Limited iron isotope variation among tissues of a marine fish: a case study of wild chub mackerel (Scomber japonicus)

Iron homeostasis in marine organisms operates under chronically low iron bioavailability, which may shape the strategies of iron uptake and storage in fish. Stable iron isotope ratios (δ⁵⁶Fe) have emerged as tracers of iron storage and uptake in terrestrial mammals, yet the physiological drivers of isotope fractionation in marine organisms remain poorly understood. Here, we investigated δ⁵⁶Fe variation and iron speciation across eight tissues of wild chub mackerel (Scomber japonicus), along with pool size estimation of key Fe species, including ferritin-bound (ferric) and heme-bound (mainly ferrous) Fe, using Fe K-edge X-ray Absorption Near Edge Structure (XANES) spectroscopy. In all the specimens, the liver δ⁵⁶Fe values were higher than the average value of all tissues, with an apparent isotopic shift between ferritin- and heme-bound Fe (Δ⁵⁶Fe) in the liver averaging 2.72 ‰ ± 3.03 ‰ (2 S.D.). In contrast to the liver, no enrichment of heavy Fe isotope was observed in the ovary and red muscle despite their high ferritin-Fe contribution, suggesting a high interconversion rate between ferritin- and heme-bound Fe pools in these tissues. The overall range of δ⁵⁶Fe variation among tissues was smaller than that reported in mammals. Our results suggested that muscular δ⁵⁶Fe in marine teleost is primarily governed by source signatures and intestinal uptake efficiency, while tissue heterogeneity due to heavy Fe storage by ferritin exerts only a minor influence. These findings highlight the potential of δ⁵⁶Fe as a proxy for intestinal iron acquisition in fish and provide new geochemical perspectives on iron cycling through marine food webs.

1 Introduction

Iron is an essential element for life, functioning in electron transfer through the redox cycling between Fe²⁺ and Fe³⁺ (Georgiadis et al., 1992; Hoffbrand et al., 1976; Robbins and Pederson, 1970). While soluble Fe²⁺ is highly bioavailable, insoluble Fe³⁺ dominates under atmospheric conditions (Crichton and Boelaert, 2001), resulting in dissolved iron concentrations below 2 nmol L⁻¹ in the open ocean (Hatta et al., 2015; Johnson et al., 1997; Martin and Gordon, 1988; Nishioka and Obata, 2017). This scarcity constitutes a major factor limiting primary production by phytoplankton (Boyd et al., 2000; Martin et al., 1989; Martin and Gordon, 1988).

Although iron limitation in primary producers is well established, its implications for higher organisms such as fish remain less understood. Unlike phytoplankton, tissue iron concentrations in fish are not directly constrained by seawater iron availability (Galbraith et al., 2019). This is based on long-term iron retention strategies via ferritin (iron storage protein) and efficient intestinal absorption mechanisms that specifically exist in marine fish (Bury et al., 2001; Carriquiriborde et al., 2004; Kwong and Niyogi, 2008). However, direct quantification of intestinal iron uptake has been largely restricted to laboratory animals using enriched isotopes (van den Heuvel et al., 1998; Fiorito et al., 2012), an approach that is not feasible for wild species especially marine fish.

Natural iron stable isotope ratios (δ⁵⁶Fe) offer a broadly applicable, non-lethal proxy for assessing iron metabolism. δ⁵⁶Fe values are sensitive to intestinal absorption processes (Albalat et al., 2021; Flórez et al., 2017; Van Heghe et al., 2013; Hotz and Walczyk, 2013; Krayenbuehl et al., 2005; Stenberg et al., 2003) as well as to variation in iron storage and homeostasis, with ferritin-bound iron often implicated as a key driver of tissue-specific fractionation in mammals (Van Heghe et al., 2014; Hotz et al., 2012; Jaouen and Balter, 2014). Extending this framework to fish requires evaluating both intestinal absorption and ferritin-associated storage effects.

