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Mercury and Selenium Balance in Endangered Saimaa Ringed Seal Depend on Age and Sex Merja Lyytikäinen, Juuso Pätynen, Heikki Hyvärinen, Tero Sipilä, and Mervi Kunnasranta Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b01555 • Publication Date (Web): 15 Sep 2015 Downloaded from http://pubs.acs.org on September 19, 2015

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Environmental Science & Technology

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Mercury and Selenium Balance in Endangered Saimaa Ringed Seal Depend on Age and Sex

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Merja Lyytikäinen*, Juuso Pätynen, Heikki Hyvärinen, Tero Sipilä†, Mervi Kunnasranta

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Department of Biology, University of Eastern Finland, P.O. Box 111, FIN-80101

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Joensuu, Finland

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†

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Finland

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Parks & Wildlife Finland of Metsähallitus, Akselinkatu 8, FIN-57130 Savonlinna,

*corresponding author; Tel. +358 50 360 6382, E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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The endangered Saimaa ringed seal (Pusa hispida saimensis) is exposed to relatively high

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concentrations of mercury (Hg) in freshwaters poor in selenium (Se), a known antagonist of

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Hg. The impact of age and sex on the bioaccumulation of Hg and Se was studied by

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analyzing liver, muscle and hair samples from seals of different age groups. Adult females

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were found to accumulate significantly more Hg in the liver (with ca. 60% as HgSe), and less

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Hg in the muscles compared to adult males, which may be explained by accelerated

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metabolism during gestation and lactation. In adult seals, molar Se:Hg ratios in the muscles

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fall below one, which is considered a threshold for the emergence of adverse effects. As a

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result, Saimaa ringed seals may be at risk of developing health and reproductive problems.

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According to mass balance calculations, the pups are exposed to considerable amounts (µg/d)

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of mercury during gestation, although lactation is their main exposure route. In lanugo pups,

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Hg concentrates in the hair, and molting serves as a main detoxification route. For other age

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groups, demethylation followed by the formation of HgSe is the main detoxification route,

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and the demethylation capability develops in pups by the time of weaning.

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Introduction

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Mercury (Hg) has been a global environmental problem for decades and environmental

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concentrations of Hg have not significantly decreased in recent years despite the regulatory

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restrictions implemented, as re-emissions of previously released and deposited Hg contribute

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up to 60% of total emissions today1. In Finland, industrial emissions of Hg to waterways

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have decreased by 75% since 19952, and the average deposition has decreased by 18%3.

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Nevertheless, Hg concentrations in Finnish fish species, such as perch (Perca fluviatilis) have

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not decreased since the mid-1990’s4. It has been found that clear-cutting and other soil

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treatments increase the runoff of Hg from soil where it has been deposited over decades5. In

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Finland, the recipient lakes are shallow and fragmented, with small surface areas in relation

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to the catchment areas, and therefore they have a low dilution capacity.

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Mercury has been connected with reproductive problems such as spontaneous abortions

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and stillbirths6 and has been suspected to play a role in lowering the viability of the

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developing fetus7. Other toxic effects of Hg include e.g. neurological alterations8, liver

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abnormalities9, reduced immunity10, 11 and cardiomyopathy12. Selenium (Se) is a universal

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antagonist to Hg in various organisms, ranging from bacteria to mammals13. Selenium’s

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central role in detoxification is based on the affinity of Hg to nucleophilic selenols. Bonding

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of Hg and Se leads to irreversible formation of various organic and inorganic compounds,

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including selenoproteins and biominerals such as tiemmanite (HgSe)13, 14. HgSe, which is a

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chemically and toxicologically inert compound, is thought to be the end product in the

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detoxification process15. In mammals, the demethylation of methylmercury (MeHg) and the

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consequent accumulation of HgSe is known to occur mainly in the liver and kidneys. It has

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been suggested that a selenoamino acid is the factor initiating the demethylation reaction, but

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the exact reaction pathway(s) by which MeHg is converted into HgSe is not known13, 15.



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Ikemoto et al. (2004a,b) have speculated that in the liver, Hg-Se complexes bind to high

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molecular weight substances (proteins), which are transported to the lysosomes and finally

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degraded, resulting in the precipitation of crystalline HgSe16, 17. It has been suggested that the

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demethylation reaction is a concentration-sensitive process starting when a species-specific

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threshold concentration in the liver is exceeded, and prior to that Hg is stored as MeHg18.

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Young individuals of many marine mammals have been found to be less effective in

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demethylating compared to adults19, 20. Selenium’s role as a scavenger of toxic Hg also has

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negative consequences. By binding to selenol-containing proteins, Hg disturbs their normal

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functioning, for example thyroid hormone regulation or antioxidant defense21. Also,

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inhibition of glutathione peroxidase, which protects cells against oxidative stress, has been

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observed in the presence of Hg22. It seems that, if there is enough Se for detoxification as well

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as for ensuring the vital bodily functions (i.e. molar Se:Hg ≥ 1), adverse effects are expected

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to be minimal23.

