BUTE REVIEW [CEAA] Subject: Bute Proposal Comment Colette Spagnuolo Project Assessment Analyst Hi Colette, Thanks for Your Interest

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From: andy sinats Sent: Wednesday, February 25, 2009 2:48 PM To: BUTE REVIEW [CEAA] Subject: bute proposal comment Colette Spagnuolo Project Assessment Analyst Hi Colette, thanks for your interest. Here are the comments you requested. cheers, Andy

From: Subject: bute proposal comment Date: February 18, 2009 11:59:45 PM PST (CA) To: [email protected] Cc: [email protected]

February 18, 2009

Marie-France Therrien Panel Manager, Canadian Environmental Assessment Agency 160 Elgin Street, 22nd Floor, Ottawa, ON K1A 0H3

Comment on Draft Terms of Reference for establishing a federal review panel for the proposed Bute Inlet Hydroelectric Project

From: Andy Sinats Director BCEN ED Society Targeting the Overuse of Pesticides

Thank you the opportunity to comment on the Draft Terms of Reference for establishing a federal review panel for the proposed Bute Inlet Hydroelectric and the draft federal-provincial environmental impact statement guidelines. I am a concerned citizen of British Columbia.

The cat is out of the bag. The president of the proponent Plutonic power's Bruce Ripley stated a week ago, "For my government in British Columbia, I think that they are making a big mistake by precluding nuclear." This project for whose ecological impact these comments are being solicited cannot stand on its own and is an investment scam. Not only can this project not do what it purports to do, provide power in a reliable and consistent fashion, but like the pyramid scheme that it is, it require s innumerable add-ons, some of the totally uncertain. a) it requires yet another transmission line for the Bute project, parallel to one already at Toba Inlet, because Plutonic for sees its own need poorly b) it requires rain at the right time of year in a quasi-predictable amount and at present there are droughts in Mexico, California, Argentina, Australia, the southern US, China and I could go on. Planners here have been very slow in acknowledging any changes in warming or the economy and particularly the climate on which this project depends. c) it requires flow thru of four billion dollars in a time of economic uncertainty when all mega projects are tanking and infrastructure in the tar sands for one example is being abandoned to rust on the ground. When the cash from GE stops, all this prattle about PPPs will also be abandoned and the tax payer of BC will again be the bailer out of last resort just as the Olympic Village in Vancouver, the entire auto industry, and half the banks. d) it requires Nuclear power because Mr Ripley has himself acknowledged his project needs a prop.

Therefore the terms of reference to review this projects feasibility have to reference, GE's future financial viability, world climactic uncertainty, the impact or additional transmission line corridors and the damage to the wilderness they entail;and the potential impact of industries that will be pulled along by this project such as nuclear which I remind the panel is under a moratorium in British Columbia.

As to the ecological impact, what this project suffers from is an untenable distance from its consumer. This untenable distance between supply and demand is unsupportable except by a fracturing of the entire wilderness by tentacles and webs of transmission line corridors, bridges, roads, and structures all build with toxic materials.

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Recent research by Nathanael Scholz of the NOAA, and Dr. Keith Tierney of the University of Windsor has proven beyond reasonable doubt that salmon are adversely impacted by the copper, chromium, and arsenic compounds in pressure treated wood used in bridges, poles, and wooden structures. Although runoff may not constitute a lethal dose for salmon and other fish species, in fact a sub-lethal dose is all it takes to permanently impair the olfactory ability of salmon to evade predators, reproduce, and find their spawning river.

It is obvious that industrialization of the wilderness entails considerable use of toxic materials. In particular the electric grid currently maintained by BC Hydro and BC Transmission consists of 1,100,000 poles treated with CCA. It is expected that run of the river projects and alternate energy projects that require transmission corridors to connect to this existing grid will have to apply for Pest Management Permits, and it is unlikely that the standards currently in place regarding NTZ's or No Treatment Zones, will be any more rigorous than the thoroughly insufficient setbacks in use today.

It is only very recently that research has revealed that pulses in copper due to rain events particularly in the vicinity of road crossings at streams and rivers contribute to sub-lethal impairment of the olfactory ability of all vertebrates and have negative implications prejudicial to the entire web of life in the affected areas. Please see the attached studies by Scholz, Baldwin, et al.

Other studies have shown Roundup and other glysophosates (Tierney, et al) typical of the herbicides used for weed control and maintenance of right of way corridors for heli-landing pads, railway beds, transmission line corridors, and other similar uses is also very prejudicial to salmon survival. In February 2009, First Nations fisherman report the salmon runs have never in their experience been as low as they are right now. Each of the two lower mainland dumps has at present 400-500 eagles, never seen there in such numbers, searching thru garbage because there are no salmon in our rivers. The entire web of life in British Columbia, without exaggeration, is at risk, Dr. Scholz, whose study is attached and who has studied copper leaching into the aquatic environment and its effect of migratory salmonids states: "Increasing runoff in areas of growing urban and rural development may explain why salmon are disappearing from streams throughout the West."

Thank you for your consideration of these comments. I appreciate your attention to the concerns and recommendations expressed here, as we seek to reduce the planet’s toxic load of persistent bioaccumulative toxic chemical, and chemicals with other long-term toxic effects on health and the environment, to which road building, transmission lines, and the industrialization which accompanies IPP's, and logging is a major and unnecessary contributor. sincerely,

Andy Sinats addendum:

St. Francis--patron saint of ecology. Hymn to brother sun and sister moon

Be praised, my Lord, of all your creature world, And first of all Sir Brother Sun, Who brings the day, and light you give to us through him, And beautiful is he, agleam with mighty splendor: Of you, Most High, he gives us indication.

Be praised, my Lord, through Sisters Moon and Stars: In the heavens you have formed them, bright and fair and precious.

Be praised, my Lord, through Brother Wind, Through Air, and cloudy, clear, and every kind of Weather, By whom you give your creatures sustenance.

Be praised, my Lord, through Sister Water, For greatly useful, lowly, precious, chaste is she.

Be praised, my Lord, through Brother Fire, Through whom you brighten up the night, And fair he is, and gay, and vigorous, and strong.

Be praised, O Lord, through our sister Mother Earth,

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For she sustains and guides our life, And yields us diverse fruits, with colored flowers, and grass.

Be praised, my Lord, through those who pardon give for love of you, And bear infirmity and tribulation: Blessed they who suffer it in peace, For of you, Most High, they shall be crowned.

Be praised, my Lord, through our Brother Death of Body, From whom no one among the living can escape. Woe to those who in mortal sins will die; Blessed those whom he will find in your most holy graces, For the second death will do no harm to them.

Praise and bless my Lord, and thank him too, And serve him all, in great humility.

file://S:\Panels\Bute Inlet\Public Comments\bute proposal comment.htm 2/26/2009 Environmental Toxicology and Chemistry, Vol. 22, No. 10, pp. 2266±2274, 2003 Printed in the USA 0730-7268/03 $12.00 ϩ .00

SUBLETHAL EFFECTS OF COPPER ON COHO SALMON: IMPACTS ON NONOVERLAPPING RECEPTOR PATHWAYS IN THE PERIPHERAL OLFACTORY NERVOUS SYSTEM

DAVID H. BALDWIN,² JASON F. S ANDAHL,³ JANA S. LABENIA,² and NATHANIEL L. SCHOLZ*² ²National Oceanic and Atmospheric Administration, Northwest Fisheries Science Center, 2725 Montlake Boulevard East, Seattle, Washington 98112, USA ³Environmental and Molecular Toxicology, Oregon State University, 333 Weniger Hall, Corvallis, Oregon 97331, USA

(Received 11 September 2002; Accepted 15 February 2003)

AbstractÐThe sublethal effects of copper on the sensory physiology of juvenile coho salmon (Oncorhynchus kisutch) were evaluated. In vivo ®eld potential recordings from the olfactory epithelium (electro-olfactograms) were used to measure the impacts of copper on the responses of olfactory receptor neurons to natural odorants (L-serine and taurocholic acid) and an odorant mixture (L-arginine, L-aspartic acid, L-leucine, and L-serine) over a range of stimulus concentrations. Increases in copper impaired the neurophysiological response to all odorants within 10 min of exposure. The inhibitory effects of copper (1.0±20.0 ␮g/L) were dose- dependent and they were not in¯uenced by water hardness. Toxicity thresholds for the different receptor pathways were determined by using the benchmark dose method and found to be similar (a 2.3±3.0 ␮g/L increase in total dissolved copper over background). Collectively, examination of these data indicates that copper is broadly toxic to the salmon olfactory nervous system. Consequently, short-term in¯uxes of copper to surface waters may interfere with olfactory-mediated behaviors that are critical for the survival and migratory success of wild salmonids.

KeywordsÐSalmon Oncorhynchus kisutch Copper Olfaction Biotic ligand model

INTRODUCTION importantly, olfaction plays a key role in the recognition and Water and sediment pollution are currently a concern for avoidance of predators [9,10], the recognition of kin [11], and the management of declining Paci®c salmon populations in in the reproductive synchronization of prespawning animals the western United States. These declines have resulted, in [7]. Moreover, a salmon's sense of smell determines, in part, part, from the deterioration or loss of critical freshwater and the long-term genetic integrity of wild and geographically dis- estuarine habitat [1]. Salmon and steelhead need cool, clean tinct populations. Salmon rely on chemical cues to form ol- water in adequate supply to grow, migrate, and spawn in fresh- factory memories [12] and return to their natal river system water systems. In the past decade, numerous salmon popula- to spawn [13]. Critically, the migratory patterns of adult salm- tions have been listed for protection under the Endangered on are disrupted when olfactory function is lost [13]. Collec- Species Act in California (USA) and the Paci®c Northwest. tively then, environmental contaminants that damage the ol- The widespread contamination of surface waters in western factory system could potentially impact salmonids at higher river systems [2,3] may be a limiting factor in the recovery scales of biological organization. of some of these stocks listed under the Endangered Species An established technique for measuring peripheral olfac- Act. tory function in ®sh is the use of ®eld potential recordings, or For salmon, the olfactory nervous system provides a po- electro-olfactograms (EOGs), to monitor the effects of pol- tential link between sublethal measures of neurotoxicity and lutants on the active (or odor-evoked) properties of primary biological consequences at the scale of individual animals or sensory neurons in the olfactory epithelium [14,15]. The EOG even natural populations. Salmon have a highly developed is a large, negative voltage transient and is measured with an sense of smell, and previous studies have shown that direct electrode positioned near the surface of the sensory epithelium. measures of olfactory capacity are reliable indicators of sub- The amplitude of the EOG re¯ects the summated electrical lethal neurotoxicity (reviewed by Klaprat et al. [4]). Use of response of olfactory receptor neurons (ORNs) as they bind the salmon olfactory system has several speci®c advantages to odor molecules in the surrounding environment [16]. There- for sublethal toxicity screening. First, primary olfactory re- fore, the EOG provides a direct measure of receptor neuron ceptor neurons are in direct contact with the surrounding en- function in the intact animal. vironment and therefore are more vulnerable to the toxic im- In the present study we evaluated the effects of copper on pacts of dissolved chemicals. Second, the olfactory epithelium odor-evoked EOGs from the olfactory epithelium of coho is easily accessible for the purpose of measuring odor-evoked salmon (Oncorhynchus kisutch) by using a computer-driven voltage potentials [5]. Third, the olfactory nervous system is exposure and data acquisition system. Copper is a widespread highly sensitive to dissolved chemicals in the animal's sur- source of water pollution in salmon habitat. Although the high- rounding environment (detection thresholds of 10Ϫ7 to 10Ϫ11 est levels of copper pollution are generally found in river sys- M, depending on the odorant [6±8]). Finally, and perhaps most tems that have been impacted by mining activities, copper also can be transported to salmon habitat from several other an- * To whom correspondence may be addressed thropogenic sources, including nonpoint sources. Copper is an ([email protected]). important pollutant in urban stormwater runoff [17], in part

2266 Effects of copper on coho olfactory neurons Environ. Toxicol. Chem. 22, 2003 2267

because of the use of copper in vehicle brake pads. Copper leaches out of treated wood placed in rivers and estuaries [18] and copper compounds are widely used as algicides in waterways and as fungicides on agricultural crops. As an example of surface water contamination, copper was the most frequently detected trace element at agricultural and mixed-use sites in the Willamette River Basin (Oregon, USA) [19]. Copper has previously been shown to impair the olfactory nervous system and olfactory-mediated behaviors in salmonids [15,20±22] and other ®sh species [23]. The aim of the present investigation was to extend these earlier studies by quantifying the effects of copper on the sensitivity of the salmon olfactory Fig. 1. Electophysiological recording system used to measure odor- evoked electro-olfactograms from the sensory epithelium of juvenile system to odorants that activate different classes of receptor coho salmon. See the Materials and Methods section for more in- neurons in the sensory epithelium, mixtures of natural odor- formation. (A) Schematic diagram showing the major components of ants, and odorants presented over a range of different stimulus the apparatus. The dashed box denotes the area shown in more detail concentrations. We also examined the in¯uence of water hard- in (B). (B) Photograph showing the rostrum of a coho salmon and the positioning of the perfusion tube and glass microelectrodes. ness on sublethal copper toxicity. We used this approach in (LabVIEW, National Instruments, Austin, TX, USA). combination with the benchmark dose method [24] to calculate thresholds for sublethal neurotoxicity to different peripheral olfactory pathways. dechlorinated city water (the source water for the Northwest Fisheries Science Center hatchery). The same water (without MATERIALS AND METHODS odorant) was used for baseline and wash perfusions. For ex- Experimental animals periments to test the in¯uence of hardness on copper toxicity, the total hardness of the water was increased by the addition Coho salmon eggs were obtained from the University of of CaCl2 before adding the odorants. All solutions were ad- Washington hatchery (Seattle, WA, USA) and raised in the justed to pH 7.5 to 7.6 with NaOH. hatchery facility at the Northwest Fisheries Science Center. City water was passed through activated charcoal ®lters to Copper solutions provide dechlorinated source water for the hatchery. Fish were A concentrated stock of copper chloride in distilled water maintained in 2,400-L ®berglass tanks supplied by a ®ltered, (3.4 g/L) was prepared each week and refrigerated. The copper Њ recirculating water system (11±13 C, pH 7.1, buffered to 120 stock was adjusted to pH 3.0 with HCl to maintain the copper ppm total hardness as CaCO3). The experiments reported here in ionic form. For exposures, copper solutions at different were performed on 77 juvenile animals with a weight of 143 concentrations were prepared daily by diluting the copper stock Ϯ Ϯ 8 g (mean standard error of the mean [SEM]) and fork into ®ltered, dechlorinated city water (source water) and ad- Ϯ length of 22.7 0.4 cm. Fish were anaesthetized with tricaine justing each solution to a pH of 7.5 to 7.6 with NaOH. For methane sulfonate (MS-222; 50 mg/L in hatchery water for some experiments, the hardness of the water was increased by 20 min) and then injected intramuscularly with the paralytic the addition of CaCl2 before adding the copper and adjusting gallamine triethiodide (0.3 mg/kg body mass). The skin over- the pH. lying the olfactory chamber (containing the olfactory rosette) To determine the concentrations of total dissolved copper was removed and the ®sh were placed in a Plexiglas holder in exposure solutions, triplicate samples of the source water on a vibration isolation table (Newport, Irvine, CA, USA). and each nominal copper exposure solution (1, 2, 5, 10, and Њ Chilled (12 C), oxygenated hatchery water containing MS-222 20 ␮g/L) were collected from the output of the perfusion sys- (50 mg/L) was delivered to the gills through a mouthpiece at tem and submitted to an outside laboratory (Frontier Geosci- a rate of 120 ml/min. A schematic diagram and photograph of ences, Seattle, WA, USA) for analysis by inductively coupled the perfusion and electrophysiological recording systems are plasma mass spectrometry (detection limit of 0.03 ␮g/L). Sam- shown in Figure 1. ples were passed through a precleaned 0.45-␮m ®lter before analysis. The source water used in this study contained total Stimulus solutions dissolved copper at 3.0 ␮g/L. The measured concentrations of Concentrated stock solutions of the bile salt taurocholic acid dissolved copper in the exposure solutions were 87% Ϯ 5% (TCA), the amino acid L-serine, and a mixture of the amino (mean Ϯ 1 SEM) of the expected concentrations (i.e., nominal acids L-arginine, L-aspartic acid, L-leucine, and L-serine (Sig- plus a background of copper at 3.0 ␮g/L in the source water ma, St. Louis, MO, USA) were prepared each week in distilled for the Northwest Fisheries Science Center hatchery). Expo- water and refrigerated at 4ЊC. These three solutions were used sures are reported as nominal copper concentrations, or an as odorants to stimulate the coho olfactory epithelium. Amino increase over background. acids and TCA have previously been shown to evoke electrophysiological and behavioral responses in salmonids [16]. Pre- Copper and odorant delivery to the olfactory epithelium vious studies have shown that TCA and each of the four amino A continuous ¯ow of chilled source water (12ЊC) was de- acids used in this study act on nonoverlapping populations of livered to the exposed rosette at a rate of 7 ml/min through a ORNs in ®sh [25±29]. Within a mixture, the four amino acids perfusion tube (Fig. 1B). Solutions containing source water were applied at the same concentration (i.e., a 10Ϫ8 M mixture only, source water plus odorant, source water plus copper, or contained 10Ϫ8 M L-arginine, 10Ϫ8 M L-aspartic acid, 10Ϫ8 M source water plus odorant and copper were stored in amber L-leucine, and 10Ϫ8 M L-serine). Odorant stimulus solutions bottles and gravity fed via separate, Te¯on tubes to a series were prepared daily by dilution of the stocks into ®ltered, of computer-controlled manifolds (Neptune Research, West 2268 Environ. Toxicol. Chem. 22, 2003 D.H. Baldwin et al.

Caldwell, NJ, USA). One input line to the manifolds was re- at a single copper concentration, and n refers to the number served for source water. The remaining input lines were varied of individual ®sh that were evaluated in each experiment. as needed for each experiment. The manifolds allowed for automated, rapid (10-ms) switching between the different so- Data analysis lutions. The single output from the manifolds passed through Electro-olfactogram responses were quanti®ed by measur- a custom-designed thermoelectric chiller just before delivery ing the peak negative amplitude relative to the preodorant Њ of the selected solution (at 12 C) to the rosette. With this baseline. In pilot experiments, the coef®cient of variance for approach, a custom computer program in LabVIEW (National between-animal olfactory responses was found to be 25.1% Instruments, Austin, TX, USA) was used to select a perfusion Ϫ (mean response to 10 5 M L-serine, n ϭ 9 ®sh). This was source from as many as 10 different copper- and odorant- slightly higher than the mean within-animal coef®cient of var- containing solutions. iance for the same ®sh (21.4%, 4±15 responses per ®sh, n ϭ Electrophysiological recordings 9 ®sh) and similar to the variability seen in the ensuing experiments (e.g., Fig. 2). The effects of copper were determined Electro-olfactograms were obtained by using an experi- by comparing the pre- and postexposure responses of each ®sh mental technique modi®ed from established methods [5,14]. to the various odorants. Thresholds for effect were determined Odor-evoked EOGs were recorded by using a pair of glass by following the benchmark dose method [24]. The threshold microelectrodes ®lled with 2% agar-saline and bridged to Ag- values obtained are referred to as benchmark concentrations, AgCl electrodes by 3 M KCl. The recording electrode was because they re¯ect exposure concentrations and not admin- placed along the midline of the rosette at the base of the large, istered doses. Statistical analyses were performed with posteriormost lamella [5] with the aid of a stereomicroscope StatView௡ (SAS, Cary, NC, USA). Kaleidagraph (Synergy (SMZ645, Nikon Instruments, Melville, NY, USA) mounted Software, Reading, PA, USA) was used for plotting nonlinear on a boom stand. A reference electrode was placed in the skin regressions and the production of graphs. above the rosette (see Fig. 1B). A separate ground (a hypo- dermic needle) was placed in the muscle near the tail. The RESULTS differential signal was ampli®ed (500ϫ) and ®ltered (100-Hz low-pass) with a direct current (DC) ampli®er (A-M Systems, Optimizing odor-evoked EOGs from the coho olfactory Carlsborg, WA, USA). The signal was then digitized at 240 epithelium samples/s by using a computerized data acquisition system To evaluate the effects of different pulse parameters on the (PowerMac G4, Apple Computer, Cupertino, CA, USA, and amplitude of odor-evoked EOGs in juvenile coho, we varied 6035E and DIO-96, National Instruments). As with the per- several aspects of the stimulus delivery. These included the fusion system, the collection of odor-evoked EOGs was con- duration of a single pulse, the interval between two pulses, trolled by using a custom data acquisition program in Lab- and the concentration of each odorant presented during a pulse. VIEW. The aim was to empirically determine the appropriate conditions for delivering stimulus solutions before, during, and after Testing procedure copper exposures. The EOG amplitudes varied as each of these After the placement of electrodes, ®sh were allowed to pulse parameters was varied (Fig. 2). acclimate for 15 min before tests began. The rosette was con- The in¯uences of odor pulse duration on peak amplitude tinuously perfused with source water or test solutions through- and the time to peak for EOGs evoked by 10Ϫ5 M L-serine are out each experiment. The EOGs were elicited by brie¯y shown in Figure 2A and B. A 10-s pulse was necessary to switching from source water (with or without added copper) evoke a maximal response for L-serine at 10Ϫ5 M. Shorter to an odorant-containing solution. Blank (or control) responses pulses produced smaller peaks or reduced the time to peak. were obtained by switching between source water delivered Longer pulse durations did not increase peak amplitude, but through the dedicated line and source water from a line nor- did increase the duration of ORN activity (and thus the po- mally used for odor-containing solutions. Blank pulses were tential for adaptation). Based on these results, pulse durations used to monitor for responses to any residual odorants in the of 10 s were used for all subsequent experiments. lines or manifolds. The consequences of varying the interval between two puls- A standard set of odor pulses was used for the copper es (10Ϫ5 M L-serine) on the amplitude of the second pulse are exposure experiments. The pulse duration, pulse interval, and shown in Figure 2C and D. When odor pulses were presented odor concentrations were determined from preliminary ex- in sequence, the amplitude of the second pulse increased with periments (see the Results and Fig. 2). Eight odor pulses (each the duration of the interpulse interval (Fig. 2C and D). As 10 s in duration and separated by 120 s) were presented in the indicated in Figure 2D, the responses of the olfactory epithe- following order: 10Ϫ5 M L-serine, 10Ϫ6 M TCA, 10Ϫ8 M amino lium to identical odorant pulses were equivalent if the pulses acid mixture, 10Ϫ7 M amino acid mixture, 10Ϫ6 M amino acid were separated by at least 120 s. Therefore, interpulse intervals mixture, 10Ϫ5 M amino acid mixture, and 10Ϫ5 M L-serine. of 120 s were used in the experiments that followed. The source water perfusing the rosette was then switched to Dose±response data for different odorants (Fig. 2E and F) source water plus copper for the duration of the exposure (30 were collected to determine appropriate stimulus concentra- or 60 min). During the copper exposure, 10-s pulses of 10Ϫ5 tions for the copper exposure experiments. Three different M L-serine (in copper plus source water) were presented every odorants were chosen for these experiments. As shown in Fig- 5 min to track the time to effect. After the copper exposure, ure 2E, perfusion of L-serine over the olfactory epithelium the perfusion was switched back to source water alone and the evoked a dose-dependent increase in the amplitude of the mea- preexposure sequence of eight odor pulses was again presented sured EOGs. At 10Ϫ5 M, L-serine stimulated an EOG response to the rosette. Additional 10-s pulses of 10Ϫ5 M L-serine then approximately three times the amplitude of the blank control were presented to track recovery. Each ®sh was tested once (Fig. 2F). Although larger EOG responses could be elicited Effects of copper on coho olfactory neurons Environ. Toxicol. Chem. 22, 2003 2269

by a higher concentration of L-serine (e.g., 10Ϫ4 M), 10Ϫ5 M used to follow the recovery of the olfactory epithelium after L-serine was chosen as a test odorant to minimize the possi- the rosette was returned to source water. At least some recovery bility of receptor adaptation. The dose±response data for TCA, was appparent after 30 min of wash (Fig. 4A). However, as is shown in Figure 2F, indicated that 10Ϫ6 M TCA produced an evident from the 10 ␮g/L trace in Figure 4A, ORNs did not EOG response that was comparable, in terms of peak ampli- recover completely within the time course of these experi- tude, to that of 10Ϫ5 M L-serine. Therefore, 10Ϫ6 M TCA was ments. Therefore, the inhibitory effects of a short-term copper chosen as the second test odorant. The ®nal set of test pulses exposure persist for hours and possibly longer. consisted of the amino acid mixture (see the Materials and To evaluate the potential role of hardness in copper's sub- Methods section) presented at four concentrations. Figure 2F lethal toxicity to the coho nervous system, the hardness of the shows that mixture concentrations of 10Ϫ8 Mto10Ϫ5 M evoked source water delivered to the rosette was adjusted by adding

