Improving our understanding of the long term effects of exposure to ionising radiation
We need to be able to derive dose rates and associated environmental activity concentrations below which we can be confident that there will be no effects on wildlife. Furthermore we need to be able to better understand at what dose rate effects will be seen. This will inform our current risk assessment methods and provide confidence in their use. We know empirical studies of impacts of chronic radiation at low doses in the natural environment are rare and often provide contradictory information which can undermine our risk assessments. Our studies will contribute to the growing data on radiation effects and hopefully start to address the concerns over the contradictory information.
Over the last century, dose rate criteria (i.e. ‘acceptable’ levels of exposure) have been established for humans. ICRP have proposed Derived Consideration Reference Levels (DCRLs) values for RAPs (Copplestone 2012); acknowledging that they are based on limited data dominated by laboratory studies. And yet, despite decades of research into radiation effects on wildlife, controversy remains concerning the dose rate at which significant impacts occur (Beresford & Copplestone 2011).
The relevance of much of the available data is questionable as they are usually derived from short-term laboratory experiments conducted at doses/dose rates often orders of magnitude higher than those observed in contaminated environments (such as in the Chernobyl Exclusion Zone (CEZ) and at Fukushima). Uncertainty also remains because of contradictory findings between laboratory and (unrelated) field studies (Garnier-Leplace et al 2013). For example a recent study of aquatic invertebrate populations in Chernobyl-contaminated lakes observed no association between radiation dose rate (up to 0.75 mGy d-1) and species abundance or diversity (Murphy et al 2011), but did not consider individual-level effects which may impact on long-term population health. However, a number of high-profile studies on terrestrial ecosystems at Chernobyl have reported observed population- and individual- level effects on birds and insects (Møller & Mousseau 2007; Møller & Mousseau 2009) in field studies at dose rates as low as 0.024-0.24 mGy d-1. A possible reason for these differences is that animals in the natural environment are often at sub-optimal levels of nutrition and condition and consequently are less resistant to radiation than animals studied in laboratory conditions (Garnier-Leplace et al 2013).
We will study the effects of chronic low doses in contaminated environments with parallel laboratory studies and at a sufficiently wide range of field sites to adequately control for the many confounding factors which impact on plant and animal health in natural systems. We will also study if and how radiation effects are transferred between generations and how results of biomarker tests of exposure can be extrapolated to indicate population health.
Our hypotheses will be tested using laboratory studies conducted over dose rates encompassing the ICRP DCRLs (1-100 mGy d-1) typical for our test species (bees, earthworms, fruit flies, sticklebacks and freshwater invertebrates) thereby contributing to the derivation of more robust benchmarks. Tests will be conducted in the CEZ, where existing dose rates range from typical background levels to those exceeding the suggested thresholds above which population level effects may be observed (i.e. 10s of mGy d-1 (Chesser et al 2000)).
We will identify and apply a range of radiation biomarkers for each species and we will use measures of longevity, reproduction and immune health Sazykina & Kryshev) to determine if the biomarker tests can be used to predict health effects in the field. Biomarker tests like the single and double strand Comet assay, chromosome aberration measures, reactive oxygen species, anti-oxidant capacity and micronucleus assay will be used. Results of all tests will be compared with effects on physiological parameters (e.g. somatic and reproductive development) to investigate any potential links. Further, as ionising radiation-induced DNA damage has been reported to result from altered transcription of genes involved in cell cycle, cell death, DNA repair, DNA metabolism and RNA processing (Oh et al 2012; Rhee et al 2012), transcriptomes from the exposed and non-exposed fish will be compared to identify candidate mRNA biomarkers; RNA-sequencing has been established as a sensitive method for searching for mRNA biomarkers (Riedmaier & Pfaffi 2013). We will be able to establish differences in transcriptomic profiles in treated and untreated individuals.
As an example, we will study the sensitive lifestages (embryos – juveniles) of the 3-spined stickleback will be exposed to Cs under controlled temperature and light conditions in the laboratory. Embryos will be examined daily to assess development, time to hatch and percent hatching success. Post-hatch all juveniles will be sacrificed and lengths, weights and morphological abnormalities recorded. RNA will be isolated from the livers of fry from each treatment for the assessment of differential gene expression using transcriptomic analysis.
