Environmental Toxicology and Chemistry, Vol. 24, No. 4, pp. 836±845, 2005 ᭧ 2005 SETAC Printed in the USA 0730-7268/05 $12.00 ϩ .00

ASSESSING TRACE-METAL EXPOSURE TO AMERICAN DIPPERS IN MOUNTAIN STREAMS OF SOUTHWESTERN BRITISH COLUMBIA, CANADA

CHRISTY A. MORRISSEY,² LEAH I. BENDELL-YOUNG,² and JOHN E. ELLIOTT*³ ²Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia V5A 1S6, Canada ³Canadian Wildlife Service, Environment Canada, 5421 Robertson Road, Delta, British Columbia V4K 3N2

(Received 1 March 2004; Accepted 8 September 2004)

AbstractÐTo develop a suitable biomonitor of metal pollution in watersheds, we examined trends in exposure to nine trace elements in the diet (benthic and ®sh), feathers (n ϭ 104), and feces (n ϭ 14) of an aquatic , the (Cinclus mexicanus), from the Chilliwack watershed in British Columbia, Canada. We hypothesized that key differences may exist in exposure to metals for resident dippers that occupy the main river year-round and altitudinal migrants that breed on higher elevation tributaries because of differences in prey metal levels between locations or possible differences in diet composition. Metals most commonly detected in dipper feather samples in decreasing order were Zn Ͼ Cu Ͼ Hg Ͼ Se Ͼ Pb Ͼ Mn Ͼ Cd Ͼ Al Ͼ As. Resident dipper feathers contained signi®cantly higher mean concentrations of mercury (0.64 ␮g/g dry wt), cadmium (0.19 ␮g/g dry wt), and copper (10.8 ␮g/g dry wt) relative to migrants. Mass balance models used to predict daily metal exposure for dippers with different diets and breeding locations within a watershed showed that variation in metal levels primarily was attributed to differences in the proportion of ®sh and invertebrates in the diet of residents and migrants. In comparing predicted metal exposure values to tolerable daily intakes (TDI), we found that most metals were below or within the range of TDI, except selenium, aluminum, and zinc. Other metals, such as cadmium, copper, and arsenic, were only of concern for dippers mainly feeding on insects; mercury was only of concern for dippers consuming high ®sh diets. The models were useful tools to demonstrate how shifts in diet and breeding location within a single watershed can result in changes in exposure that may be of toxicological signi®cance.

KeywordsÐMetals Feathers Feces American dipper Ecological risk assessment

INTRODUCTION model for monitoring metal pollution in mountain streams be- Trace metals are present in aquatic systems worldwide, cause they integrate contaminant sources from their aquatic largely from underlying substrates, natural erosion, volcanism, diet over time and space. and hydrological cycles. However, mining processes [1], urban Previous studies in the Chilliwack watershed of British Co- and agricultural runoff [2], industrial emissions [3], and de- lumbia revealed that American dippers have distinct altitudinal forestation [4] also can cause increased metal loads to water- patterns of migration, which include seasonal movement up- sheds. Although mountain streams appear remote from indus- stream and downstream within a watershed [9]. Resident and trialization and urbanization, many still contain signi®cant altitudinal migrants shared common wintering grounds on the concentrations of heavy metals from natural and anthropogenic river, but most migrants moved upstream onto higher elevation sources [5]. With concerns over environmental impacts of met- creeks in the spring while residents remained on the river to als to freshwater ecosystems, it is important to be able to breed. Eggs of resident dippers had higher levels of mercury monitor the degree of metal exposure to organisms occupying and chlorinated hydrocarbons relative to creek migrants as a mountain streams. result of higher cumulative downstream loadings and differ- The American dipper (Cinclus mexicanus) is a potentially ences in the proportion of ®sh and invertebrates in the diet useful biomonitor of stream pollution because it is a year-round [10]. Dipper diets consisted of 0 to 71% ®sh, with river res- resident of freshwater streams and has an exclusively aquatic idents consuming signi®cantly more ®sh (42%) compared to diet comprised of benthic macroinvertebrates, small ®sh, and creek migrants (22%) [10]. Therefore, we hypothesized that ®sh eggs. Many taxa have the ability to bioac- trace-metal concentrations in feathers and feces of resident and cumulate metals to high concentrations without inherent tox- migrant American dippers also may re¯ect the ' migratory icity to the host species [6]. Freshwater ®sh also can bioac- status or speci®c diet. cumulate organometallic compounds, particularly methylmer- Our main objective was to determine if any differences exist cury (MeHg) due to the high assimilation ef®ciency and the in metal exposure for resident dippers occupying the main river slow elimination rates of this compound [7]. Therefore, pred- and migrant dippers breeding on watershed tributaries using ators feeding on metal- contaminated biota, including the dip- feathers and feces as bioindicators. We further attempted to per, are at risk for elevated exposure from its aquatic diet. identify the major sources of metal contamination to resident Strom et al. [8] con®rmed adult and nestling American dippers and migrant dippers via their ®sh and invertebrate diet. This were exposed to lead through their invertebrate prey in a mine- permitted us to quantify the magnitude of exposure from the impacted river system. Therefore, dippers can be an effective major prey groups and potentially relate it to levels observed in feathers. Although some elements biologically are essential, * To whom correspondence may be addressed all are toxic at high enough concentrations, with some having ([email protected]). a very narrow window of essentiality and toxicity [11]. Thus,

