J Comp Physiol B (2008) 178:735–743
DOI 10.1007/s00360-008-0262-z

ORIGINAL PAPER

Antioxidants in eggs of great tits Parus major from Chernobyl
and hatching successAnders Pape Møller · Filis Karadas ·Timothy A. Mousseau

Received: 28 October 2007 / Revised: 17 March 2008 / Accepted: 19 March 2008 / Published online: 8 April 2008
© Springer-Verlag 2008

Abstract Antioxidants are powerful protectors against
the damaging eVects of free radicals that constitute theinevitable by-products of aerobic metabolism. Growing
embryos are particularly susceptible to the damaging eVectsof free radicals produced during rapid growth, and mothersof many species provide protection against such damage by
allocating antioxidants to their eggs. Birds living in radioactively contaminated areas use dietary antioxidants tocope with the damaging eVects of radiation, but females
also allocate dietary antioxidants to eggs, potentiallyenforcing a physiological trade-oV between self-maintenance and reproductive investment. Here we tested whether
female great tits Parus major breeding in radioactively contaminated study areas near Chernobyl allocated less dietary
antioxidants to eggs, and whether such reduced allocationof dietary antioxidants to eggs had Wtness consequences.
Concentrations of total yolk carotenoids and vitamins A
and E were depressed near Chernobyl compared to concentrations in a less contaminated Ukrainian study area and a
French control study area, and all antioxidants showed
dose-dependent relationships with all three dietary antioxi-

Communicated by G. Heldmaier.
A. P. Møller (&)
Laboratoire de Parasitologie Evolutive, CNRS UMR 7103,
Université Pierre et Marie Curie, Bât. A, 7ème étage,
7 quai St. Bernard, Case 237, 75252 Paris Cedex 05, France
e-mail: amoller@snv.jussieu.fr
F. Karadas
Department of Animal Science,
University of Yüzüncü Yil, Van, Turkey
T. A. Mousseau
Department of Biological Sciences,
University of South Carolina, Columbia, SC 29208, USA

dants decreasing with increasing level of radiation at nest
boxes. These eVects held even when controlling statisticallyfor potentially confounding habitat variables and covariation among antioxidants. Laying date was advanced andclutch size increased at nest boxes with high dose rates.
Hatching success increased with increasing concentrationof vitamin E, implying that hatching success decreased atboxes with high levels of radiation, eventually eliminatingand even reversing the higher potential reproductive output
associated with early reproduction and large clutch size.
These Wndings are consistent with the hypothesis that radioactive contamination reduced levels of dietary antioxidants
in yolks, with negative consequences for hatching successand reproductive success.
Keywords Antioxidants · Clutch size · Dose rate ·Hatching success · Laying date

Introduction
Aerobic metabolism produces energy at a cost of production of free radicals that are highly reactive biochemical
agents (e.g. Halliwell and Gutteridge 1999; LeZer 1993).
Free radicals are a natural outcome of aerobic metabolism,with mitochondrial metabolism producing free radicals that
if uncontrolled can damage DNA and other molecules (e.g.
Halliwell and Gutteridge 1999; LeZer 1993). Such damage
has to be repaired or it will have somatic consequences for
self-maintenance or germline consequences by inducing
deleterious mutations that can be transmitted to the next
generation. Anti-oxidants such as vitamins A and E, carotenoids and several others are either synthesized or ingested
as a means of reducing oxidative stress (e.g. Møller et al.
2000; Surai 2002). Females of many animals provision

