In the case of accidents, there is also a need to account for historical dose and radionuclide decay, since observed effects may be a legacy of high levels of exposure after the accident. These high exposures can also be a source of confounding factors, since the initial damage may lead to indirect ecosystem changes (such as the replacement of pine trees by less sensitive species) [44]. While much of the focus in studying the environmental impacts of radiation has been on the uncertainties in effects measurement, it is important to stress that there are also uncertainties in dosimetry.

# **9.3 Impacts of Ionizing Radiation on Nonhuman Biota**

Following the discovery of X-rays by Wilhelm Roentgen in 1895 and of radioactivity by Henri Becquerel in 1896, studies on its effects started immediately. The detonation of the atomic bombs over Hiroshima and Nagasaki in 1945 raised the concern about the health impacts of radioactive contamination and the behavior of radionuclides in the environment [45]. Therefore, a great number of studies using a variety of plants and animals have been performed since then.

The frst harmful effects caused by the exposure to ionizing radiation occur at the molecular and cellular level. If these effects are severe enough, they can impact tissues, organs, individual organisms, populations, and entire communities. However, even though an individual organism may suffer from severe damage at the molecular and cellular level, it does not necessarily mean that entire populations and communities will be affected [46]. It seems that individual organisms are able to sustain a certain level of effects before they are refected at a population level [46]. However, when an effect is seen at the population level or at higher levels of organization (i.e., communities or ecosystems), it means that effects at individual organisms are expected to be occurring (Fig. 9.3) [45].

There can be two types of effects caused by ionizing radiation. They can be stochastic or non-stochastic (deterministic). Stochastic effects are effects that occur by chance and the higher the dose the higher the probability of its occurrence. However, the severity of those effects is not dependent on radiation dose. The main stochastic effects related to ionizing radiation exposure are cancer and genetic damage/ alterations (i.e., mutations) [47]. For non-human biota, stochastic effects that occur at germinal cells will be the ones that will have a higher impact, as they will have a higher probability of being inherited and, therefore, of affecting the next generations, impacting populations and communities [47]. Deterministic effects depend on time of exposure, doses and type of radiation. They are adverse tissue reactions that result from the damage or killing of many cells in an organ or tissue. The severity of these effects increases with dose when radiation levels reach a threshold, below which harmful effects to tissues/organs do not occur. The deterministic effects that are most important at a population level are mortality (which affects density, age distribution, and death rate), fertility (birth rate) and fecundity (which affects birth rate, age distribution, size of the population) [45] (Fig. 9.3). As for other stressors (i.e., chemicals), exposure to ionizing radiation can be acute or chronic. Acute exposures are short-term exposures to relatively high doses of radiation that usually last minutes or hours. Chronic exposures are long-term exposures or lifetime exposures to usually low doses of ionizing radiation. Doses in acute exposures are often reported as total absorbed doses, whereas for chronic exposures doses are often reported as dose rates (i.e., mGy/day, Gy/year, or mGy/h) [46, 48]. For a given dose of ionizing radiation, acute

**Fig. 9.3** Exposure and effects of different radiation types on organisms

exposure induces higher injury than chronic exposure [46]. The higher the dose the lower the ability of cells to correctly and rapidly repair the damage and also the lower the ability of healthy cells to divide and regenerate the damaged tissue [46]. Depending on the dose received by cells or organisms, several types of effects can occur, namely genetic damage, DNA lesions that can induce teratogenic effects (malformations) on embryos when occurring in germinal cells (i.e., gametes), cell transformation in somatic cells and cell death (Fig. 9.3). In some cases, DNA damage can be so severe that it becomes incompatible with the survival of the cell or of the entire organism. Depending on the kind of cells that are affected (germ cell or somatic cells), there can be different consequences. Severe damage (i.e., DNA double strand breaks, gross mutation like duplications, deletions, translocations, and chromosome gain or loss) will cause cell death potentially leading to the death of the organism or, for example, to its sterility if it occurs in germ cells (Fig. 9.3). If the damage is not enough to cause cell death, it can cause cell transformation and cancer in somatic cells or it can affect the ftness of the organisms and entire populations if it affects germ cells. Mutations can cause a reduction in the production of viable embryos or viable gametes and also, they can be passed and accumulated throughout generations reducing the population's ftness. Therefore, DNA alterations can have an important impact on fertility and fecundity and consequently in reproduction [46].

Also, there can be effects on the homeostasis of organisms (Fig. 9.3), namely depression of the immune system, alterations in normal metabolism, oxidative stress, and disturbances in the endocrine system [49]. The majority of the studies performed so far are focused on the determination of the acute effects of high doses of radiation, and only few studies are focused on chronic exposures to low doses of ionizing radiation.

The younger the organisms (namely fetuses and embryos) the more sensitive they are to the deleterious effects of radiation exposure. This is due to the higher sensitivity of cells that frequently undergo mitosis (which occurs frequently in young organisms for each tissue/organ as it is part of the growing process). Also, tissues/organs that have the ability to regenerate or that are constantly producing new cells like the hepatic tissue, the skin, the bone marrow, germinal cells, and gut lining are more sensitive to radiation (Fig. 9.3). The higher the cell division rate in an organism the more sensitive it will be to radiation's harmful effects.

Regarding the sensitivity of parameters like mortality and reproduction, in general the reproductive capacity is a more sensitive parameter to the effects of radiation exposure both for terrestrial and aquatic invertebrates and vertebrates, than life expectancy (mortality) [45]. Negative effects on reproduction rate can occur at less than 10% of the radiation dose required to induce direct mortality in mammals [45].

All organisms evolved in the presence of radiation, being cosmic radiation or natural radiation emitted by NORs present in the earth crust [50]. The studies performed so far, on the effects of ionizing radiation, showed that there is a considerable variation in the response of organisms from the same or different species, due to intra- and interspecies variability in sensitivity. In general, it is widely accepted that mammals are the most sensitive organisms, followed by birds, fsh, and reptiles and that invertebrates and other less complex organisms have the highest radiation resistance (Fig. 9.4) [46, 50]. However, it has to be noted that most of the knowledge gathered so far comes from laboratory exposures of specifc strains of these organisms and that results may differ signifcantly from what happens to their wild counterparts.

# **9.3.1 Basic Molecular Efects of Low and High Linear Energy Transfer (LET) Radiation**

The majority of the existing studies on the effects of ionizing radiation in cells are focused on DNA as the main target, making it clear that there is a cause-effect relationship between DNA damage with cytotoxicity and mutagenicity associated with ionizing radiation exposure. However, the cascade of molecular effects that lead to the induction of biological effects in exposed organisms is complex and involves, frstly, the interaction of radiation with water molecules and structural and functional biological molecules inside the cells. This interaction will induce the formation of ions, radical species, and excited molecules that will move from the site where they were formed to other cell compartments, causing damage to other biological molecules. This will trigger several signaling cascades, activating cell responses that will change the normal metabolic state of the cell, including changes in gene expression, enzyme recruitment and activities, DNA methylation patterns, and other stress-induced signaling events. When DNA is damaged, the cell cycle is interrupted allowing for DNA integrity check. DNA can be damaged directly through direct ionization or indirectly through the attack of free radicals that are formed when radiation interacts with water molecules of the cell [51]. Given the high content of water in cells, IR interacts with water in a process called radiolysis, generating free radicals as H∙ or OH∙, which trigger a cascade of events giving rise to other ROS as hydrogen peroxide and the superoxide anion [52] and references quoted. If not neutralized these products may diffuse within cells, as well as between cells, affecting other biomolecules such as DNA, proteins, and lipids, both in target and non-target cells (i.e., cells not directly irradiated) [53, 54]. Regarding DNA, ROS may oxidize bases or cause single and double strand breaks (SSB and DSB) [55]. Also, post-irradiation DNA lesions can be formed as a conse-

**Fig. 9.4** Schematic representation of overall sensitivities of different taxa to acute gamma radiation exposure. (Reproduced with permission of UNSCEAR, adapted from UNSCEAR 2008 report, Annex E)

quence of the attempt of the cell to repair sugar and base residues, which can be converted to SSBs (Single Strand Breaks) and DSBs (Double Strand Breaks) [51]. If DNA is correctly repaired, the cell will continue its cycle normally, if not, the cell can undergo transformation as mutations and chromosome aberrations may occur or if the damage is too severe, programmed cell death (apoptosis) will occur. The repairability of the damage and the repair accuracy will depend on damage severity and complexity. Low LET (beta particles, gamma and X-rays) and high LET (alpha particles and neutrons) radiation exposure can cause several types of DNA damage that are usually repairable, like SSBs, abasic and apurinic and apyrimidinic sites and DSBs (Fig. 9.5). However, the fraction of irreparable DNA damage depends strongly on LET. High and low LET radiation exposure can cause complex DNA damage, but this type of damage is more frequently associated with high LET radiation. Complex DNA damage is composed by closely spaced DNA lesions that form clusters [51]. Clusters contain two or more DNA lesions of the same or different origins, close to each other and on opposite strands (bistranded lesions). These lesions can be DSBs or non-DSBs oxidative clustered DNA lesions like SSBs, oxidized base lesions, and oxidized apurinic/apyrimidinic sites (AP sites) [51] (Fig. 9.5). These clustered lesions have a high mutagenic and carcinogenic potential since they are considered repair-resistant or even unrepairable due to the relative ineffciency of DNA repair systems to process such closely spaced and complex lesions. As there are several DNA repair systems in the cells and each of them is specialized in the processing of specifc lesions, when several types of lesions are closely spaced in the DNA molecule, the different repair systems cannot act properly, retarding the repair and often generating other lesions. High LET radiation is mostly associated with the generation of DSB's clustered DNA lesions and low LET radiation to non-DSB's oxidative clustered DNA lesions [51], but this is not completely clear and needs further studies. High LET radiation is also associated with increased frequency of chromosome aberrations, and also to a high frequency of unrejoined DSBs and consequently with a higher cell killing effciency, as unrejoined DSBs are a cause of cell death.

# **9.3.2 Efects on Microorganisms**

Microorganisms, including fungi, can be seen as good indicators of the ecosystem's "health." They include ubiquitous and taxonomically diverse microorganisms that play important key roles on diverse ecosystems' function. Specifcally, with regard to radiation, microorganisms play a very important role in the health of these systems and in their cleaning and decontamination.

