Water Qual. Res. J. Canada, 2003Volume 38, No. 4, 647–666Copyright © 2003, CAWQ

* Corresponding author; sylvie.st-jean@ec.gc.ca† Present address: Aquatic Ecosystem Protection Research Branch, National

Water Research Institute, Environment Canada, P.O. Box 5050, Burlington,Ontario L7R 4A6

Immunomodulation in Blue Mussels (Mytilus edulis)Exposed to a Pulp and Paper Mill Effluent

in Eastern Canada

SYLVIE D. ST-JEAN,1*† SIMON C. COURTENAY1 AND ROY W. PARKER2

1 Fisheries and Oceans Canada, Gulf Fisheries Centre, P.O. Box 5030, Moncton, NewBrunswick E1C 9B62 Environmental Protection Branch, Environment Canada, 77 Westmorland Street,Fredericton, New Brunswick E3B 6Z3

Blue mussels (Mytilus edulis) were caged at three sites situated at increasingdistance from the point of discharge of a pulp and paper mill effluent from Julyto October 1998. Two additional cages were deployed: one inside and one at themouth of the adjacent industrialized Pictou Harbour. After 90 d exposure, wemeasured growth, survival, haemocyte counts (HC), phagocytic activity (PA),lysosome retention (LR) and bacterial clearance (BC). There was a small but sig-nificant difference in growth between cages. Mussels closest to the mill efflu-ent grew the most while those at the mouth of the harbour grew the least.Mussels from three cages showed similar difficulty in clearing bacteria; thecage inside the harbour, the cage nearest to the pulp mill effluent and the cagefurthest from the pulp mill, receiving a mixture of both pulp mill and harboureffluents. The mussels from those cages also showed the highest heavy metalburdens and conversely, the cage showing the most rapid clearance, outsideboth effluents, also showed the lowest heavy metal burden. Mussels caged inthe pulp mill effluent showed lower PA and LR and higher mortality during thebacterial clearance test than other mussels. These results suggest that immuno-logical biomarkers might be a useful and more sensitive adjunct to endpointspresently being measured from caged bivalves in environmental effects moni-toring (EEM) programs, and assessments of aquatic environmental quality.

Key words: mussels, immune, pulp mill effluent, bacterial clearance

Introduction

In 1992, new regulations required all pulp and paper mills in Canadato conduct aquatic environmental effects monitoring (EEM) studies in theareas of their effluent discharge to assess potential impacts to the healthof fish and fish habitat. As part of their 1998 EEM requirements,Kimberly-Clark Nova Scotia (KCNS), who operate a bleached kraft pulp

648 ST-JEAN ET AL.

mill in Pictou (N.S.), evaluated an alternative approach to the mandatoryfish survey, in collaboration with Environment Canada (Andrews andParker 1999). The alternative approach involved caging blue mussels(Mytilus edulis) for 90 d at three sites situated at increasing distance fromthe point of discharge. Furthermore, two additional cages were placed;one within the adjacent industrialized Pictou Harbour and the other out-side the harbour. The cage outside Pictou Harbour was intended to be arelatively pristine reference site against which to compare the cagesexposed to pulp mill effluent or the municipal and industrial effluents ofPictou Harbour. Endpoints to be assessed were survival and growth. Aparallel study was also conducted to determine if variations could bedetected in the immune function of the mussels from each cage after 90 dexposure and if those variations could be related to the growth and sur-vival of the mussels.

Blue mussels show several characteristics well suited to measureenvironmental effects (Salazar and Salazar 1995). They are sessile filter-feeders widely distributed in coastal and estuarine areas. They have a lim-ited ability to metabolize contaminants and therefore tend to accumulatethem to levels exceeding those found in the ambient seawater (Widdowsand Donkin 1992; Coles et al. 1994). Previous studies have indicated thepotential for the immune system of bivalves to act as an early warningsystem of stress in marine environments (Anderson 1990; Coles et al. 1994;Pipe et al. 1995a, 1999; Pipe and Coles 1995; Dyrynda et al. 1997, 1998;Oubella 1997; St-Jean et al. 2002a,b). Pipe et al. (1999) suggested that envi-ronmental contamination effects might result from direct toxic action onthe tissue or from more subtle alterations in homeostatic mechanisms,such as the immune system. Mussels have an open circulatory system inwhich mobile haemocytes are the primary effector in identifying andeliminating potential pathogens or irritants. It has also been suggestedthat because of the open circulatory system, haemocytes are among thetargets of many contaminants which in turn might interfere with the mus-sel’s capability to maintain homeostasis (Oubella 1997). Several studieshave shown that modulations in immune parameters could be associatedwith increased disease susceptibility (Pipe and Coles 1995; Dyrynda et al.1997; St-Jean et al. 2002a).

This paper describes observations and results of experimental workdesigned to develop immunological biomarkers to be used in environ-mental effects monitoring programs and to ultimately aid in assessingmarine environmental health. The objective of the present study wastherefore to investigate immune parameters of the mussel Mytilus edulisexposed along a gradient of a treated pulp mill effluent and to comparethe results to growth and survival parameters. In addition, the studyinvestigated whether any links could be made between exposure to pulpmill effluent or to contaminated harbour waters, immunomodulationsand the ability of mussels to clear bacteria from their haemolymph.Parameters measured included total haemocyte counts (HC), phagocyticactivity (PA), lysosome retention (LR) and bacterial clearance (BC).

