Environmental Toxicology and Chemistry—Volume 39, Number 10—pp.

2008–2017, 2020
Received: 9 February 2020 | Revised: 6 March 2020 | Accepted: 14 July 2020 2008

Environmental Toxicology

Mercury Uptake Affects the Development of Larus fuscus Chicks

Cátia S.A. Santos,a,b,* Alejandro Sotillo,a,b Trisha Gupta,a Sergio Delgado,c Wendt Müller,d Eric W.M. Stienen,e Liesbeth de Neve,a
Luc Lens,a Amadeu M.V.M. Soares,b Marta S. Monteiro,b and Susana Loureirob
a
Terrestrial Ecology Unit, Department of Biology, Ghent University, Ghent, Belgium
b
Department of Biology and Center for Environmental and Marine Studies, University of Aveiro, Campus de Santiago, Aveiro, Portugal
c
Department of Ornithology, Aranzadi Sciences Society, Donostia, Spain
d
Behavioral Ecology and Ecophysiology Group, Department of Biology, University of Antwerp, Campus Drie Eiken, Antwerp, Wilrijk, Belgium
e
Research Institute for Nature and Forest, Brussels, Belgium

Abstract: Current emission and mobilization rates of mercury (Hg) in the environment pose extensive threats to both wildlife
and human health. Assessing the exposure risk and effects of Hg contamination in model species such as seabirds is essential
to understand Hg risks at the population and ecosystem levels. The lesser black‐backed gull (Larus fuscus), a generalist
seabird species, is an excellent model species because it forages in both marine and terrestrial habitats, which in turn differ in
their Hg exposure risk. To identify possible deleterious effects of Hg exposure on developing L. fuscus chicks, a dietary
experiment was carried out and chicks were provided a marine, terrestrial, or mixed diet. The effects of embryonic and
dietary Hg exposure on chick body condition and physiological state were assessed at different developmental stages until
fledging age (30 d). Overall physiological condition was lower in chicks fed a predominantly marine diet, which coincided
with higher Hg loads in blood and primary feathers. However, no effect of dietary uptake of Hg was observed on body
condition or in terms of genotoxic damage. Body condition and genotoxic damage correlated instead with Hg exposure
during embryonic development, which seems to indicate that embryonic exposure to Hg may result in carry‐over effects on
later chick development. Environ Toxicol Chem 2020;39:2008–2017. © 2020 SETAC

Keywords: Biomarkers; Bioaccumulation; Birds; Body condition; Dietary uptake; Metal accumulation

INTRODUCTION increase above the maximum levels recommended for human

consumption by the European Commission (2006) regulation
Each year, approximately 2000 metric tons of mercury (Hg)
(0.5 ng/mg wet wt Hg; ~2.5 ng/mg dry wt).
are emitted to the environment due to anthropogenic activ-
Mercury exposure in wildlife occurs mainly via consumption
ities that, added to natural emission sources, have significantly
of fish, making piscivorous marine birds particularly susceptible
increased environmental Hg levels (Krabbenhoft and
to Hg exposure and its toxic effects (Carravieri et al. 2014;
Sunderland 2013). In addition, climate change (e.g., via tem-
Ackerman et al. 2016). The toxicity of Hg occurs primarily via
perature and hydrology alterations) is predicted to interfere
disruption of neurotransmission mechanisms and consequent
with Hg biogeochemical cycles, increasing its mobilization
impairment of nervous system functions, which may sub-
and altering rates and/or patterns of methylmercury formation
sequently lead to changes in behavior, as well as in motor and
in aquatic ecosystems (Krabbenhoft and Sunderland 2013).
sensory functions (Wolfe et al. 1998; Presley et al. 2010;
These changes are expected to increase Hg levels in the
Nabi 2014). In birds, Hg exposure has been observed to induce
environment and impose threats to human and wildlife
deleterious effects on behavior (Julie and Frederick 2005;
health due to the high toxicity of methylmercury and its
Kobiela et al. 2015), reproduction (Evers et al. 2003; Goutte
ability to biomagnify in aquatic food webs (Ackerman et al.
et al. 2014), and survival (Wolfe et al. 1998; Wiener et al. 2002).
2016; Eagles‐Smith et al. 2016; Science for Environment
Monitoring exposure risk and the effects of Hg contamination
Policy 2017). This, in turn, could push Hg levels in fish to
in susceptible target species, such as seabirds, is therefore
of utmost relevance to evaluate Hg risks at population and
This article includes online‐only Supplemental Data. ecosystem levels.
* Address correspondence to catiasantos@ua.pt As a generalist species, the lesser black‐backed gull (Larus
Published online 17 July 2020 in Wiley Online Library
(wileyonlinelibrary.com). fuscus) has a broad dietary spectrum at the population level,
DOI: 10.1002/etc.4823 from predominantly marine, over urban to terrestrial foraging