836 Trace-metal exposure to American dippers Environ. Toxicol. Chem. 24, 2005 837

Fig. 1. Map of the study area: The Chilliwack River watershed located near the Canadian±U.S. border in southwestern British Columbia, Canada.

in the interest of using the dipper as a biomonitor, we also chinook salmon (ϳ20%) also were included. All samples were modeled the potential toxicological risks of metal exposure to collected during the dipper breeding season before the spring a dipper population with different migratory strategies and freshet over a one-week period in late April 2000 and repeated diets. again in 2001. Samples subsequently were washed three times with distilled deionized water to remove any surface contam- MATERIALS AND METHODS ination or stream water and then transferred and stored frozen Collection of samples in acid-rinsed glass vials until preparation for trace-metal anal- ysis. Samples were collected from the Chilliwack River water- Several breast feathers were removed from individual after- shed (49Њ10ЈN, 121Њ04ЉW), located in the Cascade Mountains hatch-year dippers at the time of capture and banding for metal in southwestern British Columbia, Canada (Fig. 1). The wa- analysis. Contour (body) feathers are known to have low var- tershed drains an area of 1,274 km2 with elevation ranges from iability and should provide a good measure of individual metal near sea level to over 2,000 m at several mountain peaks. levels across samples [12]. Individual dippers were sexed at Composite samples of benthic invertebrates and salmon fry the time of capture using wing chord measurements [13]. were collected at eight different sites spaced at 4- to 5-km Through the use of color banding, only birds of known mi- intervals along the main stem of the Chilliwack River. Ad- gratory status (river resident or creek migrant) were used for ditional composite samples of invertebrates were collected the metal analysis. We made the assumption that the majority from seven different tributaries in the watershed. Aquatic lar- of birds molted on their breeding site and that feather metal ϳ val invertebrates ( 1-g dry wt) were collected either by kick pro®les would be indicative of the contaminants accumulated sampling in the stream (disturbing the rocks directly upstream primarily at that site in the preceding year [10]. Each individual of a Surber sampler) or by turning over rocks by hand. The sample of seven to 10 feathers (average mass ϭ 13 Ϯ 4 mg) sample represented a mixture of insect taxa that dippers nat- was stored in polyethylene bags and refrigerated until analyses. urally would prey upon, including approximately equal pro- We used 104 adult feather samples for Hg analysis and 82 of portions of ephemeropteran, plecopteran, and tricopteran lar- these also were analyzed for additional multiple elements. Fe- vae in addition to a much smaller fraction by mass of cole- cal samples (n ϭ 14) were collected from nestlings (n ϭ 5) opteran and dipteran larvae. Up to 10 individual salmon fry opportunistically during banding of chicks at 12 to 14 d of (Oncorhynchus spp.) (age 0ϩ) that each weighed 100 to 200 age or by following adults (n ϭ 9) and collecting fresh feces mg fresh weight, were captured live from the Chilliwack River off rocks, with care taken to avoid contamination from the using a dip net and represented a composite sample of pre- substrate. Fecal samples were stored frozen in acid-washed dominantly coho and chum salmon fry (ϳ80%), but pink and plastic containers until analysis. 838 Environ. Toxicol. Chem. 24, 2005 C.A. Morrissey et al.