In this study, we examined δ⁵⁶Fe distributions across tissues of marine fish chub mackerel (Scomber japonicus). Additionally, X-ray Absorption Near-edge Structure (XANES) spectroscopy provided a non-destructive and oxidation state–sensitive approach to quantify ferritin-bound iron, allowing direct estimation of the proportion of Fe³⁺ species in tissues without chemical extraction. Using this method, we quantified the relative contributions of ferritin- and heme-bound iron and assessed their roles in isotopic variability. By further considering physiological factors such as sex and reproductive status, our study provides new insights into the mechanisms governing δ⁵⁶Fe fractionation in fish and its potential as a tracer of intestinal iron acquisition in marine ecosystems.

2 Materials and Method

2.1 Sample Collection and Preparation

Six adult chub mackerel (females: Mk-1 to Mk-3, males: Mk-4 to Mk-6) were collected by longline fishing around Tsushima Island, East China Sea, on 27 April 2022. Fish were frozen onboard at −30 °C, stored at −20 to −25 °C for eight days, and subsequently transported to the laboratory at −15 °C. Body weight and fork length were recorded prior to dissection. Total blood volume was estimated as 60 mL kg⁻¹ according to teleost values (Brill et al., 1998; Itazawa et al., 1983). Each individual was dissected into eight tissues (red and white muscle, liver, gonad, spleen, heart, gills, and blood). All tissue samples were stored at −80 °C until the analysis. Wet weight was measured, and hepatosomatic index (HSI) and gonadosomatic index (GSI) were calculated as follows (Allaya et al., 2013; Shiraishi et al., 2008):

$$\begin{array}{}\text{(1)}& {\displaystyle }\text{HSI}={\displaystyle \frac{\text{Liver weight}}{\text{Total body weight}}}\times \mathrm{100}\text{(2)}& {\displaystyle }\text{GSI}={\displaystyle \frac{\text{Gonad weight}}{\text{Somatic weight}}}\times \mathrm{100}\end{array}$$

Tissues with high iron content (red muscle, liver, gonad, blood, and heart) were selected for Fe K-edge XANES analysis. Small frozen fragments were immediately sealed in oxygen-impermeable polycarbonate film (ASONE, Japan) after dissection and stored until measurement.

2.2 Iron Concentration and Stable Isotope Measurement

Analytical procedures followed Hasegawa et al. (2023). Tissues were freeze-dried, and lipids removed from liver and gonad using chloroform : methanol mixture (2:1, $v/v$). Tests with certified reference materials confirmed no significant isotopic alteration during lipid removal (<0.07 ‰, n=4). Dried tissues (0.3–2.5 g) were digested in perfluoroalkoxy (PFA) vials using 68 % HNO₃ and 35 % H₂O₂ (both Tamapure AA-100, Tama Chemical Corp., Japan) at 120 °C. The digests were evaporated to dryness and re-dissolved in 7 M HCl containing 0.001 % H₂O₂. Each sample was split for iron concentration analysis by inductively-coupled plasma mass spectroscopy (ICP-MS; Agilent 7700) and another aliquot for isotope analysis. Iron concentrations were determined from a single aliquot of each tissue sample.

Iron purification was performed using anion-exchange chromatography following Maréchal et al. (1999). Interfering elements (Ni, Cr, Cu) were removed with 7 M HCl, and Fe was subsequently eluted with 2 M HCl. The purified Fe fraction was dissolved in 2 % HNO₃ at a concentration of 1 µg mL⁻¹, with Cu added for mass bias correction.

Stable isotope ratios were determined by multicollector-ICP-MS (Neptune Plus, ThermoFisher Scientific) at The University of Tokyo. Instrumental settings were: RF power 1200 W; Ar gas flow 15 L min⁻¹ (cool), 0.7 L min⁻¹ (auxiliary), 1.0 L min⁻¹ (nebulizer). Signal intensity was 5–8 V for 1 µg mL^{−1 56}Fe⁺, with blanks below 0.004 V. Each tissue sample was measured once for iron isotope composition with 60 analytical cycles. The δ⁵⁶Fe value was expressed relative to IRMM-014 as:

$$\begin{array}{}\text{(3)}& \mathit{\delta }{}^{\mathrm{56}}\mathrm{Fe}=\left[{\displaystyle \frac{({}^{\mathrm{56}}\mathrm{Fe}/{}^{\mathrm{54}}\mathrm{Fe}{)}_{\text{sample}}}{({}^{\mathrm{56}}\mathrm{Fe}/{}^{\mathrm{54}}\mathrm{Fe}{)}_{\text{IRMM-014}}}}-\mathrm{1}\right]\times \mathrm{1000}\left[\mathrm{‰}\right]\end{array}$$

