Exposure of caged mussels to metals in a primary-treated municipal wastewater plume

Chemosphere 62 (2006) 998–1010

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Exposure of caged mussels to metalsin a primary-treated municipal wastewater plume

Christian Gagnon a,*, Francois Gagne a, Patrice Turcotte a, Isabelle Saulnier a,Christian Blaise a, Michael H. Salazar b, Sandra M. Salazar b

a St. Lawrence Centre, Environment Canada, 105 McGill St., 7th floor, Montreal, Quebec, Canada H2Y 2E7b Applied Biomonitoring, Kirkland, WA 98034, USA

Received 8 February 2005; received in revised form 25 May 2005; accepted 10 June 2005Available online 6 September 2005

Abstract

The biological availability of metals in municipal wastewater effluents is strongly influenced by the physical andchemical conditions of both the effluent and the receiving water. Aquatic organisms are exposed to both dissolvedand particulate (food ingestion) forms of these metals. In the present study, the distribution of metals in specific tissueswas used to distinguish between exposure routes (i.e. dissolved vs. particulate phase) and to examine metal bioavailabil-ity in mussels exposed to municipal effluents. Caged Elliptio complanata mussels were deployed at sites located between1.5 km upstream and 12 km downstream of a major effluent outfall in the St. Lawrence River. Metals in surface watersamples were fractionated by filtration techniques to determine their dissolved, truly-dissolved (<10 kDa), total-partic-ulate and acid-reactive-particulate forms. At the end of the exposure period (90 days), pooled mussel soft tissues (diges-tive gland, gills, gonad, foot and mantle) were analyzed for several metals. The results showed that gills and digestivegland were generally the most important target tissues for metal bioaccumulation, while gill/digestive gland metal ratiossuggest that both exposure routes should be considered for mussels exposed to municipal effluents. We also found thatAg and Cd in the dispersion plume nearest the outfall, in contrast to other metals such as Cu and Zn, are more closelyassociated with colloids and were generally less bioavailable than at the reference site in the St. Lawrence River.Crown Copyright � 2005 Published by Elsevier Ltd. All rights reserved.

Keywords: Metal speciation; Bioavailability; Colloids; Effluent

1. Introduction

Urban effluents represent a major source of chemicalcontaminants for the aquatic environment (Chamberset al., 1997). Because effluents from municipal wastewa-ter treatment plants are derived from both domestic and

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* Corresponding author. Tel.: +1 514 496 7096; fax: +1 514496 7398.

E-mail address: [email protected] (C. Gagnon).

industrial sources, they contain a wide variety of naturaland industrial products. Surveys in Canada have shownthat metals and organic chemicals are present in munici-pal effluents (OMOE, 1988; Rutherford et al., 1994;Ministere de l�Environnement du Quebec and Environ-ment Canada, 2001) and a number of relatively smallindustrial plants release metals directly into municipalsewer systems (Ministere de l�Environnement du Quebecand Environment Canada, 2001). Contaminants re-leased in urban wastewater discharges may be taken

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C. Gagnon et al. / Chemosphere 62 (2006) 998–1010 999

up by the aquatic biota, leading to toxic effects (Ruther-ford et al., 1994; Wong et al., 1995; Gagne et al., 2002).

Predicting the environmental impact of the metalscontained in municipal effluent discharges requires anunderstanding of how their physical and chemical char-acteristics, and those of the receiving waters, affect metaluptake by aquatic organisms and the associated toxicity.Although the toxicity of a municipal effluent is depen-dent on factors related to the treatment processes itundergoes, the physical and chemical characteristics ofthe receiving waters can also influence the toxicity ofcontaminants once they are released to the environment(Lijklema et al., 1993). These same properties also affectthe speciation of both dissolved and particulate metals(Lijklema et al., 1993; Gagnon and Saulnier, 2003).The biological availability of metals is also influencedby their speciation in both the dissolved and particulatephases (Luoma, 1983; Campbell et al., 1988; Wang et al.,1997; Gagnon and Fisher, 1997b).

Physico-chemical parameters such as pH, organiccarbon, suspended particulate matter (SPM) and tem-perature have an effect on the fate of metals and theirbiological uptake by both dietary and waterborne vec-tors (Luoma, 1983; Roditi et al., 2000). Metal partition-ing occurs between the dissolved and particulate phasesin effluent discharged to the aquatic environment, withgeochemical processes playing a major role (Gagnonand Saulnier, 2003; Karvelas et al., 2003). The distribu-tion of particulate metals is considerably influenced byorganic matter as well as by reactive particulate iron(Fe) and manganese (Mn) (Gagnon and Saulnier,2003). Various metal associations with both particulateand dissolved phases are determined by the concentra-tion and strength of specific ligands. Thus, aquaticorganisms are exposed to a variety of chemical formsof a given metal (Campbell et al., 1988; Luoma et al.,1992; Fisher and Reinfelder, 1995; Wang et al., 1997).Metal bioaccumulation, toxicity and exposure routesare probably all influenced by the concentration of eachform of the metal. It has been suggested that metal con-centrations in the gill and digestive gland can be used toestimate waterborne and dietary exposures, respectively(Fisher et al., 1996). Such differences in metal tissue dis-tribution were documented for mussels exposed towaterborne and dietary metals. For example, Longand Wang (2005) recently reported that metal concen-trations in mussel gills were higher than those in theremaining tissues after exposure to waterborne metalsand reversed patterns after dietary exposure.

