Comparative Biochemistry and Physiology, Part A 155 (2010) 205–210

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Comparative Biochemistry and Physiology, Part A
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a

Increased oxidative stress in barn swallows from the Chernobyl region
Andrea Bonisoli-Alquati a,⁎, Timothy A. Mousseau b, Anders Pape Møller c,d, Manuela Caprioli a, Nicola Saino aaDipartimento di Biologia, Sez. Ecologia, Università degli Studi di Milano, via Celoria 26, 20133 Milano, Italy
Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USAc
Laboratoire d′Ecologie, Systématique et Evolution, CNRS UMR 8079, Université Paris-Sud, Bâtiment 362, F-91405 Orsay Cedex, FrancedCenter for Advanced Study, Drammensveien 78, NO-0271 Oslo, Norwayb

a r t i c l e

i n f oArticle history:
Received 20 June 2009
Received in revised form 27 October 2009
Accepted 29 October 2009
Available online 5 November 2009
Keywords:
Antioxidant capacity
Barn swallow
Chernobyl
Oxidative stressRadioactive contamination
Reactive oxygen speciesa b s t r a c t
The Chernobyl nuclear accident produced the largest unintended release of radionuclides in history, withdramatic consequences for humans and other organisms. Exposure to ionizing radiation is known to reduce
circulating and stored levels of speciﬁc antioxidants in birds and humans, thus potentially increasingoxidative stress. However, overall effects of radioactive exposure on oxidative status have never been
investigated in any free ranging vertebrate. We measured plasma antioxidant capacity and concentration ofreactive oxygen metabolites in adult barn swallows (Hirundo rustica) from colonies with variablebackground radiation levels in the Chernobyl region in Ukraine and Belarus. We predicted that antioxidants
would decrease while reactive oxygen metabolites would increase with exposure to increasing levels ofradiation at the breeding sites. Consistent with this expectation, radiation level positively predicted plasma
concentration of reactive oxygen metabolites, whereas no signiﬁcant covariation was found with nonenzymatic plasma antioxidant capacity. An index of oxidative stress was also larger in barn swallows
exposed to high contamination levels. Thus, radioactive contamination appeared to be responsible for theincreased generation of reactive oxygen metabolites and the imbalance between reactive oxygen metabolites
and non-enzymatic plasma antioxidant capacity.
© 2009 Elsevier Inc. All rights reserved.

1. Introduction
Oxidative stress arises any time endogenous reactive oxygen
species (ROS) are left unquenched by enzymatic and non-enzymatic
antioxidant defences (Halliwell and Gutteridge, 2007). Organisms
need to balance their energy requirements for fuelling biologicalprocesses against the potential toxicity of ROS generated by any
activity as by-products of metabolic processes (Beckman and Ames,
1998; Finkel and Holbrook, 2000; Halliwell and Gutteridge, 2007).
However, no prediction can readily be drawn from metabolic rate toinfer oxidative damage, as organisms have evolved a complex set ofantioxidant mechanisms to face oxidative challenges (Halliwell andGutteridge, 2007; Costantini, 2008; Monaghan et al., 2009).
A promising ﬁeld of research for testing mechanistic and functional hypotheses concerning natural variation in antioxidant
defences is investigation of the consequences of exogenous sourcesof oxidative stress (e.g. pollutants, radioactive contamination;
Monaghan et al., 2009). Radioactive contamination can deplete
antioxidants because of their utilization for quenching ROS arising
as a consequence of radiation (Iliakis, 1991), or because of theirutilization for repairing DNA damage due to radiation exposure⁎ Corresponding author. Dipartimento di Biologia, Università degli Studi di Milano,
Via Celoria 26, 20133 Milano, Italy. Tel.: +39 0250314716; fax: +39 0250314713.
E-mail address: andrea.bonisoli@unimi.it (A. Bonisoli-Alquati).
1095-6433/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpa.2009.10.041(Moody and Hassan, 1982; Imlay and Linn, 1988). However, noattempt has been made to assess the consequences of radioactive
contamination for overall antioxidant status of wild populations ofanimals or humans. On the other hand, studies using the accident at
Chernobyl power plant in 1986 as a source of potentially deleterious
effects on antioxidant levels have shown that barn swallows (Hirundo
rustica) living in radioactively contaminated areas have reduced
circulating and stored levels of speciﬁc antioxidants (i.e. carotenoids
and vitamins A and E; Møller et al., 2005a). These results substantiallyconﬁrmed evidence from medical studies showing that individual
exposure to radioactive contamination negatively impacted antioxidant defences of humans exposed to radioactive contamination in
Chernobyl (Ben-Amotz et al., 1998; Ivaniota et al., 1998; Neyfakh
et al., 1998a,b; Kumerova et al., 2000). In the barn swallow,
concentrations of carotenoids and vitamins in eggs were also depleted
in Chernobyl compared to an uncontaminated site (Møller et al.,2005a), thus suggesting that a reduction in maternal antioxidant
levels may have trans-generational effects on offspring development
and ﬁtness. These ﬁndings were also consistent with results from anest-box experiment with great tits (Parus major), where concentrations of total yolk carotenoids and vitamins A and E were reduced in adose-dependent manner with increasing level of radiation at the nest
boxes (Møller et al., 2008). Finally, an inter-speciﬁc survey of species
richness and abundance in the Chernobyl region further showedthat species using a large amount of antioxidants for sexual signalling,206