IMMUNOMODULATION IN CAGED BLUE MUSSELS 649

Materials and Methods

Study Area

Pictou Harbour, located in northern Nova Scotia, occupies a lowlandarea bordering the Northumberland Strait and adjacent to the Pictou-Antigonish Highlands (Fig. 1) (Painter and Stewart 1992). It is an impor-tant commercial estuary and has a history of environmental contamina-tion, including fecal coliform bacteria, metals, nutrients, PCB and PAHfrom domestic sewage, pulp mill effluent, agricultural runoff, and conta-minants from a shipyard, rail car works and a foundry (Painter andStewart 1992). Tides are mixed, mostly semi-diurnal with a small range(<1 m) (Krauel 1969). The harbour has a low flushing rate and the mixingis ensured mainly through wind and tidal action (Krauel 1969).

Habitat Description

The sediments of the Pictou Road area (i.e., southern shore, immedi-ately outside Pictou Harbour, which receives pulp mill effluent from Boat

Fig. 1. Sampling sites.

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Harbour) consist mostly of sand, cobble and gravel mixed with mud pre-senting a red colour while the sediments inside Pictou Harbour consistmostly of light brown silty mud (Andrews and Parker 1999; Beak 2000). Astudy of the heavy metal loads in the Pictou Harbour/Road sediments con-ducted in 1991 showed dry weight concentrations of Cu (1–56 mg/kg), Hg (0.01–0.85 mg/kg), Pb (5–57 mg/kg) and Zn (7–231 mg/kg) wereslightly above natural levels (Dalziel et al. 1993). In the case of Hg, thehigh concentrations occurred beneath 10 cm and were near or exceededthe ocean disposal regulated limit of 0.75 mg Hg/kg (Canada Gazette2001). Of 159 cores, Cd levels (0.02–0.93 mg/kg) exceeded natural back-ground levels in 102 cores and 84 exceeded the oceans disposal regulatedlimit of 0.60 mg/kg.

The pulp mill effluent is the most important influence in Pictou Roadarea, while untreated sewage from the Town of Pictou, as well as treatedsewage and industrial effluents from the industrialized East River, are thepredominant influences in the Pictou Harbour (Fig. 1). Salinity in PictouRoad varies from 24 to >26‰ and from 22 to 26‰ in Pictou Harbour(Kimberly-Clark Nova Scotia, unpublished results).

Pulp Mill Process and Effluent Treatment

All information on the mill process and effluent treatment was takenfrom Beak (1998). In 1997, Kimberly-Clark (N.S.) mill produced an average631 air-dried tonnes per day (ADt/d) of bleached kraft pulp and the kraftpulping processes consisted of the following. Softwood chips were cookedin the presence of steam, caustic soda and sodium sulphide and thenwashed with liquor, deknotted, and screened to create a clean bleachablefibre. By 1998, chlorine usage was 100% substituted with chlorine dioxideto reduce organochlorine content in the mill effluent. Spent cooking chem-icals from the kraft digester, known as “black liquor,” were sent to thechemical recovery boiler and a recausticizing plant. All effluent from themill was sent to the Boat Harbour Wastewater Treatment Facility. Afterspending approximately 12 hours in two earthen settling ponds, the efflu-ent flowed into an aerated lagoon. After an 8-d retention period, mill efflu-ent entered the Boat Harbour stabilization basin for another 20 to 30 d.Treated effluent then flowed into the final aeration cell before being dis-charged into the Pictou Road area. Total effluent discharge rates averaged82,448 m3/d in 1997. At the time of this study, the final mill effluentshowed no acute toxicity to rainbow trout (Oncorhynchus mykiss, 96-h test)or Daphnia magna (48-h test) and no impact on survival or growth of 7- to11-d old inland silversides (Menidia beryllina, 7-d test) but did show sub-lethal toxicity in tests with algal (Champia parvula) sexual reproduction(geometric mean IC25: 5.4%) and sea urchin (Strongylocentrotus droe-bachiensis) fertilization (geometric mean IC25: 5.5%) (Beak 2002).

Study Design

Timmons Long Pond Mussel Farm in Port Hastings, N.S., suppliedthe certified blue mussels, Mytilus edulis. Upon receipt from the supplier,