We related the metal forms at different distanceswithin the dispersion plume with those found in the tis-sues of mussels caged in the plume. The objectives of thestudy were to: (1) measure the distribution of variousmetals in mussel tissues; (2) estimate waterborne versusdietary exposure by comparing uptake in the gill versusthe digestive gland; (3) compare the relative uptake from

water and food by using gill/digestive gland ratios forvarious sites above and below the outfall; and (4)compare metal accumulation in tissue with metals inoperationally dissolved (<0.45 lm), truly-dissolved(<10 kDa), particulate and reactive-particulate phases.Metal accumulation was assessed in mussels placed inthe dispersion plume of an effluent whose dilution byreceiving water can influence the bioavailability and dis-tribution of metals among target tissues. In parallel,changes in the physico-chemical form of metals werestudied in the effluent plume by measuring the dissolved,truly-dissolved, total-particulate, and reactive-particu-late fractions of metals. Moreover, the biomarkers ofvitellogenin and metallothionein were used to confirmmussel exposure to the effluent and its associated estro-genicity and metal exposure, respectively.

2. Methods

2.1. Water sampling

The effluent dispersion plume was previously charac-terized by the determination of total and fecal coliformsor chemical contaminants as markers for municipaleffluents (Gagne et al., 2001a; Gagnon and Saulnier,2003) (Fig. 1). Surface water and suspended matter weresampled at several sites ranging from 0.5 to 15 kmdownstream of a major municipal outfall in the St. Law-rence River (Fig. 1). Samples were also collected at a ref-erence station 1.5 km upstream of the effluent outfall, inorder to document the initial river water conditions. Sur-face water samples were collected using a Teflon pumpfollowing clean sampling procedures for trace metals,as described in Cossa et al. (1996).

2.2. Mussel exposure experiment

Elliptio complanata mussels were collected during thefirst week of May 2001 at Lake de L�Achigan in the Lau-rentians, Quebec, Canada, where mussels have been col-lected for previous studies. The animals were placed inexperimental cages according to standard protocols(ASTM, 2001). Briefly, mussels were sorted by size(weight and shell length) and placed in cylindrical nets,which were attached to a PVC frame (1 m2 surface area).The frames were attached to 20 kg blocks and markedwith a submerged buoy. Two cages were deployed ateach of four sites: 1.5 km upstream (reference station)and 5, 8 and 12 km downstream of the municipal efflu-ent outfall (Fig. 1). The cages could have not been de-ployed any closer to the outfall due to the high currentand suspended matter loads in this stretch of the river.Caged mussels (30 per cage) were submerged in the St.Lawrence River for 90 days, from the beginning of Juneuntil the beginning of September 2001. Unfortunately,

Ontario

Verchères

Repentigny

LAVAL

2 km0

20 km0

Laval

Reference Site

8 km

12 km

MONTREALISLAND

MONTREALISLAND

Ottawa River

Quebec

des Prairies River

des Mille Îles River

Assomption River

Ship channel

Sainte-ThérèseIsland

Verchères Island

LEGEND

Effluent plume

Source: MUC.

Fig. 1. Map of the study area showing the location of mussel cages along the effluent dispersion plume.

1000 C. Gagnon et al. / Chemosphere 62 (2006) 998–1010

all mussels at the site 5 km downstream of the outfalldied, probably by being smothered by high concentra-tions of SPM or by being partially covered with bottomsediment. Nevertheless, the three remaining sites pro-vided a metal contamination gradient and, especially,high upstream–downstream differences in water metalconcentrations. At the end of the exposure period, thecaged mussels were collected and brought to the labora-tory within 5 h for shell length, shell weight, and softtissue weight measurements. In prior biomarker experi-ments, mussels were kept at 15 �C for 24 h in dechlori-nated and aerated tap water to purge the tissues ofrecently ingested material. Mussel soft tissues (digestivegland, gills, gonad, foot and mantle) were collected sep-arately for biomarker and metal determinations andstored at �85 �C until analysis. Mussel tissues for metaldetermination were freeze-dried prior to analysis. Metalsin soft tissues were determined by ion-coupled plasmamass spectrometry following an acid digestion (NationalLaboratory for Environmental Testing (NLET), 1994).

2.3. Water and particle analyses

Water samples (200–500 ml) were filtered through0.45 lm Teflon filters in a class-100 clean laboratoryto obtain the dissolved and particulate fractions. The

filtered samples were then acidified with grade QHNO3 1% (Cossa et al., 1996; Gagnon and Saulnier,2003). Samples of particulate matter (>0.45 lm) ob-tained by filtration were stored at �20 �C until analysis.In addition, 0.45 lm filtered samples were also ultra-filtered within 2 h using a cross-flow tangential fil-tration system to obtain the high-molecular-weight,colloidal (>10 kDa) and truly-dissolved low-molecular-weight (<10 kDa) fractions. The cross-flow ultrafil-tration (CFUF) system was composed of a peristalticpump and a Millipore Prep/ScaleTM-TFF 1 ft2 car-tridge regenerated cellulose membrane with a cut off of10 kDa. The pressure on the membrane was maintainedbetween 14 and 16 psi during the operation. The waterflow rate was adjusted to about 6 l/min before the begin-ning of the ultrafiltration procedure. Ultrafiltered watersamples were, then, acidified (1% Q-HNO3) and storedin acid-clean LDPE bottles until the metal analysis. Dis-solved trace metals were determined by ion-coupledplasma mass spectrometry (National Laboratory forEnvironmental Testing (NLET), 1994). Following theirdigestion with concentrated acids HNO3–HCl–HF, totalparticulate metals were determined by graphite furnaceatomic absorption spectrometry and ion-coupled plasmaemission spectrometry (Dumouchel et al., 1997). Todetermine their reactivity and document their potential


