# Parental blood mercury levels are correlated, and predictive of those in eggs in a long-lived seabird

Justine Bertram, Nathalie Kürten, Coraline Bichet, Maria Moiron, Peter J

Schupp, Sandra Bouwhuis

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Justine Bertram, Nathalie Kürten, Coraline Bichet, Maria Moiron, Peter J Schupp, et al.. Parental blood mercury levels are correlated, and predictive of those in eggs in a long-lived seabird. Environmental Research, 2025, 275, pp.121437. 10.1016/j.envres.2025.121437. hal-05040005

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# Parental blood mercury levels are correlated, and predictive of those in eggs in a long-lived seabird

Article in Environmental Research · March 2025

DOI: 10.1016/j.envres.2025.121437

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# Parental blood mercury levels are correlated, and predictive of those in eggs in a long-lived seabird

Justine Bertram<sup>a, *, ◐</sup>, Nathalie Kürten<sup>a</sup>, Coraline Bichet<sup>b</sup>, Maria Moiron<sup>a, c ◐</sup>, Peter J. Schupp<sup>d, e</sup>, Sandra Bouwhuis<sup>a ◐</sup>

<figure><img src="image_7.png" /></figure>

## ARTICLE INFO

Keywords:

Mercury contamination

Mercury transfer

Parental effect

Transgenerational effect

## ABSTRACT

Mercury (Hg) is a pollutant that does not only biomagnify along the food chain, but can also accumulate in long-lived top-predators, such as many seabirds, and be transferred to the next generation during reproduction. To better understand the transfer of Hg from parents to offspring, as well as its potential negative consequences, we used seven years of data on total mercury (THg) levels in the blood of common terns (*Sterna hirundo*) breeding at the German North Sea coast, and their eggs. We assessed whether (i) THg levels of pair members correlate, (ii) (changes in) parental THg levels correlate with those in their eggs, (iii) egg THg levels are repeatable within a given clutch and within parents across years, and (iv) parental and egg THg levels correlate with embryonic development, hatching success and hatch mass. Blood THg levels of pair members were positively correlated, but only maternal THg levels correlated with those in shells of hatched eggs, with within-individual increases over time leading to increases in shell THg levels as well. THg levels in shells of hatched eggs showed within-clutch and across-year repeatability. THg levels in shells of unhatched eggs were higher than those of hatched eggs, and were lower when embryos had developed for longer before they died, suggesting Hg absorption from the eggshell. THg levels in the contents of unhatched eggs were positively correlated with those of both parents. Interestingly, hatching success and hatch mass correlated positively, rather than negatively, with maternal THg levels, suggesting that a Hg-rich maternal diet (e.g., large fish or prey of high trophic levels) may enhance reproductive investment, thereby offsetting potential negative effects of Hg, at least under current pollution levels. Testing for downstream effects of transferred Hg on chicks will, however, be important.

## 1. Introduction

Among the toxic substances that enter the environment due to human activities, mercury (Hg) is one of particular concern due to its persistence (Al-Sulaiti et al., 2022) and biomagnification along the food chain (Atwell et al., 1998; Lavoie et al., 2013). Seabirds, especially long-lived ones that forage at high trophic levels, are particularly exposed to Hg through their diet (Chételat et al., 2020; Mills et al., 2022). Although birds are able to excrete substantial amounts of their ingested Hg via their feathers as they moult (Whitney and Cristol, 2017), excretion is likely incomplete, as both short- and long-term accumulation are evident, for example in blood (Bertram et al., 2024a). Such

accumulation may increase the risk of detrimental effects, including reduced flight endurance (Ma et al., 2018), increased oxidative stress (Hoffman et al., 2011; Soldatini et al., 2020), a compromised immune response (Hawley et al., 2009; Lewis et al., 2013; Ibañez et al., 2024), and deteriorated reproductive performance (Heinz and Hoffman, 1998; Evers et al., 2008; Goutte et al., 2014a; Mills et al., 2020; Ibañez et al., 2024; Leaphart et al., 2025).

During the breeding season, female birds generally exhibit lower blood Hg levels than male birds (Bond and Diamond, 2009; Ackerman et al., 2020; Mills et al., 2022; Bertram et al., 2024a). Additionally, Hg levels have been observed to be lower in birds with more eggs (Bertram et al., 2024a), in which Hg levels decrease along the laying order

<footnote><sup>a</sup> Institute of Avian Research, An der Vogelwarte 21, 26386, Wilhelmshaven, Germany</footnote>

<footnote><sup>b</sup> Centre d'Etudes Biologiques de Chizé (CEBC), CNRS-La Rochelle Université, Villiers-en-Bois, France</footnote>

<footnote><sup>c</sup> Department of Evolutionary Biology, Bielefeld University, 33615, Bielefeld, Germany</footnote>

<footnote><sup>d</sup> Carl von Ossietzky Universität Oldenburg, Department for Chemistry and Biology of the Marine Environment, Terramare, 26382, Wilhelmshaven, Germany</footnote>

<footnote><sup>e</sup> Helmholtz Institute for Functional Marine Biodiversity at the University of Oldenburg, 26129, Oldenburg, Germany</footnote>