IMMUNOMODULATION IN CAGED BLUE MUSSELS 651

the mussels were placed in mesh bags and kept on ice during transporta-tion, cage preparation and cage loading. These measures were taken tominimize stress, preserve animal health and ensure the overall integrityof the animals. The mesh bags containing the test organisms were tem-porarily moored at 4-m depth at a location in the Northumberland Straitaway from the industrial influences of Pictou Harbour, but close enoughthat the mussels could be easily retrieved and provided to the cagepreparation crew as needed. The study design was described in Andrewsand Parker (1999). Briefly, five cages, each containing 400 individuallymeasured and weighed juvenile blue mussels measuring between 2.5and 3.5 cm, were deployed in early July 1998, in Pictou Harbour/PictouRoad area for 90 d (Fig. 1). Each cage was cube-shaped (1 m3) and con-structed of 11/4 -inch diameter rigid polyvinylchloride (PVC) pipe, gluedand riveted together to ensure durability. Mussels were held in musselsocks (10-cm diameter plastic mesh with 5-mm holes) attached to thecages and secured with plastic tie wraps. Mussel socks held 10 mussels,each of which was individually separated by constricting the musselsock with a plastic tie wrap between each mussel but leaving enoughspace for growth (i.e., 8–10 cm between cable ties). Ten mussel socks weretied to each of the four vertical faces of the cage. Recording of the posi-tion of each mussel in each sock permitted measurements of individualgrowth over the 90-d exposure. The cages were moored 1 m off the bot-tom in 3 to 12 m of water.

A 90-d exposure period, from early July to early October, 1998, wasselected in order to maximize the exposure period of the mussels to theeffluent constituents and minimize reproductive interference from springspawning. Three cages were placed along a 1-km gradient originating500 m from the mouth of Boat Harbour (Fig. 1). These cages were all with-in the area of the 1% plume of pulp mill effluent (Beak 2000). A fourthcage was placed on the north side of the mouth of Pictou Harbour, beyondthe influence of pulp mill effluent and a fifth cage was placed withinPictou Harbour (Fig. 1). A VEMCO MINILOG-TRTM temperature datalogger was attached to each cage and water temperature was recordedhourly for the duration of the exposure period. After retrieval of the cages,the data loggers were downloaded into a computer and daily averagetemperatures were calculated for each cage. Temperatures varied from 13to 20°C over the exposure period and were similar among cages except forthe cage situated inside the harbour which was consistently a few degreeswarmer except during the last few weeks.

Physical and Chemical Measurements

Before and after exposure (days 0 and 90), each mussel was weighed(whole animal wet weight or WAWW to the nearest 0.01 g) and measured(shell length along the anterio-posterior axis to the nearest 0.01 mm).Digital vernier calipers were used to measure length and digital electron-ic balances were used to measure the WAWW. Instruments were calibrat-ed daily to ensure the accuracy of the measurements. Approximately 10%of the lengths and weights of the mussels were verified randomly by a dif-

652 ST-JEAN ET AL.

ferent person against the recorded measurements prior to loading on thecage to check for accuracy and precision.

Background laboratory chemical analyses were conducted on pre-exposure composite tissue samples (n = 50) and on post-exposure com-posite samples from each cage (n = 50). Duplicate composite samplesfrom each cage were also submitted as a blind replicate for quality con-trol. Final effluent samples from Boat Harbour, collected at the beginning,the midpoint and the end of the exposure period, were analyzed.Analyses of tissue and effluent samples targeted parameters typical ofbleached kraft mill effluent (BKME), including chlorophenolic com-pounds, resin acids, plant sterols and metals. Percent moisture and lipidcontent of the tissue samples were also measured for use in interpretingthe results. Composite mussel tissue samples were placed in polyethylenebags and frozen to –20˚C. Water samples for metal analyses by ICP-MSwere preserved with nitric acid. Analysis of mussel tissue and effluentsamples were conducted with replicates by Philip Analytical ServicesCorporation (PSC, Halifax, Nova Scotia) and several subcontracting labo-ratories immediately following collection and preservation. A completelist of specific analytes, analytical methods, and detection levels achievedfor tissue and effluent analyses is provided in Andrews and Parker (1999).

Solutions used in Immunological Assays

All chemicals were either of certified grade for the dyes or HPLCgrade for the solvents and were purchased from Sigma Chemical Co.(Mississauga, Ont., Canada). The fixative used was Baker’s formol-calci-um and consisted of 4% (v/v) formaldehyde, 2% (w/v) sodium chlorideand 1% (w/v) calcium acetate (Lowe and Pipe 1994). The extraction solu-tion used to solubilize the neutral red consisted of 1% (v/v) acetic acidplus 50% (v/v) ethanol in distilled water (Grundy et al. 1996).

Haemolymph Extraction

At the termination of the 90-d caging exposure, 10 mussels were ran-domly selected from each cage to assess haemocyte function and another 10mussels were selected for a bacterial challenge (described below). Thehaemolymph extraction, performed on site, followed the protocol describedin Pipe et al. (1995b) with the following modifications. Five hundredmicrolitres of haemolymph were withdrawn from the posterior adductormuscle sinus with a 23-g sterile needle attached to a 3-mL sterile syringe con-taining 500 µL of tris-buffered saline (TBS), pH 8.4 and 930 mOs. The result-ing 1000 µL of haemolymph/TBS solution was immediately transferred into2-mL Eppendorf tubes, allowing for triplicates of each assay (3 x 100 µL forphagocytosis and 3 x 100 µL for lysosome retention) and 50 µL for totalhaemocyte counts (HC), using an improved Neubauer haemocytometer.

Cell Function Parameters

The techniques used in this study followed Pipe et al. (1995b) and St-Jean et al. (2002a) with some modifications as follows.