bioavailability, metals associated with suspended parti-cles (retained on the 0.45 lm filter) were also partly ex-tracted using 1 N HCl (Huerta-Diaz and Morse, 1990;Raiswell et al., 1994). This dilute acid solution has beensuggested as one of the best simple extractants for esti-mating extractable metals and assessing their bioavail-ability (Luoma and Bryan, 1978). HCl-extractablemetal analyses were performed on samples of particulatematter as described by Gagnon and Saulnier (2003). Sus-pended particulate matter (SPM), total organic carbon(dissolved and particulate) and other ancillary parame-ters were determined using standard methods describedelsewhere (Cossa et al., 1998; Gagnon and Saulnier,2003).

2.4. Biomarker analyses

Exposure of bivalves to environmental estrogens inmunicipal effluent was confirmed by the vitellogenin bio-marker (Kagi, 1991). Vitellin-like proteins (Vn) weredetermined in mussel gonads using the organic alkali-labile phosphate (ALP) procedure (Blaise et al., 1999;Gagne et al., 2001b; Blaise et al., 2002). For the ALPprocedure, the gonad homogenate was prepared in ice-cold 125 mM NaCl containing 10 mM Hepes-NaOH,pH 7.5, 1 mM dithiothreitol and 1 mM EDTA using aTeflon pestle apparatus. The homogenate was centri-fuged at 15000g at 4 �C for 20 min to obtain the solubleprotein fraction. Then, acetone was added to give 35%v/v and centrifuged at 10000g for 5 min to precipitatehigh molecular weight proteins. The pellet was resus-pended in 200 ll of NaOH 1 M and allowed to standat 60 �C for 30 min. The release of inorganic phosphatewas determined by phosphomolybdenum assay. The re-sults were expressed as lg phosphate per mg gonadprotein.

Induction of metallothionein-like proteins related tothe detoxification of divalent metals (Kagi, 1991; Bau-drimont et al., 1997) were analyzed in digestive gland,gonad and gills by the spectrophotometric thiol assay(Viarengo et al., 1997). Briefly, the tissues were homog-enized in the homogenization buffer and the supernatantseparated at 15000g. Metallothioneins were selectivelyisolated from high molecular weight proteins and lowmolecular weight thiol compounds by ethanol and chlo-roform extractions. The levels of thiols were determinedby the 5,5 0-dithiobis(2-nitrobenzoic acid) reagent withreadings taken at 412 nm. Standard solutions of reducedglutathione were used for calibration. The data wereexpressed as lg of thiol equivalents per mg proteins.

2.5. Quality assurance and quality control

All filtrations and analyses of water samples for tracemetals were performed in a class-100 clean room (Cossaet al., 1996). Quality assurance activities included regu-

lar processing of reagent blanks and uses of referencematerials. The precision and accuracy of metal analysesin the dissolved and ultra-filtrated phase were evaluatedusing internal reference material. The precision for allmetal, expressed as coefficient of variation was 12% orbetter. The accuracy for all metal, expressed as % ofrecovery, was comprised between 82% and 100%.

The reference sediment material MESS-3 (NationalResearch Council of Canada) was used for the evalua-tion of the precision and accuracy of total particulateanalyses. The coefficient of variation for Cr, Cu, Zn,Ni, Cd and Pb was 10% or better and the recovery forthese metals was between 85% and 106%. For Ag, itsconcentration in MESS-3 was to low, thus internal refer-ence material was used and the coefficient of variationand the accuracy were 5% and 109%, respectively. Forreactive metals (HCl 1 N extraction), the MESS-3 wasnot used because is not certified for this chemical extrac-tion. For the evaluation of the analysis precision, aninternal reference material was used and the precision,expressed as coefficient of variation, was 7% or better.

The reference material DORM-1 (NRCC) was usedfor the quality control of mussel tissue analysis. Thecoefficient of variation for Cr, Ni, Cu, Zn, Ag and Cdwas 5% or better and 18% for the Pb. The recoveryfor Cr, Ni, Cu, Zn, Cd and Pb were between 90% and105%. The reference material DORM-1 was not certifiedfor Ag.

Internal reference materials were used for the evalua-tion of the variability and recovery of the SPM, DOCand POC. For these three parameters the coefficient ofvariation was 5% or better and the recovery was between85% and 95%.

2.6. Data analysis

Differences between various metrics at the cage siteswere assessed using Student�s t test when the varianceproved to be homogeneous. In the case of variance het-erogeneity, a non-parametric Mann–Whitney U test wasperformed. Significance was set at p < 0.05. A correla-tion analysis of the metal fractions in receiving watersand in mussels was performed using a stratified versionof Kendall�s tau test (s).