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Finnish metamorphic bedrock from the Middle Precambrian era consists mainly of Se-

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poor gneiss, granite, granulite and granodiorite24. Selenium concentrations in Finnish soils

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range from 0.005 to 1.25 mg/kg, which is below the global average25. Further, in acidic soils

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rich in iron and aluminum hydroxides, which are typical in Finland, Se tends to precipitate,

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becoming insoluble and thus poorly available24, 26. Finland was the first country to add

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selenite to artificial fertilizers, but this has not affected the Se concentrations in lake waters27.

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The critically endangered ringed seals (Pusa hispida saimensis) inhabiting Lake Saimaa in

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Finland are exposed to high concentrations of Hg in a Se-poor environment, which makes

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them a unique and interesting species for ecotoxicological studies. The size of the population

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is presently ca. 300 individuals, and 50–60 pups are born annually28, 29. In the past, the Saimaa

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ringed seal has suffered from severe Hg contamination, which is suspected to have

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contributed to the decline of the population30. The Hg concentrations in the seals peaked in


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the late 60’s, with an extreme concentration of 190 µg/g fw found in the muscles of a sick

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individual31. Ever since the use of Hg to control slime in the wood industry was prohibited in

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1963, the concentrations has slowly decreased. Hepatic concentrations in seals dropped by

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78% and muscular concentrations by 43% from the 1970’s to the 1990’s (Table S1)32, 30. Even

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though incidental by-catch mortality and changing climate pose the highest risk to the Saimaa

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ringed seal population today28, 33, 34, chronic exposure to environmental contamination may

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impact the health of Saimaa ringed seals. Adverse effects of mercury (and other

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contaminants), such as low immunity and reproductive defects, may increase the

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susceptibility of the seals to anthropomorphic or environmental stressors. Also, additive and

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negative synergistic effects may arise. There is no previous knowledge as to whether

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environmental Se concentrations in Finland are high enough to protect the seals, and

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specifically vulnerable pups, from the effects of Hg.

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Saimaa ringed seals (both males and females) prey on small schooling fish species with

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an average size of 9 cm and 11 g35, 36. The Hg concentration in Finnish freshwater perch

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(Perca fluviatilis) and vendace (Coregonus albula) of this size is on average 130–150 ng/g

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fw30, 37, 38. Methylmercury, which is the main form of Hg in fish39, is almost totally (90–95%)

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absorbed in the gastrointestinal track40. From the bloodstream it is distributed throughout the

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body within low molecular weight thiol complexes (e.g. cysteine)41, with the liver, muscles,

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hair and skin being the main target organs in seals30, 42, 43. The half-life of MeHg in ringed

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seals is 20–500 days44, and it is eliminated in the feces, urine and molted hair. The part that is

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not excreted, remains mainly in the muscles as MeHg or is demethylated and stored in the

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liver and kidneys as inorganic Hg. There is little knowledge about the relative importance of

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molting and demethylation in the detoxification of Hg in ringed seals. Since Saimaa ringed

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seal males eat on an average 2.7 kg fish per day35, it can be estimated that they are exposed to

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ca. 140 mg of Hg annually. Ryg & Øritsland (1991) estimated that the energy requirements



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for sexually mature ringed seal females is 30% higher than that of males45. Thus, assuming

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that females consume 30% more, they would be exposed to ca. 180 mg of Hg annually. WHO

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has set the provisional tolerable weekly intake (PTWI) of MeHg for humans at 1.6 µg/kg bw,

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which for a 60-kg person is 96 µg of Hg per week46. For comparison, Saimaa ringed seals of

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the same size can be exposed to five times as much per day. Females transfer a portion of

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their Hg burden to their offspring during gestation and lactation. A higher accumulation of

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Hg in males has been reported in several studies conducted with marine mammals47, 48 , but

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contradictory results have also been obtained19, 20, 49, 50. In all cases the results were explained

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by the differences between the sexes regarding feeding rates, habits or prey items. Among the

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females, pregnant and lactating individuals have been found to have the highest Hg

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concentrations19, 20, which has been explained by increased food consumption and, as a

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consequence, increased MeHg uptake during these energy-consuming processes. Knowledge

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about the sex-specific accumulation of Hg in ringed seals is, however, lacking.

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The aims of this study were 1) to determine the present-day Hg and Se concentrations in

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the main target tissues in different age classes (lanugo pups, weaned pups, 1 – 3 years old sub

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adults, and adults) of Saimaa ringed seal, 2) to estimate the potential toxic effects of Hg to

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seals on the basis of the muscular Se:Hg ratios, 3) to determine the role of demethylation and

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molting in the Hg detoxification of the seals at different phases of their lifespan from young

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to adulthood, and 4) to study the impact of sex and the role of reproduction on the

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bioaccumulation of Hg by comparing the accumulation patterns in adult female and male

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seals.