© 2020 SETAC wileyonlinelibrary.com/ETC

Hg uptake effect on development of Larus fuscus chicks—Environmental Toxicology and Chemistry, 2020;39:2008–2017
(Camphuysen et al. 2015; Sotillo et al. 2019), thus varying in
their degree and type of contaminant exposure. As marine
foraging increases, the risk of Hg exposure is also likely to in-
crease (Polito et al. 2016; Santos et al. 2017a), with potential
negative fitness consequences. A previous study on L. fuscus
showed that increased exposure of breeding females to Hg
reduced investment in egg size, yet with no clear effect on the
development and body condition of the offspring (Santos
et al. 2017a). To further understand whether and how Hg ex-
posure during pre‐ and posthatch periods affects offspring
development and physiological condition, it is necessary to
study the effects of Hg accumulation on different proxies of
chick health simultaneously, such as body condition and bio-
chemical markers of health.
Birds have developed several mechanisms to cope with
environmental stressors such as Hg, within moderate thresh-
olds, including feather moult, maternal deposition in egg, and
antioxidant defences. Feather moult allows Hg elimination from
the bloodstream by sequestration into the growing feather,
thus reducing its retention in internal tissues (Condon and
Cristol 2009; Whitney and Cristol 2017). Similarly, females may
excrete Hg through maternal deposition in eggs during their
formation (Evers et al. 2003). Antioxidant defences comprise
another mechanism to minimize deleterious effects of metals
such as Hg (Patrick 2002). These include catalase (CAT) activity,
which detoxifies the hydrogen peroxide generated during ox-
idative stress, and glutathione S‐transferase (GST) activity,
which catalyzes the conjugation of glutathione (GSH) with
reactive oxygen species, contaminants, and their metabolic
byproducts to detoxify them (Andreoli et al. 1992; de la Casa‐
Resino et al. 2015).
Mercury exposure affects various physiological processes,
which may be reflected in changes in other blood biomarkers
such as cholinesterase (ChE) activity, which has previously been
used as a marker of Hg‐induced neurotoxicity (Dieter and
Ludke 1975; Elumalai et al. 2007; Frasco et al. 2007). Lactate
dehydrogenase (LDH) levels in plasma, used as a marker of
tissue damage, have also been reported to increase in re-
sponse to Hg exposure (Dieter 1974; Barata et al. 2010).
Finally, biomarkers of energy metabolism such as total protein,
carbohydrate, and lipid contents have been used to demon-
strate contaminant‐induced alterations in energy metabolism
(De Coen and Janssen 1997; Heiss et al. 2009; Ferreira et al.
2010).
The present study explores the pathways of dietary Hg up-
take in developing L. fuscus chicks, and the effects of Hg ex-
posure on offspring development and physiological condition.
To achieve this, we assessed: 1) to what extent different diets
reflect different Hg concentrations in chick blood and feathers;
2) whether prehatch exposure and posthatch dietary uptake of
Hg relate to the body condition of chicks; and 3) whether
prehatch exposure and posthatch dietary uptake of Hg relate
to prefledging physiological condition and biochemical bio-
markers known to be affected by Hg. We expected that chicks
fed on a predominantly marine diet would have higher Hg
levels in blood and feathers, and therefore lower body con-
dition, development, and physiological processes.
wileyonlinelibrary.com/ETC
2009
MATERIALS AND METHODS
Experimental set‐up
Eggs of L. fuscus were collected in 2015 at a breeding colony
in the harbor of Ostend, Belgium (51°21′N, 03°11′E), on 3 dif-
ferent dates (31 May and 7 and 14 June). Because chicks
hatching from larger eggs have a higher predicted chance of
fledging, only first or second laid pipping eggs were collected
from randomly selected 3‐egg clutches to avoid clutch rank ef-
fects connected to the smaller size of the third laid eggs. Eggs
were then taken to the aviary facilities of Ghent University hosted
at the Ostend Wildlife Rescue Centre (Ostend, Belgium) and in-
cubated (temperature 37.5 °C, humidity 62%) until hatching. Gull
chicks (n = 30) were then tagged with numbered insulation tape
around the tarsus for individual identification, and hand raised.
Each chick was randomly assigned to a diet treatment using a
blind sorting system, thus promoting a homogenous distribution
of hatching dates across treatments. Chicks within the same diet
treatment were housed together under a heat lamp until 3 d after
hatching, when they were transferred to individual cages when
30 d old, which is their approximate fledging age (Del Hoyo
et al. 1996). Each individual cage was 2 m long by 1 m wide and
0.5 m high, and was partially covered by plastic to protect against
rain and wind. An individual shelter was provided in each cage in
the form of an L‐shaped polyvinylchloride tube. All individual
cages were located inside a large aviary.
Chicks were fed diets with varying proportions of marine and
terrestrial food fractions, to provide a varying degree of Hg
exposure. The marine food consisted of an equal mix (in dry wt)
of Atlantic cod (Gadus morhua), Atlantic mackerel (Scomber
scombrus), and whiting (Merlangius merlangus), whereas the
terrestrial food consisted of an equal mix of broiler chicken
(Gallus gallus domesticus), mealworms (Tenebrio molitor
larvae), and fried potato (Solanum tuberosum) chips. The 2 food
fractions were blended and processed into gelatine‐bound
pellets with 3 diet compositions: 1) marine, 4:1 proportion of
marine and terrestrial food, respectively; 2) terrestrial, 4:1 ratio
of terrestrial and marine food; and 3) mixed, 1:1 proportion of
both marine and terrestrial foods. All chicks were fed ad libitum.
During the first 3 d after hatching, a simplified version of the
diet was provided to facilitate food deglutition. During this
period, the marine diet consisted of ground cod fillets (100%
marine diet), whereas the terrestrial diet was ground chicken
(100% terrestrial diet), and the mixed diet consisted of an equal
proportion in wet weight of ground cod and chicken. Aliquots
of the food provided in each diet treatment were sampled
