OPENSUBJECT AREAS:ECOLOGICALEPIDEMIOLOGYECOPHYSIOLOGY

Abundance and genetic damage of barnswallows from Fukushima
A. Bonisoli-Alquati1, K. Koyama2, D. J. Tedeschi3, W. Kitamura4, H. Sukuzi5, S. Ostermiller1, E. Arai6,
A. P. Møller7 & T. A. Mousseau1
1

Received
19 May 2014
Accepted
5 February 2015
Published
2 April 2015

Correspondence andrequests for materials
should be addressed to
A.B.-A. (alquati@
mailbox.sc.edu) or T.
A.M. (mousseau@sc.
edu)

Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA, 2Japan Bird Research Association,
Fuchu, Tokyo, Japan, 3Department of Physics and Astronomy, University of South Carolina, Columbia, SC 29208, USA, 4Faculty ofEnvironmental Studies, Tokyo City University, Yokohama City, Japan, 5Value Frontier Co., Ltd., Minato, Tokyo, Japan, 6Division of
Ecology and Evolutionary Biology, Graduate School of Life Sciences, Tohoku University, Sendai, Japan, 7Laboratoire d’Ecologie,
Systématique et Evolution, CNRS UMR 8079, Université Paris-Sud, Bâtiment 362, F-91405 Orsay Cedex, France.

A number of studies have assessed or modeled the distribution of the radionuclides released by the accident
at the Fukushima-Daiichi Nuclear Power Plant (FDNPP). Few studies however have investigated its
consequences for the local biota. We tested whether exposure of barn swallow (Hirundo rustica) nestlings to
low dose ionizing radiation increased genetic damage to their peripheral erythrocytes. We estimatedexternal radiation exposure by using thermoluminescent dosimeters, and by measuring radioactivity of thenest material. We then assessed DNA damage by means of the neutral comet assay. In addition, we
conducted standard point-count censuses of barn swallows across environmental radiation levels, andestimated their abundance and local age ratio. Radioactivity of nest samples was in the range 479–
143,349 Bq kg21, while external exposure varied between 0.15 and 4.9 mGy. Exposure to radioactive
contamination did not correlate with higher genetic damage in nestlings. However, at higher levels ofradioactive contamination the number of barn swallows declined and the fraction of juveniles decreased,
indicating lower survival and lower reproduction and/or fledging rate. Thus, genetic damage to nestlings
does not explain the decline of barn swallows in contaminated areas, and a proximate mechanism for thedemographic effects documented here remains to be clarified.

O

n March 11 2011, a tsunami caused by the Great East Japan Earthquake seriously damaged the electricaland the cooling systems of the Fukushima-Daiichi Nuclear Power Plant (FDNPP), causing hydrogen
explosions at the Unit 1, 2 and 3 reactors. These explosions released large amounts of high volatility
fission products, including 129mTe, 131I, 133Xe, 134Cs, 136Cs, and 137Cs1–3 . Although the estimates of the release varyconsiderably4–10, the accident is universally regarded as the second largest release of radionuclides in history after
the Chernobyl accident, with estimates of total radioactivity released in the range of up to hundreds of PBq10. Such
massive discharge of radionuclides raises concern about its possible consequences for environmental and humanhealth11, particularly given the persistence of 137Cs in the environment.
Predictably, large efforts have since been devoted to model the atmospheric release, the deposition of radionuclides and their redistribution2,12. Several other studies have assessed the concentration of the radionuclides inthe biological tissues of animals and plants (mammals:13,14; fish:7,15; birds:16; plants17–19).
Few studies so far have examined the potential biological consequences of exposure to radionuclides releasedby the accident. A study on the pale blue grass butterfly (Zizeeria maha) that coupled field sampling and rearing ofindividuals under common garden conditions showed an increase in aberrations in the coloration and patterns ofwings20,21. A study on earthworms also demonstrated that animals from sites where radiation level was as low as
2.8 mSv/h had higher DNA damage than animals from control sites22. A recent study of wild Japanese macaques(Macaca fuscata) found that individuals from Fukushima had lower white blood cell (WBCs) and red blood cellcounts (RBCs), lower hemoglobin concentration and lower hematocrit values than those sampled in the
Shimokita peninsula, at a distance of 400 km from the FDNPP23. Vitamin A levels of streaked shearwaters(Calonectris leucomelas) sampled in colonies exposed to contamination from the FDNPP were lower than in
animals from a colony that was not reached by the plume24.
Ecological studies conducted in the Chernobyl Exclusion Zone have indicated that radiation levels comparable
to those found around Fukushima can be associated with deleterious genetic, physiological and life-history
consequences for exposed wildlife25. Low-dose radiation in the Chernobyl region was associated with higher
DNA damage in adult barn swallows26, higher frequency of morphological abnormalities and tumors27,28, and a
SCIENTIFIC REPORTS | 5 : 9432 | DOI: 10.1038/srep09432