EOG amplitudes comparable to the dose-response data from CaCl2. The water perfusing the gills (hatchery water) was not L-serine or TCA alone. The EOG responses to the mixture are changed. The source water used in previous experiments (e.g., presumed to re¯ect the summed activity of four nonoverlap- Fig. 4A) was soft, with a total hardness of 20 ppm. Amending ping classes of ORNs [29]. the source water to an intermediate hardness of 120 ppm or a In summary, 10Ϫ5 M L-serine, 10Ϫ6 M TCA, and an amino high hardness of 240 ppm did not signi®cantly alter the in- acid mixture presented at four concentrations (10Ϫ8 M, 10Ϫ7 hibitory effects of 10 ␮g/L copper on L-serine±evoked EOGs M, 10Ϫ6 M, and 10Ϫ5 MofL-arginine, L-aspartic acid, L-leu- (Fig. 4C). Therefore, water hardness does not seem to in¯uence cine, and L-serine) were chosen as model odorants for the the toxicity of copper to coho sensory neurons. copper exposure experiments. In addition, odorant pulses lasting 10 s with interpulse intervals of 120 s were selected. Over- Thresholds for sublethal copper neurotoxicity all, the experimental sequence of odorant stimulus, copper One of the goals of this study was to determine thresholds exposure, and odorant stimulus required continuous recordings for sublethal, copper-induced neurotoxicity in coho salmon. from the olfactory epithelium for a period of at least 1.5 h. To this end, dose±response data were collected for the three To ensure that EOG amplitudes were stable and that the prep- odorants (L-serine, TCA, and the amino acid mixture) at six aration was otherwise healthy for this interval, odor-evoked exposure concentrations for each. The exposure groups con- EOGs were recorded from unexposed ®sh for 3 h. The am- sisted of a blank or control group and 30-min copper exposures Ϫ plitudes of the EOGs evoked by either 10 5 M L-serine or a at nominal concentrations of 1, 2, 5, 10, and 20 ␮g/L (n ϭ 6 Ϫ 10 6 M amino acid mixture were stable and did not decline ®sh per exposure group). For the control group, the source within a three-hour interval (n ϭ 6 ®sh, data not shown). water perfusion was switched from the dedicated line to a line normally used to deliver copper-containing solutions. Notably, Copper inhibits multiple olfactory pathways in coho a slight reduction occurred in the amplitude of the evoked salmon EOGs for the control animals (mean Ϯ 1 SEM: L-serine, 0.84 A short-term copper exposure at a nominal concentration Ϯ 0.09; TCA, 0.83 Ϯ 0.08; amino acid mixture, 0.97 Ϯ 0.09). of 10 ␮g/L reduced the responsiveness of the sensory epithe- These reductions in evoked EOGs were not signi®cantly dif- lium to all three model odorants (Fig. 3A to C). For the ®sh ferent from each other (one-way analysis of variance, df ϭ 2, shown in Figure 3, a 30-min exposure reduced the L-serine F ϭ 1.186, p ϭ 0.330) and none were signi®cantly different response by 57%, the TCA response by 67%, and the response from 1 (one-group t test, hypothetical mean ϭ 1, p Ͼ 0.05). to the amino acid mixture by 35% (all relative to pre-exposure The reductions were likely due to the presence of residual EOG amplitudes). The effect of copper on the response to the copper in the line used to deliver the copper exposures. amino acid mixture (Fig. 3C) was measured as a shift in the The inhibitory effects of copper for all animals were nor- dose±response curve (Fig. 3D). To quantify the copper-induced malized to the mean reduction in odor-evoked responses of shift across the range of mixture concentrations, the pre-ex- the control animals. The resulting dose±response data are posure response amplitudes were plotted against the postex- shown in Figure 5. A two-way analysis of variance showed posure amplitudes (Fig. 3E). The effect of copper was ex- that copper had a signi®cant, dose-dependent effect on the pressed as the slope of the regression line, with a slope of one responsiveness of the sensory system to the odorants (df ϭ 4, indicating no impact of copper on mixture-evoked EOGs. The F ϭ 14.246, p Ͻ 0.001), but no signi®cant differences were L-serine and TCA responses are point estimates (i.e., a single found between the odorants (df ϭ 2, F ϭ 0.890, p ϭ 0.415). olfactory pathway at a single stimulus concentration), whereas Thresholds (benchmark concentrations) for copper toxicity the mixture data capture the effects of copper on four olfactory were estimated by following the benchmark dose method [24]. pathways over three log units of stimulus intensity. The data for each odorant were ®t with a sigmoid logistic The time to effect for copper is shown in Figure 4A. L- model Serine pulses were delivered every 5 min during 30-min ex- y ϭ 1/[1 ϩ (x/k)]n posures to copper at 1, 2, 5, 10, and 20 ␮g/L nominal concentrations (n ϭ 6 individual ®sh for each exposure concen- where y is the relative EOG amplitude, x is the copper con- tration). A reduction in EOG amplitude was evident within 10 centration, k is the copper concentration at half-maximum rel- min of exposure for all but the lowest copper concentration. ative EOG amplitude (inhibition concentration of 50%), and Moreover, a 30-min exposure was suf®cient to produce a max- n is the slope. Figure 5 shows that the model was a good ®t imal reduction in EOG peak amplitude at a given concentration for all three odorants. The benchmark concentration for each of copper. For example, no additional reduction in the ampli- odorant was then determined based on the nominal concen- tude of the odor-evoked EOG was observed when the duration tration at which each curve crossed a criterion level. The of the copper exposure (5 ␮g/L) was increased to 60 min (Fig. benchmark criterion was set to 0.75, a level very close to the 4B; n ϭ 3). bottom of the 95% con®dence interval for each odorant from Repeated pulses of 10Ϫ5 M L-serine at 5-min intervals were the control group (L-serine ϭ 0.73, TCA ϭ 0.77, mixture ϭ 2270 Environ. Toxicol. Chem. 22, 2003 D.H. Baldwin et al.

Fig. 2. Varying the duration, timing, and concentration of odorant pulses changes the waveform and amplitude of evoked electro-olfactograms (EOGs). The left column (A, C, and E) shows superimposed EOGs obtained from the same ®sh. The right column (B, D, and F) shows data averaged from multiple ®sh (mean Ϯ 1 standard error). (A) The EOG responses to 10Ϫ5 M L-serine pulses delivered with durations of 1, 3, 5, 10, and 20 s. The dashed vertical line illustrates the peak amplitude (as measured from the baseline) for the EOG evoked by a 1-s pulse. (B) Double y-axis plot showing the effect of pulse duration on peak amplitude and time to peak (n ϭ 4 ®sh). Time to peak was measured from the initial de¯ection from the baseline to the peak of the EOG. (C) Traces showing pairs of 10Ϫ5 M L-serine pulses (each 10 s long) presented with interpulse intervals of 20, 60, 90, and 120 s. For clarity, the intervening data have been deleted and only the evoked EOGs are shown. The ®rst pulses (four total) are superimposed and the second pulses have been arbitrarily positioned horizontally and aligned vertically by their initial downward de¯ections. (D) Pooled data (n ϭ 4 ®sh) showing the effect of pulse interval on the amplitude of the second pulse relative to the ®rst pulse. (E) The amplitudes of EOGs evoked in response to 10-s L-serine pulses increase with increasing odorant concentration (10Ϫ7 M, 10Ϫ6 M, 10Ϫ5 M, and 10Ϫ4 M). (F) Dose±response data (n ϭ 6 ®sh) for three odorants (L-serine, taurocholic acid [TCA], and an amino acid mixture). The dotted line shows the mean response to a blank control pulse (source water; n ϭ 4 ®sh).

0.76). When using this approach, the benchmark concentra- DISCUSSION tions for copper were found to be similar for all three odorants. The estimates (Ϯ 1 SE) for the benchmark concentrations were Identi®cation of the functional or mechanistic relationships 2.7 Ϯ 0.4 ␮g/L for L-serine, 2.3 Ϯ 0.6 ␮g/L for TCA, and 3.0 between chemical habitat quality and the viability of at-risk Ϯ 0.7 ␮g/L for the mixture. species is one of the major conceptual and empirical challenges Effects of copper on coho olfactory neurons Environ. Toxicol. Chem. 22, 2003 2271

Fig. 3. Short-term copper exposures diminish the responsiveness of the olfactory epithelium to natural odorants. A 30-min exposure to copper at 10 ␮g/L reduced the electro-olfactogram (EOG) evoked by 10Ϫ5 M L-serine by 57% (A) and the response to 10Ϫ6 M taurocholic acid (TCA) by 67% (B). Similarly, the EOG responses to all four concentrations of the amino acid mixture (C) were reduced. (D) The odorant dose±response curves before and after copper exposure show a consistent decrease in EOG amplitude. (E) Scatter plot comparing EOG amplitudes in response to the amino acid mixture before and after copper exposure. Filled circles are data from a single control ®sh and open circles are the data shown in C from a single exposed ®sh. Regression lines indicate a change in evoked EOGs after a 30- min perfusion of source water (dashed line) or source water containing copper at 10 ␮g/L (solid line). that currently confronts ecotoxicologists, conservation biolo- gists, and natural resource managers [30]. The consequences of habitat degradation must ®rst be measured in terms of the health or performance of exposed animals, and then extrap- olated to natural populations. However, this is not a trivial task [31]. It requires that toxicological endpoints be measured at scales below the level of the individual animal and, at the same time, have clear signi®cance for processes at higher scales of biological complexity. For many years, the olfactory system of ®sh has been recognized to be particularly vulnerable to the neurotoxic effects of copper and other dissolved pollutants in the aquatic envi- Fig. 4. The inhibitory effects of copper as a function of concentration, ronment. This includes the olfactory nervous system [4,32] as exposure duration, and water hardness. For all three panels, the postexposure electro-olfactogram (EOG) of an individual ®sh is normal- well as olfactory-mediated behaviors [23]. However, the extent ized to the pre-exposure response for that animal before averaging of this vulnerability is not well understood. Are all olfactory (mean Ϯ 1 standard error) within a treatment group. (A) Responses pathways and, by extension, all olfactory-mediated behaviors, to 10Ϫ5 M L-serine pulses were obtained at intervals during and after impaired by copper? This question has been dif®cult to answer copper exposures at concentrations ranging from 1 to 20 ␮g/L (n ϭ for salmonids, in part because previous copper studies have 6 animals for each copper concentration). The horizontal bar denotes the 30-min exposure. (B) The effects of perfusing the olfactory epi- generally used a single odorant, delivered at a single concen- thelium with copper at 5 ␮g/L for 60 min (n ϭ 3 ®sh). The horizontal tration, as a standard stimulus in toxicity evaluations bar denotes the 60-min exposure and the dashed line indicates the [15,22,33]. In the present study, we show that copper has maximal EOG reduction after the shorter (30-min) exposure in (A). similar inhibitory effects on ORNs that respond to different (C) The in¯uence of water hardness (as CaCl2) on the inhibitory effects of copper (n ϭ 4±6 ®sh per hardness category). The horizontal classes of olfactory stimuli (bile salts vs amino acids). More- bar denotes a 30-min exposure to copper at 10 ␮g/L. over, for mixtures of amino acids that stimulate nonoverlapping 2272 Environ. Toxicol. Chem. 22, 2003 D.H. Baldwin et al.

levels above the background concentrations in base ¯ows. In the present study, the source water for the hatchery contained a small amount of copper (3 ␮g/L), and this may have con- tributed to the observed toxicity in the nominal exposures. When juvenile coho salmon were transiently exposed to copper at concentrations above this background, short-term increases of 3 ␮g/L or more signi®cantly impaired the sensory physiology of the exposed animals. It should be noted that the U.S. Environmental Protection Agency's water-quality criterion for dissolved copper (at a hardness of 100 mg/L) in freshwater systems is 13 ␮g/L for a 1-h average maximum concentration. This is approximately equivalent to our 30-min nominal exposures at 10 ␮g/L. Thus, based on our present results, a stormwater pulse containing copper at 13 ␮g/L could be expected to cause a Ͼ50% loss of sensory capacity among resident coho in freshwater habitats. Copper's toxicity to the salmonid olfactory system mani- Fig. 5. Dose±response curves and threshold determinations for sub- fests on a time scale of minutes. Although the precise mode lethal copper neurotoxicity. Data were obtained from six treatment of action in sensory neurons is not known [4], the inhibitory groups (control and ®ve copper exposures; n ϭ 6 ®sh per group). The effects of a brief (30-min) copper exposure are at least partially evoked electro-olfactogram (EOG) amplitudes for all copper expo- reversible. However, previous studies on other salmonid spe- sures were normalized to the mean response of the controls and expressed as a group (mean Ϯ 1 standard error). The dashed line in- cies have shown that exposures lasting 4 h or longer cause dicates a benchmark criterion of 0.75, or a 25% reduction in evoked ORNs to undergo cell death [22,35,36]. In chinook salmon, a response (relative to controls). Three vertical lines in the upper right loss of ORNs is accompanied by a loss of sensitivity to amino show the lower limits of the 95% con®dence interval for the control acids [22] and an inability to detect and avoid copper-con- response to the three different odorants. Filled circles indicate the taining water [21]. Therefore, olfactory function will be im- benchmark concentrations for the different olfactory pathways (L- serine, taurocholic acid [TCA], and the amino acid mixture). Note paired if salmon are unable to avoid copper pollution within that the benchmark values are nominal concentrations, or a change the ®rst few minutes of exposure. If copper levels subsequently (increase) from the copper present at approximately 3 ␮g/L in the exceed a threshold for sensory cell death, it may be weeks source water for the Northwest Fisheries Science Center hatchery before the functional properties of the olfactory system recover (Seattle, WA, USA). [37]. For salmon and steelhead, olfactory cues convey impor- populations of ORNs, the inhibitory effects of copper are ev- tant information about habitat quality (e.g., pollution), pred- ident across three log units of stimulus intensity. Consequently, ators, conspeci®cs, mates, and the animal's natal stream. all olfactory-mediated behaviors in Paci®c salmon could be Therefore, a substantial copper-induced loss of olfactory ca- affected by sublethal copper exposures if, as examination of pacity is likely to impair behaviors that might be considered the present data suggests, the toxicological impacts of copper essential for survival or reproductive success. For example, are similar for all ORNs (irrespective of their af®nities for copper exposures will shift the detection thresholds for nat- different natural odorants). Although the peripheral olfactory ural odorants to higher concentrations (Fig. 3D), and juvenile pathways involved in imprinting, predator avoidance, and salmonids may fail to respond to pheromones [8] and other homing behaviors have not been identi®ed in salmon, a general cues that naturally occur at levels near the lower detection copper-induced loss of olfactory capacity could be expected limits for unexposed animals. If the pheromone signals the to disrupt these and other higher-order processes. proximity of a predator [9,38], a failure to respond could It is important to note that salmonids will actively avoid result in ecological death. Of course, in the natural environ- copper [21,34]. Where distinct copper gradients are present in ment, the magnitude of behavioral impairment will depend salmon habitat (e.g., near a point-source discharge), salmon on the olfactory cue and the site-speci®c ecological context. may use their sense of smell to detect and avoid contaminated We cannot infer speci®c behavioral impacts from our neu- surface waters. As a consequence, low levels of copper pol- rophysiological results because, to our knowledge, neuro- lution could serve as a barrier to migration or exclude salmon physiological and behavioral thresholds for sublethal copper from habitats that are otherwise productive [34]. However, toxicity have never been directly compared in ®sh. Clearly, behavioral avoidance may not be an adequate defense against salmon and steelhead cannot detect and respond to environ- stormwater runoff, agricultural runoff, or other nonpoint sourc- mental cues in the absence of a functional sensory system. es of copper. In the absence of de®ned spatial gradients, it is However, evaluating the effects of a partial loss of sensory unlikely that salmon and steelhead will be able to avoid non± capacity on behavioral function is considerably more com- point-source inputs of copper to lakes, rivers, and estuaries. plex, and this is an important area for future research. The copper exposures used in this study were environ- Water hardness, expressed as the sum of Ca2ϩ and other mentally realistic. They were designed to emulate non±point- divalent cations in solution, is known to affect the acute tox- source copper pollution in salmon habitat arising from surface icity of copper and other metals to ®sh [39]. Dissolved copper water runoff. In the Willamette River study, for example, cop- is more acutely lethal to salmonids as water hardness decreas- per was detected in 19 of 23 ®ltered water samples at con- es. According to the biotic ligand model, the bioavailability centrations ranging up to 21 ␮g/L [19]. Thus, during storm of copper can be decreased by increasing the concentrations events, juvenile salmonids in urban and agricultural water- of cations that compete for metal binding sites on gills and sheds are likely to be exposed to short-term increases in copper other tissues [40]. The biotic ligand model is generally applied Effects of copper on coho olfactory neurons Environ. Toxicol. Chem. 22, 2003 2273 to data from copper exposures at higher concentrations and 7. Moore A, Waring CP. 1996. Electrophysiological and endocri- longer durations than those used in the present study [40]. nological evidence that F-series prostaglandins function as prim- ing pheromones in mature male Atlantic salmon (Salmo salar) However, our results indicate that water hardness may not parr. J Exp Biol 199:2307±2316. in¯uence the sublethal impacts of copper on coho sensory 8. Sveinsson T, Hara TJ. 2000. Olfactory sensitivity and speci®city neurons. For example, a 10-fold increase in the Ca2ϩ content of Arctic char, Salvelinus alpinus, to a putative male pheromone, of the perfusion solution did not alter the inhibitory effects of prostaglandin F-2 alpha. Physiol Behav 69:301±307. copper on ORNs (Fig. 4C). Presumably, copper and Ca2ϩ do 9. Brown GE, Smith RJ. 1997. Conspeci®c skin extracts elicit antipredator responses in juvenile rainbow trout (Oncorhynchus my- not compete for binding sites in the olfactory epithelium. kiss). Can J Zool 75:1916±1922. Based on these preliminary data, it should not be assumed that 10. Hiroven H, Ranta E, Piironen J, Laurila A, Peuhkuri N. 2000. the neurotoxicity of copper is proportionally less in harder Behavioural responses of naive Arctic charr young to chemical surface waters. cues from salmonid and non-salmonid ®sh. Oikos 88:191±199. The present study used a benchmark dose method [24] to 11. Quinn TP, Busack CA. 1985. Chemosensory recognition of sib- lings in juvenile coho salmon (Oncorhynchus kisutch). Anim Be- calculate sublethal thresholds for functional neurophysiolog- hav 33:51±56. ical impairment in coho. Compared with the no-observable- 12. Nevitt GA, Dittman AH, Quinn TP,Moody WJ Jr. 1994. Evidence effect concentration, the benchmark concentration is a more for a peripheral olfactory memory in imprinted salmon. Proc Natl precise determination of threshold, because it is not speci®cally Acad Sci USA 91:4288±4292. 13. Wisby WJ, Hasler AD. 1954. Effect of occlusion on migrating constrained by a single exposure concentration. Although the silver salmon (Oncorhynchus kisutch). J Fish Res Board Can 11: sample sizes for the dose±response experiments were relatively 472±478. small (n ϭ 6 ®sh per exposure concentration), the measure- 14. Baatrup E, Doving KB, Winberg S. 1990. Differential effects of ments obtained were suf®cient to calculate inhibitory effects mercurial compounds on the electroolfactogram (EOG) of salmon thresholds for copper on different olfactory pathways. Thus, (Salmo salar L.). Ecotoxicol Environ Saf 20:269±276. 15. Winberg S, Bjerselius R, Baatrup E, Doving KB. 1992. The effect the in vivo electrophysiological recording methods used in this of Cu(II) on the electro-olfactogram (EOG) of the Atlantic salmon study, in combination with benchmark concentration statistics, (Salmo salar L.) in arti®cial freshwater of varying inorganic car- provide an empirical and reproducible approach to determining bon concentrations. Ecotoxicol Environ Saf 24:167±178. thresholds for sublethal neurotoxicity in salmonids. 16. Hara TJ. 1992. Mechanisms of olfaction. In Hara TJ, ed, Fish In summary, we have shown that transient exposures to Chemoreception. Chapman & Hall, London, UK, pp 150±170. 17. City of Bellevue. 1995. Characterization and source control of copper signi®cantly impair the sensory physiology of juvenile urban stormwater quality, Vol 1. Technical Report. Bellevue, WA, coho salmon. These exposures are typical of copper concen- USA. trations that have been measured in surface waters from urban 18. Weis JS, Weis P. 1996. The effects of using wood treated with and agricultural watersheds. The sublethal thresholds for cop- chromated copper arsenate in shallow-water environments: A re- per toxicity are very similar for different olfactory pathways. view. Estuaries 19:306±310. 19. Anderson CW, Rinella FA, Rounds SA. 1996. Occurrence of se- Therefore, copper may interfere with many (or all) olfactory- lected trace elements and organic compounds and their relation mediated behaviors in coho that cannot avoid storm water and to land use in the Willamette River basin, Oregon, 1992±94. Water other non±point-source inputs to salmon habitat. More work Resources Investigations Report 96-4234. U.S. Geological Sur- is needed to de®ne the behavioral consequences of this sensory vey, Portland, OR. neurotoxicity. 20. Hara TJ, Law YMC, Macdonald S. 1976. Effects of mercury and copper on the olfactory response in rainbow trout. J Fish Res Board Can 33:1568±1573. AcknowledgementÐWe wish to thank Kathi Lefebvre, Andy Dittman, 21. Hansen JA, Marr JCA, Lipton J, Cacela D, Bergman HL. 1999. Tracy Collier, and John Stein for comments that improved the man- Differences in neurobehavioral responses of chinook salmon (On- uscript and Brad Gadberry for the care and maintenance of coho corhynchus tshawytscha) and rainbow trout (Oncorhynchus my- salmon. This work was supported by an Academies of Science and kiss) exposed to copper and cobalt: Behavioral avoidance. En- Engineering postdoctoral associateship to D.H. Baldwin and a Na- viron Toxicol Chem 18:1972±1978. tional Institute of Environmental Health Science Training grant 22. Hansen JA, Rose JD, Jenkins RA, Gerow KG, Bergman HL. 1999. T32ES07060 to J.F. Sandahl. We also thank Frontier Geosciences for Chinook salmon (Oncorhynchus tshawytscha) and rainbow trout their analysis of copper concentrations. (Oncorhynchus mykiss) exposed to copper: Neurophysiological and histological effects on the olfactory system. Environ Toxicol REFERENCES Chem 18:1979±1991. 1. National Marine Fisheries Service. 1996. Factors for decline: A 23. Beyers DW, Farmer MS. 2001. Effects of copper on olfaction of supplement to the notice of determination of west coast steelhead Colorado pikeminnow. Environ Toxicol Chem 20:907±912. under the Endangered Species Act. Protected Species Branch, 24. U.S. Environmental Protection Agency. 1995. The use of the Portland, OR, USA. benchmark dose approach in health risk assessment. EPA 630/R- 2. Domagalski J. 1996. Pesticides and pesticide degradation prod- 94/007. Of®ce of Research and Development, Washington, DC. ucts in stormwater runoff: Sacramento River Basin, California. 25. Hara TJ. 1982. Structure±activity relationships of amino acids as Water Res Bull 32:953±964. olfactory stimuli. In Hara TJ, ed, Chemoreception in Fishes. El- 3. Wentz DA, Bonn BA, Carpenter KD, Hinkle SR, Janet ML, Ri- sevier, Amsterdam, The Netherlands, pp 135±157. nella FA, Uhrich MA, Waite IR, Laenen A, Bencala K. 1998. 26. Hara TJ, Macdonald S, Evans RE, Marui T, Arai S. 1984. Mor- Water quality in the Willamette River basin, Oregon, 1991±95. pholine, bile acids and skin mucus as possible chemical cues in Circular 1161. U.S. Geological Survey, Washington DC. salmonid homing: Electrophysiological re-evaluation. In Mc- 4. Klaprat DA, Evans RE, Hara TJ. 1992. Environmental contam- Cleave JD, Arnold GP, Dodson JJ, Neill WH, eds, Mechanisms inants and chemoreception in ®shes. In Hara TJ, ed, Fish Che- of Migration in Fishes. Plenum, New York, NY, USA, pp 363± moreception. Chapman & Hall, London, UK, pp 321±342. 378. 5. Evans R, Hara TJ. 1985. The characteristics of the electro-olfac- 27. Caprio J, Byrd RP Jr. 1984. Electrophysiological evidence for togram (EOG): Its loss and recovery following olfactory nerve acidic, basic, and neutral amino acid olfactory receptor sites in section in rainbow trout (Salmo gairdneri). Brain Res 330:65± the cat®sh. J Gen Physiol 84:403±422. 75. 28. Sorensen PW, Hara TJ, Stacey NE. 1987. Extreme olfactory sen- 6. Hara TJ. 1972. Electrical responses of the olfactory bulb of Paci®c sitivity of mature and gonadally-regressed gold®sh to a potent salmon Oncorhynchus nerka and Oncorhynchus kisutch. J Fish steroidal pheromone, 17 alpha, 20 beta-dihydroxy-4-pregnen-3- Res Board Can 29:1351±1355. one. J Comp Physiol A 160:305±313. 2274 Environ. Toxicol. Chem. 22, 2003 D.H. Baldwin et al.