3. Results

3.1. Chemical and physical water characteristics

Concentrations of various parameters such as sus-pended particulate matter (SPM) and dissolved and par-ticulate organic carbon (DOC, POC) were measured tocharacterize the receiving waters and determine theirinfluence on the fate and effects of metals released inurban effluent (Table 1). The results demonstrate the

Table 1Physico-chemical parametersa in wastewater effluent dispersion plumeb

Distance from outfall SPMc (mg/l) POCd (mg C/l) C in SPMc (%) DOCe (mg/l)

Upstream 3.0 (0.2) 0.25 (0.01) 8.3 (0.6) 2.3 (0.1)8 km downstream 3.4 (0.2) 0.34 (0.02) 10.0 (0.7) 2.5 (0.1)12 km downstream 2.9 (0.2) 0.25 (0.01) 8.6 (0.6) 2.4 (0.1)

a Values in parentheses are standard deviations.b Water samples were collected on August 14, 2001.c Suspended particulate matter.d Particulate organic carbon.e Dissolved organic carbon.

Fig. 3. Distribution of particulate trace metals in the dispersionplume.


relative enrichment of urban effluent compared to unaf-fected receiving waters, with SPM, DOC and POC con-centrations being, respectively, 13%, 7% and 36% higherthan those at the reference site. After mixing with receiv-ing waters, SPM and POC concentrations at the site12 km from the outfall dropped to levels similar to thosemeasured upstream (reference station). These variationsreflect dilution in the dispersion plume. Around 75% ofthe effluent was reported to be diluted 5 km down-stream, with 90% dilution occurring in the first 10 kmdownstream of the outfall (Gagnon and Saulnier, 2003).

3.2. Trace-metal distributions in receiving waters

Dissolved-metal concentrations decreased rapidlyalong the effluent dispersion plume and were typicallyhigher downstream than at the upstream site (Fig. 2).At 8 km downstream (i.e. the site nearest the outfall),enrichment factors reached values of 10, 1.3, 20, 1.1,1.2, 0.5, respectively, for dissolved Ag, Cd, Cr, Cu, Niand Zn.

In contrast, particulate concentrations of certainmetals (i.e. Cr, Ni) tended to increase within the first5 km downstream, while others (e.g. Ag, Cu and Zn)were similar to levels for dissolved metals (Fig. 3). Par-ticulate concentrations tended to stabilize further down-

Fig. 2. Distribution of dissolved trace metals in the dispersionplume.

stream. Concentrations of metals associated with SPMwere generally higher at the downstream sites than atthe reference site. At 8 km downstream of the outfall,enrichment factors reached values of 55, 1.5, 0.6, 1.3,0.5 and 0.6, respectively, for Ag, Cd, Cr, Cu, Ni andZn associated with particles.

Municipal effluents release a substantial amountof colloids, which can decrease the relative proportionof metals in the truly-dissolved phase. Distributions ofthe truly-dissolved metal fraction (i.e. <10 kDa), ex-pressed as a proportion (%) of the total dissolved phase(<0.45 lm), are shown in Fig. 4. Truly-dissolved propor-tions of Ag and Cd were considerably lower at the site8 km downstream than at the reference site. Cadmiumand Ag were the two metals with the lowest truly-dis-solved fraction at the site 8 km, with proportions aslow as 30% and 58%, respectively. The other metals pre-sented different trends: the truly-dissolved fraction, forexample, was rather stable or decreased along the dis-persion plume. Much higher proportions of colloidalmetals were found at sites closer to the outfall. Forexample, 65% of dissolved Cd, 95% of Ag and, to a les-ser extent, half of the dissolved Cu and Zn, were mea-sured as colloids at a site 0.5 km downstream of theoutfall (unpublished data). These high colloidal metalfractions observed would be the result of their associa-tion with colloidal material released from the municipal

Fig. 4. Truly dissolved (<10 kDa) metal fractions in the effluentdispersion plume expressed as a percentage of the 0.45 lm-dissolved phase.

Fig. 6. Levels of vitellin-like proteins (Vn) in mussel gonads.


wastewater effluent. Despite the influence of the effluentoutfall on metal speciation, the colloidal metal propor-tions observed are comparable to values found in the lit-erature. For example, Wen et al. (1997, 1999) reportedhigh proportions of metal in colloidal forms: Cd: 45%,Cu 55%, Ni: 36%, Pb: 64%, Ag: 89% and Zn: 91%.The relatively low proportions of the truly-dissolvedfraction observed for some metals in the plume highlightthe influence of colloids in the distribution of dissolvedmetals and their low potential bioavailability.

Municipal effluent is a major source of particulatemetals that are potentially labile or weakly associatedwith suspended particles. The distributions of reactive/labile particulate metals (partial extraction with 1 NHCl), expressed as a proportion (%) of total particulatemetal concentration, are shown in Fig. 5. Partial chem-ical extractions on suspended particles indicate thatreleased particulate metals are generally more reactivein the effluent dispersion plume than at the reference site.Reactive metal fractions in the dispersion plumeexceeded 50% for all metals except Pb. The reactivityof particulate Pb was very low (<20%) at all sites. The

Fig. 5. Distribution of 1 N HCl-extractable: total particulatetrace-metal ratio in the dispersion plume.

highest reactivity value for all metals, except Ag, was ob-served at the site 8 km downstream of the outfall. Gen-erally, the reactive fraction was higher at this site than atthe reference site, and decreased along the dispersionplume. A contrasting trend was observed for Ag andPb, probably explained by their strong affinity for POC.