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self-maintenance or maternal allocation to eggs might be more
susceptible to the damaging effects of low-level environmental
radiation in terms of population decline (Møller and Mousseau,
2007a). Thus, reductions in antioxidant level might have serious
consequences for reproductive decisions in terms of clutch size andeven whether to reproduce or not, and in determining the fate ofnatural populations in perturbed ecological conditions (Møller et al.,2005b, 2008; Møller and Mousseau, 2007b).
However, previous results obtained while studying the consequences of radioactive contamination in Chernobyl were based on
only a few components of the antioxidant defence system, such as
carotenoids or antioxidant vitamins. The antioxidant system is amulti-faceted complex, with different components responding differently to the same oxidative challenges (Monaghan et al., 2009). A
recent analysis of the covariation among three groups of antioxidants(uric acid, vitamin E and four carotenoids) and an overall measure ofantioxidant capacity (Trolox-equivalent antioxidant capacity or TEAC)
found that the degree to which each antioxidant predicted total
antioxidant capacity in adult individuals varied signiﬁcantly bothwithin and among species (Cohen and McGraw, 2009). The authors
concluded that ecological studies attempting to examine antioxidant function should therefore use multiple measures and avoid
the assumption that dietary antioxidants are essentially the most
crucial defence against oxidative damage (Cohen and McGraw, 2009;
Monaghan et al., 2009). To avoid one-sided evaluations and tocorrectly assess the balance between damage prevention by the antioxidant system and ROS generation, a more integrated approach istherefore needed (Finkel and Holbrook, 2000; Halliwell and Gutteridge, 2007; Monaghan et al., 2009).
In this study, we measured antioxidant capacity, oxidative damage
and oxidative stress in barn swallows from different sites in the
Chernobyl region. We tested the hypothesis that radioactive contamination can impair overall antioxidant status in wild populations ofbarn swallows living in this region. The barn swallow is a longdistance migratory species and is therefore an ideal model species fortesting the relationship between radiation exposure and antioxidant
status. In fact, migratory behaviour is known to impair antioxidant
defences owing to intense metabolic activity required by ﬂight effort(Costantini et al., 2007) and it has been shown that migratory species
are experiencing a stronger decline in abundance in radioactively
contaminated areas around Chernobyl compared to resident species,
likely because of the depletion of antioxidants defences required toneutralize free radicals (Møller and Mousseau, 2007a).

sampling sites as low-, intermediate- or high-level. Mean backgroundradiation level was 0.074 (range 0.02–0.15) μSv/h in the low-levelcontamination sites (Farm 1 (1), Farm 2 (2), Farm 3 (3), Rudnia (6),Dytiaku (8), Pysky (9) and Voronkov (10)), 0.455 (range 0.45–0.46)
μSv/h in the two intermediate-level sites (Farm 4 (4) and Farm 5 (5)),
and 2.9 μSv/h in the high-level site (Vesniane (7)) (numbers inparentheses refer to the numbers used in Fig. 1). Background radiationin uncontaminated sites in Ukraine is around 0.03 μSv/h.

