# Distribution and trophic transfer of mercury and selenium in European hake (Merluccius merluccius) from the Northeast Atlantic Ocean: A stable isotope approach

Yiou Zhu, Julien Barre, Arved Staby, Martin Wiech, Zoyne Pedrero, Emmanuel Tessier, Sylvain Bérail, Amund Maage, Livar Frøyland, Michael Bank

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Yiou Zhu, Julien Barre, Arved Staby, Martin Wiech, Zoyne Pedrero, et al. Distribution and trophic transfer of mercury and selenium in European hake (Merluccius merluccius) from the Northeast Atlantic Ocean: A stable isotope approach. Journal of Hazardous Materials, 2025, 494, pp.138564. 10.1016/j.jhazmat.2025.138564. hal-05085492

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# Distribution and trophic transfer of mercury and selenium in European hake (*Merluccius merluccius*) from the Northeast Atlantic Ocean: A stable isotope approach

Yiou Zhu<sup>a,1</sup>, Julien Barre<sup>b,c</sup>, Arved Staby<sup>a,●</sup>, Martin Wiech<sup>a,2,●</sup>, Zoyne Pedrero Zayas<sup>c</sup>, Emmanuel Tessier<sup>c</sup>, Sylvain Bérail<sup>b,c</sup>, Amund Maage<sup>a,●</sup>, Livar Frøyland<sup>a</sup>, Michael S. Bank<sup>a,d,3,* ●</sup>

## HIGHLIGHTS

* Mercury (Hg) stable isotopes (SI) in European hake (EH) fillets varied along the Norwegian coast.

* Hg in EH fillet tissue was ≥ 91 % methylmercury (MeHg) and served as a good proxy.

* EH Hg SI, Selenium (Se), Se:Hg molar ratio, and δ¹³C/δ¹⁵N differed in polluted and reference areas.

* EH bioaccumulated Hg that previously underwent MeHg photodegradation.

* Hg point sources modulate EH Se-Hg dynamics in the Northeast Atlantic Ocean.

## GRAPHICAL ABSTRACT

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

## Conclusions

Different Hg sources underwent similar photodegradation processes

Different trophic niches

At the polluted site:

* Higher Hg & Se

* Lower MDF and odd MIF Hg isotopes

* Higher trophic magnification of Hg. and Hg isotopes

Good proxies for Hg source apportionment between natural and point sources

## ARTICLE INFO

### Keywords:

Mercury

Stable isotopes

European Hake

Methylmercury

Selenium

## ABSTRACT

European hake (EH - *Merluccius merluccius*) are a commercially valuable, high trophic position marine fish species inhabiting the Northeast Atlantic Ocean (NEAO). Here, we investigated total mercury (Hg) bioaccumulation and trophic transfer dynamics, and selenium (Se) Hg interactions in EH fillets at a historic waste disposal site with a known Hg point source (n = 25) and at reference sites (n = 763) throughout Norway. Fillet concentrations of Hg (≥ 91 % methylmercury - MeHg) and Se, were elevated in samples from the polluted site (Hgpolluted = 0.271 ± 0.153, Hgreference = 0.114 ± 0.096, Sepolluted = 0.425 ± 0.033, and Sereference = 0.321 ± 0.044 mg/kg wet weight). The opposite was found for Se:Hg molar ratios (Se:Hgreference = 12.1 ± 7.91, Se:Hgpolluted =

<footnote><sup>a</sup> Institute of Marine Research, Bergen, Norway</footnote>

<footnote><sup>b</sup> Advanced Isotopic Analysis, Pau, France</footnote>

<footnote><sup>c</sup> Université de Pau et des Pays de l'Adour, E2S UPPA, CNRS, IPREM, Institut des Sciences Analytiques et de Physico-chimie pour l'Environnement et la Matériaux, Pau, France</footnote>

<footnote><sup>d</sup> University of Massachusetts Amherst, Amherst, MA, USA</footnote>

<footnote>* Correspondence to: Institute of Marine Research, PO Box 1870, Bergen 5817, Norway
E-mail address: Michael.Bank@hi.no (M.S. Bank).</footnote>

<footnote n="1">¹ https://orcid.org/0000-0003-4919-6094</footnote>

<footnote n="2">² https://orcid.org/0000-0002-3987-6566</footnote>

<footnote n="3">³ https://orcid.org/0000-0001-5194-7171</footnote>
6.39 ± 4.96), however when controlling for Hg concentrations, the EH Se:Hg ratio from the polluted site was higher compared to reference conditions. We further investigated trophic niches using stable carbon, nitrogen, and Hg isotopes and found different isotopic niches between reference and polluted sites. EH from both site types had different Hg isotopic signatures and were enriched in heavier isotopes at reference sites (e.g., δ²⁰²H<sub>reference</sub> = 0.964 ± 0.177, δ²⁰²H<sub>polluted</sub> = 0.714 ± 0.115 ‰). Hg enriched in fillet tissue primarily underwent MeHg photodegradation at both sites. However, due to the local Hg point source, lower MDF isotope values and odd MIF isotope values (e.g., Δ¹⁹⁹H<sub>reference</sub> = 0.981 ± 0.105, and Δ¹⁹⁹H<sub>polluted</sub> = 0.829 ± 0.110 ‰) were observed at the polluted site. Overall, our findings indicate good discriminability regarding Hg source apportionment and highlight the role of point source pollution as an important modulator of the EH Hg bioaccumulation regime in the NEAO study system.