<footnote>* Corresponding author.
E-mail address: justine.bertram@ifv-vogelwarte.de (J. Bertram).</footnote>
(Becker, 1992; Ackerman et al., 2016a). This suggests dose-dependent transfer of Hg from mothers to their eggs, potentially leading to positive intergenerational correlations (Evers et al., 2003; Heinz et al., 2010; Ackerman et al., 2020; Lemesle et al., 2024). Interestingly, a positive correlation between paternal and egg Hg levels has been found as well as (Ackerman et al., 2016b), although it is much more scarcely investigated. Such a pattern may be expected even in the absence of a direct transfer mechanism, particularly in species with strong assortative mating (i.e., pairing of individuals with similar phenotypes) for traits related to Hg exposure (e.g., foraging behaviour) and/or courtship feeding before and during egg formation. As such, it is informative to assess assortative mating with respect to Hg levels, and to compare the correlations between paternal and maternal Hg levels and those in the eggs the females laid.

Hg transfer from parents to eggs likely is influenced by variation in parental Hg levels, which themselves are shaped by environmental variation, e.g., in prey availability (Smith et al., 2021; Tartu et al., 2022), as well as individual characteristics, such as age (Bertram et al., 2024b). If Hg transfer to eggs is passive and proportional, changes in parental Hg levels over time (a within-parental effect) should lead to corresponding changes in Hg levels in their eggs. In addition, there can be variation among parents, for example if these parents differ in the proportion of circulating Hg they transfer. To assess both the within- and among-parental effect of Hg on egg Hg levels, a longitudinal analysis approach using data from individuals with known Hg levels in blood and eggs across years is required (van de Pol and Wright, 2009). Moreover, such longitudinal data allows for calculating the repeatability of Hg levels in eggs at both a clutch- and parental level (i.e., the short-vs long-term parental consistency in Hg transfer to the eggs). Such repeatability would be expected if egg Hg levels are influenced by traits that vary among, but are stable within individuals during the laying phase (short-term), or have a genetic basis, ensuring stability within individuals across years (long-term) (Bertram et al., 2024b). An example of the latter may be foraging strategy (Renedo et al., 2018; Dodino et al., 2022), or the capacity for Hg excretion and transfer (Lewis and Furness, 1993; Ackerman et al., 2020).

Once in the egg, Hg can be detrimental for embryonic health and survival. Studies in which Hg was injected directly into eggs reported an increased risk of embryonic deformation and malpositioning (Heinz and Hoffman, 2003; 2011; Herring et al., 2010), as well as mortality (Heinz et al., 2009a; Kenow et al., 2011). Experimental studies have also reported reduced hatching success of eggs from Hg-dosed parents (Heinz and Hoffman, 1998; Albers et al., 2007; Paris et al., 2018), which fits with findings from field studies that reported negative correlations between Hg levels in parental blood and hatching probability in wandering albatrosses (Diomedea exulans; Goutte et al., 2014b) and tree swallows (Tachycineta bicolor; Hallinger and Cristol, 2011). These studies, however, could not differentiate between direct effects of transferred Hg in the offspring (as levels in the eggs were not measured) or indirect effects of altered parental behaviour caused by Hg (Taylor et al., 2018). Moreover, it is important to realize that although Hg contamination may impair development and hatching success, the causality could also be reversed, with slow growth or death leading to reduced embryonic Hg absorption (Peterson et al., 2017). As such, combining field data at both the parental and egg level, and taking into account embryo development, could offer an improved opportunity to assess the effects of Hg on reproductive parameters during early development.

In our study, performed between 2017 and 2023, we assessed Hg levels in a wild population of common terns (Sterna hirundo), using known-sex parents, whose eggs were artificially incubated under standardized conditions. Our objectives were to investigate (i) whether blood Hg levels of breeding pair members are correlated, (ii) how (changes in) parental blood Hg levels correlate with those in their eggs, while correcting for effects of laying order observed in previous studies (Becker, 1992; Ackerman et al., 2016a), (iii) whether Hg levels in eggs of the same parents show repeatability within clutches and across years,

and (iv) how parental and/or egg Hg levels correlate with embryo development, hatching success and chick hatch mass.

## 2. Methods

### 2.1. Study species and site

Common terns are socially monogamous, long-lived seabirds, mainly feeding on fish of a length up to 15 cm (Becker and Ludwigs, 2004). They mate age-assortatively (Bouwhuis et al., 2015) and typically form life-long pair bonds, which are reinforced each breeding season by extensive courtship feeding (González-Solís et al., 2001). Starting their first breeding attempt at an average age of three years, common terns usually lay one to three eggs a year (Zhang et al., 2015). During the breeding season, males and females do not differ in body mass and share incubation, which, on average, takes 22 days (Becker and Ludwigs, 2004).

