Effects of acid mine drainage from an abandoned copper mine, Britannia Mines, Howe Sound, British Columbia, Canada, on transplanted blue mussels (Mytilus edulis)

Marine Environmental Research 51 (2001) 265±288 www.elsevier.com/locate/marenvrev

E€ects of acid mine drainage from an abandoned copper mine, Britannia Mines, Howe Sound, British Columbia, Canada, on transplanted blue mussels (Mytilus edulis) J.A. Grout *, C.D. Levings Fisheries and Oceans Canada, Science Branch, West Vancouver Laboratory, 4160 Marine Drive, West Vancouver, BC, Canada V7V 1N6 Received 20 May 1999; received in revised form 10 February 2000; accepted 19 March 2000

Abstract Juvenile mussels (Mytilus edulis) were transplanted to Howe Sound, British Columbia, Canada, along an apparent pollution gradient of acid mine drainage (AMD) from an abandoned copper (Cu) mine. Cages containing 75 mussels each were placed at a total of 15 stations and were exposed to concentrations of dissolved Cu in surface waters ranging from 5 to 1009 mg/l for a period of 41 days. Mussels located at stations closer to the source of AMD at the mouth of Britannia Creek bioaccumulated higher concentrations of Cu and zinc (Zn) in their tissues. Mussel growth was adversely a€ected by Cu tissue concentrations above 20 mg/g dry wt., while declines in survival and condition index occurred in mussels that bioaccumulated greater than 40 mg/g dry wt. Cu. Tissue Zn concentrations (117±192 mg/g dry wt.) were likely not high enough to have a direct impact on mussel health. Reduced survival of transplanted mussels was supported by an absence of natural mussels in contaminated areas. Phytoplankton was also severely reduced in areas contaminated by mine waters. Based on the weight of evidence, AMD from the Britannia mine had a deleterious impact on mussel survival in a zone extending at least 2.1 km to the north and 1.7 km to the south of Britannia Creek on the east shore of Howe Sound. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Mytilus edulis; Transplant; Bioaccumulation; Metals; Cu; Zn; Mining; Acid mine drainage; Caged mussels; Howe Sound; British Columbia; Canada

* Corresponding author. Fisheries and Oceans Canada, Science Branch, c/o School of Resource Management, Simon Fraser University, Burnaby, BC, Canada V5A 1S6. Tel.: +1-604-666-2373; fax: +1604-666-1995. E-mail address: [email protected] mpo.gc.ca (J.A. Grout). 0141-1136/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(00)00104-5

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1. Introduction From 1905 until its closure in 1974, the Britannia copper (Cu) mine operated at Britannia Beach on the shore of Howe Sound in southwestern British Columbia, Canada (Fig. 1). During production, the mine produced 52.7 million tons of ore containing Cu, zinc (Zn) and small amounts of other metals. A by-product of the mining operations has been continued generation of acid mine drainage (AMD) from derelict mine workings. There are two point sources of AMD into Howe Sound from the Britannia Mine: the 2200 foot and 4100 foot portals. AMD from the 2200 foot portal (a mine tunnel 2200 feet from the top of the mountain, 700 m above sea level) drains into Britannia Creek which ¯ows into Howe Sound (Goyette & Ferguson, 1985; Price, Schwab & Hutt, 1995). AMD in Britannia Creek is characterized by low pH and high levels of dissolved metals, especially Cu and Zn (Dunn, Percival, Hall & Murdoch, 1992; Goyette & Ferguson, 1985). AMD from the 4100 foot portal (67 m above sea level) is carried through a pipeline and released directly into Howe Sound through a submerged outfall at 30 m in depth, about 50 m o€shore and directly in front of the mouth of Britannia Creek (Goyette & Ferguson, 1985; Price et al., 1995). Because discharges from the 4100 foot portal are thought to remain trapped below 10 m depth at most times of the year (Chretien, 1997) the most likely source of pollution of the near-shore zone is AMD from Britannia Creek. Howe Sound is a coastal fjord that has the oceanographic characteristics of a large estuary. The surface circulation pattern in Howe Sound is driven by a major out¯ow

Fig. 1. Location of Britannia Creek and stations where mussel cages were transplanted in Howe Sound, British Columbia, Canada.

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of fresh, turbid water of glacial origin from the Squamish River (mean annual discharge: 242 m3 sÿ1, Stockner, Cli€ & Buchanan, 1977) at the north end of Howe Sound (Fig. 2). The out¯ow of fresh water into the marine environment produces a thin layer (<5 m depth) of fresh, cold surface water overlying warmer, higher salinity waters at depth. This salinity pro®le gives rise to a pronounced pycnocline that inhibits vertical mixing and isolates the surface layer. The low salinity surface layer moves south from the Squamish River through Howe Sound and the amount of suspended sediment and fresh water decrease with distance from the river mouth (Syvitski & Murray, 1981). Localized salinity reductions typically occur throughout Howe Sound near estuaries at the mouths of creeks, including Britannia Creek. In general, metal concentrations in surface waters decrease seawards in an estuary due to increasing dilution of the metal source with more saline water and other removal processes (Windom et al., 1991). Several studies have shown that increasing

Fig. 2. The current patterns of surface waters in Howe Sound (modi®ed from Thomson, 1981).