IMMUNOMODULATION IN CAGED BLUE MUSSELS 653

a) Lysosome retention (LR)Lysosome retention was evaluated using neutral red dye. Neutral

red is a cationic dye, which accumulates in the lysosomal compartment ofcells, illustrating the detoxifying mechanism (Lowe and Pipe 1994). It istaken up into cells by membrane diffusion or pinocytosis and thereforealteration in its uptake may reflect damage to the plasma membrane andconsequent changes in the volume of the lysosome (Lowe and Pipe 1994).One hundred µL of neutral red solution, consisting of 10 µL of stock solu-tion (20 mg neutral red diluted into 1 mL DMSO) diluted into 5 mL TBSwere added to 100 µL of haemolymph/TBS suspension in Eppendorftubes for 30 min. The tubes were then centrifuged (800 g for 5 min),drained, then washed with TBS and finally 100 µL of fixative was addedto the tubes. The fixed haemocytes were refrigerated (4°C) for less than24 hours and transported on ice to the laboratory. Upon arrival, the neu-tral red was extracted by displacing it from the haemocytes with the addi-tion of 100 µL of extraction solution for 30 min. The supernatant was gen-tly transferred into multiwell plates and read at 490 nm using a UV Maxspectrophotometer (Molecular Devices, U.S.A.). Data reported are meanoptical densities standardized to 106 cells.

b) Phagocytic activity (PA)Aliquots of 100 µL of haemolymph/TBS were added to sterile 2-mL

Eppendorf tubes. Twenty microlitres of neutral-red stained zymosan type1 yeast, 2.3 x 108 particles/mL, were added to each tube for 30 min incu-bation, after which 100 µL of fixative was added for 30 min. The tubeswere then stored and transported as previously described. Upon arrivalat the laboratory, the neutral red and/or zymosan not absorbed by phago-cytes in each tube were removed by two washes with TBS (centrifuge for5 min at 2000 g), along with a control sample (fixed haemocytes andstained zymosan), to adjust for surface adsorption of the dye. The neutralred dye within phagocytes was then solubilized by adding 100 µL ofextraction solution for 30 min, the supernatant was transferred intomicroplate wells and the plates were read at 490 nm, using a UV Maxspectrophotometer (Molecular Devices, U.S.A.). Data reported are meanoptical densities standardized to 106 cells.

c) Bacterial challengeThe preparation of the stock bacteria, the inoculation of the mussels

and the bacterial clearance experiment were conducted exactly asdescribed in St-Jean et al. (2002a). Briefly, a pure strain of Listonella(=Vibrio) anguillarum was progressively rendered resistant to two antibi-otics: streptomycin and rifampicin, and then maintained on marine agarcontaining 500 mg/L of streptomycin and 100 mg/L rifampicin. Prior toinoculation of the mussels, an inoculum of L. anguillarum was transferredinto an antibiotic-free solution of marine broth (Difco) and left to grow atroom temperature for 24 hours. An injection of 0.5 mL of the bacterial cul-ture (5 x 106 per mL) was then made directly into the haemolymph of eachof 10 mussels. The mussels were then closed tightly with elastic bands to

654 ST-JEAN ET AL.

avoid any leaking and left out of the water, at room temperature for2 hours. The mussels were then freed and permitted to purge themselvesin 15-L aquaria (one per group) containing cooled (room temperaturemaintained at 12ºC), aerated water from the vicinity of each cage. Musselsare intertidal and may be exposed to air for several hours every day, dur-ing which they seal their valves to avoid dessication. The resistence ofmussels to oxygen deficiency was measured by Theede (1973) to be wellabove 10 d and more recently and under varying salinity conditions (16 to32 ppt) was shown to range between 17.2 and 9.8 d (Barbarro and deZwaan 2002). In addition, Zurburg et al. (1982) showed that musselsexposed to air for 48 hours recovered within hours after reimmersion.Bacterial clearance was evaluated 1, 4 and 7 d after inoculation by draw-ing 0.5 mL of haemolymph from each individual. From each sample, threealiquots of 20 µL were used to assess the bacterial levels remaining in eachmussel using the most probable number (MPN) technique and the meanof these samples was used for the statistical analysis.

Statistical Analysis

Statistical analysis was carried out with Systat 10® software (SPSSInc., Chicago) for personal computers. Analysis of variance (ANOVA)assumptions were verified graphically by both box and probability plotsand further verified by Levene’s and Lilliefor’s tests. Differences ingrowth among cages were determined by one-way ANOVA on theabsolute change in length or weight. When differences were detected, aFisher’s Least Significance Difference test was performed to detect whichcages differed from one another. Survival was calculated as a percentagefor each cage after the 90-d exposure. Results for LR, and PA were logtransformed and standardized to one million cells and HC was square-root transformed. Differences among cages were compared by one-wayANOVA followed by a Tukey a posteriori test. Significant differences werereported when P < 0.05. Power analyses (Green 1979) were conducted toestimate sample size required to detect a 20% difference among the cagesand to test the power of tests.