The common terns of this study are part of a monospecific breeding colony located at the Banter See in Wilhelmshaven, at the German North Sea coast (53°30'40" N, 08°06'20" E), where adults predominantly forage on clupeids (Clupea harengus and Sprattus sprattus) and smelt (Osmerus eperlanus; Wendeln and Becker, 1996). This colony has been the focus of a long-term research project since 1992, with each nest monitored three times per week. All locally hatched birds are ringed and, just prior to fledging, implanted with subcutaneous transponders (Becker and Wendeln, 1997). The colony site consists of six artificial islands (10.7 × 4.6 m), surrounded by 60 cm high walls. These walls sustain 44 platforms, each equipped with an antenna, that allow the automatic and remote detection of transponder-marked birds. Additionally, mobile antennas are placed around each nest for at least 24 h, enabling the identification of breeders and assessment of reproductive performance at the individual level. The sex of the studied birds is determined molecularly from blood keels of feathers, following the method described by Becker and Wink (2003).

### 2.2. Data collection

As part of a project investigating the migratory behaviour of common terns (see Kürten et al., 2022), eggs of tracked birds were artificially incubated to prevent potential damage from leg-mounted tracking devices (Kürten et al., 2019). Hereto, the nests of tracked birds were visited at least twice a day, enabling egg collection within 24 h of laying and allowing the precise determination of laying order. Collected eggs were replaced by fake eggs, and marked with a permanent marker according to their nest number and laying order. Eggs were transferred to digital incubators (Rcom max 50 and Rcom pro 20; Autoelex Co., Ltd., South Korea) set to a temperature of 37.5 °C and a relative humidity of 50 % (following Murk et al., 1994), and automatically turned by approximately 90° along their axis every hour (Vedder et al., 2017). After 18 days of incubation, eggs were transferred to a hatching incubator set to 37.0 °C and 60 % relative humidity, without egg turning. In this incubator, eggs were placed in individual compartments made of wire mesh to ensure that each hatched chick could be linked to its egg.

For the mercury project, of the 704 eggs collected from 271 nests with at least one parent of known sex and age (see Table S1), 578 hatched, resulting in a hatching rate of 82 %, which was slightly higher than that of unmanipulated eggs in this study population (77 %; Vedder et al., 2017). Upon hatching, eggshells were collected and individually stored in plastic cups, and chicks were ringed, weighed and returned to their original nest, in which they were readily accepted by their parents (also see Vedder et al., 2017). Fake eggs were removed upon chick return, or 1–2 days after the last chick was returned if not all eggs from a clutch had hatched. Eggs that did not hatch within 26 days of incubation were assessed with respect to the development of the embryo by candling the egg. For fully developed embryos, death was confirmed by measuring whether there was a heartrate using an Egg Buddy (Avitronics, Truro, United Kingdom). In total, we collected 126 unhatched
eggs and stored them at -18 °C until further processing.

For 102 mothers and 116 fathers of the 704 artificially incubated eggs, blood samples were collected using Mexican kissing bugs (*Diptalogaster maxima*; Becker et al., 2006). For this, a bug was placed inside an artificial hollow egg with small holes along the exterior. This egg was placed into the nest of the target bird once it was detected incubating and left there for 20–30 min, during which the bug was able to suck the bird's blood. During this time, the bird was closely observed from observation huts within the colony to prevent sample contamination from partner switches. If the bug successfully obtained a blood meal, the blood was extracted from the bug's abdomen using a syringe and stored at -18 °C until analysis. Hg concentrations measured in blood samples obtained using this method showed a strong correlation (r = 0.93) with those obtained from samples collected from the brachial vein of the same birds using needles and capillaries, confirming the validity of using bugs for measuring blood parameters, such as Hg concentrations (Bertram et al., 2024a).

## 2.3. Mercury analysis

Prior to analysis, unhatched eggs were carefully opened to separate the egg content and the eggshell. Eggshells (including the membrane) of both hatched and unhatched eggs were dried for 24 h in an incubator at 50 °C, and subsequently grounded to a fine powder, used to measure the dry weight (dw) Hg concentration. The egg content of unhatched eggs was homogenized using an IKEA Ultra Turrax T25 Homogenizer (Janke & Kunkel GmbH, Germany), and blood samples did not need any preparation, such that the wet weight (ww) Hg concentration was measured for these two tissues.

Total mercury (THg) concentrations, which act as a reliable proxy for methylmercury levels in both eggs (Ackerman et al., 2013) and blood (Renedo et al., 2021), were quantified using a Direct Mercury Analyzer (DMA-80, MLS Mikrowellen-Labor-Systeme GmbH, Germany). Either 20 µl of egg content, one spatula tip of eggshell powder (c. 30 mg), or 20 µl of whole blood were inserted into a nickel sample boat and weighed. In the DMA-80, the respective sample was then dried and thermally decomposed in a stream of oxygen at 750 °C, leading to combustion of the compounds other than THg, which itself was trapped by gold amalgamation. By subsequent heating to 850 °C, the trapped THg was released and quantified by atomic absorption spectrometry at 253.7 nm. Each measurement batch included an analytical blank to verify the absence of contamination of the device, and a certified reference material (DORM-3 or DORM-4; fish protein from National Research Council, Canada, 382 or 412 µg·kg⁻¹ dry weight; with a mean percent recovery ± standard deviation of 108 ± 6 % or 102 ± 5 %, respectively) to ensure the device's accuracy. Additionally, as part of quality assurance and quality control (QA/QC) protocols, the laboratory regularly participates in the QUASIMEME international performance study program, with consistently satisfactory results. The detection limit of THg was 0.0003 µg·g⁻¹, and all samples had values above this threshold. To further evaluate the reliability of our measurements, 532 eggshells and 53 egg contents were analyzed twice, showing a relative difference among duplicate samples of 9.1 ± 0.4 % and 1.1 ± 0.2 %, respectively. A Pearson correlation test showed a highly significant correlation of 0.97 (95 % CI [0.96–0.97], p < 0.001) for eggshells and of 0.98 for egg contents (95 % CI [0.96–0.99], p < 0.001; Fig. S1), such that all further samples were measured only once. Eighty-eight blood samples were also analyzed in duplicate, showing a highly significant correlation of 0.88 (95 % CI [0.81–0.92]; also see Bertram et al., 2024a).