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reduction in brain size29. These and other physiological and genetic
consequences of radiation exposure in Chernobyl30 have been indicated as the likely cause underlying the higher mortality and the
populations declines of many bird species living in the Chernobyl
region, as inferred from point count censuses31 and age ratios frommist netting studies32.
In spite of differences between the two accidents in the quality andamount of contaminants scattered and the number of generations of
exposure, early studies suggest that similarities also exist in the response of natural populations to radioactive contamination. Pointcount censuses conducted around Fukushima in 2011 have foundthat bird population in radioactively contaminated areas havedeclined similarly as in Chernobyl33. Later surveys validated thisfinding and concluded that the contamination might have had aneven larger negative effect during 201234.
Here, we describe the results of a study on barn swallow nestlings
during May-June 2012 to investigate whether exposure to radiation isaffecting their genetic integrity prior to fledging. We estimatedexternal radiation exposure of nestlings by attaching thermoluminescent dosimeters (TLDs) to their nest, and by collecting a sample ofnest material whose activity concentration we measured using
gamma spectrometry in the lab.
We also describe the results of a survey of barn swallows that we
conducted in July 2011-2013 across gradients of radioactive contamination spanning almost two orders of magnitude. Part of this database (2011 and 2012) has been previously published in studies
relating the abundance of birds in the Fukushima region to the levelof radioactive contamination33,34. In addition to presenting an additional year of data, here we focus the analyses on the local abundanceof barn swallows. We also present an analysis of the age ratio of barnswallows, which can be readily determined from plumage characteristics, predicting that higher levels of radiation would lead to a lower
fraction of juveniles due to egg infertility and death of nestlings35.
With a few notable exceptions16,23,24, all studies conducted so far
have at most analyzed the concentrations of radioisotopes in thetissues of organisms, but neglected the assessment of markers of theirpotential biological effects. The results that we describe represent thefirst extensive investigation of the potential genotoxicity of measured
radiation exposure in any wild population of birds from the
Fukushima region.

Results
Radioactivity of nest samples and radiation exposure of nestlings.
The average exposure measured by the TLDs was 0.59 mGy(0.79 mGy SD; range: 0.15–4.9 mGy; N 5 43), corresponding to
an average dose rate of 0.90 m Gy h21 (1.24 m Gy h21 SD; range:
0.23–7.52 m Gy h21).
The activity concentrations measured in the nest samples were
10,730 Bq kg21 dry weight (d.w.) (18,276 SD; range 318–82,409 Bq
kg21d.w.; N 5 45) for 137Cs, and 8,656 Bq kg21 d.w. (14,433 SD;
range 128–60,940 Bq kg21d.w.; N 5 45) for 134Cs. When we combined the activities measured for each radionuclide in a single estimate, the total radioactivity was 19,386 Bq kg21 d.w. (32,681 SD; range
478–143,349 Bq kg21d.w.; N 5 45). Total radioactivity of the nest
material significantly positively predicted the radiation dose received
by the TLDs (t39 5 6.74, p , 0.0001, R2 5 0.54, N 5 40;
Supplementary Fig. 1). Environmental radiation levels significantly
positively correlated with the dose received by the TLDs (t42 5 4.88, p, 0.0001, R2 5 0.37, N5 43; Supplementary Fig. 1). Environmental
radiation levels also positively correlated with the specific activity of137
Cs (t44 5 2.22, P 5 0.032, R2 5 0.08, N 5 45), and of 134Cs (t44 5
2.37, P 5 0.022, R2 5 0.09, N 5 45).
Radiation exposure and genetic damage of nestlings. The average
DNA damage, as indexed by the percentage of DNA in the comet tail,was 10.04 (4.86 SD; range: 2.83–23.41). The total activity concenSCIENTIFIC REPORTS | 5 : 9432 | DOI: 10.1038/srep09432