**Fig. 9.5** High and low LET radiation DNA damage effects

# **<IP_ADDRESS> An Overview on Microbial Radiobiology: Radioresistance and Radiotolerance**

Microorganisms play a key role in the biogeochemical cycle of elements. In soils, they are important for organic matter turnover and maintenance of soil structure and fertility. As such, changes in the structure of microbial communities, by either metals or radionuclides, can have indirect effects on the above processes. Prokaryotes (bacteria and Archaea) have dominated a large part of the history of our planet, occupying virtually every "inhabitable" niche on earth. To be able to do that they have adapted to withstand large ranges in: (1) temperature, e.g., the hot temperatures found in hot springs and fumaroles, and the contrasting cold temperatures found on sea ice and polar regions, (2) pressure, e.g., deep sea, (3) salinity, e.g., hypersaline lakes, (4) pH, e.g., acid mine drainage sites, and (5) radiation, e.g., naturally occurring (deserts and high mountains, mining sites) and from nuclear contaminated sites [56]. Microorganisms that have adapted to such environments are referred to as extremophiles or polyextremophile (the latter being capable of withstanding different extreme conditions simultaneously), and these conditions are a requirement for their normal metabolic and biochemical operation. Most of these microorganisms belong to the domains Bacteria and Archaea although some fungal species have also been described. To survive these harsh conditions, extremophiles produce various primary and secondary metabolites, such as extremolytes, enzymes, and pigments [57]. Extremolytes, for example, are known to protect extremophiles cell structures and macromolecules from their harsh environments by forming protective water layers (e.g., ectoine), which is a co-solvent that shields proteins and cell membranes from UV light, heat, and dryness [58] around them or acting as chemical scavengers (e.g., carotenoids), protecting cells and their structures from UV radiation and oxidative stress [58]. Ultimately, the exceptional properties of these biomolecules fnd possible applications in various industrial sectors, in human healthcare, and well-being [59].

With regard to radioactively contaminated sites, microorganisms play an essential role on the mobility, toxicity, and distribution of radionuclides, through processes that include reduction, uptake, and accumulation by the cells, biosorption, and biomineralization with phosphates and carbonates [16].

Culture dependent and culture-independent approaches have shown the effects of long-term exposure to metals or radionuclides on individual species and on microbial communities. In addition, they have allowed those specifc genes and cell functions mostly affected by radiation and metals to be identifed, thus contributing to a better understanding of the molecular mechanisms behind microbial metal/radioresistance. Furthermore, the acquisition of genetic determinants by horizontal gene transfer contributes to shape microorganisms and microbial communities occupying these sites. More recently, refned metagenomic approaches focusing on prokaryotic communities have been employed and are expected to shed more light on the cells' strategies to overcome radiation stress to remain operational.

The following section addresses in more detail some of the mechanisms that contribute to the survival and maintenance of microorganisms in these environments. We will end by referring to the impact of more recent methodologies, such as metagenomics and other omics technologies, and their contribution to clarify aspects such as the impact of these contaminants on the microorganisms and communities that exist in these sites.

# **<IP_ADDRESS> Mechanisms Underlying Microbial Radiation Resistance: Cell Damage and Repair**

It has been reported that when radiosensitive microorganisms are subjected to multiple high IR exposures, their resistance increases [60]. This was recently demonstrated by experimental evolution, where populations of *Escherichia coli* very resistant to IR were generated in the laboratory, after 100 selection cycles, and to which the dose needed to kill 99% of the population increased from 750 Gy to about 3000 Gy [61]. Likewise, radioresistant species can become even more resistant with repeated exposure [62]. This "memory" adaptation is associated with smooth genetic alterations that affect DNA repair and metabolic functions. During this process of adaptation, other physiological characteristics of the microorganisms are profoundly affected as, for example, growth which is slowed down, because the microorganism must direct its energies to other processes, such as effectively repairing the damaged DNA.

The association between genome size and radiosensitivity between taxa has long been suggested. For instance, for the same chronic exposure to IR, fungi, for which genome sizes range between 12 and 20 Mbp, suffer more DSBs per unit time than bacteria with their smaller genomes (3–6 Mbp). However, this is not true for *Shewanella oneidensis* and *Deinococcus radiodurans* whose genomes are practically the same size, but while the former is killed after exposure to a radiation dose causing one DSB, the latter manages to recover from hundreds of DSBs. This is probably due to the fact that *D. radiodurans* has up to ten identical copies of its genome per cell and uses this genetic information to repair its DNA. In addition, there is also evidence for the interference of non-enzymatic antioxidants such as manganese complexes, which protect proteins from IR-induced oxidation, facilitating the maintenance of cell homeostasis and DNA repair. Although in many radioresistant bacteria and yeasts, the most common DNA DSB repair pathway is similar to homologous recombination (HR),1 in fungi, non-homologous end joining (NHEJ)2 is the preferred, as in other eukaryotes, despite being error-inducing. Melanin pigments also seem to be involved in protection against multiple stressors, including IR as it can act as an oxygen radical scavenger [62].

Radioresistant microbial extremophiles have developed strategies to survive and withstand dose rates that to the majority of organisms, including humans, would result in acute health effects [63]. It is believed that radioresistant microorganisms possess highly effcient processes to repair DNA damage. However, it has recently been demonstrated that the repair mechanisms and the proteins involved are common to those found in radiation sensitive microorganisms [64].

The genus *Deinococcus* is probably the most well studied and characterized and there is a great deal of information, to what its radioresistance is concerned. Metabolically active Deinococci vegetative cells can tolerate chronic radiation levels of more than 100 Gy/h, whereas other bacteria, Archaea, and fungi can be resistant to several kGy of acute IR. *D. radiodurans* exhibits resistance to acute IR up to 15 kGy, to 60 Gy/h of chronic radiation, and also to high levels of resistance to UV-C irradiation (100–295 nm), desiccation and oxidative stress. Thus, regarding the example of *Deinococcus radiodurans*, it can be argued that it effciently and rapidly repairs DNA damage caused by IR. A number of genes have been identifed whose expression is activated after irradiation, namely those encoding proteins associated with (1) effcient DNA repair, (2) protection against oxidation and (3) DNA supercoiling, which helps to maintain DNA integrity after irradiation [65]. More recently, it was demonstrated that in this organism, the adaptation to dryness and desiccation is at the basis of its radioresistance [64].

Nonetheless, it has been reported that *Deinococcus'* ability to repair DNA damage results from a selective pressure other than ionizing radiation, because there are no terrestrial environments subjected to the levels of radiation it tolerates. Still, the information gathered, albeit with some degree of uncertainty, has contributed to a better understanding of the mechanisms of radioresistance in other organisms, making this an excellent model organism to unravel these mechanisms [66].

Studies have shown that the DNA repair systems used by *D. radiodurans* are less complex than those of radiation sensitive bacteria, namely *Bacillus subtilis*, a spore-former species and *Escherichia coli*. Transcriptomics studies revealed that in response to γ-radiation, specifc genes involved in damage response are activated (*ddr*A, *ddr*B, and *irr*E (*ppr*I)). PprI, for instance, regulates the expression of the recombi-

<sup>1</sup>Homologous recombination (HR) repair: while in eukaryotes the process occurs during meiosis and requires homologous DNA sequences, in bacteria HR is a major DNA repair mechanism that facilitates the incorporation of exogenous DNA.

<sup>2</sup>Non-homologous end joining (NHEJ) repair: in eukaryotic cells, DSB are repaired predominantly by this pathway. Broken double-stranded ends are repaired by direct ligation without the need for a homologous template.

nase recA and pprA, which is a protein involved in DNA ligation that is essential for the radiation resistance exhibited by *D. radiodurans*. Strains lacking pprI show impaired genome recovery [66]. Another important DNA repair system involves the synthesis of long and single-stranded overhangs, a process referred to as "Extended Synthesis-Dependent Strand Annealing" (ESDSA).3 The process allows the reconstruction of a functional genome from the chromosome fragments produced by the exposure to radiation. Accordingly, the process is used by the RecFOR pathway to repair DNA double strand breaks. To support these observations, strains mutated in the genes involved in the RecFOR pathway are susceptible to γ-radiation [67].

Laboratory experiments with *Escherichia coli*, and other mesophilic bacteria, have shown that these may become resistant to the chronic exposure to IR just by adding Mn2+ and orthophosphate to its growth medium, which spontaneously form potent Mn-antioxidant complexes. Another important factor associated with radioresistance is cell density. For example, in *D. radiodurans* high cell concentrations seem to exert a protective effect against a radiation dose of 67 Gy/h [60]. Still, further and more complete studies are required until we know all the phenomena that contribute to the radioresistance exhibited by microorganisms. One thing is certain, it results from the interplay of several factors.

# **9.3.3 Multiomic Approaches Applied to the Study of Radioresistant Microorganisms**

Undoubtedly, multi-omics approaches (genomics, transcriptomics, proteomics, and metabolomics) will shed light and will further contribute to our understanding of the mechanisms involved in microbial radioresistance and detoxifcation. In order to contribute to a better understanding of the mechanisms involved in uranium resistance/tolerance, a recent high-throughput proteogenomic study was applied to bacteria of the genus Microbacterium, isolated from Chernobyl U contaminated soils and from natural U rich soils. The approach allowed the identifcation of proteins involved in membrane transport (e.g., ABC transporters and effux pumps), phosphate (e.g., phosphatases involved in biomineralization) and iron metabolism (e.g., siderophores), in addition to a large percentage of proteins of unknown function, which reveals the complexity of this mechanism [68]. Still, in another study carried out with a member of the genus Geobacter exposed to 100 μM U, proteins involved in DNA protection, in effux pumps of the RND family and in oxidative stress responses (e.g., SOD and superoxide reductase), were also identifed. Exploring these recent approaches will certainly allow us to gain knowledge that will contribute to clarify this complex intricate process. Furthermore, they will allow the selection for the best microorganism(s) with the potential to clean-up these contaminated sites by more eco-friendly processes. So far, in addition to the above study, genomic approaches proved useful in the identifcation of key genes and their respective products, encoded in the genomes of microorganisms resistant/tolerant to radionuclides/metals and which are, therefore, involved in the detoxifcation of this contaminants. With this approach, U-resistant bacteria of the genus Burkholderia and fungi of the genus Penicillium have been identifed. Transcriptomics studies, by giving access to the analysis of gene expression and regulation, have gained relevance in the area of bioremediation. The information gathered from this comprehensive analysis,

and also from future studies employing these methodologies, will surely shed light on the mechanisms of microbial resistance/tolerance to radionuclides/metals, while helping in the identifcation and selection of microorganisms that can be employed for bioremediation purposes of radionuclide/metals contaminated sites [69].

# **<IP_ADDRESS> Contribution of Metagenomics Approaches to Understanding Microorganisms' Radioresistance**

Unlike most laboratory studies, environmental exposure to radionuclides, (e.g., NORM sites and nuclear power plant accident sites), includes different radiation types (α and β, as well as γ) combined with many other stressors (e.g., temperature, nutrients, toxic chemicals like metals, etc.) over long periods. Thus, in polyextremophiles, the response to the adaptation/resistance should be broader and involve an intricate crosstalk between the different cellular processes [70].