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Materials and methods

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Samples


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Ringed seals inhabit Lake Saimaa, which is a fragmented lake located in south-eastern

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Finland (61° 05’ to 62° 36’ N, 27° 15’ to 30° 00’E). The surface area of the lake is ca. 4400

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km² and the mean depth is 12 m51. Some parts of the lakes have been exposed to effluents

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from the pulp and paper industry, which for decades used Hg as a means for controlling

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slime, whereas other parts are pristine (Figure 1). The University of Eastern Finland and

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Parks & Wildlife Finland of Metsähallitus maintain a Saimaa ringed seal tissue bank, where

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seals found dead in nature are delivered and stored in a freezer. The samples for this study

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were obtained from this bank.collection and storage of tissue samples were carried out under

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the permit of the Center for Economic Development, Transport and the Environment (permit

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nr: ESAELY/567/07.01/2011). Hair, liver and muscle samples were collected during

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necropsies from 20 adult seals, 10 sub-adults (1–3 year old) and 57 pups found dead between

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the years 2002 and 2012. Depending on the stage of decay, not all samples could be taken

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from all the individuals. The ages of the adults and sub-adults were determined from the

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growth layers in the dental cement. The age range of the adults was from 4 to 30 years, the

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median being 12 years. For the majority (70%) of the adults, the cause of death was unknown

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with by-catch mortality explaining the remaining 30%. Approximately 60% of the sub-adults

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died as a result of by-catch mortality. The pups were categorized as weaned pups (n = 33) or

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lanugo pups (n = 24). The weaned pups were 3–11 months old and they had all died in

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fishing gear. The lanugo pups included stillborn pups (n = 14) as well as newborn (< 2 weeks

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old) pups (n = 10). The mercury concentrations of the stillborn pups did not differ from those

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of the newborn pups for any of the tissues, and therefore they were combined as the same

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group.

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Chemical analyses



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The hair samples were washed with boiling Milli-Q water spiked with a drop of

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detergent, and then rinsed three times with boiling Milli-Q water. Hair, liver and muscle

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samples were dried in an oven at 105 oC for 24 h, and the dry samples were digested in conc.

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HNO3 and H2O2 in a microwave oven (Mars 5®, CEM Corporation). The filtered samples

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were analyzed using ICP-OES (IRIS Intrepid II XSP, Thermo Scientific) for total Hg and Se

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concentrations. The dry weights of the tissues (%) were determined by weighing

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representative samples before and after the drying. The dry weights were 28 ± 4% (n = 13)

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for liver and 30 ± 6% (n = 8) for muscle. The measured Hg and Se concentrations were

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transformed to mg/g fresh weight (fw) to allow them to be compared with results from the

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literature.

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In order to determine the speciation of Hg (organic Hg and HgSe) in the liver, samples

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from ten females were analyzed using a modified method of Wagemann et al. (2000)52. The

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liver samples were homogenized in a mixture of acidic NaBr and CuSO4 followed by

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extraction with dichloromethane-hexane (3:2 v/v). The organic phase containing organic Hg

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was re-extracted with conc. HNO3. Thereafter, the organic phase was discarded and the acidic

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phase was subjected to microwave digestion as described above. The acidic NaBr and CuSO4

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phase was also discarded, and the pellet containing the insoluble Hg (HgSe) was digested in

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conc. HNO3 and H2O2 in a microwave oven, then filtered and analyzed using ICP-OES.

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Each set of the samples taken for microwave digestion included a blank and a standard

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sample, in order to verify the success of the digestion process. Every tenth seal sample was

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analyzed as a duplicate, and the relative standard deviation (RSD) was found to be 21%. The

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detection limits were 0.36 µg Hg/g fw and 0.19 µg Se/g fw. The reproducibility of Hg

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analysis was determined in an accredited GLP-laboratory by AFS analysis and was found to


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be 84%. The recovery of Se was found to be 95% when determined using certified reference

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material, SRM 2976 (NIST, USA).

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Morphometry and mass balance calculations

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The proportion of hair, liver and muscle in the total body mass were determined to enable

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calculation of the total average Hg loads of the organs and the mass balance of Hg in seals

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(Table S3, Figure S1). The pelt (n = 5 for each age group) was photographed and the area of

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the pelt was determined using ImageJ 1.48v software. Hair was cut from predetermined,

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representative areas (2 x 2 cm) of the pelt and weighed. The weight of the sampled hair was

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used for calculating the total weight of hair in the pelt, which was then proportioned to the

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total body mass available in the post-mortem report. The weights of the livers (n = 80) were

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also available in the post-mortem reports, and the average proportion of liver per body mass

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was calculated for each age class of seals. The muscle mass was calculated on the basis of the

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data of Lydersen et al. (1992)53, who determined the proportions of various organs in

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different-sized ringed seals.