tration of the nest material, obtained by combining the estimates for134
Cs and 137Cs, did not significantly predict genetic damage ofnestlings (F1,6.24 5 0.51, p 5 0.502, N 5 49, Fig. 1a), nor did thedose to the TLDs attached to the nest (F1,9.81 5 0.33, p 5 0.577, N 5
49, Fig. 1b). We tested if there was a difference in variance in DNA
damage at high radiation levels by splitting our dataset in two groups
using median radioactivity of the nest sample or median dose to the
TLDs as the cutting points. Variance in DNA damage did not differ
significantly between nestlings from more radioactive nests andthose from less radioactive nests (Levene’s test: F1,47 5 0.015, p 5
0.904). Similarly, variance in DNA damage did not differ significantly between nests where the TLDs received a higher dose andthose where the dose to the TLDs was lower (Levene’s test: F1,47 5
0.039, p 5 0.844).
In none of these analyses did body mass and estimated age of thenestlings significantly predict genetic damage of the nestlings (seeTable 1 in Electronic Supplementary Material). The effect of the nestof origin was never significant in predicting genetic damage of thenestlings (Table 1 in Electronic Supplementary Materials).
Abundance and age ratio of barn swallows. Radiation levels at thebreeding bird census points ranged from 0.18 to 38.11 m Sv/h [mean(SD) 5 7.16 m Sv/h (7.90), N 5 1100]. The abundance of barnswallows, as inferred from our point count censuses, significantly

Figure 1 | Radiation measurements and genetic damage. The relationshipbetween genetic damage of nestlings and (a) the activity concentrations of
the nest material (Bq/kg d.w., summing activities of 134Cs and 137Cs)or (b) external radiation dose rate, as measured by the TLD (m Gy h21).
The lines are simple regression lines interpolated to the log-transformeddata.
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Figure 2 | Barn swallows abundance and radioactive contamination. The
abundance of barn swallows declined with increasing levels of radioactive
contamination as measured during our multi-year point-count census.

Figure 3 | Age ratio of barn swallows and radioactive contamination. The
proportion of barn swallows being juveniles declined with increasing levelsof radioactive contamination.

declined with increasing environmental radiation levels (F1,1093 5
105.81, p , 0.0001; slope (SE) 5 21.18 (0.12); Fig. 2). The number ofbarn swallows increased with increasing farmland (F1,1093 5 12.53, p
5 0.0004; slope (SE) 5 6.82 3 1023 (1.37 3 1023)), and decreased
with increasing ground coverage by grass (F1,1093 5 320.78, p ,0.0001; slope (SE) 5 24.75 3 1022 (2.18 3 1023)) and coniferous
forest (F1,1093 5 4.98, p 5 0.0256; slope (SE) 5 21.74 (0.39)). In
addition, the abundance of barn swallows differed among years(F2,1093 5 103.83, p , 0.0001). There were significantly fewer barnswallows in 2012 than in 2011 (t 5 23.1, p , 0.0001) and fewer in2013 compared to 2012 (t 5 7.85, p 5 0.005), or the two previous
years combined (t 5 34.07, p , 0.0001).
The probability of a barn swallow being a juvenile decreased significantly with increasing environmental radiation levels (F1,1069 5
13.50, p 5 0.0002; slope (SE) 5 21.84 (0.57); Fig. 3). In addition,
juvenile barn swallows were more common where adults were moreabundant, as expected from the fact that the adults produced theoffspring (F1,1069 5 32.84, p , 0.0001; slope (SE) 5 1.00 (0.18)).
There was also a significant variation among years in the probability
that a bird was a juvenile (F2,1069 5 15.44, p 5 0.021). This probability was higher in 2012 than in 2011 (t 5 6.04, p 5 0.014), or 2013(t 5 6.04, p 5 0.014).