A two-way ANOVA with time (1, 4 and 7 d post inoculation) andcages as independent variables was performed to test for effects of timeand site on bacterial clearance. Where significant effects (P < 0.05) weredetected, we compared cages on each of days 1, 4 and 7 by a one-wayANOVA followed by Tukey test. Rates of bacterial clearance for each cagewere examined separately by a one-way ANOVA followed by Tukey test.

Results

Survival rates for caged mussels over the 90-d exposure ranged from93% to 97% and did not differ among cages (Table 1). Although thegrowth differences among cages were small, they were statistically signif-icant. Cage 1 showed a significantly greater increase in WAWW than allother cages which did not differ significantly (F4,1881 = 9.41, p < 0.0001;

IMMUNOMODULATION IN CAGED BLUE MUSSELS 655

p < 0.05 Fisher’s LSD test) (Fig. 2). This result was further verified usingan ANCOVA, with T = 0 shell length as a covariate, and the power waslarge (1.00) and followed by a Bonferroni corrected multiple comparisontest. Cage 4 experienced lower growth, in terms of shell length, than cages1, 2 and 3 (F4,1881 = 3.69, p = 0.0054; p < 0.05 Fisher’s LSD test). Growth,expressed as an absolute increase in centimetres over the original length,ranged from 1.17 at cage 4 to 1.27 at cage 1 while growth, expressed as anabsolute increase in grams over the original WAWW, ranged from 5.23 atcage 4 to 5.99 at cage 1 (Fig. 2). Power analysis revealed that 19 and78 mussels were required to detect a 20% and 10% absolute growth dif-ference, respectively.

Chemical analyses of mussel tissues following 90-d exposure con-firmed differences among cage sites in concentration of certain metals, butnot chemicals generally associated with BKME. Twenty-three metals wereanalyzed by ICP-MS with detection limits ranging from 0.02 to 5 mg/Kg.Total plant sterols were lower in cage 3 than all other cages (1050 versus

Fig. 2. A) Trends in Growth as Measured by WholeAnimal Wet Weight. ■ Day 0, ■■ day 90. B) Trends inGrowth as Measured by Shell Length. ■ Day 0 ■ day 90.Bars represent pooled sampled (n = 380–400) + 1 SD. SeeFig. 1 for cage locations.

656 ST-JEAN ET AL.

1580–1670 µg/g) and percent lipids and moisture were similar among cages(Table 1). Chemical analyses of tissue samples showed that chlorophenolsand resin acids were non-detectable in all samples post-exposure. Table 1reports only metals found in concentrations above baseline levels.

Mussels caged for 90 d within a gradient of pulp mill effluent cages(cages 1–3; Fig. 3) showed lower PA (F4,45 = 8.79, p < 0.001) and LR(F4,45 = 6.75, p < 0.001) than mussels caged either within Pictou Harbour(cage 5) or just outside the harbour (cage 4) (Fig. 3). Haemocyte countswere higher in mussels from cages 1 to 3 than 4 to 5 but not significantlyso (Fig. 3).

Bacterial Challenge Test

By 7 d post-inoculation, all mussels from cage 4 had either clearedthe Vibrio (6/10) or had only low (<1000) Vibrio counts remaining (4/10)(Table 2). Mussels from other cages fared less well. At least some musselsfrom cages 1 to 3 and 5 still showed high Vibrio (≥10,000/mL) counts 7 dpost-inoculation and 2 of 10 mussels from each of cages 1 to 3 had died.

Bacterial counts (BC) in haemolymph decreased over time in musselsfrom all cages (F2,135 = 31.75, P < 0.001) in similar fashion (no interactionwith site) and BC also differed among cages (F4,135 = 6.91, P < 0.001). Onday 1, cages 1, 3 and 5 showed higher BC than cage 4 and cage 2 showedlower BC than cage 3 (Fig. 4). Cages 1, 3 and 5 continued to show higherBC than cage 4 on days 4 and 7, however, these differences were no longersignificant. When looking at how rapidly each cage cleared bacteria, cages2, 3 and 4 showed a significant reduction of BC by day 4 (F2,27 = 5.13–9.17,

Table 1. Survival rates and chemical analysis on composite tissue sample ofmussels (n = 40–50) from each cage after 90 d exposurea,b

Survival Cage 1 Cage 2 Cage 3 Cage 4 Cage 5rates MDLc 94% 93% 94% 96% 97%

Al 2.5 31 25 24 19 21As 2.3 3.1 3.5 3.2 3.6 3.2Cu 0.5 2.3 2.5 4 2.3 3.4Fe 5 68 57 55 45 61Mn 0.5 9.6 7.5 7.0 6.6 7.4Zn 0.5 18 19 17 21 26% lipids 1.28 2.28 2.46 2.09 2.27 2.04% moisture 82 77 77 81 79 79

a Results are expressed in µg/g (ww).b See Fig. 1 for cage locations.c MDL; Method detection limit.

IMMUNOMODULATION IN CAGED BLUE MUSSELS 657

p = 0.013 to <0.001) while cages 1 and 5 only showed a significant reduc-tion in BC by day 7 (F2,27 = 4.62–5.96, p = 0.019–0.007).