In the field fish will be sampled from 10 surface waters with a range of contamination within and outside the CEZ. This will include the Chernobyl Cooling Pond and Glubokoye Lake; two of the most radioactively contaminated aquatic systems in the world. In addition to applying the laboratory tests, we will measure somatic growth and reproductive status, and record the presence of external parasites/viral lesions. RNA will be isolated from the livers of each fish to measure differences in expression profiles of the genes identified in the laboratory study. Confounding factors which could also impact fish health such as the presence of heavy metals and organic pollutants will be measured.
Our work on freshwater species will be complemented by our Japanese project partner, MERI, who have a programme of research in the marine environment close to the Fukushima Dai-ichi NPP. They will apply of our biomarkers on marine species.
For terrestrial species we will study earthworms and bumblebees which will be irradiated using an external 137Cs source under controlled temperature and light conditions in the laboratory. These two species are key to ecosystem function. For the earthworm, we will study development under varying levels of radiation dose rate to determine survival and growth rates and their reproductive success. Cocoons will be hatched and bred on to determine any trans-generational effects. We will study the development of young bumblebee nests to quantify the response of a population to varying levels of radiation dose rate to understand the effect of radiation exposure on survival, growth and reproductive success. Offspring will be used to investigate any trans-generational effects.
We will collect earthworms and bumblebees from field sites with a range of dose rates within the CEZ and a Ukrainian site in an area which received little Chernobyl fallout. A range of genetic, developmental and reproductive endpoints will be measured together with the biomarkers applied in the laboratory studies. As we are unaware of any effects studies on large mammals within the CEZ we will take advantage of WP3 activities and take samples for evaluation using our identified suite of biomarkers.
All biological samples will be assessed using histopathological techniques to evaluate hyperplasia, necrosis and multinucleated cells along with the biomarker tests. In addition, some of our fish and mammalian tissue samples from the CEZ will be measured for: (i) Immune system function using oxidative burst measurements of macrophage activity (Belosevic et al 2006); (ii) DNA:RNA, protein:DNA and protein:RNA ratios in muscle and gonad samples to give biochemical growth indexes, which have the potential to inform on reproductive fitness and metabolic activity including any evidence of food deprivation by our project partner McMaster University. Another project partner, IRSN, has complimentary on-going work in terrestrial ecosystems in Japan contaminated by the Fukushima accident; focusing on short-term impacts.
The experimental work described above will detect impacts of the immediate effects of radiation exposure on organism physiology and on the offspring of exposed organisms. However, the effects could be subtle and contingent on particular field environmental conditions. We will therefore investigate the evolutionary history of animal populations living in contaminated habitats within the CEZ. Evolutionary change in response to an environmental stress occurs because that stress impairs the reproductive fitness of organisms. By investigating whether evolutionary change has occurred in animal populations living close to Chernobyl we can infer whether these conditions have had detrimental effects when averaged over nearly 27 years. We will use the fruit fly as a ‘model organism’ to assess genome-wide genetic diversity at least 250 generations since the Chernobyl explosion. To study evolutionary change we will sequence DNA from pools of individuals collected at sites with different radiation exposure histories to estimate the frequency of different mutational variants. Our sequencing will estimate the frequency and distribution of rare mutational variants which may result from radiation exposure.
We will also investigate the impact of long-term radiation exposure on populations in the laboratory by irradiating the parents only, or both parents and their offspring, to assess the ‘trans-generational impacts’ hypotheses using Drosophila spp.. In addition we will gain some information for B. terrestris and L. rubellus offspring from the laboratory studies described above.
We will conduct parallel laboratory and field studies to evaluate a range of effects on organisms from the level of genome (using novel transcriptomics techniques) to whole-animal to support the expansion of the international radiological protection framework to consider wildlife (ICRP 2008)
For further information contact: Prof. David Copplestone (Stirling University)