periodically and stored at –20 °C for further Hg determination.
Growth measurements and body condition
At days 1 and 30 after hatching, chick body mass (digital
scale, to the nearest g) and total head length (digital calliper, to
the nearest 0.01 mm) were measured. The scaled mass index
(SMI; Peig and Green (2009) was calculated to express body
condition of chicks at day 1 (henceforth referred as SMIHATCH)
and day 30 (henceforth referred as SMIDAY 30). Using total head
© 2020 SETAC
2010
length and body mass as a proxy for body size and mass, re-
spectively, SMI was calculated as follows:
⎡ L ⎤bSMA
SMI = mi ⎢ 0 ⎥
⎣ Li ⎦
where mi = body mass of individual i and Li = head length of
individual i, bSMA = slope of the regression of ln body mass on
ln head length of all individuals, and L0 = arithmetic mean head
length for the study population. Then SMIDAY 30 was used as a
response variable to test the effect of Hg on body condition
considering both maternal input and dietary uptake. Higher
SMI values indicated better individual body condition, whereas
lower SMI values indicated worse individual body condition.
Sampling procedures
After hatch (1 d old), down feathers were collected for Hg
determination. Blood samples (±0.5 mL) were drawn from the
brachial vein at 12, 21, and 30 d of age (±2 d) using a sterile
1‐mL Terumo syringe with 25‐ga needle, and collected
into capillary tubes with ethylenediamine tetraacetic acid
(Microvette® CB 300; Sarstedt) to allow separation of plasma
and blood cells in the laboratory. The volume of blood col-
lected per chick was below 1% of their body weight to ensure
no impact was exerted on the health of chicks. Samples were
then divided into different aliquots and centrifuged in an
Eppendorf 5417 R centrifuge (rotor radius 9.5 cm, 3824 g,
10 min, 4 °C), and plasma and blood cells were separated into
different Eppendorf vials and stored at −80 °C for biochemical
analysis. When possible, 2 aliquots of whole blood were div-
ided into 2 different Eppendorf vials and stored at −20 or
−80 °C for determination of Hg content and proteins, and
carbohydrate and lipid levels, respectively.
At 30 d of age, an additional aliquot of blood was extracted
to prepare 2 blood smears in situ, for scoring of erythrocyte
nuclear abnormalities (ENAs). The slides were fixated with
methanol, air‐dried overnight, stained with Giemsa (5%), air‐
dried again, and stored dry until scoring of ENAs. The outer-
most primary feather (P10) was collected for Hg determination.
Biochemical analysis
Plasma and blood cell samples were processed as described in
Santos et al. (2019). Briefly, plasma aliquots were used for the
determination of butyryl (B)ChE and LDH activity, whereas blood
cells were used for the determination of GSH, CAT, GST, and lipid
peroxidation (LPO) levels. For the determination of energy re-
serve assays, whole blood aliquots were used. For further details
on procedures used for biochemical analysis, see the Materials
and Methods section of the Supplemental Data (Section S.1.1).
ENA assay
The ENA assay, as adapted by Pacheco and Santos (1996),
was used to assess DNA damage in mature peripheral
© 2020 SETAC
Environmental Toxicology and Chemistry, 2020;39:2008–2017—C.S.A. Santos et al.
erythrocytes. For each smear, slides were coded and scored
blindly, and 500 erythrocytes (in total, 1000 erythrocytes/chick)
were counted using a Novex (Holland) microscope, under a
1000× magnification. For each smear, the frequency of the
following nuclear abnormalities was determined (Supplemental
Data, Figure S1): micronuclei, lobed nuclei, kidney‐shaped
nuclei, segmented nuclei, and vacuolated nuclei. Cells with
signs of necrosis or rupture of cell membrane were not con-
sidered for scoring purposes, whereas nuclei with no signs of
abnormalities were scored as normal. All smears were scored
by a single operator to reduce scoring bias connected to
multiple operators.
Hg analysis
Total Hg concentration in feathers, food, and blood was
quantified by atomic absorption spectrophotometry with
thermal decomposition (Costley et al. 2000) using an advanced
mercury analyzer (LECO 254) with a detection limit of 0.02 ng
Hg, as described by Santos et al. (2017a). Sample preparation
and analysis were carried out following procedures described
by Santos et al. (2017a), as further detailed in the Supplemental
Data (Section S.1.2).
Precision and accuracy of the analytical method were eval-
uated by analysis of certified reference materials TORT‐3 and
ERM‐DB001 (lobster hepatopancreas and human hair, re-
spectively). All sample measurements were above the equip-
ment's limit of detection. Further details on the analytical
accuracy of analyses can be found in the Supplemental Data
(Section S.1.2 Hg analysis).
Integrated biomarker response calculation
To gain a general overview of the responses at the bio-
chemical level and to integrate all the results from the different
biomarkers assessed, we calculated the integrated biomarker
response (IBR; Beliaeff and Burgeot [2002], as adapted by
Devin et al. [2014]). This procedure uses a permutation to allow
the calculation of IBR in relation to every possible circular se-
quence of the 9 biomarkers tested along the radar diagram
(40 320 sequences in the present study), thus accounting for
possible variability in IBR value connected to biomarker se-
quence on radar plots. To plot radar IBR plots, we opted to
select a single permutation (permutation no. 66, out of 40 320
combinations). This permutation was preferred because this
sequence allowed us to present biomarkers tested in the IBR
radar diagrams according to type of stress response tested,
using the following order: neurotoxicity, tissue damage, oxi-
dative stress and energy metabolism markers.
Statistical analysis
Data outliers were initially identified using graphical in-
spection (i.e., box plots) and the calculation of standard re-
siduals exceeding ±2 standard deviation (SD). This enabled the
detection of 3 data outliers on the SMIDAY 30 variable. Because
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Hg uptake effect on development of Larus fuscus chicks—Environmental Toxicology and Chemistry, 2020;39:2008–2017 2011