Sample preparation and metal analysis only reported as means of all samples. Pearson product mo- Methods for sample preparation and digestion were adapted ment correlation coef®cients (r) were used to test for corre- and modi®ed from Canadian Wildlife Service method MET- lations among metal concentrations in both feathers and feces. ௡ CHEM-AA-02 [14] and U.S. Environmental Protection Agen- Statistical tests were performed using JMP IN Version 4.0 cy method 200.3 [15]. Feathers were washed with pure ace- (SAS Institute, Cary NC, USA) and the signi®cance level was ␣ϭ tone, 1% Triton-X solution alternated with several rinses of set at 0.05. distilled deionized water to remove any external surface con- tamination. Samples were then air-dried for 48 h and ®nally Exposure models oven-dried for 12 h. Invertebrate, ®sh, and fecal samples were A mass balance approach was used to calculate daily metal freeze-dried for 24 to 48 h until constant weight was achieved. exposure to American dippers depending on migratory status Samples were then weighed accurately into acid-washed glass (river resident or creek migrant) and the relative contributions ¯asks to the nearest 1 mg. The digestion procedure involved of ®sh and invertebrates to the diet. Models incorporated geo- adding 5 ml of 70% ultrapure nitric acid (HNO3), slow heating metric mean metal concentrations detected in invertebrates and to reduce volume, adding an additional 2 ml HNO3 while heat- ®sh collected from the main river and tributaries of the Chil- ing, and ®nally adding 1 ml of 30% ultrapure hydrogen per- liwack watershed in addition to estimated daily intake of each Ͻ oxide (H2O2). All samples were reduced by heat to 1 ml, prey item using published annual average energy requirements diluted to 10 ml with distilled, deionized water, and then stored for dippers [16]. Given the importance of body mass in com- refrigerated in polypropylene vials until metal analysis. A min- paring daily exposure among species [17], we further corrected imum of two certi®ed reference materials (Dolt-2 and Tort-2; the daily exposure for average dipper body mass (55 g). We National Research Council Canada, Ottawa, ON) and two pro- assumed that the primary route of exposure would be through cedural blanks were digested simultaneously with every batch oral ingestion. Some metals may be taken up through the water of samples and analyzed for quality assurance. In addition, a directly by drinking, but this was not accounted for. Therefore, standard calibration curve, analytical blanks, and spiked sam- a conservative metal-exposure model for American dippers in ples were run with each analysis. the Chilliwack River watershed was calculated as follows: Metal analysis was performed using an inductively cou- ϫ ϩ ϫ pled plasma mass spectrophotometer (Levelton Engineering, ϭ (WffC ) (W iiC ) Emetal (1) Richmond, BC, Canada) for feathers and fecal samples or an BW inductively coupled plasma atomic emission spectrophotom- where E ϭ exposure to metal x (␮g/g body wt/d), W ϭ eter (Cavendish Analytical Laboratories, Vancouver, BC, metal f weight of ®sh eaten per day (g/d), C ϭ geometric mean con- Canada) for invertebrates and ®sh. More than 25 different f centration of metal in ®sh (␮g/g), W ϭ weight of invertebrates elements were obtained from these analyses, but we report i eaten per day (g/d), C ϭ geometric mean concentration of only the data for Hg, Cd, Pb, Se, Mn, Cu, Zn, Al, and As, i metal in invertebrates (␮g/g), BW ϭ body weight of dipper hereinafter referred to as metals. All metal concentrations are (mean ϭ 55 g). Weight of ®sh (W ) and invertebrates (W ) expressed in ␮g/g dry weight (ppm). Recoveries of reference f i consumed on a daily basis were calculated using the following materials were within 10% of the certi®ed values or were equations: recovery corrected if outside this range (invertebrates and ϭ ϩ ϫ ϫ ®sh from 2000 only). Wff[(P DEE) AE] ED f (2) ϭ ϩ ϫ ϫ Data analysis Wii[(P DEE) AE] ED i (3) Ϯ Both the arithmetic ( standard error) and geometric mean where P ϭ proportion of ®sh or invertebrates in the diet, DEE concentrations of metals detected in the diet, feathers, and ϭ average daily energy required by dippers (estimated ϳ48.04 feces were calculated and reported to facilitate comparison kcal/d) [16], AE ϭ assimilation ef®ciency correction factor for with other studies. In addition, we report the proportion of ®sh diet (85% or 1.15) or invertebrate diet (70% or 1.3), and samples detected for each metal as a measure of prevalence. ED ϭ energy density of juvenile salmon (5.7 kcal/g dry wt) In the case of prey Hg concentrations, where detection fre- [18] or aquatic invertebrates (4.8 kcal/g dry wt) [19]. These quency was low, we used a value of one-half the detection estimates for daily food ingestion averaged 11.8 g/d dry ϭ ␮ ϭ ␮ limit (invertebrates 0.005 g/g and ®sh 0.01 g/g) to weight, which closely matched the allometric equation of daily permit statistical analysis and to provide a conservative value food ingestion rate for (12.0 g/d dry wt) given by to use in the exposure models. Metal concentrations generally Nagy [20]. exhibited a nonnormal distribution (Shapiro-Wilk W test) and, Each metal-exposure model was compared to a tolerable therefore, were log-transformed to improve normality before daily intake (TDI) calculated using the Canadian Tissue Res- performing statistical comparisons. We used a two-way anal- idue Guidelines for the Protection of Wildlife Consumers of ysis of variance followed by a Tukey multiple comparison Aquatic Biota protocol [21]. The TDI is calculated from the procedure to compare the metal concentrations among river results of avian chronic toxicity tests in which the substance invertebrates, creek invertebrates, and ®sh by year. A three- was administered orally and sensitive endpoints were mea- way analysis of variance (generalized linear model) was used sured (Appendix 1). Tolerable daily intake is calculated using to analyze feathers for effects of migratory status: River res- the geometric mean of the no-observable-adverse-effect level ϭ ϭ ident (n 42) and creek migrant (n 40), collection year and the lowest-observable-adverse-effect level and dividing (1999, 2000, 2001), sex (male or female), and interaction by an uncertainty factor (typically 10±100) to account for dif- terms. Nonsigni®cant interaction terms were removed sequen- ferences in sensitivity between species. tially from the analysis and nondetectable samples were not used. Given the limited number of fecal samples (n ϭ 14), the (LOAEL ϫ NOAEL)0.5 TDI ϭ (4) power for statistical comparisons was weak and, therefore, is UF Trace-metal exposure to American dippers Environ. Toxicol. Chem. 24, 2005 839