3.3. Biomarkers

The exposure of mussels to the effluent wasconfirmed by the estrogenic effects observed in the dis-persion plume. Mussels exposed to the municipal waste-water plume had higher levels of vitellin-like proteins(Vn) (Fig. 6). The mean value of Vn in mussel gonadswas 0.05 lg alkali-labile phosphate (ALP)/mg proteinat the reference site, while mean values of 0.13 and0.12 were measured at the sites 8 and 12 km downstreamof the outfall, respectively.

Levels of metallothionein-like proteins (MT) ana-lysed in the digestive gland, gonad and gills also helpedconfirm effluent exposure (Fig. 7). The highest MT

Fig. 7. Levels of metallothioneins (MT) in mussel tissues.

Fig. 8. Metal distribution in mussel tissues following a 90-dayexposure to wastewater. Significant differences (p < 0.05)among sites along the effluent dispersion plume are indicatedas follows: (a) upstream and 8 km; (b) upstream and 12 km and(c) 8 and 12 km downstream.


values were observed in gonads, followed by digestiveglands and then gills. Differences in MT levels for eachtissue were significant (ANOVA, p < 0.05) among sites.For a given tissue, the highest MT value was observedat 8 km downstream, with maximum values of 0.09,0.07 and 0.04 lmol thiols/mg proteins, respectively, ingonads, gills and digestive glands. MT values measuredin the tissues of mussels at the site 12 km downstream ofthe outfall were comparable or even lower than those atthe reference site. These results indicate the presence ofMT-inducing agents in the vicinity of the effluent outfall.

3.4. Metal bioaccumulation in mussels

Mussels that were exposed to the effluent dispersionplume accumulated all the metals measured in their tis-sues (Fig. 8). Accumulated metal concentrations wereabove or similar to beginning-of-test (T = 0) concentra-tions (Table 2). Based on published metal efflux rates formussel (1–5% daily: Fisher et al., 1996; Wang et al.,1997), the duration of our exposure experiment wouldhave allowed caged mussels to depurate from initial con-tamination (i.e. at T = 0). The highest concentrations ofaccumulated metals in exposed mussels were generallyobserved gill and digestive gland. Copper and Zn werethe most abundant metals in the effluent dispersionplume and the most concentrated in gills, with concen-trations exceeding 15 and 350 mg/kg dw, respectively.However, metal levels were not higher in the gills ofmussels at the downstream sites, nor were metal accu-mulations in gills significantly different (p > 0.05) be-tween upstream and downstream sites. The othermetals (Ag, Cd, Cr, Ni, Pb) were also found in musselgills, but at much lower concentrations (<7 mg/kg dw).The levels were significantly lower (p < 0.05) in musselgills at the 8 km site than at the reference site (Fig. 8).Metal concentrations were significantly higher at the site12 km downstream and similar to those at the referencesite.

Based on the observed concentrations of bioaccumu-lated metals, the digestive gland also appears to be animportant target tissue. Concentrations of Cu and Znin the digestive gland reached 20 and 150 mg/kg dw,respectively, while concentrations of the other metalswere similar to levels in gills. Concentrations of Ag,Cr, Ni and Pb in digestive glands were significantly dif-ferent (p < 0.05) among sites. Concentrations of thesemetals were significantly lower (p < 0.05) in the digestivegland of mussels placed nearest the outfall (8 km site)than further downstream (12 km) or at the upstream ref-erence site.

Gill/digestive gland metal ratios were determined toestablish the relative importance of these two main tar-get tissues and to help estimate waterborne and dietarymetal exposures. Although ratios were often higher than1 at the reference station, the ratios were close to 1 for

most metals (except Ni and Zn), suggesting comparableexposures from water and food ingestion at the 8 km site(Table 3).

Concentrations of metals were generally lower in go-nads and in the two non-target tissues (i.e. foot andmantle) than in gills and digestive glands (Fig. 8). Thesedata support the hypothesis that the gill and the diges-tive gland, at a lesser extend, are the primary target tis-sues for metal accumulation. Concentrations of Cu werebelow 15 mg/kg in the non-target tissues, while Zn con-centrations were less than 150 mg/kg. Accumulation of

Table 3Gill/digestive gland metal ratio in mussels located at differentsites

Metal Upstream 8 km downstream 12 km downstream

Ag 1.12 0.60 1.07Cd 1.48 0.75 1.18Cr 4.14 1.06 1.10Cu 0.91 0.75 0.81Ni 0.23 0.08 0.77Pb 0.82 0.72 0.69Zn 2.50 2.91 2.35

Table 2Beginning-of-experiment (T = 0) metal concentrations (mg/Kg) in mussel tissues