Whole-body δ⁵⁶Fe (δ_WB) was calculated as:

$$\begin{array}{}\text{(4)}& {\mathit{\delta }}_{\text{WB}}=\sum _i(\mathit{\delta }{{}^{\mathrm{56}}\mathrm{Fe}}_i\times T_i)=\sum _i(\mathit{\delta }{{}^{\mathrm{56}}\mathrm{Fe}}_i\times C_i\times M_i)\end{array}$$

where i represents the ith tissue, and T_i, C_i, and M_i represent the Fe burden, Fe concentration, and tissue mass, respectively.

Analytical accuracy was verified using certified reference materials (BCR-414, DORM-4, DOLT-5, ERM-CE464). Results were consistent with reported values: BCR-414 (−0.10 ‰ ± 0.03 ‰), DORM-4 (−0.24 ‰ ± 0.06 ‰), DOLT-5 (−2.32 ‰ ± 0.02 ‰), ERM-CE464 (−0.55 ‰ ± 0.02 ‰).

2.3 XANES Spectroscopy

Iron K-edge XANES was analyzed at BL-12C, Photon Factory (KEK, Japan) following the protocol of Hasegawa et al. (2023). Frozen tissue fragments were analyzed in fluorescence mode using a Si detector, with energy calibration against hematite (7110.2 eV). The number of independent spectral components was determined by principal component analysis (PCA).

The dominant Fe species in animal tissues include heme (Fe–porphyrin), ferritin (storage), and transferrin (Fe transport). Transferrin-bound Fe was excluded from further analysis due to minor abundance and negligible isotope fractionation (Walczyk and von Blanckenburg, 2005). Thus, subsequent analyses focused on ferritin- and heme-bound Fe species. Linear combination fitting (LCF) was performed to quantify their relative proportions in each tissue.

To account for possible post-mortem oxidation of hemoproteins, spectra of oxyHb, deoxyHb, and metHb were included in the fitting. Horse spleen ferritin (Hs-Ft) and bovine blood metHb (Bb-metHb) were used as reference standards, while oxyHb and deoxyHb were prepared from metHb following Di Iorio (1981) and Wilson et al. (2013). Pre-edge features were extracted and fitted using pseudo-Voigt functions implemented in Python. The quality of spectra fits was evaluated using the R-factor, defined as:

$$\begin{array}{}\text{(5)}& R={\displaystyle \frac{\sum \mathit{\left\{}{\mathit{\chi }}_{\text{obs}}\right(E)-{\mathit{\chi }}_{\text{cal}}(E){\mathit{\}}}^{\mathrm{2}}}{\sum \mathit{\left\{}{\mathit{\chi }}_{\text{obs}}\right(E){\mathit{\}}}^{\mathrm{2}}}}\end{array}$$

2.4 Statistical Analysis

Statistical analyses were conducted in Python (ver. 3.9.19). Sex differences in physiological traits, Fe concentration, and isotope ratios were tested using the Mann–Whitney U test.