Culture-independent feld studies have shown that radionuclide contaminated environments host a wide diversity of bacteria and that radionuclides strongly impact community function and structure. Recently, a metagenomics approach carried out in surface soil samples from Chernobyl and Fukushima, over a gradient of radionuclide concentrations (137Cs 1680—0.4 and 90Sr 209.1—1.9 kBq/kg), revealed that samples clustered according to the level of radiological contamination, irrespective of the collection site [71]. Nonetheless, a lower microbiota diversity was found in Chernobyl samples, which was expected as Chernobyl soils are more contaminated. The following were reported to be the most common phyla: Proteobacteria, Acidobacteria and Actinobacteria. Furthermore, as expected, the functions encoded by the genes identifed seem to be related with stress, metal and radiation tolerance. For instance, genes

<sup>3</sup>Extended Synthesis-Dependent Strand Annealing (ESDSA): a type of homologous recombination where the sequence around a DNA doublestrand break (DSB) is replaced by a copy of a homologous DNA template, while the original confguration of the fanking regions is maintained.

involved in decontamination, DNA repair, information storage and processing, cellular processes and signaling and metabolism. A comprehensive listing of the function of the genes responsive to this type of contaminants has been recently reviewed by Hoyos-Hernandez and co-workers [71].

A similar approach was employed in a study performed by Theodorakopoulos and colleagues [72], in Chernobyl, which demonstrated the high diversity of bacteria in those contaminated sites. The same authors isolated cultivable bacteria of the genus Microbacterium that were employed in laboratory exposure studies, contributing to a better understanding of the mechanisms of tolerance to radionuclides/ metals in those bacteria. The identifed mechanisms involve biosorption, effux and biomineralization [68].

Although further studies are required to better understand how radiological contamination exerts a selective pressure and how it shapes the structure of the microbial community, the sensitivity of the various organisms to radioactive contamination under environmental conditions generally exceeds the sensitivity of the same organisms to experimental laboratory exposures [62]. It is clear though that communities from soils of these contaminated sites have functional profles that allow them to deal with this type of radiological and chemical contamination. Furthermore, these environments constitute a genetic pool from which the phylogenetic affliation of cultivable and non-cultivable microorganisms can be determined, thus allowing the identifcation of new genes involved in the resistance to these contaminants, in addition to further contributing to clarify those mechanisms.

# **9.3.4 Efects on Plants**

Plants are sessile organisms that cannot leave the surrounding environment if the ecological factors are not suitable for their growth. Thus, under unfavorable circumstances, plants have only the choice to perish or adapt to changing environments. The extreme physiological plasticity of plants allowed their diffusion in all ecosystems of the Earth and today we may have a comprehensive vision of the multitude of adaptations carried out by these organisms in diverse places. Indeed, plants such as other living organisms can adapt to cyclical natural disturbances over time, developing the capacity for endurance (resistance) and self-repair (resilience) in different ecosystems.

Laboratory and feld studies showed that ionizing radiation may exert different effects on plant metabolism, growth and reproduction, depending on plant developmental stage at the time of exposure, plant physiological and morphological traits, as well as genetic characteristics [73, 74]. Moreover, depending on the dose or radiation type (low or high-LET), ionizing radiation induces detrimental outcomes at high doses, harmful consequences at intermediate levels and stimulatory effects at low doses.

In some cases, ionizing radiation exposure increases embryo lethality, induces dwarf architecture and modifes foral elements [74] and literature herein. Other studies indicated that some irradiated crops showed a taller architecture, increased yields and reproductive success and the ability to endure water shortage [75, 76]. As for many other organisms, within plant cells, the nucleus is considered the primary site of injury by ionizing radiation, which is responsible for random DNA damage and generates different kinds of mutations, such as deletions, base substitutions and chromosomal alteration [74, 77]. There is a direct relationship between the radiosensitivity of a plant and the average volume occupied by a chromosome in the cell nucleus. If the chromosome volume is large, the plant will be more sensitive and, therefore, the dose of ionizing radiation causing severe damages is less. Hence, polyploid species exhibit a minor sensitivity to radiation damage because gene redundancy protects polyploidy from the deleterious effect of mutations [78]. Besides plant cells, it is noteworthy that ionizing radiation may have different impacts on organs and tissues. Generally, more complex tissue architecture is less sensitive to damage; thus, young tissues are more vulnerable than old [73, 79]. At functional level, many studies have evidenced that radiation is dangerous for the photosynthetic apparatus. Generally, a decline of photosynthesis often implicates damage to photosystem II (PSII) and in particular to D1 protein, implicated in the right functioning of photosynthetic electron transport. Together with the impairment of PSII, a signifcant decrease of photosynthetic pigments and enzymes of the carbon assimilation cycle was also detected [73].

The majority of information on the impacts of radioactivity on plants comes from studies carried out by scientists after the nuclear disasters of Chernobyl (Ukraine) in 1986 and Fukushima (Japan) in 2011 [80].

Since 1986 the Chernobyl red forest has represented a living laboratory for biologists to study for long-lasting plant behavior in response to acute and chronic radioactive contamination. The name "Red Forest" comes from the gingerbrown color of the pine trees as a result of the high radiation levels immediately after the explosion of the nuclear plant. Studies continued in the post-accident period and enlarged the knowledge on the effects of acute and chronic radiation on plants [81]. Generally different plant species show diverse sensitivity to radiation, being shrubs more resilient than conifers. The sensitivity of the pine compared to other tree species was most apparent in the Chernobyl exclusion zone and trees showed dramatic alterations in the morphology of trunks and branches, indicating damage at meristems level [82]. Following the Fukushima accident, despite the much lower exposure levels, Japanese red pine (Pinus densifora Siebold & Zucc.) and Japanese fr (Abies frma Siebold & Zucc.) species showed developmental anomalies similar to those observed in Chernobyl [83, 84]. However, it is uncertain if the aberrations observed in Chernobyl are due to direct effects of radiation on the trees or multiple stresses due to biotic and other abiotic factors.

It is noteworthy that the quantity of radionuclides absorbed by plants depends on their phenological stage and growth status which, in turn, varies with the pedo-climatic conditions and cultivation factors. Once deposited on the vegetation and in particular on the leaf surface, the radioisotopes are absorbed through stomata and then transported to the other organs including fruits, thus possibly entering the food chain through edible leaves and fruits [85].

Today the Red Forest remains one of the most contaminated sites globally, and the surrounding forest area also represents an area of active research and scientifc interest because of the return of wildlife in the exclusion zone. Here, the understory vegetation and deciduous (silver birch) trees have reappeared, but radioactive dust still remains stored in plant biomass and soil, for the very slow matter cycle.

The occurrence of revegetation has proven to be remarkably resilient to the intense radiation around the nuclear disaster zone. The exclusion zone is now dominated by grasslands and shrublands, while the most representative trees are Scots pine and silver birch Betula pendula [74] and literature herein.

Recent studies suggest that plants subjected to not-lethal doses of ionizing radiation show an increased resistance to other environmental stresses. Two strategies have been hypothesized, namely the production of ROS-mediated cell signaling and/or a boost of secondary metabolites [86].

The resilience to radiation in plants of the Chernobyl exclusion zone and from most contaminated sites at Fukushima is due to different mechanisms to protect the genetic material, improving the plant radioresistance [80]. Generally, plants are more radioresistant than animals because they present integrated adaptation mechanisms at genetic, anatomical, and physiological levels.

At genetic level, mechanisms include the regulation of expression of some genes encoding for radical scavenging and DNA-repair enzymes, homologous and non-homologous recombination, and the activation of scavengers. The higher stability induced by polyploidy, typical among plant kingdom, enhances radioresistance thanks to the presence of several copies of the same genes, which may serve as additional wild type copies in the case of radiation-induced injuries [87]. At the structural and metabolism level, plant cells present some traits such as thickened cell walls, cuticles, pubescence, increased deposition of phenolic compounds around membranes [88, 89]. At the anatomical level, complex tissue organization is associated with high resistance to mutagenic effects and the capability to adopt repair mechanisms.

Non-lethal doses of ionizing radiation may also induce hormesis improving plant defense against stressors, through the stimulation of the production of antioxidant enzymes (SOD, CAT, APX) or morpho-anatomical and photosynthetic changes that favor plant growth and metabolism [74, 90, 91].

Radiation-induced hormesis is still an unclear phenomenon in plants because it strongly depends on species intrinsic characteristics. At present, further studies are in progress to understand if it is a sort of compensation to irradiation damage or a transitory change, not enough to induce permanent injuries.

# **9.3.5 Efects on Invertebrates**

Invertebrates have been considered a relevant group of organisms for studying the effects of ionizing radiation, both focusing on mechanisms of action and on previewing impacts in natural communities. Several reasons can be enumerated for choosing aquatic and terrestrial invertebrates as model organisms for IR studies, namely:

Aquatic invertebrates as benthic organisms and invertebrates living burrowed in soils or dwelling at the surface are among the group of organisms that may receive the highest radiation doses, since these environmental compartments are relevant environmental sinks of radionuclides. The mechanisms of action and the subsequent effects of ionizing radiation in invertebrates have been addressed mainly since the seventies, with a limited number of species, through laboratorial exposures to gamma radiation of single species, frequently at high-dose rates, with few environmental relevance for chronic exposure scenarios [98]. Real conditions include exposures to industrial radionuclides in areas affected by nuclear accidents, nuclear power plants, or in nuclear test sites, as well as through exposures to natural occurring radionuclides (NORs), as those found in uranium mining areas. In the later areas, the effects of radionuclides, mainly alphaemitters, cannot be distinguished from that of metals, also present at high levels in the affected environmental matrices. The same diffculty exists in areas of nuclear accidents as the Chernobyl exclusion zone, where the release of different artifcial radionuclides has occurred, although data available for activity concentrations in biota are almost limited to 90Sr, 137Cs, and some few other radionuclides [38].

Invertebrates are among the least sensitive organisms to ionizing radiation [62, 99]. Cassidy and co-authors [100] suggested that the reasons for these differences in sensitivity, between organisms of different taxonomic groups, may include differences in DNA content, DNA repairing processes, and kinetics of cell cycle, within other aspects. The doses able to cause mortality or decrease life span are species dependent and frequently very high: as for example above 1000 Gy for *Caenorhabditis elegans* [101]. However, differences in sensitivity of different life stages were also reported (i.e., Johnson and Hartman [101]), with reproduction effects being seen at much lower doses (i.e., 4 mG/h for earthworm).