29. Sveinsson T, Hara TJ. 1990. Multiple olfactory receptors for ami- in the olfactory epithelium of rainbow trout exposed to a low no acids in Arctic char (Salvelinus alpinus) evidenced by cross- copper level. Tissue Cell 28:367±377. adaptation experiments. Comp Biochem Physiol A 97:289±293. 36. Moran DT, Rowley JC, Aiken GR, Jafek BW. 1992. Ultrastruc- 30. Hansen LJ, Johnson ML. 1999. Conservation and toxicology: The tural neurobiology of the olfactory mucosa of the brown trout, need to integrate the disciplines. Environ Toxicol Chem 18:2121± Salmo trutta. Microsc Res Tech 23:28±48. 2122. 37. Zielinski BS, Hara TJ. 1992. Ciliated and microvillar receptor 31. Johnson LL, Collier TK. 2002. Assessing contaminant-induced cells degenerate and then differentiate in the olfactory epithelium stress across levels of biological organization. In Adams SM, ed, of rainbow trout following olfactory nerve section. Microsc Res Biological Indicators of Aquatic Stress. American Fisheries So- Tech 23:22±27. ciety, Bethesda, MD, pp 533±564. 38. Scholz NL, Truelove NK, French BL, Berejikian BA, Quinn TP, Casillas E, Collier TK. 2000. Diazinon disrupts antipredator and 32. Sutterlin AM. 1974. Pollutants and the chemical senses of aquatic homing behaviors in chinook salmon (Oncorhynchus tshawyts- animalsÐPerspective and review. Chem Sens Flav 1:167±178. 2ϩ cha). Can J Fish Aquat Sci 57:1911±1918. 33. Bjerselius R, Winberg S, Winberg Y, Zeipel K. 1993. Ca pro- 39. Sorensen EMB. 1991. Copper. In Sorensen EMB, ed, Metal Poi- tects olfactory receptor function against acute Cu(II) toxicity in soning in Fish. CRC, Boca Raton, FL, USA, pp 235±284. Atlantic salmon. Aquat Toxicol 25:125±138. 40. Meyer JS, Santore RC, Bobbit JP, Debrey LD, Boese CJ, Paquin 34. Saunders RL, Sprague JB. 1967. Effects of copper±zinc mining PR, Allen HE, Bergman HL, Ditoro DM. 1999. Binding of nickel pollution on a spawning migration of Atlantic salmon. Water Res and copper to ®sh gills predicts toxicity when water hardness 1:419±432. varies, but free-ion activity does not. Environ Sci Technol 33: 35. Julliard AK, Saucier D, Astic L. 1996. Time-course of apoptosis 913±916. NOAA Technical Memorandum NMFS-NWFSC-83

An Overview of Sensory Effects on Juvenile Salmonids Exposed to Dissolved Copper: Applying a Benchmark Concentration Approach to Evaluate Sublethal Neurobehavioral Toxicity

October 2007

U.S. DEPARTMENT OF COMMERCE National Oceanic and Atmospheric Administration National Marine Fisheries Service NOAA Technical Memorandum NMFS-NWFSC Series The Northwest Fisheries Science Center of the National Marine Fisheries Service, NOAA, uses the NOAA Techni- cal Memorandum NMFS-NWFSC series to issue scientiﬁc and technical publications. Manuscripts have been peer reviewed and edited. Documents published in this series may be cited in the scientiﬁc and technical literature.

The NMFS-NWFSC Technical Memorandum series of the Northwest Fisheries Science Center continues the NMFS- F/NWC series established in 1970 by the Northwest & Alaska Fisheries Science Center, which has since been split into the Northwest Fisheries Science Center and the Alaska Fisheries Science Center. The NMFS-AFSC Techni- cal Memorandum series is now being used by the Alaska Fisheries Science Center.

Reference throughout this document to trade names does not imply endorsement by the National Marine Fisheries Service, NOAA.

This document should be referenced as follows: Hecht, S.A., D.H. Baldwin, C.A. Mebane, T. Hawkes, S.J. Gross, and N.L. Scholz. 2007. An overview of sensory effects on juvenile salmonids exposed to dissolved copper: Applying a benchmark concentration approach to evaluate sublethal neurobehavioral toxicity. U.S. Dept. Commer., NOAA Tech. Memo. NMFS-NWFSC-83, 39 p. NOAA Technical Memorandum NMFS-NWFSC-83

An Overview of Sensory Effects on Juvenile Salmonids Exposed to Dissolved Copper: Applying a Benchmark Concentration Approach to Evaluate Sublethal Neurobehavioral Toxicity

Scott A. Hecht,1 David H. Baldwin, Christopher A. Mebane,2 Tony Hawkes,1 Sean J. Gross,3 and Nathaniel L. Scholz

Northwest Fisheries Science Center Ecotoxicology and Environmental Fish Health Program 2725 Montlake Boulevard East Seattle, Washington 98112

1 National Marine Fisheries Service Ofﬁce of Protected Resources, Endangered Species Division 510 Desmond Drive Southeast Lacey, Washington 98506

2 U.S. Geological Survey NMFS Liaison 10095 West Emerald Street Boise, Idaho 83704

3 National Marine Fisheries Service Washington State Habitat Ofﬁce 7600 Sand Point Way Northeast Seattle, Washington 98115

October 2007

U.S. DEPARTMENT OF COMMERCE National Oceanic and Atmospheric Administration National Marine Fisheries Service Most NOAA Technical Memorandums NMFS-NWFSC are available online at the Northwest Fisheries Science Center web site (http://www.nwfsc.noaa.gov)

Copies are also available from: National Technical Information Service 5285 Port Royal Road Springﬁeld, VA 22161 phone orders (1-800-553-6847) e-mail orders ([email protected])

ii Table of Contents

List of Figures...... v List of Tables ...... vii Executive Summary...... ix Acknowledgments...... xi Introduction...... 1 Previous Studies on the Effects of Copper...... 3 Application of the Benchmark Concentration Analysis...... 9 Results of the Benchmark Concentration Analysis...... 11 Discussion of Site Specific Considerations for Sensory System Effects...... 14 Impairment from Short-term Increases of dCu...... 14 Calculating an Acute Criterion Maximum Concentration...... 14 Salmonids Are Typically Exposed to Multiple Stressors ...... 15 Bioavailability of dCu ...... 15 Olfactory Toxicity in Saltwater ...... 16 Avoiding Short-term Increases in dCu ...... 17 Coho Salmon–derived BMCs Should Apply to Other Salmonids...... 17 Conclusions...... 18 Glossary ...... 19 References...... 21 Appendix A: Other Salmonid Sensory Effects of dCu ...... 27 Migratory Disruption...... 27 Laboratory Avoidance Studies ...... 28 Other Adverse Effects ...... 31 Appendix B: Supplementary Bibliography...... 33 Copper Sources...... 33 Benchmark Concentration/Dose...... 33 Copper and Neurobiology ...... 33 Reviews ...... 35 Studies with Other Metals ...... 36 Selected Behavioral Studies ...... 37 Recovery of Olfactory Epithelium after Damage...... 38 Additional Endpoints...... 38

iii

iv List of Figures

Figure 1. Recording methods and features of the salmon peripheral olfactory system...... 6 Figure 2. Copper-induced reductions in juvenile salmonid olfactory response and behavior are significantly correlated...... 8 Figure 3. Using a benchmark concentration approach to estimate a threshold for dCu toxicity in the salmonid olfactory system...... 12 Figure A-1. Reduction in downstream migration of yearling coho salmon following 6-days exposure to copper at various concentrations...... 29

vi List of Tables

Table 1. Selected examples of adverse effects with copper to salmonids or their prey...... 4 Table 2. Benchmark responses and benchmark concentrations for juvenile salmon exposed to dCu for 3 hours...... 13

vii

viii Executive Summary

Dissolved copper (dCu) is a ubiquitous surface water pollutant that causes a range of adverse effects in fish as well as in aquatic invertebrates and algae. This technical memorandum is a summary and targeted synthesis regarding sensory effects to juvenile salmonids from low- level exposures to dCu. As such, the material presented here serves to summarize scientific research on dCu and its impacts on salmonid sensory systems. In addition, this document provides a benchmark analysis of empirical data generated in recent National Marine Fisheries Service investigations that have focused on salmon olfactory function. The review section, Appendix A, discusses peer reviewed and gray literature on the effects of dCu on salmonid sensory systems, associated sensory-mediated behaviors, and physiology. It is intended to facilitate understanding of the effects of dCu on sensory system–mediated behaviors that are important to survival, reproduction, and distribution of salmonids. The review does not address the effects of dCu on salmonid habitats, although copper is also highly toxic at low µg/L concentrations to aquatic primary producers and invertebrates (i.e., the aquatic food web). Undoubtedly, new information will become available that enhances our current understanding of copper’s effect on threatened and endangered salmonids and their supporting habitats.

A large body of scientific literature has shown that fish behaviors can be disrupted at concentrations of dCu that are at or slightly above ambient concentrations (i.e., background). In this document, background is operationally defined as surface waters with less than 3 µg/L dCu, as experimental water had background dCu concentrations as high as 3 µg/L dCu. Sensory system effects are generally among the more sensitive fish responses and underlie important behaviors involved in growth, reproduction, and (ultimately) survival (i.e., predator avoidance). Recent experiments on the sensory systems and corresponding behavior of juvenile salmonids contribute to more than four decades of research and show that dCu is a neurotoxicant that directly damages the sensory capabilities of salmonids at low concentrations. These effects can manifest over a period of minutes to hours and can persist for weeks.

To estimate toxicological effect thresholds for dCu in surface waters, benchmark concentrations (BMCs) were calculated using a U.S. Environmental Protection Agency methodology. This paper presents examples of BMCs for juvenile salmonid olfactory function based on recent data. BMCs ranged 0.18–2.1 µg/L, corresponding to reductions in predator avoidance behavior of approximately 8–57%. The BMC examples represent the dCu concentration (above background) expected to affect the ability of juvenile salmonids to avoid predators in freshwater. These concentration thresholds for juvenile salmonid sensory and behavioral responses fall within the range of other sublethal endpoints affected by dCu such as behavior, growth, and primary production, which is 0.75–2.5 µg/L.

The paper also discusses the influence of water chemistry on the bioavailability and toxicity of copper to fish sensory systems. Studies exploring behavioral avoidance as well as representative studies of other effects to salmonids are also summarized. Salmon may be able to

ix avoid dCu in environmental situations where distinct gradients occur. However, avoidance of dCu originating from nonpoint sources appears unlikely. Given the large body of literature on copper and responses of aquatic ecosystems, we focused on a subset of fish sensory system studies relevant to anadromous salmonids.

Point and nonpoint source discharges from anthropogenic activities frequently exceed these thresholds by one, two, and sometimes three orders of magnitude, and can occur for hours to days. The U.S. Geological Survey ambient monitoring results for dCu representing 811 sites across the United States detected concentrations ranging 1–51 µg/L, with a median of 1.2 µg/L. Additionally, typical dCu concentrations originating from road runoff from a California study were 3.4–64.5 µg/L, with a mean of 15.8 µg/L. Taken together, the information reviewed and presented herein indicates that impairment of sensory functions important to survival of juvenile salmonids is likely to be widespread in many freshwater aquatic habitats. Impairment of these essential behaviors may manifest within minutes and continue for hours to days depending on concentration and exposure duration. Therefore, dCu has the potential to limit the productivity and intrinsic growth potential of wild salmon populations by reducing the survival and lifetime reproductive success of individual salmonids.

x Acknowledgments

The authors thank Jim Meador, Lyndal Johnson, and Karen Peck for insightful and critical reviews of prior versions of this document. We also thank Jenifer McIntyre, Neil Rickard, and Scott Anderson for fruitful discussions and comments.

xii Introduction

Copper, a naturally occurring element, is an essential micronutrient for plants and animals. However, copper is also recognized as a priority pollutant under the U.S. Clean Water Act. Historical and current anthropogenic activities have mobilized significant quantities of copper. Vehicle emissions and brake pad dust (Drapper et al. 2000), pesticides (USEPA 2005), industrial processes, municipal discharges, mining, and rooftops (Good 1993, Thomas and Greene 1993) are a few of the sources of copper in the environment. These various human activities may lead to the unintended and, in some circumstances, intended introduction of copper into aquatic ecosytems (Sansalone and Buchberger 1997, Wheeler et al. 2005). Once in the aquatic environment, copper is detected in multiple forms. It can be dissolved, or bound to organic and inorganic materials either in suspension or in sediment. This so called speciation of copper is dependent on site specific abiotic and biotic factors. As an element, copper will persist and cycle through ecosystems. Copper in its dissolved state is worthy of particular scrutiny as it is highly toxic to a broad range of aquatic species including algae, macrophytes, aquatic invertebrates, and fishes. The latter include anadromous salmon and steelhead within the Oncorhynchus and Salmo genera that are, in part, managed by the National Marine Fisheries Service.

Currently, anadromous salmonid populations inhabit waters of Alaska, Oregon, Washington, California, Idaho (Oncorhynchus spp.), and Maine (Atlantic salmon [Salmo salar]). Dissolved copper (referred to as dCu herein) is consistently detected in salmonid habitats including areas important for rearing, migrating, and spawning (Alpers et al. 2000, Soller et al. 2005). Dissolved copper is known to affect a variety of biological endpoints in fish (e.g., survival, growth, behavior, osmoregulation, sensory function, and others, as reviewed in Eisler 1998). More than three decades of experimental results have shown that the sensory systems of salmonids are particularly vulnerable to the neurotoxic effects of dCu. Recent experimental evidence showed that juvenile sensory system–mediated behaviors are also affected by short- term exposures to dCu.

Given the ecological significance of these behaviors to salmonids, it is important to characterize the potential effects from dCu. The growing body of scientific literature indicates that dCu is a potent neurotoxicant that directly damages the sensory capabilities of salmonids at low concentrations (see the Previous Studies on the Effects of Copper section). These concentrations may stem from anthropogenic inputs of dCu to salmonid habitats. Salmonid sensory systems mediate ecologically important behaviors involved in predator avoidance, migration, and reproduction. Impairment of these behaviors can limit an individual salmonid’s potential to complete its life cycle and thus may have adverse consequences at the scale of wild populations.

The purpose of this paper is to: (1) summarize information on the effects of dCu to the sensory systems of juvenile salmonids in freshwater (also see Appendix A), (2) conduct a benchmark concentration analysis to generate examples of dCu effect thresholds, and (3) to discuss site-specific considerations for sensory system effects. As such, it focuses on a single contaminant (dCu), two relevant sensory system endpoints (olfaction and alarm response behavior), and a single salmonid life stage (juvenile, <10 months old).

2 Previous Studies on the Effects of Copper

Examples of copper’s effects on a suite of selected biological endpoints from laboratory and field exposures are presented in Table 1. Additionally, Appendix A contains a targeted review and summary of some of the previous studies showing copper’s effect on salmonid behavior, including avoidance and migratory disruptions. Appendix B is a supplementary bibliography that provides further information sources on salmonid sensory systems. The following analysis of sensory effects on juvenile salmonids primarily emphasizes recent and ongoing research conducted at the National Marine Fisheries Service’s Northwest Fisheries Science Center. However, the phenomenon that copper and some other trace metals can interfere with chemoreception, alter behaviors, and influence the movements of fish was first described at least 40 years ago, and a large body of knowledge on the adverse effects of dCu has subsequently developed (Table 1).

The salmonid olfactory sensory system relies on olfactory receptor neurons (ciliated ORNs) to detect and respond to cues in the aquatic environment. The receptors are in direct contact with the aqueous environment. Olfactory receptors detect chemical cues that are important in finding food, avoiding predators, navigating migratory routes, recognizing kin, reproducing, and avoiding pollution. The architecture of the salmon olfactory system consists of a pair of olfactory rosettes, each positioned within an olfactory chamber near the midline of the fish’s rostrum (Figure 1A). Each rosette contains ORNs that respond to dissolved odorants as water passes through the olfactory chamber (Figure 1B) and over the surface of the rosette in which the receptor neurons are embedded (Figure 1C). These chemical cues convey important information about the surrounding aquatic environment.

Direct exposure to dCu can impair and destroy olfactory sensory neurons, although the precise mechanism by which dCu interferes with the normal function of ORNs remains unknown (Hansen et al. 1999b, Baldwin et al. 2003, Sandahl et al. 2006, Sandahl et al. 2007). Impairment of olfaction (i.e., smell) can be measured by an electrophysiological technique called the electro-olfactogram (EOG) (Figure 1) (Scott and Scott-Johnson 2002, Baldwin and Scholz 2005, Sandahl et al. 2006). The EOG measures olfactory response of a population of receptor neurons in fish. Reductions in the EOG amplitude of copper-exposed fish compared to unexposed fish reflect functional losses in sensory capacity. Dissolved copper’s toxic effect to olfactory sensory neurons is observable as a reduction in or elimination of the EOG amplitude to a recognizable odor (Figure 1D).