3 Results

3.1 Physiological Parameters of Chub Mackerel

The six chub mackerel analyzed had fork lengths (mean ± 2 S.D.) of 37.7 ± 2.41 cm in females and 40.9 ± 4.00 cm in males, and corresponding body weights of 0.71 ± 0.30 kg and 0.89 ± 0.24 kg, respectively (Table 1). Although females tended to be slightly smaller, the difference in body length was not significant (p=0.06). Based on the growth curve of Shiraishi et al. (2008), all individuals were estimated to be at least four years old and sexually mature. The hepatosomatic index (HSI) ranged from 1.05–2.14 in females and 0.74–1.26 in males, and the gonadosomatic index (GSI) ranged from 4.26–10.1 in females and 7.25–10.0 in males, consistent with the spawning season in the East China Sea (Shiraishi et al., 2008). Among tissues, white muscle represented the largest biomass fraction (31 %–52 % of total body weight; Fig. S1 in the Supplement), followed by gonads (3.8 %–8.9 %), red muscle (4.5 %–7.7 %), gills (2.1 %–3.9 %), liver (0.4 %–1.9 %), heart (0.2 %–0.6 %), and spleen (0.2 %–0.4 %). No significant sex-related differences were observed (p>0.05). Red and white muscles, as well as gills, exhibited strong positive correlations with body weight (r>0.82, p<0.04), whereas gonads showed a positive but non-significant trend (r=0.77, p=0.07; Fig. S2).

Table 1 Physiological parameters of chub mackerels.

3.2 Tissue Iron Concentrations and Stable Isotope Ratios

Mean iron concentrations (±2 S.D.) were highest in the spleen (3100 ± 860 $\mathrm{\mu }\mathrm{g}(\mathrm{g}\mathrm{d}.\mathrm{w}.{)}^{-\mathrm{1}}$), followed by blood (1300 ± 480 $\mathrm{\mu }\mathrm{g}(\mathrm{g}\mathrm{d}.\mathrm{w}.{)}^{-\mathrm{1}}$), heart (720 ± 580 $\mathrm{\mu }\mathrm{g}(\mathrm{g}\mathrm{d}.\mathrm{w}.{)}^{-\mathrm{1}}$), liver (630 ± 510 $\mathrm{\mu }\mathrm{g}(\mathrm{g}\mathrm{d}.\mathrm{w}.{)}^{-\mathrm{1}}$), gills (290 ± 90 $\mathrm{\mu }\mathrm{g}(\mathrm{g}\mathrm{d}.\mathrm{w}.{)}^{-\mathrm{1}}$), red muscle (250 ± 84 $\mathrm{\mu }\mathrm{g}(\mathrm{g}\mathrm{d}.\mathrm{w}.{)}^{-\mathrm{1}}$), gonads (37 ± 24 $\mathrm{\mu }\mathrm{g}(\mathrm{g}\mathrm{d}.\mathrm{w}.{)}^{-\mathrm{1}}$), and white muscle (15 ± 10 $\mathrm{\mu }\mathrm{g}(\mathrm{g}\mathrm{d}.\mathrm{w}.{)}^{-\mathrm{1}}$) (Fig. 1A). Male spleens tended to have higher Fe concentrations than those of females, and liver and gonads also tended to be higher in males.

Figure 1 (A) Iron concentration, and (B) Tissue iron burden of the chub mackerels (Mk-1 to Mk-3: female, Mk-4 to Mk-6: male). Total blood contents were assumed to be 30 mL kg⁻¹ with a specific gravity of 1.05. No uncertainty is reported as multiple sample replicates were not measured. The measurement uncertainty was assumed to be zero.

Assuming a blood volume of 30 mL kg⁻¹ and 1.05 as the specific gravity of fish blood (Davison, 2011), the total iron content in blood was estimated to be between 3.2 and 10.3 mg (Table S1 in the Supplement). Red muscle contributed the next largest Fe pool (2.3–6.1 mg), followed by gills (1.3–3.7 mg), white muscle (0.9–2.0 mg), spleen (0.8–2.6 mg), liver (0.5–2.0 mg), gonads (0.3–1.0 mg), and heart (0.2–0.9 mg). The total body iron inventory was therefore estimated to be approximately 17–26 mg kg⁻¹. Although the liver is the principal iron storage tissue, it accounted for only 4 %–8 % of total body iron. In contrast, blood and red muscle together comprised 51 %–67 % of the total iron burden.