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their oVspring with antioxidants as a means of reducing thenegative eVects of free radicals during embryonic and postnatal growth, causing antioxidants to be a unique suite ofmaternal eVects with potential trans-generational eVects on
oVspring performance (Mousseau and Fox 1998). Birds are(together with bats) unique among animals in having
extremely high metabolic rates (and therefore high rates of
production of free radicals), relatively high rates of adultsurvival for their body size and low rates of senescence(Holmes et al. 2001). In addition, birds are responsible forsome of the most remarkable bi-annual migrations, with therecord being held by Arctic terns Sterna paradisaea that
migrate up to 44,000 km per year and up to more than one
million kilometers in lifetime (the longevity record for thatspecies is a remarkable 32 years, Berthold 2001). The combination of these facts suggests that birds have evolved arange of physiological mechanisms that allow them to successfully cope with high levels of oxidative stress. In fact,
low production of free radicals per unit of oxygen consumption by mitochondria may explain these eVects (Barjaet al. 1994).
There has been increasing recent interest in ecologicaland evolutionary eVects of free radicals and antioxidants.
These recent studies include experiments designed tomanipulate the antioxidant status of mothers or oVspringwith the objective of elucidating eVects on performance or
Wtness (e.g. Saino et al. 2003; Biard et al. 2007; Costantini
et al. 2007). A second approach has been to manipulate theworkload of animals and subsequently investigate eVects
on free radical load and antioxidant status (e.g. AlonsoAlvarez et al. 2004). Yet another approach has been toinvestigate natural variation in free radical load and antioxidant status, under the assumption that natural variationmust have Wtness consequences if free radicals and antioxidants play signiWcant proximate roles in optimizing Wtness(e.g. Costantini et al. 2006).
Radioactively contaminated areas may constitute a previously under-exploited model system for studies of theeVects of antioxidants and free radicals and their eVects on
individual performance and Wtness. Both humans and birds
living in radioactively contaminated areas suVer physiological consequences in terms of reduced levels of dietary antioxidants because a higher load of free radicals depletes theamounts of circulating and stored antioxidants (Chaialo
et al. 1991; Bazhan 1998; Ben-Amotz et al. 1998; Ivaniota
et al. 1998; Neyfakh et al. 1998a, b; Lykholat and Chernaya
1999; Kumerova et al. 2000; Møller et al. 2005b). Such
reductions in levels of circulating and stored antioxidants
can have important consequences for reproductive decisions, including whether or not to reproduce (up to a quarter of females not reproducing in areas with high levels ofradiation), clutch size (fewer eggs being produced in areas
with high levels of radiation) and antioxidant investment in

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eggs (reduced amounts of dietary antioxidants allocated byfemale birds to eggs in areas with high levels of radiation,
Møller et al. 2005a). Møller et al. (2005b) analyzed antioxidant levels in barn swallows Hirundo rustica around Chernobyl in relation to local levels of background radiation,and found that circulating concentrations of dietary total
carotenoids and vitamins A and E in blood, stores of thesethree dietary antioxidants in liver and concentrations in egg
yolk were severely depressed compared to control areas.
These reduced concentrations of dietary antioxidants predicted levels of abnormal sperm that increased from lessthan 5% in areas with high levels of antioxidants to morethan 40% in areas with the lowest levels of antioxidants(Møller et al. 2005b). These Wndings are consistent with the
hypothesis that mutations as reXected by abnormal spermmorphology increase in the absence of antioxidants.
The objective of this study was to investigate how egg
quality in terms of antioxidants was related to backgroundradiation level in a study area in the highly contaminated
Red Forest, Ukraine, a weakly contaminated study area
near Izumrudnoe, Ukraine, and an uncontaminated control
study area in France. The second objective was to investigate the Wtness consequences of antioxidant levels in yolks.
This was done using great tits Parus major breeding in nest
boxes as a model system. Because availability of antioxidants may depend on habitat features, we described habitats
surrounding nest boxes in detail and included these habitatfeatures as predictor variables in our models. Previous studies of great tits in contaminated study areas in Ukraine haveshown that tits avoid breeding in the most contaminatedstudy areas, despite the fact that there is little evidence of
reduced reproductive success in these study areas (Møller
and Mousseau 2007a). A comparative study of interspeciWc
diVerences in the response of birds to radioactivity around
Chernobyl revealed that species with carotenoid-based
plumage coloration, long migration distance, long dispersal
distance, and heavy eggs (all traits closely associated withhigher levels of use of antioxidants) all independently predicted decrease in population density with increasing levelsof background radiation (Møller and Mousseau 2007b).
Therefore, these studies suggest that antioxidant levels play
an important role in the response of birds to radioactive
contamination.

