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 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. Studies conducted during TREE contributed to the growing data on radiation effects and have started to address the concerns over the contradictory information.
Background
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 Chornobyl 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 Chornobyl-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 Chornobyl 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).
During TREE we studied 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 also studied if and how radiation effects are transferred between generations and how results of biomarker tests of exposure can be extrapolated to indicate population health.
Hypotheses
- Laboratory exposure data can be used to inform on the effects of radiation exposure in wild populations
- Current environmental doses of radiation in the CEZ impact the health (somatic and reproductive development) of exposed individuals
- Trans-generational impacts caused by radiation exposure affect populations
Our approach
Our hypotheses was 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 were 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 identified and applied a range of radiation biomarkers for each species and used 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 were used. Results of all tests were 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 were compared to identify candidate mRNA biomarkers; RNA-sequencing has been established as a sensitive method for searching for mRNA biomarkers (Riedmaier & Pfaffi 2013). We were able to establish differences in transcriptomic profiles in treated and untreated individuals.
As an example, we studied 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 were examined daily to assess development, time to hatch and percent hatching success. Post-hatch all juveniles were sacrificed and lengths, weights and morphological abnormalities recorded. RNA was isolated from the livers of fry from each treatment for the assessment of differential gene expression using transcriptomic analysis.
In the field fish were sampled from 10 surface waters with a range of contamination within and outside the CEZ. This included the Chornobyl Cooling Pond and Glubokoye Lake; two of the most radioactively contaminated aquatic systems in the world. In addition to applying the laboratory tests, we measured somatic growth and reproductive status and recorded the presence of external parasites/viral lesions. RNA was 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 were measured.
Our work on freshwater species was complemented by our Japanese project partner, MERI, who had 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 studied earthworms and bumblebees which were 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 studied development under varying levels of radiation dose rate to determine survival and growth rates and their reproductive success. Cocoons were hatched and bred on to determine any trans-generational effects. We studied 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 were used to investigate any trans-generational effects.
We collected earthworms and bumblebees from field sites with a range of dose rates within the CEZ and at a Ukrainian site in an area which received little Chornobyl fallout. A range of genetic, developmental and reproductive endpoints were 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 tookl advantage of work package 3 activities and took samples for evaluation using our identified suite of biomarkers.
All biological samples were 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 were 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.
Trans-generational impacts
The experimental work described above was designed to 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 therefore investigated 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 Chornobyl we can infer whether these conditions have had detrimental effects when averaged over nearly 27 years. We used the fruit fly as a ‘model organism’ to assess genome-wide genetic diversity at least 250 generations since the Chornobyl explosion. To study evolutionary change we sequenced DNA from pools of individuals collected at sites with different radiation exposure histories to estimate the frequency of different mutational variants. Our sequencing estimated the frequency and distribution of rare mutational variants which may result from radiation exposure.
We also investigated 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 gained some information for B. terrestris and L. rubellus offspring from the laboratory studies described above.
We conducted 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 email: Prof. David Copplestone (Stirling University)