Physiological effects of gamma irradiation in the honeybee, Apis mellifera.

Terrestrial ecosystems are exposed to various kinds of pollutants, including radionuclides. The honeybee, Apis mellifera, is commonly used in ecotoxicology as a model species for evaluating the effects of pollutants. In the present study, honeybees were irradiated right after birth for 14 days with gamma rays at dose rates ranging between 4.38 × 10-3 and 588 mGy/d. Biological tissues (head, intestine and abdomen) were sampled at D3, D10 and D14. Ten different physiological markers involved in nervous (acetylcholinesterase (AChE)), antioxidative (catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST)), immune system (phenoloxidase (PO)) and metabolism (carboxylesterases (CaEs) and alkaline phosphatase (ALP)) were measured. Univariate analyses were conducted to determine whether each individual biomarker response was positively or negatively correlated with the dose rate. Then, multivariate analyses were applied to investigate the relationships between all the biomarker responses. Although no mortality occurred during the experiment, several biomarkers varied significantly in relation to the dose rate. Globally, the biomarkers of antioxidant and immune systems decreased as the dose rate increased. Reversible effects on the indicator of the neural system were found. Concerning indicators of metabolism (carboxylesterases), variations occurred but no clear pattern was found. Taken altogether, these results help better understand the effects of ionizing radiation on bees by identifying relevant physiological markers of effects. These results could improve the assessment of the environmental risk due to ionizing radiation in terrestrial ecosystems.

Uptake, depuration, dose estimation and effects in zebrafish exposed to Am-241 via dietary route.

Zebrafish were chronically exposed to Am-241, an alpha-emitting radionuclide via daily consumption of an enriched artificial diet. Am-241 uptake was quantified in Danio rerio after 5 and 21 days of exposure via daily Am-spiked food ingestion and after 21 days of exposure followed by 5 days of depuration. Americium accumulates mostly in digestive tract, muscle, rest of the body but the accumulation levels and trophic transfer rate (0.033-0.013%) were low. Corresponding cumulative doses were calculated for the whole body (9 mGy) and for the digestive tract (42 mGy) with internal alpha radiation contributing to more than 99% of the total dose. Genotoxic effects (gamma-H2AX assay) and differential gene expressions of main biological functions were examined. Although fish were exposed to a low dose rate of 13 µGy h-1, DNA integrity and gene expression linked to oxidative stress, hormonal signaling and spermatogenesis were altered after 21 days of Am-241 exposure. These results underline the higher toxicity of alpha emitter Am-241, as compared to other studies on gamma radiation exposure.

Radiosensitivity and transgenerational effects in non-human species.

The ALLIANCE working group on effects of ionising radiation on wildlife brings together European researchers to work on the topics of radiosensitivity and transgenerational effects in non-human biota. Differences in radiation sensitivity across species and phyla are poorly understood, but have important implications for understanding the overall effects of radiation and for radiation protection; for example, sensitive species may require special attention in monitoring and radiation protection, and differences in sensitivity between species also lead to overall effects at higher levels (community, ecosystem), since interactions between species can be altered. Hence, understanding the mechanisms of interspecies radiation sensitivity differences may help to clarify mechanisms underpinning intraspecies variation. Differences in sensitivity may only be revealed when organisms are exposed to ionising radiation over several generations. This issue of potential long-term or hereditary effects for both humans and wildlife exposed to low doses of ionising radiation is a major concern. Animal and plant studies suggest that gamma irradiation can lead to observable effects in the F1 generation that are not attributable to inheritance of a rare stable DNA mutation. Several studies have provided evidence of an increase in genomic instability detected in germ or somatic cells of F1 organisms from exposed F0 organisms. This can lead to induced radiosensitivity, and can result in phenotypic effects or lead to reproductive effects and teratogenesis. In particular, studies have been conducted to understand the possible role of epigenetic modifications, such as DNA methylation, histone modifications, or expression of non-coding RNAs in radiosensitivity, as well as in adaptation effects. As such, research using biological models in which the relative contribution of genetic and epigenetic processes can be elucidated is highly valuable.