Materials and methods
Study areas
S. Gaschak, APM and TAM investigated nest boxes in astudy area in the Red Forest, Ukraine, where radioactive
contamination reaches levels greater than 2,000 times thenatural background level, in a contaminated study area inJ Comp Physiol B (2008) 178:735–743

Izumrudnoe, Ukraine, with levels around 100 times normal
background radiation levels, and in a second control study
area in Foret d’Orient, France. In 2002–2003 we erected
232 nest boxes of a standard size (20 £ 20 £ 40 cm) with a
32 mm diameter hole in the Ukrainian study areas. All theboxes were placed at a distance of at least 50 m between
boxes, and they were located at a height of 1.5–2.0 m. Highlevels of spatial heterogeneity in radiation levels (Shestopalov 1996) provided an ideal setting for investigating therelationship between reproduction and radiation, making it
unlikely that results would be confounded by spatial autocorrelation in food abundance, habitat, or other features.
Thus, high heterogeneity in levels of radiation is unlikely to
exactly match high heterogeneity in food abundance, habitat, or other features. Møller and Mousseau (2007a) provide
a map showing this high degree of spatial heterogeneity inlevels of background radiation.
The habitat surrounding nest boxes was forest with amixture of deciduous and coniferous trees and shrubs, andthe oldest trees were less than 100 years old. We classiWed
habitats surrounding each box along four diVerent axes: (1)Habitat [(a) birch Betula sp., (b) birch with aspen Populus
sp. and alder Alnus sp., (c) pine Pinus sp.]. (2) Age ofstands [(a) young trees without natural holes, (b) older trees
with natural holes]. (3) Undergrowth [(a) absent, (b) sparse,(c) dense)]. (4) Habitat humidity [(a) dry, (b) humid, (c)wet with open water]. We analyzed one egg from each of
45 nests of great tits from 2003 to 2005 from the Red Forestand 26 nests from 2006 from Izumrudnoe.
A control study area in Foret d’Orient, France(48°17⬘N, 4°18⬘E, Biard 2004; Biard et al. 2006) (about
250 ha) contained 400 nest boxes evenly distributed
among homogenous deciduous old woodland composed
mainly of oak (Quercus spp.), hornbeam (Carpinus betulus) and beech (Fagus sylvatica). We analyzed one egg
from each of 42 nests of great tits from 2003 to 2004 fromthis study area.
Recording nest box contents
We visited nest boxes during April–May, 2003–2006 tocheck for occupancy, identifying species from adults, eggs
and nests present. We estimated laying date from the presence of clutches that had not been completed, assuming that
one egg was laid per day (Cramp and Perrins 1988–1993).
Likewise, we used the age of nestlings to estimate date oflaying of the Wrst egg, assuming an incubation period of
13.9 days and a nestling period of 18.9 days (Cramp andPerrins 1988–1993).
Clutch size was estimated as the number of eggs in completed clutches, and brood size as the number of young in
the nest after hatching, but before Xedging. Hatching success was deWned as brood size at hatching divided by clutch