Several recent studies highlight some important aspects of copper olfactory toxicity (Baldwin et al. 2003, Sandahl et al. 2004, 2007). Baldwin et al. (2003) found that the neurotoxic effects of copper in coho salmon (Oncorhynchus kisutch) manifest over a timescale of minutes. At 10 minutes, EOG amplitude reductions were observed in juvenile coho exposed to 2, 5, 10, and 20 µg/L dCu above experimental background (3 µg/L). After 30 minutes at 2 µg/L dCu above experimental background, the EOG amplitude from juvenile coho to odors was reduced by approximately 25% compared to controls; in 20 µg/L dCu after 30 minutes by approximately

3 80%. Sandahl et al. (2004) found similar effects following 7 days of exposure (both in EOG reductions and copper concentrations). This result indicated that the juvenile olfactory system does not appear to be able to adapt or otherwise compensate for continuous copper exposure for durations up to 7 days.

Table 1. Selected examples of adverse effects with copper to salmonids or their prey.a

Effect Species concentra- Effect Hardness Exposure (lifestage) Effect tion (µg/L)b statistic (mg/L)c duration Source

Sensory and behavioral effects

Coho salmon Reduced olfaction and 0.18–2.1 EC10 to 120 3 hours Sandahl et al. (juvenile) compromised alarm EC50 2007 response Chinook salmon Avoidance in laboratory 0.75 LOEC 25 20 minutes Hansen et al. (O. tshawytscha) exposures 1999a (juvenile) Rainbow trout Avoidance in laboratory 1.6 LOEC 25 20 minutes Hansen et al. (O, mykiss) exposures 1999a (juvenile) Chinook salmon Loss of avoidance ability 2 LOEC 25 21 days Hansen et al. (juvenile) 1999a Atlantic salmon Avoidance in laboratory 2.4 LOEC 20 20 minutes Sprague et al. (juvenile) exposures 1965 Atlantic salmon Spawning migrations in 20 LOEC 20 Indefinite Sprague et al. (adult) the wild interrupted 1965 Chinook salmon Spawning migrations in 10–25 LOEC 40 Indefinite Mebane 2000 (adult) the wild apparently interrupted Coho salmon Delays and reduced 5 LOEC 95 6 days Lorz and downstream migration of McPherson 1976, dCu-exposed juveniles 1977 Rainbow trout Loss of homing ability 22 LOEC 63 40 weeks Saucier et al. 1991 Ecosystem effects NAd Ecosystem function: 2.5 LOEC 49 ≈ 1 year Leland and Carter Reduced photosynthesis 1985 NAd Ecosystem structure: loss 5 LOEC 49 ≈ 1 year Leland et al. 1989 of invertebrate taxa richness in a mountain stream Other sublethal effects

Chinook salmon Reduced growth 1.9 EC10 25 120 days Chapman 1982 (as weight)

Rainbow trout Reduced growth 2.8 EC10 25 120 days Marr et al. 1996 (as weight)

Table 1 continued. Selected examples of adverse effects with copper to salmonids or their prey.a

Effect Species concentra- Effect Hardness Exposure (lifestage) Effect tion (µg/L)b statistic (mg/L)c duration Source

Other sublethal effects (cont.) Coho salmon Reduced growth 21–22 NOEC 24–32 60 days Mudge et al. 1993 (as weight) Steelhead Reduced growth 45 to >51 NOEC 24–32 60 days Mudge et al. 1993 (O. mykiss) (as weight) Direct lethalitye

Chinook salmon Death 19 LC50 24 96 hours Chapman 1978 (fry)

Coho salmon Death 28–38 LC50 20–25 96 hours Lorz and (fry) McPherson 1976

Steelhead/rain- Death 9–17 LC50 24–25 96 hours Chapman 1978, bow trout (fry) Marr et al. 1999

Coho salmon Death 46 LC50 20 96 hours Chapman and (adult) Stevens 1978

Steelhead Death 57 LC50 42 96 hours Chapman and (adult) Stevens 1978 Coho salmon Death 21–22 NOEC 24–32 60 days Mudge et al. 1993 (juvenile) Steelhead Death 24–28 NOEC 24–32 60 days Mudge et al. 1993 (juvenile)

Steelhead Death 11.9 EC10 25 120 days Chapman 1982 (egg-to-fry) a Abbreviations: LOEC = Lowest observed adverse effect concentration (and most LOEC values given are not thresholds, but were simply the lowest concentration tested); NOEC = No observed adverse effect concentration; LC50 = the concentration that kills 50% of the test population; ECp = effective concentration adversely affecting (p) percent of the test population or percent of measured response, e.g., 10% for an EC10, etc.; and Indefinite = field exposures without defined starting and ending times. NA = not applicable. b Effects and exposure durations stem from laboratory and field experiments, therefore in some experiments multiple routes of exposure may be present (i.e., aqueous and dietary) and water chemistry conditions will likely differ (see reference for details). c Hardness is reported, as it can influence the toxicity of copper. d This study examined ecosystems consisting of a number of species or unidentified species. e Acute sensitivity of salmonids to copper probably varies by life stage, and the swim-up fry stage is probably more sensitive than older juvenile life stages such as parr and smolts or adults.

5 Figure 1. Recording methods and features of the salmon peripheral olfactory system. A) Photograph showing the rostrum of a coho salmon during the recording of electro-olfactograms (EOGs). The mouthpiece provides chilled, anaesthetized water to the gills, while the perfusion tube delivers odor- containing solutions to the olfactory chamber. The recording electrode in the olfactory chamber and reference electrode in the skin monitor the response of the olfactory system to an odor. B) Scanning electron micrograph showing a rosette, located within an olfactory chamber of a juvenile coho salmon. Each rosette consists of lamellae (lobes) covered by an epithelium containing regions of sensory neurons. The open circle denotes the location and approximate size of the tip of the recording microelectrode. C) Scanning electron micrograph showing a cross section from a region of sensory epithelium of a lamella. In the upper left is the apical surface containing the cilia and microvilli of the olfactory receptor neurons (ORNs). The dendrites and somata of the ORNs appear in the center within the epithelium, while the axons of the ORNs emerge from the basal surface at the lower right to produce the olfactory nerve. D) Typical odor-evoked EOGs obtained from a salmon before and after exposure to copper. A 10-second switch to a solution containing 10-5 M L-serine is shown with a horizontal bar. The EOG evoked by the odor pulse consists of a negative deflection in the voltage. A 30-minute exposure to copper reduced the amplitude of the EOG evoked in the same fish by 57%. (Photos courtesy of Carla Stehr. Figure adapted from Baldwin and Scholz 2005).

6 Recently, using EOG measurements in combination with a predator avoidance assay, Sandahl et al. (2007) presented the first evidence that impaired olfaction (smell) resulted in a direct suppression of predator avoidance behavior (alarm response) by juvenile coho salmon at environmentally relevant dCu exposures (≥2.0 µg/L; 3 hr exposure). Unexposed juveniles (control treatment) reduced their swimming speed on average by 74% (alarm response) in response to an alarm odor (conspecific skin extract). A reduction in swimming speed is a typical predator avoidance response for salmonids and many other fish. In unexposed fish, the alarm odor elicited a mean EOG response of 1.2 mV. Juvenile coho salmon exposed to 2-20 µg/L copper exhibited measurable reductions in both EOG (50–92%) and alarm response (47 to >100%) (derived from data in Figure 2 of Sandahl et al. 2007). Juvenile coho exhibited statistically significant decline in antipredator behavior at 5, 10, and 20 µg/L dCu (Figure 2).

Importantly, concentrations of dCu below 2 µg/L were not tested in Sandahl et al. (2007). This is notable because all concentrations tested (between 2 and 20 µg/L) significantly affected olfaction with reductions in EOG ranging ≈50–92%. Because individual juvenile coho were significantly affected at the lowest concentration tested (2 µg/L), uncertainty remains with respect to the precise threshold for olfactory impairment. The results of this last study provide evidence that juvenile salmon exposed to sublethal dCu concentrations at 2 µg/L (resulting in approximately 50% reductions in EOG), and likely even lower, might not recognize and respond to a predation threat, and therefore have an increased risk of being eaten by other fishes or birds (a form of ecological death, Kruzynski and Birtwell 1994).

Typically dCu concentrations in road runoff are well within the range affecting antipredator behavior, for example, 3.4–64.5 µg/L, with a mean of 15.8 µg/L (Soller et al. 2005). A 3 hour exposure is also likely to be environmentally relevant, as stormwater runoff durations from roads typically range from a few minutes to several hours (Sansalone and Buchberger 1997). Fish may regain their capacity to detect odors fairly quickly in some cases; physiological recovery of olfactory neuron function is dose-dependent and occurs within a few hours at low copper concentrations (i.e., <25 µg/L dCu, Baldwin et al. 2003). However, long-term damage to the sensory epithelia has also been documented. Where cell death occurs (i.e., ≥25 µg/l copper, Hansen et al. 1999a, 1999b) recovery is on the order of weeks (Moran et al. 1992) and in some cases months (Evans and Hara 1985).

Interestingly, another fish sensory system, the lateral line, is also a target for the neurotoxic effects of dCu. It is composed of mechanosensory neurons (hair cells) that respond to surface water vibrations, flow, and other types of mechanical cues in the aquatic environment. The lateral line system thereby mediates shoaling, pursuit of prey, predator avoidance, and rheotaxis (orientation to flow). In a recent study, dCu (i.e., ≥20 µg/L; 3 hour exposure) killed 20% of hair cells in zebrafish (Danio rerio) (Linbo et al. 2006). As mentioned earlier, juvenile salmon ORNs may also be killed at higher concentrations of dCu, highlighting the similar sensitivity of olfactory and lateral line receptors to this toxic metal. Consequently, dCu may damage or destroy either or both of these important sensory systems. Currently, we are not aware of any research on the effects of dCu to the lateral line of salmonids, although the comparable sensitivity of the olfactory system across species suggests that the salmon lateral line is likely to be vulnerable as well.

Figure 2. Copper-induced reductions in juvenile salmonid olfactory response and behavior are significantly correlated. Fish exposed to dCu (3 hours) showed reduced olfactory sensitivity and corresponding reduction in predator avoidance behavior. Values represent treatment means (with copper exposure concentration labeled to the right); error bars represent one standard error; n = 8–12 individual coho salmon; asterisk (*) represents a statistically significant difference in olfactory response (EOG data) compared to controls (one-way ANOVA with Dunnett post hoc test, p < 0.05); †represents statistically significant difference in behavioral response to skin extract (% reduction in swimming) compared to controls (one-way ANOVA with Dunnett post hoc test, p < 0.05). The line represents a statistically significant linear regression based on treatment means (n = 5; p < 0.0001; r2 = 0.94). 1 ppb = 1 µg/l. (Adapted from Figure 2C in Sandahl et al. 2007.)

In this paper, a benchmark dose (concentration) analysis (USEPA 1995) is applied to recent data from dose-response experiments on juvenile salmonids exposed to dCu (Sandahl et al. 2007) to determine the exposure concentrations that may adversely affect salmonid sensory systems. In previous studies, benchmark concentrations (BMCs) were determined for olfactory responses, however, concomitant behavioral responses were not measured (Baldwin et al. 2003, Sandahl et al. 2004). The BMC analysis conducted herein determined concentrations of dCu that could be expected to affect juvenile salmonid olfaction and, by extension, alarm response behavior involved in predator avoidance.

8 Application of the Benchmark Concentration Analysis

The BMC, also referred to as a benchmark dose, is a method that has been used since 1995 by agencies such as the U.S. Environmental Protection Agency (EPA) to determine no observable adverse effect level (NOAEL) values. The method statistically fits dose-response data to determine NOAEL values (EPA 1995). This is in contrast to other methods (e.g., using an analysis of variance) that rely on finding a no observable effect concentration (NOEC) and lowest observable effect concentration (LOEC) to establish the NOAEL. Multiple difficulties arising from the traditional approach of selecting a NOAEL from dose-response data were previously identified by the EPA. Specific shortcomings associated with traditional methods included: 1) arbitrary selection of a NOAEL based on scientific judgments; 2) experiments involving fewer animals produced higher NOAELs; 3) dose-response slopes were largely ignored; and 4) the NOAEL was limited to the doses tested experimentally (EPA 1995). These as well as other concerns with selection of a NOAEL led to the development of an alternative approach, the BMC analysis. The BMC approach uses the complete dose-response data set to identify a NOAEL, thereby selecting an exposure concentration that may not have been tested experimentally.

The BMC is statistically defined as the lower confidence limit for a dose that produces a predetermined adverse effect relative to controls. This effect is referred to as the benchmark response (BMR) (EPA 1995). Unlike the traditional method of selecting the NOAEL (e.g., establishing a NOEC), the BMC takes into account the full range of dose-response data by fitting it with an appropriate regression equation. These can be linear, logarithmic, sigmoidal, etc. The BMR is generally set near the lower limit of responses (e.g., an effect concentration of 10%) that can be measured directly in exposed or affected animals.

In the present context, a BMC approach was used to estimate thresholds for dCu’s sublethal effects on the chemosensory physiology and predator avoidance behaviors of juvenile coho salmon (Sandahl et al. 2007). An example of this approach is shown in Figure 3. This methodology has been used previously to determine toxicity thresholds in Pacific salmon (Sandahl and Jenkins 2002, Baldwin et al. 2003, Sandahl et al. 2004). The dose-response relationship for copper’s effect on the EOG was described by fitting the data with a sigmoid logistic model:

y = m/[1+(x/k)n]

where m is maximum EOG amplitude (fixed at the control mean of 1.2 mV), y is EOG amplitude, x is copper concentration, k is copper concentration at half-maximum EOG amplitude (EC50), and n is slope.

9 For this nonlinear regression, the average olfactory response of the control fish to a natural odor was used to constrain the maximum odor evoked EOG (m in the above equation). Consequently, the control fish were not used in the regression other than to set m. The regression incorporated the individual response of each exposed fish (n = 44 total) rather than the average values for each exposure group. As shown in Figure 3, the sigmoid logistic model was a very good fit for both the sensory and behavioral data (r2 = 0.94, p < 0.0001). Benchmark concentrations were then determined based on the concentration at which the estimated curve intersected benchmark responses.

10 Results of the Benchmark Concentration Analysis

Examples of benchmark concentrations and responses are presented in Figure 3 and Table 2. The EPA methodology recommends using the concentration that represents a 10% reduction in response compared to controls when limited biological effects data are available (EPA 1995). This is the BMC10 and is synonymous with the concentration producing an effect of 10% (EC10), in this case a 10% reduction in the recorded amplitude of the salmon’s chemosensory response (EOG). Since the predicted fish EOG response at the BMC10 falls well within the olfactory response of unexposed juveniles, that is, 95% CI (control fish, Figure 3), it is more than likely that this individual response (1.08 mV) at the BMC10 (0.18 µg/L) would not be detectable or biologically significant as an adverse response.

Other BMCs were derived using statistical criteria to determine benchmark responses. For example, Table 2 shows two BMCs that were determined using the statistical departure of the lower-bound confidence interval (CI) of the control mean (unexposed fish), 1.2 mV (either the 90 or 95% CI). The selection of different CIs results in different BMCs. The CI-derived BMCs represent a reasonable estimate of when an individual salmonid is likely to have a biologically significant reduction in olfaction and a concomitant reduction in predator avoidance behavior. The relative departures from controls in Table 2 are equivalent to effective concentrations for olfactory inhibition, that is, at the lower-bound 90% CI a BMC of 0.59 µg/L equates to a BMC24.2. Put another way, the BMC analysis predicts a substantial 24.2 % reduction in olfaction (i.e., EOG amplitude) at 0.59 µg/L dCu. At the lower-bound 95% CI a 29.2% reduction in olfaction is predicted to occur at 0.79 µg/L.

The BMC50 is equivalent to the EC50 for olfactory responses (2.1 µg/L) and is very similar to the lowest observable effect concentration (LOEC) of 2 µg/L. Since the EC50 approximately equals the LOEC, it is almost certain that effects to juvenile salmonid olfaction will occur at lower concentrations than those measured. Therefore it is appropriate and useful to apply a BMC analysis to these data to predict effects occurring between 0 and 2 µg/L dCu. The predicted effect thresholds for sensory responses in juvenile coho salmon ranged 0.18–2.1 µg/L, which corresponded to reductions in predator avoidance behavior (i.e., reduced alarm response) of 8–57%. Comparatively, the other two studies that conducted a BMC approach with salmon olfaction data sets (e.g., EOG measures) estimated dCu BMCs of 3.6–10.7 µg/L (BMC20– BMC50) (Sandahl et al. 2004) and 2.3–3.0 µg/L (BMC25) (Baldwin et al. 2003).

Together these three studies highlight that different experimental conditions including age of fish, exposure duration, and experimental background of dCu may influence BMCs. Importantly, of the three experiments that derived BMCs for olfactory impairment, the data set used in this technical memorandum from Sandahl et al. (2007) empirically linked impaired olfaction to an ecologically relevant behavior, that is, reduced alarm behavior (Figure 2).

11 Therefore, we believe that the dCu BMC analysis herein is derived from the most ecologically relevant of the three studies.

Figure 3. Using a benchmark concentration approach to estimate a threshold for dCu toxicity in the salmonid olfactory system. Filled circles represent treatment means; error bars represent the 95% confidence interval for each mean (n = 8–12 individual coho salmon). An asterisk (*) indicates a statistically significant difference in the size of the olfactory response (EOG data) compared to controls (one-way ANOVA with Dunnett post hoc test, p < 0.05). The line represents a statistically significant nonlinear regression based on individual fish (n = 44, p < 0.0001, r2 = 0.55). The gray shading shows the 95% confidence band for the nonlinear regression. The regression used a standard sigmoid function with the maximum constrained to the control mean (1.2 mV, indicated by the upper horizontal dashed line). Therefore, the control fish were not included in the nonlinear regression. The lower bound of the 95% confidence interval of the control mean (0.85 mV) is indicated by the lower horizontal dashed line and is an example of a BMR. The large open circle shows where the regression line crosses the BMR and denotes the corresponding BMC, which in this case is a dCu concentration of 0.79 µg/L. Horizontal and vertical lines through the open circle highlight the 95% confidence intervals for the BMC based on the results of the nonlinear regression. The small open circle shows where the regression line crosses the BMR (1.08 mV) and denotes the corresponding BMC10 (0.18 µg/L) at which a 10% reduction in olfactory capacity is expected. (Data from Sandahl et al. 2007.)

12 Table 2. Benchmark responses and benchmark concentrations for juvenile salmon exposed to dCu for 3 hours. Benchmark response values represent a reduction in olfactory response to an alarm pheromone as measured via EOG recordings. Behavioral impairment indicates a predicted decrease in predator recognition and avoidance as indicated by a reduced alarm response. CI = confidence interval; NA = not applicable. Benchmark Behavioral impairment b Benchmark responsesa concentrations (predicted)c Departure from mean Departure from mean of controls of controls Statisticald Relativee Relativeh f g (CI of control (% reduction in Value 95% CI (% reduction in alarm mean) olfactory response) (µg/l) (µg/l) response) NA 10.0 0.18 0.06–0.52 8.3 Lower 90% 24.2 0.59 0.30–1.16 25.6 Lower 95% 29.2 0.79 0.44–1.42 31.8 NA 50.0 2.10 1.60–2.90 57.2

a The predetermined level of altered response or risk at which the benchmark dose (concentration) is calculated (EPA/630/R-94/007, 02/1995). b The dose (concentration) producing a predetermined, altered response for an effect (EPA/630/R-94/007; 02/1995). c Based on the linear regression shown in Figure 2; note behavioral responses were determined by inputting the Benchmark response value (EOG, mV) into the regression equation. d Location of the value with respect to a confidence interval of the mean of the controls. e Amount of reduction in the olfactory response represented by the value relative to the mean of the controls. f Corresponding concentration; see Figure 3 and text for calculation method. g Confidence interval for the value based on the nonlinear regression. h Amount of reduction in alarm response represented by the value relative to the mean of the controls.

13 Discussion of Site Specific Considerations for Sensory System Effects

Below we identify several issues to consider when using the BMCs to evaluate dCu concentrations under natural conditions. Impairment from Short-term Increases of dCu

These BMCs reflect expected impairment of chemosensory systems from short-term increases of dCu above ambient concentrations (defined here as < 3 µg/L) (Baldwin et al. 2003, Sandahl et al. 2004, 2007) and are not expected to be alleviated by homeostatic mechanisms. Specifically, the BMCs are predicated on increases of dCu in salmon habitats that result from specific human activities. Effects to juvenile salmonid olfaction are expected following a few minutes of exposure. Salmonids are capable of regulating the amount of internal copper via uptake and elimination processes. These so called homeostatic mechanisms (such as metallothionein induction) can reduce copper’s toxic effects and may result in acclimation. Consequently, fish may tolerate certain dCu exposures without showing overt toxicological responses; however, at higher levels these mechanisms could ultimately fail.