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salinity facilitates removal of Cu from estuaries of low pH, acid mine streams (Featherstone & O'Grady, 1997; Foster, Hunt & Morris, 1978; Johnson & Thornton, 1987). Recent observations in Howe Sound indicate that Cu concentrations are also high in low salinity surface waters and uniformly low in deeper waters (Chretien, 1997). High surface water concentrations of Cu and Zn in Howe Sound near the mouth of Britannia Creek result from high metal concentrations in the Britannia Creek discharge (Chretien, 1997; Dunn et al., 1992). Thus, marine organisms found in inter-tidal areas near Britannia Creek may be exposed to AMD-contaminated waters. Elevated concentrations of Cu and Zn can be toxic to marine organisms (Eisler, 1997). High concentrations of these metals have been measured in surface water, inter-tidal biota, and beach sediments near Britannia Beach (Dunn et al., 1992; Harding, 1992). Cu and Zn are generally acknowledged to be more-than-additive in toxicity to a wide variety of aquatic organisms (Eisler, 1997). However, a causal relationship between AMD exposure and biological e€ects in marine organisms has not been quantitatively investigated in Howe Sound. The Mytilus sp. complex are commonly used as biological indicators for monitoring the impacts of human activities on the marine environment, because mussels are sedentary and thus representative of the area where they are located, have a high concentration factor for many metals in the water column, and tolerate a wide range of salinities characteristic of estuarine environments. They have also been widely used and this allows a comparison of results in the literature. Natural populations of Mytilus edulis around the world have been used in Mussel Watch studies to track large-scale trends in environmental contamination (e.g. Goldberg, Koide, Hodge, Flegal & Martin, 1983; Lauenstein, Robertson & O'Connor, 1990). In addition, caged mussels have increasingly been used in transplant studies to de®ne areas of metal pollution (Peven, Uhler & Querzoli, 1996; Salazar & Salazar, 1991, 1995). However, few ®eld studies have attempted to link contaminant exposure in water and contaminant concentration in mussel tissues to mussel health indicators, such as survival, growth or condition (notable exceptions: Salazar & Salazar, 1991, 1995; Widdows & Johnson, 1988). As part of a general ecosystem assessment of the impacts of AMD from the abandoned Britannia mine on marine organisms, we deployed M. edulis in cages along the suspected pollution gradient in Howe Sound. Our goals were: (1) to quantify the relationship between bioaccumulation of Cu and Zn and mussel health indicators (e.g. survival, growth and condition index); and (2) to de®ne the zone of impact of AMD on mussel health. 2. Materials and methods 2.1. Study area Juvenile M. edulis were transplanted from an aquaculture operation located at Quadra Island, BC, to cages at 15 stations in Howe Sound. Quadra Island is about

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150 km northwest of Howe Sound in an unpolluted area. Stations were located on an AMD pollution gradient centered on the source at the mouth of Britannia Creek (Fig. 1). Stations 1±12 were placed on the eastern side of Howe Sound to de®ne the extent of the pollution gradient along the inter-tidal area. Stations 13±15 were placed on the western side of Howe Sound in areas not contaminated by heavy metals to control for the north to south gradient in environmental factors (e.g. salinity, suspended sediments and chlorophyll a) a€ected by the Squamish River. 2.2. Experimental design A total of 15 stations were monitored during the test. At each station, one mussel cage, containing 75 mussels, was attached 1 m below a ¯oat that was anchored in approximately 10 m of water; generally less than 50 m o€shore. Mussels were exposed to ambient conditions in Howe Sound beginning on 28 April and were removed on 8 June 1998, a total of 41 days. The experiment was ended after signs of high mortality in test mussels raised concerns that not enough living mussel tissue would be available for analysis of metals. The timing of the study was chosen to coincide with high mussel growth potential associated with spring plankton blooms (Stockner et al., 1977) and high discharges of metals in water from Britannia Creek (Robert McCandless, Environment Canada, North Vancouver, BC, personal communication). The Squamish River also began a spring freshet (high ¯ows) sometime during the ®rst 2 weeks of the study. Juvenile M. edulis were used to maximize potential growth and metal accumulation (Davenport & Redpath, 1984). Test mussels were chosen from a narrow range in shell lengths (21.0±31.4 mm; mean: 26.2 mm) to minimize initial di€erences in size-dependent growth and bioaccumulation (Peven et al., 1996; Salazar & Salazar, 1995). Initial whole weights including shells ranged from 0.69 to 3.26 g (mean: 1.71 g). The mussels were less than 1 year old and not expected to spawn until their second year of life (Ronnie Rombo, Kanish Bay Shell®sh, Quadra Island, BC, personal communication). Mussels were placed in mesh tubes and divided into individual compartments to allow individuals to be followed through the experiment. Mesh tubes were hung inside barrel-shaped cages constructed of PVC mesh for deployment in the ®eld. We used the same protocols as Salazar and Salazar (1995) for the mussel selection, measurement, and deployment. Three additional replicates, each containing the tissues from 75 mussels (mean length: 26.8 mm; weight: 1.75 g), were set aside to obtain data on concentrations of metals in the tissues before they were transplanted to Howe Sound. At the end of the test, mussels with intact tissues that had closed shells or shells that closed upon physical stimulation were considered survivors. Shell lengths and whole wet weights were recorded for each surviving mussel. After shucking each surviving mussel, shell and tissue wet weights were recorded. Because of small tissue weights, all tissues were pooled together for single measurements of metal concentrations at each station. Shucking equipment was plastic or stainless steel and was acid washed (5% nitric acid) after each station to avoid cross-contamination of tissue samples. Mussel tissue was analyzed for concentrations of Cu and Zn using