Metal Gill Digestive gland Gonads Remainder

Mean SD Mean SD Mean SD Mean SD

Ag 1.21 0.39 2.35 0.79 1.56 0.50 2.08 0.66Cd 5.79 0.82 8.73 1.77 4.89 1.03 9.45 1.36Cr 6.76 1.30 6.71 2.91 2.46 0.43 11.37 4.20Cu 14.33 1.12 15.42 0.67 7.28 1.18 5.67 0.37Ni 1.13 0.87 3.04 1.60 1.06 0.20 3.87 1.63Pb 2.69 0.59 4.23 1.77 3.14 0.98 4.76 1.61Zn 292.5 14.7 134.2 18.6 82.8 3.9 200.5 24.8

SD: Standard deviation.


the other metals in non-target tissues and gonads wasgenerally observed at levels slightly lower than or similarto levels in gills and digestive glands. Data on gonadsand non-target tissues were given little consideration inthis study on metal exposure because metal concentra-tions in these tissues are the result of re-distributionamong all tissues. On the other hand, more attentionwas paid to the metal accumulation in gills and digestiveglands, as they are expected to have more direct contactwith metals, respectively, in the dissolved phase and par-ticulate phase through particle ingestion.

4. Discussion

The exposure of mussels to the municipal wastewaterplume was confirmed through their estrogenic response,i.e. higher levels of Vn (Fig. 6). The synthesis of Vn,which is estrogen-dependent, can be modulated by envi-ronmental chemicals known to interact with the estrogenreceptor (Palmer and Selcer, 1996; Gagne et al., 2001b).

Mussels at the downstream sites had elevated levelsof MT in tissues (Fig. 7). Indeed, there was significant(p < 0.05) induction of MT in mussel tissues, with thehighest MT value being observed at the site 8 km down-stream of the outfall. However, those MT values mea-sured could not be explained by metal uptake intissues at this downstream site. This raises the issue of

the specificity of the MT biomarker towards heavy met-als in complex mixtures. Although MT is generally rec-ognized as a biological marker for metal exposure andmetal detoxification in less complex contaminated aqua-tic environments (Roesijadi, 1992; Couillard et al.,1995), the biomarker does not appear to be specific en-ough for assessing metal exposure in this municipal efflu-ent which releases various domestic, clinical andindustrial contaminants. Indeed, this protein was shownto be induced in aquatic species by other stressors thanmetals such as inflammation from infection (Regalaand Rice, 2004) and oxidative stress (Viarengo et al.,1997; Wright et al., 2000). A previous study at the samesites, showed an increased phagocytic activity which isassociated to infection/inflammation conditions after60 days (Blaise et al., 2002). Lipid peroxidation in mus-sels exposed for 1 year to the municipal effluent plumewas increased in gills and gonad tissues (Gagne et al.,2004). MT induction in aquatic environments contami-nated by a large municipal effluent seems highly depen-dent on the duration of exposure to urban pollutantsand its specificity towards metals is difficult to appraisein such chemically complex environments. To best ofour knowledge, this is the first study showing that MTinduction in aquatic environments contaminated by alarge municipal effluent could be associated to other fac-tors than metals. Hence the value of measuring metalspeciation and bioavailability to tailor out the causativeagents underlying MT induction are of values in envi-ronments contaminated by complex urban pollutants.

4.1. Metal bioaccumulation vs. dissolved phase

Based on the relative tissue levels of metals, the gillsappear to be the major target tissue (Fig. 8). These find-ings support the dissolved phase as an important expo-sure route for mussels (Carpene and George, 1981).The relatively high Cu and Zn accumulation in gills is re-lated to the relative abundance of these metals in thereceiving waters (Figs. 2 and 3). Metal accumulation,however, was generally lower (p > 0.05) for Ag, Cd,Cr, Ni and Pb in mussel gills at the site nearest the

Table 4Correlation analysis (sa) of metal fractions in receiving waters and mussel tissues

Metal fraction Gill Digestive gland Gonads Remainder

s Pb s P s P s P

Dissolved �0.62 0.0041 0.05 0.6959 0.05 0.6950 �0.14 0.4332Truly-dissolved �0.20 0.3544 0.20 0.3544 �0.07 0.6451 �0.07 0.6451Particulate �0.24 0.2377 0.33 0.1130 0.33 0.1138 0.52 0.0153Labile particulate �0.56 0.0159 0.22 0.2890 0.22 0.2890 0.00 0.8332

a Stratified version of Kendall�s tau test (s).b P > Abs (s).


outfall than at the reference site or further downstream(Fig. 8). The low bioaccumulation observed at the8 km site could not be explained by the total dissolvedmetal concentrations in the water. In fact, mussel gillswere not exposed to lower dissolved metal concentra-tions. Rather, they were exposed to concentrations sim-ilar to or higher than those at the upstream site (Fig. 2).As mussel gill can be seen as the target tissue for metalbioaccumulation through the dissolved phase (Longand Wang, 2005), dissolved metal concentrations inthe receiving water were compared to levels in this mus-sel tissue.