Discussion

The premise of the caged bivalve study is that if a pulp mill effluentis having a negative impact on the marine biota of the receiving environ-ment, those effects will be reflected in reduced survival and/or changes

Fig. 3. Phagocytic Activity (PA), LysosomeRetention (LR) and Haemocyte Counts (HC) inmussels caged for 90 d in the Pictou Road/PictouHarbour area. LR and PA were standardized to 1 ×106 cells. Cages not differing significantly(ANOVA) share a common letter a–c. Scale isOptical Density (OD) for LR and PA and millions ofcells for HC. ■ PA ■■ LR ■ HC. Bars represent amean (n = 10) + 1 SD.

Table 2. Bacterial counts (BC) in 10 mussels inoculated with Vibrio anguillarum1, 4 or 7 d previouslya

Day 1 Day 4 Day 7

Cages 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

Cleared 0 0 0 1 0 1 3 1 5 1 3 3 1 6 4Low BC 1 5 0 5 2 5 3 4 3 3 2 4 3 4 3High BC 9 5 10 4 8 4 2 4 2 6 3 1 4 0 3Dead 0 0 0 0 0 0 2 1 0 0 2 2 2 0 0

a Low BC = <1000 bacteria/mL, high BC = >10,000 bacteria/mL.

658 ST-JEAN ET AL.

in growth of mussels caged there relative to otherwise comparable areasnot receiving pulp mill effluent. There was no evidence in this study ofreduced survival or growth of mussels caged within the 1% effluentplume relative to mussels caged elsewhere in Pictou Harbour/Road area,and therefore no indication of negative impact of the pulp mill effluent onthe marine biota. In fact, slight nutrification effects of pulp mill effluentwere noted at cage 1. The average overall growth rates exhibited by thetransplanted mussels in this study (11.75–12.77 mm) are comparable togrowth rates of indigenous populations. Shell lengths of indigenous mus-sels were monitored by Freeman (1974) in two Nova Scotia embayments.For the same July to October time frame, those mussels of the 25- to35-mm size class grew at most 10 to 12 mm.

The rate of mussel growth depends on a number of factors includingtemperature, salinity, water depth and food availability. There was littledifference in water temperatures among the cage locations. The tempera-tures recorded at cage 5 were consistently higher by a couple of degreesthan the temperatures recorded at all other cage locations. This cage waslocated in shallow water (3 metres) and was the farthest upriver of all ofthe cages. Its location would be expected to be more influenced by thewarm fresh water flowing down the East River into Pictou Harbour. Thetemperatures at cage 4, which was located near the narrow inlet to PictouHarbour remained between those recorded at cage 5 and the outermost

Fig. 4. Bacterial Counts (BC) in mussels previouslycaged for 90 d in the Pictou Road/Pictou Harbour area1, 4 and 7 d after inoculation with Vibrio anguillarum.Cages not differing significantly (ANOVA) on day 1share a common letter a–c. No significant differenceswere found among cages on days 4 and 7. Brackets joindays on which bacterial counts did not differ signifi-cantly for each cage. Bars represent a mean (n = 10) +1 SD. ■■ Day 1 ■ day 4 ■ day 7.

IMMUNOMODULATION IN CAGED BLUE MUSSELS 659

cages, but exhibited variation and fluctuation on a more frequent basis.This may have been attributable to the twice-daily tidal exchange andassociated cold water influence of the Northumberland Strait.Temperatures recorded at cages 1, 2 and 3 were very similar for the dura-tion of the exposure period.

Tidal influence reaches well inside Pictou Harbour and extends upthe East, Middle and West Rivers. All cage locations were within theboundaries of the tidal influence and salinity does not vary significantlyamongst sites (24–28‰; St-Jean et al. 2001). It is not likely that salinity lev-els had an effect on growth rates.

Comparisons of increases from T = 0 to T = 90 in mean absolutelength and WAWW indicated that, although only marginally differentfrom other sites, cages 1 and 5 exhibited the greatest amount of growth.Of the four cages located outside Pictou Harbour, cage 1 was located inshallow water closest to shore and also closest to the pulp mill effluentdischarge location. Nutrients were detected in the effluent chemicalanalysis (Andrews and Parker 1999) and it is possible that these nutrientsstimulated productivity in the area near cage 1 resulting in increased foodavailability. The abundance of mussel spat attached to the mooring linesand cage observed during cage retrieval indicated that there were natur-al indigenous populations of mussels near this location. Cage 5 was locat-ed inside Pictou Harbour near an area with a high population of indige-nous mussels, aptly named Mussel Point. The presence of the indigenouspopulations near these areas attests to the presence of an abundant foodavailability. A sewage treatment plant for several of the municipalitiesaround Pictou Harbour discharges treated municipal waste to the EastRiver 8 kilometres upstream from the location of cage 5 and is a likelysource of elevated levels of nutrients to this section of the estuary.

