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Environmental Origins of Methylmercury Accumulated in Subarctic Estuarine Fish Indicated by Mercury Stable Isotopes Miling Li, Amina Traore Schartup, Amelia P. Valberg, Jessica D. Ewald, David P. Krabbenhoft, Runsheng Yin, Prentiss H. Balcom, and Elsie M. Sunderland Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03206 • Publication Date (Web): 02 Oct 2016 Downloaded from http://pubs.acs.org on October 4, 2016

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Environmental Science & Technology

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Environmental Origins of Methylmercury Accumulated in Subarctic Estuarine Fish

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Indicated by Mercury Stable Isotopes

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Miling Li1,2*, Amina T. Schartup1,2, Amelia P. Valberg1,2, Jessica D. Ewald2, David P.

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Krabbenhoft3, Runsheng Yin4, 5, Prentiss H. Balcom2, Elsie M. Sunderland1,2

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Boston, MA, USA

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2

Department of Environmental Health, Harvard T.H. Chan School of Public Health,

Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard

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University, Cambridge MA, USA

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U.S. Geological Survey, Middleton, WI, USA

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Department of Civil and Environmental Engineering, University of Wisconsin-Madison,

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WI, USA

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Academy of Sciences, Guiyang 550002, China

State Key Laboratory of Ore Deposit Geochemistry, Institute of Geochemistry, Chinese

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*Corresponding author. E-mail: [email protected]. Phone: 617-496-5492. Fax:

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617-496-4551. Address: 29 Oxford Street, Cambridge, MA 02138, USA.

ACS Paragon Plus Environment

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Abstract

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Methylmercury (MeHg) exposure can cause adverse reproductive and

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neurodevelopmental health effects. Estuarine fish may be exposed to MeHg produced in

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the terrestrial environment, benthic sediment and the marine water column but the

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relative importance of each source is poorly understood. We measured stable isotopes of

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mercury (δ202Hg, ∆199Hg, and ∆201Hg), carbon (δ13C), and nitrogen (δ15N) in fish with

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contrasting habitats from a large subarctic coastal ecosystem to better understand MeHg

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exposure sources. We identify two distinct food chains exposed to predominantly

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freshwater and marine MeHg sources but do not find evidence for a benthic marine

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MeHg signature. This is consistent with our previous research showing benthic sediment

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is a net sink for MeHg in the estuary. Marine fish display lower and less variable ∆199Hg

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values (0.78‰ to 1.77‰) than freshwater fish (0.72‰ to 3.14‰) and higher δ202Hg

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values (marine: 0.1‰ to 0.57‰; freshwater: -0.76‰ to 0.15‰). We observe a shift in the

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Hg isotopic composition of juvenile and adult rainbow smelt (Osmerus mordax) when

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they transition between the freshwater and marine environment as their dominant

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foraging territory. The Hg isotopic composition of Atlantic salmon (Salmo salar)

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indicates they receive most of their MeHg from the marine environment despite a similar

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or longer duration spent in freshwater regions. We conclude that stable Hg isotopes

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effectively track fish MeHg exposure sources across different ontogenic stages.

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Introduction Methylmercury (MeHg) is a bioaccumulative neurotoxicant produced from

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divalent inorganic mercury (HgII) in aquatic ecosystems that adversely affects the health

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of humans and wildlife.1-3 Benthic sediment, rivers, wetlands, and the marine water

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column are all potential locations for MeHg formation and uptake into estuarine

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foodwebs.4-8 The origin of environmental MeHg sources accumulated by fish affects their

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responses to changes in HgII inputs. For example, there may be a substantial temporal lag

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between atmospheric Hg inputs and MeHg levels in benthic sediment but a more rapid

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response in surface seawater.9 Tracing the environmental origins of fish MeHg is

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challenging due to their diverse foraging preferences and migration patterns. Stable Hg

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isotopes show promise as a new empirical tracer for fish foraging behavior and

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corresponding MeHg exposure sources.10-13 Here we use stable isotopes of Hg, carbon

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and nitrogen in subarctic estuarine fish with diverse habitats to characterize their

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predominant environmental MeHg exposure sources.