Ionizing radiation hormesis has been reported in a number of studies with invertebrates (dipterans, coleoptera), exposed to low doses from different sources (X-ray, gamma radiation, 137Cs) (see review by Vaiserman et al. [97]). Reduced mortality rates and long-life spans were highly dependent on the exposure conditions [102], for example, life-extended effects were only observed in house fies (*Musca domestica*) reared in groups, and thus under high locomotor activity and exposed to a 10 Gy dose. Several hypotheses were then postulated and tested to unveil the factors responsible for modulating radiation hormesis, using *Drosophila melanogaster*, as model species, as for example: increased IR resistance, IR-induced sterility in females, apoptosis induction and changes in DNA repair genes and life-stage differential sensitivity were some of the proposals [97] and references quoted herein. X-ray irradiation of *D. melanogaster* eggs with 0.75 Gy, decreased the amount of DNA segments, by cleavage of S1 nuclease sensitive sites (<3 kb), resulting in a great DNA stability, changing the repair and/or transcription processes and thus affecting lifespan and the resistance of adults to IR [103]. Based on all the studies conducted, the radiation hormesis model proposes that the exposure to low doses of IR could induce several adaptive responses, which in turn will prevent environmental-induced health effects [97].

At the cellular level, oxidative stress and the activation of oxidative stress-response mechanisms have been reported as the major indirect consequences of exposures to IR of aquatic and terrestrial invertebrates. Won and Lee [104] observed a signifcant increase in the activation of several enzymes, as for example, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione-S-transferase (GST) in the marine copepod *Paracyclopina nana*, exposed to gamma radiation doses equal or greater than 10 Gy (at a dose rate of 2 Gy/min). However, in this study, no data from additional molecular parameters, as those related with DNA damage or lipid peroxidation were provided, preventing us to infer if the activation of these enzymes was suffcient or not to prevent cellular damages. A dose-dependent increase in ROS was also recorded in another marine invertebrate species, as for example, the copepod *Tigriopus japonicus* and the rotifer *Brachionus koreanus*, for a range of concentrations from 50 to 200 Gy (irradiated at a dose rate of 2 Gy/min) [105, 106]. Concomitantly, the antioxidant response system was activated, and GST and GR activities were signifcantly increased for the copepod, while for the rotifer the same was recorded for the activity of GST. A cellular and lipid peroxidation (LPO) related ROS was dose-dependent overproduction was also recorded in the freshwater cladoceran *Daphnia magna* after 8-day exposure to a dose rate of 100 mGy/h of gamma radiation. The overproduction of mitochondrial ROS was signifcantly enhanced at 40 and 100 mGy/h [1]. Dose rates of the same order of magnitude (10.7 and 42.9 mGy/h) were also able to cause lipid peroxidation in daphnids, after both 24 and 48 h of exposure. However, at the highest dose rate tested (106 mGy/h), the same effect was only registered after 48 h of exposure [55]. This observation, which was consistent with other studies (i.e., Fuller et al. [107]), gave rise to the hypothesis that ROS may also act as a signaling molecule, requiring a certain level within the cell to activate antioxidant defense mechanisms. Neutral lipid catabolism was also observed in the nematode *C. elegans* independently of the different doses and dose rates tested (7 and 52 mGy/h), and this effect was associated with a reduced longevity, as lipid homeostasis is responsible for endocrine signaling of longevity [108]. In fact, the up-regulation of different hormone receptors in daphnids was suggested as a signal of disruption of normal endocrine functions in response to IR exposure [1].

Regarding the interaction of ROS with proteins, Won and Lee [104] registered an upregulation of the *hsp* gene in the copepod *P. nana*, which was interpreted as being related with a possible response to protect key proteins (probably those involved in DNA repair signaling pathways) through the synthesis of chaperones.4 In the cascade of events promoted by

<sup>4</sup>Chaperones—are proteins that assist other proteins folding.

ionizing radiation, Song and co-authors [1] recorded an enhanced expression of the *Ube2* gene in *D. magna*, involved in the degradation of proteins, suggesting the activation of a mechanism responsible for the elimination of proteins damaged by ROS. A highly effcient antioxidant protection system may not be able to protect DNA from damage but it can delay protein carbonylation.5 Therefore, protecting cellular components involved in the repair of DNA double-strand breaks (DSB) was proposed as a main factor to explain the resistance of bdelloid rotifers to ionizing radiation [109].

DNA damage is a frequently reported effect in invertebrates exposed to IR from different sources. These damages can be either caused indirectly, mediated by ROS, or by direct deposition of radiation energy in DNA [1, 104]. In response to DNA damage, the expression of genes related with DNA repair systems (e.g., *p53*, RAD50, *Mre11* coding for the DSB repair protein, *Ku70*, *Ku80,* and *DNA-PK*) was recorded in different invertebrate species, frequently with a non-monotonic response, but always with a signifcant and differential expression at low and higher dose rates (at 4, 100, and 200 mG/h) [1, 55, 104, 106]. In summary, genes involved in nucleotide excision, base excision, homologous recombinant, and non-homologous recombination repair pathways have been found to be all involved in the response of cells to IR. At high IR dose/dose rates, ROS may also induce DNA methylation,6 leading to the accumulation of damages, by silencing some genes. Song et al. [1] recorded an enhanced expression of DNA (cytosine-5)-methyltransferase 1 (Dnmt1), DNA cytosine-5 methyltransferase 3A2 (Dnmt3a2) genes, involved in maintenance of DNA methylation and in de novo DNA methylation, respectively, in *D. magna*.

The disruption of energy metabolism under the exposure to IR is another reported effect at the cellular level, once again in different invertebrate species [1, 55]. Direct interference with proteins of the electron transport chain, mitochondria ultrastructural changes caused by ROS and modulation of oxidative phosphorylation are within some of the mechanisms proposed, based on observations made in *D. magna* exposed to gamma radiation [55]. Genes encoding NADH dehydrogenase (*Nd*), succinate dehydrogenase subunit A (*SdhA*) of complex II, different cytochrome oxidase subunits (*COX1*, *COX2,* and *COX3*), cytochrome c oxidase copper chaperone (*COX17*) of complex IV and ATP synthase subunit mitochondrial (*sun*) of complex 4 were some of the genes involved in the electron transport chain found to be suppressed by gamma radiation [55]. At the end of the cascade of events triggered by gamma-radiation, the regulation of different apoptotic signaling pathways was observed in freshwater Cladocera, in parallel with DNA damage and regulation of repair mechanisms, cell cycle disruption and mitochondrial dysfunction [1, 55]. Although not signifcant, an increasing trend in apoptotic cell death with increasing dose rates of radiation was recorded in crustaceans, namely daphnids and in the Norway lobster (*Nephrops norvegicus)* cell cultures exposed to 60Co gamma-radiation [110]. Apoptosis is a downstream event, to oxidative stress and DNA damage occurrences, that is activated to eliminate damaged cells in an ultimate effort for protecting organisms.

The effects of ionizing radiation at the population level are poorly documented and it has been demonstrated that equal levels of effect at similar individual endpoints (e.g., growth or reproduction) may have different impacts on population dynamics [111]. Furthermore, it is still diffcult to link the results of biomarkers of oxidative stress and genotoxic damage with phenotypic consequences (changes in morphology, growth, reproductive output, and viability of offspring) [112]. Data available allowed a tentative hierarchization of individual endpoints based on their radiosensitivity: mutation > reproduction > morbidity and mortality [113]. One step forward, modeling population responses it was shown that they differed depending on the affected individual reproduction endpoint (juvenile or adult survival, delay in maturity, or reduction in fecundity) [114]. Hatching was shown to be the most sensitive endpoint to chronic exposures to gamma radiation for aquatic invertebrates (EDR107 of 830 mGy/h for the polychaete worm *Neanthes arenaceodentata*) and fecundity for terrestrial invertebrates (EDR10 of 2600 mGy/h for *Porcellio scaber*). These species displayed similar EDR10 values for individual and population level endpoints (net reproduction rate). This was observed for the species that had a particularly sensitive individual endpoint.

The most concerning consequences of genotoxicity, that may support inferences about potential effects on natural populations, are those that affect the reproductive ftness of organisms. Reproduction has shown to be the most sensitive parameter in invertebrates (collembolans, worms, tardigrades, chironomids, and polychaetes) exposed to IR, when compared with survival or other endpoints at the individual level [109, 115–120]. It was suggested that the decrease in fecundity is not caused by the number of DNA DSB, but by the inactivation of the DNA repair systems [109]. In fact the incomparable ability of bdelloid rotifers to remain fertile, after extensive DNA damage, was attrib-

<sup>5</sup>Protein carbonylation—Reaction of hydroxyl radicals with side chains of certain aminoacids causing irreversible oxidation of proteins.

<sup>6</sup>DNA methylation—DNA methylation of eukaryotic cells is an epigenetic signaling mechanism characterized by the transfer of a methyl group onto the C5 position of the cytosine to form 5-methylcytosine, by DNA methyltransferase enzymes. DNA methylation regulates gene expression by recruiting proteins involved in gene repression or by inhibiting the binding of transcription factor(s) to DNA.

<sup>7</sup>EDR10—effective dose rate inducing an effect of 10%.

uted to high effciency of repair systems and to mechanisms that protect proteins of these repair systems [121]. These authors also associated the resistance to ionizing radiation of these organisms with their resistance to desiccation resulting from their adaptation to ephemeral ponds. Desiccation, similarly to radiation, increases ROS production and DNA breakage.

Harrison et al. [112, 122], also working with the polychaeta *N. arenaceodentata*, hypothesized that chromosomal aberrations caused by gamma radiation doses of 2.0 and 4.0 Gy were responsible for gametal cell death and subsequent decreases in brood sizes of this species. In opposition, under laboratory conditions, signifcant effects were recorded in sperm quality, but not on sperm numbers, of males of the crustacean *Echinogammarus marinus* chronically exposed to doses rates of 1 and 10 mGy/day provided by the betta emitter 32P, for two weeks. Signifcant DNA damage was recorded in spermatozoa cells only at the highest dose rate. Furthermore, only a weak correlation was found between sperm quality parameters, fecundity, and embryo parameters analyzed [107]. Effects on ovary structure and oocyte development were also reported in the freshwater cladoceran *D. magna* in response to exposure to 1 and 100 mGy/h gamma radiation, dose rates.

Another possible cause of reproduction impairment in invertebrates, under exposure to ionizing radiation, may be related with the allocation of energy to molecular response mechanisms (e.g., activation of antioxidant defense system, DNA repair mechanisms) rather than to reproduction, with consequences on the fecundity of organisms [117].

