1   Coho salmon spawner mortality in western U.S. urban watersheds: bioinfiltration prevents 2   lethal stormwater impacts 3   4   5   6   7   8   9   10   11   12   13   14   Julann A. Spromberg1, Julann.spromberg@noaa.gov David H. Baldwin2, David.Baldwin@noaa.gov Steven E. Damm3, Steve_Damm@fws.gov Jenifer K. McIntyre4, jen.mcintyre@wsu.edu Michael Huff5, mhuff@suquamish.nsn.us Catherine A. Sloan2, Catherine.a.sloan@noaa.gov Bernadita F. Anulacion2, bernadita.anulacion@noaa.gov Jay W. Davis3, jay_davis@fws.gov Nathaniel L. Scholz2, nathaniel.scholz@noaa.gov 15   16   2 17   18   3 19   20   4 21   5 22   23   24   25   26   27   28   29   30   31   32   33   34   35   36   37   38   39   40   41   Corresponding author: 1 Ocean Associates, under contract to Northwest Fisheries Science Center, National Marine Fisheries Service, NOAA, 2725 Montlake Blvd. E., Seattle, WA 98112 USA Environmental and Fisheries Science Division, Northwest Fisheries Science Center, National Marine Fisheries Service, NOAA, 2725 Montlake Blvd. E., Seattle, WA 98112 USA U.S. Fish and Wildlife Service, Washington Fish and Wildlife Office, 510 Desmond Dr. S.E., Lacey, WA 98503 USA Washington State University, Puyallup Research and Extension Center, 2606 W. Pioneer Ave., Puyallup, WA 98371 USA Suquamish Tribe, P.O. Box 498, 18490 Suquamish Way, Suquamish, WA 98392 USA Nathaniel L. Scholz, Ph.D. Environmental and Fisheries Science Division Northwest Fisheries Science Center National Oceanic and Atmospheric Organization 2725 Montlake Blvd. E. Seattle, WA 98112 Voice: (206) 817-1338 Fax: (206) 860-3335 Email: Nathaniel.Scholz@noaa.gov Running Head: Toxic urban runoff and salmon survival. Word Counts: Summary - 319 Main Text - 4959 Acknowledgements - 121 References - 1100 Table Legends - 264 Figure Legends - 235 3 Tables, 3 Figures 35 References   1     42   Summary 1. Adult coho salmon (Oncorhynchus kisutch) return each autumn to freshwater spawning 43   44   habitats throughout western North America. The migration coincides with increasing 45   seasonal rainfall, which in turn increases stormwater runoff, particularly in urban 46   watersheds with extensive impervious land cover. Previous field assessments in urban 47   stream networks have shown that adult coho are dying prematurely at high rates (>50%). 48   Despite significant management concerns for the long-term conservation of threatened 49   wild coho populations, a causal role for toxic runoff in the mortality syndrome has not 50   been demonstrated. 51   2. We exposed otherwise healthy coho spawners to 1) artificial stormwater containing 52   53   mixtures of metals and petroleum hydrocarbons, at or above concentrations previously 54   measured in urban runoff; 2) undiluted stormwater collected from a high traffic volume 55   urban arterial (i.e., highway runoff); and 3) highway runoff that was first pre-treated via 56   bioinfiltration through experimental soil columns to remove pollutants. 57   3. We find that mixtures of metals and petroleum hydrocarbons – conventional toxic 58   59   constituents in urban stormwater – are not sufficient to cause the spawner mortality 60   syndrome. By contrast, untreated highway runoff collected during nine distinct storm 61   events was universally lethal to adult coho relative to unexposed controls. Lastly, the 62   mortality syndrome was prevented when highway runoff was pretreated by soil 63   infiltration, a conventional green stormwater infrastructure technology.   2     64   65   4. Our results are the first direct evidence that 1) toxic runoff is killing adult coho in urban 66   watersheds, and 2) inexpensive mitigation measures can improve water quality and 67   promote salmon survival. 68   5. Synthesis and applications. Coho salmon, an iconic species with exceptional economic 69   70   and cultural significance, are an ecological sentinel for the harmful effects of untreated 71   urban runoff. Wild coho populations cannot withstand the high rates of mortality that are 72   now regularly occurring in urban spawning habitats. Green stormwater infrastructure or 73   similar pollution prevention methods should be incorporated to the maximal extent 74   practicable, at the watershed scale, for all future development and redevelopment 75   projects, particularly those involving transportation infrastructure. 76   77   Keywords: habitat restoration, non-point source pollution, Pacific salmon, runoff, stormwater, 78   urban ecology, urban streams   3     79   Introduction In recent decades, non-point source runoff has become the leading pollution threat to 80   81   aquatic habitats in the United States and similarly developed countries. In highly built 82   watersheds, the transport of toxic chemical contaminants via stormwater contributes to the well 83   documented “urban stream syndrome”, as evidenced by various indicators of biological and 84   ecological degradation (Walsh et al., 2005). These include declines in species abundance, 85   species diversity, and the proliferation of non-native, pollution-tolerant taxa. Nevertheless, field assessments in urban watersheds rarely report fish kills or similar 86   87   acute mortality events for aquatic life. A notable exception is the recurring die-off of adult coho 88   salmon that return from the ocean to spawn each year in large metropolitan areas of northern 89   California, western Oregon and Washington in the United States, and southern British Columbia 90   in Canada. The coho mortality phenomenon has been studied most extensively in lowland 91   streams of the greater Seattle area of Puget Sound. Coho begin the freshwater phase of their 92   spawning migration with the onset of autumn rainfall. Typically within days of arriving at 93   stream reaches suitable for spawning, affected fish become stricken with symptoms that progress 94   from a loss of orientation (surface swimming) to a loss of equilibrium and death on a timescale 95   of a few hours (Supplemental Videos 1 & 2; Scholz et al., 2011). Year-to-year mortality rates 96   within and across urban watersheds are typically high (~ 50-90%), as measured by the proportion 97   of unspawned females for an entire annual run (Scholz et al., 2011). As might be expected, initial modeling indicates that such high mortality rates at the 98   99   100   critical spawner life stages pose a significant extinction risk for wild coho populations (Spromberg & Scholz, 2011). Coho distinct population segments, or evolutionarily significant   4     101   units (ESUs; Waples, 1991), are comprised of metapopulations that span large river basins with 102   varying degrees of urban and suburban land use (e.g., Bilby & Mollot, 2008; Pess et al., 2002). 