POLYCHLORINATED BIPHENYLS AND THEIR HYDROXYLATED METABOLITES IN WILDFISH FROM WHEATLEY HARBOUR AREA OF CONCERN, ONTARIO, CANADA

EVE A.M. GILROY,y DEREK G.C. MUIR,*y MARK E. MCMASTER,y COLIN DARLING,z LINDA M. CAMPBELL,§

SHANE R. DE SOLLA,k JOANNE L. PARROTT,y SCOTT B. BROWN,**y and JAMES P. SHERRYyyAquatic Contaminants Research Division, Water, Science, and Technology Directorate, Environment Canada, Burlington, Ontario, Canada

zFreshwater Institute, Fisheries and Oceans Canada, Winnipeg, Manitoba, Canada

§Faculty of Environmental Studies, Saint Mary’s University, Halifax, Nova Scotia, Canada

kEcotoxicology and Wildlife Health Division, Wildlife and Landscape Science Directorate, Environment Canada, Burlington, Ontario, Canada

(Submitted 29 January 2012; Returned for Revision 29 May 2012; Accepted 20 August 2012)

Abstract—Whole-body polychlorinated biphenyls (SPCBs) and plasma hydroxylated PCBs (OH-PCBs) concentrations were deter-mined in brown bullhead (Ameiurus nebulosus) from Wheatley Harbour, Ontario, Canada. Elevated SPCBs in Wheatley Harbour aresuspected to have originated from industrial waste disposal and/or discharges from nearby fish processing through discarding of fishremains. Mean SPCB concentrations in brown bullhead from Wheatley Harbour were approximately 250 ng/g wet weight comparedwith approximately 40 ng/g wet weight for brown bullhead from the reference sites, Hillman Marsh and Turkey Creek (both in Ontario,Canada). A significant relationship was found between the concentrations of non-ortho and mono-ortho PCB concentrations (toxicequivalents) and liver mixed-function oxygenase in brown bullhead (r¼ 0.74, p< 0.001). Plasma OH-PCB concentrations were greaterin Wheatley Harbour brown bullhead than in those from Hillman Marsh (3.6 vs 1.5 ng/g wet wt, p< 0.01), and were detectedinfrequently in those from Turkey Creek (0.1 ng/g wet wt, n¼ 2). The OH-PCB congeners most frequently detected were 40-OH-CB172,30-OH-CB180, 4-OH-CB187, 4-OH-CB146, 3-OH-CB138, and 4-OH-CB130, which are structurally similar to the thyroid hormones.To test the hypothesis of fish waste as the cause of the observed PCB contamination of Wheatley Harbour brown bullhead, a principalcomponent analysis (PCA) was used to compare the brown bullhead PCB congener data with equivalent data for Lake Erie walleye,Lake Erie sediment, and industrial Aroclor mixtures. The relative proportions of each Aroclor mixture were estimated using theconjugated gradient method. The high similarity between the congener signatures for Lake Erie walleye and Wheatley Harbourbrown bullhead supports the hypothesis of contamination from the fish processing industry. Environ. Toxicol. Chem. 2012;31:2788–2797. # 2012 Crown in the Right of Canada

Keywords—Areas of concern Polychlorinated biphenyls Hydroxylated polychlorinated biphenyls FishPrincipal component analysis

INTRODUCTION

Following the industrial development of the last century,several regions of the Great Lakes basin have suffered fromhabitat degradation. Under the 1987 protocol of the Interna-tional Joint Commission, 43 of those regions have been des-ignated areas of concern (AOCs). In 2002, Environment Canadainitiated studies readdressing fish health issues in the CanadianAOCs. Studies were developed for investigation of the phys-iological state of wild fish populations, using various indicatorsof overall fish health, as well as a suite of measurements of theirendocrine function, to determine whether wild fish from theCanadian Great Lakes AOCs suffered from endocrine disrup-tion brought about by exposure to contaminants.

Wheatley Harbour AOC is a shallow harbor located on thenorthwest shore of Lake Erie, which includes Wheatley Har-bour and nearby Muddy Creek. Wheatley Harbour has sufferedfrom habitat degradation since at least the 1970s due to indus-trial discharges (e.g., from fish and vegetable processing plants)[1], and is contaminated by polychlorinated biphenyls (PCBs)and other organic contaminants that have accumulated in thesediments. Due to their shallow waters, the sediment contam-

inants in the harbor and Muddy Creek are constantly resus-pended both by dredging and by seiche activity (wind-causedoscillations of the water surface that can last a few minutes to afew hours) from Lake Erie [2], thus increasing their bioavail-ability. In contrast to other PCB-contaminated AOCs, WheatleyHarbour is the site of an important fish processing facility,which, until plant upgrades in 1983, disposed of high volumesof fish waste and other discharges into the harbor waters. ThePCB contamination ofWheatley Harbour is thought to be due tothese historical disposal practices [3,4], a hypothesis that hasnot been previously tested.

Polychlorinated biphenyls are industrial chemicals widelyused as heat transfer fluids, hydraulic lubricants, flame retard-ants, plasticizers, and dielectric fluids. Due to their chemicaland thermal stability and resistance to breakdown, PCBs are notonly ideal chemicals for these applications, they have becomeubiquitous environmental contaminants [5]. The productionof PCBs was halted in the United States in 1978, but use ofPCBs in closed systems continued after the ban on production.Consequently, PCBs remain a major environmental concerndue to their stability, volatility, and persistence.

Polychlorinated biphenyls that were used in North Americawere mostly produced in the United States under the commer-cial name of Aroclor, each consisting of a unique and identi-fiable mixture of individual congeners. In total, 95% of eachAroclor mixture is composed of 110 to 120 of the possible209 different congeners, adding complexity to their chemical

Environmental Toxicology and Chemistry, Vol. 31, No. 12, pp. 2788–2797, 2012# 2012 Crown in the Right of Canada

Printed in the USADOI: 10.1002/etc.2023

* To whom correspondence may be addressed(derek.muir@ec.gc.ca).

**Deceased.Published online 1 October 2012 in Wiley Online Library

(wileyonlinelibrary.com).