An ED508 for reproduction of 21.9 Gy, one order of magnitude lower than that recorded for growth (144 Gy) was found for the collembolan *Folsomia candida*, under exposure to 137Cs gamma radiation at a constant dose rate of 8.3 Gy/ min. Song et al. [1] also observed a non-monotonic reduction in the total number of offspring of the cladocera *D. magna*, concomitantly with no effects on survival, molting or ovulation frequency (at dose rates of 1 and 100 mGy/h). At the lowest dose, the effect on the cumulative reproduction output was mainly associated with an increase in the number of days needed to deliver four broods, while at the highest dose rate, the reproductive cycles were accelerated but the size of the broods was reduced. A similar observation was made by Parisot et al. [123] in the same organisms exposed to dose rates of 0.07–35.4 mGy/h of gamma-radiation, for 23 days. The same non-monotonic response was recorded *D. magna* representing 38 different genotypes collected in lakes located inside the Chernobyl exclusion zone with a range of dose rates between 0.1 and 181.2 mGy/h.

In a study conducted by Alonzo and collaborators [111], the freshwater species *D. magna* and the terrestrial earthworm *Eisenia fetida*, two species with different life history strategies (short lived/parthenogenic versus more long-term life/sexually reproducing hermaphrodite, respectively) were selected: (1) to model population growth in response to individual effects caused by the exposure to IR and (2) to investigate populations susceptibility using two different models to take into account single generation and multiple generation exposures. It was shown that in daphnids, the population growth was 1.5-fold more sensitive to changes in fecundity than in mortality. Daphnids population growth was also highly affected by delays in reproduction. Earthworms' population growth was more sensitive to delays in reproduction, while effects in fecundity and mortality have a similar and lower impact on populations. Despite the different life strategies, the intrinsic rates of population increase were equivalent for both species, because the greater reproductive rate of daphnids is compensated by a shorter life span relative to earthworms.

After disturbances of great magnitude, the recovery of natural populations of cladocera may rely on the banks of resting eggs in the sediments of lentic systems. These resting eggs if irradiated may have its performance compromised, affecting the dynamic of the natural populations. Zadereev et al. [124] observed that although doses up to 100 Gy (variable dose rates) did not affect the survival and hatching of resting eggs of *Moina macrocopa*, the size and the structure of populations initiated from resting eggs exposed to this highest dose of gamma-radiation, were affected. Therefore, subsequent effects on the dynamic of the populations of this cladocera may be expected in lakes with highly contaminated sediments.

Under a real scenario of radionuclides contamination, no correlation was found between different reproduction endpoints (proportion of breeding females, fecundity, brood mass, maternal body mass) of the crustacean *Asellus aquaticus*, sampled at different lakes in the Chernobyl affected area, with the gradient of dose rates between 0.064 and 27.1 mGy/h registered at these ecosystems [107]. Also, upscaling to populations and communities, Murphy et al. [125] focused on the diversity of littoral macroinvertebrates communities at eight natural lakes in *Belarus*, with a range of external dose rates from 0.066 to 10.22 mGy/h once again did not fnd any correlation between population endpoints (abundance, taxon richness, Shannon-Wiener diversity index, and the Berger-Parker dominance index) and the range of external dose rates registered in the sampled lakes. This study suggested that the IR dose rates recorded had no detectable effects on the littoral macroinvertebrate communities of these lakes.

Impacts on natural populations of invertebrates may be also caused by other mechanisms rather than those affecting gamete production, eggs viability, fecundity, or reproduction delays. For example, the exposure of fourth-instar nymphs to IR from a 137Cs source up to doses of 12 Gy (at a dose rate of 0.25 Gy/min) has shown to affect the acous-

<sup>8</sup>ED50—effective dose causing a 50% effect.

tic signaling of male crickets (*Acheta domesticus*) and subsequently their ability to fnd mates, due to morphological changes in their wings [126]. In fact, few is known about other direct and indirect effects that may affect the ftness individuals, its biotic relationships, and subsequently the dynamics of natural populations and communities at IR contaminated scenarios, rather than those effects identifed based on commonly used biomarkers. The complexity of the biotic interactions, as well as the role of dominant abiotic factors determines the type and the impact of the indirect effects on ecosystems, whose responses can be unpredictable [98]. In a birch forest in South Urals, the contamination of litter with 90Sr (doses reaching up 70 Gy) compromised the development of pupae of tachinid fies (Tachinid sp.). This accounted for an increased survival of the host caterpillars of the gypsy moth (*Lymantria dispar dispar* L.) with increasing IR levels [127] in Geras'kin [98]. Møller et al. [128] also linked the reduction in the set of fruits produced by trees and bushes, at the Chernobyl exclusion zone with the local reduction of pollinator insects. The role of other biotic factors in the radiosensitivity of invertebrates also needs to be investigated, as it may be relevant under specifc environmental or industrial scenarios. It was shown that the ability of marine mussels (*Mytilus galloprovincialis*) to respond to genotoxic induced effects by tritiated water, released by cooling operations of nuclear power plants, was limited by enhanced temperatures [129].

Invertebrates also have a key role in several ecosystem functions, as, for example, the degradation of wood, organic matter, and nutrients recycling. Mousseau et al. [130] conducted a study in forest areas within the Chernobyl exclusion zone, at different distances of the nuclear plant and with levels of background radiation differing by several orders of magnitude (range 0.09–240.25 mSv/h). A signifcant effect of background radiation in the mass loss of litter bags buried in the surface of the forest soils was registered. The mass loss of litter bags from the sites with high levels of background IR was 40% lower than that recorded at the sites with lower levels of radiation. However, no signifcant infuence of the mesh size of litter bags was found, suggesting that decrease in the decomposition of litter at that site was not only caused by impacts on soil invertebrates' communities, but also on soil microbiome. Soil invertebrates' assemblages from pit falls and wood slices from the same area showed that the abundance of taxonomic groups displayed a different relationship with background radiation and with wood contamination with radionuclides, being positively, negatively, or not affected at all [131, 132]. This was consistent with a previous observation of a general loss of diversity in sub-surface and fying invertebrates with increasing concentration activities of 90Sr and 137Cs in the litter of forest sites within the Chernobyl exclusion zone [133], as well as by the apparent decrease in the feeding activity of these organisms measured by the bait-lamina test.9 However, such changes were not followed by changes in total biomass of organisms. These results suggest that chronic environmental exposures to IR may exert their effects on natural communities, through structure and functional diversity simplifcation, with possible impacts on ecosystem's functions.

In a frst attempt to estimate risk limits for chronic g-radiation exposures, predicted no effect dose rates (PNEDR) of 10 mGy/h (0.24 mGy/day) for freshwater ecosystems and of 67 mGy/h (1.61 mGy/day) for terrestrial ecosystems were obtained, using assessment factors and species sensitivity distribution methods, respectively. The estimated values were found to be highly protective as they were about ×50 to ×100 times higher than the upper bound of the range of natural background concentrations and of the lower dose rates causing effects at contaminated sites [134]. Later, and by applying an assessment factor (AF) of 3 to the HDR5 10 estimated for invertebrates, a PNEDR of 170 mGy/h for IR was obtained. However, and considering that no suffcient data was available for applying probabilistic methods to estimate PNEDR for specifc groups of organisms or for environmental compartments, Garnier-Laplace et al. [135] derived a generic HDR5 from a species sensitivity distribution using data from controlled laboratorial chronic exposures to low dose rates of gamma-radiation, and applied an AF of 5, obtaining a PNEDR of 1.5 mG/h which was considered to be protective for the conditions found at Chernobyl exclusion zone.

# **9.3.6 Efects on Vertebrates**

#### **<IP_ADDRESS> Terrestrial Organisms**

#### **Mammals**

Among all the vertebrates, mammals are organisms on which the effects of radiation exposure were most extensively studied in radiobiological experiments. Negative effects on these organisms, due to radiation exposure at high doses (i.e., 10–50 Gy), are primarily due to effects at the hematopoietic system and the gastro-intestinal mucosa [45, 46]. The time needed for death to occur varies widely within species. The dose of radiation needed to cause lethality, due to gastrointestinal syndrome, to 50% of the exposed organisms (LD50) is approximately as follows for dog—8 Gy, mouse—12 Gy, rat—11 Gy, and rhesus monkey—9 Gy [46]. However, these values were estimated for particular species of these organisms, so there can be wide variations for other species. These

<sup>9</sup>Bait-lamina test—a feld test performed with baited lamina which are buried in the soil to measure the feeding activity of edaphic fauna (for more details, please see [181, 182]; ISO 18311:2016).

<sup>10</sup>HDR5—the hazardous dose rate for 5% of the species.

variations are normally related with specifc intestinal morphologies, which are related to diet (i.e., herbivores, carnivores, and omnivores). Regarding bone marrow damage, the weight of the animals receiving the dose appears to have a signifcant role in the bone marrow radioresistance, being weight inversely proportional to radiation sensitivity, as LD50 values are greater for smaller mammals (6–10 Gy approximately) than for larger ones (1.2–3.9 Gy). A reduction in life span is also related to the type of radiation to which animals are exposed, being high LET radiation more effective than low LET radiation. Also, acute exposures are substantially more effective by a factor of 7 in causing mortality than chronic exposures [46]. Signifcant life span shortening occurred in dogs and mice exposed to low LET radiation (gamma radiation) at dose rates between 100 and 1000 mGy/h and the same happened for mice exposed to neutrons (high LET radiation) at the same dose rates [136]. In general, a signifcant reduction in life span of several mammal species was observed at dose rates higher than 1000 mGy/h [50, 136, 137]. Chronic exposures of less than 100 mGy/h have a low probability of inducing signifcant effects on most terrestrial organisms [45, 46, 136]. Particularly, a dose rate of less than 40 mGy/h has a low probability of inducing effects on the fertility, fecundity, and the production of viable offspring of a mammalian population [45]. This is true for low LET radiation, however for high LET radiation, this dose rate value is lower, as this type of radiation has a much higher relative biological effectiveness (RBE) [45]. An experiment performed in mice irradiated with neutrons, at dose rates lower than 100 mGy/h for at least 475 days, led to a signifcant increase of mortality in mice in comparison with the control [136].

Reproduction is a more radiosensitive parameter than mortality, and effects of radiation may appear at radiation levels that apparently do not induce other observable responses. The magnitude of the effects depended also on the developmental stage in which the animal was irradiated [136]. A good example are mice, as the LD50 occurs at a radiation dose approximately between 6 and 10 Gy; however, at a radiation dose of 0.08 Gy, the production of oocytes was reduced to 50% in newborn mice (the most radiosensitive stage in mice) [45, 46]. However, this does not necessarily mean that there will be a decline in fecundity, since mice produce much more oocytes than the amount effectively used for reproduction, but there could be a reduction in the offspring [46]. In adult males, fertility is temporarily impaired after a 10 Gy exposure; however, in young mice (3–5 days old), it can cause permanent sterility. Mice in the second week after birth are also especially sensitive to the detrimental effects of radiation on reproduction [136]. The differences between males and females are mostly a consequence of the differences in the gametogenesis process. There are also differences between species, being mice one of the least radioresistant. Chronic irradiation affects mainly the time needed for oogonial cell division and the size of stem cell pool [46]. In males, the spermatogenic process is maintained, although at lower levels than unexposed organisms [46].