Initial evidence indicates that homeostatic mechanisms are not likely to reduce copper toxicity to the olfactory sensory system for pulsed or short-term exposures lasting less than a week (Hansen et al. 1999a) or for chronically exposed fish (McPherson et al. 2004). Moreover, lateral line neurons exposed continuously to dCu for 72 hours showed no signs of acclimation within this exposure interval (Linbo et al. 2006). For other measures of copper toxicity from long-term exposures, evidence suggests that olfactory acclimation may not occur (Table 1, Appendix A). Fish exposed to higher dCu concentrations for longer periods may lose much of their olfactory function. For example, field evidence suggests that wild fish living in heavy metal contaminated lakes where total copper concentrations ranged 9.7–15 µg/L showed reduced olfactory-mediated predator avoidance behavior; that is, homeostatic mechanisms appeared insufficient to alleviate metal toxicity, including copper (McPherson et al. 2004). Calculating an Acute Criterion Maximum Concentration

The EPA sets acute water quality criteria by calculating an acute criterion maximum concentration (CMC) (Stephan et al. 1985). The CMC is an estimate of the highest concentration of a substance in surface water to which an aquatic community can be exposed briefly without resulting in an unacceptable effect (EPA 2002).We calculated an acute CMC using the Biotic Ligand Model (BLM) (EPA 2007). Interestingly, the estimated acute CMC based on the BLM using measured and estimated water quality parameters from Sandahl et. al. (2007) was 0.63 µg/L with a range from 0.34 to 3.2 µg/L, while the EPA hardness-based acute CMC (EPA 2002) was 6.7 µg/L. Because the BLM-based acute criterion is sensitive to pH and

14 DOC, the range of measured test pH values (6.5–7.1) and the range of estimated DOC values (0.3–1.5 mg/L) produced this range of BLM-based acute criterion values. It is also interesting that the acute CMC range (0.34–3.2 µg/L) overlapped with the olfactory-based BMC range (0.18–2.1 µg/L). Salmonids Are Typically Exposed to Multiple Stressors

These BMCs are specifically focused on the impact of dissolved copper alone on olfaction and predator avoidance behavior. Salmonids are rarely exposed to dCu only under natural conditions. In fact, exposure to complex environmental mixtures of other toxic compounds (e.g., metals, pesticides, PAHs, etc.) in conjunction with other stressors (e.g., elevated temperatures, low dissolved oxygen, etc.,) is the norm for many salmonid-bearing habitats. Equally important are exposure routes other than the water column, such as consumption of contaminated prey items (dietary) or direct contact with contaminated sediments. Threshold examples (BMCs) presented here are based solely on juvenile salmonids exposed to dCu. Presently, these thresholds do not take into account multiple routes of exposure or the potential impacts of complex mixtures of contaminants on olfaction. That said, several studies have shown a greater than expected toxicity (i.e., nonadditive) to other fish endpoints from mixtures of metals (Sprague et al. 1965, Norwood et al. 2003). For example, mixtures containing zinc and copper were found to have greater than additive toxicity to a wide variety of aquatic organisms including freshwater fish (Eisler 1998). Other metal mixtures also yielded greater than additive toxic effects at low dissolved concentrations (Playle 2004). The toxic effects of metals to salmonids may also be exacerbated by other types of contaminants such as pesticides (Forget et al. 1999). While interactions among multiple stressors, including contaminant mixtures, are beyond the scope of this document, they warrant careful consideration in site-specific assessments. Bioavailability of dCu

These BMCs were derived from experiments using a single freshwater source (dechlorinated, soft municipal water). Hardness, alkalinity, and dissolved organic carbon (DOC) are known to alter the bioavailability of dissolved copper in surface waters to ligands in the fish gill. These water chemistry parameters can therefore influence the potential for dCu exposure in the field to cause an acute fish kill. Acute copper lethality mediated via the gill route of exposure is typically estimated using the Biotic Ligand Model (BLM; reviewed by Niyogi and Wood 2004). However, recent unpublished research by McIntyre et al. (in press) suggest that these parameters may have less of an influence on salmonid olfactory function across environmentally realistic ranges of hardness, alkalinity, and DOC.

To date, the U.S. Geological Survey (USGS) has monitored hardness, alkalinity, and DOC for more than 10 years in many West Coast river basins including the Willamette River basin, Puget Sound basin, Yakima River basin, and the Sacramento-San Joaquin River basin (USGS no date). Several at-risk species of anadromous salmonids inhabit these basins. The monitoring data indicate that surface waters within these basins typically have very low hardness and alkalinity and seasonally affected DOC concentrations. Hardness, alkalinity, and DOC levels found in most freshwater habitats occupied by Pacific salmonids would be unlikely to

15 confer substantial protection against dCu olfactory toxicity (Winberg et al. 1992, Bjerselius et al. 1993, Baldwin et al. 2003, McIntyre et al. in press).

Recent experimental results suggest that significant amelioration of olfactory toxicity due to hardness is unlikely in typical Pacific salmonid freshwater habitats. The experiment showed that hardness at 20, 120, and 240 mg/L Ca (experimentally introduced as CaCl2) did not significantly protect juvenile coho salmon from olfactory toxicity following 30 minute laboratory exposures to 10 µg dCu/L above an experimental background of 3 µg/L (Baldwin et al. 2003). In another experiment, a 20 µg dCu/L exposure (30 minutes) in water with low hardness and alkalinity and no DOC produced an 82% inhibition in juvenile coho olfactory function (McIntyre et al. in press). A hardness of ≥82 mg/L Ca was needed to reduce the level of olfactory inhibition to ≤50% at 20 µg/L dCu ( McIntyre et al. in press). However, 82 mg/L was never exceeded in any of the surface water samples from USGS-sampled NAWQA basins (McIntyre et al. in press).

Typical alkalinity values from Pacific Northwest and California freshwater surface waters are also unlikely to protect salmonids from olfactory toxicity (USGS no date). Some reduction in dCu olfactory toxicity was observed in a recent study (McIntyre et al. in press). However, only 0.4% of stream samples contained alkalinity levels sufficient to reduce olfactory toxicity of dCu by half (McIntyre et al. in press). Bjerselius et al. (1993) and Winberg et al. (1992) also found that hardness and alkalinity provided limited amelioration of olfactory responses in juvenile Atlantic salmon exposed to dCu.

Increases in DOC showed greater protection to dCu compared to increases in alkalinity and hardness. Twenty-nine percent of USGS surface water samples from West Coast basins had a DOC concentration sufficient to limit olfactory impairment to 50% or less at 20 µg dCu /L (McIntyre et al. in press). Only a small fraction (6%) of all samples contained DOC levels (greater or equal to 6 mg/L) sufficient to completely protect the olfactory responses of juvenile coho salmon from the toxic effect of 20 µg dCu /L (McIntyre et al. in press). This information underscores the importance of evaluating site-specific DOC data to address the potential influence of this water quality parameter on olfactory toxicity.

Because the typical range of hardness, alkalinity, and DOC concentrations are unlikely to confer substantial protection against dCu toxicity, we expect that the BMC thresholds presented in this document will be applicable for most of the freshwater environments that provide migrating, spawning, and rearing habitats for salmonids. Olfactory Toxicity in Saltwater

Dissolved copper’s effect on salmonid olfaction in saltwater environments remains a recognized data gap and it is presently uncertain whether the BMC thresholds derived in this document apply to salt water environments. Estuarine and nearshore salt water environments, despite their higher salinity (in part due to increased cation concentrations) and hardness may or may not confer protection against dCu-induced olfactory toxicity. One source of this uncertainty is whether or not free copper (Cu2+) is the sole species of copper responsible for olfactory toxicity. In freshwater, evidence suggests that Cu2+ is not the only toxic species that adversely affects olfaction in fish (McIntyre et al. in press) as well as more conventional endpoints such as

16 mortality (Niyogi and Wood 2004). Other copper species (e.g., CuOH; Cu1+) will also bind to the gill, thereby causing toxicity (Niyogi and Wood 2004). While the physiological basis for salmonid olfaction is well characterized, the transition to saltwater may involve important changes in olfactory receptor neuron function that ultimately influence the expression of the as yet unidentified ligands for dCu. Avoiding Short-term Increases in dCu

Salmonids may or may not avoid short-term increases in dCu. Salmonids will actively avoid water containing dCu if they can detect it. As a consequence, fish may not use otherwise high quality rearing and spawning habitats. In addition, the presence of dCu may affect migratory routes of juveniles and adults. Smith and Bailey (1990) and Mebane (2000) derived regulatory “zones of passage” around wastewater discharges that were based on salmonid avoidance responses. However, in areas with diffuse, nonpoint source pollution, or multiple point source discharges, it may be difficult to apply “zones of passage”, and in some cases available zones of passage may not exist. Despite a fish’s preference to avoid dCu, circumstances may force migrating juveniles and adults to be exposed. For dCu contaminated, high quality rearing habitats, juveniles could either remain and be exposed or move to lower quality habitats. Juveniles could therefore suffer either reduced predator avoidance or reduced growth. For contaminated spawning habitats, adult salmon may either remain and be exposed as well as their offspring or move to lower quality habitats. Both of these scenarios result in potential reductions in reproductive success. Coho Salmon–derived BMCs Should Apply to Other Salmonids

These BMCs were derived using data from juvenile coho salmon, but should apply to other fish species. The examples of BMC thresholds were derived from data based on juvenile coho salmon (4–5 month old, mean of 0.9 grams wet weight). However, we expect these BMC examples to be generally applicable to other species of salmon, trout, and steelhead in freshwater habitats. For example, 3 hour exposures of 4-month-old steelhead to a similar range of dCu produced comparable olfactory toxicity to that reported for 4-month-old coho salmon (Baldwin et al. in prep.). Studies on 10-month-old juvenile coho had similar reductions in olfaction compared to 4-month-old fish (Baldwin et al. 2003, Sandahl et al. 2004). Juvenile chum salmon (O. keta) (2–3 month old) also showed a dose dependent reduction in EOG amplitude following exposure to dCu (3–58 µg/L) (Sandahl et al. 2006). Taken together these findings suggest that the BMC threshold derived herein should be applicable to juvenile life stages of coho, Chinook, sockeye (O. nerka), and pink salmon (O. gorbuscha) as well as steelhead, bull trout (Salvelinus confluentus), and other members of the family Salmonidae. As noted earlier, the toxicity of dCu to other life stages (particularly marine phases of life) remains to be determined.

17 Conclusions

Dissolved copper (dCu) is a ubiquitous, bioavailable pollutant that can directly interfere with fish sensory systems and by extension important behaviors that underlie predator avoidance, juvenile growth, and migratory success (see Appendix A). Recent research shows that dCu not only impairs sensory neurons in a salmonid’s nose, but also impairs juvenile salmonids’ ability to detect and respond to predation cues. A juvenile salmonid with disrupted predator avoidance behaviors stands a greater risk of mortality and by extension a reduction in the likelihood of surviving to reproduce. The degree to which effects on individual behavior and survival impact a given population will depend in part on the number of the individuals affected and the status of the population (numbers, distribution, growth rate, etc.).

In this report, BMCs were calculated using an EPA methodology to provide examples of effect thresholds of dCu’s impacts on salmonid sensory biology and behavior. The BMC examples represent increases in the dCu concentration above background or ambient levels (where background is less than or equal to 3 µg/L) expected to affect juvenile salmonid ability to avoid predators in fresh water. Benchmark concentrations ranged 0.18–2.1 µg/L, corresponding to reductions in predator avoidance behavior (alarm reaction) that ranged approximately 8–57%. Taking into account the olfactory responses of unexposed fish, a more biologically relevant range of BMCs is 0.59–2.1 µg/L (Table 2). This second range of BMC thresholds is similar to or slightly less than documented effects to other copper-affected sublethal endpoints such as behavior and growth that range 0.75–2.5 µg/L (see Table 1).

The primary objective of this report was to present examples of threshold concentrations for effects of dCu on a critical aspect of salmonid biology: olfaction. A secondary objective of this paper was to summarize a selection of recent and historical information related to the effects of dCu on salmonid sensory systems. This document is based on the current state of the science. Importantly, this overview is not a comprehensive summary of the myriad effects of copper to anadromous salmonids. As such, new information will undoubtedly become available that enhances our understanding of copper’s effect on salmonid populations and their supporting habitats. The information reviewed and presented herein indicates that significant impairment of sensory functions important to survival of threatened and endangered juvenile salmonids is likely to be widespread in many freshwater aquatic habitats. Impairment of these essential behaviors may occur following 10 minutes of exposure and continue for hours to weeks depending on concentration and duration.

18 Glossary

Acute exposure. Short-term continuous exposure usually lasting 96 hours or less.

BLM. Biotic Ligand Model

Chronic exposure. Longer-term continuous or pulsed exposures generally lasting greater than 96 hours.

Confidence interval (CI). A random interval constructed from data in such a way that the probability that the interval contains the true value can be specified before the data are collected. dCu. dissolved copper.

DOC. dissolved organic carbon.

ECp. Effective concentration adversely affecting (p) percent of the test population or percent of measured response, for example, 10% for an EC10 and so forth.

EOG. electro-olfactogram.

LC50. The aqueous concentration of a substance that kills 50% of the test population.

Lower-bound 90% confidence interval. The lower half of the 90% confidence interval of the mean.

Lower-bound 95% confidence interval. The lower half of the 95% confidence interval of the mean.

LOEC. lowest observable effect concentration.

Mean. The average of the response values in a treatment population. Numerically the mean represents the sum of the individual response values divided by the number of individuals in a treatment. mV. millivolts.

NOAEL. no observable adverse effect level.

NOEC. no observable effect concentration.

ORN. olfactory receptor neuron. ppb. part(s) per billion, equivalent to µg/L.

19 Relative departure from control response. A user selected level of response compared to control response; for example, a 10% reduction from the control response (unexposed individuals).

Statistical departure from control response. Uses statistical methods to select a response based on the distribution of responses seen in unexposed individuals. For example, the 95% lower bound confidence interval of the mean response from controls (unexposed individuals).

20 References

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26 Appendix A: Other Salmonid Sensory Effects of dCu

In this appendix, results are highlighted from several studies that we thought were particularly relevant, including comparing the concentrations that have caused sensory effects to concentrations causing lethality or growth reductions in field and laboratory experiments. As such, the following review is not an exhaustive summary of copper’s adverse effects to anadromous salmonids. We emphasize studies that were conducted in waters with low alkalinity and hardness (<50 mg/L as calcium carbonate), and if reported, low concentrations of dissolved organic material. These conditions were emphasized since we believe these are the most relevant water quality conditions for an area of particular concern to us—freshwater habitats used by juvenile salmonids in the Pacific Northwest and California. Migratory Disruption

Laboratory and field experiments with salmonids have shown avoidance of low concentrations of copper, disruption of downstream migration by juvenile salmonids, loss of homing ability, and loss of avoidance response to even acutely lethal concentrations of copper following long-term habituation to low level copper exposure. Saucier et al. (1991) examined the impact of a long-term sublethal copper exposure (22 µg/L, 37–41 weeks in duration) on the olfactory discrimination performance in rainbow trout (Oncorhynchus mykiss). When controls were given a choice between their own rearing water or other waters, they significantly preferred their own rearing water, whereas both copper-exposed groups showed no preference. They concluded that their results demonstrate that a long-term sublethal exposure to copper, as it commonly occurs under “natural” conditions, may result in olfactory dysfunction with potential impacts on fish survival and reproduction.

Field studies have reported that copper impairs both upstream spawning migration of salmonids and downstream outmigration of juveniles. Avoidance of copper in the wild has been demonstrated to delay upstream passage of Atlantic salmon (Salmo salar) moving past copper- contaminated reaches of the river to their upstream spawning grounds, cause unnatural downstream movement by adults away from the spawning grounds, and increase straying from their contaminated home stream into uncontaminated tributaries. Avoidance thresholds in the wild of 0.35 to 0.43 toxic units were about seven times higher than laboratory avoidance thresholds (0.05 toxic units), perhaps because the laboratory tests used juvenile fish rather than more motivated spawning adults. For this study 1.0 toxic unit was defined as an incipient lethal level (ILL, essentially a time independent LC50), of 48 µg/L in soft water (Sprague et al. 1965, Saunders and Sprague 1967). Studies of home water selection with returning adult salmon showed that addition of 44 µg/L copper to their home water reduced the selection of their home stream by 90% (Sutterlin and Gray 1973). Releases of about 20 µg/L from a mine drainage into a salmon spawning river resulted in 10–22% repulsion of ascending salmon during four consecutive years compared to 1–2% prior to mining (Sutterlin and Gray 1973). The upstream

27 spawning migration of Chinook salmon (O. tshawytscha) in Panther Creek, Idaho, may have been interrupted during the 1980s and early 1990s when the fish encountered dCu concentrations of 10–25 µg/L. In Panther Creek, the majority of spawning habitat and historical locations of Chinook salmon spawning were high in the watershed, upstream of copper discharges. However, Chinook salmon were only observed spawning below the first major diluting tributary, a point above which copper concentrations averaged about 10–25 µg/L during the times of the spawning observations (Mebane 1994, 2000).

Sublethal copper exposure has been shown to interfere with the downstream migration to the ocean of yearling coho salmon (O. kisutch). Lorz and McPherson (1976, 1977) and Lorz et al. (1978) evaluated the effects of copper exposure on salmon smolts’ downstream migration success in a series of 14 field experiments. Lorz and McPherson (1976, 1977) exposed yearling coho salmon for six to 165 days to nominal copper concentrations varying from 0–30 µg/L. They then marked and released the fish during the normal coho salmon migration period and monitored downstream migration success. The fish were released simultaneously, allowing for evaluation of both copper exposure concentrations and exposure duration on migration success. All dCu exposures resulted in reduction of migration compared with unexposed control fish. Migration success decreased with both increasing copper concentrations and increased exposure time for each respective concentration. Exposure to 30 µg/L dCu for as little as 72 hours caused a considerable reduction in migration (≈60%) compared to control fish. The reductions in migration following short-term exposures to dCu are illustrated in Figure A-1. Following exposure to 30 µg/L dCu, 80% of coho did not reach the migratory point in 49 days. These concentrations (5-20 µg/L) were one-tenth to one-third the 96-hour LC50 for the same stock of juvenile coho salmon in the same water. Lorz et al. (1978) further tested downstream migration with yearling coho salmon previously exposed to copper, cadmium, copper-cadmium mixtures, zinc, and copper-zinc mixtures. Copper concentrations in all tests were held at 10 µg/L. In all cases, the copper exposed fish again had poorer migratory success than did controls. The other metals did not show the dose-dependent result found for copper. These studies suggest that exposure to copper concentrations at levels found in streams subject to nonpoint copper pollution may impair downstream migration, a result of direct and indirect effects to salmon smolts, including reproductive success. Laboratory Avoidance Studies

Studies have shown that salmonids can detect and avoid copper at low concentrations when tested in troughs or streams that allow them to choose between concentration gradients. To our knowledge, the lowest copper concentration reported to cause avoidance in laboratory conditions was 0.1 µg/L (Folmar 1976). However, these results may have low applicability to ambient conditions because copper exposure concentrations were not analytically verified. Avoidance thresholds of 2 µg/L copper have been reported for Atlantic salmon (Salmo salar), concentrations that are less than one-tenth of acute LC50 values (Saunders and Sprague 1967). Giattina et al. (1982) reported that rainbow trout appeared to detect copper concentrations down to 1.4–2.7 µg/L, because declines in residence time started to occur at these lower concentrations. However, the responses were only statistically significant at 4.4 to 6.4 µg/L depending on whether fish were exposed to a gradually increasing or abruptly increasing concentration gradient respectively. At exposure to extremely high dCu levels, for example,

28 0 µg/L 5 µg/L 10 µg/L 20 µg/L 30 µg/L

100

20 Downstream migration (%) migration Downstream

0 0 5 10 15 45 50 Days post release Figure A-1. Reduction in downstream migration of yearling coho salmon following 6 days of exposure to copper at various concentrations. (Redrawn from Lorz and McPherson 1977, their Figure 19.)

330–390 µg/L, trout showed diminished avoidance and sometimes attraction to acutely lethal concentrations (Giattina et al. 1982, Hansen et al. 1999a, Chapman unpubl. data).

Chapman (unpubl. data) reported that long-term sublethal copper exposures had impaired the avoidance performance of salmonids. Steelhead (O. mykiss), acclimated to low copper levels by surviving about 3 months early life stage toxicity testing, subsequently failed to avoid much higher, acutely lethal concentrations. Following about 3 month continuous exposure to 9 µg/L copper (from fertilization to about 1 month after swim up) the copper-acclimated fish and control fish with no previous copper exposure were exposed to a range of copper concentrations from 10 to 80 µg/L in avoidance-preference testing. The tests used the same counter flow avoidance- preference test chambers described by Giattina et al. (1982). The acclimated steelhead failed to avoid even the highest copper concentrations while most of the unexposed fish avoided all concentrations.

Hansen et al. (1999a) and Marr et al. (1995) conducted a variety of behavioral and other toxicity studies with Chinook salmon and rainbow trout exposed to copper. In these studies they used well water that was diluted with deionized water and spiked with copper to obtain a hardness, alkalinity, and pH that simulated those in Panther Creek, a mine-affected stream in Idaho. The avoidance response of the Chinook salmon was statistically significant for 0.8 and 2.8–22.5 µg/L copper but was not significant for a 1.6 µg/L copper treatment. Since the avoidance responses (percent time spent in test water) were similar between the 0.8, 1.6, and 3 µg/L treatments, but the 1.6 µg/L treatment had fewer replicates than the other treatments (10 vs. 20), the lack of statistical significance for the 1.6 µg/L treatment was probably an artifact of the

29 different sample sizes rather than a true lack of response. Rainbow trout consistently avoided copper at concentrations of 1.6 µg/L and above. To simulate avoidance responses that might result on exposing fish to background levels of copper, Hansen et al. (1999a) acclimated both Chinook salmon and rainbow trout to 2 µg/L copper for 25 days, and repeated the avoidance experiments. They observed that the avoidance response of Chinook salmon was greatly dampened such that no copper treatments resulted in statistically significant responses. In contrast, the avoidance response of rainbow trout was unaffected by the acclimation. This dramatic difference between Chinook salmon and rainbow trout avoidance was so unexpected that Hansen et al. (1999a) ran a second set of experiments that yielded the same results. Background dCu concentrations (<4 µg/L) are commonly observed in natural waterways, yet Chinook salmon failed to avoid any higher dCu concentrations following an acclimation to a nominal 2 µg dCu/L. Importantly, if Chinook salmon will not avoid any dCu concentrations following acclimation to low dCu concentrations, the behavioral defense against chronic and acute exposures to dCu is lost, and high mortality or chronic physiological effects are probable if subsequent higher levels of dCu exposure occur. Unlike Chinook salmon, dCu-acclimated rainbow trout preferred clean water and avoided higher dCu concentrations. Other differences between Chinook salmon and rainbow trout avoidance responses to copper were that addition of 4 and 8 mg/L dissolved organic carbon (DOC) did not appreciably affect the avoidance response of Chinook salmon to copper, nor did altering pH across a range of 6.5 to 8.5. In contrast, the addition of DOC (4 and 8 mg/L) did reduce the avoidance response of rainbow trout to copper. Although variable, avoidance responses of rainbow trout were slightly stronger at pH 7.5 and 8.5 than at 6.5 (Marr et al. 1995).

A further repeated finding from these laboratory avoidance tests was that although rainbow trout, steelhead, and Chinook salmon avoided low concentrations of dCu, they were apparently intoxicated and sometimes attracted to very high concentrations (Giattina et al. 1982, Hansen et al. 1999a, Chapman unpubl. data). The direct relevance of laboratory avoidance studies to the behaviors of fish in the wild is debatable since in natural waters fish likely select and move among habitats based on myriad reasons such as access to prey, shelter from predators, shade, velocity, temperature, and interactions with other fish. In contrast, laboratory preference/avoidance tests are commonly conducted under simple, highly artificial conditions to eliminate or minimize confounding variables other than the water characteristic of interest. Laboratory tests may overestimate the actual protection this behavior provides fish in heterogeneous, natural environments (Hartwell et al. 1987, Korver and Sprague 1989, Scherer and McNoil 1998).