## 2.4. Statistical analyses

All analyses were performed, and figures created, using R (version 4.3.3, R Core Team, 2023). Models were run using “lme4” (Bates et al., 2015) and p-values obtained using “lmerTest” (Kuznetsova et al., 2017) with the level of significance set at p < 0.05. Data were visualized using

“ggplot2” (Wickham, 2016). THg data were log-transformed to obtain a Gaussian error distribution, visually confirmed with histograms.

### 2.4.1. Assortative mating and parental effects

We first split the adult blood THg data to obtain two datasets, one for males and one for females, and ran a linear mixed model for each sex separately with ‘blood THg concentration’ (in µg·kg⁻¹ ww) as the dependent variable and ‘ID’ and ‘year of sampling’ as random effects. Second, we ran these models while additionally adding ‘age’ as a covariate. We then ran two Pearson correlation tests using the residuals of those models to (i) test for assortative mating with respect to blood THg levels, and (ii) corroborate that any such assortative mating was not (only) driven by the positive correlation in age across the two pair members (Bouwhuis et al., 2015). These analyses included data for the 102 and 116 birds mentioned to have been sampled above, along with an additional 610 blood samples collected between 2017 and 2023 from 530 birds whose eggs were not analyzed for THg concentrations. In total, we therefore included 930 samples from 274 pairs (of which 94 pairs were sampled in 2–7 of the sampling years). As we found a strongly positive correlation (see Results), we prevented problems of collinearity by proceeding to conduct all parental effect models for mothers and fathers separately. Additionally, running separate models for mothers and fathers maximized sample sizes, avoiding the loss of 234 eggshell THg data points that would occur if only pairs with both parents sampled were included.

To investigate how THg concentrations of egg components vary with parental THg concentrations, we ran 10 linear mixed models, 5 for each parental sex (see Table 1). All models included ‘laying order’ as a three-level class variable. As random effects, we included ‘year’ of sampling to account for potential variation in THg concentrations among years, a composite variable combining ‘year’ and ‘nest’ (‘year_nest’) to account

<caption>**Table 1**</caption>

Overview of ten models run to explain variation in the total mercury (THg) concentration of eggshells and egg contents, in hatching success of these eggs and in the hatch mass of chicks, including their fixed effects, random effects and error distribution.

<table><thead><tr><th>Model</th><th>Response variable</th><th>Fixed effects</th><th>Random effects</th><th>Error distribution</th></tr></thead><tbody><tr><td>a</td><td>egg content THg</td><td>laying order + parental blood THg</td><td>ID + year_nest + year</td><td>Gaussian</td></tr><tr><td>b</td><td>eggshell THg of unhatched eggs</td><td>laying order + parental blood THg</td><td>ID + year_nest + year</td><td>Gaussian</td></tr><tr><td>c</td><td>eggshell THg of hatched eggs</td><td>laying order + parental blood THg</td><td>ID + year_nest + year</td><td>Gaussian</td></tr><tr><td>d</td><td>eggshell THg of hatched eggs</td><td>laying order + average THg + delta THg</td><td>ID + year_nest + year</td><td>Gaussian</td></tr><tr><td>e</td><td>hatching success (0/1)</td><td>laying order + parental blood THg</td><td>ID + year_nest + year</td><td>Binomial</td></tr><tr><td>f</td><td>hatch mass</td><td>laying order + parental blood THg + egg volume</td><td>ID + year_nest + year</td><td>Gaussian</td></tr><tr><td>g</td><td>hatching success (0/1)</td><td>laying order + parental blood THg + eggshell THg</td><td>ID + year_nest + year</td><td>Binomial</td></tr><tr><td>h</td><td>hatch mass</td><td>laying order + parental blood THg + egg volume + eggshell THg</td><td>ID + year_nest + year</td><td>Gaussian</td></tr><tr><td>i</td><td>egg content THg</td><td>laying order + parental blood THg + embryonic death age</td><td>ID + year_nest + year</td><td>Gaussian</td></tr><tr><td>j</td><td>eggshell THg of unhatched eggs</td><td>laying order + parental blood THg + embryonic death age</td><td>ID + year_nest + year</td><td>Gaussian</td></tr></tbody></table>
for the fact that eggs of the same clutch are not independent from each other, as well as parental 'identity' to control for repeated sampling of 58 fathers and 57 mothers across 2-6 years (Table S1). First, we ran a general linear mixed model with the 'egg content THg' (in µg·kg⁻¹ ww; (a)) or 'eggshell THg' (in µg·kg⁻¹ dw) of either unhatched (b) or hatched (c) eggs as a dependent variable and parental 'blood THg' as a covariate. For 'shell THg concentration' of the hatched eggs, we had sufficient sample size to run an additional model (d), in which we partitioned the 'blood THg' of each parent into an 'average THg' and 'delta THg' component (following van de Pol and Wright, 2009). An individual's average THg concentration was calculated as the average of all THg concentrations we measured for that individual, while its delta THg concentration was calculated for each year as the difference between its THg concentration in that year ('actual THg') and its average THg concentration (i.e., 'delta THg' = 'actual THg' - 'average THg'). When including both 'average THg' and 'delta THg' as covariates in the model, 'average THg' tested whether parents with high levels of blood THg produced eggs with high levels of THg in their shells, whereas 'delta THg' quantified the within-parental change in shell THg concentration as their blood THg concentrations changed (van de Pol and Wright, 2009).