The developing embryo is particularly sensitive to radiation, due to the high number of cells proliferating, reducing fecundity and postnatal survival, potentially infuencing population size [46]. Acute radiation exposure, before the implantation of the embryo, causes its early death and can also cause post implantation and postnatal death [46]. This has a good correlation with the occurrence of DNA damage in the form of chromosome aberrations in the blastomeres (cells that result from the cleavage during the early development of the embryo) [46]. Radiosensitivity is strongly infuenced by cell cycle stages and mitotic cycle in the very early developmental stages [46]. During organogenesis, the most typical response to acute radiation is the occurrence of malformations (teratogenic effects), which can occur during embryonic and fetal growth and may or may not be fatal. The occurrence of teratogenic effects in a particular organ is related to a high level of cell proliferation in the precursor tissue [46]. Although this has been observed for the several species studied (mouse, hamster, cattle, pig, monkey rabbit, etc.), the responses to specifc radiation doses will depend on the species and on its developmental stage at the time of exposure [46]. There are not many studies on the effects of chronic radiation exposure during organogenesis, however a study performed on mice showed doses of 0.01 Gy/day in pregnant mice 6–9 days after conception induce a signifcant impairment of the offspring's learning ability [46]. Also, dose rates of 420 mGy/h reduced neonatal brain weight, with unknown effect at the functional and behavioral levels [45].

A direct relationship between DNA damage and radiation dose is expected at high doses of radiation; however below 100 mGy, it is not clear. In reindeer, a tenfold increase in the number of chromosome aberrations was observed at dose rates between 100 and 1000 mGy/h [136]. For rodent species acutely exposed to low LET radiation, mutations in the form of reciprocal translocations (exchange of DNA between homologous chromosomes) occur in stem cell spermatogonia when organisms are exposed to between 0.01 and 0.03 Gy at total doses from 3 Gy [46]. High LET radiation exposure (in the form of alpha particles emitted by 239Pu), delivered at a dose rate of 36 mGy/h signifcantly increased the occurrence of translocations and acentric fragments (chromosome fragments without a centromere) in spermatogonia and spermatocytes, respectively [46]. In primates, the dose interval is 0.01–0.078 Gy at doses from 1 Gy [46]. Translocations, ring chromosomes (aberrant chromosomes whose ends were broken and then fused together to form a ring) and dicentric chromosomes (the result of two broken chromosomes that fused together) are used for radiation dosimetry in human and non-human biota for a long time, as their frequency increases with radiation dose [138]. In rodents, this is more easily seen at total absorbed doses higher than 0.5 Gy, suggesting that their use as a biomarker of radiation exposure is more effective at high dose exposure than at low doses (below 100 mGy). Regarding carcinogenicity, there is a wide variation in the sensitivity for tumor formation among tissues and species. The induction of cancer, even at high radiation exposure doses (>100 mGy) will also vary according to the age of exposure. Dogs exposed to doses higher than 7 Gy showed soft tissue cancers when exposed in utero but not when exposed as young adults [46]. In rodent species, there were limited carcinogenic effects on animals that were exposed to doses between 0.1 and 1 Gy [138].

#### **Birds**

The effects of radiation exposure in birds are apparently similar to the ones observed in small mammals [45]. The LD50 for wild birds is in the same range as small mammals (5–12 Gy). For poultry, the LD50 determined experimentally for mortality is of 7–11 Gy in 3–4-day-old individuals when irradiation lasts for less than 1 h and of 12–20 Gy when irradiated for 24 h. Egg production is affected in white leghorn chicken at a total absorbed dose of 4–8 Gy and at higher doses, effects are more severe and long lasting [45]. A limited number of experiments performed in artifcially incubated chicken embryos showed a LD50 of 12–13 Gy, which apparently indicates a higher radioresistance than adults [46]. In white leghorn chickens, eggs hatchability is affected at a total absorbed dose of 8 Gy, but the progeny is unaffected [48]. The International Commission on Radiation Protection also reported dose ranges for which long-term effects on developing embryos were reported (100– 1000mGy/day), reduced reproductive success (1–10mGy/ day) and increased morbidity (10–100 mGy/day) [139]. Recently, it was reported a decrease in species abundance at a dose range of (from 0.3 to 97 μGy/h) in the Fukushima exclusion zone, which is consistent with the dose ranges reported for increased morbidity and decreased reproductive success [140]. The existing knowledge on DNA damage/alterations on birds exposed to ionizing radiation results from the evaluation of effects of radioactive environmental contamination resulting from the Fukushima and Chernobyl accidents [141].

#### **Reptiles and Amphibians**

The information gathered so far for reptiles and amphibians suggest that their radiosensitivity is similar to that of mammals and birds. The LD50 values recorded for frogs, salamanders, turtles and snakes vary between 2 and 24 Gy [46]. The main cause of death identifed was damage to the hematopoietic system [46]. In two separate experiments performed on lizards, two very different LD50 doses ranges were obtained (10–12 and 17–22 Gy). The possible reasons for this marked difference are associated with the fact that these values may vary according to radiation type and quality, the dose rate to which the organisms were exposed and their maintenance conditions at the laboratory [46]. An acute exposure to 50 Gy caused temporary sterility in males, but recovery was well in process after 48 days post irradiation and irradiation of gonads in males and females to an absorbed dose of 4.5 Gy leads to a substantial decrease in the production of offspring [46].

Regarding amphibians, different life stages showed different radiosensitivities. For adult toads, the LD50 value is of 24 Gy, for juveniles it is of 10 Gy and for tadpoles it is of 17 Gy [46, 139]. The life stage more sensitive to radiation exposure was the fertilized egg with an LD50/40 (LD50 after 40 days of exposure) of 0.6 Gy [33]. There is evidence that the exposure of male toads to 3–20 Gy caused a reduced survival and increased induction of abnormalities to the offspring [46, 139]. Although these LD50 values for amphibians seem slightly higher than the ones recorded for mammals, time after exposure optimal for the recording of LD50 values seem to be an important factor [33]. Reptiles and amphibians are poikilothermic organisms; therefore, their metabolism is quite variable and different from mammals and birds [33]. A study performed on 4 species of amphibians showed that if the assay period was extended a decrease in the LD50 to values that ranged between 0.8 and 7 Gy would be recorded [33].

Chronic irradiation exposure (5.5 years duration) of common side blotched lizard, western whiptail, long nosed leopard lizard and long nosed lizard showed that at ranges from 285 to 570 μGy/h, radiation exposure caused lack of reproduction, female ovaries regression and some degree of male sterilization [46].

Regarding the induction of DNA damage, it was observed by Ulsh and co-authors [142] that the exposure of turtles from the species *Trachemys scripta* to 0–8 Gy 137Cs gamma radiation, given at a dose rate of 0.55 Gy/h induced the occurrence of signifcant levels of chromosome translocations in lymphocytes. Studies on the induction of DNA alterations in amphibians and reptiles have been performed in Fukushima and Chernobyl exclusion zones, as well as in areas contaminated with NORM.

#### **Aquatic Vertebrates**

Among non-mammalian aquatic organisms, fsh are the most sensitive to the exposure to ionizing radiation [45, 46]. Although these organisms are also poikilothermic (as amphibians and reptiles), and therefore, apparently more radioresistant than mammals, there is a substantial overlap in radiosensitivities [46]. Until now, there is no substantial data on effects of ionizing radiation on marine mammals, however, there is no reason to believe that their radiosensitivity is substantially different from that of terrestrial mammals. Data on acute exposures exist mainly for bony and freshwater fshes, with a small number of studies on cartilaginous and marine and anadromous species.

The LD50 determined for six marine species after 40–50 days of exposure was of 9–23 Gy [46, 139]. Fish developing embryos are, however, more sensitive than adults, as for silver salmon their LD50 after 50 days of exposure is of 0.30 Gy at hatching and 0.16 Gy at a post-hatching larval stage of 90 days [46]. A study performed on sharks (*Triakis scyllium* and *Heterodontus japonicus*) exposed to 20 Gy showed that mortality occurred after 20 days of exposure, due to hematopoietic and gastrointestinal damage [33]. This suggests that the radiosensitivity of cartilaginous fsh may be similar to that of teleost fsh.

Regarding reproduction, an acute exposure to 10 Gy reduced the total number of germ cells at all developmental stages of medaka fsh (*Oryzias latipes*) [46]. A similar radiosensitivity was found in rainbow trout, with an induction of more than 50% sterility in organisms exposed late in embryonic development [46]. This leads to the conclusion that as in mammals, the newly hatched fry and the primordial gonads in fsh embryos are more sensitive to the acute radiation exposure than in adult fsh [46]. Irradiation of mature medaka fsh at acute doses of 5–10 Gy only induced temporary sterility, being completely recovered at 60 days after irradiation [46]. On the other hand, chronic irradiation of males from the fsh species *Ameca splendens* for 5.4 days at a dose rate from 137Cs gamma rays of 7300 mGy/h disrupted spermatogenesis and render the animals sterile at an accumulated dose of 9.7 Gy (8 weeks of exposure) [46]. There was 60–70% recovery, 236 days after irradiation [46]. Another freshwater fsh, the guppy (*Poecilia reticulata*), when exposed to gamma dose rates from 1700 to 13,000 mGy/h showed a signifcant reduction in fecundity, but no negative effects on survival and sex ratio, as well as no signifcant higher incidence of abnormalities in the offspring were observed [33, 136]. The marine fshes *Pleuronectes platessa* and the eelpout (*Zoarces viviparus*) exposed to 240 and 2000 mGy/h gamma radiation, respectively, showed a signifcant reduction of testes when compared to the control [136].

There are some fndings also on the effects of the exposure to ionizing radiation in the immune system of these organisms. A signifcant reduction in the humoral immune response in the rainbow trout (*Oncorhynchus mykiss*) exposed to tritium beta-particles for 20 days at a dose rate as low as 8.3–83 mGy/h during embryogenesis was evidenced through a reduction in antibody titer following a specifc challenge [46].

Regarding DNA damage there are very few studies on which some conclusion can be taken on this matter. On a study on medaka fsh, at larval stages, there was a signifcant induction of vertebral anomalies after irradiation at dose rates from 137Cs gamma rays higher than 18,000 mGy/h and also to beta particles from 3 H at dose rates higher than 35,000 mGy/h [46]. There is also a report on the occurrence of minor morphological abnormalities in the operculum of salmons exposed to a gamma radiation dose rate of 200 mGy/h that may affect latter survival [136].