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standard procedures (Puget Sound Estuary Program, 1997). Instrumental analysis of metal concentrations in mussels tissues was done by inductively coupled plasmaoptical emission spectrophotometry (ASL Laboratories, Vancouver, BC, US EPA Method 6010B 1996). Results are reported on a dry weight basis to allow comparison across stations on an equal basis. 2.3. Surface water parameters At each station, surface water parameters were measured at the start, end and on a biweekly basis during the test period. Temperature, salinity, pH and turbidity were determined using conventional procedures (Grout, Levings, Piercey & Mossop, 1999). Due to a lab error, turbidity measurements were probably overestimated but are included to allow comparative analysis between stations since all samples were handled with the same technique. Water samples were collected at each station to measure chlorophyll a and dissolved metal concentrations in the lab. Chlorophyll a samples were ®ltered in the lab with 0.45 mm Millipore polycarbonate membrane ®lters. Chlorophyll a concentration on each ®lter was determined using a ¯uorometer and standard methods (Parsons, Maita & Lalli, 1984). Analytical methods for metal analyses followed procedures described in Environment Canada (1998). Water samples for metal analyses were ®ltered in the ®eld through 0.45-mm ®lters, collected in acid-washed polyethylene bottles and preserved with 1 ml of analytical grade concentrated nitric acid. Water samples were analyzed for Cu and Zn using inductively coupled argon plasma atomic emission spectrometry (ICP-AES). High concentrations of salts in the marine and estuarine water samples created matrix interference and necessitated dilution of the samples. As a result, Cu and Zn fell below detection limits of ICP-AES (e.g. Cu <500 mg/l and Zn <200 mg/l) in many instances. Due to cost constraints, samples were re-analyzed for Cu but not Zn by graphite furnace atomic absorption spectrometry (GF-AAS) which had lower detection limits (e.g. Cu <5 mg/l). 2.4. Data analysis The following calculations were performed on the biological data: 1. Weekly growth rates were calculated for mussels that survived the duration of the test by dividing the di€erence between the initial and end shell lengths or whole wet weights by the duration of the test (5.86 weeks). 2. Condition index was calculated for each surviving mussel using the ratio of the wet weights of whole tissues to end-of-test shell weights (Burbidge, Macey, Webb & Talbot, 1994; Pridmore, Roper & Hewitt, 1990). 3. Mean values for variables are reported with 1 standard error where appropriate in text and ®gures. 4. To describe the expected relationship between surface water concentrations of Cu (CuW) and concentrations of Cu in mussel tissues (CuT) we ®t a MichaelisMenten equation to the data:

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CuT ˆ

c…Cuw ÿ e† : ‰…d ÿ e† ‡ …Cuw ÿ e†Š

271

…1†

In this equation, c is the asymptotic Cu concentration in mussel tissues, d is the surface water concentration at 50% of the maximum Cu concentration in mussel tissues (e.g. CuS at 0.5c), and e is the x-intercept. This equation is commonly used to describe the regulation of biochemical reactions by enzymes and may be useful for describing the uptake of metals by mussels which are known to regulate accumulation of metals in their body tissues (Phillips, 1977). Non-linear regression was used to ®t this model. 5. In order to describe the relationship between concentrations of Cu or Zn in mussel tissues (CuT or ZnT) and mussel health indicators (H) (e.g. survival, growth, and condition index), we compared the ®t of a log-linear model: H ˆ a ÿ b  ln…CuT †;

…2†

where a is the y-intercept and b is the slope of the relationship, to a threshold (or a step function) model:  Y2 ; if CuT 5k ; …3† Hˆ Y1 ; if CuT < k where k is a threshold metal concentration in mussel tissues, Y1 is the mean mussel health below the threshold and Y2 is the mean mussel health above the threshold. Linear regression was used to ®t Eq. (2) and non-linear regression was used to ®t Eq. (3) to the data. We then compared model ®ts using R2 to assess whether mussel health indicators responded gradually over a wide range of Cu and Zn concentrations in mussel tissues (linear model) or abruptly over a much narrower range of metal concentrations (threshold model). 6. Measurement errors associated with measuring shell lengths may have resulted in growth rates being biased on average by ÿ0.01 mm/week. Re-measurements of shell lengths for quality control purposes indicated that shell lengths that were re-measured tended to be longer than the original measurements. These errors can be explained by the diculty with precisely measuring the longest axis on a curved, irregularly shaped shell where only one shell axis is the longest. If calipers are not placed exactly along this axis then measurements will underestimate length. Measurement errors in whole weights of mussels were negligible. Therefore, we considered negative growth rates in terms of weight to be indicative of actual decreases in mussel weight over time. 3. Results Mussels from 13 of the 15 stations were recovered from the ®eld. Cages from stations 12 and 14 were not recovered because they were lost due to strong currents,

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debris or unknown causes. Mussels from station 13 were excluded from the analysis because no mussels survived the duration of the test even though physical conditions were comparable to other stations and Cu and Zn concentrations in the water were below detection limits suggesting an unmeasured factor may have been responsible. Although biological data for mussels at these three stations were not available, surface water measurements made during the test period are included for comparison to the other stations and to provide additional insight into the causes for observed biological e€ects. 3.1. Surface water parameters Low pH and high dissolved Cu concentrations in surface waters at station 7 indicate AMD was entering surface waters of Howe Sound from Britannia Creek (Fig. 3). Surface waters were more acidic at station 7 (mean pH: 6.4) relative to all other stations (mean pH >7.2). Dissolved Cu concentrations were also up to 100-fold higher at station 7 and up to 10-fold higher at adjacent stations 8 and 9 than concentrations at other stations throughout the study period (Table 1, Fig. 3). Cu concentrations at other stations varied between 5 and 31 mg/l. Cu concentrations were below detectable limits on all sampling intervals at stations 13, 14 and 15. Temperatures during all sample periods ranged from 5 to 13 C and mean values varied by less than one-fold across all stations (Fig. 4a). However, salinity, turbidity and chlorophyll a had strong seasonal variations associated with spring runo€ (i.e. freshet) from the Squamish River and to a lesser extent Britannia Creek. When the test began on 29 April, runo€ from the Squamish River was low (i.e. pre-freshet)

Fig. 3. Spatial variability in mean Cu concentrations and pH in surface waters at 15 mussel stations in Howe Sound over four sample intervals. Error bars represent 1 standard error about the mean.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Station

7.5±8.0 7.2±7.8 7.2±7.7 7.1±7.8 7.0±7.8 7.1±7.6 6.1±7.0 7.0±7.6 6.9±7.6 7.1±7.8 7.1±7.6 7.3±7.6 6.9±7.7 7.1±7.9 7.4±7.8