However, at least one study suggested that experimental avoidance responses observed with salmonids are relevant to fish behaviors in the wild. From 1980 to 1982, sublethal levels of a contaminant (fluoride) from an aluminum mill at the John Day Dam on the Columbia River were associated with a significant delay in salmon passage and decreased survival (Damkaer and Dey 1989). Salmon took an average of 36 hours to pass up the fish ladder at the Bonneville and McNary dams compared to 157 hours delay at the John Day Dam. Greater than 50% mortality occurred between the Bonneville and McNary dams (above and below the John Day dam), compared to about 2% mortality associated with the other dams. Damkaer and Dey (1989) introduced similar levels of the contaminant in streamside test flumes alongside a salmon spawning stream (Big Beef Creek, Washington). Significant numbers of adult Chinook salmon failed to move out of their holding area and continue upstream; those that did move upstream

30 chose the noncontaminated side of the flume. By adjusting the dose, Damkaer and Dey (1989) predicted a threshold detection limit for avoidance by salmon. The mill subsequently reduced its release of the contaminant to below these experimental threshold levels, which did not show a response in the streamside tests. Afterwards, fish passage delays and salmon mortality between the dams decreased to 28 hours and <5%, respectively (Damkaer and Dey 1989). This study suggested that the delay due to avoidance of a chemical affected the spawning success of migrating adult salmonids. These results are also consistent with the field studies of salmon migration in copper-contaminated streams and from laboratory avoidance/preference testing. Experimental avoidance/preference testing thus appears to be relevant to fish behavior in nature. Other Adverse Effects

The focus of this literature synthesis is sensory effects of copper on juvenile salmonids. However, other adverse effects of copper to salmonids reported in the literature include weakened immune function and disease resistance, increased susceptibility to stress, liver damage, reduced growth, impaired swimming performance, weakened eggshells, and direct mortality (McKim and Benoit 1971, Stevens 1977, Schreck and Lorz 1978, Waiwood and Beamish 1978a, 1978b, Chapman 1982, Farag et al. 1994, Marr et al. 1996, Farag et al. 2003). While a comprehensive review of other adverse effects of copper on fish is beyond the scope of this synthesis, we discuss several studies of interest below.

Stevens (1977) reported that preexposure to sublethal levels of dCu interfered with the immune response and reduced the disease resistance in yearling coho salmon. Juvenile coho salmon were vaccinated with the bacterial pathogen Vibrio anguillarum prior to copper exposure to investigate the effects of copper upon the immune response and survival. Following copper exposure (9.6–40 µg/L), surviving juveniles were challenged under natural conditions to V. anguillarum, the causative agent of vibriosis in fish. Vibriosis is a disease commonly found in wild and captive fish from marine environments and has caused deaths of coho and Chinook salmon. Coho salmon were exposed to constant concentrations of dCu for about one month at levels that covered the range from no effect to causing 100% mortality, 9.6–40 µg/L. The antibody titer level against V. anguillarum was significantly reduced in fish exposed to 13.9 µg/L of dCu when compared to that developed in control fish. The survivors of the dCu bioassays were then exposed in saltwater holding ponds for an additional 24 days to the V. anguillarum pathogen. The unvaccinated, non-dCu exposed control fish had 100% mortality and the vaccinated, non-dCu exposed fish had the lowest mortality. The vaccinated, dCu-exposed fish had increasing mortality corresponding to the lower antibody titer levels which in turn corresponded to the increasing dCu exposure levels. Therefore, dCu exposure can significantly reduce a fish's immune function and disease resistance at concentrations as low as 13.9 µg/L following 30 days of exposure (Stevens 1977).

Schreck and Lorz (1978) studied the effects of copper exposure to stress resistance in yearling coho salmon. Fish that were exposed for 7 days to 15 µg/L dCu and unexposed control fish were subjected to severe handling and confinement stress. Copper-exposed fish survived this additional stress for a median of 12–15 hours while control fish experienced no mortality at 36 hours. Schreck and Lorz (1978) concluded that exposure to copper placed a sublethal stress on the fish which made them more vulnerable to handling and saltwater adaptation. Further,

31 they hypothesized that dCu exposure may make salmonids more vulnerable to secondary stresses such as disease and pursuit by predators.

Exposure of brook trout (Salvelinus fontinalis) eggs to 17.4 µg dCu/L for 90 days resulted in weakened chorions (eggshells) and embryo deformities. After hatching, poor yolk utilization and reduced growth were demonstrated. These overall weakened conditions may reduce survival chances in the wild (McKim and Benoit 1971, McKim 1985). Copper accumulation in the liver of rainbow trout caused degeneration of liver hepatocytes, which resulted in reduced ability to metabolize food, reduced growth, or eventual death (Leland and Carter 1985, Farag et al. 1994, Meyer 2005). Waiwood and Beamish (1978a), Chapman (1982), Seim et al. (1984), McKim and Benoit (1971), and Marr (1996) have also observed reduced growth of salmonids in response to chronic copper exposures as low as 1.9 µg/L. Waiwood and Beamish (1978b) reported that rainbow trout exposed to copper levels had reduced swimming performance (10, 15, 20, 30 µg/L dCu) and reduced oxygen consumption (25, 40 µg/L dCu) apparently due to gill damage and decreased efficiency of gas exchange.

In sum, there is a large body of literature showing that behavior of salmonids and other fishes can be disrupted at concentrations of dCu that are only slightly elevated above background concentrations. Further, dCu stress has been shown to increase the cost of maintenance to fish and to limit oxygen consumption and food metabolism. Reduced growth may result in increased susceptibility to predation, and impaired swimming ability may result in reduced escape reaction and prey hunting, with a possible consequence of reduced survival at the population level. We summarize selected examples of effect concentrations reported with copper for several different types of effects in Table 1 of this technical memorandum. In general, typical copper exposures probably do not kill juvenile salmonids directly until concentrations greater than about 10 times that of sensory thresholds, and then only if the concentrations are sustained for at least several hours. In selecting these examples, we sought to list representative effects and concentrations rather than extreme values that could be gleaned from the literature. However, the selected examples do not constitute an exhaustive review of the effects of copper to fish; more general reviews of effects of copper to fish and other aquatic organisms are available elsewhere (Leland and Carter 1985, Sorensen 1991, Eisler 1998, USEPA 2007).

32 Appendix B: Supplementary Bibliography

Copper Sources

Good, J. C. 1993. Roof runoff as a diffuse source of metals and aquatic toxicity in storm water. Water Sci. Technol. 28:317–322. Thomas, P. R., and G. R. Greene. 1993. Rainwater quality from different roof catchments. Water Sci. Technol. 28:291–297.

Benchmark Concentration/Dose

Crump, K. S. 1995. Calculation of benchmark doses from continuous data. Risk Anal. 15:79–89. Sandahl, J. F., and J. J. Jenkins. 2002. Pacific steelhead (Oncorhynchus mykiss) exposed to chlorpyrifos: Benchmark concentration estimates for acetylcholinesterase inhibition. Environ. Toxicol. Chem. 21:2452–2458. Slikker, W., K. S. Crump, M. E. Andersen, and D. Bellinger. 1996. Biologically based, quantitative risk assessment of neurotoxicants. Fundam. Appl. Toxicol. 29:18–30. U.S. Environmental Protection Agency. 1995. The use of the benchmark dose approach in health risk assessment. EPA 630/R-94/007. EPA Office of Research and Development, Washington, DC.

Copper and Neurobiology

Baldwin, D. H., J. F. Sandahl, J. S Labenia, and N. L. Scholz. 2003. Sublethal effects of copper on coho salmon: Impacts on nonoverlapping receptor pathways in the peripheral olfactory nervous system. Environ. Toxicol. Chem. 22:2266–2274. Baldwin, D. H., and N. L. Scholz. 2005. The electro-olfactogram: An in vivo measure of peripheral olfactory function and sublethal neurotoxicity in fish. In G. K. Ostrander (ed.), Techniques in aquatic toxicology, Volume 2, p. 257–276. CRC Press, Boca Raton, FL. Bettini, S., F. Ciani, and V. Franceschini. 2006. Recovery of the olfactory receptor neurons in the African Tilapia mariae following exposure to low copper level. Aquat. Toxicol. 76:321–328 Beyers, D. W., and M. S. Farmer. 2001. Effects of copper on olfaction of Colorado pikeminnow. Environ. Toxicol. Chem. 20:907–912. Bjerselius, R, S. Winberg, Y. Winberg, and K. Zeipel. 1993. Ca2+ protects olfactory receptor function against acute copper (II) toxicity in Atlantic salmon. Aquat. Toxicol. 25:125–138. Carreau, N. D., and G. G. Pyle. 2005. Effect of copper exposure during embryonic development on chemosensory function of juvenile fathead minnows (Pimephales promelas). Ecotoxicol. Environ. Saf. 61:1–6.

33 Chai, M., and X. Chen. 1999. Inhibition of Cd2+, Hg2+ and Pb2+ on EOG responses of Tilapia sp. Journal of Fishery Sciences of China/Zhongguo Shuichan Kexue Beijing 6:89–92. Chai, M., and Z. Huang. 1996. Effect of Ca2+ on the EOG of Tilapia sp. and exploration of detoxifying feasibility. Journal of Xiamen University Natural Science/Xiamen Daxue Xuebao Xiamen 35:941–946. Chai, M., and L. Pan. 1996. Effects of heavy metal (Cu2+, Zn2+) on the EOG response of fish (Tilapia sp.). Journal of Xiamen University Natural Science/Xiamen Daxue Xuebao Xiamen 35:94–99. Goldstein, J. N., D. F. Woodward, and A. M. Farag. 1999. Movements of adult Chinook salmon during spawning migration in a metals-contaminated system, Coeur d’Alene River, Idaho. Trans. Am. Fish. Soc. 128:121–129. Hansen, J. A., J. C. A. Marr, J. Lipton, D. Cacela, and H. L. Bergman. 1999. Differences in neurobehavioral responses of Chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (Oncorhynchus mykiss) exposed to copper and cobalt: Behavioral avoidance. Environ. Toxicol. Chem. 18:1972–1978. Hansen, J. A., J. D. Rose, R. A. Jenkins, K. G. Gerow, and H. L. Bergman. 1999. Chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (Oncorhynchus mykiss) exposed to copper: Neurophysiological and histological effects on the olfactory system. Environ. Toxicol. Chem. 18:1979–1991. Hara, T. J., Y. M. C. Law, and S. Macdonald. 1976. Effects of mercury and copper on the olfactory response in rainbow trout. J. Fish. Res. Board Can. 33:1568–1573. Hernández, P. P., V. Moreno, F. A. Olivari, and M. L. Allende. 2006. Sublethal concentrations of waterborne copper are toxic to lateral line neuromasts in zebrafish (Danio rerio). Hear. Res. 213:1–10. Julliard, A. K., D. Saucier, and L. Astic. 1993. Effects of chronic low-level copper exposure on ultrastructure of the olfactory system in rainbow trout (Oncorhynchus mykiss). Histol. Histopathol. 8:655–672. Julliard, A. K., D. Saucier, and L. Astic. 1995. Metal X-ray microanalysis in the olfactory system of rainbow trout exposed to low level of copper. Biol. Cell 83:77–86. Julliard, A. K., D. Saucier, and L. Astic. 1996. Time-course of apoptosis in the olfactory epithelium of rainbow trout exposed to a low copper level. Tissue Cell 28:367–377. Kasumyan, A. O. 2001. Effects of chemical pollutants on foraging behavior and sensitivity of fish to food stimuli. J. Ichthyol. 41:76–87. Kasumyan, A. O., and A. M. H. Morsi. 1998. Effect of heavy metals on the feeding activity and taste behavior responses of carp Cyprinus carpio: 1. Copper, cadmium, zinc, and lead. J. Ichthyol. 38:393–409. Klima, K. E., and F. M. Applehans. 1990. Copper exposure and the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss). Chem. Speciat. Bioavailab. 2:149–154. Linbo, A. O., C. M. Stehr, J. P. Incardona, and N. L. Scholz. 2006. Dissolved copper triggers cell death in the peripheral mechanosensory system of larval fish. Environ. Toxicol. Chem. 25:597–603.

34 Little, E. E., R. D. Archeski, B. A. Flerov, and V. I. Kozlovskaya. 1990. Behavioral indicators of sublethal toxicity in rainbow trout. Arch. Environ. Contam. Toxicol. 19:380–385. Little, E. E., and S. E. Finger. 1990. Swimming behavior as an indicator of sublethal toxicity in fish. Environ. Toxicol. Chem. 9:13–19. McPherson, T. D., R. S. Mirza, and G. G. Pyle. 2004. Responses of wild fishes to alarm chemicals in pristine and metal-contaminated lakes. Can. J. Zool. 82:694–700. Moran, D. T., J. C. Rowley, and G. Aiken. 1986. Trout olfactory receptors degenerate in response to waterborne ions: A potential bioassay for environmental neurotoxicology? Chem. Senses 11:642. Moran, D. T., J. C. Rowley, G. R. Aiken, and B. W. Jafek. 1992. Ultrastructural neurobiology of the olfactory mucosa of the brown trout, Salmo trutta. Microscopy Res. Tech. 23:28–48. Rehnberg, B., and C. B. Schreck. 1986. Acute metal toxicology of olfaction in coho salmon: Behavior, receptors, and odor-metal complexation. Bull. Environ. Contam. Toxicol. 36:579–586. Sandahl, J. F., D. H. Baldwin, J. J. Jenkins, and N. L. Scholz. 2007. A sensory system at the interface between urban stormwater runoff and salmon survival. Environ. Sci. Technol. 41:2998–3004. Sandahl, J. F., D. H. Baldwin, J. J. Jenkins, and N. L. Scholz. 2004. Odor-evoked field potentials as indicators of sublethal neurotoxicity in juvenile coho salmon (Oncorynchus kisutch) exposed to copper, chlorpyrifos, or esfenvalerate. Can. J. Fish. Aquat. Sci. 61:404–413. Saucier, D., and L. Astic. 1995. Morphofunctional alterations in the olfactory system of rainbow trout (Oncorhynchus mykiss) and possible acclimation in response to long-lasting exposure to low copper levels. Comp. Biochem. Physiol. A Physiol. 112:273–284. Saucier, D., L. Astic, and P. Rioux. 1991. The effects of early chronic exposure to sublethal copper on the olfactory discrimination of rainbow trout, Oncorhynchus mykiss. Environ. Biol. Fishes 30:345–351. Saunders, R. L., and J. B. Sprague. 1967. Effects of copper-zinc mining pollution on a spawning migration of Atlantic salmon. Water Res. 1:419–432 Starcevic, S. L., and B. S. Zielinski. 1997. Neuroprotective effects of glutathione on rainbow trout olfactory receptor neurons during exposure to copper sulfate. Comp. Biochem. Physiol. C 117C:211–219. Tjalve, H., and J. Henriksson. 1999. Uptake of metals in the brain via olfactory pathways. Neurotoxicology 20:181–195. Weis, J. S., and P. Weis. 1996. The effects of using wood treated with chromated copper arsenate in shallow-water environments: A review. Estuaries 19:306–310. Winberg, S., R. Bjerselius, E. Baatrup, and K. B. Døving. 1992. The effect of copper (II) on the electro- olfactogram (EOG) of the Atlantic salmon (Salmo salar L.) in artificial freshwater of varying inorganic carbon concentrations. Ecotoxicol. Environ. Saf. 24:167–178.

Reviews

Brown, G. E. 2003. Learning about danger: Chemical alarm cues and local risk assessment in pre fishes. Fish Fish. 4:227–234.

35 Døving, K. B. 1991. Assessment of animal behaviour as a method to indicate environmental toxicity. Comp. Biochem. Physiol. C 100:247–252. Eisler, R. 1998. Copper hazards to fish, wildlife, and invertebrates: A synoptic review. Report No. USGS/BRD/BSR--1998-0002. U.S. Geological Survey, Biological Resources Division. Halpern, B. P. 1982. Environmental factors affecting chemoreceptors: An overview. Environ. Health Perspect. 44:101–105. Hara, T. J. 1972. Electrical responses of the olfactory bulb of Pacific salmon Oncorhynchus nerka and Oncorhynchus kisutch. J. Fish. Res. Board Can. 29:1351–1355. Hara, T. J. 1992. Mechanisms of olfaction. In T. J. Hara (ed.), Fish chemoreception, p. 150–170. Chapman & Hall, London. Kats, L. B., and L. M. Dill. 1998. The scent of death: Chemosensory assessment of predation risk by prey animals. Ecoscience 5:361–394. Klaprat, D. A., R. E. Evans, and T. J. Hara. 1992. Environmental contaminants and chemoreception in fishes. In T. J. Hara (ed.), Fish chemoreception, p 321–342. Chapman & Hall, London. Scott, J. W., and P. E. Scott-Johnson. 2002. The electroolfactogram: A review of its history and uses. Microscopy Res. Tech. 58:152–160. Scott, G. R., and K. A. Sloman. 2004. The effects of environmental pollutants on complex fish behaviour: Integrating behavioural and physiological indicators of toxicity. Aquat. Toxicol. 68:369–392. Shumway, C. A. 1999. A neglected science: Applying behavior to aquatic conservation. Environ. Biol. Fishes 55:183–201. Smith, R. J. F. 1992. Alarm signals in fishes. Rev. Fish Biol. Fish. 2:33–63. Sorensen, E. M. B. 1991. Chapter VII: Copper. In E. M. B. Sorensen (ed.), Metal poisoning in fish, p. 235–284. CRC Press, Boca Raton, FL. Sutterlin, A. M. 1974. Pollutants and the chemical senses of aquatic animals—perspective and review. Chem. Senses Flavor 1:167–178.

Studies with Other Metals

Baatrup, E. 1991. Structural and functional effects of heavy metals on the nervous system, including sense organs, of fish. Comp. Biochem. Physiol. C 100:253–257. Baatrup, E., and K. B. Døving. 1990. Histochemical demonstration of mercury in the olfactory system of salmon (Salmo salar L.) following treatments with dietary methylmercuric chloride and dissolved mercuric chloride. Ecotoxicol. Environ. Saf. 20:277–289. Baatrup, E., K. B. Døving, and S. Winberg. 1990. Differential effects of mercurial compounds on the electroolfactogram (EOG) of salmon (Salmo salar L.). Ecotoxicol. Environ. Saf. 20:269–276. Baker, C. F., and J. C. Montgomery. 2001. Sensory deficits induced by cadmium in banded kokopu, Galaxias fasciatus, juveniles. Environ. Biol. Fishes 62:455–464.

36 Beauvais, S. L., S. B. Jones, J. T. Parris, S. K. Brewer, and E. E. Little. 2001. Cholinergic and behavioral neurotoxicity of carbaryl and cadmium to larval rainbow trout (Oncorhynchus mykiss). Ecotoxicol. Environ. Saf. 49:84–90. Chakrabarti, P., M. Ghosal, and D. K. Mandal. 1994. Microanatomical and histopathological sequels of cadmium intoxication on the olfactory epithelium of the fish Mystus vittatus (Bloch). Environ. Ecol. 12:540–544. Hernadi, L. 1993. Fine structural characterization of the olfactory epithelium and its response to divalent cations Cd2+ in the fish Alburnus alburnus (Teleostei, Cyprinidae): A scanning and transmission electron microscopic study. Neurobiology 1:11–31. Scott, G. R., K. A. Sloman, C. Rouleau, and C. M. Wood. 2003. Cadmium disrupts behavioural and physiological responses to alarm substance in juvenile rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 206:1779–1790.

Selected Behavioral Studies

Berejikian, B. A., R. J. Smith, E. P. Tezak, S. L. Schroder, and C. M. Knudsen. 1999. Paired chemical alarm signals and complex hatchery rearing habitats affect predator behavior and survival of Chinook salmon (Oncorhynchus tshawytscha) juveniles. Can. J. Fish. Aquat. Sci. 56:830–838. Brown, G. E., J. C. Adrian Jr., T. Patton, and D. P. Chivers. 2001. Fathead minnows learn to recognize predator odor when exposed to concentrations of artificial alarm pheromone below their behavioral-response threshold. Can. J. Zool. 79:2239–2245. Brown, G. E., and R. J. Smith. 1997. Conspecific skin extracts elicit antipredator responses in juvenile rainbow trout (Oncorhynchus mykiss). Can. J. Zool. 75:1916–1922. Døving, K. B., H. Westerberg, and P. B. Johnsen. 1985. Role of olfaction in the behavioral and neuronal responses of Atlantic salmon, Salmo salar, to hydrographic stratification. Can. J. Fish. Aquat. Sci. 42:1658–1667. Hatfield, C. T., and J. M. Anderson. 1972. Effects of two pesticides on the vulnerability of Atlantic salmon (Salmo salar) parr to brook trout (Salvenius fontinalis) predation. J. Fish. Res. Board Can. 29:27–29. Hatfield, C. T., and P. H. Johansen. 1972. Effects of four insecticides on the ability of Atlantic salmon parr (Salmo salar) to learn and retain a simple conditioned response. J. Fish. Res. Board Can. 29:315–321. Hiroven, H., E. Ranta, J. Piironen, A. Laurila, and N. Peuhkuri. 2000. Behavioural responses of naive Artic charr young to chemical cues from salmonid and nonsalmonid fish. Oikos 88:191–199. Kruzynski, G. M., and I. K. Birtwell. 1994. A predation bioassay to quantify the ecological significance of sublethal responses of juvenile Chinook salmon (Oncoryhynchus tshawytscha) to the antisapstain fungicide TCMTB. Can. J. Fish. Aquat. Sci. 51:1780–1790. McLennan, D. A., and M. J. Ryan. 1997. Responses to conspecific and heterospecific olfactory cues in the swordtail Xiphophorus cortezi. Anim. Behav. 54:1077–1088. Mirza, R. S., and D. P. Chivers. 2001. Chemical alarm signals enhance survival of brook charr (Salvelinus fontinalis) during encounters with predatory chain pickerel (Esox niger). Ethology 107:989–1005.