# **9.4 The Particular Case of NORM Contamination**

Anthropogenic activities of concern related to the environmental release of natural uranium isotopes (mainly 238U and 235U) and other radionuclides from their decay chains, namely 226Ra and 223Ra, 222Rn, and 210Po, include mainly the production of phosphate fertilizers, uranium mining and milling and the incorrect disposal of tailings, uranium conversion and enrichment, the production of uranium fuel, production of coal, oil and gas, extraction of rare earths, extraction and purifcation of water, extraction of minerals for building materials and the generation of geothermal energy [3, 143]. All of these industrial activities increase the concentration of these elements in all environmental matrices, thereby posing a risk to human and non-human biota as many of them have not been regulated for NORM release [3, 143]. Another important issue is the fact that the contaminated areas that result from these anthropogenic activities do not only present high levels of certain natural radionuclides, like 226Ra, 222Rn, and 210Po but also other important stressors, namely metals like manganese, zinc, iron, aluminum, etc. [143]. These are usually multiple exposure scenarios, which contain several kinds of contaminants that may act synergistically and increase the risk of the occurrence of biological effects on human and non-human biota and even of modifying the susceptibility of cells/organisms to the biological effects of ionizing radiation exposure [144].

# **9.4.1 Chronic Exposure and Interaction with Uranium and Metals**

The accumulation of small amounts of radionuclides and metal over long periods is translated in chronic exposure to radiation. Naturally contaminated sites harbor a diversity of microbial species that become resistant or tolerant to these contaminants by bioaccumulating radionuclides and metals either by biosorption to their cell surfaces and biomolecules or by internalization into their cells. Briefy, under environmental conditions, chronic IR effects are very complex, particularly when compared to those from laboratory exposures because (1) radiation emitted by the different radionuclides present has different biological effects, (2) radiation from the same location is absorbed differently by different microorganisms, (3) abiotic factors (e.g., temperature, nutrients, pH, other stressors) are present and can interfere with radiation, (4) cooperation/interaction between microbial communities, including diversity and/or abundance can all be modulated by radiation [62]. Regarding uranium, probably the most well studied radionuclide, and for which a lot of information is available, interaction with microbial cells involving solubility by biomineralization (bioprecipitation) depends on all the above factors and also on the presence of affnity groups generated by microorganisms' cell metabolism, like hydroxides, phosphates, and carbonates. Uranium toxicity is both chemical and radiological. In the environment, uranium exists in its reduced insoluble form U(IV), and/or the oxidized form U(VI), which is soluble and toxic. Microorganisms interact with uranium by changing its redox state, aerobically, through oxidation (biolixiviation), or anaerobically by reduction. In order to do that, microorganisms need to be highly tolerant to uranium and to radiation. Other processes of microbial interaction with metals, involve biosorption, where contaminants passively concentrate through binding to cell structure constituents (e.g., lipopolysaccharides, teichoic acids, peptidoglycan), and biomineralization, which leads to the formation of biominerals using organic phosphate sources and phosphatases.

Unless disturbance occurs, NORM sites have a characteristic microbiome, which is specifc for a given site, but may share common microbial genera and species, regardless of location and/or chemical contamination. It includes nitrate-reducing bacteria that tolerate acidic and low-nutrient conditions, while being highly resistant to metals. Members of the Proteobacteria (Alpha-, Beta-, Delta- and Gamma- proteobacteria), Acidobacteria, Actinobacteria, Bacteroidetes, and Firmicutes are generally associated with uranium transformation and are therefore found in these environments. Most represented bacterial genus include Geobacter, Thiobacillus, Arthrobacter, Bacillus, Actinobacteria, Desulfovibrio, and Microbacterium. Most of the studies are focused on bacteria and bacterial communities. Although little information exists regarding fungi, they are particularly resistant to radiation and thus play a role in the process of detoxifcation of radionuclides. For instance, an isolate of the genus Paecilomyces, was found to detoxify U(VI) through bioprecipitation of the metal, and the reduction was promoted by phosphate. Also, the yeast *S. cerevisiae* was able to reduce U(VI) toxicity by biomineralization [60].

Accordingly, the survival, abundance, and maintenance of a given species or community diversity depend on its adaptability to the existing conditions. Furthermore, several studies suggest that in those radionuclide-rich natural sites, resistance to high levels of chronic IR may occur among taxa that tolerate a wide range of environmental conditions and, therefore, have an advantage over other more sensitive species [62].

#### J. Lourenço et al.

# **9.4.2 Efects of NORM and Metals on Eukaryotes**

#### **<IP_ADDRESS> Invertebrates**

There have been some studies in aquatic organisms, namely in *Daphnia magna, Daphnia longispina,* and *Moinodaphnia macleayi* at NORM sites [145, 146]. When testing several percentages of a uranium mine effuent containing metals and radionuclides from 238U and 235U decay chains, the Antunes et al. [145] study recorded an EC5011 for daphnids immobilization at 50.4% for *D. magna* and at 28.4% for *D. longispina*, showing that *D. magna* was less sensitive than *D. longispina*. However, regarding fertility, *D. magna* was more sensitive than *D. longispina*, as this last species did not show signifcant effects in the offspring produced at effuent concentrations lower than 30.38%. Regarding *M. macleayi*, when a natural population of these organisms, living adjacent to a uranium mine in Australia, was challenged with a concentration of uranium ranging from 0 to 700 μg/L, it was shown that this population comparing to other populations tested, was the one that presented the highest sensitivity as it evidenced the lowest NOECs and LOECs.12 It was shown that although this population lived in a water containing already considerable amounts of uranium, there was no tolerance to higher levels of uranium, when compared to the other tested populations. This probably shows that it was an already very stressed population that suffered "genetic erosion" [147] and because of that, it had lower capacity to deal with additional stresses, such as a single high dose of uranium.

When *D. magna* was exposed to uranium and to a uranium mine effuent [148, 149], signifcant genotoxic effects (DNA strand breaks) were detected in neonates and <5 days old daphnids after exposure to 55.3 μg/L of uranium and 2% of a uranium mine effuent. Moreover, in this same study, bystander effects, in the form of DNA damage, were detected in unexposed organisms when placed in contact with organisms directly exposed to uranium and to uranium mine effuent. In another paper [149], published by the same authors, on a transgenerational study performed on *D. magna* exposed to the same concentrations of uranium and uranium mine effuent as the study previously referred*,* it was observed that DNA damage was transmitted only to the frst broods of the exposed organisms. By the third brood, DNA damage was no longer detected. This study showed that although short-term exposure to low concentrations of uranium and uranium

<sup>11</sup>EC50 is the concentration of a substance in water causing death to 50% of the tested population.

<sup>12</sup>LOEC is the lowest concentration where an effect has been observed in chronic or acute ecotoxicity studies. NOEC is the highest concentration at which there is no statistically signifcant difference from the control condition in an acute or chronic ecotoxicity study.

mine effuent induces DNA damage to exposed organisms, it seems that it was not enough to signifcantly affect life history traits of *D. magna* populations in a long-term scenario. Nevertheless, the interpretation of these results is limited to the response observed for the endpoints here analyzed (DNA strand breaks). As such, other endpoints for genotoxicity assessment (i.e., mutation detection) and also the analyses of the epigenome of these organisms should be performed, as these molecular changes do not refect a loss of DNA's structural integrity [149].

As for terrestrial invertebrates, most of the studies conducted so far were on the annelid *Eisenia andrei* [150–155]. Gene expression alterations were reported in earthworms exposed to sludge from a uranium mine decantation pond. These genes were mainly related with metabolism, oxidoreductase activity, redox homeostasis, and response to chemical stimulus and stress [152]. In these studies, the occurrence of DNA damage in the form of DNA strand breaks and changes in cell's DNA content in exposed organisms was also detected. Alterations in earthworm's immune system were also reported, in terms of the frequency of each cell compartment, as it was observed a decrease in the number of effector cells (amebocytes) and an increase of the cells responsible for the maintenance of the organism's homeostasis (eleocytes) [153, 154]. In parallel with a signifcant bioaccumulation of metals and radionuclides from uranium's decay chain (238U, 234U, 235U, 226Ra, 230Th, and 210Pb), it was also observed a signifcant decrease in earthworms' biomass, a reproduction inhibition, and signifcant histological alterations, namely in earthworm's body wall (epidermis, circular, and longitudinal muscles) and gastrointestinal tract (chloragogenous tissue and intestinal epithelium) [153–155].

Under a real scenario of contamination, all of these effects may explain the lower biodiversity of soils contaminated with NORM, and the subsequent loss of their functions, if the contamination is perceived by the organisms. By using an avoidance assay (a standard ecotoxicological assay), to study earthworms' behavioral responses to soils collected in a uranium mine area, it was shown that earthworms actively avoided several contaminated soils. Earthworm's avoidance responses allowed it to discriminate highly to moderately toxic soils. On the other hand, on another study published by the same authors, using the analyses of oxidative stress enzymatic biomarkers (catalase, glutathione peroxidase) and lipid peroxidation biomarkers (through the quantifcation of thiobarbituric acid reactive substances), in earthworms exposed to soils nearby a uranium mine, showed no response for none of the biomarkers analyzed [150].

#### **<IP_ADDRESS> Vertebrates**

Although there have been a wide number of studies performed on the effects of gamma radiation exposure on vertebrates, very few were performed so far for NORM exposure. Regarding aquatic vertebrates, fsh have been the most used model organisms. On a study performed in former uranium mines from the Limousin region of France, where *Rutilus rutilus* specimens were caged on a pond contaminated with NORM and metals, immune, oxidative stress, biotransformation, neurotoxicity, and physiological parameters were measured [156]. The results obtained showed a stimulation of the immune parameters, the occurrence of oxidative stress and a decrease of acetyl choline esterase-AChE in the fsh caged in the contaminated pond [156]. Zebrafsh (*Danio rerio*) specimens exposed to uranium mill tailings leaching solution also showed alterations for the oxidative stress biomarkers used (superoxide dismutase—SOD, catalase—CAT, malondialdehyde—MDA and Na+–K+–ATPase) but specially for Na+–K+–ATPase and also evidenced that the organs most susceptible to oxidative stress were the gills [157]. In another study performed on a uranium milling operation in Northern Saskatchewan, Canada, *Pimephales promelas* specimens (adults and 5-dayold larvae) were exposed to contaminated water and contaminated sediment [158]. Results indicated effects on reproduction (reduced hatching) and larvae development (increase of skeletal deformities) and an increase in metal body burdens. However, the effects detected on the offspring, when considering the increase in egg production, were not signifcant in the level of deformities between treatments [158]. The effects on reproduction on the same species have already been observed under an exposure to effuent waters also from a uranium mining site in Saskatchewan, Canada. A signifcant decrease in eggs hatching time and hatching success was registered when early life stages of fathead minnows were exposed [159]. Nevertheless, metals and radionuclides are not the only stressor responsible for the effects caused by effuent waters from NORM sites. Lourenço et al. [160] performed an exposure of zebrafsh eggs to a uranium mine effuent, barium chloride-treated mine effuent, and settling ponds sludge elutriates and showed that pH of the mine effuent strongly affected hatching success. After eliminating the effect of pH, this study also showed some teratogenicity associated with the uranium mine effuent, the occurrence of DNA damage, mainly associated with the exposure to treated mine effuent and sludge elutriates and mild effects on growth observed mainly on embryos exposed to the mine effuent and sludge elutriates. This study showed that the use of the Fish Embryo Toxicity Test (FET) test is suitable to test uranium mining wastes to determine and discriminate the risk of discharge. It also showed that the inclusion of the evaluation of genotoxicity endpoints in the FET test prevented the underestimation of risks, when only looking at chemical and radiological benchmark values defned by national and international directives, for the determination of risks, due to the chemical complexity of these wastes.