103   This population structure and the highly migratory life histories of salmonids have generally 104   constrained ecotoxicological studies (Ross et al., 2012). Nevertheless, if urban runoff is killing 105   adult coho, ongoing regional development pressures may present an important obstacle to the 106   recovery of coho ESUs, including those designated as threated (Lower Columbia River) or a 107   species of concern (Puget Sound) under the U.S. Endangered Species Act. To date, the evidence linking urban stormwater runoff and coho spawner mortality has 108   109   been indirect. The uniform nature of the symptoms, over many years and across many streams, 110   is consistent with a common and prevalent form of toxicity. A forensic investigation spanning 111   nearly a decade ruled out several other potential causes, including conventional water quality 112   parameters (e.g., dissolved oxygen, temperature), habitat availability, poor spawner condition, 113   and disease (Scholz et al., 2011). Moreover, an initial geospatial land cover analysis found a 114   significant positive association between the severity of the coho die-off phenomenon and the 115   extent of impervious surface within a watershed (Feist et al., 2011). The aim of the present study was to explore the connection between water quality and 116   117   coho mortality more directly by experimentally exposing freshwater-phase spawners to both 118   artificial and actual highway runoff. Although urban stormwater is chemically complex, field 119   collected samples consistently contain motor vehicle-derived mixtures of metals and polycyclic 120   aromatic hydrocarbons (PAHs), many of which are toxic to salmon at other life stages (e.g., 121   copper, McIntyre et al., 2012; Sandahl et al., 2007; PAHs, Meador et al., 2006; Heintz et al., 122   2000). If the mortality syndrome could be reproduced with an environmentally realistic mixture 123   of metals and PAHs, it would then be possible to identify the causal agents by removing different   5     124   components of the mixture. To account for the possibility that some other contaminant(s) is 125   causal, we also exposed adult coho to stormwater collected from a dense urban arterial (i.e., 126   highway runoff). Lastly, we exposed adult coho to highway runoff which was pre-treated with a 127   conventional green stormwater infrastructure (GSI) technology (bioinfiltration through soil 128   columns) to remove pollutants, with the aim of lessening or eliminating any overtly harmful 129   impacts of unmitigated stormwater. 130   131   Materials and Methods 132   Animals Adult coho salmon were collected at the Suquamish Tribe’s Grovers Creek Hatchery near 133   134   Poulsbo, Washington. Hatchery coho are an appropriate surrogate for wild coho given that field 135   observations have documented the mortality syndrome in spawners of both wild and hatchery 136   origins (Scholz et al., 2011). At Grovers Creek, returning coho migrate less than 4 km in 137   freshwater from Miller Bay in Puget Sound to a hatchery pond via a fish ladder. The pond was 138   seined on Monday, Wednesday and Friday of each week, and thus the fish were in the pond for a 139   maximum of 72 hours prior to capture. The coho were strays from a net-pen operation designed 140   to provide a terminal fishery to the south of Miller Bay. When available, females were used for 141   the controlled stormwater exposures. For trials with an insufficient number of females, males 142   were also included, as the urban mortality syndrome affects males and females alike (Scholz et 143   al., 2011). Only fish exhibiting normal behavior and with no obvious signs of trauma, disease, or 144   poor condition were included. One set of exposures was conducted on a given day.   6     Each individual coho spawner was placed in a holding tube constructed of PVC, of either 145   146   15.2 cm x 76.2 cm (diameter x length) or 20.3 cm x 106.7 cm with 1.1 cm thick polyethylene 147   gates fitted into slots at either end. Ventilation was provided by six 2.5 cm diameter holes on 148   either side of the anterior (head) end of each tube and five 1.75 cm diameter holes in each gate. 149   For each trial, four separate coho holding tubes were placed in a large polyethylene tank 150   containing 440 L of clean well water, artificial stormwater, highway runoff, or runoff pretreated 151   with soil infiltration. A ventilation hose attached to a pump (for 2011-12, a Flotec Tempest 1/6 HP, 4.5 m3/h; 152   153   for 2013-14, a Lifegard Aquatics Quiet One 3000, 3.1 m3/h) submerged in the polyethylene tank 154   supplied a minimum of 4 L/min flow through the forward gate and over each fish in an anterior- 155   posterior direction. Adult coho were exposed for 4-48 hrs depending on the treatment (see 156   below). Aeration was provided with air stones attached to an air pump (Coralife 05146 Model 157   SL-38 Super Luft Air Pump). Exposure waters were maintained at temperatures below 14oC by 158   flow-through (2011) and Aqua Logic® Cyclone® Drop-In Titanium Chillers (2012). Smaller 159   ventilation pumps that produced less heat were used in 2013-14, and thus chillers were not 160   needed. 