2788

analysis [6]. Typically, PCBs are analyzed by gas chromatog-raphy as individual congeners instead of measuring Aroclors,because the composition of PCBs in the environment changesover time due to ‘‘weathering’’ from various chemical andbiological processes including volatilization, chemical trans-formation, degradation, and bioaccumulation, the rates of whichvary among congeners [7]. Congener-based analysis allowscharacterization of samples based on toxicity, which is a moremeaningful estimate for risk assessment purposes; however,many of the congener-specific toxicity data are unavailable [7].

Hydroxylated metabolites of PCBs have emerged as animportant class of environmental contaminants in aquatic ver-tebrates, wildlife, and humans [8]. These metabolites areformed through cytochrome P450 enzyme-mediated metabo-lism of PCBs, via either direct OH insertion in the meta position,or, more commonly, via the formation of an epoxide in themeta-para position, which can be followed by a 1 to 2 shift, thusproducing meta or para OH-PCB metabolites [8,9]. Polychlori-nated biphenyls and their hydroxylated metabolites have beendetected in fish blood from several locations around the GreatLakes [10,11], as well as in the blood of wildlife species andhumans [8,12–15]. Due to structural similarities to thyroidhormones, OH-PCBs have been shown to be retained in blood,associated with thyroid binding proteins, for which they havehigh affinity [8], and are known to act as thyroid hormonedisruptors in mammals [16]. It has been hypothesized that thewidespread presence of PCBs and their hydroxylated metabo-lites, as well as other halogenated phenolic compounds, maycontribute to the thyroid microfollicular hyperplasia observed insalmonids from the Great Lakes (S.B. Brown, unpublisheddata). However, despite the widespread presence of halogenatedphenolic compounds and their potential for disruption of thethyroid and immune systems, their distribution and biochemicaleffects in fish are not well understood.

The sediments from the western basin of Lake Erie containsome of the highest concentrations of PCBs among the GreatLakes, particularly highly chlorinated congeners from Aroclor1260, thought to originate from the Detroit River [17]. Becausethe relative contribution of a contaminant would be expected todecrease with increasing distance from the point of origin,samples from a site close to the source would be expected toexhibit congener patterns most similar to Aroclor 1260, whereasproportions of lower chlorinated congeners would increasewith increasing distance. However, as previously mentioned,Wheatley Harbour has been the site of extensive processing offish products from Lake Erie, and disposal of fish residue isthought to have been an additional source of PCBs until 1983,when plant upgrades were implemented. If fish processing was asignificant source of PCB contamination in Wheatley Harbour,then the relative contribution of contaminants from the DetroitRiver would be lower, and congener patterns would be expectedto differ from those of fish from a local reference site in amanner that cannot only be explained by the distance from thesource.

Several studies have described PCB patterns in environ-mental samples using various multivariate approaches [18–21].Principal component analysis (PCA) has been used to describeand differentiate patterns of contamination in various environ-mental samples, including sediments, fish, birds, and eggs[18,22,23], whereas predictive approaches such as mixingmodels and conjugated gradient models have been used topredict the relative composition of Aroclors [18–20].

The main objective of the present study was to quantify,compare, and contrast whole-body concentrations of SPCBs

and plasma hydroxylated PCBs in brown bullhead (Ameiurusnebulosus) from Wheatley Harbour, Hillman Marsh (a nearbyconservation area on Lake Erie included as a reference site), andTurkey Creek (a tributary of the Detroit River, included as anadditional contaminated site). Furthermore, whole-body PCBconcentrations were used in a PCA to investigate the hypothesisof fish-borne contamination of Wheatley Harbour from fishprocessing. The underlying assumption was that if the contam-ination of Wheatley Harbour was due to the fish processingindustry, then the congener patterns of the brown bullheadsamples from the harbor would most likely resemble thoseof Lake Erie fish, as opposed to those of an industrial PCBsource (e.g., Aroclor mixtures), and would differ from those offish from Hillman Marsh. Thus, additional samples were ana-lyzed for PCB congeners, including walleye (Stizostedionvitreum) collected in the western basin of Lake Erie in 2002,representing a commercial fish species commonly harvestedand processed in Wheatley Harbour, and sediments collectedin Wheatley Harbour in 2002 [4]. Data for rainbow trout(Oncorhynchus mykiss) from the eastern basin of Lake Eriecollected in 2002 and Lake Erie sediments collected in 1998were also added to the multivariate analysis. Data for relativecongener patterns for Aroclors 1016, 1242, 1248, 1254, and1260 [6,24] were also added to the analysis, representingcongener patterns of potential industrial contamination.

MATERIALS AND METHODS

Sampling protocol

The fish sampling protocol is outlined in further detail in ourcompanion paper [25]. Briefly, brown bullhead (298� 135.3 g,4� 1.3 years old [mean� standard deviation], n¼ 42 selectedfor this particular study) were collected using overnight hoopnets between September 11 and 18 (Wheatley Harbour andHillman Marsh) and between October 1 and 9 (Turkey Creek),2002. Figure 1 shows the location of each site. Fish wereanesthetized in a clove oil bath (�0.05þ�0.025% ethanolto aid emulsification). Blood was sampled by caudal puncture,collected in heparinized vials kept on ice, and centrifugedwithin 1 h of collection. The plasma was removed, frozen inliquid nitrogen, and stored at �808C until analysis. Fish weresacrificed by a blow to the head, the various samples and organswere collected and stored as required [25], and the remainingcarcasses were kept on dry ice until return to the laboratory,where they were frozen at �208C until analysis.

Five walleye (2,251� 119.5 g [mean� standard deviation],all 5 years old, collected in 2002) homogenate samples from the

Fig. 1. Location of the three sampling sites from the present study.

PCBs and their metabolites in fish from Wheatley Harbour Environ. Toxicol. Chem. 31, 2012 2789

western basin of Lake Erie were provided by EnvironmentCanada’s National Aquatic Biological Specimen Bank (Envi-ronment Canada, Burlington, ON). The fish were frozen in dryice on collection and transported to the laboratory where theywere partially thawed, measured, weighed, and sexed. Entirefish were homogenized and frozen at�808C [26]. Five archivedsediment samples collected in Muddy Creek in 2002 [4] wereobtained for analysis of PCB congeners. The archived sedimentsamples were frozen at �208C until analysis.