Next, we assessed the repeatability of eggshell THg concentrations of hatched eggs within clutches and within parents, across years. To obtain the within-clutch repeatability, which cannot differ for mothers and fathers, we used the maternal version of model (d) and performed 1000 bootstrapped simulations using the "arm" R-package to calculate the proportion of the total variance explained by the random effect of 'year_nest'. To obtain the within-parent, across-year repeatability, which can differ for mothers and fathers due to effects of widowing or divorce, we used model (d) to estimate the proportion of total variance explained by 'parental ID' for each parental sex.

Finally, to assess how parental THg levels correlate with hatching success, we ran a generalized linear mixed model (e) with 'hatching success' (hatched (1), or not hatched (0)) as the dependent variable and assuming a binomial error structure, with parental 'blood THg' as a covariate. Lastly, for the hatched eggs, we ran another linear mixed model (f) with the 'hatch mass' of the resulting chicks as the dependent variable. We assumed hatch mass to follow a Gaussian distribution and added parental 'blood THg' as well as 'egg volume' as covariates; the latter because it is known that hatch mass is positively correlated with egg size (Vedder et al., 2023). The random effect structure of these models was identical to those described above (a – d).

### 2.4.2. Egg THg effects

After assessing the association between parental blood THg and hatching probability and hatch mass, we also assessed whether THg measured in the shells of hatched eggs was correlated with these traits, using four more (generalized) linear mixed models that were identical to models (e) and (f) above, except for additionally including log-transformed 'eggshell THg' as a covariate ((g) and (h)).

For unhatched eggs, we assessed the correlation between the 'egg content THg' and 'eggshell THg', before testing whether variation in embryonic development was related to variation in either 'egg content THg' or 'eggshell THg'. For this, we ran two more linear mixed models ((i) and (j)) that were identical to models (a) and (b) above, except for additionally adding the approximate 'embryonic death age' as a 4-level class variable (group '1': embryo died between 0 and 5 days of development, group '2': embryo died between 6 and 10 days of development, group '3': embryo died between 11 and 16 days of development, group '4': embryo died after 17 days of development).

## 3. Results

We measured THg concentrations in 205 blood samples from 102 mothers and 221 blood samples from 116 fathers (Table 2), as well as successfully quantified the eggshell THg concentration for 702 of the

<caption>**Table 2**</caption>

Sample sizes, mean total mercury (THg) concentrations, standard errors (SE) and minimum and maximum values for egg components of hatched and unhatched common tern eggs, as well as parental blood samples.

<table><thead><tr><th></th><th></th><th>n</th><th>mean THg</th><th>SE</th><th>min THg</th><th>max THg</th></tr></thead><tbody><tr><td rowspan="2">hatched eggs</td><td>shells</td><td>578</td><td>0.019</td><td>0.002</td><td>0.002</td><td>0.912</td></tr><tr><td>shells</td><td>124</td><td>0.165</td><td>0.017</td><td>0.003</td><td>0.969</td></tr><tr><td rowspan="2">eggs</td><td>content</td><td>116</td><td>0.481</td><td>0.017</td><td>0.114</td><td>1.040</td></tr><tr><td>maternal</td><td>205</td><td>0.781</td><td>0.011</td><td>0.288</td><td>1.491</td></tr><tr><td rowspan="2">parental blood</td><td>maternal</td><td>221</td><td>1.123</td><td>0.017</td><td>0.263</td><td>3.896</td></tr></tbody></table>

704 incubated eggs, and 116 contents of the 126 unhatched eggs. Both eggshell and egg content THg concentrations varied considerably (Table 2). A significant amount of this variation was explained by laying order, with concentrations being lower in later laid eggs (Fig. S2).