161   Exposures to artificial stormwater In the fall of 2011, returning adult coho were exposed to artificial stormwater containing 162   163   mixtures of PAHs and metals. The mixtures were comprised of individual compounds at 164   concentrations at or above those measured during fall storm events in Seattle-area urban streams 165   (Seattle Public Utilities 2007), or at levels representative of urban stormwater runoff more 166   generally (Stein, Tiefenthaler & Schiff 2006; Gobel, Dierkes & Coldewey   2007; Tiefenthaler,   7     167   Stein & Schiff 2008). The PAH profile of urban runoff is compositionally similar to that of crude 168   oil, particularly for toxic three- and four-ring compounds (McIntyre et al., 2014). The exception 169   is a lack of dissolved pyrene and fluoranthene in crude oil (Incardona et al., 2009). Thus, the 170   PAH portion of the mixture was generated from a water-accommodated fraction (WAF) of 171   Alaska North Slope crude oil, to which pyrogenic pyrene and fluoranthene were added 172   (Supplemental Information Table S1). The WAFs were prepared in a 3-speed commercial 173   blender with a 3.8 L stainless steel container (Waring CB15, Waring Commercial, Torrington, 174   CT), following a protocol developed to yield fine oil droplets and bioavailable PAHs in the 175   dissolved phase (Incardona et al., 2013). In brief, the stainless steel container was cleaned with 176   acetone and dichloromethane, the rubber lid was lined with dichloromethane-rinsed heavy-duty 177   aluminum foil and the container was filled with 1 L of de-ionized water. The volume of crude 178   oil added to the WAF (1 mL) was intended to produce a final maximum phenanthrene exposure 179   concentration of 0.384 µg/L (Stein, Tiefenthaler, & Schiff 2006). Pyrene and fluoranthene were 180   then added to coequal final target exposure concentrations of 0.584 µg/L. Water and oil were 181   blended for 30 s on the lowest speed four times. The oil-water mixture was then poured into a 1 182   L separatory funnel and allowed to sit for 1 h. With care to leave the surface slick undisturbed, 183   the bottom 789.3 mL were then drawn off and added to the exposure chamber. 184   The metals fraction of the PAHs/metals mixture consisted of cadmium, nickel, lead, 185   copper and zinc (anhydrous CdCl2, NiCl2, PbCl2, CuCl2, and ZnCl2; Sigma-Aldrich, > 98% 186   purity) added to clean well water at nominal concentrations (Table S1) that were in the upper 187   range of metal- detections in urban streams (Stein, Tiefenthaler & Schiff 2006; Seattle Public 188   Utilites 2007; Gobel, Dierkes & Coldewey   2007; Tiefenthaler, Stein & Schiff 2008). 189   Moreover, the concentrations of metals in urban runoff are transiently elevated during the first   8     190   flush interval (Kayhanian et al., 2012). To capture this exposure scenario, experiments in the fall 191   of 2012 used relatively higher nominal concentrations of metals only (Table S1). Temperature, 192   pH and dissolved oxygen were measured (data not shown) and water samples were collected for 193   analytical verification of exposure concentrations. 194   Exposures to highway runoff Stormwater was collected from the downspouts of an elevated urban principal arterial in 195   196   Seattle, WA. The downspouts receive runoff from the on-ramp to a four-lane (70 m wide) 197   highway over which approximately 60,000 motor vehicles travel each day (WA DOT 2013a). 198   The highway, paved with Portland cement concrete (WA DOT 2013b), is a conventional urban 199   impervious surface. All of the flow to the downspouts originated from precipitation falling on 200   the active arterial. 201   The captured runoff was transported to the hatchery facility in either covered glass 202   carboys or in a stainless steel tank. The holding interval prior to exposures varied with the 203   timing and intensity of fall storm events, but did not exceed 72 h. While some collections took 204   place after an extended antecedent dry interval and therefore included the first flush for a given 205   storm, others spanned periods of intermittent rainfall. Daily and cumulative rainfall for each 206   autumn season is shown in Fig. 1, with each stormwater collection interval superimposed (solid 207   rectangular boxes). Collected runoff was used for one exposure only. Temperature, pH, and 208   dissolved oxygen were measured at the outset of each exposure, and water samples were 209   collected for chemical analyses to quantify concentrations of metals and PAHs (2012-13 but not 210   2014 storms). After exposures, the runoff was transported to a Kitsap County Wastewater Pump 211   Station for disposal.   9     212   Exposures to filtered runoff In the fall of 2013, four 200 L bioretention columns were constructed and plumbed with 213   214   outflow drains following conventional guidelines for green stormwater infrastructure (WA DOE, 215   2012). The filtration columns were composed of a 30.5 cm drainage layer of gravel aggregate 216   overlain by 61 cm of bioretention soil media (60% sand: 40% compost) and topped with 5 cm of 217   mulched bark. In the fall of 2014, the bioretention columns were emptied and fresh media 218   installed. In each year, the bioretention media was conditioned by passing seven pore volumes 219   (660 L) of clean well water from the hatchery facility through each column at a rate of 2 220   L/minute, equivalent to two months of summer rainfall on a contributing area 20x the treatment 221   area. Urban highway runoff was collected as described above, and the homogenized volume was 222   evenly divided to flow through one of the four bioretention columns at a rate of 2 L/minute, with 223   the outflows from the four columns recombined into a single post-treatment exposure volume. 224   Adult coho spawners were exposed to either untreated urban stormwater or the same runoff post- 225   filtration for 4–24 h. Water quality was measured and samples were collected for chemical 226   analyses as described above. 