737

size, and Xedging success as brood size at Xedging divided
by brood size at hatching.
Recording background radiation levels at nest boxes
As an estimate of background radiation levels we relied ona dataset of air-gamma-survey of the Chernobyl Exclusion
Zone, including geo-positioned data of 137Cs and 90Sr deposition in a grid of 100 £ 100 m as of 1 January 1992. Using
corresponding software (MapInfo Prof.) we sampled fromthe database all the data that were within a radius of 500 m
from each nest box. The choice of a radius of 500 m wasbased on the assumption that most birds remained within anarea with a radius of 500 m during breeding as suggestedby the size of the home range of tits during breeding (Perrins 1979). Radioactive isotopes decay at a characteristic
rate, implying that the radiation data from 1992 should behigher than, but positively correlated with the data from2006. We cross-validated these data from 1992 using ourown Weld measurements of background radiation at the
ground level using a hand-held Inspector (Model: Inspector, SE International, Inc., Summertown, TN, USA) dosimeter at a sample of 28 nest boxes during May–June 2006.
As expected, there was a strong positive relationshipbetween these two series of measurements [F = 210.99,
d.f. = 1,26, r2 = 0.89, P < 0.0001, slope (SE) = 1.15 (0.08)](Møller and Mousseau 2007a).
Antioxidant analyses
We have shown previously that if the concentrations of
total carotenoids and vitamins A and E in yolks of one egg
are at a speciWc level, they are at a similar level in subsequent eggs produced by the same female (Biard 2004). All
the antioxidant analyses were done by FK blindly withrespect to the origin of nests and hence the level of background radiation. Eggs were separated into egg white andyolk, and the egg white was used for other analyses, while
the yolk was used for androgen and antioxidant analyses.
Entire eggs were frozen as soon as possible after collection, and then maintained at ¡20°C until analysis in 2006.
All the samples from the same area were stored for thesame period of time, eliminating the possibility that storage
under oxygen could have produced the diVerences reported
here. Antioxidants were extracted from 0.1 to 0.2 g of yolk.
Samples were homogenized with 0.7 ml NaCl (5%) and
1 ml ethanol, after which we added 2 ml hexane followed
by further homogenization, centrifugation and collection of
the hexane phase (extraction repeated twice). Hexane
extracts were pooled and evaporated at 60–65°C under
nitrogen Xow, and the residue was dissolved in dichloromethane and methanol (v:v). Carotenoid and vitamins A andE concentrations were determined following previously

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published procedures (Surai 2000; Hõrak et al. 2002).
Total carotenoid concentration was determined by HPLC
with a Spherisorb type S5NH2 normal-phase column
25 cm £ 4.6 mm (Phase separation, Clwyd, UK) with amobile phase of methanol-distilled water (97:3), at a Xow
rate of 1.5 ml/min. Lutein was used for calibration (Sigma,
Poole, UK). Concentrations of vitamins A and E were
determined by injection of samples onto a Spherisorb type
ODS2 3- C18 reverse-phase column, 15 cm £ 4.6 mm(Phase Separation, Clwyd, UK) with a mobile phase of
methanol/distilled water (97:3), at a Xow rate of 1.05 ml/
min using Xuorescence detection by excitation and emission wavelength of 295 and 330 nm, respectively, for vitamin E and 330 and 480 nm for vitamin A. Peaks of retinol,-, - and -tocopherol were identiWed by comparison withthe retention time of standards (Sigma, Poole, UK). All thesampled yolks were analyzed for total carotenoids, vitamins A and E concentrations. Vitamin E was calculated as
the summed concentrations of -, - and -tocopherol.
Concentrations rather than quantities of antioxidants wereused as the variable of interest in statistical analysis
because concentration is the main factor determining physiological action of antioxidants at the level of developing
tissues that are subject to damage caused by free radicals(Surai 2002). The inter-assay CVs for retinol and alphatocopherol determinations were 3.9 and 4.4% respectively(Surai 2000).
Statistical analyses
We used the French control study area only in an analysis
of variation in antioxidant levels among study areas, but
excluded this study area from the analyses of eVects of radiation and Wtness consequences because radiation level
never reached more than the common background radiationlevel in the French study area.
Hatching success and Xedging success were square-root
arcsine-transformed, while dose rate was log-transformed
to meet requirements for normally distributed data.