Mean iron isotope compositions (δ⁵⁶Fe ± 2 S.D.) of each tissue across individuals were as follows: red muscle, −1.54 ‰ ± 0.20 ‰; white muscle, −1.46 ‰ ± 0.21 ‰; liver, −1.19 ‰ ± 0.16 ‰; gonads, −1.23 ‰ ± 0.42 ‰; spleen, −1.37 ‰ ± 0.15 ‰; heart, −1.43 ‰ ± 0.19 ‰; gills, −1.37 ‰ ± 0.22 ‰; and blood, −1.39 ‰ ± 0.20 ‰ (Fig. 2). The net δ⁵⁶Fe in whole mackerel bodies weighted by tissue Fe contents ranged from −1.50 ‰ to −1.35 ‰ with the liver in all individuals showing higher values than the net values. No sex differences were detected, although ovarian δ⁵⁶Fe was lower than testicular values.

Figure 2 Iron stable isotope ratio in tissues of the six Scomber japonicus individuals (Mk1–3: females, Mk4–6: males). Error bars represent 2 S.E. during a single measurement (60 analytical cycles).

3.3 Proportion of Ferritin-Bound Iron

Iron K-edge XANES spectra identified the dominant chemical forms of iron in each tissue (Wilke et al., 2001). Pre-edge peak positions for all samples were distributed between ferritin (most oxidized) and hemoglobin (reduced) reference standards (Fig. 3A). We note that the XANES spectrum of MK-2 liver was unavailable because its quality was not good.

Figure 3 (A) Fe K-edge XANES spectra of Scomber japonicus tissues (Mk-1 and Mk-3: female, Mk-4 to Mk-6: male). Mk-1 and Mk-3 represent females, and Mk-4 to Mk-6 represent males. The standard spectra were obtained from ferritin from horse spleen and hemoglobin derivatives from bovine blood. Blue lines indicate the spectra of the standard materials, while black lines represent the tissue samples. Red dashed lines show the results of linear combination fitting (LCF) using the four standard spectra. Gray solid lines correspond to the first derivative of the normalized XANES spectra, which highlights the peak top energies, and gray dashed lines mark the peak top energy of Ferritin at 7129 eV. (B) Estimated proportions of each standard materials with linear combination fitting (LCF) of XANES spectra. R value is a goodness-of-fit parameter described in Eq. (4). Each number on the pie charts represents proportions (%) of each component.

Linear combination fitting (LCF) using Hs-Ft and Bb-Hb derivatives revealed that red muscle and heart were dominated by heme-bound Fe, whereas ferritin accounted for 8 %–31 % of total Fe in the liver (Fig. 3B). One individual exhibited extremely low liver ferritin iron (8 %). The liver ferritin fraction was comparable to those reported for chub mackerel from other regions (28 %; Hasegawa et al., 2023) but substantially lower than those of mouse liver (66 %; Chen and Chen, 2018).

Sex-related differences were also apparent: females exhibited lower ferritin-bound Fe proportions in the liver, while higher proportions in the red muscle and gonads than males. In ovaries, ferritin represented the predominant Fe form (70 %–80 %), whereas testes showed highly variable proportions (3 %–59 %).

4 Discussion

4.1 Elevated δ⁵⁶Fe in Liver and Contribution of Ferritin-Bound Iron

First, we focused on the liver because it is a central tissue for iron storage in vertebrates and is expected to exhibit iron isotope fractionation through the synthesis of ferritin. All the chub mackerel individuals showed higher δ⁵⁶Fe values in the liver than δ_WB across eight tissues. Because ferritin preferentially incorporates heavier Fe isotopes during the oxidation in the storage process according to human studies (Albarède et al., 2011), this enrichment most likely reflects the contribution of ferritin-bound iron within the liver. It should be noted that human ferritin consists of H and L subunits responsible for iron oxidation and mineralization (Plays et al., 2021). While teleost ferritin includes an additional M-type subunit with intermediate functional properties, its effect on iron oxidation and isotope fractionation remains unclear and is not considered further here (Dickey et al., 1987; Andersen et al., 1995).