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Bioaccumulation modeling for MeHg suggests dietary ingestion accounts for

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>90% of the body burden in fish.14, 15 Stable isotopes of carbon (reported as δ13C) and

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nitrogen (reported as δ15N) provide information on fish habitat type and trophic

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position.16 However, δ15N and δ13C change more rapidly in fish than MeHg because their

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half lives (5-173 days) are generally much shorter than persistent and bioaccumulative

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contaminants like MeHg (~1-4 years).17-22 Understanding long-term foraging patterns is

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particularly important for anticipating MeHg levels in species such as salmon and trout

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that shift their diet and/or foraging territory over different ontogenic stages. Thus,


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naturally occuring Hg stable isotopes in fish can potentially provide complementary

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information to δ15N and δ13C.10, 12, 23

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All seven Hg stable isotopes undergo mass-dependent fractionation (MDF,

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reported as δ202Hg) as the result of many physical, chemical, and biological processes.24-

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predominantly during photochemical reactions, wherein the odd-mass-number isotopes

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are either enriched or depleted in reaction products relative to the even-mass-number

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isotopes.26, 29, 31 MIF is widely observed in aquatic organisms.26, 32, 33 However, no

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substantial isotopic fractionation has been observed between MeHg in plankton and

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higher trophic level fish.11, 34, 35 This allows MIF signatures to serve as a conservative

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tracer for environmental MeHg sources accumulated by fish.

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Mass-independent fractionation (MIF, reported as ∆199Hg and ∆201Hg) occurs

Prior work suggests different sources and/or reaction pathways of Hg species

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drive the variable Hg isotopic composition measured in terrestrial, coastal, and oceanic

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biota. Tsui et al.13 found riverine and terrestrial food webs have distinct δ202Hg values,

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which they attributed to different MeHg sources. In aquatic ecosystems, MIF is primarily

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a function of photochemistry and varies with the extent of light penetration in different

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foraging regions. Blum et al.12 showed pelagic marine fish foraging in deeper waters with

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limited light penetration had lower ∆199Hg values relative to those feeding in surface

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waters. Senn et al.10 showed the ∆199Hg values in pelagic fish were greater than coastal

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fish from the Gulf of Mexico, presumably due to reduced water turbidity, enhanced

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photochemistry and faster demethylation in the open ocean water column.

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The main objective of this paper is to investigate whether stable Hg isotopes can elucidate the environmental origins of MeHg accumulated in fish from coastal


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ecosystems with multiple potential exposure sources (rivers, benthic sediment, marine

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water column). Prior work has focused on mid-latitude ecosystems and demonstrated

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differences in Hg isotopic signatures of biota from two contrasting food webs (oceanic

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vs. coastal, terrestrial vs. riverine)10, 13 but has not extended these findings to the diversity

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of MeHg exposure sources and contrasting food-webs within a single coastal ecosystem.

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Here we report new measurements of Hg, C, and N stable isotopes and tissue Hg and

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MeHg burdens in fish and shellfish with contrasting benthic, benthopelagic, and pelagic

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habitats from the region within and surrounding Lake Melville, a large subarctic fjord in

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Labrador, Canada (Supporting Information (SI), Figure S1).

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Methods

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Sample collection

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Table 1 lists the fish and shellfish species analyzed in this study and their

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predominant habitats. We reviewed the literature on the indigenous composition of

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benthic, benthopelagic, and pelagic marine and freshwater fish commonly found in the

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Lake Melville region to identify species that represent each habitat type. Inuit community

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members harvested 15 types of fish and shellfish (n=202) from the main freshwater

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tributary to Lake Melville (lower Churchill River), the estuary (Lake Melville), and the

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Labrador Sea between June and August of 2014 and 2015 (Table 1). Smaller fish were

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captured by minnow traps and larger fish were obtained by rod or directly from

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commercial fishing vessels in Lake Melville. Standard length and weight of each whole

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fish was measured in the field or lab. Each fish was frozen after collection and shipped to

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Harvard University for analysis.


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In the laboratory, we collected subsamples of axial muscle tissue and freeze-dried

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and homogenized samples prior to analysis for Hg, MeHg and stable isotopes. Minnow

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species (stickleback, juvenile smelt, and chub) were freeze dried and whole fish samples

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were homogenized using a blender because they were too small to fillet muscle tissue.

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Surface sediment samples (0-3 cm) were taken from a box corer or stainless steel gravity

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corer in Goose Bay, Groswater Bay, and the Churchill river (September 2012 and June

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2013) (SI, Figure S1), as described in Schartup et al.5, and analyzed for Hg, MeHg and

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Hg isotopes in this work.