breeding sites. Alternatively, differences in the mixture of radionuclides scattered by the two disasters could explain the difference inthe effect, if different mixtures have different associated risks due todifferences in particle emission. While 134Cs and 137Cs are the dominant radionuclides dispersed by the Fukushima disaster12, 137Cs, 90Sr,
241
Am and several radioisotopes of plutonium are the ones currently
present around the Chernobyl Exclusion Zone36. The greater abundance of Pu isotopes and other actinides in Chernobyl could thus beresponsible for the difference between the two disasters, as alphaemitters have large health effects. Finally, differences in historicalexposure and associated trans-generational accumulations of deleterious effects could be responsible for the differences between thetwo disasters. Swallow populations in the Chernobyl region havebeen chronically exposed to radioactive contamination for over 20
years at the time of sampling for our 2010 study of genetic damage.
Conversely, exposure to radioactive contamination only lasted little
more than one year when we sampled barn swallows in the contaminated areas around Fukushima for the present study. While thisdifference in historical exposure is expected to affect mutation accumulation, predictions regarding the resistance of natural populations
to radiation-induced genetic damage are less clear, as genetic damage
is not inherited, and natural populations are expected to evolve resistance to radiation-induced oxidative damage over generations37.
Given that the nestlings examined here belonged to the second generation after the disaster, and the first one from parents that werethemselves exposed throughout development, we do not expect thelack of an increase in genetic damage to be due to selection foradaptation to ionizing radiation.
At present, the interpretation of the variation among nestlings intheir levels of DNA damage is deemed to be largely speculative.
Differences in individual growth rates or in the intensity of competition with siblings could account for such variation through an effecton the oxidative status of nestlings. Consistent with this, resistance of
red blood cells to free radicals has been found to negatively correlatewith growth rate in zebra finch (Taeniopygia guttata) nestlings38. In
addition, magpie (Pica pica) nestlings begging more intensively wereshown to have higher levels of lipid peroxidation, as indexed by theirmalondialdehyde levels39. Future studies where nestlings are repeatedly measured will allow controlling for these confounding factors,
uncovering potential more subtle effects of radiation.
Due to restriction in access to more highly contaminated areas
during our sampling of nestlings, we could not access sites where
nestlings might have received considerably higher doses, including
the towns of Okuma, Futaba and Namie. Thus, the data presented
here should be cautiously interpreted when addressing whether