Chemical analysis

Polychlorinated biphenyls. Analysis of brown bullhead andwalleye homogenate samples were performed by the GreatLakes Institute for Environmental Research according to theprocedures outlined in Lazar et al. [27]. Briefly, the fishcarcasses were thawed and homogenized, and 4 to 5 g of fishhomogenates were dried with 60 to 80 g of anhydrous sodiumsulfate extracted in 1:1 dichloromethane:hexane, after spikingwith an internal standard consisting of 100 ng each of 13C12-CB52 and 13C12-CB 153 (multi-ortho PCBs; Cambridge Isotopes),and 20 ng each of 13C12-CB 77, 13C12-CB 126, and 13C12-CB169 (non-ortho PCBs; Wellington Laboratories). The extractswere reconstituted to 10ml, and neutral lipids were determinedgravimetrically [28]. Lipids and coextracted high-molecular-weight biogenic molecules were removed by gel permeationchromatography, and the collected extracts were then concen-trated and applied to a fully activated Florisil column in hexane.A first fraction (multi-ortho PCBs) was collected by elution withhexane. The second fraction (non-ortho PCBs) was collected byelution with 15/85% dichloromethane/hexane, and further sep-arated on a carbon column [27]. Each fraction was reconstitutedin 5ml and stored at 48C until analysis.

Analysis of congener-specific, multi-ortho PCBs was con-ducted using an Agilent 5890 gas chromatograph equipped witha DB-5 column (60m� 0.25mm i.d.� 0.10mm film thickness;Chromatographic Specialties), an Agilent 7673 auto-sampler,and an Agilent 5973mass-selective detector. Helium (1ml/min)was used as a carrier gas. Further details on instrument con-ditions are provided in O’Rourke et al. [29].

Analysis of non-ortho PCBs was performed using an Agilent6890 gas chromatograph, a 7673B auto-sampler equipped witha DB-5 column (30m� 0.25mm i.d.� 0.10mm film thickness,1ml/min helium used as carrier gas), and coupled with aWatersPremier orthogonal acceleration time-of-flight mass spectrom-eter operated in EI- mode. The injector temperature was main-tained at 2758C in splitless mode. The oven program was asfollows: 908C held for 1min, ramped at 308C/min to 2008Cheld for 2min, and ramped at 38C/min to 2808C held for 10min.Postprocessing of the gas chromatograph–mass selectivedetector output was performed using QuanLynx software.The three dominant ions were extracted from the total ionchromatogram over a window of� 10 s from the expectedanalyte retention time. Raw areas (non-ortho PCBs) were thenquantified using the analyte response relative to the externalstandard calibration curve. The sum of PCBs (SPCBs) calcu-lation included the following congeners: 17, 18, 28/31, 33,44, 49, 52, 70, 74, 82, 87, 95, 99, 101, 105 110, 118, 128,138, 149, 151, 153/132, 156, 158, 170, 171, 177, 180, 183, 187,191, 194, 195, 201, 205, 206, 208, and 209. The methoddetection limits for the PCB congeners were calculated using3� SD from eight laboratory blanks, or for undetectablecongeners, from the lowest instrument response at 3� signalto noise [30].

The five Muddy Creek sediment samples were extracted asoutlined in Drouillard et al. [31], and analyzed as outlined abovefor the brown bullhead and walleye samples.

Plasma OH-PCBs and PCBs. Plasma samples from brownbullhead were analysed in Environment Canada (Burlington)laboratories, according to methods developed from Sandauet al. [15,32] with minor modifications outlined in Campbellet al. [10]. Plasma OH-PCB and PCB congeners were analyzedusing an Agilent 6890 gas chromatograph equipped with a 7673automatic injector and a DB5-MS column (30m� 0.25mm,0.25mm thickness; J&W Scientific), and coupled to a high-resolution mass spectrometer (Micromass Ultima; Waters).

All laboratory blanks had undetectable concentrations ofOH-PCBs; hence, a method detection limit was derived fromthe instrument response curves for each OH-PCB congenerbased on a four-point calibration curve at 3� signal to noise[30]. This yielded an average method detection limit of0.0002 ng/g for all 42 individual congeners (range, 0.00018–0.00022 ng/g). The Wheatley Harbour and Hillman Marshsamples were analyzed by standard addition in which theywere spiked with a low-level standard, which was subtracted.

Statistical analysis

Differences among sites were evaluated using Kruskal–Wallis tests. A nonparametric test was selected because thedata did not meet the assumptions of the normal distribution ofresiduals and homogeneity of variance, and because of thepresence of values below the level of detection. Plasma con-centrations of PCBs were expressed in toxic equivalents (TEQs)using the coplanar congeners 77, 81, 126, and 169, and themono-ortho substituted congeners 118, 105, and 156, and therelative coefficients for fish from Van den Berg et al. [33].The relationship between plasma concentrations of PCBs(expressed in TEQs) and liver mixed-function oxygenaseactivity (reported in our companion paper [25]) was testedusing a linear regression.

The homogenized tissue data for 39 PCB congeners from thebrown bullhead from Wheatley Harbour, Hillman Marsh, andTurkey Creek were combined with the data for walleye from thewestern basin of Lake Erie, rainbow trout (2,980� 680.1 g, ageunknown, n¼ 5, collected in 2002) from the eastern basin ofLake Erie (S. Bhavsar, Ontario Ministry of the Environment,Toronto, Ontario, Canada, personal communication), sedimentsfrom Wheatley Harbour [4], and sediments from offshoredepositional areas of the western basin and the southern areaof the central basin of Lake Erie. Data was collected in 1998(sites 947, 1082, 1084, 1086, 1097, and 1111 described inPainter et al. [34]; C. Marvin, Environment Canada, Burlington,Ontario, Canada, personal communication). Data were con-verted to percentages and used in a PCA to compare thecongener profile of various sources with that of the samplesfrom the present study, as well as that of Aroclor 1016, 1242,1248, 1254, and 1260 [6,24], representing unweathered tech-nical mixtures. The first four principal components wereretained following the examination of a Scree plot. Kaiser’smeasure of sample adequacy was used to determine the use-fulness of each variable. The PCAwas refined by the removal ofvariables with a low measure of sample adequacy (below 0.7)one at a time, until the measure of sample adequacy values forthe remaining variables was greater than 0.7 [35]. Variableswith final communality estimates below 0.7 were also removedone by one, until all remaining variables had measure of sampleadequacy and final communality estimate values greater than

2790 Environ. Toxicol. Chem. 31, 2012 E.A.M. Gilroy et al.

0.7. Differences in the first four principal components weretested by multivariate analysis of variance.

The relative contribution of each Aroclor was calculatedusing the conjugated gradient method [36] and by minimizingthe mean square error between the congener pattern in the fishand a theoretical mixture of each Aroclor, as described inde Solla et al. [19]. Differences in the various proportions ofAroclor were tested using a multivariate analysis of variance.