Effects of in situ exposure to tritiated natural environments: A multi-biomarker approach using the fathead minnow, Pimephales promelas.

Aquatic ecosystems are chronically exposed to radionuclides as well as other pollutants. Increased concentrations of pollutants in aquatic environments can present a risk to exposed organisms, including fish. The goal of this study was to characterize the effects of tritium, in the context of natural environments, on the health of fathead minnow, Pimephales promelas. Fish were exposed to tritium (activity concentrations ranging from 2 to 23,000Bq/L) and also to various concentrations of several metals to replicate multiple-stressor environments. Fish were exposed for 60days, then transferred to the tritium background site where they stayed for another 60days. Tritium, in the forms of tritiated water (HTO) and organically bound tritium (OBT), and a series of fish health indicators were measured in fish tissues at seven time points throughout the 120days required to complete the exposure and the depuration phases. Results showed effects of environmental exposure following the increase of tritium activity and metals concentrations in water. The internal dose rates of tritium, estimated from tissue HTO and OBT activity concentrations, were consistently low (maximum of 0.2µGy/h) compared to levels at which population effects may be expected (>100µGy/h) and no effects were observed on survival, fish condition, gonado-somatic, hepato-somatic, spleno-somatic and metabolic indices (RNA/DNA, proteins/DNA and protein carbonylation (in gonads and kidneys)). Using multivariate analyses, we showed that several biomarkers (DNA damages, MN frequency, gamma-H2AX, SFA/MUFA ratios, lysosomal membrane integrity, AChE, SOD, phagocytosis and esterase activities) were exclusively correlated with fish tritium internal dose rate, showing that tritium induced genotoxicity, DNA repair activity, changes in fatty acid composition, and immune, neural and antioxidant responses. Some biomarkers were responding to the presence of metals, but overall, more biomarkers were linked to internalized tritium. The results are discussed in the context of multiple stressors involving metals and tritium.

External gamma irradiation-induced effects in early-life stages of zebrafish, Danio rerio.

In the general context of validation of tools useful for the characterization of ecological risk linked to ionizing radiation, the effects of an external gamma irradiation were studied in zebrafish larvae irradiated for 96 h with two dose rates: 0.8 mGy/d, which is close to the level recommended to protect ecosystems from adverse effects of ionizing radiation (0.24 mGy/d) and a higher dose rate of 570 mGy/d. Several endpoints were investigated, such as mortality, hatching, and some parameters of embryo-larval development, immunotoxicity, apoptosis, genotoxicity, neurotoxicity and histological alterations. Results showed that an exposure to gamma rays induced an acceleration of hatching for both doses and a decrease of yolk bag diameter for the highest dose, which could indicate an increase of global metabolism. AChE activity decreased with the low dose rate of gamma irradiation and alterations were also shown in muscles of irradiated larvae. These results suggest that gamma irradiation can induce damages on larval neurotransmission, which could have repercussions on locomotion. DNA damages, basal ROS production and apoptosis were also induced by irradiation, while ROS stimulation index and EROD biotransformation activity were decreased and gene expression of acetylcholinesterase, choline acetyltransferase, cytochrome p450 and myeloperoxidase increased. These results showed that ionizing radiation induced an oxidative stress conducting to DNA damages. This study characterized further the modes of action of ionizing radiation in fish.

Combined effects of depleted uranium and ionising radiation on zebrafish embryos.