The proportion of ferritin-bound Fe in mackerel liver (8 %–31 %) was notably lower than that reported for mammalian liver (∼60 %; Chakrabarti et al., 2015; Chen and Chen, 2018), suggesting that a smaller fraction of hepatic Fe is stored as ferritin in teleost. Consequently, the δ⁵⁶Fe enrichment in the mackerel liver appears more moderate than in mammals, possibly due to a greater influence of isotopically lighter Fe pools such as circulating hemoglobin iron.

Because blood contains little ferritin-bound iron, the elevated liver δ⁵⁶Fe cannot be explained by residual blood contamination. Given that iron cycling within the body is a semi-closed system, in which the liver acts as the central exchange site between storage (ferritin) and transport (hemoglobin and transferrin) pools (Slusarczyk and Mleczko-Sanecka, 2021), hepatic δ⁵⁶Fe can be interpreted as a mixture of these isotopically distinct components. This relationship can be expressed as:

$$\begin{array}{}\text{(6)}& {\mathit{\delta }}_{\text{Liv}}=p{\mathit{\delta }}_{\text{Ft}}+(\mathrm{1}-p){\mathit{\delta }}_{\text{Hm}}\end{array}$$

where p is the fraction of ferritin-bound Fe in the liver, and δ_Ft and δ_Hm represent the isotopic compositions of ferritin- and heme-bound Fe, respectively.

By assuming that the lowest δ⁵⁶Fe observed here in red muscle represents δ_Hm (−1.66 ‰), the isotopic offset between ferritin- and heme-bound Fe in the liver ($\mathrm{\Delta }={\mathit{\delta }}_{\text{Ft}}-{\mathit{\delta }}_{\text{Hm}}$) was estimated to be 2.72 ‰ ± 3.03 ‰ (2 S.D.) in all specimens, whereas it was 4.15 ‰ ± 2.84 ‰ (2 S.D.) in females and 1.76 ‰ ± 0.88 ‰ in males. These estimated values are higher than previous observations in skipjack tuna (Katsuwonus pelamis, female, Δ=1.52 ‰, n=1; Hasegawa et al., 2023) and another chub mackerel specimen (Δ=1.41 ‰, male, n=1; Hasegawa et al., 2023), and, in females, appear higher than the equilibrium isotope fractionation between Fe(II) and Fe(III) (∼2.8 ‰, Johnson et al., 2002; Welch et al., 2003). The two females exhibited a lower ferritin-bound iron fraction than the three males, while their liver δ⁵⁶Fe values were comparable to those of the males, resulting in a tendency for their estimated Δ values to be higher. Because of the limited sample size, it cannot be definitively concluded that a sex difference exists. However, one possible explanation is that the release of isotopically lighter Fe from hepatic ferritin following Rayleigh fractionation leads to significant enrichment of heavy Fe in the residual ferritin and results in Δ values larger than those expected under isotopic equilibrium. As the sample consists of mature individuals during the spawning season, it cannot be ruled out that physiological changes relating to spawning may have temporarily altered iron metabolism, particularly in females.

4.2 δ⁵⁶Fe Variation Across Tissues

Outside the liver, some tissues did not exhibit elevated δ⁵⁶Fe values despite high ferritin content. In particular, ovaries and red muscle in females exhibited high ferritin-to-heme ratios but δ⁵⁶Fe values comparable to other tissues. This pattern suggests that ferritin-bound Fe in these tissues is not retained over long periods but is actively mobilized and recycled in response to metabolic activity and oxygen consumption. For example, ferritin in skeletal muscle can be quickly mobilized to meet oxygen demand (Robach et al., 2007; Ryan et al., 2021), preventing the accumulation of a heavy isotope signature and maintaining δ⁵⁶Fe values close to those of the whole-body iron pool dominated by heme iron. Moreover, δ⁵⁶Fe has been associated with oxidative stress markers including superoxide dismutase and glutathione peroxidase (Sauzéat et al., 2025), indicating that tissues prone to oxidative stress, such as gonads, may display isotopic variability due to redox-related iron turnover.