2. Materials and methods

2.3. Reactive oxygen metabolites (ROMs) and oxidative stress2.1. Field procedures

Reactive oxygen metabolites (ROMs) are peroxidation products(primarily hydroperoxides, ROOH) of reactive oxygen species (ROS).
They can be considered markers of early oxidative damage, as theircleavage by metals leads to the generation of two highly reactive
prooxidants, the alkoxyl (R–OU) and alkylperoxyl (R–OOU) radicals,
which in turn can promote an oxidative cascade (Costantini, 2008).
ROMs are relatively more stable than ROS (that can last as short as afew seconds) and therefore they can be detected and quantiﬁed byanalytical procedures (Iorio, 2004). The plasma concentration ofROMs was measured by the d-ROMs test (Diacron). The plasma(10 µL) was diluted with 1 mL of a solution containing an acetate
buffer (pH 4.8). Then 20 µL of an alkyl-substituted aromatic amine (N,N-diethyl-p-phenylenediamine) solubilised in a chromogenic mixture
were added. The solution was incubated for 75 min at 37 °C. During
incubation, the acidic pH of the acetate buffer favours the release ofiron from plasma proteins. Iron ions (Fe2+ and Fe3+) catalyse thecleavage of ROOH generating alkoxyl (R–OU) and peroxyl (R–OOU)
radicals. Such radicals are able to oxidize the alkyl-substituted
aromatic amine (N,N-diethyl-p-phenylenediamine) solubilised in
the chromogen, thereby producing a pink-coloured derivative

Using mist nets, we captured barn swallows at the peak of reproduction just before and during the laying period for mostindividuals (Møller, 1994; Møller et al., 2005b). Captures wereconducted in 2007–2008, between June 1st and 15th, during daytime(ca. 10:00 h. to 15:00 h.).
We measured several phenotypic characters in adult males andfemales (including beak length, wing length, tail length, tarsus length,
keel length, and body mass) with a ruler, a digital caliper, or a Pesola
AG (Baar, Switzerland) spring balance, as described in Møller (1994).
We also carefully checked all females for a fully developed broodpatch. A blood sample was collected by puncturing the ulnar vein
immediately after capture and collecting the blood in heparinizedcapillary tubes. Blood samples were then centrifuged for 10 min at
16,000 g and stored at − 80 °C to serve for oxidative stress measurement. Background radiation level was measured using a handheld dosimeter (Inspector, SE International, Summertown, Tennessee,
USA) and later cross-validated with ofﬁcial measurements (Shestopalov, 1996). Based on our ﬁeld measurements, we classiﬁed