Range (n=4)

Range (n=4)

7.5±13.0 7.0±12.0 7.0±10.5 6.0±11.5 6.0±10.0 7.0±10.0 6.0±10.5 7.0±13.0 7.0±10.5 7.0±10.0 7.0±12.0 7.0±10.5 5.0±11.0 7.0±11.0 7.0±12.0

pH

Temperature ( C)

26.0 28.0 26.0 21.0 23.0 25.0 14.0 27.0 27.0 28.0 26.0 20.0 14.0 23.0 24.0

Pre-freshet (n=1)

Salinity (%)

5.5±9.0 4.5±7.0 4.0±6.0 3.0±6.0 3.0±7.0 4.0±6.0 2.5±3.5 4.0±6.0 2.5±6.0 4.0±5.0 5.0±6.0 7.0±8.5 2.5±3.0 6.0±8.0 4.0±8.0

Freshet (n=3) 0.5 0.5 0.9 0.8 1.0 0.8 5.8 1.6 0.9 0.6 0.6 0.8 1.7 0.7 0.9

Pre-freshet (n=1) 1.4±3.9 2.5±5.8 4.5±5.2 5.1±7.5 4.4±7.2 4.3±6.0 7.8±9.4 5.9±6.8 5.8±7.7 5.0±5.9 4.5±5.1 3.6±3.6 5.5±9.4 3.8±7.8 2.9±3.7

Freshet (n=2)

Turbidity (NTU)

7.43 8.46 3.40 4.49 3.34 4.43 0.19 0.45 6.10 5.74 7.41 6.54 5.51 5.48 6.88

Pre-freshet (n=1) 0.50±0.94 0.17±0.98 0.49±1.31 0.20±0.58 0.01±0.19 0.04±0.48 0.00±0.03 0.00±0.02 0.00±0.01 0.00±0.20 0.00±0.27 0.12±0.57 0.07±0.38 0.23±0.71 0.25±1.32

Freshet (n=3)

Chlorophyll a (mg/l)

7.4 9.8 6.8 8.8 5.5 8.8 765.2 95.6 67.5 13.0 12.3 6.3 <5.0 <5.0 <5.0

Mean (n=4)

<5.0±11.7 7.0±12.0 <5.0±11.0 <5.0±15.0 <5.0±6.0 <5.0±18.0 431.3±1009.0 32.3± 189.0 <5.0±185.0 <5.0±31.0 <5.0±19.0 <5.0±8.0 <5.0 <5.0 <5.0

Range (n=4)

Surface water [Cu] (mg/l)

Table 1 Ranges in temperature, salinity, pH, chlorophyll a, turbidity, and Cu concentrations in surface waters at mussel stations in Howe Sound during four sampling events (29 April, 15 May, 28 May, and 8 June 1998)

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Fig. 4. (a) Mean surface water temperatures by station during the test period, (b) mean salinity, (c) turbidity and (d) chlorophyll a concentration by station during the freshet period. Error bars represent 1 standard error about the mean. The dashed line in all panels represents the distance of each station from the mouth of Britannia Creek (station 7).

and surface waters in Howe Sound had relatively high salinity, low turbidity and high chlorophyll a concentrations (Table 1). Chlorophyll a levels were depressed at stations 7 and 8 near the mouth of Britannia Creek. The remaining samples (15 May, 28 May, and 8 June) were collected when the Squamish River was in freshet. This period was characterized by relatively low salinity, high turbidity and low chlorophyll a concentrations associated with fresh water run-o€ carrying high sediment loads from the Squamish River. Surface salinities varied less than three-fold across stations with lowest salinities recorded on average at stations 7 and 13 at the mouth of Britannia Creek and closest to the Squamish River, respectively (Fig. 4b). Turbidity also varied less than three-fold across stations and was higher at stations 7 and 13, re¯ecting higher sediment inputs from nearby sources of freshwater runo€ (Fig. 4c). Chlorophyll a concentrations were negligible at stations 7, 8 and 9 but showed dramatic 50-fold increases for stations located furthest away from the mouth of Britannia Creek (Fig. 4d). 3.2. Mussel health indicators We grouped stations into one of two distinct zones for illustrative purposes: high survival (585%) for mussels at stations 1, 2, 3 and 15 and low survival (<45%) for mussels at stations 4±11 (Table 2). Stations in the low survival zone were on the east side of Howe Sound up to 1.7 km to the south of Britannia Creek and 2.1 km to the north of Britannia Creek (Fig. 5a). Stations in the high survival zone were located greater than 1.7 km to the south of Britannia Creek.

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Table 2 Mean di€erences in length growth rate, weight growth rate, condition index and concentrations of Cu and Zn in mussels from high and low survival zonesa Station

Survival Mean length Mean weight Mean conditon Tissue Cu Tissue Zn rate index (mg/g dry wt.) (mg/g dry wt.) Growth rate Growth rate (mm/week) (g/week)

Exposure references NA

NA

NA

1.24

9.4

123

High survival zone 1 2 3 15

0.97 0.88 0.85 0.91

0.36 0.12 0.06 0.06

0.13 0.06 0.03 ÿ0.02

1.42 1.34 1.33 1.10

14.9 24.3 33.2 21.7

124 117 144 126

Low survival zone 4 5 6 7 8 9 10 11 13

0.25 0.18 0.25 0.11 0.42 0.21 0.04 0.36 0.00

0.00 0.04 0.02 ÿ0.03 0.00 ÿ0.01 0.01 ÿ0.01 NA

0.00 ÿ0.01 ÿ0.04 ÿ0.07 ÿ0.05 ÿ0.09 ÿ0.02 ÿ0.02 NA

0.91 0.87 0.90 0.93 0.88 0.83 0.82 0.96 NA

44.0 61.0 61.0 115.0 89.9 91.0 90.0 102.0 NA

132 139 184 192 182 186 172 179 NA

a Survival rates are based on 75 mussels. Growth rates and condition index are mean values based only on mussels that survived for the duration of the test period. Tissue concentrations of metals are single estimates based on analyses of tissues from all surviving mussels for a given station.