The assumptions of normal distribution of residuals andhomogeneity of variance were verified using the Shapiro–Wilktest and by observing a plot of residual versus predicted values,respectively. The level of significance selected was a¼ 0.05,except when multiple comparisons were made, and where aBonferroni correction was added, such that a value of a¼ 0.05/nwas used, where n is the number of comparisons. Statisticalanalyses were performed using SAS (Version 9.2, The SASInstitute). The relative contribution of each Aroclor was deter-mined using Mathcad (Version 11, PTC).

RESULTS AND DISCUSSION

Polychlorinated biphenyls analyses

All fish analyzed had detectable PCB whole-body concen-trations, with an average of more than 250 ng/g wet weightSPCBs detected in fish collected in Wheatley Harbour (Fig. 2).The penta-, hexa-, and hepta-homologs contributed 76 to 90%of the total PCB body burdens. In contrast, SPCBs were clearlylower in fish from Hillman Marsh and Turkey Creek, averagingapproximately 40 ng/g wet weight. The concentrations of non-ortho and mono-ortho PCBs followed similar trends. The mostprevalent congeners among the non-ortho PCBs analyzed wereCB-77 and CB-126, which were highest in fish collected inWheatley Harbour (p< 0.0001; Fig. 3A). The sum of non-orthoPCBs represented at most 0.02% of SPCBs. The concentrationsof mono-ortho PCBs were considerably greater than those ofnon-ortho PCBs (Fig. 3B; note the different scale), but followedthe same general trend, with brown bullhead from WheatleyHarbour having whole-body concentrations 5 to 10 timesgreater than those from Hillman Marsh and Turkey Creek.When these seven congeners were expressed in tetrachlorodi-benzo-p-dioxin equivalents (TCDD TEQs), the average PCBconcentrations in fish from Wheatley Harbour were 0.2 ng/kgTCDD TEQs, whereas those in fish collected from HillmanMarsh and Turkey Creek were 0.02 ng/kg (data not shown).

Both of those values are below Environment Canada’s guidelinevalue of 0.71 and 4.75 ng/kg TEQs for the protection ofmammals and birds consuming aquatic biota, respectively[37]. Although these calculated values did not include poly-chlorinated dibenzo-p-dioxins/dibenzofurans, dioxin-like PCBstypically contribute more than 70% of the total TEQs in theGreat Lakes [38], suggesting that the addition of polychlori-nated dibenzo-p-dioxins/dibenzofurans would not likely resultin levels greater than the guidelines.

Increased mixed-function oxygenase activity is caused bythe binding of chemicals to the aryl hydrocarbon receptor,which initiates a series of events, including increased synthesisof cytochrome P450, a mixed-function oxygenase catalystprotein. Increased mixed-function oxygenase activity is widelyused to indicate exposure of organisms to specific classes ofplanar organic compounds (e.g., chlorinated dibenzo-p-dioxins,dibenzofurans, polycyclic aromatic hydrocarbons, PCBs) thatcan bind to the aryl hydrocarbon receptor [39]. The liver mixed-function oxygenase activity data from our companion paper[25] were combined with the whole-body concentrations ofplanar PCBs (non-ortho and mono-ortho, expressed as TEQs)from the present study. A significant relationship was foundbetween liver mixed-function oxygenase activity and TEQs(r¼ 0.74, p< 0.001; Fig. 4), suggesting that the twofoldincreased ethoxyresorufin-O-deethylase activity detected in fishfrom Wheatley Harbour likely resulted from exposure to theseplanar PCBs, although the additional contribution of othercontaminants cannot be excluded.

Plasma hydroxylated PCBs

Analytical standards are commercially available for fewerthan 10% of the 837 possible mono-hydroxylated PCB con-geners. Hence, the identification of specific OH-PCBs in envi-ronmental samples is challenging. Gas chromatography withhigh-resolution mass spectrometry can detect congeners withunknown structure that have the same mass and fragmentation

Fig. 2. Whole-body concentrations of polychlorinated biphenyls (PCBs)homologs (ng/g wet wt, mean� standard deviation) and most prevalentcongeners in brown bullhead (Ameiurus nebulosus) from Hillman Marsh,WheatleyHarbour, andTurkeyCreek.Asterisk (�) indicatesvaluessignificantlygreater than other sites (p� 0.007, Kruskal–Wallis nonparametric test witha¼ 0.025 to adjust for multiple comparisons).

Fig. 3. Whole-body concentrations of non-ortho (A) and mono-ortho (B)polychlorinated biphenyls (PCBs) (ng/g wet wt, mean� standard deviation)in brown bullhead (Ameiurus nebulosus) from Hillman Marsh, WheatleyHarbour, andTurkeyCreek.Asterisk (�) indicates values significantly greaterthan other sites (p� 0.001, Kruskal–Wallis nonparametric test witha¼ 0.025 to adjust for multiple comparisons).

PCBs and their metabolites in fish from Wheatley Harbour Environ. Toxicol. Chem. 31, 2012 2791

as OH-PCB standards, thus permitting the detection of uniden-tifiable congeners, in addition to those for which analyticalstandards exist. Analysis by gas chromatography with high-resolution mass spectrometry provides a complete character-ization of all OH-PCB homolog groups and congeners, asdiscussed in further detail in Ueno et al. [30].

Hydroxylated PCBs were detected in the plasma of all brownbullhead collected in Wheatley Harbour and Hillman Marsh,but only in two fish from Turkey Creek. Plasma OH-PCBconcentrations were higher in brown bullhead from WheatleyHarbour and Hillman Marsh than in those from Turkey Creek(SOH-PCBs of 3.6 and 1.5 ng/g wet wt, respectively, comparedwith 0.1 ng/g wet wt in Turkey Creek; p� 0.004; Table 1).Those observations were paralleled by the total concentrationsof plasma PCBs, which were also elevated inWheatley Harbour(SPCBs of 39� 6.7 ng/g wet wt, compared with 11� 3.1 and0.1� 0.02 ng/g in brown bullhead from Hillman Marsh andTurkey Creek, respectively; p� 0.004). Most plasma samplescontained a mixture of penta-, hexa-, and heptachloro OH-PCBs. Mono or dichloro OH-PCBs were not detected, andtrichloro OH-PCBs were only detected in one fish from HillmanMarsh and another from Turkey Creek. Octa- and nonachloroOH-PCBs were not detected.

