used to develop tools for evaluating stormwater treatment effectiveness is premature. For the remainder of the project, our focus will be on elucidating the underlying physiological changes that occur in coho leading from health to death within hours of exposure to stormwater. What opportunities for training and professional development has the project provided?Training for 5 graduate students and one postdoc at WSU How have the results been disseminated to communities of interest?Results have been shared primarily through presentations at local and national meetings of professionals, and also through outreach to citizens active in conservation in their watersheds around Puget Sound. What do you plan to do during the next reporting period to accomplish the goals?We have additional studies planned to elucidate the target tissues of the novel contaminant identified as the primary toxicant in stormwater.

Impacts
What was accomplished under these goals? We learned more about the underlying mode of action of stormwater on juvenile coho salmon, including their sensitivity in terms of amounts and durations of exposure that cause acute mortality, as well as what systems in the fish are the target of stormwater contaminants, and the identity of the primary toxicant causing acute mortality. By diluting roadway runoff with clean water, we found that coho exposed for as little as one hour could suffer delayed mortality within 24 h from concentrations as low as 12% stormwater. During 24-h exposures, coho died in concentrations of stormwater as low as 5%. We found that blood is not the direct target of contaminants in stormwater. We developed a method to quantitate methemoglobin in coho that can account for the instability of their hemoglobin and found that stormwater does not cause methemoglobin to be formed. This means evidence of cardio-respiratory distress in exposed coho is not due to an inability of hemoglobin to transport oxygen. We learned that a leachate made from running clean water over tire particles was sufficient to recreate the acute mortality of coho salmon, including their unique sensitivity relative to co-occurring chum salmon. Finally, using effects-directed-analysis, we were able to isolate and identify the contaminant in tire leachate primarily responsible for the acute mortality as a transformation product of an anti-oxidant added to tires to protect them from ozone.

Publications

• Type: Theses/Dissertations Status: Other Year Published: 2019 Citation: Prat, J. 2020. ASSESSING JUVENILE COHO SALMON SENSITIVITY TO URBAN STORMWATER RUNOFF. M.S. Thesis for Washington State University, School of the Environment.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Blair, S., C. Barlow, E. Martin, R. Schumaker & J. McIntyre (2020). Methemoglobin determination by multi-component analysis in coho salmon (Oncorhynchus kisutch) possessing unstable hemoglobin. MethodsX, 7: 100836.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: Tian, Z. Y., H. Zhao, K. T. Peter, M. Gonzalez, J. Wetzel, C. Wu, X. Hu, J. Prat, E. Mudrock, R. Hettinger, A. E. Cortina, R. G. Biswas, F. V. C. Kock, R. Soong, A. Jenee, B. Du, F. Hou, H. He, R. Lundeen, A. Gilbreath, R. Sutton, N. L. Scholz, J. W. David, M. C. Dodd, A. Simpson, J. K. McIntyre & E. P. Kolodziej (2020). Ubiquitous tire rubber-derived chemical induces acute mortality in coho salmon. Science, Accepted Nov 5, 2020.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Presenations that included the research were given during the reporting period to the following audiences: Conference on Stormwater organized by the International Erosion Control Association, Vancouver, BC Annual national meeting of the American Ecological Engineering Society, Asheville, NC Conference on Storwmater organized by the Northwest Environmental Business Council, Tacoma, WA National Urban Extension Conference, Seattle, WA Annual meeting of the Pacific Northwest chapter of the Society of Environmental Toxicology and Chemistry, Vancouver, WA Salmon Recovery Conference, Tacoma, WA Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Presentations as decribed in Target Audience What do you plan to do during the next reporting period to accomplish the goals?I plan to hire a postdoctoral researcher with the necessary experience in analysis of RNA sequencing and pathway analysis.

Impacts
What was accomplished under these goals? We have learned more about the sensitivity of zebrafish to stormwater chemicals including that tire wear particles appear to be a major source of the chemicals that produce toxic effects in this model species. This work is done in preparation for RNAseq.

Publications

Progress 02/27/18 to 09/30/18

Outputs
Target Audience: Nothing Reported Changes/Problems:We were not able to begin the project during the reporting period for the following reasons: The post-doc who was going to work on this took another job 3 months after project approval. Our zebrafish colony was initiated in January 2018, but is still not reproducing reliably - an issue I am told is unfortunately common in new colonies. We are consulting with other zebrafish colony owners to troubleshoot this issue. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?In the absence of a dedidcated post-doc and reliably producing zebrafish, technicians will conduct exposures of zebrafish from a different colony to stormwater runoff during the winter of 2018-2019 and sample tissues for analysis. We are seeking an appropriate candidate who will be able to conduct the RNA-Seq and pathway analysis.

Impacts
What was accomplished under these goals? We were not able to begin the project during the reporting period for the following reasons: The post-doc who was going to work on this took another job 3 months after the projejct was approved. Our zebrafish colony was initiated in January 2018, but is still not reproducing reliably - an issue I am told is unfortunately common in new colonies.

Publications