One of the weaknesses with the experimental design used for thisstudy was the lack of true replication at the exposure locations. With onlyone cage holding 400 individual mussels at each exposure location, thelevel of replication is the cage and not each individual mussel. This lackof replication reduces the robustness of the statistical analyses. A morerobust design would have been achieved by placing 100 individual mus-sels in each of four separate cages at each exposure location. This wouldprovide an “n” equal to 4 for the statistical tests and should increase theconfidence in the conclusions obtained. A recent Environment Canadaguidance document on conducting caged bivalve studies (JacquesWhitford Environment Limited 2001) recommends a minimum of 20 indi-vidual mussels per cage and a minimum of 5 replicate cages at each expo-sure location. Notwithstanding, the power analysis provides confidencethat the number of individuals was sufficient to determine significant dif-ferences between the cages. In addition, our laboratory has deployedcaged mussels, with true replication, at these locations for three subse-quent years and the immunomodulations observed were consistent eachyear and did not differ between replicates (St-Jean 2001).

660 ST-JEAN ET AL.

All immune parameters measured, except for haemocyte count,showed variations between cages, suggesting that the immune system hasthe potential to be used as an effective early indicator of marine environ-mental health. One of the most striking results of this study is the differ-ence in bacterial clearance between the cages. Although Vibrio spp. are notrecognized as a pathogen of adult mussels, there is evidence suggestingthat they can cause serious damage to mussels by inhibiting filtration(McHenery and Birkbeck 1986), inhibiting chemiluminescence (Lambertand Nicholas 1998) and inducing haemocyte rounding, thus preventinglocomotion (Lane and Birkbeck 1999). The cages showing the most diffi-culty in mounting a defence, cages 1, 3 and 5 (Table 2), also showed thehighest heavy metal burden (Table 1) and conversely, the cage showing themost rapid clearance (cage 4) also showed the lowest heavy metal burden(Table 2). To our knowledge, there have been no in situ studies that exam-ined the relationships between chemical burden, immunomodulations andbacterial clearance in bivalves. Some in situ studies have related haemo-cyte function to body burdens of metals (Pipe et al. 1995a; Cajaraville et al.1996; Dyrynda et al. 1998), organotins (Oubella 1997) or PAHs (Cajaravilleet al. 1996; Dyrynda et al. 1997). These authors showed that the greatestdegree of immunomodulations was found at sites with the greatest chem-ical burden, consistent with our results. In addition, the levels of contami-nation present at the sites studied by those authors, are comparable to thelevels found in Pictou Harbour (Table 3). It is interesting to note that onday 1 cages 1, 3 and 5 showed the highest BC and that cages 1 and 5 onlyshowed a significant reduction in BC 7 d after inoculation, suggesting thatcages 2 and 4 (Fig. 4) were healthier. Cage 4 was situated outside the maincurrents carrying effluents originating from both the harbour and the pulpmill (Kimberly-Clark, unpublished data), suggesting that this cage wasless exposed than others to both sources. It is also reasonable to think thatcage 2 was marginally exposed to effluent from the harbour and due to itsdistance from the pulp mill effluent and position in the water column (8 m)only exposed intermittently to the pulp mill effluent. However, cage 5, wassituated near the untreated municipal wastewater of the town of Pictouand within the area that would also receive waters from the heavily indus-trialized East River, while cage 1 was situated closest to the pulp mill efflu-ent and cage 3 would receive a mixture of waters from the pulp mill as wellas from the harbour. The position of the cages and the heavy metal burdenmay help explain the results of the bacterial challenge. Even though themean BC was much lower for cage 4 than for the other cages, no signifi-cant differences were found between the cages on days 4 and 7. This couldbe explained by the high BC variation found between individuals fromeach of cages 1, 2, 3 and 5; while some mussels were still heavily burdenedby the bacteria others had cleared most or all bacteria (Table 2).

Our study showed no significant difference among sites in HC. Thisresult is similar to that reported by Dyrynda et al. (1998) who showed nosignificant differences in HC in mussels from different sites but contra-dictory to those of Oubella (1997) who showed an augmentation of circu-

IMMUNOMODULATION IN CAGED BLUE MUSSELS 661

Tab

le 3

.Ran

ges

of h

eavy

met

al c

once

ntra

tion

s in

biv

alve

s so

ft ti

ssue

s (µ

g/g

dw

)

Ref

eren

ceSp

ecie

sC

uZ

nFe

Mn

Cr

Cd

Pb

Pipe

et a

l. (1

995a

) M

ytilu

s ga

llopr

ovin

cial

is11

.7–3

8.6

62–1

5974

1–18

3322

.4–3

9.5

1.7–

6.1

2.9–

9.6

12.6

–19

Ven

ice

Lag

oon,

Ita

ly

Dyr

ynd

a et

al.

(199

8)M

ytilu

s ed

ulis

8.19

–14.

969

.8–4

36.5

—0.

4–4.

91.

8–13

.91.

6–29

.9Ta

we

Est

uary

, Red

Riv

er,

Hol

es B

ay U

.K.

Nas

ci e

t al.

(199

9)a

Mer

cena

ria

mer

cena

ria

1.63

–3.5

515

.2–2

0.2

18.8

–27.

210

.1–1

7.5

0.08

–0.1

10.

17–0

.20.