The six OH-PCB congeners most frequently detected were40-OH-CB172, 30-OH-CB180, 4-OH-CB187, 4-OH-CB146,3-OH-CB138, and 4-OH-CB130 (Table 2). All six congenersare structurally similar to thyroxine in that their OH-group is ina meta or para position, adjacent to two chlorine atoms, and theyalso have a chlorine atom in the para position on the other ring.Hence, these congeners are susceptible to competitive bindingto transthyretin and other transport proteins [8,40]. These sixcongeners were also reported in a survey of hydroxylatedphenolic contaminants in lake trout from various Ontario lakes[10], and from rainbow trout exposed to a mixture of PCBs inthe laboratory [41]. Four of these congeners were also reportedin the plasma of benthic fish species from the Detroit River,at concentrations as high as 65 ng/g for 30-OH-CB138 [11].However, the identified congeners represented only approxi-mately 17% of penta-OH-PCBs, 84% of tetra-OH-PCBs, and

88% of hexa-OH-PCBs at Wheatley Harbour, but 100% oftetra-, penta-, and hexa-OH-PCBs in Hillman Marsh. Studies inhumans, polar bears, seals, and birds have also reported adominance of penta- to heptachlorinated hydroxylated homo-logs and the presence of specific congeners similar to thosedetected in the present study [8,9,12]. Metabolites reported inthe present study have also been noted in marine birds andmammals [9,12–14,32].

Ratios of plasma OH-PCB/PCBs were 0.04 to 0.53 in fishfromWheatley Harbour and Hillman Marsh, whereas they weremuch lower in Turkey Creek—from nondetectable to 0.04. Themetabolism of PCBs and retention of hydroxylated congeners isthought to vary among species and individuals, or both [8], butthese also depend on the exposure profile [12]. The study of Liet al. [11] reported OH-PCB/PCB ratios of 0.01 to 1.51 in theDetroit River, the lowest value being for brown bullhead, whichis similar to that of the Turkey Creek fish from the present study.The highest ratio reported was for lake sturgeon. In comparison,ratios of 0.11 and 0.33 have been reported in human populations[15], of 0.39, 0.57, and 0.90 in albatross [8,12], and of 1.03 to19.47 in polar bear [13]. Although the ratios of plasma OH-PCB/PCBs from the present study are among the lowestreported values, these results appear to indicate some levelof biotransformation of PCBs.

Fish were originally thought to have poor capability tobiotransform PCBs [42], and detected OH-PCBs were pre-sumed to be bioaccumulated from other sources such as abioti-cally or microbially transformed PCBs. However, laboratorystudies have confirmed the ability of rainbow trout to formOH-PCBs via cytochrome P450 2B-like activity, and haveshown that metabolite synthesis may be influenced by watertemperature, as well as enzyme induction through higher expo-sure [41,43]. Moreover, in a survey of Ontario snow, rain,surface water, and particulate organic carbon, Ueno et al. [30]reported detectable OH-PCB concentrations in all samplesanalyzed, with the greatest concentrations near highly popu-lated areas, especially in samples collected near sewage treat-ment plants. The congener patterns varied considerably fromthose of the plasma samples in the present study in that themajority of congener homologs reported were mono- to tetra-chloro OH-PCBs. Thus, the OH-PCBs detected in the plasmafrom brown bullhead, which mainly contained heavier homo-logs (no mono- or di-, mainly penta-, hexa-, and heptachloroOH-PCBs), are likely not from abiotic sources, but rather frombiotransformation by fish themselves, as reported in laboratorystudies with rainbow trout [41,43]. Further studies are needed toconfirm and quantify the biotransformation of PCBs in benthicspecies, such as the brown bullhead, to better assess andunderstand the risks associated with exposure to contaminatedsediments.

Source of PCB contamination: Multivariate analysis

Principal component analysis. The western basin of LakeErie has been reported to contain the highest sediment concen-trations of PCBs among the Great Lakes, particularly highlychlorinated congeners from Aroclor 1260, reported to haveoriginated from the Detroit River, with additional sources ofAroclor 1242 from Lake Huron and the St. Clair River [17].Previous studies have shown that the concentrations of PCBsfrom the western basin of Lake Erie, particularly those ofheavier (more chlorinated) PCB congeners representative ofAroclor 1260, decrease with increasing distance from theDetroit River, which is considered one of the main sourcesof contamination [17,22]. If the main source of contamination

Fig. 4. Liver mixed-function oxygenase activity (expressed asethoxyresorufin-O-deethylase [EROD] activity, pmol/mg/min) as a functionof whole-body concentrations of coplanar (polychlorinated biphenyls [PCBs]77, 81, 126, and 169) and mono-ortho substituted (PCBs 118, 105, and 156)PCBs, expressed in toxic equivalents (TEQs; ng/kg) using the relativecoefficients for fish from Van den Berg et al. [33]. Equation: EROD¼7.722�TEQþ 0.559; r¼ 0.74, p< 0.001).

2792 Environ. Toxicol. Chem. 31, 2012 E.A.M. Gilroy et al.

originated from the Detroit River, the relative proportion ofAroclor 1260 to the congener pattern of the fish from the threesites examined would be expected to decrease with increasingdistance from the point of origin. If dilution only were to explainthe relative Aroclor patterns observed in the present study, andnotwithstanding weathering processes known to take place,brown bullhead from Turkey Creek would have congenerpatterns most similar to Aroclor 1260, whereas brown bullheadfrom Wheatley Harbour and Hillman Marsh, which are approx-imately 60 km east of the mouth of the Detroit River, would beexpected to contain higher proportions of lighter congeners thanthose of Turkey Creek, but quite similar to each other, these twosites being only 10 km apart. Conversely, if fish processing hadbeen an additional source of PCB contamination in WheatleyHarbour (until plant upgrades in 1983), brown bullhead wouldexhibit congener patterns that differ from those of fish fromHillman Marsh and Turkey Creek in a manner that cannot beexplained solely by distance from the point source. To confirmthe origin of the PCBs detected in the fish collected at Wheatley

Harbour, a PCA was performed, in which data from up to 31congeners or groups of congeners were combined from anumber of the following sources: the fish body burden datafrom the present study, data on walleye from the western basinof Lake Erie, rainbow trout from the eastern basin of Lake Erie,sediments collected inWheatley Harbour in 2002 [4], Lake Eriesediments collected in 1998, and relative congener patterns forAroclors 1016, 1242, 1248, 1254, and 1260 [6,24].