In the environment, living organisms are exposed to a mixture of stressors, and the combined effects are deemed as multiple stressor effects. In the present work, the authors studied the multiple stressor effect in embryos of the zebrafish (Danio rerio) from simultaneous exposure to alpha particles and depleted uranium (DU) through quantification of apoptotic signals at 24 h post-fertilisation (hpf) revealed by vital dye acridine orange staining. In each set of experiments, dechorionated zebrafish embryos were divided into 4 groups, each having 10 embryos: Group (C) in which the embryos did not receive any further treatment; Group (IU) in which the embryos received an alpha-particle dose of 0.44 mGy at 5 hpf and were then exposed to 100 µg l(-1) of DU from 5 to 6 hpf; Group (I) in which the embryos received an alpha-particle dose of 0.44 mGy at 5 hpf and Group (U) in which the dechorionated embryos were exposed to 100 µg l(-1) of DU from 5 to 6 hpf. The authors confirmed that an alpha-particle dose of 0.44 mGy and a DU exposure for 1 h separately led to hormetic and toxic effects assessed by counting apoptotic signals, respectively, in the zebrafish. Interestingly, the combined exposure led to an effect more toxic than that caused by the DU exposure alone, so effectively DU changed the beneficial effect (hormesis) brought about by alpha-particle irradiation into an apparently toxic effect. This could be explained in terms of the promotion of early death of cells predisposed to spontaneous transformation by the small alpha-particle dose (i.e. hormetic effect) and the postponement of cell death upon DU exposure.

Establishing relationships between environmental exposures to radionuclides and the consequences for wildlife: inferences and weight of evidence.

Ecological risk assessments for radioactive substances are based on a number of inference rules to compensate for knowledge gaps, and generally require the implementation of a weight-of-evidence approach. Until recently, dose (rate)-response relationships used to derive radioprotection criteria for wildlife have mainly relied on laboratory studies from a limited number of species as representatives of biodiversity. There is no doubt that additional knowledge, combined with advanced conceptual and mathematical approaches, is needed to develop general rules and increase confidence when extrapolating from test species to complex biological/ecological systems. Moreover, field data sets based on robust sampling strategies are still needed to validate benchmark values derived from controlled laboratory tests, and to indicate potential indirect ecological effects, if any. This paper illustrates, through several examples, the need for implementing a combined laboratory-field-model approach to obtain science-based benchmark doses (or dose rates) (e.g. screening benchmarks for ecological risk assessments or derived consideration reference levels), based on robust meta-analysis of dose-effect relationships covering ecologically relevant exposure time scales, species, and endpoints.

Soil nematode assemblages as bioindicators of radiation impact in the Chernobyl Exclusion Zone.

In radioecology, the need to understand the long-term ecological effects of radioactive contamination has been emphasised. This requires that the health of field populations is evaluated and linked to an accurate estimate of received radiological dose. The aim of the present study was to assess the effects of current radioactive contamination on nematode assemblages at sites affected by the fallout from the Chernobyl accident. First, we estimated the total dose rates (TDRs) absorbed by nematodes, from measured current soil activity concentrations, Dose Conversion Coefficients (DCCs, calculated using EDEN software) and soil-to-biota concentration ratios (from the ERICA tool database). The impact of current TDRs on nematode assemblages was then evaluated. Nematodes were collected in spring 2011 from 18 forest sites in the Chernobyl Exclusion Zone (CEZ) with external gamma dose rates, measured using radiophotoluminescent dosimeters, varying from 0.2 to 22 µGy h(-1). These values were one order of magnitude below the TDRs. A majority of bacterial-, plant-, and fungal-feeding nematodes and very few of the disturbance sensitive families were identified. No statistically significant association was observed between TDR values and nematode total abundance or the Shannon diversity index (H'). The Nematode Channel Ratio (which defines the relative abundance of bacterial- versus fungal-feeding nematodes) decreased significantly with increasing TDR, suggesting that radioactive contamination may influence nematode assemblages either directly or indirectly by modifying their food resources. A greater Maturity Index (MI), usually characterising better soil quality, was associated with higher pH and TDR values. These results suggest that in the CEZ, nematode assemblages from the forest sites were slightly impacted by chronic exposure at a predicted TDR of 200 µGy h(-1). This may be imputable to a dominant proportion of pollutant resistant nematodes in all sites. This might result from a selection at the expense of sensitive species after the accident.

An invitation to contribute to a strategic research agenda in radioecology.