We modeled antioxidant levels in relation to log-transformed dose rate of boxes, using Generalized Linear Models. These models included study area and year as factors.
We modeled laying date, clutch size, hatching successand Xedging success by using study area, year, dose rate,
antioxidant levels and four habitat variables (main tree species, age of stands, undergrowth, habitat humidity) as predictor variables. Habitat was used as a factor, while thethree other habitat variables were used as continuous variables. In addition, because clutch size may depend on laying date, we included laying date and its interaction withdose rate in the analyses. For hatching and Xedging success
we included laying date, clutch size, year and study area as
additional variables.
Data were missing for some variables due to complete
nest failure, causing sample sizes to diVer among tests.
Variables reported are means (SE).

Results
Antioxidant levels in yolks diVered signiWcantly among thethree study areas, with reduced levels in the Red Forest andto some extent in Izumrudnoe compared to the uncontaminated study area in France (Table 1). While total carotenoid
levels in the Red Forest were 62% of the level in France,
and the level of vitamin A was 59%, the level of vitamin E
was only 11% (Table 1). These patterns could potentiallybe due to diVerences among study areas caused by many
diVerent factors. Therefore, we investigated patterns within
the two Ukrainian study areas by including study area as afactor.
Antioxidant levels in yolks decreased with increasing
dose rate at nest boxes for all three antioxidants in analysesof data from the Red Forest and Izumrudnoe (Fig. 1). ThiseVect was independent of potentially confounding variables
such as year, habitat and study area (Table 2). Likewise,
there was a similar negative relationship within the data set
from the Red Forest (F > 4.43, d.f. = 1,43, r2 > 0.08,

Table 1 Mean (SE) concentration (g/g) of antioxidants in yolks of great tits from Red Forest, Izumrudnoe (Ukrainian control study area) andFrance (external control study area)
Variable

Red Forest

Total carotenoids (g/g)

15.49 (1.07)aa

Izumrudnoe

FranceRelative concentration
in Red Forest

25.76 (2.33)b

25.93 (1.90)b

0.60

2.88 (0.18)c

0.59
0.11

b

Vitamin A (g/g)

1.69 (0.13)

2.12 (0.19)

Total vitamin E (g/g)

15.49 (1.07)a

25.76 (2.33)b

137.08 (8.58)c

N

45

26

42

The relative concentration in Red Forest was the mean concentration in Red Forest relative to France. DiVerences among the three study areas weresigniWcant (total carotenoids: F = 14.58, d.f. = 2,110, r2 = 0.21, P < 0.0001; vitamin A: F = 13.05, d.f. = 2,110, r2 = 0.20, P < 0.0001; vitamin E:
F = 33.42, d.f. = 2,110, r2 = 0.38, P < 0.0001). Means with diVerent superscripts diVered signiWcantly (P < 0.05) from each other in ScheVe F tests

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739
Table 2 Antioxidant levels in yolks of great tits in relation to year,
dose rate, habitat features and study area
Variable

Sum ofsquares

d.f.

F

P

Slope (SE)Vitamin E

0.28

1

7.51

0.00780.31 (0.11)

Dose rate

0.22

1

5.73

0.019

¡0.09 (0.04)

Error

2.56

68

Dose rate

0.25

1

5.49

0.022

¡0.08 (0.03)

Error

3.17

69

Vitamin A

0.30

1

8.56

0.00470.31 (0.11)

Total carotenoids

0.17

1

4.96

0.029

0.25 (0.11)

Dose rate

0.46

1

13.43

0.0005¡0.12 (0.03)

Error

2.31

67

Total carotenoids

Vitamin A

Vitamin E

Year, habitat and study area never entered as predictors. Antioxidant
levels and dose rates were log10-transformed before analysis. The relationships between antioxidant levels and dose rates are shown in Fig. 1.
The reported models had the statistics F = 15.32, d.f. = 2,68, r2 = 0.31,
P < 0.0001, F = 5.49, d.f. = 1,69, r2 = 0.07, P = 0.022 and F = 19.03,
d.f. = 3,67, r2 = 0.46, P < 0.0001