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The mass balance of Hg uptake and excretion was calculated for a Saimaa ringed seal

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male (60 kg, 15 years old) and a female (57 kg, 13 years old), on the basis of the following

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equation:

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muptake = mliver + mmuscle + mhair + m foetus + mlactation + mother (1)

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Where muptake indicates the total mass of Hg taken up by a seal per year (mg/year), which

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was estimated, separately for females and males, on the basis of the literature-derived

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consumption rates35, 45 and Hg concentrations in the prey fish species 30, 37, 38,. The average



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annual accumulation of Hg in liver (mliver) was calculated, separately for adult females and

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males, based on the association between the hepatic Hg concentrations and age (Figure 3).

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The total quantity of Hg in muscles (mmuscle) and in hair (mhair) of the adult seals was

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calculated by multiplying the average Hg concentrations by the organ masses (Table S3). For

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simplicity it was assumed that the muscular Hg concentrations remain constant throughout

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the year. The last variable (mother), which includes Hg allocated in other tissues (mainly skin,

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kidneys and brain)43 and Hg excreted in feces and urine, was determined for the male,

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assuming that all ingested Hg is assimilated. Thereafter, it was assumed that this proportion

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(mother) of the intake is equal for the female. The quantities of Hg transferred from the mother

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to the pup via gestation (mfoetus) and lactation (mlactation) were estimated using the

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morphometry of a stillborn lanugo pup (5 kg) and a newly weaned pup (19 kg, 3 months old),

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and the average Hg concentrations in their muscle, liver and hair. It was assumed that

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bioaccumulation of Hg in tissues other than liver, muscle and hair of the pups is insignificant

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and that a 3-mo old pup does not forage before weaning (i.e. lactation is the only source of

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exposure).

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Statistical analyses

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The data were tested for normality using the Shapiro-Wilk normality test, and due to the

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general lack of normality in the data, non-parametric tests were used for further testing. The

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accumulation patterns at different ages was studied by comparing Hg concentrations in liver,

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muscle and hair of different age classes (adults, sub adults, weaned pups and lanugo pups)

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using the Kruskall-Wallis test with Dunn’s multiple comparison test. The non-parametric

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Spearman correlation analysis was used to survey the age-dependence of Hg and Se

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accumulation, and the co-dependence of Hg and Se. The impact of sex on the


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bioaccumulation was studied by comparing the Hg concentrations in adult females and males

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using the two-tailed Mann–Whitney U-test. To estimate the potential toxic effects of Hg to

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seals, the deviation of muscular Se:Hg ratios from the unity was tested using the Kruskall-

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Wallis test with Dunn’s multiple comparison test. The one-phase decay model was used to

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model the proportion of organic Hg of total Hg, and a five-parameter logistic equation was

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used to model both HgSe and total Hg concentrations.

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Results

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Mercury concentrations

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The adult Saimaa ringed seal females had the highest hepatic concentrations, ranging

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from 16.8 to 245.7 µg Hg/g fw, whereas the concentrations in adult males were 12.3–52.0 µg

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Hg/g fw (Table S2 and Figure 2a). The difference was statistically significant (p = 0.006)

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according to the Mann-Whiney U-test. In females (n = 13), the concentrations correlated

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strongly with age (rs = 0.82, p = 0.0007), while for males (n = 7) the correlation with age was

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statistically insignificant (rs = 0.51, p > 0.05). Old unpublished data from the 1980’s (Table

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S2) verified the observed differences in the accumulation of Hg between sexes. In the both

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lanugo pups and weaned pups, the hepatic concentrations were expectedly lower (p < 0.05)

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compared to those of adults (Figure 2a and Table S2).

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Adults had the highest muscular Hg concentrations (Figure 2b and Table S2), but the

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concentrations did not correlate with age (p = 0.569). The highest concentrations (ca. 3.5 µg

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Hg/g fw) were found in 5-year old and 28-year old males. Males had higher average muscular

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concentrations than females but the difference was statistically insignificant (p = 0.059). The



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concentrations in both lanugo pups and weaned pups were significantly (p < 0.05) lower than

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in adults, and they were detectable in only ca. 50% of the muscle samples.

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The highest Hg concentrations in hair were found in adults (Figure 2c) but the

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concentrations did not correlate with age (p = 0.467). Males and females were not found to

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differ from each other (p = 0.085), which may be due to the high variation in the results for

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males. An 11-year old male had an approximately ten times higher concentration of Hg (59

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µg/g dw) compared to other males. The lowest concentrations were found in the hair of

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weaned pups, and they differed significantly (p < 0.0001) from those of lanugo pups and

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adults.