these points were clearly aberrant values compared with the significance that would be biologically meaningless. The IBR
rest of the data, which was probably due to a measurement sample size was calculated by setting the magnitude of the
error during chick measurement and/or weighing, they were effect size to 1, which was biologically meaningful in this
removed. Normality in the distribution of variables was tested context, and the statistical power to 0.8.
using the Kolmogorov–Smirnov and Lilliefors test, whereas Whenever relevant, a post hoc Tukey test was used to
heteroscedasticity across treatments was tested using Levene's compare mean differences between diet groups following the
test. All variables that did not fit a normal distribution were GLM analysis. A Pearson correlation coefficient was calculated
either log‐transformed (parameter: total carbohydrates) or between HgPF and Hg in blood (all ages) and between re-
square‐rooted (parameters: HgPF [primary feathers], HgDAY 12, sponse variables. Information pertaining to intermediate values
HgDAY 21, or HgDAY 30, micronuclei, and total ENA frequencies) of univariate tests of significance for GLMs modeled is further
to meet normality. To assess whether chicks were randomly available in the Supplemental Data, Table S6.
distributed across diet treatments with respect to hatching Hg Statistical analyses were conducted using Statistica 8 (Stat-
burden (HgDF [down feathers]) and SMI (SMIHATCH), individual Soft) and R Ver 3.4.0 (R Core Development Team 2018) with the
linear models were fitted with hatching date and chick sex as integrated development software RStudio Ver 1.0.143. All
covariates. In the model with SMIHATCH, HgDF was added as a graphs were made using Statistica 8, except the radar plots, for
covariate. A logistic regression with diet as the explanatory which SigmaPlot Ver 12.5 (Systat Software) was used.
variable and logit as s link function was fitted to check whether
the male/female ratio in each diet was homogenous.
To test whether diet treatments induced different Hg con- Ethics statement
centrations in blood at 12, 21, and 30 d (HgDAY 12, HgDAY 21,
All experimental manipulations were in accordance with the
and HgDAY 30, respectively), a repeated measures analysis of
European/FELASA ethical guidelines (Directive 2010/63/EU) and
variance (RM ANOVA) was run. The relationship between Hg
approved by the Ethical Committee of Ghent University (ECD
load in primary feathers (HgPF) and diet treatment was tested
number 2015‐017). Further details on the ethical guidelines
by means of a generalized linear model (GLM) including
used to ensure animal wellbeing are detailed in the Animal
hatching date, sex, and HgDF as covariates.
welfare section in the Supplemental Data (Section S.1.5).
To assess whether and to what extent prefledging con-
dition (SMIDAY 30) was affected by diet treatment and prehatch
Hg exposure (HgDF), a GLM with condition at hatching RESULTS
(SMIHATCH), chick sex, and the interaction between HgDF and Diet and Hg variation in blood and feathers
diet as explanatory variables was tested. Moreover, to check
The average Hg concentration in food was 0.191 ± 0.021 ng/mg
whether Hg burden through maternal input (HgDF) and diet
dry weight for the marine diet, 0.088 ± 0.009 ng/mg dry weight
treatment explained variation in different biochemical markers
for the mixed diet, and 0.011 ± 0.005 ng/mg dry weight for the
assessed in 30‐d‐old chicks, a GLM was run with chick sex
terrestrial diet.
and the interaction between HgDF and diet treatment as ex-
The average, minimum, and maximum Hg concentrations
planatory variables. To evaluate whether Hg burden accu-
detected in blood, down, and primary feathers are depicted in
mulated through maternal input and dietary uptake resulted
Table 1. The Hg in down feathers was not related to sex or
in different ENA frequencies, a GLM with chick sex, SMIHATCH,
hatching date (GLM, both p > 0.10). No differences in Hg
SMIDAY 30, and the interaction between HgDF and diet treat-
measured in down feathers, body condition at hatch (linear
ment as covariates was tested. The interaction between HgDF
model, all p > 0.4), or sex (linear model, p = 0.67) were de-
and diet treatment was included in all models to account
tected across diets. The male/female ratio across diet treat-
for possible variation within treatments driven by maternal
ments was homogenously distributed (Wald test = 0.78,
input of Hg.
df = 2, p = 0.68).
Due to a limited sample size, data on biochemical markers
At all ages, chicks fed a marine diet had significantly higher
from days 12 and 21 after hatching are provided as Supple-
levels of Hg in the blood than chicks fed a mixed diet; the
mental Data without further analysis. The similarity of IBR
lowest values were measured in those raised on a terrestrial
scores between diets was assessed from samples taken at 30 d
diet (F2,20 = 9.08, p = 0.002; Table 1 and Figure 1A). A similar
of age: 100 random samplings of 10 IBR values among those
pattern across diet treatments was observed for Hg in primary
calculated for each diet treatment were run and, for each
feathers (HgPF; GLM, F2,25 = 1036.39, p < 0.001; Figure 1B).
sampling, a one‐way ANOVA and post hoc Tukey test were
The Hg levels in blood were positively correlated with HgPF
performed. The p value of each Tukey test was stored in a
at all developmental stages (12, 21, and 30 d; all p > 0.01;
vector and corrected for multiple comparisons following
Supplemental Data, Table S1).
Benjamini and Hochberg (1995), and the mean of the
100 corrected p values was obtained. This procedure was
carried out to remove the bias in the statistical analyses in-
duced by the large number of IBR values produced during the Body condition
permutation procedure (N = 40 320 IBR values), which would The SMIHATCH did not differ between diet treatments and
otherwise become too powerful and allow for statistical was not related to HgDF (GLM, both p > 0.12), but was