A statistical analysis (Kendall�s tau test) was per-formed on data for all the metals combined (Table 4).The analysis showed very few meaningful relationshipsbetween metal exposure and mussel responses and thatmetal concentrations in mussel gills were significantly(p < 0.05) inversely correlated to the levels in the dis-solved phase of the receiving water. This suggests thatlow- and high-molecular-weight dissolved matter con-tribute differently to metal availability in gills. Thus,the dissolved metal concentration in receiving waters isnot directly proportional with metal accumulation inmussel gills. Fig. 9 provides examples (Ag and Cd) ofhow total dissolved concentrations of metals cannot pre-dict the metal bioaccumulation in mussel gills. Otherfield studies on the effects of environmental conditionson metal uptake by mussels showed poor relationshipbetween total metal exposure and metal tissues concen-trations (Gundacker, 1999). The chemical conditions

Fig. 9. Metals in mussel gills vs. concentrations in water total measuand particulate phases do not explain metal accumulation in mussel

of the water, rather than its metal concentrations, ap-pear to influence metal uptake by gills, as speciation ofdissolved metals can change along the dispersion plume.

4.2. Metal bioaccumulation vs. particulate phase

The digestive gland can be viewed as the target tissuethrough particle ingestion. Therefore, metal concentra-tions in this tissue were compared against those in parti-cles. No significant correlation (p > 0.05) was foundbetween metal concentrations in the digestive glandand concentrations in suspended particles (Table 4).Thus, total concentrations of particulate metals alonecannot predict metal bioaccumulation in the digestivegland. However, the metals in the remainder of tissues(after gills, gonad and digestive gland removal) is ex-plained by the particulate phase of metals in the plume(s = 0.52; p = 0.01). As the plume is characterized bysuspended particles rich in organic matter (Table 1), thebioavailable fraction of particulate metals may changealong the dispersion plume. This is shown in Table 3,where the gill/digestive gland metal ratio steadilydropped for most metals along the dispersion gradient.

4.3. Metal bioaccumulation vs. truly dissolved phase

The high concentration of dissolved organic carbonmay be related to colloidal enrichment close to the out-fall. The effluent contains large amounts of organic mat-ter, and relatively high concentrations of particulate

red concentrations of Ag and Cd, for example, in the dissolvedgills.


(POC; 0.34 mg C/l) and dissolved (DOC; 2.47 mg C/l)organic carbon were measured at the 8 km site (Table1). Organic carbon favors the formation of colloidalmetals (Wen et al., 1999), which can considerablychange the speciation of dissolved metals. The relativelyhigh concentrations of POC and DOC can therefore de-crease the fraction of truly-dissolved (<10 kDa) metals.Since total dissolved (<0.45 lm) metal concentrationsdo not provide adequate information on the availabilityfor metal uptake by gills, these tissue/metal ratios wereinstead compared to the results of water ultrafiltrations(Fig. 4). The truly-dissolved (i.e. <10 kDa) fractionwas low for Ag and Cd at the site 8 km from the outfalland increased along the dispersion plume. The relativelylow proportions observed in the plume highlight theimportance of colloids to the distribution of certain dis-solved metals. Cadmium and Ag were the two metalspresenting the lowest truly-dissolved fractions at the8 km site, and this may explain the results reported formetal bioaccumulation in gills at this site. Because natu-ral colloids can influence metal uptake by mussels differ-ently, distinctive metal bioaccumulation behaviors wereobserved (Wang and Guo, 2000; Pan and Wang, 2004).No relationship (p > 0.05) was observed between metalconcentrations in tissue and truly-dissolved phase (Table4). Both truly-dissolved and colloidal metals are bio-available to mussels, but with a relative importance thatis metal-specific (Pan and Wang, 2004). This corrobo-rates the statement that it is the binding properties ofcolloids rather that the concentration of metals in thisfraction that drives the bioavailability in this system.The speciation of metals within the tissues or cells is alsoof consequence for toxicity. For example, metals associ-ated to high molecular weight proteins were related totoxicity in trout hepatocytes (Gagne et al., 1990). Inanother study, the level of the soluble fraction of Cd(cytosolic fraction) was closely related with oxidative-mediated damage in gill tissues (Giguere et al., 2003).The presence of calcium concretions in gill tissues wasalso shown to act as sink for metals although seemsnot to be inducible by site quality (Bonneris et al., 2005).

4.4. Metal bioaccumulation vs. labile particulate phase

The partial extraction of metals on suspended parti-cles with 1 N HCl (Fig. 5) provides key informationfor determining the reactivity and lability of particulatemetals and documenting their potential bioavailability(Luoma and Bryan, 1978; Gagnon and Saulnier, 2003).Iron and manganese hydr(oxides), which are generatedby the mixing of wastewater (more reduced than up-stream waters) with receiving waters and their oxygena-tion, were reported as important carriers of trace metalsand the metals associated with these mineral phases re-mained reactive (Gagnon and Saulnier, 2003). Metalsassociated with Fe-rich particles were reported as bio-

available for freshwater and marine mussels (Luomaand Bryan, 1978; Gagnon and Fisher, 1997b). The highratios of HCl-extractable metal/ total metal observed atthe 8 km site help explain the reactivity of most metals inthe particulate phase (Fig. 5). The ratios of many metals,including Cr and Zn, were higher at the downstreamsites than at the reference station, and remained highalong the dispersion plume. In contrast, the reactivityof Ag and Cd was low at the 8 km site, with ratios reach-ing values comparable to those at the reference site,which indicates that the acid reactivity of suspendedmatter changes spatially in the plume. Interestingly,the bioaccumulation of these metals in the digestivegland showed a similar trend, with minimum values atthe site 8 km downstream albeit was not correlated withall the tissues examined in mussels (Table 4). Neverthe-less, the reactivity of particulate metals suggests thatfood ingestion can also be considered a potential expo-sure pathway for mussels. However, particulate acidreactivity for Ag (lower at 8 km) and Cd (higher at8 km) does not seem to contribute to the gill/digestivegland metal ratio.