2.2. Plasma antioxidant capacity (TAC)
The plasma antioxidant barrier includes both exogenous (e.g.
ascorbate, tocopherols, carotenoids) and endogenous (e.g. uric acid,enzymes) compounds. The total antioxidant capacity (TAC) of plasma
was measured using the OXY-Adsorbent test (Diacron, Grosseto, Italy)
that uses a colorimetric determination to quantify the ability of theplasma antioxidant barrier to cope with the oxidant action of
hypochlorous acid (HClO). Similarly to the Trolox-equivalent antioxidant capacity or TEAC (Cohen and McGraw, 2009), TAC reﬂects levelsof circulating antioxidants including vitamin C and E, carotenoids anduric acid, but does not account for levels of enzymatic antioxidants.
The plasma (10 µL) was diluted 1:100 with distilled water. A 10 µL
aliquot of this solution was added to 1 mL of a known-titre HClO
solution in plastic photometer cuvettes. The solution was gently
mixed and incubated at 37 °C for 10 min. At the end of the incubation
time, 10 µL of an alkyl-substituted aromatic amine (N,N-diethyl-pphenylenediamine) solubilised in a chromogenic mixture wereadded. Such amine is oxidized by the residual HClO and converted
into a pink-coloured derivative. Colour intensity is directly proportional to the HClO excess and inversely related to the antioxidant
capacity of tested plasma. The absorbance of the coloured solution
was measured at 546 nm using a dedicated photometer (Free,
Diacron). One standard sample of known TAC was used as reference.
The absorbance of a blank sample (10 µL of distilled water) was
subtracted from that of the tested samples. Antioxidant capacity perunit volume of plasma was expressed in µM of HClO neutralised by the
antioxidants contained in the sample. Measurement repeatability wasestimated on 15 samples (12 samples run in duplicate and threesamples run in triplicate) by means of a one-way ANOVA withindividual as the main effect. This analysis showed that TAC measures
were highly consistent within individual (F14,32 = 28.79, P b 0.001,
adjusted R2 = 0.926). Concentration for samples assayed more thanonce were calculated as the mean of the measures.

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207

Fig. 1. Location of the study sites and levels of radioactive contamination in Ukraine and Belarus. 1: Farm 1 (53° 04′ N, 31° 18′ E), 2: Farm 2 (52° 47′ N, 31° 27′ E), 3: Farm 3 (52° 45′ N,
31° 31′ E), 4: Farm 4 (52 34′ N, 31° 32′ E), 5: Farm 5 (52° 32′ N, 31° 22′ E), 6: Rudnia (51° 17′ N, 29° 47′ E), 7: Vesniane (51° 18′ N, 29° 37′ E), 8: Dytiaku (51° 07′ N, 30° 09′ E), 9: Pisky(51° 05′ N, 30° 03′ E), and 10: Voronkov (50° 12′ N, 30° 49′ E). Modiﬁed from: European Union, 1998. Atlas of Caesium Deposition on Europe after the Chernobyl Accident. EU
Publication EUR 16733, Bruxelles.

whose colour intensity is directly proportional to the concentration ofROMs. Absorbance was measured at 505 nm using a dedicatedphotometer (Free, Diacron). One standard sample and one blanksample (i.e. 10 µL of distilled water) were processed and used as
reference for the tested samples.
The results of d-ROMs test are expressed as mM of H2O2
equivalents. Measurement repeatability as estimated by means of aone-way ANOVA with individual as the main effect on showed that
ROMs values were repeatable within individual (F 8,9 = 2.87,
P = 0.069, adjusted R2 = 0.468). Concentration values for samples
assayed twice were calculated as the mean of the two measures.
The ratio of ROMs concentration to the TAC was used as an index ofoxidative stress (Costantini and Dell'Omo, 2006; Costantini et al.,2006, 2007). Oxidative stress was calculated as ROMs/TAC × 1000,
according to Costantini et al. (2006). Higher values indicate higher
oxidative stress.
2.4. Statistical analyses
We mainly relied on linear mixed model analyses of variance
where sex and radiation level (as a three-level factor) were included
as ﬁxed effects. Collection site was included in all analyses as random

factor. Year and individual identity were also entered as random
factors in the analyses of TAC to account for differences between years
and for non-independence of nine individuals that were sampled inboth years. Parameters were estimated using restricted maximum
likelihood (REML) and degrees of freedom were computed using
Satterthwaite's approximation. Data on ROMs concentration andoxidative stress were log-transformed before the analyses to attainnormality and homoscedasticity. Statistical tests were performed
using SPSS 13.
3. Results
Data on TAC were obtained for a total of 247 individuals from sites
with low contamination levels, 45 individuals from sites withintermediate contamination levels, and 30 individuals exposed tohigh level of contamination (see Fig. 2). These data were normally
distributed and homoscedastic, and were analyzed in linear mixed
models in relation to contamination level (3-levels factor) and sex as
ﬁxed effects, with year and site as random effects. Individual identity
was also included as a random factor to account for non-independence of TAC data obtained from 9 individuals that were sampled inboth years.