With intentions of integrating a portion of their respective research efforts into a trans-national programme that will enhance radioecology, eight European organisations recently formed the European Radioecology ALLIANCE (www.er-alliance.org). The ALLIANCE is an Association open to other organisations throughout the world with similar interests in promoting radioecology. The ALLIANCE members recognised that their shared radioecological research could be enhanced by efficiently pooling resources among its partner organizations and prioritising group efforts along common themes of mutual interest. A major step in this prioritisation process was to develop a Strategic Research Agenda (SRA). An EC-funded Network of Excellence in Radioecology, called STAR (Strategy for Allied Radioecology), was formed, in part, to develop the SRA. This document is the first published draft of the SRA. The SRA outlines a suggested prioritisation of research topics in radioecology, with the goal of improving research efficiency and more rapidly advancing the science. It responds to the question: "What topics, if critically addressed over the next 20 years, would significantly advance radioecology?" The three Scientific Challenges presented within the SRA, with their 15 associated research lines, are a strategic vision of what radioecology can achieve in the future. Meeting these challenges will require a directed effort and collaboration with many organisations the world over. Addressing these challenges is important to the advancement of radioecology and in providing scientific knowledge to decision makers. Although the development of the draft SRA has largely been a European effort, the hope is that it will initiate an open dialogue within the international radioecology community and its stakeholders. This is an abbreviated document with the intention of introducing the SRA and inviting contributions from interested stakeholders. Critique and input for improving the SRA are welcomed via a link on the STAR website (www.star-radioecology.org).

Uranium-induced sensory alterations in the zebrafish Danio rerio.

The effect of chronic exposure to uranium ions (UO(2)(2+)) on sensory tissues including the olfactory and lateral line systems was investigated in zebrafish (Danio rerio) using scanning electron microscopy. The aim of this study was to determine whether exposure to uranium damaged sensory tissues in fish. The fish were exposed to uranium at the concentration of 250 µg l(-1) for 10 days followed by a depuration period of 23 days. Measurements of uranium uptake in different fish organs: olfactory rosettes and bulbs, brain, skin, and muscles, were also determined by ICP-AES and ICP-MS during the entire experimental period. The results showed that uranium displayed a strong affinity for sensory structures in direct contact with the surrounding medium, such as the olfactory and lateral line systems distributed on the skin. A decreasing gradient of uranium concentration was found: olfactory rosettes>olfactory bulbs>skin>muscles>brain. At the end of the experiment, uranium was present in non-negligible quantities in sensory tissues. In parallel, fish exposed to uranium showed severe sensory tissue alterations at the level of the olfactory and lateral line systems. In both sensory systems, the gross morphology was altered and the sensory hair cells were significantly damaged very early after the initiation of exposure (from the 3rd day). At the end of the experiment, after 23 days of depuration, the lateral line system still displayed slight tissue alterations, but approximately 80% of the neuromasts in this system had regenerated. In contrast, the olfactory system took more time to recover, as more than half of the olfactory rosettes observed remained destroyed at the end of the experiment. This study showed, for the first time, that uranium is able to damage fish sensory tissues to such an extent that tissue regeneration is delayed.

Developmental energetics of zebrafish, Danio rerio.

Using zebrafish (Danio rerio) as a case study, we show that the maturity concept of Dynamic Energy Budget (DEB) theory is a useful metric for developmental state. Maturity does not depend on food or temperature contrary to age and to some extent length. We compile the maturity levels for each developmental milestone recorded in staging atlases. The analysis of feeding, growth, reproduction and aging patterns throughout the embryo, juvenile and adult life stages are well-captured by a simple extension of the standard DEB model and reveals that embryo development is slow relative to adults. A threefold acceleration of development occurs during the larval period. Moreover we demonstrate that growth and reproduction depend on food in predictable ways and their simultaneous observation is necessary to estimate parameters. We used data on diverse aspects of the energy budget simultaneously for parameter estimation using the covariation method. The lowest mean food intake level to initiate reproduction was found to be as high as 0.6 times the maximum level. The digestion efficiency for Tetramin™ was around 0.5, growth efficiency was just 0.7 and the value for the allocation fraction to soma (0.44) was close to the one that maximizes ultimate reproduction.