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2012 Environmental Toxicology and Chemistry, 2020;39:2008–2017—C.S.A. Santos et al.

significantly higher in females (GLM, F1, 31 = 4.80, p = 0.04) and

7.778
0.335

0.118
0.007
0.004
MAX
increased with hatching date (GLM, F1, 31 = 14.99, p < 0.001).
Similarly, female chicks displayed significantly higher SMIDAY 30
than males (GLM, F1, 28 = 6.42, p = 0.02). Moreover, variation in
2.104 SMIDAY 30 was unrelated to diet treatment or SMIHATCH (GLM,
0.217

0.006
0.003
0.001
MIN

all p > 0.90), but tended to decrease with increased HgDF levels
(GLM, F1, 27 = 3.71, p = 0.06; Figure 2).
[0.003, 0.005]
[0.001, 0.003]
Terrestrial

[2.54, 5.60]
[0.25, 0.31]

[0.01, 0.08]
CI

ENA assay and biochemical markers

Total ENA and micronuclei frequencies are depicted in
4.077 ± 0.676 (11)
TABLE 1: Mercury (Hg) levels in down feathers (DF), blood (12‐, 21‐, and 30‐d‐old), and primary feathers (PF) of Larus fuscus chicks with varying dieta

0.280 ± 0.014 (9)

0.036 ± 0.018 (6)

0.004 ± 0.001 (7)
0.002 ± 0.001 (9)

Table 2. Overall, micronuclei frequency was unrelated to ENA

Hg ± SE (no.)

(r = –0.03, p = 0.87) and low across all treatments, never ex-

ceeding 1.2% of micronuclei/1000 cells. The ENA frequency
increased with increased HgDF load (GLM, F1, 28 = 5.17, p = 0.03;
SE = standard error; CI = confidence interval of mean, presented as [–95%, 95%]; MIN = minimum; MAX = maximum; no. = sample size (number of chicks).

Figure 3), but was unrelated to diet, sex, SMIHATCH, or

SMIDAYS 30 (GLM, all p > 0.32).
5.924
1.954

0.058
0.049
0.035
MAX

Levels of biochemical markers in plasma (LDH and ChE),

Data are presented as the mean ± standard error. Values in feathers and blood are presented in ng/mg of dry weight and ng/mL, respectively.

blood cells (LPO, CAT, GST, and GSH), and whole blood (total
proteins, carbohydrates and lipids) for all sampling times are
0.683
1.199

0.016
0.020
0.019

presented in the Supplemental Data, Tables S2, S3, re-

MIN

spectively. The LPO levels in 30‐d‐old chicks were negatively

related to HgDF (GLM, F1, 26 = 9.05, p = 0.006; Figure 4), but
[2.14, 4.40]
[1.77, 1.20]

[0.02, 0.05]
[0.02, 0.04]
[0.02, 0.03]

did not vary according to diet or sex. The variation in all other
oxidative stress markers and LDH was unrelated to HgDF load,
Mixed

CI

diet, or sex (GLM, all p > 0.16). Butyryl (B)ChE activity did not
relate to HgDF load or diet (GLM, all p > 0.12), but was sig-
nificantly higher in males than females (GLM, F1, 26 = 5.58,
3.272 ± 0.513 (12)
1.613 ± 0.070 (10)

0.026 ± 0.002 (11)

p = 0.03).
0.035 ± 0.003 (8)
0.030 ± 0.001 (9)
Hg ± SE (no.)