### 3.1. Assortative mating and parental effects

THg concentrations in the blood of the members of 274 breeding pairs were significantly positively correlated (r = 0.245, p < 0.001, Fig. S3), providing evidence for assortative mating with respect to contamination levels. This correlation remained nearly identical when using the model residuals of age-corrected THg concentrations (r = 0.220, p < 0.001), showing that the observed positive correlation is not due to assortative mating for age. Even in the absence of direct transfer of Hg from fathers to their eggs we would, therefore, expect correlations between paternal and egg THg levels to be 22–25 % of the correlation between maternal and egg THg levels.

Among unhatched eggs, THg concentrations in the content correlated with those in the blood of both parents (p = 0.042 and p = 0.010 for mothers and fathers, respectively; Table S2 (a), Fig. 1a), in a very similar way. In contrast, THg concentrations in the shells of these eggs did not correlate with those in parental blood (Table S2 (b), Fig. 1b). THg concentrations in shells of hatched eggs, however, significantly correlated with those in the blood of their mothers (p < 0.001), but not their fathers (Table S2 (c)). Rerunning the models for the shells of the hatched eggs and splitting parental 'blood THg' into its average and delta component showed that as blood THg concentrations in mothers increased, THg concentrations in their eggshells also increased (p = 0.006), with no such effect in fathers (Table S2 (d), Fig. 1c). Furthermore, higher average blood THg concentrations in mothers, but not fathers, were associated with higher THg concentrations in their eggshells (p = 0.003; Table S2 (d), Fig. 1d).

The within-clutch repeatability of THg concentrations in eggshells quantifies to which extent shell THg levels of eggs within the same clutch are more similar to themselves than to those in other clutches. When correcting for the substantial annual variation in eggshell THg levels (Table S2 (d)), eggs within a clutch did not show a high repeatability with respect to the THg concentration in their shells ($R_{within clutch} = 0.016$ (95 % CI [0.012, 0.021]; Fig. S4a)). This means that variability within nests was not substantially smaller than among nests. When, however, rerunning the model without year as a random effect, and hence allowing this annual variation to either go to the 'year_nest' or 'residual' term, the within-clutch repeatability increased to 0.327 (95 % CI [0.281, 0.369], Fig. S4a) showing that the fact that different parents were sampled in different subsets of years masked the repeatability of this trait.

The repeatability of THg concentrations within individual parents across years quantifies the relative consistency of the transfer of Hg from a parent to the shells of its eggs across different breeding seasons. We found this repeatability to be relatively low, but significant, for both mothers ($R_{mat.} = 0.113$ (95 % CI [0.083, 0.147]), Fig. S4c) and fathers ($R_{pat.} = 0.065$ (95 % CI [0.046, 0.088]), Fig. S4d).

Lastly, parental THg concentrations did not explain variation in hatching success (Table S3 (e)) or in the hatch mass of the chicks from
<figure><img src="image_8.png" /><figcaption>**Fig. 1.** Total mercury (THg) concentrations in the content (a) and shells (b) of unhatched common tern eggs (in log, µg·kg⁻¹) in relation to paternal (black) and maternal (red) blood THg concentrations, and in shells of hatched common tern eggs in relation to paternal and maternal delta (c) or average (d) blood THg concentrations. Points represent the raw data, lines the significant parent-specific model predictions, and shaded areas the 95 % confidence intervals around the predicted relationships. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

the eggs that hatched (Table S3 (f)).

## 3.2. Egg THg effects

Hatched eggs had significantly lower THg concentrations in their shells than unhatched eggs (p < 0.001; Table S3 (g), Fig. 2). When correcting for this, a maternal, but not paternal, THg concentration effect on hatching success (not found before, see Table S3 (e)), became apparent as well (p = 0.025; Table S3 (g)), showing that eggs from mothers with higher THg concentrations in their blood exhibited a higher hatching success. Similarly, chicks that hatched with a larger body mass had significantly lower THg concentrations in their eggshells (p < 0.001; Table S3 (h), Fig. 3), and, when correcting for this, hatch mass became significantly positively correlated with maternal, but not paternal, blood THg concentrations as well (p < 0.001; Table S3 (h)).

Among unhatched eggs, there was a small, but significantly positive correlation between THg concentrations in the shell and content (r = 0.216, p = 0.020, Fig. S5). Embryos that died late in development (> day 17, group 4), had lower THg concentrations in their eggshells (p <

0.001; Fig. 4), but not in the egg content.