Table 1. Summary of trace-metal concentrations and frequency of metal detection for aquatic invertebrates (n ϭ 30, except Hg: n ϭ 15) and salmon fry (®sh; n ϭ 9, except Hg: n ϭ 17) from the Chilliwack River watershed (BC, Canada). Shown are arithmetic means (␮g/g dry wt) Ϯ standard error with geometric means in parentheses. Data for 2000 and 2001 are combined. Geometric means with the same capital letters are not signi®cantly different using one-way analysis of variance and Tukey multiple comparison procedure (␣ϭ0.05)

River Creek Signi®cance Invertebrate Fish Metal invertebrates invertebrates Fish (p) % detected % detected

Hg NDab 0.018 Ϯ 0.004b 0.035 Ϯ 0.01b (0.005)A (0.011)A,B (0.022)B 0.002 20 47 Cd 4.58 Ϯ 0.40 3.66 Ϯ 0.40 1.37 Ϯ 0.19 (4.31)A (3.35)A (1.27)B Ͻ0.0001 100 100 Pb 0.67 Ϯ 0.12 0.55 Ϯ 0.15 0.42 Ϯ 0.10 (0.58)A (0.41)A (0.33)A NSc 70 100 Se 5.83 Ϯ 0.74 6.08 Ϯ 0.79 2.68 Ϯ 0.27 (5.55)A (5.14)A (2.58)B 0.006 100 100 Cu 33.29 Ϯ 1.96 26.43 Ϯ 2.10 9.05 Ϯ 1.23 (32.48)A (25.17)B (8.39)C Ͻ0.0001 100 100 Mn 129.5 Ϯ 29.7 107.9 Ϯ 17.4 8.56 Ϯ 2.13 (99.8)A (96.1)A (7.07)B Ͻ0.0001 100 100 Zn 228.6 Ϯ 17.1 203.3 Ϯ 18.9 87.76 Ϯ 7.8 (217.6)A (190.5)A (84.9)B Ͻ0.0001 100 100 Al 1,296.4 Ϯ 216.0 1,586.0 Ϯ 303.7 165.5 Ϯ 49.4 (1,040.3)A (1,275.8)A (119.9)B Ͻ0.0001 100 100 As 3.73 Ϯ 0.50 3.77 Ϯ 0.65 0.63 Ϯ 0.10 (3.09)A (3.01)A (0.56)B Ͻ0.0001 100 100

a ND ϭ no samples with detectable concentrations. b For Hg, a value of half the detection limit was used to permit statistical analyses (detection limit ϭ 0.01 ␮g/g for invertebrates and 0.02 ␮g/ g for ®sh). c NS ϭ not signi®cant (p Ͼ 0.05).