Lipid levels in blood tended to be negatively related to

HgDF (GLM, F1,26 = 3.47, p = 0.07), but not protein or carbo-
hydrate levels (GLM, all p > 0.23). Diet did not explain a sig-
nificant part of variation in total proteins and lipids (GLM, all
p > 0.23), but chicks fed a mixed diet displayed significantly
higher levels of carbohydrates than those fed marine and ter-
7.633
4.649

0.244
0.135
0.081
MAX

restrial diets (GLM, F2, 23 = 4.87, p = 0.02). No sex‐related dif-

ferences in total proteins, carbohydrates, or lipids were found
(GLM, all p > 0.23).
1.397
3.762

0.075
0.046
0.023
MIN

[2.30, 4.91]
[4.02, 4.50]

[0.10, 0.20]
[0.06, 0.11]
[0.04, 0.07]

IBR
Marine

CI

The permutation performed for the set of biochemical

markers assessed in 30‐d‐old chicks resulted in a matrix of
40 320 IBR values, summarized in Table 3. Permutation no. 66
3.611 ± 0.584 (11)

out of 40 320 combinations was used to create the IBR plots

4.262 ± 0.104 (9)

0.151 ± 0.022 (7)

0.085 ± 0.011 (7)
0.056 ± 0.007 (7)
Hg ± SE (no.)

presented in Figure 5.
Chicks fed a marine diet showed the highest IBR scores,
followed by those with a mixed and terrestrial diet (one‐way
ANOVA, corrected p < 0.00; post hoc: Tuckey test, corrected
p < 0.0001). This tendency observed in IBR scores of chicks fed
the marine diet seemed to be mostly related to lowered levels
Tissue/Diet

12 days
21 days
30 days

of total proteins, carbohydrates, and lipids in blood, as well as

Primary
Down
Feather

to the increased levels of LDH and oxidative stress parameters

Blood

observed in this group (Table 3 and Figure 5).

a

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Hg uptake effect on development of Larus fuscus chicks—Environmental Toxicology and Chemistry, 2020;39:2008–2017 2013

FIGURE 1: Mean ± standard error mercury (Hg) levels in (A) blood at varying age and (B) primary feathers (PF) of Larus fuscus chicks for each diet
treatment. Letters represent significant differences across diets in blood (repeated measures analysis of variance [RM ANOVA], F4,20 = 2.88,
p = 0.049; post hoc: Tukey test, p < 0.02) and primary feather (generalized linear model [GLM], F4,20 = 1036.39, p < 0.001; Tukey test, p < 0.04).
MAR = marine; MIX = mixed; TER = terrestrial; * = significantly different from HgBLOOD in 12 d‐old chicks (RM ANOVA, F4,20 = 2.88, p = 0.049; post
hoc: Tukey test, p < 0.02).

FIGURE 3: Mercury down feather (HgDF) load in relation to erythrocyte

FIGURE 2: Scaled mass index30 d in relation to mercury down feathers nuclear abnormality (ENA) frequency (‰) in blood of 30‐d‐old Larus
(HgDF) in Larus fuscus chicks, per sex and diet treatment. Dotted lines fuscus chicks for each diet treatment. Dotted lines depict 95% con-
depict 95% confidence intervals of the regression line. fidence intervals of the regression line.

TABLE 2: Total erythrocyte nuclear abnormalities (ENAs) and micronuclei (MN) frequency in 1000 erythrocytes (%) in 30‐d‐old Larus fuscus chicks
with varying dieta

MN (%) ENAs (%)

Diet Mean ± SE (no.) CI MIN MAX Mean ± SE (no.) CI MIN MAX

Marine 0.04 ± 0.03 (9) [–0.03, 0.12] 0.00 0.30 3.36 ± 0.90 (9) [1.28, 5.43] 1.10 10.10
Mixed 0.19 ± 0.10 (11) [–0.04, 0.42] 0.00 1.20 3.94 ± 0.73 (11) [2.31, 5.58] 1.20 7.80
Terrestrial 0.18 ± 0.06 (11) [0.25, 0.31] 0.00 0.60 4.17 ± 0.81 (11) [2.36, 5.99] 1.00 10.60
a
Data are presented as the mean ± standard error.
SE = standard error; CI = confidence interval of mean, presented as [–95%, 95%]; MIN = minimum; MAX = maximum; no. = sample size (number of chicks).

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2014 Environmental Toxicology and Chemistry, 2020;39:2008–2017—C.S.A. Santos et al.