37 Poulin, R., D. Marcogliese, and J. McLaughlin. 1999. Skin-penetrating parasites and the release of alarm substances in juvenile rainbow trout. J. Fish Biol. 55:47–53. Wisby, W. J., and A. D. Hasler. 1954. Effect of occlusion on migrating silver salmon (Oncorhynchus kisutch). J. Fish. Res. Board Can. 11:472–478.

Recovery of Olfactory Epithelium after Damage

Evans, R. E., and T. J. Hara. 1985. The characteristics of the electro-olfactogram (EOG): its loss and recovery following olfactory nerve section in rainbow trout (Salmo gairdneri). Brain Res. 330:65–75. Zielinski, B. S., and T. J. Hara. 1992. Ciliated and microvillar receptor cells degenerate and then differentiate in the olfactory epithelium of rainbow trout following olfactory nerve section. Microscopy Res. Tech. 23:22–27.

Additional Endpoints

Ali, A., S. M. Al-Ogaily, N. A. Al-Asgah, and J. Gropp. 2003. Effect of sublethal concentrations of copper on the growth performance of Oreochromis niloticus. J. Appl. Ichthyol. 19:183–188. Beaumont, M. W., P. J. Butler, and E. W. Taylor. 2003. Exposure of brown trout Salmo trutta to a sublethal concentration of copper in soft acidic water: Effects upon gas exchange and ammonia accumulation. J. Exp. Biol. 206:153–162. Bielmyer, G. K., D. Gatlin, J. J. Isely, J. Tomasso, and S. J. Klaine. 2005. Responses of hybrid striped bass to waterborne and dietary copper in freshwater and saltwater. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 140:131–137. Brix, K. V., D. K. DeForest, and W. J. Adams. 2001. Assessing acute and chronic copper risks to freshwater aquatic life using species sensitivity distributions for different taxonomic groups. Environ. Toxicol. Chem. 20:1846–1856. Buckley, J. T., M. Roch, J. A. McCarter, C. A. Rendell, and A. T. Matheson. 1982. Chronic exposure of coho salmon to sublethal concentrations of copper. 1. Effect on growth, on accumulation and distribution of copper, and on copper tolerance. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 72:15–19. Campbell, H. A., R. D. Handy, and D. W. Sims. 2002. Increased metabolic cost of swimming and consequent alterations to circadian activity in rainbow trout (Oncorhynchus mykiss) exposed to dietary copper. Can. J. Fish. Aquat. Sci. 59:768–777. Campbell, H. A., R. D. Handy, and D. W. Sims. 2005. Shifts in a fish’s resource holding power during a contact paired interaction: The influence of a copper-contaminated diet in rainbow trout. Physiol. Biochem. Zool. 78:706–714. Darwish, A. M., D. L. Straus, and B. R. Griffin. 2005. Histologic evaluation of the safety of copper sulfate to channel catfish. N. Am. J. Aquacult. 67:122–128. De Boeck, G., A. Vlaeminck, and R. Blust. 1997. Effects of sublethal copper exposure on copper accumulation, food consumption, growth, energy stores, and nucleic acid content in common carp. Arch. Environ. Contam. Toxicol. 33:415–422.

38 Diamond, J., M. Bowersox, H. Latimer, C. Barbour, J. Bearr, and J. Butcher. 2005. Effects of pulsed contaminant exposures on early life stages of the fathead minnow. Arch. Environ. Contam. Toxicol. 49:511–519. Furuta, T., N. Iwata, K. Kikuchi, and K. Namba. 2005. Effects of copper on survival and growth of larval false clown anemonefish Amphiprion ocellaris. Fish. Sci. 71:884–888. Galvez, F., and C. M. Wood. 2002. The mechanisms and costs of physiological and toxicological acclimation to waterborne silver in juvenile rainbow trout (Oncorhynchus mykiss). J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 172:587–597. Hansen, J. A., J. Lipton, P. G. Welsh, D. Cacela, and B. MacConnell. 2004. Reduced growth of rainbow trout (Oncorhynchus mykiss) fed a live invertebrate diet pre-exposed to metal-contaminated sediments. Environ. Toxicol. Chem. 23:1902–1911. Hansen, J. A., J. Lipton, P. G. Welsh, J. Morris, D. Cacela, and M. J. Suedkamp. 2002. Relationship between exposure duration, tissue residues, growth, and mortality in rainbow trout (Oncorhynchus mykiss) juveniles subchronically exposed to copper. Aquat. Toxicol. 58:175–188. Hansen, J. A., P. G. Welsh, J. Lipton, and D. Cacela. 2002. Effects of copper exposure on growth and survival of juvenile bull trout. Trans. Am. Fish. Soc. 131:690–697. Lorz, H. W., and B. P. McPherson. 1976. Effects of copper or zinc in freshwater on adaptation to seawater and Atpase activity, and effects of copper on migratory disposition of coho salmon (Oncorhynchus kisutch). J. Fish. Res. Board Can. 33:2023–2030. Marr, J. C. A., J. Lipton, D. Cacela, J. A. Hansen, H. L. Bergman, J. S. Meyer, and C. Hogstrand. 1996. Relationship between copper exposure duration, tissue copper concentration, and rainbow trout growth. Aquat. Toxicol. 36:17–30. Rajotte, J. W., and P. Couture. 2002. Effects of environmental metal contamination on the condition, swimming performance, and tissue metabolic capacities of wild yellow perch (Perca flavescens). Can. J. Fish. Aquat. Sci. 59:1296–1304. Roch, M., and J. A. McCarter. 1984. Metallothionein induction, growth, and survival of Chinook salmon exposed to zinc, copper, and cadmium. Bull. Environ. Contam. Toxicol. 32:478–485. Sloman, K. A., D. W. Baker, C. G. Ho, D. G. McDonald, and C. M. Wood. 2003. The effects of trace metal exposure on agonistic encounters in juvenile rainbow trout, Oncorhynchus mykiss. Aquat. Toxicol. 63:187–196. Sloman, K. A., D. W. Baker, C. M. Wood, and G. McDonald. 2002. Social interactions affect physiological consequences of sublethal copper exposure in rainbow trout, Oncorhynchus mykiss. Environ. Toxicol. Chem. 21:1255–1263. Sloman, K. A., T. P. Morgan, D. G. McDonald, and C. M. Wood. 2003. Socially induced changes in sodium regulation affect the uptake of waterborne copper and silver in the rainbow trout, Oncorhynchus mykiss. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 135:393–403.

Recent NOAA Technical Memorandums published by the Northwest Fisheries Science Center

NOAA Technical Memorandum NMFS-NWFSC-

82 Helser, T.E., I.J. Stewart, C.E. Whitmire, and B.H. Horness. 2007. Model-based estimates of abundance for 11 species from the NMFS slope surveys. U.S. Dept. Commer., NOAA Tech. Memo. NMFS-NWFSC- 82, 145 p. NTIS number pending.

81 Hard, J.J., J.M. Myers, M.J. Ford, R.G. Cope, G.R. Pess, R.S. Waples, G.A. Winans, B.A. Berejikian, F.W. Waknitz, P.B. Adams. P.A. Bisson, D.E. Campton, and R.R. Reisenbichler. 2007. Status review of Puget Sound steelhead (Oncorhynchus mykiss). U.S. Dept. Commer., NOAA Tech. Memo. NMFS-NWFSC- 81, 117 p. NTIS number PB2008-100451.

80 Berntson, E.A., P.S. Levin, and P.C. Moran (editors). 2007. Conservation of North Paciﬁc rockﬁshes: Ecological genetics and stock structure. Proceedings of the workshop, March 2-3, 2004, Seattle, Washing- ton. U.S. Dept. Commer., NOAA Tech. Memo. NMFS-NWFSC-80, 80 p. NTIS number PB2007-111137.

79 Lawson, P.W., E.P. Bjorkstedt, M.W. Chilcote, C.W. Huntington, J.S. Mills, K.M.S. Moore, T.E. Nickelson, G.H. Reeves, H.A. Stout, T.C. Wainwright, and L.A. Weitkamp. 2007. Identiﬁcation of historical populations of coho salmon (Oncorhynchus kisutch) in the Oregon coast evolutionarily signiﬁcant unit. U.S. Dept. Commer., NOAA Tech. Memo. NMFS-NWFSC-79, 129 p. NTIS number PB2007-111607.

78 Ruckelshaus, M.H., K.P. Currens, W.H. Graeber, R.R. Fuerstenberg, K. Rawson, N.J. Sands, and J.B. Scott. 2006. Independent populations of Chinook salmon in Puget Sound. U.S. Dept. Commer., NOAA Tech. Memo. NMFS-NWFSC-78, 125 p. NTIS number PB2007-104920.

77 Sloan, C.A., D.W. Brown, G.M. Ylitalo, J. Buzitis, D.P. Herman, D.G. Burrows, G. Yanagida, R.W. Pearce, J.L. Bolton, R.H. Boyer, and M.M. Krahn. 2006. Quality assurance plan for analyses of environmental samples for polycyclic aromatic compounds, persistent organic pollutants, fatty acids, stable isotope ratios, lipid classes, and metabolites of polycyclic aromatic compounds. U.S. Dept. Commer., NOAA Tech. Memo. NMFS-NWFSC-77, 30 p. NTIS number PB2007-104919.

76 Gustafson R.G., J. Drake, M.J. Ford, J.M. Myers, E.E. Holmes, and R.S. Waples. 2006. Status review of Cherry Point Paciﬁc herring (Clupea pallasii) and updated status review of the Georgia Basin Paciﬁc herring distinct population segment under the Endangered Species Act. U.S. Dept. Commer., NOAA Tech. Memo. NMFS-NWFSC-76, 182 p. NTIS number PB2007-104918.

75 Keller, A.A., B.H. Horness, V.J. Tuttle, J.R. Wallace, V.H. Simon, E.L. Fruh, K.L. Bosley, and D.J. Kamikawa. 2006. The 2002 U.S. West Coast upper continental slope trawl survey of groundﬁsh resources off Washington, Oregon, and California: Estimates of distribution, abundance, and length composition. U.S. Dept. Commer., NOAA Tech. Memo. NMFS-NWFSC-75, 189 p. NTIS PB2006-111432.

Most NOAA Technical Memorandums NMFS-NWFSC are available online at the Northwest Fisheries Science Center web site (http://www.nwfsc.noaa.gov) Environ. Sci. Technol. 2007, 41, 2998-3004

at-risk aquatic species, the current conservation challenges A Sensory System at the Interface associated with toxic runoff are global in scope, complex, between Urban Stormwater Runoff expanding and poorly understood. Pavement is a universal feature of urbanized landscapes, and Salmon Survival and impervious surfaces accumulate chemical pollutants from automobile traffic as well as from other sources (5). During rainfall events, these contaminants are mobilized by JASON F. SANDAHL,† stormwater (6) and transported to rivers, lakes, and estuaries DAVID H. BALDWIN,‡ JEFFREY J. JENKINS,† AND (7). Dissolved copper is a particularly pervasive contaminant NATHANIEL L. SCHOLZ*,‡ in urban runoff. This reflects the many industrial, commercial, and residential uses of copper, including the incorporation Oregon State University, Department of Molecular and of the metal into roofing and flashing materials, treated wood, Environmental Toxicology, 333 Weniger Hall, Corvallis, Oregon 97331, and NOAA Fisheries, Northwest and various pesticide formulations. In addition, vehicle Fisheries Science Center, Ecotoxicology and Environmental emissions via exhaust and brake pad wear represent major Fish Health Program, 2725 Montlake Boulevard East, sources of copper in runoff from roads (8). Within a particular Seattle, Washington 98112 watershed, the loading of copper to surface waters will depend, in part, on site-specific hydrological characteristics, as well as land cover (e.g., percent impervious surface), vehicle traffic, and rainfall patterns. As an example of measured Motor vehicles are a major source of toxic contaminants concentrations in aquatic habitats, recent monitoring of such as copper, a metal that originates from vehicle exhaust streams in northern California following storm events found and brake pad wear. Copper and other pollutants are dissolved copper at levels that varied from 3.4 to 64.5 µg/L, deposited on roads and other impervious surfaces and with a mean of 15.8 µg/L (9). then transported to aquatic habitats via stormwater runoff. In the present study, we investigate the impact of dissolved copper on juvenile coho salmon (Oncorhynchus kisutch). Wild In the western United States, exposure to non-point stocks of coho and other species of anadromous Pacific source pollutants such as copper is an emerging concern salmon and steelhead are declining throughout much of their for many populations of threatened and endangered natural range in the western U.S. (10). Currently, 26 distinct Pacific salmon (Oncorhynchus spp.) that spawn and rear population segments (evolutionary significant units; ref 11) in coastal watersheds and estuaries. To address this concern, of coho, chinook (O. tshawytscha), sockeye (O. nerka), and we used conventional neurophysiological recordings to chum (O. keta) salmon as well as steelhead (O. mykiss) are investigate the impact of ecologically relevant copper listed as either threatened or endangered under the U.S. exposures (0-20 µg/L for 3 h) on the olfactory system of Endangered Species Act (ESA). In the case of coho, several juvenile coho salmon (O. kisutch). These recordings were historical runs have been extirpated throughout California, combined with computer-assisted video analyses of Oregon, Washington, and Idaho (12). To reverse salmon declines, federal, state, and local governments have invested behavior to evaluate the sensitivity and responsiveness of hundreds of millions of U.S. dollars in recent years to conserve copper-exposed coho to a chemical predation cue and restore the quality of freshwater and estuarine habitats (conspecific alarm pheromone). The sensory physiology (e.g., ref 13). Freshwater habitat quality is particularly and predator avoidance behaviors of juvenile coho were important for coho salmon that rear for more than a year in both significantly impaired by copper at concentrations as lowland streams and ponds before beginning their seaward low as 2 µg/L. Therefore, copper-containing stormwater migration (14). runoff from urban landscapes has the potential to cause Copper is a neurobehavioral toxicant in fish, and it has chemosensory deprivation and increased predation mortality been known for more than three decades that the metal in exposed salmon. disrupts the normal function of the fish olfactory system (15). Ultrastructural analyses have shown that dissolved copper damages the olfactory sensory epithelium (16-19), and previous studies using direct neurophysiological record- Introduction ings from the fish nose (15, 17, 20, 21) or observations of - Human population growth is increasingly concentrated along chemosensory behavior (16, 22 24) have shown that copper the coastal margins of countries such as the United States interferes with the ability of fish to detect and respond to (1, 2). Urbanization and other forms of coastal development chemical signals in aquatic environments. Chemosensory increase the runoff of pollutants from terrestrial landscapes deprivation has important implications for salmon, as these to the aquatic environment. Upon completing the most migratory animals rely on their sense of smell to find food, comprehensive review of the nation’s management of oceans, avoid predators, form social dominance hierarchies, navigate coasts, and the Great Lakes in more than three decades, the from the ocean to freshwater spawning habitats, and assess U.S. Commission on Ocean Policy recently highlighted non- the reproductive status of prospective mates. point source pollution as one of the most significant emerging To determine whether short term (3 h) exposures to threats to aquatic species (3). A similar review by the Pew dissolved copper at concentrations typical of urban storm- Ocean Commission found that non-point sources represent water runoff (0-20 µg/L) interfere with olfaction and the greatest pollution threat to oceans and coasts (4). For olfactory-mediated behaviors in juvenile coho salmon, we used a combination of in vivo neurophysiological recordings from the olfactory epithelium and three-dimensional digital * Corresponding author phone: (206) 860-3454; fax: (206) 860- 3335; e-mail: [email protected]. imaging to quantify predator avoidance behaviors that are † Oregon State University. normally triggered in juvenile salmon by a conspecific ‡ NOAA Fisheries. chemical alarm pheromone (25). For each copper exposure

2998 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 8, 2007 10.1021/es062287r CCC: $37.00  2007 American Chemical Society Published on Web 03/14/2007 concentration, recordings from olfactory sensory neurons TABLE 1. Effects of Dissolved Copper on the Swimming were matched to behavioral observations from the same a animal. This allowed us to evaluate the sublethal neurobe- Behavior of Coho Salmon havioral effects of copper at two different biological scales copper copper pre-stimulus post-stimulus freeze and to assess the extent to which classical measures of sensory nominal measured swimming swimming responses toxicity (i.e., electrophysiology) are predictive of behavioral (µg/L) (µg/L) speed (cm/s) speed (cm/s) (fraction) impairment. 0 0.3 ( 0.2 5.6 ( 0.4 1.4 ( 0.3 11/12 2 1.9 ( 0.4 6.0 ( 0.3 3.7 ( 0.7 6/12* Experimental Procedures 5 4.7 ( 0.6 5.6 ( 0.3 4.8 ( 0.7 3/12* Animals. Coho salmon eggs were obtained from the Uni- 10 10.2 ( 1.6 5.2 ( 0.5 4.1 ( 0.5 2/12* versity of Washington hatchery (Seattle, WA) at the eyed egg 20 16.8 ( 1.7 2.3 ( 0.4* 2.4 ( 0.5 1/8* stage and raised at the Northwest Fisheries Science Center’s a Measured copper values are from three composite samples for hatchery facility under natural photoperiod conditions. Coho each treatment group taken at the start of the exposure period. A freeze parr were maintained in tanks supplied with filtered, response was a 50% or greater reduction in locomotory activity (see Experimental Procedures). Data are presented as mean ( SE or as dechlorinated municipal water (hereafter referred to as fractions representing the number of responders over the total number hatchery water; 120 mg/L total hardness as CaCO3, pH 6.6, of fish tested. For pre-stimulus swimming speed and freeze responses, dissolved oxygen 8.1 mg/L, temperature 11-13 °C) on a asterisks represent a statistical difference from controls (p < 0.05, one- single-pass flow system. Fish were raised on standard way ANOVA with a Dunnett’s post hoc and Fisher’s exact test, commercial salmon pellets (Bio-Oregon, Warrenton, OR). respectively). Fish were 4-5 months of age with an average ((SD) length of 4.6 ( 0.4 cm and a weight of 0.9 ( 0.2 g. course of the exposure period, with a mean and range (in Preparation of Chemical Alarm Stimulus. A stock alarm parentheses) of 10.8 °C (10-12 °C), pH 6.7 (6.5-7.1), and 8.2 substance was prepared by homogenizing approximately 600 mg/L dO (6.5-9.6 mg/L). cm2 of skin from 16 juvenile coho in 50 mL of distilled water. Nominal exposure solutions were analyzed for total The homogenate was then filtered through polyester floss, dissolved copper by an outside laboratory using inductively diluted to a final concentration of 100 cm2 skin/L in distilled coupled plasma mass spectrometry (Frontier Geosciences, water, mixed, aliquoted into 10 mL glass vials, and stored at Seattle, WA). The background concentration of total dissolved -20 °C. Immediately before use, aliquots were thawed, copper in the hatchery water was 0.3 µg/L. Copper recovered filtered, and diluted 1:100 in hatchery water to a final from exposure tanks ranged from 84 to 102% of nominal concentration of 1 cm2 skin/L. Control blank solutions values (Table 1). Accordingly, copper exposures are hereafter consisted of hatchery water only. Although the as-yet expressed in terms of nominal concentrations. unidentified alarm substance is unlikely to be a protein (26), Quantitative Analysis of Predator Avoidance Behaviors. the pheromone is contained within specialized club cells Followinga3hexposure, the behavioral response of each that are generally distributed throughout skin tissue (reviewed juvenile coho to a chemical predation cue was monitored in ref 27). Thus, the concentration of pheromone is likely to using a computer-assisted, three-dimensional data acquisi- vary in proportion to the protein content of the skin extract. tion system (29). The experimental design was modified Moreover, protein assays are more precise and more slightly from Scholz et al. (25). For the behavioral trials, reproducible than estimates of epidermal surface area. individual fish were transferred to a clean 30 L glass aquarium Accordingly, we measured the total protein content of the filled with 25 L of hatchery water. Continuous, closed conspecific skin extract using a modified Bradford (28) assay circulation mixing in the aquarium was provided by a small (Coomassie Plus-2000 Protein Assay Reagent, Pierce, Rock- aquarium pump. Conspecific skin extract was injected into ford, IL). Odor stimulus concentrations are reported as mg the behavioral observation tank via a 50 cm length of Tygon (or µg) of protein/L. As a point of reference, 1 cm2 skin was tubing. Initial tests with dye indicated an even distribution empirically determined to be equivalent to 5 mg of protein. of odor stimulus throughout the tank within approximately Moreover, a mechanical disruption of the skin as small as 1 1 min. mm2 (50 µg of protein) would be sufficient to fill 100 L to a The three-dimensional position of fish was monitored concentration of 0.5 µg/L protein, a concentration within using two orthogonally placed Firewire digital cameras the experimental range examined here. (Fire-i, Unibrain Inc., San Ramon, CA) connected to a laptop Copper Exposures and Chemical Analysis. Copper- computer (iBook, Apple Computer, Cupertino, CA), as containing exposure solutions were constituted by dissolving previously described (29). In brief, the two cameras acquired copper chloride (Sigma Chemical Co., St. Louis, MO; 99% simultaneous images of the fish from the front and side of purity cupric chloride, dihydrate) in distilled water. A total the tank every 2 s. Each pair of images was then analyzed to of five stock solutions was prepared, such that adding 100 determine the position of the fish via triangulation, with a mL of each stock to 25 L of hatchery water produced nominal correction for refraction. The three-dimensional distance dissolved copper concentrations of 0, 2, 5, 10, and 20 µg/L between subsequent pairs of images (divided by 2 s) was in aerated, 30 L glass exposure aquaria. The exposure aquaria used to calculate the swimming speed at each time point. were visually isolated from each other. Prior to the introduc- Trials began by transferring individual control or copper- tion of fish, 100 mL water samples for dissolved copper exposed fish to the observation tank and then allowing them analysis were collected in acid-washed, Teflon bottles and to acclimate for 30 min. A baseline, pre-stimulus swimming refrigerated at 4 °C. Fish were then exposed to copper for 3 speed for each animal was subsequently recorded for a 3 h. Each fish was treated individually (n ) eight to 12 animals min interval (t )-180-0 s). Following this, a small volume per exposure concentration) in separate tanks using freshly of the chemical alarm substance (0.5 mL; 5 mg of protein/L) prepared copper exposure solutions diluted from a common was injected into the circulation system (t ) 0 s) to achieve stock. Individual exposures were staggered to maintain a a final diluted concentration of 0.1 µg of protein/L in the constant duration between the onset of the copper exposure observation chamber. The post-stimulus swimming speed and the onset of either behavioral or electrophysiological of the fish was then monitored for an additional 4 min. To trials. Different combinations of copper-exposed fish were allow for differences in odorant dispersal as well as differences tested on any given day, but at least one fish from the control in the initiation of the avoidance response among animals, group was tested on each day. Water temperature, pH, and we selected a fixed 30 s interval (t ) 45-75 s) to measure the dissolved oxygen (dO) remained relatively constant over the post-stimulus swimming speed. On the basis of initial trials,