Fig. 1 Concentration of antioxidants (g/g) in yolks of great tits inrelation to dose rates for nest boxes (mR/h). a Total carotenoids, b vitamin A and c vitamin E

P < 0.045), and analyses of the data using Kendall rankorder correlations provided a similar conclusion (Kendall < ¡0.22, P < 0.037).
Laying date diVered signiWcantly between the two study
areas [F = 17.31, d.f. = 1,117, P < 0.0001; Red Forest: 3
May (SE = 1), N = 82; Izumrudnoe: 14 May (SE = 3),
N = 37]. Laying date advanced with dose rate, independentof year, habitat and study area (Table 3; Fig. 2a). Clutch
size diVered signiWcantly between the two study areas[F = 10.56, d.f. = 1,477, P = 0.0014; Red Forest: 9.08
(SE = 0.31), N = 104; Izumrudnoe: 7.36 (SE = 0.38),
N = 104]. Clutch size increased with dose rate at nest
boxes, independent of date of egg laying, year, habitat andstudy area (Table 3; Fig. 2b). Brood size at hatching
diVered marginally between the two study areas [F = 3.86,
d.f. = 1,116, P = 0.052; Red Forest: 6.96 (SE = 0.49),
N = 75; Izumrudnoe: 5.49 (SE = 0.51), N = 43]. Brood size
at Xedging did not diVer signiWcantly between the twostudy areas [F = 0.57, d.f. = 1,104, P = 0.45; Red Forest:
4.88 (SE = 0.55), N = 66; Izumrudnoe: 5.50 (SE = 0.54),
N = 40]. Hatching success increased with increasing concentration of vitamin E in yolks, independent of date of egg
laying, year, habitat and study area (Fig. 3), while there wasno signiWcant eVect for Xedging success, independent ofdate of egg laying, year, habitat and study area (partial
eVect of dose rate: F = 0.01, d.f. = 1,71, P = 0.92). The positive relationship between hatching success and concentration of vitamin E was conWrmed in a non-parametric

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J Comp Physiol B (2008) 178:735–743

Table 3 Laying date and clutch size in relation to concentration ofantioxidants in yolks, dose rate, habitat features, study area and year
Variable

Sum ofsquares

d.f.

F

P

Clutch size5928.32

Dose rate

344.6510834.05

116

256.19Slope (SE)

1

63.47

<0.0001¡2.86 (0.36)

1

3.69

0.057

¡3.03 (1.58)

1

63.47

<0.0001¡0.12 (0.02)

13.78

0.0003

1.17 (0.31)

Laying date

Error
Clutch size
Laying date
Dose rate

55.62

1

Error

468.19116

Study area never entered as a predictor. The reported models had thestatistics F = 59.48, d.f. = 2,116, r2 = 0.51, P < 0.0001 and F = 69.39,
d.f. = 2,116, r2 = 0.54, P < 0.0001

Kendall rank-order correlation analysis (Kendall  = 0.21,
P = 0.021). Restricting these analyses to the data from the
Red Forest only provided a similar conclusion [F = 6.09,
d.f. = 1,35, r2 = 0.15, P = 0.019, slope (SE) = 0.687(0.279); Kendall rank-order correlation analysis: Kendall
 = 0.24, P = 0.040].