# 1. Introduction

European hake (EH, *Merluccius merluccius*) are widely distributed throughout the Atlantic Ocean and Mediterranean and are commercially valuable in the Northeast Atlantic Ocean (NEAO) [18,67,94]. EH are large and long-lived, occupying a high trophic position (TP) in marine food webs (TP = 4.4; [35]). EH are widely distributed along the Norwegian coast up to the Lofoten archipelago and have small-scale population structures [94], complex life histories and population dynamics [82], and a defined seasonal spawning period [93]. EH are abundant in the NEAO with a recent estimated catch of 14,000 tonnes (live weight) in 2022 from Norway (https://www.ices.dk/data/dataset-collections/Pages/Fish-catch-and-stock-assessment.aspx). EH are often fished through a non-targeted, mixed, demersal fishery in the North Sea as well as a set-net fishery in western Norwegian coastal waters [82]. Typically, EH prey on pelagic fish species (e.g., Atlantic herring, *Clupea harengus* and Atlantic mackerel, *Scomber scombrus*), demersal Norway pout (*Trisopterus esmarkii*), as well as crustaceans during the juvenile stage [92].

EH are nutrient-dense [43], yet also exposed to ocean pollution, including radioactive elements, persistent organic pollutants, PFAS and PFOA compounds, and heavy metals such as total mercury (Hg) [41,68]. Hg in marine fish occurs in different forms or species, including inorganic mercury (iHg) and methylmercury (MeHg). Both iHg and MeHg can be hazardous, but the latter poses a more significant toxicological threat to individual organisms, populations, communities, and ecosystems [21,78]. MeHg is also the dominant contaminant often identified in seafood safety surveillance efforts [5] and regional human fish consumption advisories due to its status as a powerful neurotoxin with a higher degree of bioavailability [16,40]. Long-lived apex predator species, such as EH, can also be highly contaminated by MeHg [41,101]. When analysing Hg concentrations, selenium (Se) concentrations are often assessed together to concomitantly evaluate the risk of Hg toxicological impacts because of the potential mitigating effects of Se on MeHg [7,37]. Such Se/Hg interactions are typically examined using Se: Hg molar ratios and sometimes Se health benefit values [17,3,48,58,73].

Ontogenetic diet shifts, and geographical variation in prey use and feeding strategies are common in EH [15,29,32,83], suggesting potential TP- and size-dependent drivers of biomagnification and bio-accumulation of Hg and MeHg. This was confirmed by previous studies that have investigated the trophic transfer of Hg and MeHg in EH in local food webs using stable carbon and nitrogen isotopes [22,27,28].

Hg isotopic signatures are widely recognized as powerful tools for distinguishing Hg sources in both abiotic and biotic matrices [11,38,56, 66] and for identifying ontogenetic dietary shifts [42] and tracing environmental and anthropogenic influences of Hg [100,57,87]. Mercury has seven stable isotopes ($^{196}$Hg, $^{198}$Hg, $^{199}$Hg, $^{200}$Hg, $^{201}$Hg, $^{202}$Hg, and $^{204}$Hg) and undergoes both mass dependent fractionation (MDF) and mass independent fractionation (MIF) [8,81]. The resulting Hg isotopic signatures in environmental samples have been successfully used to trace processes such as photochemical reactions, trophic transfer [69,87], inter-organ distribution [74], and Hg species transformation in fish and other biota such as seabirds, etc. [49,61,71,95].

Hg concentrations and Hg, C, and N isotope values from EH fillets from the Norwegian region of the NEAO and from a known point source

polluted site have never been modelled, nor reported, despite their growth and interest as a commercially relevant species [6]. Here, we investigated Hg and Se bioaccumulation and trophic transfer dynamics in EH collected from coastal and offshore locations (i.e., reference sites), and from a sampling area near a known pollution point source. The specific objectives of this investigation were to: 1) examine the spatial distribution of Hg in EH, 2) report EH Se concentrations and Se:Hg molar ratios, 3) investigate trophic accumulation of Hg and its isotopic trends, and 4) characterize Hg isotopic patterns across reference conditions throughout Norway and from a point-source polluted site. Specifically, we tested the hypothesis that Hg concentrations and isotopic signatures in EH fillets would differ between reference conditions and the site with a known point source of Hg.

# 2. Methods and materials

## 2.1. Study area and sample collection

EH (n = 788) were collected along the Norwegian coast (Fig. 1A) during 2019–2022. The sampling locations were divided into two categories: reference (including coastal and offshore locations) and polluted (60.43307 N, 5.09212 E, Hauglandsosen/Kollevåg, Norway, which has a known pollution point source; [91]). In reference sites, it was assumed that EH obtained Hg mainly from atmospheric and geogenic sources, while at the polluted site, Hg sources also included local anthropogenic ones. Samples from the contaminated Hauglandsosen/Kollevåg site were caught using rod and line by local fishermen. All other samples were collected by the Institute of Marine Research (IMR) reference fleet and on IMR research surveys in the North Sea and Norwegian Sea. The reference fleet consists of commercial fishing vessels operating in coastal and oceanic waters and that collect biological information / samples from catches for IMR [24]. The fishing methods deployed include bottom trawls, Danish seines, set-nets and longlines, and due to the nature of the gears, they may have varying length selectivity curves. On IMR surveys the GOV bottom trawl (chalut à Grande Ouverture Verticale), the standard trawl on International Bottom Trawl Surveys [45] was used in the North Sea and a modified Campelen bottom trawl (with rock hopper gear) on the coastal survey in the Norwegian Sea. In total, we have collected 763 samples from reference sites and 25 from the polluted site ($n_{reference}$ = 763 and $n_{polluted}$ = 25). Samples were stored frozen prior to analyses.