Discussion
In this study, we investigated genetic damage in barn swallows nestlings exposed to radioactive contamination following the accident at
the Fukushima Daiichi Nuclear Power Plant in March 2011. We also
estimated the abundance of barn swallows across sites differing inenvironmental radiation levels by almost two orders of magnitude,
while also assessing the relative frequency of juveniles and adults. To
the best of our knowledge, this is the first study at Fukushima relatinga known biomarker of radiation exposure to estimates of radiation
exposure in any wild population of animals exposed to the radioactive fallout.
We could not detect any increase in genetic damage in nestlings
exposed to a range of contamination levels during their rearing period. These results partially conflict with previous results in adult barnswallows from the Chernobyl region, where higher genetic damage
was demonstrated at levels that were comparable to the ones detectedin this study26. It could be argued that barn swallow nestlings at
Fukushima were exposed for shorter times compared to adults in
Chernobyl. The exposure period of barn swallow nestlings (averaging 26 6 5 days in our sample, due to the combined duration of
the incubation period and the rearing period) is considerably shortercompared to the months-long residence of adult barn swallows at the
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exposure to radioactive contamination is causing an increase in genetic damage in wild populations of animals in contaminated areas, as
higher contamination levels might imply more deleterious consequences. The barn swallow is a model species for investigating theeffects of radioactive contamination in Fukushima due to its abundance, philopatry to a once chosen breeding site, and availability ofcontrol as well as affected populations (e.g. in Chernobyl). However,different species may vary in their radiosensitivity and the lack of aneffect in one species does not necessarily imply that all others are
similarly unaffected1–3,30,31,33,34.
In addition, while the biomarker that we assessed did not show any
response among nestlings, our census of barn swallows confirmed
previous findings of population declines of several bird species in the
Fukushima region33,34. In addition, it suggested that the population
decline is due to lower fecundity and/or lower fledging rate, as
demonstrated by a decrease in the proportion of juveniles at higher
levels of radiation exposure. This result is consistent with the demonstrated decline in fertility, reproductive function and parental carethat we have shown in Chernobyl in the barn swallow as well as inother species35,40–42.
Human absence from highly contaminated towns, with the associated changes in the farming practice and the lack of deterrence forthe natural predators of this species (e.g. the Japanese jungle crow
Corvus macrorhynchos) is a potential alternative explanation for the
decline of this species in contaminated areas. In future studies, theassessment of biomarkers of radiation exposure will help determine
whether the decline of this species is due to a direct or indirect effectof ionizing radiation (i.e. through an effect on human presence).
The discrepancy between the decline in abundance of barn swallows and the lack of any response in the biomarker of genetic damage
that we assessed in barn swallow nestlings calls for further investigation of the potential mechanistic (i.e. physiological and genetic) links
between radiation exposure and population dynamics. Multiple
cytogenetic biomarkers of radiation exposure will have to be investigated in the future, while at the same time expanding research into
areas contaminated to a higher degree than it was assessed in thepresent study. Similarly, aural ad visual censuses of diversity andabundance will have to be complemented with mist netting of birds
in order to estimate transfer of radionuclides to birds. It should be
noted that radiation levels examined during the censuses were muchwider than the range of contamination levels where nestlings havebeen measured, as more highly contaminated areas could not besampled due to lack of access or sampling permit. Thus, the present
results should not be interpreted as indicating that no deleterious
consequence is expected over the entire area that was contaminatedby the radioactive fallout, nor should they be taken as evidence thatgenetic damage at the adult stage is not mediating the population
decline of barn swallows, as adult birds were not assessed in thepresent study. In fact, the investigation of genetic damage of nestlings
should also be expanded to more highly contaminated areas in order
to exclude the possibility that it contributed to the decline of barnswallow populations.
Overall, our radioactivity measurements are compatible withpreviously published measurements and dose estimates43,44. Higher
exposure levels for barn swallows can thus be predicted in more
highly contaminated areas. The exposure levels measured here areconsistent with the occurrence of physiological and life-history consequences (i.e. reduced survival and reproduction) in exposed organisms44. A recent analysis that inferred doses from publishedinformation on contamination levels and used official benchmarks
for dose-response also concluded that exposure to contamination
following the accident could induce sub-lethal effects on the populations of terrestrial vertebrates45. This same analysis, however, also
concluded that any population-level consequence of such individuallevel doses would be unlikely, thus raising the issue of reconcilingmeasured doses with population declines that have been shown by
SCIENTIFIC REPORTS | 5 : 9432 | DOI: 10.1038/srep09432

recent censuses33,34. At the same time, it should be noted that ourestimates of radiation exposure are conservative, as they admittedly
do not account for internal radiation exposure due to inhalation oringestion of radionuclides. Dose conversion coefficients (DCC) forinternal exposure to 134Cs and 137Cs in species ecologically similar tothe barn swallow are expected to be at least as large as the DCCs forexternal exposure46. Thus, future studies will have to improve dosimetry by assessing both internal and external radiation exposure of
local populations of barn swallows potentially impacted by the
fallout.
Similar population declines at levels of contamination that are notpredicted to have population-level consequences have also been
observed in the Chernobyl region31,32,47, prompting similar skepticism48. There is increasing evidence, however, that the benchmarks
indicated as safe by international organizations (IAEA, ICRP)49,50
might be underestimating the risk associated with exposure to ionizing radiation in the natural environment, especially under chronic
exposure51. Hazards to natural populations have recently been foundto arise at doses considerably lower than it had been shown in controlled experiments in the lab51. In addition, a recent meta-analysis
that reviewed studies conducted in very high background radiation
areas where radionuclides occur naturally found a consistent positive
relationship between environmental radiation levels and mutation
rate, DNA repair and genetics, in human as well as animal populations52. The likely explanation for the discrepancy between the lab
and an ecologically-meaningful setting is that lab conditions are far
more benign than realistic ecological conditions, where food andessential nutrients are scarcer, predators and parasites are more frequent, and other stressors may make the effects of ionizing radiation
more apparent.