Mussels in the high survival zone exhibited small increases in shell length and whole animal wet weight (Table 2). Mussels in the low survival zone did not grow well either. Increases in length were small and weights decreased. Relative to preexposure references, mussels in the low survival zone were in worse condition than at the start of the test and mussels in the high survival zone were in better condition. 3.3. Bioaccumulation of Cu and Zn in mussel tissues Across all stations, mean Cu concentrations were nearly seven-fold greater for test mussels (mean: 62.32.9 mg/g) compared to the three pre-exposure reference samples (mean: 9.40.3 mg/g) (Table 2). However, mean Zn concentrations were less than 30% higher in test mussels (mean: 156.42.4 mg/g) than in pre-exposure reference samples (mean: 123.02.6 mg/g). In test mussels, tissue Cu concentrations increased eight-fold between stations 1 and station 7 with increasing proximity to Britannia Creek. At stations 8±11 to the north of Britannia Creek, Cu concentrations were six- to seven-fold above those found at station 1 (Fig. 5b). Tissue Zn concentration data showed the same general

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Fig. 5. (a) Survival (n=75 mussels per station) at 12 stations (12, 13, and 14 not shown) in Howe Sound in relation to the distance of each station from the mouth of Britannia Creek (e.g. station 7). (b). Concentrations of Cu and Zn in tissues of mussels that survived for the duration of the test period in relation to the distance of each station from the mouth of Britannia Creek.

trend although concentrations at station 7 were only 55% higher than those at station 1 (Fig. 5b). Cu and Zn concentrations in mussel tissues were highly correlated across stations (R2=0.82, P<0.001, n=12; Fig. 6) suggesting mussels may have been exposed to similar gradients of these metals in the water. 3.4. Relationship between Cu concentrations in water and mussel tissue Mussels at stations exposed to higher concentrations of dissolved Cu in surface waters had signi®cantly higher concentrations of Cu in their tissues (R2=0.62, P=0.003, n=12; Fig. 7). This relationship was non-linear and was described by a Michaelis-Menten equation (Eq. (1)) where c=108 mg/g dry wt., d=9 mg/l, and e=3 mg/l. For concentrations of Cu in surface waters below approximately 9 mg/l (dashed line, Fig. 7) the concentration of tissue Cu increased approximately linearly with Cu concentrations in the water. As Cu in surface water increased above 9 mg/l, Cu accumulation in mussel tissues leveled o€, even as Cu concentration in the water increased nearly 100-fold.

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Fig. 6. Relationship between concentrations of Cu and Zn in mussel tissues (linear regression, R2=0.82, P<0.001, n=12).

Fig. 7. Michaelis-Menten relationship between the concentration of Cu in surface waters of Howe Sound and concentration of Cu in mussel tissues (R2=0.62, P=0.003, n=12). The dashed line at 9 mg/l indicates the surface water concentration at 50% of the estimated maximum Cu concentration in mussel tissues. x-Axis is plotted on log scale.

3.5. Relationships between metal accumulation and mussel health indicators Higher Cu concentrations in mussel tissue were associated with signi®cant declines in survival, growth, and condition index (Table 3; Fig. 8a±d). Declines in these mussel health indicators were generally consistent with a threshold model rather

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Table 3 Summary of the parameters, ®t and signi®cance of log-linear and threshold models ®t to relationships between Cu or Zn and indicators of mussel health Metal

Mussel health indicator

Log-linear model

Threshold model

A

B

R2

P-value

Cu Cu Cu Cu

Survival Length growth Weight growth Condition index

2.21 0.53 0.28 2.07

0.45 0.12 0.07 0.26

0.77 0.64 0.69 0.70

<0.001 0.002 <0.001 <0.001

Zn Zn Zn Zn

Survival Length growth Weight growth Condition index

7.23 1.85 1.24 5.12

1.24 0.36 0.25 0.81

0.52 0.40 0.59 0.49

0.009 0.030 0.003 0.010

Yl

Y2

R2

P-value

40 19 19 39

0.9 0.36 0.13 1.3

0.23 0.02 ÿ0.02 0.89

0.91 0.85 0.55 0.86

<0.001 <0.001 0.006 <0.001

130 125 125 125

0.92 0.24 0.1 1.38

0.30 0.01 ÿ0.03 0.95

0.65 0.69 0.67 0.59

0.001 <0.001 0.001 0.004

K

Fig. 8. Relationship between (a) the concentration of Cu in mussel tissues and survival (n=75 mussels per station), (b) length growth, (c) weight growth and (d) condition index. Solid lines represent the ®t of a threshold model to the observed data and dashed lines represent the ®t of a log-linear model. Error bars indicate 1 standard error about the mean.

than a log-linear model. The threshold model produced better ®ts (higher R2) for all relationships between Cu and mussel health, except for weight growth (Table 3). In particular, survival and condition index drop sharply at a tissue Cu concentration of about 40 mg/g dry wt. (k parameter, Table 3; Fig. 8a, d). Decreases in length and