These observations suggest that ferritin-bound (storage) iron in chub mackerel exhibits a high turnover rate, resulting in small δ⁵⁶Fe differences among tissues. In contrast, heme iron likely turns over more slowly. Fish erythrocyte lifespans range from ∼80 to 500 d (Götting and Nikinmaa, 2017; Avery et al., 1992), comparable or longer than those of mice (∼60 d, Dholakia et al., 2015) and humans (∼120 d; Shemin and Rittenberg, 1946). Marine mammals, by contrast, display extended erythrocyte lifespans that scale with diving capacity (Pearson et al., 2024), highlighting the link between oxygen storage and iron utilization. In migratory fish such as mackerel, the high and continuous demand for heme to support aerobic activity may promote rapid redistribution of heme iron, which may result in reduced δ⁵⁶Fe variability among organs.

4.3 Features of δ⁵⁶Fe Variation Among Marine Species

The range of δ⁵⁶Fe variation among tissues in chub mackerel was clearly narrower than that reported for mammals. For example, the differences in δ⁵⁶Fe values between muscle and liver range from 0.72 ‰ to 1.71 ‰ in mice (Balter et al., 2013), 1.64 ‰ to 2.72 ‰ in sheep (Balter et al., 2013), and 0.83 ‰ to 1.75 ‰ in humans (Walczyk and von Blanckenburg, 2002), whereas the maximum isotopic difference among tissues in the present chub mackerel specimens was 0.73 ‰. Despite this relatively small internal fractionation, δ⁵⁶Fe values in marine organisms often show species-specific ranges (Fig. 4). For example, muscular δ⁵⁶Fe values in skipjack tuna are high regardless of regional differences (−1.46 ‰ to −0.71 ‰; Hasegawa et al., 2022), whereas mammals generally show lower values (−3.79 ‰ to −1.5 ‰; Walczyk and von Blanckenburg, 2002; Balter et al., 2013). This pattern does not appear to arise from the enrichment of lighter isotopes in specific tissues, but rather from the limited influence of internal isotopic fractionation on whole-body δ⁵⁶Fe. This interpretation is consistent with our previous study on a single individual (Hasegawa et al., 2023), and the limited inter-tissue δ⁵⁶Fe variation observed across multiple individuals in the present work supports that this characteristic is generally applicable to chub mackerel.

Figure 4 Comparison of δ⁵⁶Fe values in seawater (upper figure), and marine organisms and mammals (lower figure). Literature data in the upper figure are indicated by reference numbers: seawater from the Pacific Ocean (1–4: Radic et al., 2011; Conway and John, 2015; Pinedo-González et al., 2020; Kurisu et al., 2024), North Atlantic Ocean (5: Conway and John, 2014), and South Atlantic–Southern Ocean (6–8: Abadie et al., 2017; Ellwood et al., 2020; Sieber et al., 2021). The data of chub mackerel includes this study and Hasegawa et al. (2023), while values for zooplankton (whole body), squid (mantle), and mammals (harbor porpoise, melon-headed whale, striped dolphin, human, sheep, and mouse) are compiled from previous studies (Walczyk and von Blanckenburg, 2002; Balter et al., 2013; Hasegawa et al., 2022).

As discussed in the previous section, hepatic ferritin iron likely undergoes isotopic fractionation on the order of approximately 1.5 ‰–3 ‰, based on the Δ values estimated in this study and Hasegawa et al. (2023). However, because ferritin constitutes only a small fraction of total iron, its contribution to whole-body δ⁵⁶Fe variation is limited. Consequently, heterogeneous distribution of δ⁵⁶Fe across tissues is likely to have only a minor influence on the δ⁵⁶Fe of indicator tissues representing whole-body δ⁵⁶Fe values (e.g. muscle with a large Fe pool size). The primary determinant of tissue δ⁵⁶Fe in chub mackerel therefore appears to be iron uptake process from external sources rather than internal redistribution.