Discussion
The main Wndings of this study of antioxidants in yolks of aresident passerine bird were (1) antioxidant concentration
diVered among populations, with severely depressed levelsin the Red Forest near Chernobyl, (2) these eVects showed
dose-dependent negative relationships, and (3) fecundity
was positively related to dose rate, while hatching success
was negatively related to concentration of vitamin E inyolks. We will brieXy discuss these Wndings and theirimplications.
Antioxidant levels have been shown to decrease in
response to exposure to radiation in both humans and birds(Chaialo et al. 1991; Bazhan 1998; Ben-Amotz et al. 1998;
Ivaniota et al. 1998; Neyfakh et al. 1998a, b; Lykholat andChernaya 1999; Kumerova et al. 2000; Møller et al.
2005b). While some of these studies show an eVect of radiation exposure on levels of antioxidants (Chaialo et al.
1991; Bazhan 1998; Ben-Amotz et al. 1998; Ivaniota et al.
1998; Lykholat and Chernaya 1999; Kumerova et al. 2000;
Møller et al. 2005b), others have shown that mothers that
had been exposed to radiation produce oVspring with antioxidant deWciencies (Neyfakh et al. 1998a, b; Møller et al.
2005b), apparently because of eVects of maternal exposureon the ability of their oVspring to absorb, metabolize or use
antioxidants (Neyfakh et al. 1998a, b). Furthermore, betacarotene supplementation to humans exposed to radiation
has been demonstrated to reduce the negative eVects of123

Fig. 2 a Laying date (1 = 1 May) and b clutch size of great tits in relation to dose rate (mR/h)radiation (Ben-Amotz et al. 1998). Here we have shownlarge diVerences among populations in antioxidant levels ofyolks of a resident bird species, the great tit, with the lowest
levels occurring in the most contaminated parts of the Red
Forest. Furthermore, we have shown dose-dependent, negative relationships between levels of background radiationand concentrations in yolks of total carotenoids and vitamins A and E, respectively. This eVect of radiation on dietary antioxidants implies that radiation dose will also
increase free radical production in developing oVspring,further exacerbating the eVects of already low levels ofantioxidants of maternal origin deposited in eggs. These
Wndings have similarities with those reported for humans
by Neyfakh et al. (1998a), who showed that vitamins A andE were reduced in children in direct proportion to the exposure of their mothers to radiation in the surroundings ofChernobyl, while their own exposure was not important.

J Comp Physiol B (2008) 178:735–743

Fig. 3 Hatching success (%) in relation to dose rates for nest boxes(mR/h) of great tits. Habitat features, year and study area never enteredas signiWcant predictors

We can exclude the possibility that storage under oxygen ina freezer have changed the relationships because of oxidation of samples, because we found no evidence of signiWcant eVects of year (and hence duration of storage) on
levels of antioxidants.
We analyzed relationships between Wtness components(laying date and clutch size) and dose rate and antioxidants.
Clutch size increased with background radiation level,
while the relationship for laying date was less pronounced.
In contrast, there was no signiWcant eVect of antioxidant
concentrations in yolks on clutch size or laying date,despite the fact that experimental evidence from gulls suggests that clutch size may be limited by carotenoid availability (Blount et al. 2003). We interpret these relationships
as indicative of females being exposed to high dose rates
increasing their terminal investment in reproduction (Clutton-Brock 1984; Pärt et al. 1992) because of their reduced
residual reproductive value. Møller et al. (2005a) showedthat adult survival rate for the barn swallow is severely
depressed in Chernobyl compared to a control study area.
When we investigated hatching and Xedging success inrelation to dose rate and antioxidant levels, the relationships for dose rate were weak and non-signiWcant, while
hatching success increased with increasing concentration ofvitamin E. A possible interpretation of the latter result isthat embryo mortality increased as the concentration ofvitamin E was reduced, producing the positive relationshipbetween hatching success and vitamin E. Alternatively, theeVects of dose rate may cause hatching failure due to elevated mutation rates. However, this explanation is unlikely
because vitamin E rather than dose rate predicted hatching
success. We can exclude the possibility that reduced insect741