## 2.2. Laboratory analyses

Total length, weight (body weight and gonad weight), fat content, k-factor, and gender were measured on individual whole fish. The liver was removed before filleting, using powder-free gloves, clean stainless-steel utensils, and trace metal-clean techniques (except for the use of a metal blade food processor for homogenization of skinless fish fillet tissue). Skinned fillet samples were homogenised then lyophilised prior to laboratory analyses. A full list of reproducible methods is provided in Bank et al. [6].
## 2.2.1. Mercury and selenium concentration measurements

Total Hg and Se concentrations were measured at the Institute of Marine Research, Norway using inductively coupled plasma-mass spectrometry (ICP-MS), which is a Nordic, European and US standard [20,47]. Briefly, 0.2 g of sample was digested with 2 mL 65 % nitric acid and 0.5 mL 30 % hydrogen peroxide in a microwave oven (Milestone MLS-1200 MEGA Microwave Digestion Rotor - MDR 158 300/10). A standard curve was used to determine the concentrations. Rhodium, thallium, or yttrium was used as an internal standard to correct for instrumental drift, and gold was added to stabilise the Hg-ions. The limit of quantification (LOQ) of the method was 0.005 and 0.01 mg/kg dry weight for Hg and Se, respectively. Accuracy and trueness of these methods were determined by analysis of the certified reference materials (CRMs) CRM NIST-1566 (oyster tissue) and TORT-3 (lobster hepatopancreas) and by regular participation in lab proficiency tests. The recoveries of CRMs were CRM NIST-1566: mean Se = 102 % and mean Hg = 84 %, and TORT-3: mean Se = 95 % and mean Hg = 82 %).

## 2.2.2. Bulk carbon and nitrogen stable isotope analysis

Bulk carbon and nitrogen stable isotope analysis (SIA) and carbon and nitrogen contents were conducted by the Institute for Energy Technology (Kjeller, Norway). Briefly, approximately 1 mg of sample was weighed into individual tin capsules, pelletised, and stored in well plates. Samples were then analysed by Elemental Analyser-Isotope Ratio Mass Spectrometry (EA-IRMS). The ¹⁵N:¹⁴N ratio (δ¹⁵N) was expressed relative to N₂ in air for nitrogen while that of ¹³C:¹²C (δ¹³C) was relative to Vienna Pee Dee Belemnite (VPDB) of CO₂. The accuracy and precision of the method was determined using internal standard (IFE trout) calibrated against international certified reference materials (IAEA-N-1 and IARA-N-2 for δ¹⁵N and USGS-24 for δ¹³C). The δ¹³C and δ¹⁵N values of the internal standard were: δ¹⁵N = 11.6 ± 0.2 ‰ and δ¹³C = 20.1 ± 0.1 ‰. All samples were analysed for δ¹³C, and δ¹⁵N.

## 2.2.3. Mercury stable isotope analysis

A subset of samples was selected for the mercury stable isotope analysis ($n_{reference} = 15$ and $n_{polluted} = 15$; for geographic distribution, see Fig. 1B). To avoid potential bioaccumulation of different Hg isotopes and gender-related effects, the selected individuals were all male and had similar total lengths (L) between 60 and 70 cm ($L_{reference} = 64.3$

± 16.6 cm and $L_{polluted} = 65.6 \pm 6.84$ cm; mean ± SD).

Mercury stable isotope analyses were conducted at Advanced Isotopic Analysis laboratories (AIA) in Pau, France. Approximately 250 mg of freeze-dried ground samples was used. The samples were digested on a heating block using a mixture of 3 mL HNO₃ solution and 1 mL HCl at 90 °C for 24 hours. Samples were then oxidised using 1 mL H₂O₂ solution and heated at 90 °C for 24 hours.

Mercury stable isotope analysis was performed using a cold vapor generation (CVG) with SnCl₂ reduction, coupled to a multi-collector-ICP-MS (Nu Plasma, Nu Instrument). Instrumental mass-bias was corrected by sample standard bracketing with NIST SRM 3133 standard solution [8]. Samples and standards were diluted to obtain a Hg concentration of 1 µg/L.

Quality assurance and quality control (QA/QC) of the results were performed by controlling the background blank of the reagents and analysing two certified reference materials: UM-Almaden (NIST 8610, *n* = 8) and ERM CE 464 (*n* = 3). The measured Hg isotope values of NIST 8610 were compared with its certified values, while those of ERM CE 464 were compared with the values presented in Sherman and Blum [80] apart from δ²⁰⁴Hg and Δ²⁰⁴Hg. Analytical uncertainty is expressed as 2 SD obtained for NIST 8610 to indicate the variability for the measured values of each sample (Table S2).

## 2.3. Data analysis

First, we used qqplot to evaluate data normality. For element concentration values, $log_{10}$ transformation was applied to ensure a normal distribution. All data analyses were conducted in program R (Version 4.3.0) and operated in RStudio (Version 1.4.1106).

### 2.3.1. Hg, Se, and Se:Hg molar ratio

We used analysis of variance (ANOVA) to compare $log_{10}$ transformed Hg, length-corrected Hg (i.e., Hg/total length), and $log_{10}$ transformed Se between reference and polluted sites. Statistical significance was accepted at $p \le 0.05$ [98]. We calculated Se:Hg molar ratios using molecular weight from each element (Hg: 200.59 and Se: 78.96).