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weight growth are evident at even lower Cu concentrations around 19 mg/g, although weight growth tended to decline gradually across tissue Cu concentrations which is consistent with a log-linear model (Table 3; Fig. 8b, c). Mussel health indicators also had signi®cant negative relationships with Zn in mussel tissues (Table 3; Fig. 9a±d). The threshold model produced better ®ts for all relationships between Zn and mussel health indicators compared to the log-linear model (Table 3). Abrupt declines in survival, growth, and condition index occurred at tissue Zn concentrations of about 125±130 mg/g dry wt. (Table 3; Fig. 9a±d). 3.6. Relationships between environmental conditions and mussel health indicators There was little evidence of variation in temperature or salinity as a function of distance of the station away from the mouth of Britannia Creek (Fig. 4a, b). However, turbidity and chlorophyll a appeared to be inversely and directly related to distance, respectively (Fig. 4c, d). Chlorophyll a concentrations were negatively correlated with tissue Cu concentrations in mussel tissues (R2=0.80, P<0.0001, n=12; Fig. 10a), while turbidity had a weaker positive relationship with tissue Cu concentrations (R2=0.66, P=0.001, n=12; Fig. 10b). Mussels that bioaccumulated higher amounts of Cu were located at stations that had lower chlorophyll a concentrations and higher turbidity values. Because of the high degree of correlation

Fig. 9. Relationship between (a) the concentration of Zn in mussel tissues and survival (n=75 mussels per station, (b) length growth, (c) weight growth and (d) condition index. Solid lines represent the ®t of a threshold model to the observed data and dashed lines represent the ®t of a log-linear model. Error bars indicate 1 standard error about the mean.

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Fig. 10. Log-linear relationships between concentrations of Cu in mussel tissues and (a) mean chlorophyll a concentrations and (b) turbidity during the freshet period. Regression equations and R2 values are shown. Error bars indicate 1 standard error about the mean.

between turbidity, chlorophyll a and tissue Cu data, it was not possible to further separate the in¯uences of turbidity and chlorophyll a levels on mussel health (but see discussion in Section 4). 4. Discussion 4.1. AMD contamination in Howe Sound Evidence of AMD from the 2200 foot portal entering Howe Sound at Britannia Creek was indicated by the low pH and high dissolved Cu concentrations in surface waters near the mouth of the creek (Fig. 3). These results are consistent with past observations of contamination of the surface waters in this area (Chretien, 1997; Dunn et al., 1992; Goyette & Ferguson, 1985). Higher Cu concentrations at stations north of Britannia Creek relative to southern stations (Table 1) are consistent with a northward circulation of surface waters in Howe Sound near Britannia Creek (Fig. 2). Rapid decreases in concentrations of Cu in surface waters at stations located further away from the mouth of Britannia Creek (Fig. 3) is consistent with increasing

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dilution of the metal source with more saline water and other removal processes (Featherstone & O'Grady, 1997; Foster et al., 1978; Johnson & Thornton, 1987; Windom et al., 1991). The exact processes of Cu removal for this area are complex and are detailed elsewhere (Chretien, 1997). We expect that a similar gradient of Zn contamination also existed, but concentrations of dissolved Zn were below detection limits used here. Other reports also show that Zn and Cu concentrations in water from Britannia Creek and surface waters of Howe Sound are highly correlated (Chretien, 1997; Dunn et al., 1992). 4.2. Bioaccumulation of Cu and Zn in mussel tissues Natural concentrations of Cu in the tissues of mussels living in uncontaminated marine areas are approximately 10 mg/g dry wt. (Delhaye & Cornet, 1975; Simkiss, Taylor & Mason, 1982). Cu concentrations measured in the tissues from these mussels before the start of the test (i.e. pre-exposure reference samples from Quadra Island) were the same as those from uncontaminated areas (Table 2). Thus, higher tissue Cu concentrations in mussels from all of our stations relative to pre-exposure references may re¯ect background concentrations of Cu in Howe Sound, as found by others (Dunn et al., 1992). Mussels exposed to higher concentrations of dissolved Cu in surface waters bioaccumulated signi®cantly higher concentrations of Cu in their tissues (Fig. 7). Higher bioaccumulation of Cu and Zn in mussels at stations to the north of Britannia Creek (Fig. 5b) was not unexpected since the prevailing circulation patterns move AMD-contaminated surface waters northwards from Britannia Creek (Fig. 2). Bioaccumulation of Cu and Zn in mussel tissues was also signi®cantly correlated across stations (Fig. 6) and supports our contention that mussels were exposed to a Zn pollution gradient in surface waters similar to that of Cu. Based on the non-linear relationship between bioaccumulation of Cu in mussel tissues and Cu in surface waters (Fig. 7), M. edulis appears to be an extremely sensitive indicator of Cu contamination. Even at relatively low Cu concentrations (<9 mg/l) mussels bioaccumulated Cu. Thus, despite dramatic declines in Cu concentrations in surface waters more than 0.5 km away from Britannia Creek, environmental contamination was indicated by bioaccumulation of Cu in mussel tissues (Fig. 5b). This result is consistent with other ®ndings that bioaccumulation provides a better measure of exposure to metal contaminants because it is a time-integrated measurement of the water concentrations of bioavailable metals (Salazar & Salazar, 1991). Above 9 mg/l Cu in surface waters, mussels did not bioaccumulate more than 115 mg/g Cu in their tissues, even for Cu concentrations in the water that were two orders of magnitude higher (Fig. 7). The fact that mussels do not continue to bioaccumulate Cu in a linear fashion as Cu concentrations in the water increase above 9 mg/l is likely attributable to mussel mortality (Section 4.3) or a mechanism for metal detoxi®cation and elimination (Raspor & Pavicic, 1991; Viarengo, Pertica, Canesi, Mazzucotelli, Orunesu & Bouquegneau, 1989). Although we could not de®ne a relationship between Zn concentrations in surface waters and its bioaccumulation in mussel tissues, Zn bioaccumulation did not exceed 192 mg/g and varied less than two-fold across stations