DISCUSSION
Diet and Hg in developing chicks
As expected, Hg loads in lesser black‐backed gull chicks
increased with the proportion of marine food in their diet. This
finding is in accordance with our initial expectation, because
consumption of fish, in particular predatory fish, has been
previously considered to be the principal pathway of Hg ex-
posure in marine wildlife (Wiener et al. 2002; Carravieri
et al. 2014; Ackerman et al. 2016). The Hg levels in blood
decreased with age, especially in chicks fed a marine diet, and
thus with the highest Hg load. This was likely related to
feather growth, which allows Hg sequestration from the blood
into the growing feather, a key mechanism of Hg detox-
ification in birds (Condon and Cristol 2009; Whitney and
Cristol 2017). Chicks of L. fuscus start growing their primary
FIGURE 4: Mercury down feather (HgDF) load in relation to lipoprotein
feathers at approximately 10 d after hatching (Sotillo et al.
(LPO) levels in red blood cells (RBCs) of 30‐d‐old Larus fuscus chicks for
each diet treatment. Dotted lines depict 95% confidence intervals of 2019), which coincided with the period when Hg in blood was
the regression line. TBARS = thiobarbituric acid reactive substances. highest in all treatments. The Hg sequestration into the
growing feathers is further evidenced by the positive rela-
TABLE 3: Mean, median, minimum, and maximum integrated bio- tionship between Hg in blood and Hg in primary feathers, the
marker response (IBR) values in chicks 30 d after hatching (N = 40 320 latter thus constituting an appropriate noninvasive matrix to
permutations)a assess dietary exposure of chicks during rearing. Decreased
Hg concentration in blood due to the increase in blood
Diet Mean ± SE Median MIN MAX
volume with growth could also have contributed to this effect,
Marine 4.895 ± 0.002 A 4.841 4.125 6.091 because increased size and blood volume in older chicks may
Mixed 1.937 ± 0.003 B 1.895 0.371 3.677
dilute Hg burden in blood, thus contributing to a decreased
Terrestrial 0.958 ± 0.003 C 0.941 0.063 2.189
overall blood Hg concentration.
a
Different capital letters denote statistically significant differences across treat-
ments (one‐way analysis of variance, corrected p < 0.0001; post hoc: Tukey test,
corrected p < 0.0001).
SE = standard error; MIN = minimum; MAX = maximum.
Hg effects on body condition and DNA damage
Contrary to our expectations, posthatching uptake of Hg
was unrelated to prefledging body condition. In contrast, we
noticed a nearly significant negative relationship between Hg
load in down feather and prefledging body condition. The Hg
load in down feathers, which is sequestered in the feather
during embryonic development, is of maternal origin and thus
connected to the maternal diet (Santos et al. 2017a). Previous
studies have reported a negative relationship between Hg
input and body condition of adult birds (Wayland et al. 2003;
Ackerman et al. 2012). This effect may be more acute for young
individuals, because survival up to adult age positively relates
to body condition at fledging (Souchay et al. 2013; Arizaga
et al. 2015). Our results suggest that Hg exposure during em-
bryonic development, rather than dietary input post hatching,
may influence chick development and negatively affect off-
spring quality, with long‐lasting effects on chick development.
In the present study, however, the levels of Hg in the diet we
provided to chicks (0.191 ng/mg dry wt Hg in marine diet) were
FIGURE 5: Radar plot of the integrated biomarker response (IBR) in
below the maximum recommended for human consumption by
30‐d‐old Larus fuscus chicks for each diet treatment. Values correspond
to biomarker score (S) following standardization and Z‐score European regulations (0.5 ng/mg wet wt Hg; ~2.5 ng/mg dry wt;
calculations. The IBR representation corresponds to permutation no. European Commission 2006). It is therefore possible that Hg
66 among the 40 320 ordination combinations possible. MAR = concentrations in the diets provided, which were representative
marine diet; MIX = mixed diet; TER = terrestrial diet; BChe =
of levels observed in the diet of wild populations, were too low
butyrylcholinesterase; LDH = lactate dehydrogenase; LPO = lipoprotein;
CAT = catalase; GST = glutathione‐S‐transferase; GSH = glutathione; to induce deleterious effects on the body condition of L. fuscus
PROT = protein; SUG = sugar; LIP = lipids. chicks.