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Fig. 2. Mean (+ SE) (a) antioxidant capacity (TAC), (b) reactive oxygen metabolites(ROMs) concentration and (c) ratio of the two variables (ROMs/TAC × 1000) for eachlevel of radioactive contamination (low, intermediate and high-level) in male andfemale barn swallows. See Materials and methods section for further details. Sample
size is indicated above bars for each group. For variables where the effect of radiationlevel was signiﬁcant, different Greek letters indicate signiﬁcant differences between
levels of radioactive contamination, after Bonferroni correction for multiple testing.

We found no signiﬁcant differential effect of radiation level on TAC
of males or females (interaction between sex and contamination level:
F2,314 = 2.10, P = 0.124), nor a signiﬁcant main effect of radiation level(F2,7.77 = 0.03, P = 0.971). However, females had lower TAC levels thanmales, independently of radiation level (F1,314 = 5.13, P = 0.024;
Fig. 2).
Measures of ROMs (log-transformed to ensure normality andhomoscedasticity) were obtained for 84 individuals from sites withlow contamination levels, 19 individuals from sites with intermediatecontamination levels, and 13 individuals from highly contaminatedsites (see Fig. 2). Data were analysed using linear mixed model
analysis with contamination level and sex as ﬁxed effects and site as arandom factor. As all individuals were sampled only once in 2008,
neither individual identity nor year were included in this model. The
analysis disclosed no signiﬁcant sex by contamination level effect(F2,110 = 0.27, P = 0.765). However, contamination level had a highly
signiﬁcant effect on ROMs values (F2,110 = 7.43, P = 0.0009; Fig. 2).
Post-hoc comparisons with Bonferroni correction showed that ROMs
values were signiﬁcantly higher in individuals from sites with highcompared to low or intermediate radiation levels (P b 0.03 in bothcomparisons). No signiﬁcant difference was found between sites withlow compared to intermediate radiation levels (P = 0.061). The difference between mean (untransformed) ROMs values exposed to highradiation levels compared to those exposed to intermediate or low
radiation levels pooled corresponded to 1.2 standard deviations of thevalues recorded among the latter. In this analysis, sex had no signiﬁcant effect on ROMs concentrations (F1,110 = 0.39, P = 0.532).