227   Observations of symptomatic fish Hallmark characteristics of the adult coho spawner mortality syndrome include a 228   229   progression from lethargy to a loss of orientation, a loss of equilibrium, followed by death 230   (Scholz et al., 2011). Individual fish were examined for these symptoms during and after each 231   exposure. Fish were moved from the large exposure tank and released from their holding tubes 232   into an observation tank containing clean well water at a minimum depth of 50 cm. Swimming 233   ability and evasiveness (responses to light and gentle prodding) were recorded over a 1-3 min   10     234   observation interval. For the trials using artificial stormwater, symptomology was assessed at 24 235   h and then again at the end of the exposure. Coho exposed to highway runoff were visually 236   examined at 2, 4 and 24 h. Live fish at 2 h and 4 h were returned to their holding tubes and 237   exposure chambers for the remainder of the trial. 238   Water quality analyses Conventional water quality parameters, including pH, dissolved oxygen, alkalinity, total 239   240   suspended solids, N-ammonia, nutrients, and organic carbon were measured for selected trails 241   using standard instrumentation or by outside laboratories using U.S. EPA-approved methods 242   (Analytical Resources Inc., Tukwila, WA or Am Test Inc., Kirkland, WA). Total and dissolved 243   concentrations of cadmium, copper, nickel, lead and zinc were determined by inductively 244   coupled plasma mass spectrometry (ICP-MS) by Frontier Global Sciences (Bothell, WA; EPA 245   method 1638) or Am Test Inc. (EPA method 200.8). Briefly, samples were preserved in 1% 246   (v/v) nitric acid (total metals) or passed through a 0.45 µm filter (dissolved metals) and then 247   oven digested prior to analysis by ICP-MS. Duplicate samples and laboratory blanks were 248   included to ensure quality control. Selected water samples for PAH determinations were 249   preserved with 10% dichloromethane and stored at 4oC in amber glass bottles until analysis at 250   the NOAA Northwest Fisheries Science Center by gas chromatography/mass spectrometry (GC- 251   MS) with additional selected ion monitoring for alkyl-PAHs (Sloan et al., 2014). 252   Tissue sampling and analyses At the conclusion of each exposure, fish length, weight, reproductive status, and origin 253   254   were assessed. To confirm the bioavailability of PAHs in exposure waters, bile was screened for 255   PAH metabolites in a subset of 2011 and 2012 trials with both artificial stormwater and highway   11     256   runoff. Fish were sacrificed and bile was collected from the gall bladder and stored in amber 257   glass vials at -20°C until analysis for PAH metabolites using high-performance liquid 258   chromatography with fluorescence detection (Krahn et al., 1986, da Silva et al., 2006). The 259   concentrations of fluorescent PAH metabolites in bile are determined using naphthalene (NPH), 260   phenanthrene (PHN) and benzo[a]pyrene (BaP) as external standards and converting the relative 261   fluorescence response of bile to NPH, PHN and BaP equivalents, and reported as ng/g bile or 262   ng/mg biliary protein. Coho gills were sampled to confirm uptake of metals in selected 2011 and 2012 artificial 263   264   and collected stormwater exposures. Tissues were excised with Teflon or titanium scissors and 265   plastic forceps, placed in plastic Whirl-paks, and stored at -80°C. Metals analyses were 266   determined by inductively-coupled mass spectroscopy (ICP-MS) at the Trace Elements Research 267   Laboratory (TERL; College Station, TX) using standard methods (TERL Method Codes 001, 268   006). Briefly, gill tissues were wet digested with nitric acid, freeze-dried, and homogenized by 269   ball-milling in plastic containers. Samples were ionized in a high temperature argon plasma and 270   positively-charged ions were separated on the basis of their mass:charge ratios by a quadrupole 271   mass spectrometer. Student-t tests assessed differences in the tissue concentrations between 272   exposures and their respective paired control. 273   Results 274   Adult coho responses across treatments Coho spawners exposed for 24 h to mixtures of PAHs and metals at concentrations 275   276   slightly higher than those previously measured in urban runoff were asymptomatic – i.e., 277   behaviorally indistinguishable from controls exposed to clean well water (Table 1). Although   12     278   there was some mortality across the four independent trials (n=4 of 30 fish total), this was not 279   significantly different by treatment (Fisher Exact Tests, two-tailed, P≥ 0.21) and was therefore 280   apparently attributable to handling stress. Extending the exposures to 48h did not increase the 281   incidence of mortality or symptomology (data not shown). Increasing the concentrations of 282   metals by 5- or 10-fold in metals-only mixtures was also insufficient to elicit the symptoms of 283   the pre-spawn mortality syndrome (Table 2). As with the PAHs/metals mixture, there was a 284   small but insignificant amount of mortality across treatments (n = 2 of 38 fish; Fisher Exact 285   Tests, two-tailed, P = 1). 286   Although the artificial stormwater preparations were designed to have a similar 287   composition to highway runoff for many PAHs and metals, the effects on coho spawners were 288   very different. Whereas the artificial mixtures did not elicit the distress characteristic of the 289   mortality syndrome, coho exposed to the unfiltered highway runoff rapidly became symptomatic. 290   For every discrete rainfall collection interval (n=9; 2012-2014), all of the exposed fish were 291   either symptomatic or dead within 4 hours (Fig. 1, Table 3). Those that survived the initial 4h 292   exposure were dead by 24 h. All of the paired control coho in clean well water survived, 293   showing no behavioral symptoms at 4 or 24 h (Fig. 1, Table 3). Each exposure showed a 294   statistically significant difference in mortality (Fisher Exact Tests, two-tailed, P=0.006). 