The walleye from the western basin of Lake Erie had totalPCB concentrations of 972� 359.1 ng/g wet weight (mean� standard deviation, n¼ 5), approximately three times greaterthan those of brown bullhead from Wheatley Harbour(253� 134.9 ng/g wet wt, n¼ 14; p¼ 0.002). In contrast, therainbow trout from the eastern basin of Lake Erie had totalPCB concentrations of 308� 107.1 ng/g wet weight (mean�standard deviation, n¼ 5). Refinement of the PCA required theremoval of three congeners prior to obtaining measurementsystem analysis values > 0.7. The ordination and scoringcoefficients of the first two principal components are presented

Table 1. Concentratios (mean [min–max], ng/g wet wt) of hydroxylated polychlorinated biphenyls (OH-PCBs) and polychlorinated biphenyls (PCBs) in theplasma of brown bullhead (Ameiurus nebulosus) from the Wheatley Harbour Area of Concern, Hillman Marsh, and Turkey Creeka

Hillman Marsh (n¼ 6) Wheatley Harbour (n¼ 6) Turkey Creek (n¼ 6)

Tri OH-PCBsIdentified 0.1 (<0.0002–0.66)b <0.0002 <0.0002Unidentified <0.0002 <0.0002 0.06 (<0.0002–0.34)b

Total 0.1 (<0.0002–0.66)b <0.0002 0.06 (<0.0002–0.34)b

Tetra OH-PCBsIdentified <0.0002 0.7 (<0.0002–2.37) <0.0002Unidentified 0.2 (<0.0002–0.39) 0.1 (<0.0002–0.15) 0.04 (<0.0002–0.16)Total 0.2 (<0.0002–0.39) 0.8 (<0.0002–2.47) 0.04 (<0.0002–0.16)

Penta OH-PCBsIdentified <0.0002 0.1 (<0.0002–0.75) <0.0002Unidentified 0.4 (0.13–0.93) 0.5 (<0.0002–0.89) 0.1 (<0.0002–0.23)b

Total 0.4 (0.13–0.93) 0.6 (<0.0002–1.64) 0.1 (<0.0002–0.23)b

Hexa OH-PCBsIdentified <0.0002 0.8 (<0.0002–1.41) <0.0002Unidentified 0.3 (0.10–0.53) 0.1 (<0.0002–0.18) 0.01 (<0.0002–0.04)b

Total 0.3 (0.10–0.53) 1.1 (<0.0002–1.89) 0.01 (<0.0002–0.04)b

Hepta OH-PCBsIdentified 0.4 (0.09–1.12) 1.1 (0.29–2.24) <0.0002Unidentified <0.0002 <0.0002 <0.0002Total 0.4 (0.09–1.12) 1.1 (0.29–2.24) <0.0002

SOH-PCBs 1.5 (0.51–1.88) 3.6 (2.21–4.64)c 0.2 (<0.0002–0.77)SPCBs (plasma) 10.8 (3.58–25.12) 39.1 (26.12–70.65)c 15.9 (7.91–30.24)SOH-PCBs/SPCBs 0.21 (0.04–0.53) 0.10 (0.06–0.17) 0.01 (<0.0002–0.026)

aMinimum detection limit: 0.2 pg/g. The data from Turkey Creek were excluded from the statistical analysis because OH-PCBs were only detected in amaximum of two fish.

b Detected in one fish only.c Value significantly different among sites (p� 0.004).

Table 2. Concentrations (mean [range], ng/g wet wt) and frequency of detection of the most commonly detected hydroxylated polychlorinated biphenyls(OH-PCBs) in the plasma of brown bullhead (Ameiurus nebulosus) from the Wheatley Harbour Area of Concern, Hillman Marsh, and Turkey Creeka

Congener

Hillman Marsh Wheatley Harbour Turkey Creek

Mean (range)ng/g wet weight Frequency

Mean (range)ng/g wet weight Frequency

Mean (range)ng/g wet weight Frequency

40-OH-CB 172 0.14 (0.009–0.353)b 6/6 0.35 (0.222–0.624)b 6/6 <0.0002 0/530-OH-CB 180 0.21 (0.060–0.482) 6/6 0.34 (<0.0002–0.757) 4/6 <0.0002 0/54-OH-CB 187 0.14 (<0.0002–0.281) 4/6 0.45 (<0.0002–0.856) 4/6 <0.0002 0/54-OH-CB-146 <0.0002 0/6 0.53 (<0.0002–0.90) 5/6 <0.0002 0/53-OH-CB 138 <0.0002 0/6 0.28 (<0.0002–0.456) 4/6 <0.0002 0/54-OH-CB 130 <0.0002 0/6 0.33 (<0.0002–0.510) 4/6 <0.0002 0/5

aMinimum detection limit: 0.2 pg/g.b Value significantly different among sites (p¼ 0.025).

PCBs and their metabolites in fish from Wheatley Harbour Environ. Toxicol. Chem. 31, 2012 2793

in Figure 5. An examination of the scoring coefficients for eachcongener revealed their distribution along the ordinationplot according to their number of chlorines (Fig. 5A). Thefirst principal component separated the tri-, tetra-, and penta-congeners from the hexa- to nona-congeners, whereas thesecond principal component separated the tri-, tetra-, andpenta-congeners from each other, and the hexa-congeners fromthe other heavier (hepta to nona) congeners (Fig. 5A). Hence,the lighter congeners (e.g., tri- and tetrachlorobiphenyls) weredistributed in the bottom left quadrant, pentachlorobiphenylswere in the top left/center portion of the graph, hexachlorobi-phenyls were in the top right quadrant, and the remaining,heavier chlorobiphenyls (hepta-, octa-, nona-, and decachlor-obiphenyls) were found in the bottom right quadrant. The thirdprincipal component further separated the hepta-, octa-, andnona-congeners (data not shown). None of the principal com-ponents retained appeared to separate the congeners accordingto metabolic group.