abundance in contaminated areas was the underlying mechanism, because we should then not have found increased
clutch size and we should have found increased nestlingmortality in more contaminated areas. That was not thecase (see also Møller and Mousseau 2007a).
Alternative interpretations must be considered because
this is a correlational study that does not allow inferences
about causation. Hence both directions of causality, as wellas indirect eVects through third variables, must be entertained. Here we have argued that elevated levels of background radiation reduced concentrations of antioxidants ineggs by aVecting antioxidant levels in the females that produced the eggs. However, it is also possible that reduced
levels of antioxidants are a cause of negative eVects ofradioactive contamination of birds by increasing mutation
rates. We suggest tentatively that the eVect of vitamin E
may outweigh the eVect of dose rate on hatching successbecause it lowers the mutation rate. We tested for eVects ofhabitat features, study area and year on antioxidant levels inyolks, but found no evidence consistent with the explanation that antioxidant levels in yolks could be accounted forby the eVects of habitat features, study area or year. We
cannot think of any alternative interpretation through third
variables that can have produced the negative relationships
between antioxidant levels and radiation, although this possibility that the negative relationship between antioxidant
levels and radiation was produced by a third variable cannot be excluded. We found strong population diVerences inantioxidant levels among great tits from the Red Forest, aless contaminated study area in Izumrudnoe and a French
control study area, with the less contaminated Ukrainianstudy area having intermediate levels of antioxidants. The
levels of antioxidants recorded for the Chernobyl population are well below what is commonly reported for any bird
species (review in Biard 2004). We have previously
reported a similarly depressed level of total carotenoids andvitamins A and E in the migratory barn swallow (Møller
et al. 2005b), suggesting that populations of birds from the
Chernobyl Exclusion Zone have peculiar antioxidant statusthat distinguish them from other populations. This diVerence may be due to radioactive contamination. The Wndings
reported here occurred despite extensive dispersal in greattits (Paradis et al. 1998), potentially causing Chernobyl
populations to be sinks with a net immigration to maintainbreeding populations (Møller et al. 2006). Several bird species including the great tit with reduced breeding population density due to radioactivity in the most contaminatedareas near Chernobyl are more often long-distance dispersers than expected by chance, suggesting that long-distancedispersal may exacerbate the negative eVects of radiation
on population density, and that immigration cannot compensate for the negative eVects of radiation on local population density (Møller and Mousseau 2007b).

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Although we were able to analyze the eVects of radiation
only during breeding, fecundity, breeding success and yolk
contents, we can speculate about the potential Wtness consequences of these documented eVects. First, there might beconsequences for parents. Elevated clutch size and indications of early start of reproduction in highly contaminatedareas are consistent with terminal investment (CluttonBrock 1984; Pärt et al. 1992), and such females may suVer
reduced survival and future reproduction. Second, there
might be consequences for oVspring of depressed pre-natal
and neo-natal levels of antioxidants. Previous studies ofgreat tits and chickens suggest that reduced levels of carotenoids in mothers and hence in yolks can have long-lastingeVects on the phenotype of oVspring (Hõrak et al. 2000;
Koutsos et al. 2003). A similar conclusion has been reached forhumans, where maternal exposure to radiation reduced levels of vitamins A and E in children (Neyfakh et al. 1998a).
In conclusion, maternal antioxidants were severely
depressed in study areas and nest boxes with elevated levelsof radiation, in a dose-dependent manner, and clutch size
increased with dose rate, while hatching success decreased
with concentration of vitamin E. These Wndings are consistent with the hypothesis that restrictions of antioxidants
cause mothers to invest terminally in reproduction, and that
reduced levels of antioxidants reduces the viability ofembryos as reXected by reduced hatching success.
Acknowledgments We are grateful to S. Gaschak, G. Milinevski, A.
M. Peklo, E. Pysanets, I. Chizhevsky, O. Bondarenko, and M. Bondarkov for logistic help during our visits to Ukraine. C. Biard kindly provided information on yolk composition from France. We received
funding from the CNRS (France), the University of South Carolina
School of the Environment, Bill Murray and the Samuel Freeman
Charitable Trust, the Civilian Research Development Foundation, theNational Science Foundation and the National Geographic Society to
conduct this research.

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