Methods
During May 2012, we attached thermoluminescent dosimeters (TLDs) to the innerand outer rim of 55 barn swallow nests from the Fukushima region (Fig. 4). We used
individually calibrated LiF:Mg,Cu,P TLDs (3.2 3 3.2 3 0.8 mm; GR-200A), whichhave a higher sensitivity than GR-100 TLDs53. The linearity and dose-response of the
TLDs were measured with beams produced by a medical linac and a 137Cs source. We
read the TLD response with a System 310 TLD Reader (Teledyne Brown
Engineering), in a temperature range from room temperature to 240uC, at a rate of
10uC/s. The readings were consistent with previously published results54. After an
average 28.4 days (0.4 SE; range: 25–33 days), we retrieved the TLDs from the nest.
On the occasion of retrieving the TLDs, we collected a sample (,1 g) of nest material
from the rim of the nest. From 62 chicks from 16 nests that we estimated to be at least
7–8 days old, we also collected a small blood sample (,50 mL), through puncture of
the brachial vein and collection in a heparinized capillary tube. We also transferred adrop of blood (,10 mL) to a vial containing RNAprotect (Qiagen).
All procedures were performed in accordance with relevant guidelines and regulations, and approved by the Institutional Animal Care and Use Committee (IACUC)
of the University of South Carolina (Protocol number: 2014-100237-052611).
Radioactivity measurements. In the field, we measured environmental a, b and cradiation at the ground level below the nest using a hand-held dosimeter (Model:
Inspector, SE International, Inc., Summertown, TN, USA).
We measured the activity concentrations of nest samples by conducting gamma ray
spectrometry with a SAM 940 Radioisotope Identifier (Berkeley Nucleonics, San
Rafael, CA) equipped with a 7.62 3 7.62 cm (30 3 30) sodium iodide (NaI) detector.
The spectrometer was placed vertically within a lead detector shield (Canberra
Industries, Meriden, CT, USA), with additional shielding provided by lead bricks. We
measured each sample by placing it on top of the SAM 940 Radioisotope Identifier.
We later converted the spectra to activity measurements after calibration of the
instrument using standard 137Cs and 134Cs sources. For analysis we focused on the
661 keV decay gamma from 137Cs and the 597 and 796 keV peaks from 134Cs. The
samples were dried in a heating oven with mechanical convection (Binder Inc.,
Bohemia, NY) at the temperature of 60uC for 12 h, and weighed using a Sartorius
electronic balance (Model R160P; Göttingen, Germany).
A high statistics, ‘‘empty target’’ spectrum was collected prior to the sample
readings and subtracted from all spectra to remove counts not associated withradioactive decay from the sample. A linear background function was then fit to thepeak region (490–500 keV) to remove the continuum gammas and isolate the decay
peaks. The 597 keV peak from 134Cs and the 661 keV peak from 137Cs overlapped
considerably while the 796 keV peak from 134Cs was resolved completely. A spectrum
from the 134Cs calibration source was normalized to the data spectrum by fitting to the796 keV peak. This fit was then subtracted from the entire sample spectrum to isolate
the 661 keV peak from 137Cs. Integrating and comparing the counts in the decay4

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Figure 4 | Locations of sampling sites. Locations of the sixteen nests used in the analyses of the relationship between contamination levels and DNA
damage of the nestlings. Each location may correspond to more than one nest sampled. Contamination levels are derived from official data from theJapanese Ministry of Education, Culture, Sports, Science and Technology (MEXT), and used to interpolate a map of contamination at 1-m height. The
map was created using ArgGis v10.2 (Environmental Systems Research Institute, Redlands, CA). Used with permission. Copyright E 2015 Esri, DeLorme,
NAVTEQ. All rights reserved.