<figure><img src="image_6.png" /><figcaption>**Fig. 1.** Map of sampling locations for European hake in the Northeast Atlantic Ocean, 2019–2022. A: All collected samples, B: Samples selected for mercury stable isotope analysis, and C: Distribution of mean total mercury (Hg) concentrations (mg/kg wet weight) in sampled European hake (mean, minimum, and maximum) for each sampling site. The base maps were created using Ocean Data View (version 5.6.3) [79]. In Fig. 1A and B, the red dot indicates the polluted site, and the blue dots indicate reference site sampling locations.</figcaption></figure>
### 2.3.2. Bulk δ¹³C$_{lipid-corrected}$ and δ¹⁵N

We used the mass balance arithmetic lipid correction method [84] to standardise the δ¹³C values to reduce the uncertainty introduced by the lipid content in the fillet tissue:

$$ \delta^{13}C_{\text{lipid-corrected}} = \frac{\delta^{13}C_{\text{sample}} \times C : N_{\text{sample}} + 7 \times (C : N_{\text{sample}} - C : N_{\text{protein}})}{C : N_{\text{sample}}} \quad (1) $$

where δ¹³C$_{sample}$ is the measured δ¹³C value, C : N$_{sample}$ is the ratio between the measured carbon content and nitrogen contents, and C : N$_{protein}$ with a constant of 3.7 as determined by Fry et al. [36].

To check whether the isotopic niche (as a proxy of trophic niche) differed between reference and polluted sites, we used δ¹³C$_{lipid-corrected}$ and measured δ¹⁵N values to calculate stable isotope Bayesian ellipses in program R (SIBER; [46]) and to construct sample size-corrected standard ellipses with 40 % confidence interval and then visually inspected ellipse overlap.

### 2.3.3. Trophic magnification factors

We investigated the trophic magnification factor (TMF) of Hg from reference and polluted sites by using linear regression between Hg concentrations and bulk δ¹⁵N values. The trophic magnification slope or TMS (b) of the linear regression was then used to calculate TMF using Eq. (2) assuming the trophic enrichment factor of δ¹⁵N equal to 3.4 ‰ [25].

$$ TMF = 10^{3.4 \times b} \quad (2) $$

### 2.3.4. Hg source discrimination

Hg SI profiles can successfully be used to trace Hg sources in areas with known point sources and can also be compared to biota inhabiting areas with reference conditions, with no obvious point sources of Hg (e. g., [11,56,51]). For individual Hg SIs, we compared values between the reference and polluted sites using one-way ANOVA. Then we used bi-plots of combinations of isotopes which are commonly applied to discriminate between sources of Hg in fish, including δ²⁰²Hg - Δ¹⁹⁹Hg (e.g., [56,55]) and Δ²⁰¹Hg - Δ¹⁹⁹Hg [81]. For δ²⁰²Hg - Δ¹⁹⁹Hg, we applied the SIBER approach [46] to delineate isotopic niches and to discriminate between reference and polluted sites. For Δ²⁰¹Hg - Δ¹⁹⁹Hg, we applied a linear regression to the data with zero intercept. The slope values were then compared with the slope values of the established regression lines for MMHg- predominance and HgII-predominance based on Sonke [81].

## 3. Results and discussion

We examined the Hg concentrations in EH fillets along the Norwegian coast and northern North Sea. Specifically, we compared Hg and Se between samples collected at a coastal site with a known point source (i. e., polluted site) and from offshore and other coastal sites (i.e., reference sites). We further investigated the stable carbon and nitrogen isotopic niches, trophic magnification factors, Hg stable isotopes, and associated isotopic niches between sites. Our data showed significant differences between reference and polluted sites for Hg and Se concentrations, trophic magnification of Hg, and Hg isotopic signatures. Our data also suggested that Hg stable isotopes were successfully discriminated between differential Hg baselines and were useful tools for tracking Hg and MeHg in EH fillets.

### 3.1. Mercury and selenium concentrations

Hg in EH varied across sampling sites and Hg concentrations tended to be higher at coastal sites compared to offshore sites (Fig. 1C). The highest mean Hg value, 0.397 mg/kg wet weight, was found outside of Molde area (~63.1°N, 6.8°E), while that of the polluted site ranked 6th (for details, see Table S3). For individual samples, the highest value

(0.713 mg/kg wet weight [total length = 105.5 cm]) was found at a reference site near the polluted site (~59.9°N, 3.1°E), and the second and third highest values were from the polluted site (0.554 mg/kg wet weight [total length = 72.0 cm] and 0.529 mg/kg wet weight [total length = 71.5 cm], respectively) (Fig. 1C). The high Hg values were likely due to high background Hg concentrations at or near the polluted site and/or the effect of bioaccumulation of Hg in large-bodied and older EH. Compared with current EU maximum limit (ML) for Hg in fish muscle (0.5 mg/kg wet weight) established under EU Regulation 2023/915, all sampled individuals had Hg below the ML apart from these three individuals (Fig. 2).

Across sites, both Hg and Se were higher in EH samples from the polluted site (Table 1). Compared with other commercial fish species in NEAO, EH from reference sites had mean Hg (0.114 ± 0.096 mg/kg wet weight; Table 1) similar to Atlantic wolffish, *Anarhichas lupus* (0.114 mg/kg wet weight; [102]) but much higher than other species from NEAO including Atlantic cod, * Gadus morhua*: 0.051, Atlantic herding, *Clupea harengus*: 0.039, and Atlantic mackerel, *Scomber scombrus*: 0.037 mg/kg wet weight [102]. Additionally, Se in EH from reference sites (0.321 ± 0.044 mg/kg wet weight; Table 1) were similar to haddock (*Melanogrammus aeglefinus*) and pollack (*Pollachius pollachius*) (0.333 and 0.327 mg/kg wet weight, respectively; [102]) but substantially lower than Greenland halibut (*Reinhardtius hippoglossoides*, 0.421 mg/kg wet weight; [102]).