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1±7 compared to eight-fold for Cu (Table 2). This indicates that mussels regulated the bioaccumulation of Zn in this study, as has been widely shown by others (Eisler, 1993; Phillips, 1977; Regoli & Orlando, 1994; Wang, Fisher & Luoma, 1995). 4.3. Relationships between Cu and Zn bioaccumulation and mussel health Bioaccumulation of Cu and Zn in mussel tissues provides an important link between exposure to dissolved metals in the environment and adverse e€ects on mussel health. Bioaccumulation of Cu above approximately 40 mg/g dry wt. was related to signi®cant sharp declines in both survival and condition index of study mussels (Fig. 8a, d). A critical threshold of 59 mg/g for the bioaccumulation of Cu in mussel tissues has been found to be acutely lethal to M. edulis regardless of the concentration of Cu in the water or exposure time in the lab (Martin, 1979; Widdows & Johnson, 1988). This threshold is consistent with our results (Fig. 8a). Mussels at stations in the high survival zone had tissue Cu concentrations less than 33 mg/g dry wt. whereas mussels in the low survival zone, with the exception of station 4 at 44 mg/g dry wt., all had tissue Cu concentrations greater than 61 mg/g dry wt. (Table 2). Length and weight growth in mussels were even more sensitive to bioaccumulation of Cu with declines in growth evident at Cu concentrations of approximately 19 mg/g dry wt. (Fig 8b, c). Based on this threshold, mussels as far away as 5.8 km from Britannia Creek (Fig. 5b) may have been a€ected by Cu contamination. Other work has also shown that M. edulis had signi®cantly lower scope for growth when they bioaccumulated >27 mg/g dry wt. Cu, which was roughly half of the bioaccumulation that caused e€ects on survival (Widdows & Johnson, 1988). Growth has also been shown to be more sensitive than survival to bioaccumulation of tributlytin in Mytilus sp. (Salazar & Salazar, 1996). Bioaccumulation of Zn above approximately 125 mg/g dry wt. was also related to signi®cant declines in all mussel health indicators (Fig. 9a±d). However, molluscs often bioaccumulate Zn far in excess of their immediate needs and it does not seem to a€ect normal life processes (Eisler, 1981). Tissue Zn concentrations observed in our study (Table 2) were all within a range of 50 to 241 mg/g Zn dry wt. that was observed in natural populations of mussels collected at an unpolluted site in Newfoundland, Canada (Lobel, 1986). A laboratory study also showed that a median Zn bioaccumulation of 350 mg/g dry wt. in mussels resulted in less than 10% mortality (Burbidge et al., 1994). Thus, Zn concentrations in mussels from our study were consistent with those found in an unimpacted natural population and were probably not high enough to have had a direct impact on their health. The co-occurrence of both Cu and Zn in mussel tissues (Fig. 6) prevented us from isolating the metal responsible for adverse e€ects on mussel health. However, concentrations of Cu in mussel tissues produced better ®ts (R2 values, Table 3) to the mussel health data compared to Zn that, along with the discussion above, supports our assertion that Cu was mainly responsible for the observed e€ects on mussel health. These results are consistent with Cu being more toxic than Zn for Mytilus sp. (D'Silva & Kureishy, 1978) but it is also possible Zn may have an additive e€ect on toxicity in mussels as observed in other species (Eisler, 1997).

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Our ®ndings also indicate that bioaccumulation is a better predictor of adverse e€ects on mussel health compared to using water samples. However, a large body of research has focused on the e€ects of water-borne metals on mussel health. In seawater, adverse e€ects on mussel behavior, feeding and growth are evident over a narrow range of Cu concentrations (5±10 mg/l) and these activities may cease at 20 mg/l Cu (Calabrese, MacInnes, Nelson, Greig & Yevich, 1984; Davenport & Redpath, 1984; Eisler, 1997; Manley & Davenport, 1979; Redpath, 1985; Widdows & Johnson, 1988). Our results are consistent with these data. Laboratory studies have shown that acute toxicity in mussels occurs at Cu concentrations greater than approximately 100 mg/l Cu in seawater (Davenport & Manley, 1978; Eisler, 1997) and some mortalities have occurred for concentrations as low as 20 mg/l Cu (Davenport & Redpath, 1984; Martin, 1979). However, in a laboratory study of mussels kept in brackish water (salinity: 7%), a much higher concentration (400 mg/l Cu) was acutely toxic (Sunila, 1981). Concentrations thought to be acutely toxic (>100 mg/l Cu) have not always caused high moralities in ®eld studies (Roesijadi, Young, Drum & Gurtisen, 1984). Obviously, it is much more dicult to relate Cu concentrations in the water to adverse e€ects on survival as e€ects thresholds vary widely from as low as 20 up to 400 mg/l. Concentrations of water-borne Cu measured during our study (Table 1) indicate that mortality was certainly possible at most stations in the low survival zone. In the ®eld, Cu in water samples provides only a crude approximation of what biological e€ects might be expected to occur. Cu concentrations in the water were only sampled four times during the study and probably do not accurately quantify peak levels or the daily variability of Cu concentrations. On the other hand, M. edulis is capable of concentrating metals making trace contamination levels easier to measure in mussel tissues than in the seawater itself (Phillips, 1977). Thus, using bioaccumulation of metals in mussel tissues, as was done here, is a more e€ective way to assess adverse e€ects on mussel health. 4.4. Relationship between environmental conditions and mussel health In addition to heavy metals, mussels were exposed to natural environmental conditions that may have in¯uenced mussel health during the study. Temperature, salinity and variations in food supply are all known to in¯uence bioaccumulation, survival and growth in mussels (Bayne et al., 1985; McLusky, Bryant & Campbell, 1986; Widdows et al., 1984). Average water temperatures during the study were within a 5±20 C range (Table 1) where mussels maintain physiological rates independent of temperature (Bayne et al., 1985; Widdows, 1978). Salinities varied less than three-fold across stations and were probably not responsible for the large differences observed in mussel health indicators. However, during the freshet period, point estimates of salinities at some stations (Table 1) were below the 5% lower tolerance limit suggested for mussels (Stewart, 1994) on at least one occasion. In particular, salinities at stations 7 and 13 remained below 5% during the whole freshet period (Table 1). Station 13 was also the only station located in the main surface current from the Squamish River (Figs. 1 and 2) where constant exposure to