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Figure 5 | Locations of census sites during 2011–2013. Contamination levels are derived from official data from the Japanese Ministry of Education,
Culture, Sports, Science and Technology (MEXT), and used to interpolate a map of contamination at 1-m height. The map was created using ArgGis v10.2(Environmental Systems Research Institute, Redlands, CA). Used with permission. Copyright E 2015 Esri, DeLorme, NAVTEQ. All rights reserved.
peaks from the samples to the counts in the same peaks from the known calibration
sources produced the absolute calibration. The total activity of each sample wascalculated by summing the activities estimated for 137Cs and 134Cs.
To estimate total duration of exposure for each nestling, we summed the estimatedage of the nestlings and the duration of the incubation period, which we conservatively estimated at 14 days.
Analysis of genetic damage. We estimated genetic damage using a single cell gel
electrophoresis assay, also known as comet assay, following the protocol reported inRef. 55, with minor modifications.
We prepared slides in advance by dipping single-frosted slides (VWR, Radnor, PA)
in 1.5% normal melting-point agarose. We transferred 3 mL of the solution of blood in
RNAProtect (Qiagen) to 997 mL of 13 PBS. We then mixed 50 mL of the solution
with 450 mL of 1.5% low melting-point agarose, and layered 100 mL of this mixture on
the slides, covering with a glass coverslip. We allowed the agarose to solidify for fiveminutes at 4uC. We then removed the coverslip and added another layer of 100 mL of
low melting-point agarose, and again allowed to solidify for five minutes, before
removing the coverslip. The slides were left for 1 hour at 4uC to allow the solidification of the gel, and then immersed in cold lysis buffer (1% sodium sarcosinate,
2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1% Triton X-100 added immediately
prior to use, at a final pH 5 10), where they were kept for 1 hour at 4uC. We thenrinsed the slides with cold ddH2O, and immersed them in neutral buffer (300 mM
NaOH, 100 mM Tris, pH 10.0), to allow unwinding of the DNA for 30 minutes at
4uC. We electrophoresed slides in a tank filled with the same buffer for 30 minutes at
0.7 V/cm and 150 mA at 4uC. After electrophoresis, we rinsed the slides in a neutralization buffer (0.4 M Tris, pH 7.4) three times, for five minutes each. The slides
were then fixed in 70% ethanol for 15 minutes, and left to dry overnight. We ran four
slides per each individual.
We stained the slides by immersion in a 1510,000 solution of SYBRH Gold
(Trevigen, Gaithersburg, MD) for five minutes. Slides were then de-stained throughimmersion in a bath of dd-H2O for five minutes, and left to dry. Images of individual