4.5. Environmental factors controlling metal availability

The high Corg concentrations (both POC and DOC)observed near the effluent outfall (Table 1) could be ex-pected to influence the bioaccumulation of most metals.The high binding capacity of organic carbon has beenshown to play a major role in metal availability (Sant-schi et al., 1999). Decreased bioaccumulation of mostmetals was observed in mussels located downstream ofthe effluent outfall; surprisingly, neither particulate nordissolved metal concentrations can explain these results.DOC and POC appear to be one of the key factors thatmodulate the bioaccumulation of organic-reactive met-als (Gagnon and Fisher, 1997a; Guo et al., 2001; Voetset al., 2004; Lorenzo et al., 2005).

Metal speciation within the dissolved phase, whichcan be considerably modified by DOC, might influencemetal uptake by mussel gills. The presence of dissolvedorganic carbon such as humic acids was reported toclearly damper Cu uptake in mussel gills, but did nothave effect on whole-animal uptake (Lorenzo et al.,2005). Those reported results suggest that dissolved or-ganic substances influence differently metal uptake bymussel gills and remaining tissues. We observed thatthe truly-dissolved proportion of Ag and Cd decreased,while the gill/digestive gland ratio decreased. The factthat metal concentrations in gills decrease while DOCincreases may point out DOC as a key factor controllingthe speciation of organic-reactive metals such as Ag andCd. However, other factors (such as MT expression)must also be considered in those complex aquatic sys-tems. We also observed that the reduced gill/digestivegland metal ratio appears to be caused by a decrease


in Ag and Cd levels in gills, but not in the digestivegland. As the proportion of metals in the dissolvedphase was also reduced by a concomitant rise in the par-ticulate phase, it can be suggested that the particulateform of the metals contributes to reduced gill/digestivegland metal levels.

Changes in the distribution of metals between the dis-solved and particulate phases in the effluent dispersionplume was shown to considerably influence the exposureroutes of contaminants for aquatic organisms as evi-denced by gill/digestive gland metal ratios. A compari-son of metal accumulation levels in gill and digestivegland allowed us to estimate the metal uptake pathwaysin effluent-exposed mussels. The results of gill/digestivegland metal ratios were mixed (Table 3). Cadmium,Cr, Ag and Zn showed the highest ratios (>1), suggest-ing a higher metal uptake in gills in effluent receivingwaters (i.e. upstream site). Gill/digestive gland metalratios at the 8 km site, however, were close enough to1 to suggest that metal uptake by gills and digestiveglands were equivalent at the 8 km site nearest theeffluent outfall, despite the considerable metal concen-trations measured in the total dissolved phase. Theseresults suggest that metal speciation in the effluent dis-persion plume is crucial to understand the importanceof waterborne and dietary exposure routes and theirenvironmental relevance to metal accumulation by thesefilter-feeding invertebrates.

5. Conclusions

The distribution of metals in target tissues of musselswas used to distinguish the exposure routes (dissolvedand particulate forms) for mussels exposed to municipaleffluents. Gills and digestive gland but at a lesser extentfor the latter, are target tissues for most metals in botheffluent dispersion plumes and receiving waters. Metalaccumulation was generally lower (i.e. less bioavailable)in mussels placed nearest the effluent outfall (8 kmdownstream) than at the upstream reference site,although MT was significantly induced. MT inductionis a response to overall exposure to metals, but the bio-marker appears not specific enough to metal exposure incomplex contaminated sites such as environments im-pacted by municipal effluents. The bioaccumulation ofmetals in mussel tissues (except the remainder) was notproportional to total concentrations of either dissolvedor particulate metal forms in receiving waters. Physico-chemical conditions of receiving waters (e.g. organic car-bon content) can control metal uptake in the dissolvedphase. Truly-dissolved metal fractions were relativelylow near the outfall and may reduce the bioavailabilityof specific metals in the dissolved phase for mussel gills.The results of selective metal extractions using 1 N HClindicated that the particulate metals in the dispersion

plume are in the labile form (i.e. 1 N HCl-reactive). Met-als in the particulate phase should probably be consid-ered potentially bioavailable by food ingestion, andthey contribute to metal accumulation by musselsthrough this exposure route. Although metal accumula-tion from the dissolved phase appears to be a logicalroute of exposure in most cases, both dissolved and par-ticulate sources must be considered when evaluating thebioavailability of metals released in municipal effluents.Metal accumulation in the gill and digestive gland sug-gests that metal exposure and accumulation from bothwaterborne and dietary sources in municipal effluentreceiving waters are significant and should be the subjectof further study.

Acknowledgements

We are grateful to M. Bouachrine, L. Gougeon, M.Arseneau and G. Brault for their help in the field andthe laboratory. Statistical analyses were performed byP. Gagnon and M. Bouachrine. The manuscript was edi-ted by P. Potvin. This project was funded by the St.Lawrence Centre, Environment Canada (to C.G.) andthe International Copper Institute (to M.H.S.).

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Exposure of caged mussels to metals in a primary-treated municipal wastewater plume

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