In addition, we analysed the covariation between ROMs and TAC
values. The analysis was performed by means of a linear mixed model
where TAC was the response variable, sex and contamination level
were entered as ﬁxed effects, site as a random factor, and ROMs values
as a covariate, together with the two-way interaction terms among
ROMs, sex or radiation level. None of the interaction terms
signiﬁcantly predicted TAC (interaction between: sex and ROMs:
F1,101 = 0.62, P = 0.431; radiation level and ROMs: F2,101 = 0.73,
P = 0.484; sex and radiation level: F2,101 = 0.81, P = 0.448). In a
simpliﬁed model excluding these non-signiﬁcant interactions, ROMs
values positively predicted TAC (F1,106 = 5.76, P = 0.018, slope =13.65 (5.689)), while no signiﬁcant effect of sex or contaminationlevel was detected (P N 0.17 in both cases).
Finally, we analysed estimates of oxidative stress (see Materials
and methods) obtained in 2008 on a sample of 81 individuals fromsites with low contamination levels, 17 individuals from sites withintermediate contamination levels, and 13 individuals from highly
contaminated sites. The individuals included in this analysis were
those included in TAC and ROMs analyses for which both measures
could be obtained. Measures of oxidative stress (log-transformed)
were not signiﬁcantly predicted by the sex by radiation levelinteraction (F2,102 = 0.63, P = 0.533). Oxidative stress was maximalin heavily contaminated areas and showed a signiﬁcant variation in
relation to radiation level (F2,5.22 = 8.92, P = 0.021; Fig. 2). Post-hoc
tests with Bonferroni correction disclosed a signiﬁcant difference only
between birds from sites with heavy compared to intermediatecontamination levels (P = 0.025), whereas the difference betweensites with low contamination and those with either intermediate orhigh contamination level was non-signiﬁcant (P N 0.06 in both comparisons). In this analysis, sex did not signiﬁcantly predict oxidative
stress (F1,102 = 1.38, P = 0.243).
Morphological traits (including length of bill, keel, tarsus, wing
and outermost rectrices, and body mass) of the individuals included in
the analyses of TAC did not vary among radiation levels (F values
always associated to P N 0.10; details not shown) at analyses ofvariance with year, site and individual as random factors, and sex as aﬁxed effect. In addition, body mass did not vary according to radiationlevel while controlling for the effect of body size as indexed by tarsus
or keel length. Thus, mean phenotype of the individuals did not vary
among radiation levels. Moreover, presence of a brood patch infemales (indicating breeding status) did not differ among radiationlevels (details not shown). Similar results were obtained when theanalyses of these features were restricted to individuals for which we
had information on ROMs or oxidative stress. Thus, there was no
indication that the observed signiﬁcant variation in ROMs or oxidative
stress in relation to radiation level could be caused by other sourcesof phenotypic variation, at least as assessed by the traits we could
measure under the constraints imposed by working in sites with highlevels of radioactive contamination.
4. Discussion
In this study, we analysed total non-enzymatic antioxidant capacity(TAC), concentration of reactive oxygen metabolites (ROMs) andoxidative stress, as indexed by the ratio of these two variables, in barnswallows from several sites in the Chernobyl region, with variable levelsof radioactive contamination. We found that radiation level positively
predicted ROMs values, while no signiﬁcant covariation was found with
TAC. Oxidative stress was also increased in barn swallows exposed tohigh radioactive contamination.
Contrary to our hypothesis, plasma TAC did not vary according toradiation level. This ﬁnding is in contrast with previous studies ofantioxidant defences of barn swallows and other bird species
inhabiting the radioactively contaminated area around Chernobyl(Møller et al., 2005b, 2008; Møller and Mousseau, 2006, 2007a).
However, previous conclusions about the antioxidant status of