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The mass balance calculations, which are based on numerous assumptions, give a rough

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estimate for the relative importance of gender and generation-specific distribution of mercury

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in the seal. Based on them, a 5-kg lanugo pup allocates the majority of assimilated Hg (ca.

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64%) to natal hair, followed by the muscles (ca. 26%) and the liver (ca. 10%; Figure S1). A

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19-kg (ca. 3 month old) just weaned pup allocates ca. 2% of assimilated Hg to hair, ca. 25%

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to the muscles, and ca. 5% to the liver. A 60-kg (ca.13-year old) adult seal excretes ca. 1% of

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Hg in the hair. In males, the majority of the remaining Hg (ca. 30%) is allocated to the

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muscles and only a minor part (ca. 0.3%) to the liver. Females, on the other hand, allocate ca.

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13% to the muscles, ca. 7% to the liver, and they transfer ca. 11% to their offspring via

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gestation (ca. 1%) and lactation (ca. 10%).

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Selenium concentrations and molar Se:Hg ratios

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The hepatic Se concentrations were highest in adult females (Table S2) and correlated

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with age (rs = 0.77, p = 0.002). In adult males, the concentrations were lower, similarly to Hg


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concentrations, and no correlation with age was observed. The hepatic Se concentration was

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lowest in lanugo pups and increased towards older age groups. The muscular concentrations

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were similar in all age groups, the average being 0.33 ± 0.10 µg Se/g fw. Weaned pups (2-3

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mo old) had the lowest and lanugo pups the highest concentrations in hair. The difference

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was statistically significant (p < 0.05), but other groups did not differ from each other.

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There was a strong correlation between hepatic Hg and Se concentrations for adults, sub-

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adults and weaned pups, the Spearman correlation coefficients being 0.99 (p < 0.0001), 0.87

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(p = 0.0022) and 0.94 (p < 0.0001), respectively. For the lanugo pups, the correlation was

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weaker (rp = 0.45, p = 0.02). Further, the hepatic molar Se:Hg ratio followed a 1:1 line, with

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the exception of the lanugo pups (Figure 4a). A significant negative correlation (rp = -0.88, p

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< 0.0001) was instead found between the molar Se:Hg ratio and total Hg concentration in the

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liver for the lanugo pups. When the Hg concentration rose above 2 µg/g fw, the hepatic

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Se:Hg ratio fell below one.

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The muscular Se:Hg ratio decreased along with increasing Hg concentrations for seals of

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all age groups (Figure 4b). The lowest average molar Se:Hg ratio was measured in adult

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males (0.41 ± 0.29), and it deviated from one (p < 0.05; Figure 5), which is considered to be a

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threshold for the emergence of adverse effects54.

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Speciation of mercury

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In the adult female seal liver, 62 ± 29% of Hg remained bound to the tissue pellet after

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extraction. Wagemann et al. (2000) identified the compound remaining in the insoluble

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fraction after extraction with organic solvent and acidic NaBr and CuSO4 as tiemmanite

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(HgSe)52. We found Se and Hg in the insoluble fraction to correlate strongly with each other



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(rs = 0.93, p < 0.0001), the average Se:Hg ratio being 1.28 ± 0.11 and thus confirming that

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insoluble Hg is HgSe. The concentration of HgSe was found to correlate with total hepatic

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Hg concentration (rs = 0.93, p < 0.0001). It was also found to correlate with age (rs = 0.94, p

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= 0.0002), but the annual increase in concentration was found to be 40% slower compared to

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that of total Hg (Figure S2). The concentration of organic Hg was 2.7 ± 1.2 µg/g fw in adults

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and 0.47 ± 0.04 µg/g fw in pups. The proportion of organic Hg of total Hg was found to

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decrease along with age (rs = -0.86, p = 0.0238; Figure S2). A negative correlation (rs = -0.95,

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p < 0.0001) was also observed between the proportion of organic Hg and total Hg

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concentration.

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Discussion

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The Saimaa ringed seal suffers from very high Hg contamination compared to marine

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ringed seals (Table S1) and also to the majority of other Arctic warm-blooded species55, 56.

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The concentrations are highest in the liver, and in 80% of the studied adult Saimaa ringed

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seals, the concentrations exceed the threshold for subclinical toxicity for marine mammals

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(65 mg/kg dw in liver) suggested by AMAP57. The threshold concentration for acute toxicity

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(130 mg/kg ww) also exceeds in two individuals (10%). Equally high or even higher

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concentrations of Hg have, however, been found in Steller sea lions (Eumetopias jubatus)58,

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harbor seals (Phoca vitulina)48, 59, and dolphins (Stenella coeruleoalba)18. The Hg

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concentrations of Saimaa ringed seal have not decreased since the 1990’s, even though direct

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industrial emissions to water have dropped by 75% during this time2. Apparently, the Lake

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Saimaa area is subject to continuous atmospheric deposition as well as to soil cultivation

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measures that release soil-bound Hg into the waterways. In an aquatic environment, Hg is

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converted into MeHg, the most toxic form of Hg, and the conversion rate is faster at higher


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temperatures60. Given that warming climate will also lead to increased precipitation and

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runoff of Hg from soils, the concentrations of MeHg in aquatic environments are expected to

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increase in the future.