01–0

.16

Tam

pa B

ay, F

la.,

U.S

.A.

And

rew

s an

d P

arke

r (1

999)

aM

ytilu

s ed

ulis

2.3–

4.0

17–2

645

–68

6.6–

9.6

<0.

50.

17–0

.24

<0.

18Pi

ctou

Har

bour

, N.S

., C

anad

a

aM

easu

rem

ent i

n w

w.

662 ST-JEAN ET AL.

lating haemocytes in mussels caged for 6 weeks in a polluted bay (Brest,France). Many studies have suggested that a significant increase in thetotal number of circulating haemocytes is a common response to environ-mental stressors (Cheng 1988; Renwrantz 1990; Anderson 1993; Auffretand Oubella 1994; Coles et al. 1994, 1995). Pipe et al. (1995b), suggestedthat these increases in HC occur by stimulation of the cells from tissues,rather than by blood cell proliferation but, in a parallel study in the PictouHarbour, our laboratory showed mitotic rate to increase within 4 d ofmussels being caged in Pictou Harbour. Dyrynda et al. (1998) suggestedthat studies showing stimulation of HC often involved sudden, short-term exposures and that when bivalves are exposed for longer periods,their HC returns to background levels. More studies are needed to verifythese results.

In comparison to mussels caged in the least anthropogenically impact-ed site (4), mussels exposed to pulp mill effluent (1–3) appeared immuno-suppressed as reflected in depressed PA, LR and bacterial clearance rates.Results from other field studies in contaminated environments showedphagocytic activity to be suppressed when analyzed either microscopically(Sami et al. 1992; Pipe et al. 1995b) or spectrometrically (Dyrynda et al.1998). In other field studies (Dyrynda et al. 1998; Cajaraville et al. 1996),neutral red uptake by lysosomes of mussels (M. edulis and M. trossulus) hasbeen reported to increase. Laboratory exposures to metals in either short-term or low concentration experiments show lysosome swelling, i.e., moredye retained (Kohler et al. 1992; Coles et al. 1995; St-Jean et al. 2002a,b). It ispossible in the present study that after an initial increase in permeability,lysosomes became overloaded, antioxidant protection was impaired anddamage resulted in cages 1 to 3. This would agree with other studies show-ing a pattern of elevation in dye retention following a low, chronic xenobi-otic concentration or short-time exposure (Nott and Moore 1987; Grundy etal. 1996; Moore and Farrar 1985; St-Jean et al. 2002a,b).

Although mussels from cage 5 did not show depressed LR and PA rel-ative to cage 4 they did show reduced bacterial clearance ability similar tocages 1 to 3. It is possible that mussels from cage 5 were exposed to othertypes of contaminants, whether from raw sewage discharged by the townof Pictou or from treated sewage or industrial contaminants from the EastRiver, that would have more impact on other immune parameters, such asthe release of reactive oxygen, the prophenoloxidase-activation system orthe nitric oxide systems (Renwrantz 1990; Anderson 1993, 1996; Coles et al.1994; Pipe et al. 1995b; Dyrynda et al. 1998). To resolve this apparent con-tradiction, more endpoints and reference sites would be needed.

Conclusions

This study showed that immune parameters of mussels have potentialas an early warning tool. The parameters studied provided a sensitive, mea-surable response to pulp mill effluent and suggested their potential utility as

IMMUNOMODULATION IN CAGED BLUE MUSSELS 663

additional endpoints for environmental monitoring. However, this studypoints to the importance of a larger suite of bioassays that would include cel-lular and humoral components as well as a reference site outside the stud-ied area in order to better relate effects. The use of a bacterial challenge incombination with immune parameters not only showed the immune systemto be sensitive enough to discriminate between sites situated within thesame body of water, but also proved to be more sensitive than growth andsurvival for a relatively short-term exposure. Future work should concen-trate on establishing links between sublethal changes, as seen in this study,with growth, survival or reproductive endpoints commonly measured ineffluent effects monitoring programs. This study suggests that the observedsublethal changes in the immune parameters could be related to populationeffects as revealed by the reduced ability to clear an invader and reducedvigour as expressed by the greater mortality (20%) in pulp mill effluentexposed mussels during the challenge test. This could be of importance forfuture programs such as environmental effects monitoring or environmen-tal assessment of locations potentially exposed to anthropogenic inputs.

Acknowledgements

We wish to thank Kimberly-Clark Nova Scotia for their partnershipin the caging experiment and the use of their maps and especially Joe VanBuskirk for his continued help and support. Bob Christie of the PictouHarbour Environmental Protection Project assisted with project coordina-tion and provided financial management to the study. We also wish tothank The Customs House Inn for letting us transform their conferenceroom into a field laboratory. The deployment and recovery of the musselcages was skillfully coordinated by Steve Andrews. Special thanks areextended to the field crew: Kelly Connors, Catherine Elliot, MelissaFredericks, Sarah Kinnie, Jeff Nash, Krista Page, Jaclyn Shepherd andKaren Swan. The charter vessel captain, Allan Elliot, deserves a specialthank you for his skill, patience and cooperation.

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Received: October 22, 2002; accepted: July 3, 2003.