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Chernobyl barn swallows were largely based on carotenoids and otherdietary antioxidants, rather than on integrated measures of overall
antioxidant status. The role of carotenoids in antioxidant protection
has increasingly been questioned recently by experimental (e.g. Cohenet al., 2007), correlational (e.g. Isaksson et al., 2007) and metaanalytical studies (Costantini and Møller, 2008; see also Møller et al.,
2000; Hartley and Kennedy, 2004). Although it has been shown that
supplementation with beta-carotene reduced the deleterious consequences of radiation exposure in Chernobyl children (Ben-Amotz
et al., 1998), ﬁndings based on carotenoid levels and other dietary
antioxidants should thus be interpreted cautiously. Moreover, a highantioxidant capacity may indicate not only a better antioxidant status,
but also the up-regulation of antioxidant defences as a response to anincrease in oxidant production (Monaghan et al., 2009). Several
mechanisms can account for this up-regulation. Antioxidant defences
can be enhanced through mobilization of stored antioxidants orincreased production of endogenous antioxidants (Surai, 2002). Moreover, a rather permanent, beneﬁcial up-regulation of antioxidant
defences may also arise as a consequence of a chronic exposure to
low-dose radiation (a phenomenon termed ‘hormetic effect’; Rattan,
2008). Consequently, relatively high levels of antioxidants are notnecessarily associated with a better antioxidant state, as actual oxidative
stress depends on abundance of ROS with which these defences have tocope (Finkel and Holbrook, 2000; Halliwell and Gutteridge, 2007). In ourstudy, ROMs values positively predicted plasma TAC, thus suggestingthat barn swallows from highly contaminated areas might be actively
up-regulating their antioxidant defences, rather than having themcompromised by the oxidative challenge. The mechanism throughwhich this up-regulation might take place, and particularly whether it
involves mobilization of stored antioxidants or an increased intake ofnutrients, remains to be elucidated.
Performance impairment resulting from oxidative damage canonly be determined by relating TAC to the oxidative challenge in thesame tissue (Monaghan et al., 2009). In our study, both ROMs andoxidative stress values were signiﬁcantly affected by contaminationlevel, with higher oxidative damage and oxidative stress in barnswallows associated with high radiation level.
This effect can be due to two, non-mutually exclusive phenomena.
First, radiation can affect oxidative status by increasing the productionof ROS (Iliakis, 1991), thus disrupting the prevention of their toxic
effects by the antioxidant defences. Second, radiation can increaseoxidative stress because antioxidants are allocated to repair the DNA
damage arising as a consequence of exposure to radiation (Collins andHorváthová, 2001; Møller et al., 2005a). This latter explanation is
supported in our model species by direct and indirect evidence
concerning the effects of radioactive exposure on genetic integrity. In
barn swallows from the Chernobyl region microsatellite mutation
rates were shown to be higher by a factor two to ten compared tosamples from a Ukrainian and an Italian control population (Ellegren
et al., 1997), as well as in a range of other species (review in Møller
and Mousseau, 2006). Mutation level, as indexed by sperm abnormality, negatively correlated with circulating and stored levels ofantioxidants in barn swallows from Chernobyl, suggesting thatvariation in mutation rate might be dependent on individual differences in antioxidants availability (Møller et al., 2005a). Furthermore, we recently documented that barn swallows from the
Chernobyl region have an increased level of DNA damage, as
estimated by the single cell gel electrophoresis test (or cometassay), compared to birds from relatively uncontaminated control
sites (Bonisoli-Alquati, submitted for publication). However, our test
procedure in that study did not allow us to discriminate between
different damage categories. Modiﬁcations of the protocol should thus
be implemented to recognize oxidized nitrogenous bases, thus
clarifying the mechanistic process linking genetic damage to depleted
antioxidant resources (Collins and Horváthová, 2001; BonisoliAlquati, submitted for publication).