At both reference and polluted sites, Hg and Se were positively correlated and the ellipses of Hg and Se were significantly discriminable between sites (Fig. 2A). Apart from Hg, anthropogenic influences can also provide additional Se sources to marine ecosystems [63], causing higher Hg and Se baseline concentrations (i.e., in sediments, seawater, and plankton) at the polluted site. However, baseline samples were not collected in the present study to confirm this. Additionally, the length-corrected Hg concentrations differed between reference and polluted sites (Table 1) suggesting that size/age and bioaccumulation of Hg were secondary drivers behind the observed Hg differences, especially when considering the strong influence of the point source as a modulator of Hg exposure.

Se and Hg concentrations had a weak but significant positive correlation at reference sites ($R^2_{adj} = 0.02, p < 0.001, n = 763$) and a stronger correlation at the polluted sites ($R^2_{adj} = 0.42, p < 0.001, n = 25$) (Fig. 2A). This suggests that elevated Hg loadings may lead to active accumulation of Se, which is likely linked to the trophic magnification of Se (Fig. 3), yet the underlying mechanisms remain unknown.

### 3.2. Se:Hg molar ratios

The relationships between Se:Hg molar ratios and Hg were similar between reference and polluted sites, however, when controlling for Hg concentration, samples from the polluted site had higher Se:Hg molar ratios compared to those from reference sites (Fig. 2B). This can be due to higher Se baseline values or variation in Se speciation dynamics, or in response to other factors beyond Se-Hg interactions [34,62,96] across the different sites, however this was beyond the scope of this investigation. Additionally, individuals from the polluted site did not have the highest maximum Hg concentration values or Se:Hg molar ratios compared with those from the reference sites (Fig. 2B). Although the total lengths were similar between reference and polluted sites (Table 1), the former had a wider individual length range compared to the latter (total length range = 29.0–113.5 and 54.0–84.0 cm, respectively). Further investigation of Se-Hg interactions in these larger-bodied EH individuals may be required to further understand these relationships.

All samples had Se:Hg molar ratios > 1 (i.e., Se and Hg are equal in mole) suggesting a potential protective effect against MeHg toxicity in fish and human consumers [3,72]. However, the underlying mechanism of Se-Hg interaction and the relationship between the protective effects of Se and Se:Hg molar ratios remain poorly understood and consistently
<figure><img src="image_7.png" /><figcaption>**Fig. 2.** A: Biplot of total selenium (Se) – mercury (Hg) concentrations (mg/kg wet weight) in sampled European hake from the reference and polluted sites in Norway during 2019–2022. The sample size-corrected standard ellipse was drawn using SIBER [46], and linear regression was performed for the correlation between Se and Hg. B: Biplot of selenium to mercury molar ratio (Se:Hg) – Hg concentration. The EU regulation (No 2023/915) maximum limit for Hg in fish fillet (0.5 mg/kg wet weight), and Se:Hg = 1 and 5 are illustrated.</figcaption></figure>

**Table 1**

Total mercury (Hg; mg/kg wet weight), total length (cm), length-corrected Hg (Hg/L; mg/kg/cm), and total selenium (Se; mg/kg wet weight) of all samples, and mercury stable isotope values (‰) of selected samples. All values are mean ± 1 SD, and element concentrations were based on wet weight. European hake samples were collected during 2019–2022 from reference sites and a polluted site in the Northeast Atlantic Ocean with sample sizes indicated (n). One-way ANOVA was performed to analyse the difference in the values between the reference and polluted sites with accepted statistical significance *p* < 0.05. For element concentrations, log₁₀ transformation was conducted prior to the one-way ANOVA.

<table><thead><tr><th rowspan="2">Variable</th><th>Reference</th><th>Polluted</th><th rowspan="2">p-value</th></tr><tr><th>n = 763</th><th>n = 25</th></tr></thead><tbody><tr><td>Hg</td><td>0.114 ± 0.096</td><td>0.271 ± 0.153</td><td>p < 0.05</td></tr><tr><td>Total length</td><td>64.3 ± 16.6</td><td>65.6 ± 6.8</td><td>p = 0.70</td></tr><tr><td>Hg/Total length</td><td>0.00164 ± 0.00109</td><td>0.00405 ± 0.00172</td><td>p < 0.05</td></tr><tr><td>Se</td><td>0.321 ± 0.044</td><td>0.425 ± 0.033</td><td>p < 0.05</td></tr><tr><td>Se:Hg molar ratio</td><td>12.1 ± 7.91</td><td>6.39 ± 4.96</td><td>p < 0.05</td></tr><tr><td></td><td>n = 15</td><td>n = 15</td><td></td></tr><tr><td>δ<sup>204</sup>Hg</td><td>1.400 ± 0.258</td><td>0.995 ± 0.182</td><td>p < 0.05</td></tr><tr><td>δ<sup>202</sup>Hg</td><td>0.964 ± 0.177</td><td>0.714 ± 0.115</td><td>p < 0.05</td></tr><tr><td>δ<sup>201</sup>Hg</td><td>1.500 ± 0.175</td><td>1.207 ± 0.136</td><td>p < 0.05</td></tr><tr><td>δ<sup>200</sup>Hg</td><td>0.531 ± 0.104</td><td>0.389 ± 0.074</td><td>p < 0.05</td></tr><tr><td>δ<sup>199</sup>Hg</td><td>1.220 ± 0.135</td><td>1.010 ± 0.116</td><td>p < 0.05</td></tr><tr><td>Δ<sup>204</sup>Hg</td><td>-0.035 ± 0.072</td><td>-0.071 ± 0.069</td><td>p = 0.17</td></tr><tr><td>Δ<sup>201</sup>Hg</td><td>0.776 ± 0.072</td><td>0.670 ± 0.098</td><td>p < 0.05</td></tr><tr><td>Δ<sup>200</sup>Hg</td><td>0.047 ± 0.036</td><td>0.030 ± 0.044</td><td>p = 0.26</td></tr><tr><td>Δ<sup>199</sup>Hg</td><td>0.981 ± 0.105</td><td>0.829 ± 0.110</td><td>p < 0.05</td></tr></tbody></table>

inconsistent [39] and are often dependent on speciation dynamics and the chemical forms of Hg and Se [30].