## 4. Discussion

In this study, we examined total mercury concentrations in both eggs and the blood of their parents, over seven years in a natural population of a long-lived, piscivorous seabird, the common tern. THg levels in the 704 (artificially incubated) eggs showed considerable variation, with part of this variation being explained by laying order. The first egg of a clutch had the highest THg level, followed by a significant decrease in the second egg, and the third egg exhibiting the lowest levels. This pattern supports previous findings of decreasing THg concentrations across the laying sequence (Becker, 1992; Ackerman et al., 2016a), suggesting egg laying to be a passive, but efficient pathway for excretion of circulating maternal THg. In addition to laying order, maternal blood THg levels, along with embryonic development, were key predictors of THg concentrations in both eggshells and egg contents.
<figure><img src="image_9.png" /><figcaption>**Fig. 2.** Eggshell total mercury (THg) concentrations (in log, µg·kg⁻¹) of hatched and unhatched common tern eggs. The width of the violin plots represents the raw data distribution, while boxplots show the median (horizontal black line within the boxes) and the first and third quartiles. Whiskers represent the minimum (first quartile − 1.5 × inter-quartile range) and maximum (third quartile + 1.5 × interquartile range) concentrations and outliers are represented by black dots.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**Fig. 3.** The mass of freshly hatched common tern chicks (in grams) in relation to the total mercury (THg) concentrations in the shells of the eggs from which they hatched (in log, µg·kg⁻¹, dw). Points represent the raw data, the line the model prediction, and the shaded area the 95 % confidence interval around the predicted relationship.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 4.** Eggshell total mercury (THg) concentrations of unhatched common tern eggs (in log, µg·kg⁻¹) in relation to the age of the embryos when they died. Developmental stages are categorized in four roughly equal age groups (group 1: day 0–5; group 2: day 6–10; group 3: day 11–16, group 4: > day 17). The width of the violin plots represents the raw data distribution, while boxplots show the median (horizontal black line within the boxes) and the first and third quartiles. Whiskers represent the minimum (first quartile − 1.5 × inter-quartile range) and maximum (third quartile + 1.5 × interquartile range) concentrations and outliers are represented by black dots.</figcaption></figure>

### 4.1. Assortative mating

In species where mating patterns relate to, or courtship behaviors influence, Hg exposure, correlations in Hg levels between breeding partners may exist (Hargreaves et al., 2010). Correcting for annual fluctuations in THg levels, we found a moderate correlation between the blood THg levels of breeding partners, indicating that a quarter of the variation in THg levels is shared between parents. In theory, this could have been attributed to assortative mating with respect to a trait such as age (Bouwhuis et al., 2015), which is known to be related to blood THg levels in the terms of the study population investigated here (Bertram et al., 2024a). Using age-corrected values to calculate the correlation, however, it only reduced by 3 %, to a still substantial and significant 22 %. Given that common terns engage in extensive courtship feeding before and during egg formation (Wendeln, 1997), it therefore seems most likely that male terns provide their females with prey items of the same species and size as they consume themselves, thereby aligning their females' circulating THg levels with their own. As such, if recently ingested Hg in females is transferred to the eggs (Heinz et al., 2009b; Lewis and Furness, 1993, and see below), males may play a substantial, albeit indirect, role in this process.

### 4.2. Correlation between parental and egg THg levels

THg levels in the shells of hatched eggs were significantly positively correlated with maternal blood THg levels, while the correlation with paternal blood THg levels was ~10 % of that of the maternal effect and non-significant. As such, the paternal effect was even smaller than one would expect in the light of the level of assortative mating we observed (22–25 %). The analysis of within- and among-individual components of blood THg levels further revealed that when mothers experienced within-individual increases in blood THg concentrations, they also laid eggs with higher THg concentrations in the shells. This suggests that
THg transfer from mothers to their eggs is proportional, a conclusion that aligns with that of previous studies (Heinz et al., 2010; Ackerman et al., 2020).

In unhatched eggs, surprisingly, there was no correlation between maternal (or paternal) blood THg concentrations and those in the eggshells, whereas both maternal and paternal blood THg levels were significantly positively, and very similarly strongly, correlated with THg levels in the egg contents. With respect to the eggshells, this suggests that maternal effects are detectable only after the developing chicks have absorbed THg from the eggshell. This, in turn, suggests that variation in development among the unhatched eggs, itself indeed related to eggshell THg levels, might mask these maternal effects. Here, we must admit that we did not pick up on such masking when adding ‘death age’ to the model, but this measure of embryonic development may have been too coarse a variable to do so. With respect to the egg contents, our result may be partly explained by previous findings that most Hg transferred by females is deposited into these contents (Burger, 1994; Kennamer et al., 2005; Abola et al., 2021; Daniels-Abdulahad et al., 2024), allowing for detectable paternal effects even if these were small as well. Further studies are, however, required to understand why the observed paternal effect was stronger than expected based on the observed level of assortative mating for blood THg levels.

### 4.3. Repeatability of THg transfer

To contribute to our understanding of parental effects on THg levels in eggs, we assessed the repeatability of THg concentrations of eggshells of hatched eggs (i) within clutches, as well as (ii) within parents across years. When taking into account that parents were sampled in different subsets of years, and that years varied substantially in the level of THg found in the eggshells, we found a within-clutch repeatability of c. 33 %. This relatively low to moderate value suggests that factors beyond parental identity play a large role in determining eggshell THg after hatching. One such factor might be among-egg, within-clutch variation in egg size and development not captured in the laying order effect we took into account when calculating our repeatability (see below).

Across years, we found eggshell THg concentrations of parents to be repeatable by 11 and 7 % for mothers and fathers, respectively. Given that parental blood levels themselves are repeatable by roughly 19 % across the same time scale (Bertram et al., 2024b), this implies that other factors, most likely those that would also cause variation in blood levels in breeding adults, play an important role in predicting THg concentration in eggshells. Those factors might comprise changes in diet (see Cansse et al., 2024 for such variation during the chick rearing phase) or partner change (Rebke et al., 2017), rather than factors that would lead to variable levels of excretion into the eggs, although an effect of male courtship feeding on female egg size (Wendeln, 1997) may underlie these latter effects. Notably, we only had sufficient data to assess the repeatability of THg levels in the shells of hatched eggs. Both repeatability values might have been much higher if measurements could have been taken before embryonic development, but this would require further (experimental) investigation.