where TDI ϭ tolerable daily intake, LOAEL ϭ lowest-ob- river residents compared to the creek migrants (Table 2). Alu- served-adverse-effect level, NOAEL ϭ no-observed-adverse- minum (n ϭ 13) and As (n ϭ 1) were not detected frequently effect level, and UF ϭ uncertainty factor. The no-observable- in dipper feathers. Feathers of adult dippers were further an- adverse-effect-level and lowest-observable-adverse-effect lev- alyzed to determine the effects of migratory group, sex, and el for suitable avian toxicity tests were taken from the literature year on metal concentrations. Sex effected Mn ( p ϭ 0.002) and summarized by Sample et al. [17]. Our TDI estimates use and Zn ( p ϭ 0.03) concentrations with Cu being marginally the most-conservative uncertainty factor of 10 for all metals. insigni®cant ( p ϭ 0.06). In all cases, females tended to have The TDI value is in units of ␮g/g body weight/d for direct higher feather metal concentrations relative to males. The year comparison with the values in the exposure model for Amer- of collection was important for predicting Hg levels ( p Ͻ ican dippers. 0.0001) and Mn levels ( p ϭ 0.03). In general, levels were higher in 1999 for Hg and higher in 2001 for Mn. Migratory RESULTS status was only an important effect in predicting higher Hg Metals in diet items: Invertebrates and ®sh and Cd feather concentrations in resident feathers when cor- rected for the other variables. Invertebrate samples from the river and the tributaries gen- Mean metal concentrations for adult and nestling fecal sam- erally did not differ signi®cantly in metal concentrations (Table ples were not signi®cantly different and, therefore, were pooled 1). Copper was the only metal found to be signi®cantly higher and reported as a single value (Table 3). No differences existed in the river invertebrates relative to those collected from creeks in metal concentrations by sex or migratory status; however, (t ϭϪ2.45, p ϭ 0.02), although Cd, Pb, Mn, and Zn also 28 due to small sample sizes, statistical power was limited. Metals showed similar patterns to Cu. In all cases except for Hg and were detected in 100% of the fecal samples analyzed, with the Pb, ®sh had lower concentrations of metals than both the river exception of one low-weight mercury sample. For all metals and creek invertebrate samples (Table 1). For Hg, ®sh con- except Se, the fecal concentrations (geometric means) ex- centrations were almost four times higher and were detected ceeded those in the invertebrate and ®sh prey items. more frequently than invertebrates. For Pb, there was no dif- Few metals in dipper feathers showed signi®cant positive ference in residue levels between ®sh and invertebrate samples correlations; however, all were weak (r Յ 0.35) and bordered but lead was detected at a higher frequency in ®sh relative to signi®cance. Stronger correlations were found between several invertebrates. Collection year had no effect for the majority metals in the feces, including Mn with Al (r ϭ 0.80, p ϭ of metals detected in invertebrates and ®sh. Only Se, Zn, and 0.0007) as well as for Hg and Se (r ϭ 0.73, p ϭ 0.005). Other As were signi®cantly lower (p Ͻ 0.0001) for invertebrates correlations in fecal samples included As and Al (r ϭ 0.70, p collected in 2000 relative to 2001. ϭ 0.006), Hg and Zn (r ϭ 0.66, p ϭ 0.01), Mn and As (r ϭ ϭ ϭ ϭ Metals in feathers and feces 0.66, p 0.01), and Se with Cd (r 0.56, p 0.04). Only Hg and Zn were signi®cantly correlated in both the feathers Metals most commonly detected in dipper feather samples and feces. in decreasing order were Zn Ͼ Cu Ͼ Hg Ͼ Se Ͼ Pb Ͼ Mn Ͼ Cd Ͼ Al Ͼ As. Migratory status (river resident or creek Exposure assessment migrant) was signi®cant in predicting higher feather Hg (0.64 In modeling the degree of daily metal exposure to dippers ␮g/g), Cd (0.19 ␮g/g), and Cu (10.8 ␮g/g) concentrations in in the Chilliwack watershed, two general trends emerged. First, 840 Environ. Toxicol. Chem. 24, 2005 C.A. Morrissey et al.

Table 2. Summary of mean trace-metal concentrations (only in detectable samples) and frequency of metal detection in adult feathers of resident and migrant American dippers from the Chilliwack River watershed (BC, Canada), 1999 to 2001 (Hg: n ϭ 104, other metals: n ϭ 82). Shown are arithmetic means (␮g/g dry wt) Ϯ standard error with geometric means in parentheses

Signi®cance Metal All birds % Detected River resident Creek migrant (p)