The relatively high mean δ⁵⁶Fe values in mackerel tissues may primarily reflect more efficient intestinal iron absorption than in mammals, resulting in a stronger reflection of environmental δ⁵⁶Fe values. Additionally, selective ingestion of prey with intrinsically higher δ⁵⁶Fe values could also contribute to their isotopic composition. Various prey taxa of marine fish, including zooplankton, squid, and crustaceans, are known to exhibit relatively high δ⁵⁶Fe values (−1.00 ‰ to −0.03 ‰; von Blanckenburg et al., 2013; Hasegawa et al., 2022, 2023). The δ⁵⁶Fe differences observed among fish groups such as tuna and mackerel (−1.58 ‰ to −0.71 ‰) versus sardine and herring (−2.64 ‰ to −1.73 ‰; Hasegawa et al., 2022) seemed difficult to explain solely by isotopic differences in their prey since all prey species analyzed so far showed higher δ⁵⁶Fe values than sardine and herring (Hasegawa et al., 2022). However, recent studies have suggested species-specific δ⁵⁶Fe variability even among zooplankton (Hasegawa et al., 2026) and therefore prey-derived δ⁵⁶Fe may also partially contribute to the overall isotopic composition of fish such as sardines and herrings that feed selectively on specific plankton groups. Further expansion of marine biological δ⁵⁶Fe databases may enable a more comprehensive understanding of Fe physiology in wild marine organisms.

In contrast, terrestrial mammals such as humans, mice, and sheep consistently show large δ⁵⁶Fe differences between liver and muscle (Walczyk and von Blanckenburg, 2002; Balter et al., 2013), reflecting a well-developed iron recycling system based on the long-term ferritin storage. This mechanism reduces daily iron requirements and promotes preferential uptake of lighter isotopes, thereby lowering the overall δ⁵⁶Fe values. In contrast to mammals that rely on ferritin-based iron storage and recycling, the isotopic pattern observed in fish may indicate that long-term iron storage plays a less dominant role in iron homeostasis, despite inhabiting chronically iron-limited environments. They may instead have evolved a “recycle dominant” mode of iron homeostasis that relies on intensive recycling of iron to sustain metabolic demands.

4.4 Implications for biological iron cycling in marine environments

The results of this study suggest that the δ⁵⁶Fe variability in marine fish reflects dietary characteristics and uptake efficiency more strongly than internal physiological variations. The distinctly lighter isotopic signatures of animals compared to seawater may ultimately influence the isotopic composition of iron recycled through marine food webs. Therefore, whole-body δ⁵⁶Fe values could serve as an integrated tracer of biologically available iron within marine ecosystems. Our analyses also indicate that in fish, δ⁵⁶Fe values in non-destructive tissues such as blood can represent the whole-body isotopic composition, highlighting their potential as key species for assessing iron supply and cycling in higher marine ecosystems.

In the future, applying a Rayleigh-type model may allow quantitative evaluation of the relative contributions of absorption and retention to isotopic fractionation in fish. Combined with XAFS analyses that provide species-level insights into ferritin- and heme-bound iron pools, these approaches could offer a powerful framework for linking biological iron metabolism to marine biogeochemical iron cycling. In addition, further biochemical studies examining Fe isotope fractionation during enzymatic incorporation into iron-binding proteins such as ferritin or heme in fish would help to better constrain the mechanisms controlling Fe isotope distribution in marine organisms.

5 Conclusions

This study demonstrates that δ⁵⁶Fe variations among tissues in chub mackerel are smaller than those observed in mammals. Although isotopic fractionation of around 1.5 ‰–3 ‰ may occur in liver ferritin, its effect on whole-body δ⁵⁶Fe is minimal due to the limited ferritin fraction and overall liver iron content. These results suggest that the high δ⁵⁶Fe of mackerel primarily reflects dietary iron sources and intestinal uptake rather than internal isotopic fractionation. The maximum δ⁵⁶Fe range among major tissues was 0.73 ‰, indicating relatively small tissue isotopic variability. The small variation in δ⁵⁶Fe among major tissues such as muscle and liver compared with the potential isotopic fractionation associated with ferritin formation, suggests that iron isotope pools in fish remain relatively stable. Although the number of individuals analysed in this study is limited, these δ⁵⁶Fe patterns across multiple individuals provide robust constraints on iron isotope behaviour in wild marine fish. These findings provide fundamental information for studies of iron cycling and metabolism in marine organisms and have potential applications in food-web modeling and ecological assessments.