**3.3. Carbon and nitrogen stable isotope (δ¹³C – δ¹⁵N) dynamics**

The δ¹³C – δ¹⁵N isotopic niche (sample size-corrected standard ellipse) of EH samples differed between reference and polluted sites (Fig. 3A). The samples from the polluted site had higher δ¹⁵N values with a narrower δ¹³C range compared to EH from reference sites. Similar trends were observed when we analysed all samples compared with the subset selected for Hg isotope analyses (Fig. 3B) indicating that selected samples were representative of both the pollution and reference

conditions.

δ¹⁵N baseline values in coastal and offshore areas in the NEAO vary greatly [31], however, marine environments are highly susceptible to human impacts and these impacted areas tend to have elevated δ¹⁵N baselines [1,26]. Since we did not collect baseline samples, we could not confirm whether the elevated δ¹⁵N values in the individuals from the polluted site corresponded to higher TPs than those from the reference sites. Additionally, δ¹³C often indicates different energy sources and individual food habits, and coastal δ¹³C baselines are often greater than offshore ones [19] apart from known exceptions in mangrove ecosystems where δ¹³C is often depleted [14]. The spatial distribution of reference samples was much wider compared to polluted samples and hence had more variable δ¹³C values, likely reflecting more variable dietary patterns and differences in energy sources. Overall, it is unclear whether the trophic niche of EH differed between reference and polluted sites, and isotopic baseline data are needed to verify these observations.

**3.4. Source discrimination using Hg stable isotopes**

The Hg isotopic pattern in EH samples differed between reference and polluted sites (Table 1). Regarding both, MDF and odd MIF signatures (e.g., δ²⁰²Hg and Δ¹⁹⁹Hg), higher values were observed at the reference sites compared to the polluted site (e.g., δ²⁰²Hgreference = 0.964 ± 0.177; δ²⁰²Hgpolluted = 0.714 ± 0.115 and Δ¹⁹⁹Hgreference = 0.981 ± 0.105, and Δ¹⁹⁹Hgpolluted = 0.829 ± 0.110 ‰), while there was no difference between sites for the even MIF isotopes (e.g., Δ²⁰⁰Hg). The difference between odd and even isotopes are likely due to the differential Hg isotopic values at the food web baselines, and lower reaction rates of even isotopes to MIF [9].

The isotopic niche of δ²⁰²Hg and Δ¹⁹⁹Hg also allowed for substantial discrimination of the sample size-corrected standard ellipses between them (Fig. 4A). In contrast, the linear regressions of Δ¹⁹⁹Hg – Δ²⁰¹Hg were nearly identical between reference and polluted sites (Fig. 4B) with the Δ¹⁹⁹/²⁰¹Hg slopes being 1.23 ± 0.03 and 1.26 ± 0.05, respectively (mean ± 2SE). This indicates that Hg in EH’s diet principally underwent MeHg photodegradation [8,81] although the Hg was from different sources between sites and that this process was not significantly affected by the Hg point source.

Notably, the polluted site in this study was coastal, while several of the reference sites were offshore. In general, compared with the open ocean, coastal sites exhibit lower MIF (e.g., Δ¹⁹⁹Hg; [70]) and δ²⁰²Hg
<figure><img src="image_8.png" /><figcaption>**Fig. 3.** Biplot of δ¹³C – δ¹⁵N in European hake from the reference sites and the polluted site during 2019–2022 in Norway for A) All collected samples, and B) Samples analysed for mercury stable isotopes. The sample size-corrected standard ellipse was drawn using SIBER [46]. Trophic magnification of C) Total mercury (log₁₀Hg – δ¹⁵N), D) Total selenium (log₁₀Se – δ¹⁵N), E) Trophic position (δ¹⁵N as a proxy) and F) δ¹⁵N corrected-Hg in relation to total length. Linear regression was applied to estimate the trophic magnification factor of Hg and the relationships between δ¹⁵N / δ¹⁵N corrected-Hg and length.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 4.** A: Relationships between δ²⁰²Hg and Δ¹⁹⁹Hg in European hake from the reference and polluted sites in Norway during 2019–2022. The sample size-corrected standard ellipse was drawn using SIBER [46]. B: Relationships between Δ¹⁹⁹Hg and Δ²⁰¹Hg. Linear regression with zero intercept was applied (solid line). Regression lines (long-dashed) and slopes of MMHg-predominated MIF and Hg II -predominated MIF are presented. The error bar indicates 1 × SD of replicated measurements (n = 8) of UM-Almaden (NIST 8610) secondary standard.</figcaption></figure>

values [100], and our findings were in accordance with these previously
observed trends (Table 1, Fig. 4). As MIF in EH from both sites was
related to in situ photodegradation, such observed difference may be
attributable to different basal Hg isotopic signatures between the two
sites that sediments contaminanted by point sources are often isotopi-
cally discriminable from local background isotopic baselines [11]. The
small negative Δ²⁰⁴Hg and small positive Δ²⁰⁰Hg signatures in EH from
both sites suggested important input of Hg sources from precipitation Hg
(II) [87]. This is also supported by the positive Δ¹⁹⁹Hg values at both
sites from our investigation which pointed to atmospheric Hg source
through Hg(II) photoreduction [97]. Industrial sources of Hg(II) often
have lower negative δ²⁰²Hg signatures (i.e., −1 to 0 ‰) [87], this might
have resulted in the lower δ²⁰²Hg values observed at the Kollevåg
polluted site (Fig. 1) and was likely as a result of the mixing of atmo-
spheric and local Hg point sources. However, starvation may also reduce
δ²⁰²Hg values in fish muscle [50], and therefore it is challenging to
accurately assess and fully quantify the contributions from the two
sources mentioned above.