Hg 0.69 Ϯ 0.05 97 0.79 Ϯ 0.06 0.58 Ϯ 0.06 0.05 (0.56) (0.64) (0.50) Cd 0.18 Ϯ 0.03 49 0.25 Ϯ 0.04 0.13 Ϯ 0.03 0.01 (0.15) (0.19) (0.12) Pb 0.97 Ϯ 0.15 88 1.06 Ϯ 0.23 0.88 Ϯ 0.18 NSa (0.58) (0.57) (0.59) Se 6.03 Ϯ 0.25 92 6.04 Ϯ 0.34 6.01 Ϯ 0.37 NS (5.68) (5.62) (5.75) Cu 12.12 Ϯ 1.47 98 14.45 Ϯ 2.03 9.68 Ϯ 2.08 0.04 (8.92) (10.8) (7.29) Mn 1.21 Ϯ 0.19 90 1.40 Ϯ 0.27 1.03 Ϯ 0.26 NS (0.66) (0.70) (0.63) Zn 131.8 Ϯ 2.5 100 130.9 Ϯ 2.9 132.7 Ϯ 4.2 NS (130.1) (129.7) (130.6) Al 54.9 Ϯ 20.3 16 41.8 Ϯ 17.6 66.1 Ϯ 35.5 NS (22.4) (17.7) (27.4) As 12.28 0.01 12.28 NDb NS a NS ϭ not signi®cant (p Ͼ 0.05). b ND ϭ no samples with detectable concentrations. for residents breeding on the river, the predicted exposure to daily metal exposure clearly exceeded TDIs for Se, Al, and Cd, Cu, Pb, and Zn generally was higher than for migrant birds Zn, while both Pb and Mn were well below TDI guidelines. breeding on tributaries (Table 4). For Se, Al, As, Mn, and Hg, Some models only exceeded the guideline depending on the there were very little or no differences between river and creek individual's diet or location or both (e.g., Hg, Cd, Cu, and locations, or creek values were slightly higher. Second, the As). effect of diet on metal concentrations generally exceeded that of migratory status (breeding location). For all metals except DISCUSSION Hg, increasing proportions of ®sh relative to invertebrates in Because metals frequently are excreted through the feces the diet resulted in a decrease in metal exposure (Table 4). or by deposition in the uropygial gland, salt gland, eggs, and Because Hg was more prevalent in salmon fry, greater con- molting feathers, measuring metal levels in excretory tissues sumption of ®sh resulted in higher predicted Hg exposure. is now a common tool to examine environmental pollution that Each exposure model for the metal of interest was compared is noninjurious and noninvasive to birds [12,22,23]. Through to a TDI that represents a safe daily intake level for this species analysis of prey items, feathers, and feces, we found that Amer- (based on body wt and daily dose) that should not cause any ican dippers in the Chilliwack River watershed were exposed sublethal effects to populations [21] (Appendix). Our predicted to a suite of trace metals including Se, Cu, Zn, Al, Hg, and Cd. Although this watershed is not impacted by mining or other discharge point sources, several elements may be mo- Table 3. Summary of arithmetic (geometric) mean metal concentrations (␮g/g dry wt) Ϯ standard error and frequency of metal bilized as a result of natural hydrological processes, soil ero- detection in adult and nestling American dipper fecal samples (n ϭ sion from deforestation, or long-range transport and atmo- 14) collected from the Chilliwack River watershed (BC, Canada) spheric deposition. Metal pro®les in dipper feathers generally in 2001 re¯ected predicted daily exposure for river residents and creek Fecal metal migrants. However, the effect of diet appears to exceed that Metal concentration % Detected of breeding location for most trace metals because concentra- tions in prey were similar between the main river and tribu- Hg 0.036 Ϯ 0.005 93 taries (except Cu). (0.031) Ϯ Cd 5.97 1.04 100 Signi®cance of feather and fecal metal levels with respect (4.89) Pb 3.65 Ϯ 1.03 100 to diet (2.50) Several studies have determined that Hg, Pb, and Cd among Se 4.83 Ϯ 0.43 100 (4.55) other metals are deposited signi®cantly in the feather during Cu 53.28 Ϯ 4.54 100 the period of feather growth, producing metal pro®les that (50.58) remain inert and stable [24±26]. Metal concentrations in the Mn 311.53 Ϯ 47.28 100 feathers re¯ect levels in at the time of feather growth, (259.42) Zn 396.10 Ϯ 42.09 100 either from current dietary sources or from mobilization of (370.68) metals from internal organs [12]. Assuming dippers are molt- Al 2,780.5 Ϯ 388.30 100 ing and breeding in a consistent location among years, we (2,312.07) anticipated that metal levels in feathers would re¯ect contam- Ϯ As 5.05 1.03 100 inant concentrations in prey at the breeding site either on a (4.11) river or a higher elevation tributary. Morrissey et al. [10] found Trace-metal exposure to American dippers Environ. Toxicol. Chem. 24, 2005 841

Table 4. Predicted daily exposure to metals (␮g/g body wt/d) for resident and migrant American dippers in the Chilliwack watershed with diets of increasing proportions of ®sh relative to invertebrates (i.e., 25% ®sh and 75% invertebrates). Model uses mean metal concentrations for invertebrates and ®sh collected in 2000 and 2001, food ingestion rate, and mean body mass of American dippers (see Methods section for details). Tolerable daily intakes ([TDI] ␮g/g body wt/d) are shown as guidelines for safe levels for protection of American dippers

Predicted daily exposure (␮g/g body wt/d) Metal River residents Creek migrants TDI Diet (% ®sh:% (␮g/g body invertebrates 0:100 25:75 50:50 75:25 0:100 25:75 50:50 75:25 wt/d)