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Hg uptake effect on development of Larus fuscus chicks—Environmental Toxicology and Chemistry, 2020;39:2008–2017
This may be the reason why no effect of diet was observed on
ENA frequency. On the other hand, an increased Hg load in down
feathers was found to significantly increase ENA frequency.
During replication, DNA undergoes cyclic changes in its structure,
which reduces its stability and increases susceptibility to genotoxic
damage (loss of DNA integrity, chromosome aberrations, muta-
tions, carcinogenicity, etc.) (Shugart et al. 2003; Baos et al. 2006).
More recently, the frequency of ENAs has been used as a marker
to detect genotoxic damage in birds (De Mas et al. 2015; Santos
et al. 2017b). Although its genotoxic effects in birds have been
scarcely addressed in the literature, Hg has been suggested to
have genotoxic effects (e.g., Fenstad et al. 2016). In the present
study, higher levels of Hg in down feathers were positively related
to an increase in ENA frequency, but again not with increased
dietary intake of Hg. Our results suggest that during embryonic
development individuals are more susceptible to genotoxic ef-
fects of Hg. The ENA levels we report were relatively high, for
instance higher than the ones reported for penguins exposed to
heavy metals in field conditions (De Mas et al. 2015). However,
micronuclei frequencies were below the threshold levels reviewed
in the literature (less than 2.14 micronuclei/1000 erythrocytes) as
typical for healthy birds (Zúñiga‐González et al. 2000, 2001; Quirós
et al. 2008).
Several studies have demonstrated the usefulness of as-
sessing nuclear abnormalities as a complementary tool to-
gether with micronuclei frequency to diagnose the exposure to
genotoxic agents (Braham et al. 2017; López González
et al. 2017). However, ENA frequency seems to be a more
sensitive marker of genotoxic damage than micronuclei alone
(Santos et al. 2017b). Moreover, the mechanisms underlying
the development of nuclear abnormalities remain unclear
(Oliveira et al. 2010; Braham et al. 2017), and it is not yet
possible to discern whether these abnormalities are linked to
loss of gene expression and, by extension, to fitness effects
such as survival and reproduction.
Increased maternal input of Hg during embryo development
thus seems to increase genotoxic effects in offspring, which
could contribute to an overestimation of genotoxic effects of
Hg in developing L. fuscus.
Hg input and biochemical parameters
The biochemical markers assessed 30 d after hatching were
unrelated to Hg load in down feathers or dietary uptake of Hg,
except for lipid levels in the blood and LPO levels in blood
cells, which decreased with increasing Hg exposure during
embryonic development. This could relate to the induction of
cell‐protecting antioxidant mechanisms or improved mem-
brane stabilization processes to prevent peroxidation of
membrane lipids, as reported for fish (Cappello et al. (2016).
Alternatively, changes observed in LPO measured at day 30
could also be the result of a parallel decrease of lipids in blood,
because LPO levels are dependent on lipid availability and thus
likely to decrease with lower lipid levels.
Differences between diets became apparent in IBR scores,
which were significantly higher in chicks fed the marine diet and
wileyonlinelibrary.com/ETC
2015
lowest in chicks fed the terrestrial diet. This trend was mostly
due to decreased levels of energy metabolism markers (pro-
teins, carbohydrates and lipids), increased oxidative stress, and
an increase in anaerobic metabolism (LDH activity) in chicks fed
the marine diet.
However, diet effects other than Hg exposure may also affect
resource allocation and energy metabolism (De Coen and
Janssen 1997; Ferreira et al. 2010), and disentangling these
different effects is challenging. Diet composition may have
contributed to the difference in levels of energy metabolism
markers between diet treatments, because different proportions
of terrestrial and marine prey items can result in different total
lipid, carbohydrate, and protein contents. Nevertheless, an al-
tered resource allocation due to the metabolic demands of Hg
detoxification into the growing primary feathers, or connected
to the increased activation of antioxidant defences, can be
presumed to have driven these differences to a certain extent. If
so, Hg detoxification would be responsible for a decrease in
levels of proteins, carbohydrates, and lipids in the blood.
The increased LDH and oxidative stress levels we observed
have elsewhere been found as a response to increased Hg ex-
posure (Elumalai et al. 2007; Carvalho et al. 2008; Barata
et al. 2010; Costantini et al. 2014; de la Casa‐Resino et al. 2015).
High LDH activity may be related to Hg‐induced interference in
the glycogen cycle, namely, by activation of anaerobic gly-
colysis, decreasing carbohydrates levels in blood and thus con-
tributing to lowered levels of energy metabolism markers.
CONCLUSIONS
Exposure to Hg during embryonic development decreases
body condition and increases ENA frequency of lesser black‐
backed gull chicks. Embryonic exposure to Hg may thus entail
long‐lasting health effects. Furthermore, a marine diet in-
creases Hg load in blood and primary feathers of chicks. This
may result in higher IBR scores, and hence decreased physio-
logical condition. Increased Hg load via dietary uptake in de-
veloping chicks may thus also induce negative effects on
development and physiological condition, although the evi-
dence found here remains indirect.
Taken together, the results obtained in our study could be
of great value to resource managers and risk assessors. Gulls,
and in particular lesser black‐backed gulls, can serve as study
models to understand how environmental stressors (con-
tamination, climate changes, etc.) may affect the behavior,
physiology, and reproductive success of other seabird species
and the integrity of the ecosystem they inhabit. Thus our results
could be further used to manage and promote the con-
servation of endangered seabirds and other marine wildlife.
Further research should aim at disentangling the effect of Hg
from the interacting effects of food nutritional composition, as
well as assessing the difference in deleterious effects of Hg
from maternal transfer versus dietary uptake post hatching.
Supplemental Data—The Supplemental Data are available on
the Wiley Online Library at https://doi.org/10.1002/etc.4823.
© 2020 SETAC
2016
Acknowledgment—We thank A. Ferreira and A. Alcantara for
technical support during laboratory procedures, as well as
S. Durnez for technical support during the experiment. The
present study was supported by doctoral grant B/13833/01
from the Special Research Fund of Ghent University (to C.S.A.
Santos), research grant G0E1614N from the Research Foun-
dation Flanders (to L. Lens, L. de Neve, and W. Müller), and the
European COMPETE program. The work of A. Soares is funded
by a Fundacão para a Ciência e a Tecnologia (FCT) grant (PB/
BD/113792/2015). The work of M.S. Monteiro is funded by
national funds (Orçamento de Estado‐OE), through the FCT, I.P
(Public Institution/Institute), in the scope of the framework
contract from sections 4, 5, and 6 of article 23 of the Decree
Law 57/2016, of August 29, changed by Law 57/2017, of July
19. Thanks are also due to FCT/Ministry of Science, Tech-
nology, and Higher Education for the financial support to the
Centre for Environmental and Marine Studies (UIDP/50017/
2020+UIDB/50017/2020), through national funds.
Author Contribution Statement—C.S.A. Santos and A. Sotillo
designed the experiments. C.S.A. Santos, A. Sotillo, T. Gupta,
and S. Delgado performed the experiments. C.S.A. Santos,
L. Lens, M.S. Monteiro, and S. Loureiro analyzed the data.
C.S.A. Santos wrote a first draft of the paper, and all authors
contributed to discussing and writing subsequent drafts.
Data Availability Statement—Data, associated metadata, and
calculation tools are available from the corresponding author
(catiasantos@ua.pt).
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