Depth in the water column and dissolved organic carbon (DOC) gradients may also govern the distribution of δ²⁰²Hg and odd MIF isotope values [10]. However, as EH can feed throughout the water

column [67,92], their mixed diet may mitigate the influence of depth
and DOC on photodegradation, however further research is required to
evaluate this hypothesis.

3.5. *Trophic magnification of Hg*

In both reference and polluted sites, Hg increased with TP (using
δ¹⁵N as a proxy and assuming the same δ¹⁵N baseline). The degree of
trophic magnification of Hg differed between reference and polluted
sites (Fig. 3C) with TMS values = 0.158 ± 0.014 and 0.392 ± 0.043,
respectively (mean ± 1 SE) and mean TMF values = 3.45 and 21.52,
respectively. This can be partially due to ontogenetic dietary shift of EH.
EHs from both sites were feeding on higher TPs with increasing total
length, but the degree of change was higher in the polluted site
compared to reference sites (Fig. 3E). However, when Hg was normal-
ized by δ¹⁵N, the degree of change was still higher at the polluted site
(Fig. 3F), suggesting that elevated Hg concentrations strongly influenced
the observed higher TMS values at the polluted site.

A previous meta-analysis study by Lavoie et al. [54] and later studies
suggested that higher Hg loading may lead to higher TMS values. Lavoie
et al. [54] established a TMS spectrum (-0.19-0.48) and TMS values
estimated from subsequent investigations were all within the TMS spectrum, e.g., 0.095–0.290 (Antarctica and sub-Antarctica; [33,64]), 0.147 (Southeastern Gulf of California, USA; [89]), 0.155–0.201 (Florida coral reefs, USA; [77]), 0.271 (Southwest Florida; [85,23,12]), 0.067–0.355 (Oregon coast, Sanriku coast, and Jakarta Bay; [75]) and 0.357–0.426 (northwest Spain; [23,12]). Lavoie et al. [54] reported that Hg food web biomagnification was highest in cold and low productivity systems, and that warm water, temperate ecosystems were also vulnerable due to anthropogenic pollution, for example, Bay of Biscay in northwest Spain [13,59] and the Amazon region due to effluents from gold mining (TMS = 0.19–0.21; [4]). Additionally, in contrast to our findings, Van der Velden et al. [90] found no significant difference of TMS values in Arctic char (Salvelinus alpinus) from habitats of higher (TMS = 0.14–0.22) and lower Hg loadings (TMS = 0.07–0.16), although the range of TMS values from higher Hg loading was higher than the range from lower Hg loading habitats. Therefore, it is likely that in their investigation, the elevated Hg concentrations were less influenced by anthropogenic factors. Apart from Hg loadings, different temperature regimes may yield distinctive TMS values due to potential changes in fish growth rates and possible biodilution effects [54]. Additionally, TMS values may also be affected by salinity gradients [76], who reported that higher salinity led to higher TMS values. Lastly, Lavoie et al. [54] reported very low R² values for the TMS and latitude regression relationships for both freshwater sites (≤ 13 % variance explained) and all sites (≤ 7 % variance explained), highlighting that much of the ecosystem-scale variation remains unexplained and that further research is required to understand the inherent complexities of the Hg bioaccumulation phenomenon in aquatic biota.

MeHg is efficiently transferred in marine food webs and trophic magnification dynamics in both warm and cold-water systems have been

widely reported [44,53,54]. This has important implications for seafood safety since MeHg has higher toxicity than its counterpart iHg [16]. Hg in EH fillets from the Norwegian coast was primarily in the MeHg form (≥91 %; Table S1), thus, the TMS values of Hg served as a good proxy for MeHg TMS values in EH.

Our data show that under relatively low Hg loadings in reference sites, Hg MDF (δ²⁰²Hg) demonstrated a negative δ²⁰²Hg – δ¹⁵N trend (p < 0.05; Fig. 5). In contrast, at the polluted site, an opposite trend was observed (Fig. 5). These differential trends between the two sites were also the case for Hg MIF values (Δ¹⁹⁹Hg and Δ²⁰¹Hg) (Fig. 5). Interestingly, decreasing MDF and MIF isotopic signatures in reference sites and increasing signatures in the polluted site were also noticed when these isotopic signatures were analysed against Hg concentrations (Fig. 5). This is likely indicative of the compositional dynamics of Hg isotopes when they were being biomagnified in EH muscle tissues under different Hg loadings. Further research is required to understand the underlying mechanisms behind this observed pattern.

## 4. Study limitations

Our study has a few caveats that need to be considered in the context of our study inferences and their potential limitations. The reference sites in our investigation encompassed a larger area compared to the polluted site. While this categorization was solely based on point source location, natural variation in the Hg baselines among reference sites may contribute uncertainty to our inferences. Nonetheless, our large sample size and broad geographical coverage provides a strong overview of the Hg bioaccumulation exposure regime of EH in Norway and therefore may counterbalance this potential limitation, while also recognizing that our results are context specific to the Norwegian region of the

<figure><img src="image_10.png" /><figcaption>**Fig. 5.** δ/Δ⁴⁰⁰Hg – δ¹⁵N and δ/Δ⁴⁰⁰Hg – Hg relationships for δ²⁰²Hg, Δ²⁰¹Hg, and Δ¹⁹⁹Hg in European Hake from Norway during 2019–2022. Linear regression was applied to estimate the relationships between δ/Δ⁴⁰⁰Hg and δ¹⁵N or Hg for reference and polluted sites. For regressions relationships for all other Hg stable isotopes, see Fig. S1 and Fig. S2.</figcaption></figure>
NEAO.