295   Examples of asymptomatic control fish and symptomatic, runoff-exposed spawners are shown in 296   Supplemental Video 3. For the purpose of comparing symptoms, digital movies of affected coho 297   in Seattle-area urban watersheds are shown in Supplemental Videos 1 and 2. Thus, despite the 298   event-to-event variation in rainfall duration and intensity, and a corresponding variation in water 299   chemistry (conventionals, metals and PAHs, Tables S2, S3, and S5), urban runoff was 100%   13     300   lethal to otherwise healthy adult coho salmon. The contribution of handling stress was evidently 301   minimal, as the survival rate for controls across treatments was 100%. The constructed bioretention columns effectively treated the highway runoff in terms of 302   303   both toxic chemical exposure and salmon spawner survival. Although the focal (measured) 304   contaminants were not completely removed by infiltration, the overall improvement in water 305   quality was sufficient to completely prevent the lethal effects and sublethal symptomology 306   caused by untreated stormwater. All of the adult coho exposed to filtered runoff survived and 307   showed no behavioral symptoms at either 4 h or 24 h (100% survival, n=20; Table 3; 308   Supplemental Video 3). Thus, urban stormwater contains an as-yet unidentified chemical 309   component(s) that, while lethal to salmon spawners, can be removed using inexpensive 310   bioinfiltration. 311   Measured levels of metals, PAHs, and conventional water quality parameters across treatments 312   The chemical properties of highway runoff were evaluated for the six distinct collection 313   events in the fall of 2012 and 2013. As expected, conventional water quality parameters varied 314   across stormwater collections, as did concentrations of PAHs and metals. The analytical results 315   are shown in Tables S2, S3, and S5. As expected, suspended solids (TSS 23 - 220 mg/L) and 316   organic matter (DOC = 8 - 92 mg/L) were elevated in urban runoff relative to control water (TSS 317   < 1.1 mg/L, DOC < 1.8 mg/L). In contrast, runoff had lower Mg (t(8)=6.072, P<0.001), 318   alkalinity (t(8)=6.201, P < 0.001), and phosphate (t(8) = 3.547, P=0.008). The pH values for 319   runoff were circum-neutral in runoff (6.12 - 7.47) and consistently lower than those for control 320   water (t(8)=2.691, P=0.027). Other conventional chemistry parameters were not significantly 321   different among treatments, including Ca (t(8) = -0.121, P=0.907) and hardness (t(8) = 1.159,   14     322   P=0.280). At the outset of exposures, dissolved oxygen levels ranged from 8.1 - 10.7 mg O2/L 323   and were maintained above 6.5 mg/L with additional aeration as needed. 324   Collected highway runoff had a more pyrogenic (or combustion-driven) PAH profile 325   relative to the artificial stormwater mixtures, as evidenced by a relative enrichment of higher 326   molecular weight (5- and 6-ring) compounds and fewer low molecular weight (2- and 3-ring) 327   compounds (Fig. 2). Bile PAH metabolites were not significantly different between fish exposed 328   to control well water or stormwater runoff after a 4 h exposure (Fig. 3). Although the measured 329   concentrations of PAH metabolites in the bile of fish exposed for 24 h to the PAHs/metals 330   mixture were elevated relative to paired controls, the difference was not significant (Student t- 331   test; P= 0.1, 0.14, 0.11 for phenanthrene, benzo-a-pyrene, and naphthalene metabolites, 332   respectively). This indicates that low-level PAH exposures typical of urban runoff do not 333   produce large increases in measurable bile metabolites, and is consistent with bile PAH 334   metabolite measurements from symptomatic coho previously collected during field surveys of 335   urban spawning habitats (Scholz et al., 2011). Notably, in 2012, the levels of 2- and 3-ring PAHs in the control exposure water were 336   337   unexpectedly elevated relative to all other control treatments (Fig. 2, arrow). This was attributed 338   to the recent drilling of a new well at the Suquamish hatchery facility. Measured PAH levels in 339   the well water declined sharply over a timespan of two weeks (data not shown), and adult coho 340   controls that were exposed during the interval did not exhibit behavioral symptoms (Fig. 1). Whereas the levels of dissolved-phase Cd and Pb were generally lower in collected 341   342   runoff relative to all of the artificial stormwater mixtures (Fig. 2), Cu and Ni in runoff spanned 343   the range of these two metals in the environmentally relevant mixture. Zinc levels in runoff were   15     344   higher, and within the range of corresponding Zn levels in the high-metals mixture. The 345   concentrations of metals in the gills of stormwater-exposed and unexposed coho (4h) were not 346   significantly different (Student t-tests, P > 0.05; Fig. 3) and, in both cases, were lower than gill 347   metals levels measured from symptomatic spawners collected from the field (Scholz et al, 2011). 348   Similarly, exposures to the environmentally relevant artificial stormwater mixture of 349   PAHs/metals did not produce a significant accumulation of metals in the gills, with the exception 350   of Ni (Student t-test, P=0.017). For the high metals mixture, only Cd, Cu, and Pb levels were 351   significantly elevated relative to controls (Student t-test; P= 0.002, 0.018, 0.003 for Cd, Cu, and 352   Pb, respectively). Filtering collected highway runoff through the bioretention columns reduced total PAHs 353   354   by 94% and total metals by 58%. As expected, removal efficiency varied for different 355   contaminants. For example, the soil columns removed lower molecular weight PAHs less 356   efficiently than higher molecular weight PAHs (e.g., 81-89% for 2-3 ring PAHs vs 93% removal 357   of 4-5 ring PAHs; Table S5). Notably, the medium in the bioretention columns was a source (i.e., 358   an exporter) of total Ni to the treated runoff, resulting in a 57% increase over the pre-filtration 359   input (Table S3). All other total metals decreased by an average of 48-88% across the two 360   events in the order of Cd < Pb < Cu < Zn. For each of the metals, concentrations in the dissolved 361   phase also declined after soil column infiltration (Table S3). In addition to exporting Ni, the 362   bioretention columns were also a source of DOC (post/pre-filtration increase of 164%), alkalinity 363   (+ 29%), Ca (+ 60%), Mg (+ 372%), ortho-P (+ 4000%) and increasing hardness (+ 107%). By 364   contrast, column infiltration reduced the ammonia content of stormwater by 92% (Table S2). 