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Fish species age, habitat and diet classification

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We harvested available fish scales and otoliths for age determination at NOAA’s

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Northeast Fisheries Science Center in Woods Hole, MA. We determined the age of

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Atlantic salmon and brook trout by analyzing their scales, following established

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methods.36, 37 Otoliths were used to age other species following the “thin-section and

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bake” technique.38 Stomach contents of all fish were dissected and examined prior to

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chemical analysis. Qualitative diet composition from this analysis was combined with a

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literature synthesis of the feeding ecology and life history for all fish to determine the a

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priori habitat of all species (Table 1), which we compared to information from Hg

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isotopes in this analysis.

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Total Hg and MeHg analysis

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We measured total Hg concentrations in all fish samples by thermal

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decomposition, amalgamation, and atomic absorption spectrophotometry (EPA method

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7473) using a Nippon MA-3000 Mercury Analyzer. At least one method blank and one

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certified fish tissue reference material (CRM: TORT 3) was tested every 10 samples.


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The average Hg recovery for the TORT 3 CRM was 100.7±2.2% (n=22).

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We measured MeHg concentrations in all minnows, mussels and benthic fish

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following a modified EPA 1630 method established in prior work.40-42 The tissue Hg

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burden of predatory fish (cod, salmon, trout, pike, and whitefish) is widely assumed to be

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close to 100% MeHg43 but lower trophic level organisms have more variable MeHg

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fractions.44, 45 Samples were spiked with 1 ml enriched Me201Hg (2 ng/ml) then digested

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with 5N HNO3 solution at 70 ºC overnight prior to MeHg analysis. Two CRMs (TORT 3

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and DORM 4) were included in each digestion cycle. Acid was neutralized with 8 N

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KOH, buffered with a 2M acetate buffer. Aqueous MeHg was ethylated using sodium

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tetraethylborate (NaBEt4). Ethylated MeHg was purged onto a Tenax packed column and

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separated by gas chromatography using a Tekran 2700 MeHg auto-analyzer coupled to a

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Thermo iCAP-Q ICP-MS with Teflon tubing for MeHg detection.8, 46 We analyzed

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ongoing precision and recovery (OPR) standards with different concentrations every 10

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samples. Mean recovery was 105.5±5.7% (SD; n=8). The average recoveries for TORT 3

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and DORM 4 were 92.4±5.0% (SD; n=8) and 99.7±9.8% (SD; n=8). Precision, estimated

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by replicate analysis of CRMs and duplicate fish samples, was better than 7% and 8%,

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respectively.

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Carbon and nitrogen isotope analysis

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Freeze dried and homogenized fish tissue samples were analyzed for stable

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isotopes of carbon and nitrogen at Boston University’s Stable Isotope Laboratory and the

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University of Hawaii at Manoa’s Isotope Biogeochemistry Laboratory.47 Isotopic values

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are reported in conventional δ-notation relative to international standards (C: Peedee

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Belemnite; N: atmospheric nitrogen). A secondary standard (glycine) was included to


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ensure instrumental accuracy and precision in both labs. The standard deviation around

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the expected values for the secondary standards was within 0.3% and 0.2% for δ13C and

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δ15N, respectively. As lipids have more depleted δ13C values relative to other tissues,

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aquatic samples with high lipid content (i.e., C:N ratio >3.5) are generally reported as a

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normalized δ13C value by either lipid extraction or mathematical methods.48, 49 We used

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the C:N ratio as a proxy for lipid content to estimate δ13C values of lipid-free muscle and

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whole-body tissue, following the relationships derived by Logan et al.49

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Hg stable isotope analysis

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Approximately 0.1 to 0.2 g of freeze-dried and homogenized fish (n=135) and

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sediment samples (n=7) were digested at 120°C for 6 hours using a 2 mL acid mixture

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(HCl:HNO3 = 1:3, v:v) for stable Hg isotope analysis, following the protocol established

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by others.33, 50 The digest solutions were diluted to 1 ng mL-1 for most samples. For

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samples with low available mass or low tissue total Hg concentrations, digest solutions

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were diluted to 0.4 – 0.5 ng mL-1. Samples were analysed using a Neptune Plus

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multicollector inductively coupled plasma mass spectrometry (MC-ICP-MS) housed at

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the Wisconsin State Laboratory of Hygiene.