To compare the isotopic values and niches between groups, bulk δ¹⁵N stable isotopes values are often corrected using isotopic baselines of local food webs. However, this was not possible considering the large scale and scope of this work, hence affecting the inferences from the δ¹³C – δ¹⁵N isotopic niche data. Additionally, although EH is a rather sedentary species, it likely utilizes several prey items from different primary production sources due to its ontogenetic dietary shift and vertical migration feeding habits. Thus, potential multiple δ¹⁵N baselines may be incorporated into individuals resulting in uncertainty in the precise estimation of trophic position and subsequently the derivatisation of TMS and TMF values. Yet since we used adult, commercial size classes and avoided sampling of juvenile and young of the year fish, this limitation is likely minimal. Using amino acid-specific nitrogen stable isotopes to estimate TP can further reduce such uncertainty [52] since both isotopic baseline and fractionation information can be obtained from a single consumer sample [65], thus significantly reducing sampling effort. However, trade-offs exist as the cost and time to conduct these analyses is truly quite high.

The estimation of food web-TMS values may incorporate uncertainty due to marine ecosystem community dynamics (e.g., presence or absence of top predators in the ecosystem; [60,88]) and the resolution of data points (e.g., individual- or species-based; [60]). In comparison, species-TMS value estimation can reduce such uncertainty (e.g., Arctic char from Canadian Arctic and sub-Arctic; Van der Velden et al. [90], and spotted seal, *Phoca largha*, and minke whale, *Balaenoptera acutorostrata*, from Yellow Sea, China; [86]), yet may also be highly nuanced, variable across species, and highly context-specific.

MeHg is widely recognized as the key Hg species because of its toxicological implications for seafood safety; consequently, investigating its biogeochemical cycling in different marine ecosystem compartments using MeHg specific Hg isotope analysis can aid in identifying its complex pathways of trophic transfer and bioaccumulation [100,99]. While this was not performed in the present study, our inferences are based on Hg isotope data in EH fillet tissue which was comprised of ≥ 91 % MeHg (Table S1), and therefore highly representative of MeHg-specific isotopes.

Apart from the two areas with elevated Hg concentrations identified in our data, there are other locations with potential Hg point sources in the NEAO, for example, the Hardangerfjord ecosystem [2] in western Norway. How high Hg concentrations may impact bioaccumulation dynamics in EH from these locations may warrant further investigation. Lastly, the present study indicated good discriminability between the polluted and reference sites along the Norwegian coast using Hg stable isotopes in EH. However, how Hg isotopes fractionate through the complex food web of the NEAO remains unclear and will require further investigation to gain a further understanding of these inherently complex biogeochemical and ecosystem processes.

## 5. Conclusions

We successfully differentiated Hg and Se concentrations in EH fillets collected from reference sites along the Norway coast and a known point source, Hg polluted site. Concentrations of both Hg and Se and Se:Hg molar ratios were elevated due to high Hg loadings at the polluted site. Additionally, the high Hg loading was also shown to increase the bio-accumulation and biomagnification of EH Hg and led to trophic magnification of MDF and MIF Hg isotopic signatures. Apart from Hg, stable isotopes of Hg in EH fillets were also affected by the Hg point source site. While individuals from the reference and polluted sites were both exposed to Hg which was mainly derived from photodegradation of MeHg, the isotopic signatures (MDF and MIF) were different between polluted and reference sites. This indicates good discriminability of Hg and δ¹⁵N in source differentiation and potential in source apportionment of Hg. Future work to refine our understanding of these mechanisms include investigation of Se-Hg interactions under elevated Hg loading,

and trophic magnification/elimination of compound specific Hg stable isotopes.

## Environmental implication

Point sources of Hg pose significant risks to marine ecosystems and potentially to human consumers of marine fish. Elevated Hg concentrations may shift trophic transfer dynamics of Hg, particularly the more toxic form of MeHg in marine food webs. Hg stable isotope analyses can be used to differentiate natural sources and point sources of Hg to allow for a greater understanding of trophic transfer and fate and transport dynamics of Hg in marine fish species commonly consumed by humans. Our findings have important implications for understanding how Hg point sources modulate bioaccumulation rates, Hg isotope regimes, and discriminability between natural sources of Hg in EH sampled along the Norwegian coast. Collectively, our findings have important implications for the Minamata Convention on Mercury and the relevant United Nations Sustainable Development Goals.

## CRediT authorship contribution statement

**Frøyland Livar:** Writing – review & editing, Resources, Investigation. **Bank Michael:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Zhu Yiou:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Barre Julien:** Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. **Staby Arved:** Writing – review & editing, Investigation. **Wiech Martin:** Writing – review & editing, Investigation. **Zayas Zoyne Pedrero:** Writing – review & editing, Visualization, Validation, Supervision, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Tessier Emmanuel:** Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. **Bérail Sylvain:** Writing – review & editing, Methodology, Investigation. **Maage Amund:** Writing – review & editing, Investigation.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

This project has received funding from the European Union's Horizon 2020 research and innovation MSCA-RISE program under grant agreement No. 101007962 (merfish.eu). This work was also supported by the Institute of Marine Research SeaTrace project (Project # 16086), and the Norwegian Food Safety Authority.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jhazmat.2025.138564.

## Data availability

Data will be made available upon request.

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