365     16     366   Discussion We have confirmed that controlled exposures to untreated urban runoff are sufficient to 367   368   reproduce the coho spawner mortality syndrome. Adult coho became symptomatic and died 369   within a few hours of immersion in collected stormwater. Mortality rates were 100% for 370   exposed fish versus 0% in control fish held in clean well water, and these results were consistent 371   across nine distinct rainfall intervals that spanned three consecutive autumn spawning runs. As 372   evidence that one or more toxic chemical contaminants are causal, pre-treating the highway 373   runoff with soil bioinfiltration completely prevented the acutely lethal impacts on coho 374   spawners. Surprisingly, coho did not develop symptoms in response to artificial mixtures of 375   PAHs and metals, even at concentrations that were higher than those typically measured in 376   stormwater, including the first flush. Urban runoff is chemically complex, with many chemical 377   constituents that are very poorly characterized in terms of toxicity to fish. While it may take 378   years of additional assessment to identify precisely which of these agents is killing coho, our 379   initial results suggest that simple GSI technologies hold promise as a means to improve water 380   quality and effectively prevent coho mortality in urban spawning habitats. Our finding that road runoff alone is sufficient to induce the spawner mortality syndrome 381   382   aligns with previous evidence for a positive association between the amount of impervious 383   surface within an urban watershed and the year-to-year severity of coho die-offs (Feist et al., 384   2011). It appears that other forms of water quality degradation are not necessary to produce the 385   phenomenon. Consistent with this, symptomatic spawners do not show evidence of neurotoxic 386   pesticide exposure (Scholz et al., 2011), and adult coho are not unusually vulnerable to low-level 387   mixtures of current use pesticides (King et al., 2013). The link to impervious runoff also   17     388   discounts a role for personal care products, pharmaceuticals, and other classes of compounds that 389   are transported to some urban streams via combined sewer overflows in heavy rains. As noted above, urban road runoff contains a complex mixture of chemicals, many of 390   391   which originate from motor vehicles in the form of exhaust, leaking crankcase oil, and the 392   wearing of friction materials (i.e., brake pads) and tires. We assessed the toxicity of PAH and 393   metals mixtures because these compounds are ubiquitous in stormwater and are known to be 394   disruptive to the fish cardiovascular system (PAHs; Brette et al., 2014), as well as the respiratory 395   and osmoregulatory functions of the gill (metals: Niyogi & Wood, 2004). Although bile and gill 396   tissue results suggest that PAHs and some metals are bioavailable to the coho spawners (this 397   study, Scholz et al. 2011), artificial mixtures of PAHs and metals did not produce the symptoms 398   of the mortality syndrome. Our results appear to rule out many of the PAHs that are common to 399   urban runoff and crude oil spills (e.g., phenanthrenes). However, there may be a role for the 400   higher molecular weight pyrogenic PAHs found in particulate vehicle exhaust (i.e., soot), other 401   than pyrene or fluoranthene. The remaining list of potential causal chemicals is long, and 402   includes other organic hydrocarbons such as methylphenols, quinones, thiazoles, thiophenes, 403   furans, and quinolines. Given the logistical challenges associated with adult coho exposures – 404   seasonal availability of animals, large volume assays, limited number of fish, etc. – it may be 405   years before the causal agent(s) is identified. Notably from a water resource management 406   perspective, this will likely be a chemical or chemicals for which there are no existing water 407   quality criteria. 408   Biological indicators play an important role in field assessments to document the urban 409   stream syndrome in affected watersheds worldwide. Common examples are benthic indices of 410   biological integrity (B-IBIs), which are used to characterize the health of streams based on the   18     411   diversity and abundance of macroinvertebrates (Karr, 1999). Although poor B-IBI scores are 412   diagnostic of aquatic habitat degradation, they do not necessarily differentiate between drivers 413   that may be chemical (i.e., pollution) versus physical or biological. Conversely, biological 414   indicators that are specific to toxic runoff may not have directly meaningful implications for 415   individual survival, as a basis for guiding species conservation at the population and community 416   scales. This includes, for example, the upregulation of sensitive and responsive cytochrome 417   p450 enzymes in the livers of fish exposed in situ to certain PAHs and other contaminants that 418   act via the aryl hydrocarbon receptor (van der Oost, Beyer, & Vermeulen 2003). Coho spawners, by contrast, appear to be very sensitive ecological indicators, with a 419   420   response metric that is directly attributable to toxic stormwater. Moreover, the implications of 421   widespread and recurring mortality are relatively clear at higher scales (e.g., Spromberg & 422   Scholz, 2011). Although the highway runoff used in this study (at the point of discharge) 423   presumably contained higher concentrations of chemical contaminants than surface water 424   conditions in urban spawning habitats, it is evident that runoff in