The first four principal components explained 84% of thevariance encountered within the data set, and successfullyseparated the majority of the data based on their respectiveproportions of each PCB congener (Fig. 5B). With largerproportions of the lighter biphenyls, Aroclor 1016, 1242, and

1248 occupied the bottom left quadrant of the graph, whereasAroclor 1254 was on the upper center of the graph, and Aroclor1260 was on the center right portion of the graph, closest to thebrown bullhead samples fromHillmanMarsh and Turkey Creek(Fig. 5B), as we hypothesized. The various Aroclor mixtureswere separated on at least one of the first three principalcomponents (p� 0.04). Aroclor 1242 and 1248 were the mostsimilar mixtures, and were only successfully separated on thethird principal component (data not shown). The Aroclor datarepresent unweathered technical mixtures, and were included toprovide the reader with a sense of relative positioning of eachsample in relation to the various commercial mixtures.

The PCB profiles of the brown bullhead fromHillmanMarshand Turkey Creek were included in the analysis as representa-tives of western Lake Erie and Detroit River PCB contamina-tion, respectively. These brown bullhead were successfullyseparated from those from Wheatley Harbour on the first twoprincipal components (p< 0.0001), and were located further tothe right and bottom in Figure 5B, thus exhibiting a greaterproportion of the heavier PCBs (e.g., hepta-, octa-, and nona-homologs). In fact, the congener pattern of the brown bullheadfrom Turkey Creek did not differ from that of Aroclor 1260 onthe first two principal components (p� 0.86), consistent

Fig. 5. Scoring coefficients (A) and ordination (B) of the first two principal components for brown bullhead (Ameiurus nebulosus) collected in the WheatleyHarbour Area of Concern, HillmanMarsh, and Turkey Creek, Wheatley Harbour sediments, walleye from the western basin of Lake Erie, and Aroclor mixtures.

2794 Environ. Toxicol. Chem. 31, 2012 E.A.M. Gilroy et al.

with the Detroit River being a significant source of Aroclor1260. In contrast, the congener patterns for brown bullheadfrom Hillman Marsh differed from that of Aroclor 1260 on thesecond principal component (p¼ 0.04), which is also consistentwith its location further from the source of contamination.However, the brown bullhead samples from Hillman Marshand Turkey Creek were not successfully separated on any of thefour principal components retained (p� 0.14), suggesting thatthese PCB profiles are likely representative of western LakeErie contamination, including the significant contribution ofDetroit River sediment, known to exhibit high proportions ofAroclor 1260 [17,22]. Similarly, the study of de Solla andFernie [44] reported concentrations of PCB congeners in snap-ping turtles (Chelydra serpentina) from southwestern Ontariothat resembled the profile of Aroclor 1260. It was later esti-mated that Aroclor 1260 represented 85.7% of the PCBs insnapping turtle eggs fromWheatley Harbour AOC, and that theremaining 14.3% originated from Aroclor 1254 [19].

The Lake Erie walleye and rainbow trout samples were notsuccessfully separated from Wheatley Harbour brown bullheadsamples on the first two principal components (p¼ 1.00). TheWheatley Harbour brown bullhead samples were separatedfrom the walleye and rainbow trout on the third principalcomponent (p� 0.005), whereas the walleye and rainbow troutwere separated on the fourth principal component (p¼ 0.01).The congener profile of these three sample sets exhibited alower contribution of Aroclor 1260, and thus likely proportion-ately less contamination from the Detroit River [17]. Theabsence of separation of these sample sets on the first twoprincipal components strongly suggests that fish processingwithin Wheatley Harbour was likely a significant source ofcontamination.

The data for Wheatley Harbour and Lake Erie sedimentswere also included in the analysis; these samples were separatedfrom those of Wheatley Harbour brown bullhead on the firstprincipal component (p� 0.01). As best seen in Figure 5B, thesediment samples from Lake Erie were located further towardthe center of the figure (left of the Wheatley Harbour brownbullhead samples), indicating a greater proportion of lighter(e.g., tetra and penta) congeners. Heavier PCB congeners aremore recalcitrant and are generally bioaccumulated to a greaterextent than lighter congeners (with the exception of octa- andnona-homologs) [45], thus causing an enrichment with trophictransfer. On closer inspection of the Lake Erie sediment sam-ples, three separate groups are apparent. Within the sedimentsamples from Lake Erie (Fig. 5B), the four observations locatedcentrally relative to the others, as well as two on the lower left,were from the western basin of Lake Erie, and the two isolated

samples were a sampling location adjacent to the mouth of theDetroit River. The three samples isolated on the upper left werefrom sites located on the south shore of the central basin, whichrepresent suspected additional sources of PCB contaminationfrom tributaries [34]. The sediment samples from WheatleyHarbour were located in the same general axis as the fishsamples, but toward the upper left of Figure 5B, closer toA1254, and thus exhibiting a greater proportion of lowerchlorinated congeners. Although there were no significantdifferences between Wheatley Harbour sediments and LakeErie sediments on the first two principal components (p� 0.06),the Wheatley Harbour sediments appeared to have a congenersignature that is of different origin.

Because the various chemical processes that take place insediments differ from those taking place in fish, fish from aspecific location (e.g., Wheatley Harbour) would not exhibitcongener patterns that are identical to those of the sediments. Infact, despite significant differences between sediment and fishsamples, the close position of the Wheatley Harbour sedimentsin relation to the Wheatley Harbour fish is unexpected, com-pared with that of the Lake Erie sediments and Lake Eriewalleye. This might reflect the strong association between fishand sediments specific to Wheatley Harbour, as well as the factthat until upgrades in the fish processing facility in 1983, fishprocessing was the main source of PCB contamination into theHarbour rather than the sediments being the source of contam-ination. Subsequently, continuous resuspension dynamicsbetween the fish and the sediment likely resulted in equilibra-tion of PCB concentrations, which over time would result in agreater retention of higher chlorinated congeners in fish, asobserved in the present study, yet a greater similarity betweenthe two matrices, compared with other locations in Lake Erie.

In summary, the results of the present study indicate that thecongener pattern of brown bullhead from Wheatley Harbourcannot be explained by its distance from a known source ofcontamination (the Detroit River). The nearly identical PCBcongener patterns of Wheatley Harbour brown bullhead andLake Erie walleye suggest that waste from the fish processingindustry likely contributed to PCB contamination in theharbour.