### 4.4. Consequences of THg for egg viability and chick hatch mass

Contrary to previous studies (Hallinger and Cristol, 2011; Goutte et al., 2014b), we found a significantly positive, rather than negative, correlation between maternal blood THg concentration and measures of reproductive performance: eggs from mothers with higher THg concentrations were more likely to hatch and resulted in heavier chicks. This suggests that a mother's blood THg level reflects her ability to invest in reproduction, for example because mothers with a diet consisting of larger fish or prey from higher trophic levels (Gilmour et al., 2019), could be in an improved energetic state. An additional model testing the relationship between egg volume and parental blood THg levels showed that mothers, but not fathers, with high blood THg

concentrations indeed did have larger eggs (Fig. S6, Table S4). Given that our model for hatch mass already controlled for egg volume (Vedder et al., 2023), the positive correlation we found cannot be attributed to egg size effects directly, but perhaps the transfer of specific beneficial nutrients plays a role. Either way, the finding of a positive correlation between maternal THg concentration and measures of reproductive success suggests that the current level of transfer of Hg to eggs does not come with measurable negative fitness consequences. An interesting next step would be to analyze stable isotopes in conjunction with THg levels in maternal blood to obtain insights into the link between diet and trophic level, pollution level and reproductive parameters.

Despite the positive associations between maternal blood and eggshell THg levels and between maternal blood THg levels and reproductive performance, eggshell THg levels were negatively correlated with hatching success and hatch mass. This suggests that elevated THg levels in eggshells are not a cause for, but rather a consequence of, impaired embryo development or survival. Supporting this, we found that while THg levels in the egg content did not vary strongly with the age at which the embryo died, those in the eggshells were significantly lower when embryos died during the last stage of development. The latter result aligns with that of a previous study investigating this effect in 22 bird species, although the decline in THg levels in the eggshells of those birds seemed to degrade more gradually throughout embryonic development (Peterson et al., 2017). Combined, we suggest these results indicate that embryos absorb Hg from the eggshell, along with other nutrients (Romanoff, 1960), perhaps most strongly at the last stage of development. Experimental studies, able to compare THg levels in the shells of unincubated eggs and eggs whose development has been stopped at different phases of development would be able to confirm or refute this hypothesis. With respect to the consistent THg levels in the egg content, independent of the stage of development, we suggest that the amount of Hg absorbed from the shell does not substantially contribute to the overall levels in the egg content, which are much higher (Burger, 1994; Kennamer et al., 2005; Abola et al., 2021; Daniels-Abdulahad et al., 2024).

## 5. Conclusion

Overall, our study indicates (i) assortative mating with respect to blood THg levels in common terns, and (ii) that maternal blood THg levels significantly positively correlate with THg concentrations in the content and shells of their eggs, while paternal blood THg levels are surprisingly strongly correlated with those in the contents of the eggs their partners have laid as well. In addition, we have shown that (iii) THg concentrations in the shells of hatched eggs show both within-clutch, and within-parent across-year repeatability. While (iv) materially transferred THg levels do not seem to adversely affect embryonic quality or survival at the observed levels, they rather reflect maternal investment and egg quality. Additionally, we observed that (v) unhatched eggs and those eggs from which chicks hatched at a lower mass had significantly higher THg levels in their shells. We suggest this not to be caused by detrimental effects of Hg, but to rather result from the embryo's ability to absorb Hg from the eggshell. These findings underscore the complexity of Hg transfer and absorption, and highlight the need for further, ideally experimental, research to fully understand the long-term ecological and health implications of Hg exposure in wild bird populations.

### CRediT authorship contribution statement

**Justine Bertram:** Writing – original draft, Visualization, Investigation, Formal analysis, Conceptualization. **Nathalie Kürten:** Writing – review & editing, Investigation. **Coraline Bichet:** Writing – review & editing, Investigation. **Maria Moiron:** Writing – review & editing, Formal analysis. **Peter J. Schupp:** Writing – review & editing,
Resources. Sandra Bouwhuis: Writing – review & editing, Supervision, Project administration, Conceptualization.

## Ethical approval

The study was performed under licenses of the city of Wilhelmshaven and the Lower Saxony State Office for Consumer Protection and Food Safety, Germany.

## 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.

## Acknowledgments

We would like to thank all field assistants who helped with data collection, as well as U. Pijanowska and P. Schwarz for laboratory support. Special thanks goes to P.H. Becker for setting up the long-term common tern study at the Banter See, O. Vedder for setting up the incubation project and taking part in fruitful discussions about maternal effects and mercury contamination, and G. Wagenknecht for being a backbone of the overall project throughout the sampling period. We would also like to thank three anonymous reviewers for their constructive comments that helped us improve this manuscript.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.envres.2025.121437.

## Data availability

Data will be made available on request.

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