urban waterways is not 425   sufficiently diluted to protect many or most coho from premature death (Scholz et al., 2011). By 426   establishing a direct link between non-point source pollution and the mortality syndrome, our 427   findings set the stage for future indicator studies in western North America. This includes, for 428   example, more refined predictive mapping of vulnerable habitats as a function of impervious 429   land cover, at present and with future urban growth scenarios (Feist et al., 2011). Coho survival 430   in urban streams can also indicate the success of pollution control programs, via GSI or other 431   strategies. Intensive control measures will almost certainly be necessary, across large spatial 432   scales, to 1) recover viable coho populations in the built environment, and 2) prevent the rapid   19     433   future loss of coho as a consequence of expanding impervious cover in watersheds that are 434   currently productive but primarily non-urban. In the future, it may be possible to narrow the focal list of chemicals by determining more 435   436   precisely why stormwater-exposed coho are dying. The gaping, surface swimming, and 437   disequilibrium of affected spawners suggest adverse physiological impacts on the gill, the heart, 438   the nervous system, or some combination of these. An earlier forensic study found no evidence 439   of physical injury to the gills or other tissues (Scholz et al., 2011). An alternative approach 440   would be to screen the target organs of symptomatic fish for changes in gene expression, and 441   specifically gene sets that are diagnostic for specific categories of physiological stress (e.g., 442   respiratory uncoupling). If the cause of death is ultimately found to be heart failure, for example, 443   the candidate chemicals could be screened for cardiotoxic potential. It may also be possible to 444   develop alternative exposure methods that reflect different sources of contaminants on roadways. 445   This includes, for example, large-volume suspensions of particulate soot from motor vehicle 446   exhaust, dust from brake pad wear, or fine particles from tire wear. 447   Lastly, toxic runoff is likely to represent an increasingly important conservation 448   challenge for west coast coho populations in the coming years. Extant population segments are 449   generally at historically low abundances, as evidenced by current ESA threatened designations in 450   central and northern California, as well as northwestern Oregon and southwestern Washington. 451   Land cover change has been extensive in some lowland watersheds where coho spawn, as a 452   consequence of sprawl in recent decades (e.g., Robinson, Newell, & Marzluff 2005). Over a 453   similar period of time, coho habitat use in areas affected by urbanization has declined sharply 454   (Bilby & Mollot, 2008). Resource managers have been aware of the urban pre-spawn mortality 455   syndrome among adult coho since at least the 1980s (Kendra & Willms, 1990). However, the   20     456   extent to which recurring adult die-offs have driven down wild coho numbers in urbanizing 457   watersheds is not presently known. Initial modeling has shown that local populations in 458   urbanizing watersheds cannot withstand the rates of mortality observed in Puget Sound urban 459   stream surveys since 2000 (Spromberg & Scholz, 2011). However, in terms of recovery 460   planning, this stormwater-related threat has yet to be mapped out for actual coho conservation 461   units at the sub-basin scale. In conclusion, a core objective of GSI is to slow, spread, and infiltrate stormwater. As 462   463   anticipated from recent studies (e.g., McIntyre et al., 2015), the experimental soil columns used 464   here effectively prevented the acutely lethal toxicity of runoff from a dense urban arterial. This 465   extends the range of aquatic species and life stages that demonstrably benefit from stormwater 466   bioinfiltration. These include the early life stages of zebrafish (McIntyre et al., 2014), juvenile 467   coho salmon and their macroinvertebrate prey (McIntyre et al., 2015), and adult coho spawners 468   (this study). Bioretention is therefore a promising clean water technology from the standpoint of 469   installation cost, reliability, reproducibility, and scalability. However, the science of GSI 470   effectiveness is still relatively young (Ahiablame, Engel, & Chaubey 2012), and fundamental 471   questions remain as-yet unanswered – e.g., how much treatment will be needed, over what 472   spatial scales, to ensure coho salmon survival? Whereas bioretention may work well for small- 473   footprint sites that receive modest inputs of stormwater, they are but one of many evolving non- 474   point source pollution control and prevention methods that are currently under development 475   (Hughes et al, 2014). For the urban watersheds of the future, the coexistence of humans and wild 476   coho will likely hinge on the success of these innovations. 477     21     478   Acknowledgements 479   We appreciate the technical assistance of Allisan Beck, Richard Edmunds, Tony Gill, 480   Emma Mudrock, Tiffany Linbo, Kate Macneale, Jana Labenia, Mark Tagal, Frank Sommers, 481   Gina Ylitalo, Daryle Boyd, Barb French, Ann England, Karen Peck, MaryJean Willis, Cathy 482   Laetz, Sylvia Charles, William Alexander, Ben Purser, Corey Oster, Luke Williams, and the 483   Kitsap Poggie Club. This study received agency funding from the NOAA Coastal Storms 484   Program (National Ocean Service, Coastal Services Center), the U.S. Fish & Wildlife Service, 485   the Puget Sound’s Regional Stormwater Monitoring Program (RSMP as administered by the WA 486   State Dept. of Ecology), and the U.S. Environmental Protection Agency, Region 10. 