SCIENTIFIC REPORTS | 5 : 9432 | DOI: 10.1038/srep09432

cells were captured using a Metafer System (Metasystems, Bethesda, MD), an automated system that performs detection and scoring of individual cells56. Only thenestlings for whom we had captured at least 100 cells across the different slides wereretained in the final sample57. In the final analyses we included 49 nestlings belonging
to 16 nests, representing 78% of the initial sample of 63 nestlings. On average we
captured 313 cells (147 SD; range: 111–725) from 1–4 slides. As a measure of damage
to the DNA we used the percentage of DNA in the tail, which is a measure based on
the relative fluorescence intensity of the tail compared to the head of the comet, andthe most reliable parameter for the comet assay57,58.
Data on genetic damage in each nestling were obtained by averaging percentages of
DNA in the tail of the comet across all cells.
Point count censuses. During the first week of July 2011–2013, we conducted a pointcount census of birds across clean and contaminated sites (Fig. 5). Each count lasted
five minutes, with census points located at approximately 100 m intervals. At each
census point, we classified the habitat as being agricultural, grassland, deciduous
forest or coniferous forest, and estimated ground coverage by these different habitats(to the nearest 10%) within a distance of 50 m. In total, we collected 1100 5-min point
counts (2011: N 5 300; 2012: N 5 400; 2013: N 5 400). The census points were thesame in all years, except for 2011, when 100 fewer counts were conducted due torestrictions in access. The relationship between radiation and abundance did not
qualitatively change if we restricted the analyses to the 300 points where the counts
were conducted in all three years (results not shown). At each census point, werecorded radiation levels using a hand-held dosimeter at ground level (Model:
Inspector, SE International, Inc., Summertown, TN, USA). We also recorded thegeographic coordinates and altitude (using a GPS), cloud cover at the start of eachpoint count (to the nearest eighth), temperature (degrees Celsius), and wind force(Beaufort). For each census point we recorded time of day at the start of the count (tothe nearest minute) and included it in the analyses as an explanatory variable. As
activity levels of birds peak in the morning and, to a lesser extent, in the evening, we
also included time squared in our analyses. APM conducted all censuses, thus

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preventing any issue due to inter-observer variability. All the nests that we inspectedduring 2012–2013 would be fledged by the time we conducted our census. Thus, nodifference among years would be expected based on differences in the timing of
reproduction.
In a second set of analyses, we analyzed the probability of observing a juvenile barnswallow (as identified from the short tail streamers and the pale coloration, using
binoculars) as a function of environmental radiation levels, as well as the same predictors that we included in the analysis of barn swallow abundance. We also included
the local abundance of adult barn swallows as a predictor in the analysis, as juvenile
barn swallows are the offspring of the adult barn swallows present.
Statistical analysis. For the analysis of genetic integrity of nestlings we used generallinear mixed models (GLMMs) where we included radiation exposure (either logtransformed radioactivity of the nest material or radiation dose as inferred from theTLDs) as a covariate, and the nest of origin as a random effect. In both analyses weincluded duration of exposure as a covariate. Degrees of freedom were estimatedusing the Kenward-Roger approximation. All analyses were performed in SAS 9.3(SAS Inc., Cary, NC).
In the analysis of the abundance of barn swallows, we used generalized linear
models, assuming a Poisson distribution of count data. As predictor variables, we
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above. In addition, we included temperature, cloud cover, wind, time of day and timeof day squared, the latter to account for the fact that bird activity has a peak during
early morning and a second, milder peak in the afternoon. We also included radiationlevel squared to account for non-linear relationships between species richness andabundance, respectively, and radiation. These analyses were all implemented in thestatistical software JMP (SAS Institute Inc., 2012). In the analyses of the proportion ofswallows being juveniles, we relied on general linear models with binomially distributed data and a logit link function.
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Acknowledgments
We are grateful to Claudia Benitez-Nelson and Wendy Plessinger for the calibration of ourspectra using a well det. Ken Buesseler kindly provided the 134Cs standard for instrument

SCIENTIFIC REPORTS | 5 : 9432 | DOI: 10.1038/srep09432

calibration. Ivan Frigerio, Shane Welch and Jennifer Fill all greatly helped with the
preparation of the maps used here. We are thankful to all the Japanese people who allowed
access to the barn swallow nests, often on the very top of their house door. This paper isdedicated to all those that had to leave their beloved houses – and barn swallows.

Author contributions
A.B.A., A.P.M. and T.A.M. conceived the research; A.B.A., K.K., W.K., H.S., E.A., A.P.M.
and T.A.M. collected data in the field; A.B.A. and S.O. performed the lab assays; A.B.A. and
D.J.T. collected and analyzed spectra; A.B.A. and A.P.M. performed statistical analyses; A.B.
A. wrote the first version of the paper; all authors commented on the manuscript.

Additional information
Supplementary information accompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Bonisoli-Alquati, A. et al. Abundance and genetic damage of barnswallows from Fukushima. Sci. Rep. 5, 9432; DOI:10.1038/srep09432 (2015).
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