Hg 0.0012 0.0019 0.0026 0.0033 0.0026 0.0029 0.0033 0.0036 0.002 Cd 1.14 0.85 0.57 0.29 0.79 0.60 0.40 0.20 0.54 Pb 0.14 0.12 0.10 0.08 0.10 0.09 0.08 0.07 0.64 Se 1.22 1.03 0.84 0.65 1.31 1.10 0.88 0.67 0.16 Cu 7.7 6.1 4.6 3.0 6.0 4.8 3.7 2.6 5.39 Mn 23.6 18.0 12.4 6.8 22.7 17.4 12.0 6.6 235.9 Zn 51.5 42.4 33.2 24.1 45.1 37.5 30.0 22.5 4.36 Al 246.0 192.7 139.4 86.2 301.6 234.4 167.2 100.1 26.0 As 0.73 0.58 0.43 0.28 0.71 0.57 0.42 0.27 0.62

that stable isotope ratios in American dippers feathers gen- locations, resident dippers consuming more ®sh generally will erally followed the same pattern as isotope ratios in blood and be at greater risk for exposure to Hg, though migrants mainly prey. However, some individuals had alternate isotopic sig- feeding on insects will be expected to have greater exposure natures, suggesting a movement away from the breeding site to other metals. or a possible diet switch during molt. For Hg, Cd, and Cu, Although feathers have been used since the 1960s for in- migratory status was signi®cant in predicting feather metal dicating metal exposure in birds, more recent studies show pro®les with river residents having higher concentrations than fecal matter also can be a sensitive indicator of metal contam- creek migrants. This primarily can be attributed to differences ination [23,30]. Metals were detectable in almost all fecal sam- in the diet (proportion of ®sh and invertebrates) or differences ples collected at concentrations exceeding those of the prey in prey metal levels between the main river and tributaries. items. Spahn and Sherry [31] similarly found that Little Blue Other metals, including Se, Mn, Zn, and Al, also were found heron (Egretta caerulea) fecal samples contained higher con- to differ between prey types but did not re¯ect dipper migra- centrations of metals than their prey, suggesting the feces tory status. The lack of formal statistical signi®cance may have largely represent the unabsorbed remnants of multiple food been caused by high sample variability as a result of dipper items. Because metals found in feces readily are detected, often postbreeding movements, possible changes in diet during the at higher concentrations than the diet items, they can provide molting period, or the inclusion of ®rst-year birds with un- a nondestructive and quanti®able means of monitoring food- known natal and molting origin. chain contamination from trace metals. Copper was the only element that was signi®cantly higher Correlations among metal levels for the feathers and feces in river invertebrates compared to creek invertebrates, but Cd showed only a small number of signi®cant relationships and also showed a similar trend. Therefore, the effect of breeding only Hg was correlated signi®cantly with Zn in both feathers location was likely an important predictor of feather pro®les and feces, indicating no clear patterns with respect to metal of Cu and Cd. For Hg, we did not detect any differences in excretion mechanisms. However, key correlations among met- invertebrate concentrations between breeding locations, im- als in fecal samples may be important for understanding the plying that differences in feather Hg between residents and kinetics and toxicity of metals in dippers. Aluminum and Mn migrants may be more strongly in¯uenced by diet. Species that were correlated positively, suggesting similar metal availabil- eat prey from different levels in the food chain have contam- ity or metabolism. Both elements typically are derived from inant levels that are in¯uenced strongly by diet [27]. Food natural mineral deposits. They were excreted in high concen- chain differences among marine birds were important in ex- trations in feces, indicating they are either abundant in this plaining variation in metal concentrations in eggs [28] and system or not readily bioavailable to the birds. Mercury also tissues [29]. Invertebrates from the Chilliwack watershed typ- was correlated signi®cantly with Se in feces. Inorganic Hg ically had higher concentrations of all metals, placing insec- often is bound to Se in liver and other tissues, and Hg and Se tivorous dippers at greater risk to increased metal intake. In can interact to counter the toxicity of each other [32]. Given contrast to most metals, Hg is of greater importance to aquatic the high levels of Se detected in invertebrate and dipper sam- birds on primarily ®sh diets due to the prevalence of the more ples and the correlation with Hg in feces, these two elements toxic methylmercury in ®sh tissue [7]. Juvenile salmon from may be interacting to produce ameliorative effects to dippers. the Chilliwack River were found to have almost four times higher Hg concentrations and a higher frequency of detection Tolerable daily intakes and toxicity concerns than invertebrates. Furthermore, the highest Hg levels detected Many populations may be subject to the effects of in dipper feathers (2.74 ␮g/g and 2.09 ␮g/g) were from a chronic exposure to low-level inorganic toxicants, resulting in resident pair at the ®sh hatchery. This is consistent with our reproductive dysfunction, increased susceptibility to disease previous work, which showed that resident dippers consume or other stresses, and changes in normal behavior [33]. How- higher proportions of ®sh and, subsequently, had higher Hg ever, it is dif®cult to determine critical threshold levels relevant levels in eggs compared to migrants [10]. Therefore, where to all species. Relatively few controlled laboratory studies ex- prey contaminant residues are not different among breeding amine the