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The muscular Se:Hg molar ratio in Saimaa ringed seal males is significantly (p < 0.05)

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lower than one, which is a potential threshold for adverse effects (Figure 5). Mercury stored

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in the muscles is of special importance. Firstly, it exists as toxic MeHg61, and secondly, the

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muscles contribute 30% of the total body mass of the seal53 and thus allocate the majority of

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total Hg. Since the muscles act as storage for Se62, muscular Se:Hg ratios describe the status

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of available Se. Due to the crucial role of Se in toxicity of Hg, the Se:Hg ratio is regarded as

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being a better descriptor of expected Hg toxicity than Hg concentration alone. Ralston et al.

323

(2007) found impaired growth in the Long Evans rats to correlate with the Se:Hg ratio of the

324

diet63. Bennett et al. (2001) found that healthy harbor porpoises (Phocoena phocoena), that

325

had died of physical trauma had almost twice as high Se:Hg ratios in their liver compared to

326

those that died of infectious diseases11. This indicates that low Se:Hg ratios may have

327

immunosuppressive effects. In their study with free-ranging trout (Salmo trutta), Mulder et

328

al. (2012) found that when the muscular Se:Hg ratio fell below one, thyroidhormone function

329

was disrupted54. They suggested that an Se:Hg ratio ≈ 1 is a threshold for ensuring the proper

330

functions requiring Se. Sørmo et al. (2011) found that the muscular Se:Hg ratios just below

331

one were sufficient to induce metallothionein synthesis in an attempt to scavenge Hg in the

332

absence of Se or to suppress the effects of oxidative stress caused by an excess of Hg64. In the

333

present study, a clear decreasing trend in free muscular Se concentrations along with

334

increasing Hg concentrations was observed (Figure 4b) indicating potential Hg-induced Se-

335

deficiency. The Se:Hg ratios were two times lower in males than in females, which may be

336

due to their less efficient demethylation capability observed in this study (Figure 5).

337

However, it is still possible that both sexes may suffer from Se deficiency-related conditions,



338

ranging from systemic problems in immunity or reproduction to topical problems such as

339

cardiomyopathy65.

340

Based on the mass balance calculations, Saimaa ringed seal fetuses are exposed to µg/d

341

level- concentrations of Hg during gestation. Perinatal exposure to MeHg has been shown to

342

affect thyroid hormone metabolism in the mouse brain already at low (ng/d - µg/d) MeHg

343

doses66. It has also been shown that MeHg concentrates selectively in the fetal brain40 and

344

affects their antioxidant system67. According to Dietz et al. (2013), Hg concentrations as low

345

as 3–5 µg/g dw in the brain can result in neurological alterations8. In the present study,

346

concentrations in the brain were not measured, but in the 1980’s, concentrations ranging

347

between 2.9 and 12.5 µg/g dw were detected in the brain of Saimaa ringed seal pups (n = 5;

348

Hyvärinen et al. unpublished data). These pups had ca. 50% higher muscle concentrations

349

(0.56–1.14 µg/g fw) than the pups included in the present study. Even though we found that

350

the average muscular Se:Hg ratio is above one for lanugo pups (Figure 5), their hepatic Se:Hg

351

ratios fall below one when the Hg concentrations exceed 2 µg/g fw (Figure 4a).

352

Selenoenzymes are crucial for fetal and post-natal development and growth, and Se-deficient

353

diets have been found to increase mortality at birth, decrease viability, stunt growth and

354

retard the liver development of the offspring63, 68. The pups obtain Se from their mother across

355

the placental barrier and in the milk, and the actual concentrations depend on the maternal

356

reserves69, 70. In the case of the Saimaa ringed seal, these reserves may not, however, always

357

be sufficient for ensuring the adequate Se status of the fetus or the lanugo pup.

358

Lactation is the main source of Hg for Saimaa ringed seal pups (Figure S1). This is

359

indicated by the fact that the Hg concentrations in weaned pups are on the same level or even

360

higher than in lanugo pups, even though they have multiplied their masses. Noël et al. (2015)

361

found the transfer of Hg from a harbor seal (Phoca vitulina) mother to a pup to increase


Page 16 of 38

Page 17 of 38


362

towards to the end of the gestation period and peak remarkably at the onset of lactation71.