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low salinity water may have contributed to the 100% mortality of these mussels despite no exposure to elevated metal concentrations. Mussels feed by ®ltering phytoplankton from the water column. This process can be interrupted by high levels of suspended inorganic material (high turbidity) that can cause stress by increasing the water volumes that must be ®ltered to obtain adequate food (Kiorboe, Mohlenberg & Nohr, 1981; Madon, Schneider, Stoeckel & Sparks, 1998; Stewart, 1994). High suspended sediments have also been shown to result in light attenuation in surface waters and reduced phytoplankton productivity in Howe Sound (Stockner et al., 1977). Turbidity varied inversely with chlorophyll a across stations (Fig. 4c, d) which suggests light attenuation may have been partly responsible for reduced chlorophyll a. Turbidity was also related to bioaccumulation of Cu in mussels (Fig. 10b); however, this factor varied by less than two-fold across stations and was probably not the primary factor responsible for observed adverse e€ects on health. Dramatic 50-fold reductions in chlorophyll a concentrations (i.e. a measure of phytoplankton abundance) in the vicinity of Britannia Creek were signi®cantly related to Cu bioaccumulation in mussel tissues (Fig. 10a) which is a proxy for Cu concentrations in surface waters (Fig. 7). This suggests that exposure to metal contamination from the Britannia mine had adverse e€ects on phytoplankton. Bioassay experiments using AMD-contaminated water from Britannia Creek diluted with seawater adversely a€ected growth of phytoplankton from Howe Sound at Cu concentrations above 6.4 mg/l (Chretien, 1997), and con®rms the negative impacts of Cu contamination on phytoplankton that we observed. Grout et al. (1999) also reported depressed chlorophyll a concentrations and phytoplankton abundance near the mouth of Britannia Creek relative to other areas in Howe Sound. Thus, reduced phytoplankton food supplies resulting from metal contamination likely also contributed to detrimental e€ects on mussel health in contaminated areas. 4.5. Species distributions in Howe Sound Examples from natural populations of invertebrates and plants in Howe Sound provide supporting evidence of adverse e€ects of Cu contamination on organism health in the vicinity of Britannia Creek. The low survival of study mussels in a zone extending 1.7 km to the south and 2.1 km to the north of Britannia Creek (Fig. 5a) corresponds with observed patterns in natural populations of mussels in Howe Sound. Increasing concentrations of Cu and Zn in natural populations of mussels (Mytilus trossulus) and oysters (Crassostrea gigas) in Howe Sound with proximity to Britannia Creek were measured while the mine was still operating (Goyette, 1975, cited in Harding 1992; Nelson & Goyette, 1976). Surveys of natural mussel populations (M. trossulus) showed general increases in abundance with distance from the mouth of the Squamish River but a complete absence of mussels within 1.5 km of the mouth of Britannia Creek (Levings & McDaniel, 1976). These data suggest natural mussel populations also su€er adverse e€ects from AMD contamination at Britannia Creek.

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Negative impacts of mine pollution on the common rockweed (Fucus gardneri) are also apparent. Rockweed is tolerant of low salinity and grows in abundance throughout Howe Sound (Levings & McDaniel, 1976). A study of metal concentrations in F. gardneri tissues indicated Cu concentrations were in excess of 3000 mg/g near Britannia Creek but decreased to 60±70 mg/g to the south of our station 1 in Howe Sound (Dunn et al., 1992). Similarly, concentrations of Zn up to 1540 mg/g were present near the mouth of Britannia Creek whereas they ranged from 300 to 400 mg/g to the south. As with natural mussels, the shoreline is devoid of any rockweed for 1.5 km to the north and south of Britannia Creek presumably because of its intolerance to the in¯ux of metals (Dunn et al., 1992, Marsden & deWreede, 2000). 5. Conclusion The weight of evidence from our work and related studies supports the conclusion that Cu and Zn associated with AMD from the Britannia mine have adverse e€ects on mussel health. Mussels transplanted to stations within a low survival zone extending 2.1 km to the north and 1.7 km to the south of Britannia Creek in Britannia Creek had high concentrations of Cu and Zn in their tissues which were signi®cantly related to deleterious e€ects on survival and condition index. Adverse e€ects on mussel growth were also probable at stations as far away as 5.8 km from the source of AMD. Cu contamination was also signi®cantly correlated with reduced chlorophyll a concentrations which may have resulted in food limitation and deleterious e€ects on mussel health in the contaminated zone. Finally, the low survival observed for transplanted mussels in the vicinity of Britannia Creek was corroborated by an absence of natural mussel and rockweed populations in this area. Acknowledgments This research was supported in part by funds from the Canadian Department of Fisheries and Oceans Toxic Chemicals Fund. Many thanks to R. Bahry and B. Golke of Polaris Marine Consultants for providing equipment and expertise in the ®eld. Thanks to T. Anderson, A. Coombs, D. Marsden, B. Piercey, P. Poon and S. Shong for their tireless e€orts in the laboratory and ®eld. Thanks also to N. Mehlenbacher for help producing ®gures. The Paci®c Environmental Science Center performed the water chemistry analyses and ASL Laboratories did the analyses of tissue metal concentrations. Chlorophyll a concentrations were determined in the laboratory of Dr. P.J. Harrison at the University of British Columbia. Thanks also to M. Salazar and two anonymous reviewers for their constructive comments. References Bayne, B. L., Brown, E. A., Burns, K., Dixon, D. R., Ivanovici, A., Livingstone, D. R., Lowe, D. M., Moore, N. M., Stebbing, A. R. D., & Widdows, J. (1985). The e€ects of stress and pollution on marine animals (Praeger Special Studies). Praeger Scienti®c, New York, USA.

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