209

Although both ROMs and oxidative stress values were maximal inbirds from highly-contaminated areas, the difference between lowand intermediate-level sites, although marginally non-signiﬁcant
after the Bonferroni correction, was in the opposite direction to apriori predictions. At present, we have no straightforward explanation
for this pattern. It is possible however, that variation in reproductive
behaviour or in age structure among populations exposed to different
radiation levels accounts for this counter-intuitive patterns.
We found that female barn swallows had lower TAC than males.
Barn swallows are known to provision large amounts of antioxidants
to their eggs (e.g. Saino et al., 2003). This active allocation, as indicatedby the larger concentrations of these compounds in eggs than inmaternal plasma (Saino et al., 2003), has to be traded against the useof these same compounds for protecting the tissues of adult females.
Such a trade-off has been suggested by a decline in carotenoids levelsof female barn swallows during laying (Ninni et al., 2004) and in yolk
concentration of some antioxidant compounds during the laying
sequence (Saino et al., 2003). Maternal antioxidant transfer to theeggs to buffer ROS generation during embryonic growth may thus
entail a cost for mothers in terms of a reduced capacity to deal withoxidative threats. Support for the interpretation that egg-laying might
account for a depletion of antioxidant defences in females comparedto males comes from an inter-speciﬁc survey that we conducted in the
Chernobyl region to investigate the relationship between speciﬁcabundance, life-history traits and radiation exposure (Møller andMousseau, 2007a). Relative egg mass was found to predict speciesspeciﬁc sensitivity to the deleterious effects of radiation, with species
laying relatively large eggs for a given body size declining morestrongly than others (Møller and Mousseau, 2007a).
Alternatively, this ﬁnding might be due to the sex-speciﬁc patternof dispersion in our study species. The same inter-speciﬁc survey
demonstrated that species with longer dispersal distance were more
susceptible to the deleterious consequences of radiation (Møller andMousseau, 2007a). In the barn swallow, females are the dispersing
sex, and might therefore experience larger consequences of radioactive exposure because of higher physical activity or more frequentcontact with novel antigens that would require immunostimulants
such as carotenoids and antioxidant vitamins to be allocated toimmune response rather than to antioxidant defences (Møller andMousseau, 2007a).
The effects of radiation we observed are likely to be relevant to
individual ﬁtness and to the fate of wild populations of barn swallows
in the Chernobyl region, owing to the plausible role of oxidative stressas a mediator of life-history trade-offs (Catoni et al., 2008; Monaghan
et al., 2009). Counter-strategies to defy oxidative stress are expected
to entail physiological costs for individuals (Monaghan et al., 2009).
Due to their generation by virtually any biological process, ROS havebeen suggested as likely mediators of life history trade-offs (Monaghan et al., 2009), underlying the so-called ‘cost of reproduction’(Alonso-Alvarez et al., 2004) and the deleterious consequences of
rapid growth (Metcalfe and Monaghan, 2001), as well as ensuringhonesty in sexual signalling (von Schantz et al., 1999). Therefore, anincrease in oxidative stress levels can entail negative consequences fora wide range of behavioural and life-history traits. Oxidative stress isalso known to participate in the senescence-related decline inphysiological performance (Harman, 1956; Beckman and Ames,
1998; Finkel and Holbrook, 2000; Sohal et al., 2002; Monaghan
et al., 2008), and to have a role in the onset of many pathologies(including atherosclerosis, autoimmune diseases and cancer; Halliwell and Gutteridge, 2007). Finally, correlative evidence from ourprevious studies in Chernobyl suggests that availability of antioxidants might be a determinant of reproductive success for barnswallows exposed to radioactive contamination (Møller et al., 2005b),
and species-speciﬁc requirements for antioxidants may be a contributing factor in driving population declines of some bird species in theregion (Møller and Mousseau, 2007a).

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Overall, our ﬁndings suggest that radioactive contamination in the
Chernobyl region might be damaging barn swallows through an effecton oxidative stress. Factors other than radioactive contamination areunlikely to have caused the observed patterns, as ecological conditions in the sampling sites were otherwise consistent and farming
practice has deteriorated in Ukraine and Belarus irrespective ofradiation level. However, we recently demonstrated that theabundance of invertebrates decreased with increasing radiation atforest sites around Chernobyl, in a study where we controlled forseveral ecological factors, including soil type, habitat and height ofvegetation (Møller and Mousseau, 2009). Although the taxa surveyed
in the study are not part of the barn swallow's diet, a reduction inavailable preys could contribute to explain variation in oxidative
status in the barn swallow if its main preys (namely Diptera andHemiptera; Møller, 1994) are similarly affected by radioactive
contamination. Future studies utilizing the Chernobyl area as a‘natural’ ﬁeld laboratory will relate oxidative stress levels to
behavioural and life-history traits, thus providing insight into themechanistic and functional processes linking oxidative stress to lifehistory plasticity and evolution, and clarifying the exact magnitude of
the worst nuclear accident in history.
Acknowledgments
The authors gratefully acknowledge the help of D. Costantini, G.
Milinevski, I. Chizhevsky, O. Bondarenko, A. Cavalleri, S. Ostermiller, J.
Kenney-Hunt, and the Chernobyl EcoCenter. The authors acknowledge
ﬁnancial support from the CNRS (France), The National Geographic
Society, the Samuel Freeman Charitable Trust, the NATO CLG program,
the Fulbright Program, and the School of the Environment and theCollege of Arts and Sciences of the University of South Carolina. The
manuscript greatly beneﬁted from the comments of three reviewers,
which we thankfully acknowledge here.
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