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Adult coho salmon spawner mortality following a 24 h exposure to either clean well 606   water (unexposed) or a mixture of polycyclic aromatic hydrocarbons (PAHs) and metals. Shown 607   in parentheses are the numbers of symptomatic or dead fish as a proportion of the total numbers 608   of spawners in each exposure. The PAHs/metal exposures were based on measured levels in 609   urban creeks during storm events (see Methods). Relative to environmental samples, the 610   artificial mixture contained higher concentrations of both total PAHs and metals. Each exposure 611   was conducted on a separate day. Mortality PAHs/Metal Exposure Unexposed Mixture 24 h 25% (1/4) 0% (0/4) 24 h 33% (1/3) 0% (0/3) 24 h 0% (0/4) 50% (2/4) 24 h 0% (0/4) 0% (0/4) 612   613     28     614   615   Table 2. Exposures to relatively high levels of metals in artificial mixtures are not sufficient to 616   elicit the coho spawner mortality syndrome. Similar to unexposed controls, nearly all of the 617   adults survived exposures to mixtures of metals (Cd, Cu, Pb, Ni, Zn) that were 5-fold (Low) or 618   10-fold (High) higher than measured concentrations in urban creeks where coho mortality 619   syndrome was observed. Shown in parentheses are the numbers of symptomatic or dead fish as a 620   proportion of the total numbers of spawners in each exposure. Each exposure was conducted on 621   a separate day. Mortality Low Exposure Unexposed Metals 24 h 0% (0/4) 0% (0/4) 24 h 0% (0/4) 0% (0/3) 24 h 0% (0/4) 24 h 25% (1/4) 24 h 0% (0/3) High Metals 0% (0/4) 25% (1/4) 0% (0/4) 622   623     29     624   625   Table 3. Proportion of adult coho displaying the spawner mortality syndrome after placement in 626   clean well water (unexposed) or highway runoff that was either unfiltered or filtered through an 627   experimental soil bioretention system during 2013 and 2014). Shown in parentheses are the 628   numbers of symptomatic or dead fish as a fraction of the total number of coho in each treatment. 629   Each exposure was conducted on a separate day. Mortality Exposure Unexposed 4h 24 h 24 h 24 h 24 h 0% (0/4) 0% (0/4) 0% (0/4) 0% (0/4) 0% (0/4) Unfiltered Filtered 100% (4/4) 100% (4/4) 100% (4/4) 100% (4/4) 100% (4/4) 0% (0/4) 0% (0/4) 0% (0/4) 0% (0/4) 0% (0/4) 630     30     631   632   Legends 633   Fig. 1. 634   Presence or absence of the pre-spawn mortality syndrome in adult coho salmon exposed to 635   unfiltered highway runoff (E) or clean well water (C). Paired exposures spanned three 636   consecutive fall spawning seasons, 2012-14. Shown in each panel are daily rainfall (shaded 637   bars), cumulative rainfall (dotted lines), highway runoff collection intervals for each separate 638   exposure event (black rectangles), and the presence or absence of symptomatic (or dead) fish in 639   each individual treatment (4-24 h duration; see Methods). Symptoms included lethargy, loss of 640   orientation, or loss of equilibrium. 641   Fig. 2. 642   Dissolved metal (left column) and dissolved polycyclic aromatic hydrocarbon (right column) 643   concentrations summarized by ring number for adult exposures to well water controls, 644   PAHs/metals mixtures, highway runoff, and filtered runoff. Closed symbols indicate dead or 645   symptomatic individuals were observed in the exposure. Lines connect paired highway runoff 646   and filtered runoff from the same collection. Control points are the mean of samples collected 647   each year. The number of mean values below the reporting limits is indicated by # ND. 648   Fig. 3. 649   Left column shows the relative measured concentrations of metals in adult coho salmon gill 650   tissue for Cd, Cu, Pb, Ni and Zn (ug/g). Control values are means of control tests run in 2011 and 651   2012. Closed symbols indicate dead or symptomatic individuals were observed in the exposure.   31     652   The right column shows bile fluorescent aromatic compounds (FACs) detected at naphthalene 653   (NPH), phenanthrene (PHN), benzo-a-pyrene (BAP) wavelengths shown in protein corrected 654   PAH equivalents (ng/mg). 655   Supporting Information 656   Additional Supporting Information may be found in the online version of this article. 657   Video S1. 658   Video 1 of a symptomatic adult coho spawner in a Seattle-area urban stream. 659   660   Video S2. 661   Video 2 of a field observation of a symptomatic adult coho in a Seattle-area urban stream. 662   663   Video S3. 664   Adult coho spawners exposed under controlled experimental conditions to either clean well 665   water, unfiltered urban runoff, or runoff treated using bioinfiltration. 666   Table S1. Nominal concentrations (ug/L) for metals and selected PAHs in the PAHs/metals 667   mixture and the metals-only mixture exposures. 668   Table S2. Measured conventional water chemistry parameters in treatments used in adult coho 669   experiments during 2012-2013. 670   Table S3. Measured metal concentrations in treatments used in adult coho experiments during 671   2012-2013.   32     672   Table S4. Abbreviations and Polycyclic Aromatic Hydrocarbon analytes, including sums of 673   alkyl PAH isomers measured in water samples. 674   Table S5. Measured parent and alkylated homologue PAHs (µg/L) in treatments used in adult 675   coho experiments during 2012-2013. Abbreviations are listed in Table S4. 676     33   677 678 Fig. 1. 679 5' 2012 '25 Emma;ch A 4- caama -20 unexposed controls =stormwater-exposed is 3? ?15 Q: asymtomatic 0535450? I (X251 or dead Oct. 1 Oct. 15 Nov.1 Nov.15 25' 2013 '10 5' 2014 "'20 caama 2_ _8 4_ ?15 g, gamma ?6 3- stormwater collection/cumulative rainfall I _5 0-5 daily rainfall 2 1 UH H001 Hm 0 I I Oct. 1 Oct. 15 Nov.1 Nov.15 Oct. 1 Oct. 15 Nov.1 Nov.15 680 34 (we) ?Emma eAneInwno (we) nemea eAneInwno 681 Fig. 2 10 0.1 0.01 1000 100 10 0.1 100 10 0.1 0.01 100 Dissolved Metals (pg/L) 10 0.1 1000 100 PAHs/Metals Highway Runoff Filtered Runoff 10 0.1 0.01 10 0.1 A 0.01 10 0.1 0.01 Dissolved PAHs (pg/L 0.1 0.01 0.001 0.01 0.001 0.0001 2-Ring 8 3-Ring . 9 0 4-Ring El 0 5-Ring A El 1 ND 6-Ring 36 684 Control PAHs/Metals Highway Runoff 10 20 0.01 Gill Metals (pg/g) 01 0.Bile FACs (ng PAH equivalents/mg biliary proteinNPH PHN contrOI PAHs/Metals [j O. 00 Highway Runoff .: . 683 Fig. 3.