Congener patterns. The estimation of the relative propor-tions of each congener in our samples indicated that Aroclor1254 and 1260 were the most prevalent sources of contami-nation, with a minor contribution of Aroclor 1248 (except forLake Erie sediments, for which a contribution of 21.6% waspredicted), and little to negligible contribution of Aroclor 1016and 1242 (Table 3). Undoubtedly, some weathering of the PCBstook place, which would contribute to some uncertainty in the

Table 3. Proportion of each Aroclor (mean percentage� standard deviation) in brown bullhead (Ameiurus nebulosus) from the Wheatley Harbour Area ofConcern, Hillman Marsh, a nearby reference site, and Turkey Creek, in the nearby Detroit River, walleye (Sander vitreus) from the western basin of Lake Erie,

rainbow trout (Oncorhynchus mykiss) from the eastern basin of Lake Erie, and sediments from Wheatley Harbour and Lake Eriea

No. A1016 A1242 A1248 A1254 A1260

Hillman Marsh brown bullhead 14 0.02� 0.087b ND 2.5� 4.37 A 3.7� 5.67 B 93.7� 6.51 ETurkey Creek brown bullhead 14 0.6� 1.52 ND 6.9� 6.70 A 5.2� 7.91 B 87.3� 9.05 EWheatley Harbour brown bullhead 14 ND ND 0.9� 1.53 A 34.5� 10.65 C 64.5� 10.83 FLake Erie walleye 5 ND ND ND A 33.0� 2.60 C 67.0� 2.60 FLake Erie rainbow trout 5 12.3� 4.47 0.5� 1.16b ND A 43.4� 4.84 CD 43.8� 2.89 GWheatley Harbour sediment 5 ND ND 3.3� 2.07 A 54.5� 5.45 D 42.2� 3.75 GLake Erie sediment 10 4.8� 3.67 4.8� 7.13 21.6� 6.52 35.4� 7.82 C 33.4� 6.20 G

aValues with corresponding upper case letters were not significantly different (p> 0.05).b Predicted in one fish only.ND¼ not detected/identified.

PCBs and their metabolites in fish from Wheatley Harbour Environ. Toxicol. Chem. 31, 2012 2795

contribution of different Aroclor sources. However, as we didnot have a ‘‘theoretical’’ pattern for weathered Aroclors, wewere limited to using unweathered Aroclor mixtures as refer-ence material, which likely caused some uncertainty in theestimation of the sources of Aroclors.

The multivariate analysis of variance revealed significantdifferences in relative proportions of congeners among thevarious samples (p< 0.0001). On further inspection, we notedthat our model may not accurately distinguish between A1016and A1242, due to the similarity in congener patterns betweenthese two mixtures, as has been reported previously [20]. Therelative contribution of Aroclor 1248 was greater in Lake Eriesediments than any other site (p< 0.0001). The relative pro-portions of Aroclor 1254 were highest in Wheatley Harboursediments (54.5%; greater than every other sample [p< 0.007]except for Lake Erie rainbow trout [p¼ 0.27]). The relativecontribution of Aroclor 1254 was lower in Lake Erie rainbowtrout, Lake Erie sediments, Wheatley Harbour Brown bullhead,and Lake Erie walleye (43.4, 35.4, 34.5, and 33%, respectively),and did not differ significantly (p� 0.29). Turkey Creek andHillman Marsh brown bullhead had the lowest relative con-tribution of Aroclor 1254 (5.2 and 3.7%, respectively; p¼ 1.00).Accordingly, the brown bullhead from Hillman Marsh andTurkey Creek also had the highest relative contributions ofAroclor 1260 (93.7 and 87.3%, respectively; p¼ 0.32), fol-lowed by the walleye from Lake Erie and brown bullhead fromWheatley Harbour (67.0 and 64.5%, respectively; p¼ 1.00),consistent with our hypothesis of dilution of Aroclor 1260 withincreasing distance from its source, the Detroit River. Lastly,the Lake Erie rainbow trout, Wheatley Harbour sediment, andLake Erie sediment had the lowest relative contributions ofAroclor 1260 (43.8, 42.2, and 33.4%, respectively; p� 0.22).The similarity in relative contribution of the various Aroclorsfor Hillman March and Turkey Creek brown bullhead furthersuggests similar sources of contamination, notably A1260from the Detroit River. Furthermore, the indistinguishablePCB congener patterns of Lake Erie walleye and WheatleyHarbour brown bullhead, as seen both by the PCA and thesimilar relative contribution of each Aroclor, suggest that LakeErie walleye are a plausible source of contamination, throughdumping of fish waste from fish processing plants.

CONCLUSIONS

In the present study, we report elevated body concentrationsof PCBs in brown bullhead from Wheatley Harbour, comparedwith those of fish from Hillman Marsh, a local reference site,and Turkey Creek, a contaminated site in the Detroit RiverAOC. Wheatley Harbour brown bullhead also had higher OH-PCB concentrations, which were suspected to originate frombiotransformation of PCBs by the fish. These OH-PCBsretained in the blood are structurally similar to thyroid hor-mones, with which they compete for binding to the hormonetransport protein transthyretin. Using PCA, we showed that thesimilar PCB congener patterns of Wheatley Harbour brownbullhead and Lake Erie walleye support the hypothesis that thecontamination of Wheatley Harbour comes from the historicaldisposal of fish waste and other discharges from the fishprocessing facility. This hypothesis is also supported by ourestimation of the relative proportions of Aroclors, whichshowed the highest proportions of Aroclor 1260 in brownbullhead from Turkey Creek and Hillman Marsh, comparedwith those of brown bullhead from Wheatley Harbour andwalleye from Lake Erie.

Acknowledgement—The authors thank J. Bennett, G. Tetreault, C. Tinson,and M. Villella (Environment Canada, Burlington, ON) for their technicalsupport, and J. Kraft and D. McGoldrick (Environment Canada, Burlington,ON) for contributing sediment and fish samples. Special thanks to GrazinaPacepavicius (Environment Canada, Burlington, ON) for conducting thehigh-resolution mass spectrometry analysis of OH-PCBs. The authors alsothank L. Grapentine for his comments and input on multivariate approaches.This studywas funded by theGreat Lakes Ecosystem Initiative and the GreatLakes Action Plan.

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