363

Habran et al. (2011) observed that the Hg concentration in the blood of a northern elephant

364

seal (Mirounga angustirostris) pup decreases significantly along progression of lactation with

365

milk being an insignificant source of Hg compared to gestation.69 By comparing the Hg

366

concentration of a 6-d old harp seal (Phoca groenlandica) pups to an estimated exposure via

367

milk, Wageman et al. (1988) also concluded that gestation is more important source of Hg

368

than lactation72. The results of this study indicating the opposite may be due to the 3–4 times

369

longer lactation period of the Saimaa ringed seals (of 7 – 12 weeks)73 compared to that of

370

elephant seals69 or harp seals74. Also, ringed seal mothers continue eating during lactation,

371

exposing themselves continuously, but still lose substantial amounts (ca. 30%) of weight75.

372

The mother’s hyperactive metabolism during lactation may facilitate the transfer of the

373

assimilated Hg from the blood directly into the milk and its being passed on to the suckling

374

pup rather than bioccumulating in the mother. It is also known that if the diet is protein-poor

375

during lactation, catabolism of the muscles may occur in order to guarantee an adequate

376

protein status76. As a result, muscle-bound Hg could be released into the blood stream and

377

transferred to the milk.

378

Molting serves as a significant excretion route for Hg in lanugo pups. On the basis of

379

mass balance estimates, they can excrete a significant proportion (ca. 64%) of estimated total

380

prenatal exposure via this route. The importance of molting decreases thereafter, and adult

381

Saimaa ringed seals are able to excrete only ca. 1% of their estimated annual exposure via

382

molting. Studies have revealed that organisms vary in their capability to utilize molting as an

383

excretion route, and the importance of this route has been linked e.g. to frequency of

384

molting77. Saimaa ringed seals molt in springs, while European otters (Lutra lutra) living the

385

same area and feeding on the same fish species, molt twice a year and have ca. 30–50% lower

386

Hg concentrations than the seals78, 79. The otters’ thick fur, which contributes 3.2–3.7% of



387

total body mass, serves as an insulating layer, similarly to that of the lanugo pups. Blubber

388

replaces the lanugo fur as an insulator after few weeks, and the relative weight of hair

389

compared to total body mass decreases significantly (Table S3). This, rather than the molting

390

frequency, may explain the lower importance of molting as an excretion route for seals.

391

Organisms are known to vary in their demethylation capabilities77. It has also been suggested

392

that the relative importance of demethylation-derived detoxification overcomes molting when

393

organisms are exposed to high concentrations of Hg80.

394

Saimaa ringed seal females seem to be more efficient in demethylating MeHg and storing

395

it in the liver as HgSe than males. The majority of Hg (ca. 60%) in the liver was found to

396

exist as HgSe, ca. 6% as organic Hg and the remaining 34 % as unknown inorganic Hg

397

compound(s). These numbers agree well with those of Wagemann et al. (2000) for ringed

398

seals52. Along aging of Saimaa ringed seals, the concentration of HgSe increases at ca. 40%

399

lower rate than the total Hg concentration in liver (Figure S2). This implies that HgSe

400

formation is a fairly slow multi-step process. Even though females transfer a portion of their

401

Hg burden (ca. 11%) to their offspring, their hepatic Hg concentrations increase along with

402

age (Figure 3). A 60-kg (ca. 13 year old) Saimaa ringed seal female has ca. 150 mg of Hg

403

allocated to the liver, whereas the corresponding value for a male is 45 mg. The hepatic Hg

404

load of females increases by ca. 12.5 mg annually, which is ca. 9% of the estimated exposure.

405

Males, on the other hand, are able to retain fairly constant levels of hepatic Hg despite their

406

age. The difference between the sexes cannot be explained by foraging ecology, since both

407

sexes appear to have similar diets. Higher accumulation of Hg in females, and specifically in

408

pregnant and lactating individuals, has been reported in some studies19, 20. The phenomenon

409

was explained by increased exposure due to increased food consumption during these energy-

410

consuming processes. No gender-specific bioaccumulation studies for ringed seals are found

411

in the literature, but females are estimated to have 30% higher energy needs than males45.


Page 18 of 38

Page 19 of 38


412

Assuming that the binding of Hg to muscle was one of the rate-limiting factors in

413

demethylation, a release of muscle-bound Hg into the blood stream due to potential

414

catabolism during lactation could explain the gender-based differences in hepatic and

415

muscular Hg concentrations for Saimaa ringed seals. Further, hyperactive metabolism during

416

lactation may facilitate the transfer of the assimilated Hg from the blood directly to the liver

417

(and to the pup).