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The sample introduction system and analytical methods for stable Hg isotope

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analysis follow previous studies.51, 52 Briefly, samples containing Hg were continuously

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mixed and reduced with 3% SnCl2. The cold vapor generator was custom designed, as

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described in Yin et al.52 Volatile Hg(0) was separated by a frosted glass phase separator

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and introduced to the MC-ICP-MS with Argon gas. Instrumental mass bias was corrected

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using an internal Tl standard (NIST SRM 997) and sample-standard bracketing. The Hg

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concentrations and acid matrices of the bracketing standard (NIST SRM 3133) were


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systematically matched to the neighbouring samples. Data nominations follow the

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protocols suggested by Blum and Bergquist.53 MDF is expressed using δ202Hg notation

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(Equation 1). MIF is expressed as the difference between the measured δxxxHg value, the

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value predicted based on MDF and the δ202 Hg value (Equation 2-3).53

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δ202 Hg = [202/198Hgsample /202/198 HgNIST3133 -1]×103 ‰

(1)

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∆199Hg ≈ δ199Hg － (δ202Hg * 0.2520)

(2)

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∆201Hg ≈ δ201Hg － (δ202Hg * 0.7520)

(3)

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All CRMs (TORT-2, TORT-3, DORM-2, DORM-4, MESS-1) were prepared and

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analysed in the same way as the samples. Total Hg in all solutions was monitored by

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MC-ICP-MS using 202Hg signals, which yielded mean recoveries of 94.0% for fish

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samples and CRMs. The UM-Almadén standard solution (0.5~1.0 ng mL-1, diluted in

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10% aqua regia) was measured once every 10 samples. We found the overall average and

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uncertainty of UM-Almadén and CRMs agreed well with prior work (SI, Section 1).13, 51,

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Data analysis Differences in isotopic composition (δ13C, δ15N, ∆199Hg, ∆201Hg, and δ202 Hg)

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among fish with different habitats and ecological zones were examined using two-way

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ANOVA and Tukey's Honest Significant Difference (HSD) test. The ∆199Hg /∆201Hg

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ratio was calculated from the slope of York regression which accounts for error in both

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the dependent and independent variables.55 All statistical analyses were performed using

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R.56


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Results and Discussion

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Fish habitats and hypothesized MeHg sources

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Fish species collected as part of this work span predominantly freshwater and

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marine habitats. Within these environments, we collected species that represent benthic,

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benthopelagic, and pelagic habitats. Many species are exposed to a mixture of MeHg

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sources (benthopelagic and anadromous fish). We hypothesized that the predominant

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habitat type of each fish would dictate their environmental MeHg source and, in turn,

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would be recorded by distinguishable differences in the Hg isotopic composition of their

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tissues.

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Table 1 shows the literature and δ13C derived habitats (SI, Figure S2) and prey of

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different fish and shellfish included in this study. Lake chub, northern pike, and juvenile

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rainbow smelt have low tolerance for high salinity conditions57-59 and thus primarily

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forage in the freshwater environment. Potential MeHg sources in the freshwater

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environment include benthic sediment in rivers, terrestrial soils, and/or wetlands.13, 60 In

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brackish sections of the Churchill River (the main freshwater tributary to Lake Melville

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estuary), some MeHg may also be transported inland from the estuarine river mouth

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during seasonal intrusion of tidal waters.61, 62

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Herring, capelin, and Atlantic cod are generally considered offshore marine

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species, although they may migrate into estuaries for food and/or spawning (Table 1).59

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In the marine environment, MeHg is produced both in the water column and in benthic

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sediment.5, 6, 8, 63-65 Species with a strong link to the benthic environment across different

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habitats include shorthorn sculpin (marine), flatfish (anadromous) and longnose sucker

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(freshwater).59 All other species are anadromous or benthopelagic and likely to be


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exposed to MeHg from multiple sources (Table 1).

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Distinct freshwater and marine food chains

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Distinct freshwater and marine food webs are evident from the Hg isotopic

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composition of fish and shellfish species measured in this study (Figure 1a). Marine

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species from this study (capelin, herring and cod) cluster together based on their Hg

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isotope composition and are enriched in δ202Hg relative to freshwater species (chub, pike,

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and juvenile smelt). Anadromous fish generally fall between the marine and freshwater

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food webs (Figure 1a). We expected the Hg isotopic composition of benthic species

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(shorthorn sculpin, flatfish, longnose sucker) to also be distinct. Instead, benthic species

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cluster with pelagic and benthopelagic species across marine, anadromous, and

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freshwater food webs. The lack of a distinct benthic signature suggests a common MeHg

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source across benthic and pelagic environments (Table 1, Figure 1).

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Further evidence for a common MeHg source is provided in Figure 2, which

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shows that mean ∆199Hg and δ202Hg values in species within each foraging region

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(freshwater, mixed, marine) do not differ significantly among benthic, benthopelagic and

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pelagic habitats. By contrast, mean δ13C values in benthic species are significantly higher

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(Tukey’s HSD, p

Environmental Origins of Methylmercury ... - ACS Publications

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