VOL. 41, NO. 8, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2999 sure the behavioral and physiological responses of fish to a range of skin extract dilutions. For the alarm behavior, unexposed juvenile coho salmon were presented with the skin extract at nominal concentrations of 0 (hatchery water blank), 0.04, 0.1, 0.4, and 1.0 µg of protein/L (n ) seven to 12 animals per concentration) with each fish tested with only one skin extract dilution. For the physiological response, EOGs from a separate group of unexposed fish were recorded in response to five dilutions of skin extract (0.1-10 µgof protein/L; n ) eight to nine fish per stimulus dilution). Finally, to evaluate the effects of copper on the stimulus-response relationship for the alarm substance, a third group of fish was exposed to 2 µg/L of copper for 3 h, and EOG responses to skin extract were then recorded at dilutions ranging from 0.4-40 µg of protein/L (n ) three to six fish per dilution). FIGURE 1. Odorant stimulus-response curves were determined Statistical Analysis. The electrophysiological and behav- for both alarm (closed squares) and EOG responses (closed circles) ioral measures were analyzed using either one-way analysis- to skin in control (unexposed fish) and for EOG responses to skin of-variance (ANOVA) to test for statistical differences between in fish exposed to 2 µg/L of copper (open circles). Fractions within groups (followed by a Dunnett’s test for comparisons with parentheses correspond to the proportion of fish tested in each controls), Fisher’s exact test (for freeze responses), or group that showed a >50% reduction in activity (number of fish regression analysis to test for concentration-dependent responding/total number of fish tested). A slight EOG response was relationships. Paired t-tests were used to determine differ- observed when the perfusion of the olfactory chamber was switched ences in pre-stimulus baseline activity and post-stimulus to hatchery water alone (blank stimulus). Unlike the behavioral activity for antipredator responses. Correlations were de- responses, the EOG response did not plateau at higher concentrations termined by using the Pearson correlation procedure. of skin extract. In both graphs, error bars represent one standard Statistical analyses and graphing were performed with error. Asterisks denote the skin extract concentration used in GraphPad Prism 4.0 (San Diego, CA) and SAS Institute JMP subsequent copper exposure experiments. 5.1 (Cary, NC). this interval included the behavioral responses of almost all Results the fish. The magnitude of the response was quantified by comparing the change (reduction) in swimming speed over Neurobehavioral Responses to a Chemical Predation Cue the pre- and post-stimulus intervals (Supporting Information, over a Range of Stimulus Concentrations. The onset of Figure S1A). Additionally, the reaction to alarm pheromone predator avoidance behavior usually occurred within 30-60 was scored as a predator avoidance response if the animal s after the introduction of skin extract to the observation exhibited motionlessness, as indicated by a reduction in tank. This brief delay presumably reflected variability in the swimming speed of 50% or more. To reduce inter-animal time required for the skin extract to circulate throughout the variability arising from risk-taking behavior (i.e., motivation tank, the position of the fish in the tank at the time of stimulus to forage in the face of a predation threat), we did not feed introduction, and variation in inter-animal behavior. In a juvenile coho during behavioral trials and thus did not typical response, juvenile coho oriented to the direction of monitor food strikes (25). water flow and began a rapid fanning motion of the pectoral Odor-Evoked Neurophysiological Recordings from the fins. This sculling or freezing behavior served to hold the fish Coho Olfactory Epithelium. Once the behavioral observa- in a relatively fixed position. Responsive fish also tended to tions were complete, odor-evoked EOGs were recorded from slowly settle toward the bottom of the tank (Supporting the peripheral olfactory epithelium of each juvenile coho Information, Movie S1). Although the stereotypical anti- using established procedures (30). Fish were anaesthetized predator response was a rapid onset of motionlessness, the in tricaine methanesulfonate (MS-222; 50 mg/L) and trans- duration of the response varied, with some animals freezing ferred to a vibration isolation table for electrophysiological for tens of seconds and others freezing for several minutes recordings. For each animal, the EOG evoked by an odorant (not shown). was measured in triplicate and then averaged to produce a The degree of acclimation to the observation tank was single response value. The size or amplitude of the EOG was consistent across the groups of fish, as indicated by a expressed as the negative phasic displacement (in millivolts) comparable amount of baseline (pre-stimulus) swimming of the evoked peak relative to the pre-stimulus electrical activity among groups (mean ( SE; 5.2 ( 0.2 cm/s; one-way baseline (refs 20 and 21 and Supporting Information Figure ANOVA, p > 0.5). Fish presented with hatchery water only 1B). (blank) showed no change in swimming speed over the pre- Odorant solutions were prepared daily from concentrated and post-stimulus observation period (paired t-test, p > 0.5), stocks of conspecific skin extract (alarm substance), the amino and no animals exhibited the stereotypical freezing behavior acid L-serine, and the bile salt taurocholic acid (TCA) dissolved in response to a blank stimulus. The swimming speed of fish in hatchery water. The olfactory chamber of each animal presented with skin extract at 0.04 µg of protein/L was slightly (with the nare intact) was perfused with a sequence of the diminished, with a 21 ( 14% (mean ( SE) reduction in speed three different odorants: skin extract (10 µg of protein/L), relative to the pre-stimulus interval (paired t-test, p ) 0.12). - - l-serine (10 5 M), and TCA (10 6 M). L-Serine and TCA are However, three of eight animals exhibited motionlessness well-studied odorants in salmon and were included for the or freezing. A more pronounced antipredator response purposes of comparing the results of this study to previous occurred when the alarm stimulus concentration was investigations (21). At these concentrations, all three odorants increased to 0.1 µg of protein/L. This included a 74 ( 6% elicit similar, robust EOGs from the olfactory epithelium of reduction in speed relative to controls (paired t-test, p < unexposed animals. Fish were euthanized by decapitation 0.001) and a freezing response in 11 of 12 animals. Behavioral after recording EOGs. changes were similarly pronounced at higher stimulus Initial Alarm Substance Range-Finding Experiments. concentrations (0.4 and 1.0 µg of protein/L; paired t-test, p Several range-finding experiments were performed to mea- < 0.001).

3000 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 8, 2007 Odor-evoked field potential recordings from the olfactory epithelium of coho indicated a concentration-dependent increase in EOG amplitude in response to skin extract (0.1-10 µg of protein/L; n ) eight to nine fish per stimulus concentration, Figure 1), with evoked extracellular potentials ranging from 0.2-1.7 mV, after subtraction of the response to a blank solution (hatchery water only). At a concentration of skin extract of 0.1 µg of protein/L, the EOG responses were indistinguishable from responses to the blank solution (p > 0.05, one-way ANOVA followed by Dunnett’s post hoc). Thus, for juvenile coho, the measured neurophysiological detection threshold for conspecific skin extract under these experimental conditions was between 0.1 and 0.4 µg of protein/L. Exposure to 2 µg/L of copper for 3 h reduced the EOG responses to all skin extract concentrations, effectively shifting the concentration relationship to the right (Figure 1). For copper-exposed fish, responses to concentrations of skin extract of 1 µg of protein/L or less were indistinguishable from blank responses (p > 0.05, one-way ANOVA followed by Dunnett’s post hoc), indicating an increase in response threshold by about 1 log unit. In summary, the conspecific skin extract elicited measurable electrophysiological and behavioral responses from juvenile coho salmon at concentrations either above or at 0.1 µg of protein/L. The behavioral stimulus-response curve was steep, with the skin extract evoking maximal predator avoidance behaviors at a concentration that was below the lowest concentration detectable via olfactory neurophysi- ology. Also, since motionlessness was observed in response to alarm substance at a concentration subthreshold for evoked EOGs (0.04 µg of protein/L), the behavioral measure- ment appears to be the more sensitive of the two experimental assays. On the basis of these initial observations, a stimulus concentration of 0.1 µg of protein/L was used for subsequent behavioral trials involving copper-exposed fish. To elicit a robust EOG response, the olfactory chamber was perfused with 10 µg of protein/L during neurophysiological experiments. Relative Thresholds for Neurophysiological and Behav- ioral Impairment in Juvenile Coho Exposed to Dissolved Copper. To determine the relative impacts of short-term copper exposures (3 h; 2-20 µg/L) on olfactory sensitivity and predator avoidance behavior, we exposed individual fish to copper, monitored a behavioral response to 0.1 µgof protein/L of skin extract, and then recorded odor-evoked EOGs from each animal’s olfactory epithelium using conspecific skin extract and two other natural odorants (the amino acid L-serine and the bile salt TCA) as stimuli. The Supporting Information includes examples of paired etho- grams and olfactograms for four control fish and four animals exposed to 10 µg/L of copper (Figure S2) and a movie showing the behavioral responses of a control fish and a fish exposed to 10 µg/L of copper (Movie S1). FIGURE 2. Exposure to copper diminished olfactory sensitivity and Dissolved copper inhibited olfactory responses to all three alarm behavior in juvenile coho. (A) Electro-olfactogram (EOG) - odorants (skin extract, L-serine, and TCA) in a concentration- responses to skin extract (10 µg of protein/L), L-serine (10 5 M), and dependent manner (ANOVA, p < 0.001, Figure 2A). In taurocholic acid (TCA, 10-6 M) were inhibited at increasing copper unexposed animals, the mean EOG responses to 10 µgof exposure concentrations. Note that, in contrast to Figure 1, the EOG - - protein/L of skin extract, 10 5 M L-serine, and 10 6 M TCA responses shown here were blank-subtracted. The results of were 1.2, 2.8, and 4.0 mV, respectively. At the lowest copper nonlinear regressions are shown with solid lines (see Results for exposure concentration (2 µg/L), the mean skin extract- details). (B) Copper exposure also reduced the alarm response evoked EOG amplitude was 0.6 mV, a significant reduction elicited by 0.1 µg of protein/L of skin extract in a dose-dependent relative to controls (ANOVA, Dunnett’s test, p < 0.01). At 20 manner. The result of a nonlinear regression is shown with a solid µg/L of copper, EOG responses to all three odorants were line (see Results for details). (C) Paired physiological and behavioral nearly abolished. The data for each odorant were also closely response means were highly correlated (i.e., fish with reduced fit (r2 g 0.97) by a nonlinear regression to a sigmoidal function, olfactory sensitivity showed reduced alarm behavior). Error bars slope in all graphs represent one standard error. EOG ) max/(1 + (copper/EC50) ), which was applied previously for juvenile coho (21). For each of the regressions Dissolved copper also disrupted odor-evoked predator shown in Figure 2A, the mean olfactory response of the avoidance behaviors (Figure 2B and Table 1). For juvenile control group was used to define the value of max in the coho exposed to copper at concentrations up to 10 µg/L, previous equation. pre-stimulus baseline swimming activity was indistinguish-

VOL. 41, NO. 8, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 3001 able from controls (5.6 ( 0.4 cm/s, Table 1). By contrast, fish exposed to 20 µg/L of copper for 3 h were noticeably lethargic, as indicated by a reduction in the mean baseline activity in these fish relative to controls (2.3 ( 0.4 cm/s; ANOVA, p < 0.05). The alarm pheromone triggered an average reduction in swimming speed of 74 ( 6% (mean ( SE) among unexposed animals. The behavioral change was highly significant (paired t-test, p < 0.001), with all but one of the unexposed animals (n ) 11 of 12 fish) becoming motionless during the post-stimulus interval (Table 1). While the reduction in swimming speed among fish exposed to 2 µg/L of copper (39 ( 12%) was significant (paired t-test, p < 0.01), fewer animals (n ) six of 12) became motionless. At higher copper concentrations (5, 10, and 20 µg/L), there were no significant reductions in swimming speed (paired t-tests, p > 0.1), and the majority of fish did not become motionless. The effect of copper on the alarm reaction also showed a reasonable fit to the same sigmoidal function as the EOG responses (r2 ) 0.80, Figure 2B). The duration of the alarm reaction for the few fish that did respond to the pheromone at these higher copper exposures was generally shorter than for controls (e.g., 24 s for the one 20 µg/L exposed fish versus 114 ( 27 s for the 11 unexposed fish). Overall, however, too few of the copper-exposed fish responded to allow for a comparison of duration (not shown). A direct comparison of the inhibitory effects of dissolved copper on the sensory biology and behavior of juvenile coho FIGURE 3. Conceptual model to illustrate how shifts in olfactory is shown in Figure 2C. The relationship between olfactory sensitivity can result in corresponding shifts in predator avoidance inhibition and diminished alarm response was significantly behavior. On the basis of the data in Figure 1, sigmoidal and power correlated (Pearson r )-0.97, r2 ) 0.94, p < 0.01). From the functions were used to approximate the behavioral and olfactory stimulus-response curves, respectively. In this theoretical model, slope of the correlated measures (linear regression, slope ) a threshold concentration of alarm pheromone (left vertical dashed 75 ( 11), a ∼25% decrease in olfactory function corresponds ∼ line) is required to generate an olfactory response (horizontal dashed to a 29% decrease in the magnitude of the pheromone- line) that will be sufficient to trigger an alarm response in unexposed mediated predator avoidance behavior. Consequently, the fish. Following exposure to copper, a shift in olfactory sensitivity relative impacts of dissolved copper exposure are similar at increases the strength of the stimulus needed to reach this these two different scales of biological organization. physiological and behavioral threshold (right vertical dashed line), and the previous stimulus now effectively fails to elicit the alarm Discussion behavior. Our current findings provide an important link between habitat degradation (i.e., dissolved copper exposure) and shift will increase with higher copper exposures, as evidenced changes in the sensory-mediated behavior of threatened and by the continued reduction in EOGs following exposure to endangered Pacific salmon. More specifically, we have shown 5, 10, and 20 µg/L (Figure 2A). Consequently, as the dissolved that short-term exposures to dissolved copper diminish the copper content in surface waters increases, the responsive- olfactory sensitivity of juvenile coho salmon and that this ness of the peripheral olfactory system to a predation cue loss of sensory function leads, in turn, to a failure to initiate will diminish until it falls below the threshold required to predator avoidance behaviors in response to a conspecific initiate an appropriate behavioral response (Figure 3). olfactory stimulus. For salmonids, the detection of chemical Therefore, a likely outcome in salmon habitats is that copper- alarm cues is important for predator recognition and learning exposed fish will make behavioral decisions that are inap- (reviewed by ref 31) as well as for surviving encounters with propriately risky for a particular ecological situation (33). predators (32). Notably, these neuroethological effects of The consequences of this for actual rates of predation on copper occur at concentrations that are well within the lower juvenile salmon have not been determined, and this remains range of measured copper levels in surface waters of urban an important area for future research. and urbanizing watersheds (e.g., 3-64 µg/L; ref 9). Salmon will avoid copper originating from point sources The effective range of chemical alarm pheromone- with defined environmental gradients (e.g., ref 23). However, mediated signaling in aquatic systems is likely to vary with such spatial gradients are unlikely to be present in watersheds the strength of the signal at the source (i.e., the degree of contaminated with diffuse non-point source runoff. For fish damage to the skin of another fish), the turbulent dispersal that are unable to avoid stormwater, the toxic effects of copper of the chemical cue, and the sensory capabilities of the will be reversible, with physiological recovery taking place receiver. By interfering with chemosensation in the receiver, over the course of several hours following low-dose exposures dissolved copper will effectively reduce the active space over (21). At higher concentrations, including those sufficient to which a conspecific alarm signal is effective. Moreover, trigger cell death in the sensory epithelium (i.e., g25 µg/L; copper-exposed fish may simply fail to respond to a predation ref 17), the regeneration of olfactory neurons may take place cue at concentrations that would normally trigger anti- over days or weeks. In either case, intermittent rainfall can predator behaviors in uncontaminated systems. The neu- be expected to drive a dynamic process of neurobehavioral robehavioral basis for this shift can be seen in Figure 1 and toxicity and recovery among salmon in urban creeks. is illustrated in Figure 3. In the present study, the EOG Finally, our current results in juvenile coho should be response of juvenile salmon followinga3hexposure to applicable to other fish species in urbanizing watersheds copper at 2 µg/L was reduced by ∼40% over the entire range worldwide. In addition to coho (this study and refs 20 and of odor concentrations (Figure 1), thereby shifting the 21), dissolved copper has been shown to impair olfaction in stimulus-response curve to the right nearly a log unit. This chinook salmon (17, 23), rainbow trout (15, 18, 24), brown

3002 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 8, 2007 trout (Salmo trutta; ref 19), fathead minnow (Pimephales (13) Columbia River Basin salmon and steelhead: Federal agencies’ promelas; ref 22), Colorado pikeminnow (Ptychocheilus lucius; recovery responsibilities, expenditures, and actions; United States ref 16), and tilapia (34). It is also likely that the neurotoxic General Accounting Office: Washington, DC, 2002; GAO-02- 612. effects of copper extend beyond the olfactory networks that (14) Nickelson, T. E.; Rodgers, J. D.; Johnson, S. L.; Solazzi, M. F. underlie predator avoidance behavior. For example, Baldwin Seasonal changes in habitat use by juvenile coho salmon et al. (21) found that copper reduces the sensitivity of coho (Oncorhynchus kisutch) in Oregon coastal streams. Can. J. Fish. salmon to distinct classes of natural odorants in a similar, Aquat. Sci. 1992, 49, 783-789. dose-dependent manner. This suggests that copper is a (15) Hara, T. J.; Law, Y. M. C. MacDonald, S. Effects of mercury and general-purpose inhibitor of fish olfaction and may thus copper on the olfactory response in rainbow trout. J. Fish. Res. Board Can. 1976, 33, 1568-1573. interfere with a wide range of chemosensory behaviors. Last, (16) Beyers, D. W.; Farmer, M. S. Effects of copper on olfaction of two recent studies indicate that dissolved copper is also toxic Colorado pikeminnow. Environ. Toxicol. Chem. 2001, 20, 907- to fish lateral line neurons (35, 36) and thus may also disrupt 912. mechanosensory behaviors such as shoaling, prey capture, (17) Hansen, J. A.; Rose, J. D.; Jenkins, R. A.; Gerow, K. G.; Bergman, and predator evasion. For these reasons, non-point source H. L. Chinook salmon (Oncorhynchus tshawytscha) and rainbow stormwater runoff from roads has the potential to interfere trout (Oncorhynchus mykiss) exposed to copper: Neurophysi- ological and histological effects on the olfactory system. Environ. with a wide variety of behaviors in a diversity of fish species. Toxicol. Chem. 1999, 18, 1979-1991. (18) Julliard, A. K.; Saucier, D.; Astic, L. Time-course of apoptosis in Acknowledgments the olfactory epithelium of rainbow trout exposed to a low copper level. Tissue Cell 1996, 28, 367-377. This work was supported by the NOAA Coastal Storms (19) Moran, D. T.; Rowley, J. C.; Aiken, G. R.; Jafek, B. W. Ultra- Program, a Public Health Service grant (T32ES07060) from structural neurobiology of the olfactory mucosa of the brown the National Institute of Environmental Health Sciences to trout, Salmo trutta. Microsc. Res. Tech. 1992, 23,28-48. J.F.S., and an internship from the Oak Ridge Institute for (20) Sandahl, J. F.; Baldwin, D. H.; Jenkins, J. J.; Scholz, N. L. Odor- Science and Education to D.H.B. We thank Jana Labenia, evoked field potentials as indicators of sublethal neurotoxicity Kate Macneale, and Jenifer McIntyre for comments on the in juvenile coho salmon (Oncorynchus kisutch) exposed to copper, chlorpyrifos, or esfenvalerate. Can. J. Fish. Aquat. Sci. manuscript. 2004, 61, 404-413. (21) Baldwin, D. H.; Sandahl, J. F.; Labenia, J. S.; Scholz, N. L. Sublethal Supporting Information Available effects of copper on coho salmon: Impacts on nonoverlapping Measured behavioral and EOG responses of fish (Figure S1); receptor pathways in the peripheral olfactory nervous system. Environ. Toxicol. Chem. 2003, 22, 2266-2274. examples of the behavioral and EOG responses from four (22) Carreau, N. D.; Pyle, G. G. Effect of copper exposure during control fish and four fish exposed to 10 µg/L of copper (Figure embryonic development on chemosensory function of juvenile S1); and behavioral responses of a control fish and a fish fathead minnows (Pimephales promelas). Ecotoxicol. Environ. exposed to 10 µg/L of copper (Movie S1). This material is Saf. 2005, 61,1-6. available free of charge via the Internet at http://pubs.acs.org. (23) Hansen, J. A.; Marr, J. C. A.; Lipton, J.; Cacela, D.; Bergman, H. L. Differences in neurobehavioral responses of chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (Oncorhynchus Literature Cited mykiss) exposed to copper and cobalt: Behavioral avoidance. Environ. Toxicol. Chem. 1999, 18, 1972-1978. (1) Crosset, K. M.; Culliton, T. J.; Wiley, P. C.; Goodspeed, T. R. (24) Saucier, D.; Astic, L.; Rioux, P. The effects of early chronic Population trends along the coastal United States: 1980-2008; exposure to sublethal copper on the olfactory discrimination National Oceanic and Atmospheric Administration: Silver of rainbow trout, Oncorhynchus mykiss. Environ. Biol. Fish. 1991, Spring, MD, 2004. 30, 345-351. (2) Beach, D. Coastal sprawl: The effects of urban design on aquatic (25) Scholz, N. L.; Truelove, N. K.; French, B. 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VOL. 41, NO. 8, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 3003 lowing exposure to low copper level. Aquat. Toxicol. 2006, 76, line neuromasts in zebrafish (Danio rerio). Hear. Res. 2006, 213, 321-328. 1-10. (35) Linbo, A. O.; Stehr, C. M.; Incardona, J. P.; Scholz, N. L. Dissolved copper triggers cell death in the peripheral mechanosensory system of larval fish. Environ. Toxicol. Chem. 2006, 25, 597- Received for review September 25, 2006. Revised manuscript 603. received January 27, 2007. Accepted February 6, 2007. (36) Herna´ndez, P. P.; Moreno, V.; Olivari, F. A.; Allende, M. L. Sub- lethal concentrations of waterborne copper are toxic to lateral ES062287R

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