418

On the basis of our results we suggest that for Saimaa ringed seal, demethylation and

419

HgSe formation becomes fully functional by the time of weaning. Contrary to what has been

420

suggested in some studies18, there were no signs of a threshold concentration below which Hg

421

exists as MeHg. On the other hand, we observed that Se and Hg are present in unimolar

422

concentrations in the livers of adults, sub-adults and weaned pups regardless of the

423

concentration, implying the presence of HgSe. We also found that even though the proportion

424

of organic Hg to total Hg decreases along with age (Figure S2), the actual concentrations of

425

organic Hg remain fairly constant. The decrease thus describes the accumulation of Hg over

426

time rather than indicating a limited demethylation capability in juveniles. The lanugo pups,

427

however, differ from the other age groups. Their hepatic Se:Hg ratios decrease along with

428

increasing total Hg concentrations (Figure 4a). This indicates that the hepatic Se and Hg

429

concentrations are not co-dependent, which implies that the demethylation reaction is not

430

functional during gestation.

431

To summarize, the low muscular ratio of Se and Hg imply that Saimaa ringed seal may

432

suffer from Se-deficiency, and potentially from a consequent Hg toxicity. If this were the

433

case, it could have detrimental effects on the seals’ immunity as well as in its ability to

434

reproduce normally and produce viable offspring. The implications may be significant, given

435

that the population is critically endangered, and should be further investigated. Climate



436

change will increase precipitation and, as a consequence, run-off of metals from cultivated

437

land areas. If mitigating processes, such as increase in Se run-off, will not occur, it is likely

438

that Saimaa ringed seal will continue to be exposed to Hg in the future.

439

440

Acknowledgements

441

The authors wish to thank the Elma, Eino and Veikko Jumppanen Foundation for

442

financial support. M. Noponen is acknowledged for her indispensable help in performing the

443

chemical analyses, and J. Koskela and the Metsähallitus field staff are acknowledged for

444

collecting samples during a period of over 10 years. We thank O. Stenman, P. Timonen and

445

M. Valtonen for age determination. Sari Oksanen is thanked for her valuable help with the

446

map (Figure 1). Finally, thanks are owed to the Saimaa ringed seal research group of UEF for

447

their fruitful co-operation and useful advice.

448

449

Supporting information

450

Supporting information includes a table describing present and historical Hg concentrations

451

in adult ringed seals from various locations, a table of total Hg and Se concentrations in the

452

main target organs, and a table of robust mass balance estimates and parameters used for

453

calculated them. A figure describing the results of the robust mass balance calculations, and

454

figures describing the proportion of organic Hg and HgSe of total Hg in adult seal liver,

455

proportion of organic Hg of total Hg, and concentration of HgSe in adult Saimaa ringed seal

456

liver as a function of age are also included. This material is available free of charge via the

457

Internet at http://pubs.acs.org


Page 20 of 38

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458

459

Conflict of Interest Disclosure

460

The authors declare no competing financial interest.

461

462

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713


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714

715

Figure 1. Map of Lake Saimaa. The circles indicate towns with more than 20,000

716

inhabitants, the factory symbols indicate industrial activities (mainly pulp and paper industry)

717

by the lake, and black dots indicate the sites where dead seals were found.

718 719 720 721 722



200

a

A.

µg Hg/g fw

100 50

a

40 30 20

a

10

b

b

0

4

µg Hg/g fw

b

B.

3

ab

2

ac

ac c

1 0

40

µg Hg/g dw

ab

C.

30

a 20

ab

a

10

b ps

ps go la nu

ed ea n

pu

pu

lts ad u w

su b

al es lt m

ad u

ad ul

tf em

al es

0

723 724 725

Figure 2. Concentration of total mercury in a) liver, b) muscle and c) hair of Saimaa

726

ringed seals of different age classes. The error bars represent standard deviation. Different

727

letters indicate the statistical difference among the groups (p < 0.05) analyzed using the

728

Kruskall-Wallis test followed by Dunn’s multiple comparison test.

729


Page 34 of 38

Page 35 of 38


Hg (µg/g fw)

300

females males 200

100

0 0

10

20

30

40

Age (years)

730 731 732 733

Figure 3. Mercury concentrations in the liver of adult Saimaa ringed seals as a function of age (n = 13 for females and n = 7 for males).



4

A. Se:Hg

3 2 1 0 0

2

4

6

8

10

adults sub adults

100

300

weaned pups lanugo pups

4

B. Se:Hg

3 2 1 0 0

1

2

3

4

µg Hg/g fw

734 735

Figure 4. Molar Se:Hg ratios versus mercury concentrations in a) liver and b) muscle of

736

Saimaa ringed seals. The line (y = 1) denotes the limit below which adverse effects are likely

737

to occur.


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738 739

2.5

a

Se:Hg

2.0 1.5

a

ab

* ab

1.0

b

0.5

go

pu

pu la

nu

ed w

ea n

b su

ps

ps

ts ad

ul

al es ul tm

ad

ad

ul

tf

em

al es

0.0

740 741 742

Figure 5. Molar Se:Hg ratio in the muscles of Saimaa ringed seals of different age

743

classes. The line (y = 1) denotes the limit below which adverse effects are likely to occur.

744

The error bars represent standard deviation. An asterisk denotes a statistically significant (p

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