effects of toxic metals on passerines and, because 842 Environ. Toxicol. Chem. 24, 2005 C.A. Morrissey et al. dippers belong to the unique family Cinclidae, the world's were they at levels known to cause toxicity in birds [47,48]. only truly aquatic passerines, direct comparisons of toxicity Other metals, including Cd, Cu, and Hg, were found to exceed tests from other passerine species may be inappropriate. For tolerable daily intakes depending on the diet, breeding loca- these reasons, we selected the approach of determining a tol- tion, or both. Cadmium and Cu are known to bioaccumulate erable daily intake value, which included a marginal uncer- in target organs (kidneys) in excess of the levels in the food tainty factor. supply [33,49]. For example, with continued long-term ex- The only metals to which dippers on any diet clearly ex- posure to low-level dietary Cd exposure, there is a persistent ceeded the TDI guidelines were Zn, Se, and Al. Those elements increase in renal Cd with very little excretion that can lead to are either homeostatically controlled or are essential elements renal tubular necrosis at critical concentrations of 100 to 200 where the range of essentiality and toxicity is not well un- ␮g/g [33,50]. Other sublethal effects on immature and adult derstood. Evidence of Zn toxicity to wild birds is limited pri- birds may be apparent at lower concentrations. Therefore, marily because Zn is regulated internally even when birds are monitoring bioaccumulative elements, such as Cd and Cu, by exposed to high levels of contamination [12,34]. However, insectivorous birds remains important. mortality and reproductive effects from Se (particularly in the In general, Hg levels detected in prey and feathers were form selenomethionine) have been documented, especially for below reported toxic thresholds for birds. Heinz [51] found aquatic birds in areas receiving agricultural drainage [35,36]. that dietary levels of 0.5 ␮g/g dry weight MeHg were signif- Food-chain organisms, such as benthic invertebrates and ®sh, icant to cause female mallards to lay fewer eggs and produce can accumulate high concentrations of Se without toxicity to fewer young in addition to behavioral changes in ducklings. the host; however, a dietary toxicity threshold for ®sh and Barr [52] detected reductions in egg-laying and nest site and wildlife is recommended at 3 ␮g/g dry weight [37]. Although territory ®delity in Common loons (Gavia immer) on diets we do not have any information about the concentrations of containing 0.2 to 0.3 ␮g/g wet weight. Although our inver- the more toxic organic form of Se (selenomethionine) in dipper tebrate and ®sh concentrations were well below those values, prey, all the invertebrate samples and many ®sh samples col- predicted daily exposure was considerably greater for birds lected from the Chilliwack watershed exceeded the 3-␮g/g consuming high ®sh diets, thus exceeding the TDI. Consistent guideline. Harding and Paton [38] recorded no reproductive with the model, resident dippers had higher feather Hg con- impairment with invertebrate Se concentrations of 4.2 ␮g/g centrations, but within the range of 1 to 5 ppm, considered as wet weight at a coal mine site and feather Se concentrations background exposure [34]. Henny et al. [53] found American almost identical to our study at exposed (pooled sample: 6.5 dippers at a mine-contaminated site in Oregon, USA, repro- ␮g/g dry wt) and reference streams (pooled sample: 6.3 ␮g/g duced normally even with elevated total Hg concentrations in dry wt). In our study, feather concentrations were not different dipper invertebrate prey at 0.2 ␮g/g dry weight and in feathers among migratory groups, but daily Se exposure was six times at 1.2 ␮g/g dry weight. However, the authors did not consider higher than the TDI levels for birds on exclusively invertebrate potential exposure from ®sh at sites further downstream. Given diets, indicating migrant dippers may be at a higher risk to that our exposure model and TDIs used in this study account potential toxic effects from selenium. for interspeci®c and intersite variability, TDI guidelines may Aluminum also has been reported to in¯uence reproduction be more appropriate than direct comparison of critical diet of insectivorous passerines breeding in acid-sensitive envi- concentrations for other species or locations. ronments, particularly if Ca and P are limiting [39±41]. Several orders of aquatic invertebrates, including chironomids, cad- AcknowledgementÐWe wish to thank I. Pollet, R. McKibbin, and dis¯ies, stone¯ies, and may¯ies, have exhibited high Al con- several volunteers who conducted the ®eldwork and J. Morrissey who centrations of 0.1 to 0.3% body weight (dry wt) [42]. Inver- assisted with the sample preparation and analysis. Levelton Engi- tebrates sampled in 2001 from the Chilliwack watershed had neering, Cavendish, and CanTest Laboratories conducted the metal elevated Al levels in the range of 0.05 to 0.43% (mean ϭ analyses; M. Saffari, R. Leary, and B. Massuto provided advice and assistance on sample preparation and metal analysis; F. Cooke, R. 0.12% dry wt). Aluminum is of particular concern in acid- Butler, and R. Ydenberg provided additional guidance throughout the sensitive regions, especially in ecosystems with exposed gran- study and on earlier drafts of the manuscript. This project primarily ite or other calcium-poor substrates, which are most severely was funded by the Georgia Basin Ecosystem Initiative through En- affected by acidi®cation [43]. 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