On what concerns amphibians, there are very few studies on these organisms as well. Marques and co-authors performed very important studies on amphibians, namely *Pelophylax perezi* exposed to NORM in situ. They have studied both tadpoles and adults and they have analyzed several endpoints, such as growth, survival, oxidative stress biomarkers (catalase, glutathione peroxidase, glutathione reductase, and lipid peroxidation through thiobarbituric acid reactive species (TBARS) quantifcation), gene expression alterations, histopathological changes, erythrocytic nuclear abnormalities and micronuclei, on organisms exposed to a uranium mine effuent in Portugal [161–165]. A study performed on 2008, on larvae and eggs [165] exposed to a uranium mine effuent, showed a decrease in larvae body length as well as a decrease in stimulus reactions, an increase in pigmentation along with tail deformities and metals bioaccumulation. The in situ exposure of tadpoles of the same species showed decreased survival and growth, a higher glutathione peroxidase activity and an increased lipid peroxidation [164] in organisms exposed in the mine effuent pond, when compared with organisms from a control pond. Although there may have been the infuence of NORM and metals exposure, the studies also evidenced the effects of effuent's acidity (typically seen in metal mining contexts), mainly in the growth and survival parameters and also in metal's uptake. Another study, performed by the same authors [163], on adults living on the same uranium mine pond, analyzed gene expression changes using a technique called Suppressive Subtractive Hybridization (SSH). Signifcant changes in the expression levels of genes that play an important role in protecting cells against oxidative stress were shown, evidencing once again that oxidative stress response is very important in protecting cells and in maintaining DNA integrity on organisms exposed to NORMs and metals. Another study performed by this team on *Pelophylax perezi* adults inhabiting a uranium mining pond [162], showed signifcant metals bioaccumulation in the liver and the kidneys. Signifcant histopathological alterations in the liver, the lungs and in the kidneys, mainly in the form of a slight increase in melanomacrophagic centers, a dilatation of the renal tubules, a discrete thickening along with a slight hyperplasia of the alveolar septa and a slight hypoplasia of the goblet cells, were observed. The same animals living in the mine pond also displayed a signifcantly higher number of erythrocytic abnormalities (micronuclei and notched, kidney and lobed shaped nuclei) as well as a signifcantly lower frequency of immature erythrocytes. Both observations led to the belief that the removal and replacement of abnormal blood cells might be compromised.

There are a few studies published on the uptake of NORM by mammals that were performed mainly on former uranium mining areas, but very few examined the effects of that exposure. A study performed by Cleveland et al. [166] analyzed NORM uptake and histopathological alterations in liver and kidneys of rodents (*Peromyscus maniculatus* and *P. boylii*) inhabiting former uranium mines and observed that rodents bioaccumulated elements from 238U decay chain but without exceeding literature-based effects thresholds for small rodents. The authors also observed that there were some minor lesions in the tissues (liver and kidneys) analyzed that could not, however, be attributed to U mining activities. Lourenço and co-authors [167], captured mice (*Apodemus sylvaticus*) on the surroundings of a former uranium mining site and on a control area. DNA damage and bioaccumulation of metals and radionuclides were assessed, as well as the expression and the presence of single nucleotide polymorphisms on tumor suppressor genes. Results showed that cadmium and uranium were signifcantly bioaccumulated by exposed organisms. Organisms living in the former uranium mining area also evidenced signifcantly higher levels of DNA damage when compared with control organisms and also a higher expression of TP53 tumor suppressor gene and the presence of single nucleotide polymorphisms in Rb tumor suppressor gene. These effects can cause a disturbance in the genetic material of exposed organisms causing genetic instability and changes in the genetic pool of the population, potentially affecting the population's ftness and stability. However, they cannot be attributed to any of the stressors in particular. It is known that uranium is genotoxic due to its chemical and radiological properties. Nevertheless, other metals present in uranium ore have shown greater genotoxic properties [151].

#### **<IP_ADDRESS> Plants**

As for plants, there are a few studies already performed using soil/sludge or plant species collected directly from radium production industry storage sites, uranium rich regions, but mainly uranium mining sites and uranium milling tailings, that showed NORM bioaccumulation [168– 180]. However, very few assessed the effects of that bioaccumulation. On a study performed by Evseeva et al. [170], *Vicia cracca* populations, inhabiting areas contaminated with uranium mill tailings and radium production wastes, were sampled and analyzed for the presence of chromosome aberrations, frequency of embryonic lethal mutations, seed germination and survival rate of seed sprouts. Results showed an increased frequency of embryonic lethal mutations, decreased seed germination, increased chromosome aberration counts and decreased survival rate of seed sprouts. The same authors [171], used *Allium cepa* specimens to determine the genotoxicity of an effuent from a radium production storage facility, through chromosome aberrations counting. Results showed a signifcant increase in chromosome aberration counts in the roots of exposed plants. Two studies [168, 179] using soils contaminated with metals and radionuclides from Portuguese former uranium mines were performed using *Lactuca sativ*a and *Zea mays* as test species to determine the eco (through growth inhibition) and genotoxicity (mutation analysis through the Ames test) of amended and unamended mine soils. Studies showed genotoxicity of the unamended soils containing the highest levels of metals and radionuclides, a signifcant decrease in *Lactuca sativ*a biomass and also a signifcant bioaccumulation of these elements. The soil amendment methodology used in these studies signifcantly decreased the levels of metals and radionuclides in soils leachates and the soil available fraction.

# **9.5 Exercises and Self-Assessment**

# **9.6 Exercise Solutions**

Genome sequencing, comparative genomics, and proteomics have allowed the identifcation of microbial essential genes (key players) that encode biomolecules, mainly proteins, involved in biological processes, including those involved in detoxifcation of radionuclides and metals. Furthermore, metagenomics approaches directed to the microbial communities of these contaminated environments allow for the identifcation, and characterization, of microorganisms with relevant functions in the bioremediation/ decontamination processes. It is therefore expected that these broader approaches will contribute even more to the identifcation of microorganisms and to the elucidation of the metabolic pathways and key genes involved in those processes that may be further applied in the bioremediation/decontamination of these sites.

# **References**

in developing an ecosystem approach for environmental radiation protection. Int J Radiat Biol. 2020; https://doi.org/10.1080/09553 002.2020.1793022.

adult male housefy, Musca domestica. Arch Gerontol Geriatr. 1985;4(2):169.

fndings of the United Nations Chernobyl forum. Health Phys. 2007;93:427.

ity endpoints in earthworms exposed in situ to uranium mining wastes. Ecotoxicol Environ Saf. 2012;75(1):46.

http://www.sciencedirect.com/science/article/pii/S004896971 1005675

# **Further Reading**

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons. org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **10 Space Radiobiology**

Christine Elisabeth Hellweg, Carmen Arena, Sarah Baatout, Bjorn Baselet, Kristina Beblo-Vranesevic, Nicol Caplin, Richard Coos, Fabiana Da Pieve, Veronica De Micco, Nicolas Foray, Boris Hespeels, Anne-Catherine Heuskin, Jessica Kronenberg, Tetyana Milojevic, Silvana Miranda, Victoria Moris, Sébastien Penninckx, Wilhelmina E. Radstake, Emil Rehnberg, Petra Rettberg, Kevin Tabury, Karine Van Doninck, Olivier Van Hoey, Guillaume Vogin, and Yehoshua Socol

#### **Learning Objectives**

S. Baatout

B. Baselet Radiobiology Unit, Belgian Nuclear Research Center (SCK CEN), Mol, Belgium e-mail<EMAIL_ADDRESS>
K. Beblo-Vranesevic · P. Rettberg Radiation Biology Department, Astrobiology, Institute of Aerospace Medicine, German Aerospace Center, Cologne, Germany e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>
Fabiana Da Pieve is currently employed by the European Research Council Executive Agency, European Commission, BE-1049 Brussels, Belgium. The views expressed are purely those of the authors and may not in any circumstances be regarded as stating an offcial position of the European Commission.

C. E. Hellweg Radiation Biology Department, Institute of Aerospace Medicine, German Aerospace Center (DLR), Cologne, Germany e-mail<EMAIL_ADDRESS>
C. Arena Department of Biology, University of Naples Federico II, Naples, Italy e-mail<EMAIL_ADDRESS>
Institute of Nuclear Medical Applications, Belgian Nuclear Research Centre (SCK CEN), Mol, Belgium e-mail<EMAIL_ADDRESS>
N. Caplin Human Spacefight and Robotic Exploration, European Space Agency, Noordwijk, The Netherlands e-mail<EMAIL_ADDRESS>
R. Coos Laboratory of Analysis by Nuclear Reactions, University of Namur, Namur, Belgium e-mail<EMAIL_ADDRESS>
F. Da Pieve European Research Council Executive Agency, European Commission, Brussels, Belgium

Royal Belgian Institute for Space Aeronomy, Brussels, Belgium e-mail<EMAIL_ADDRESS>
V. De Micco Department of Agricultural Sciences, University of Naples Federico II, Naples, Italy e-mail<EMAIL_ADDRESS>
N. Foray Inserm Unit 1296 "Radiation: Defense, Health, Environment", Lyon, France e-mail<EMAIL_ADDRESS>
#### B. Hespeels

Namur Research Institute for Life Sciences, Institute of Life-Earth-Environment and Research Unit in Environmental and Evolutionary Biology, University of Namur (UNamur), Namur, Belgium e-mail<EMAIL_ADDRESS>
A.-C. Heuskin Namur Research Institute for Life Sciences, Laboratory of Analysis by Nuclear Reactions, University of Namur (UNamur),

Namur, Belgium e-mail<EMAIL_ADDRESS>
J. Kronenberg Radiation Biology Department, Cellular Biodiagnostics, Institute of Aerospace Medicine, German Aerospace Center, Cologne, Germany

e-mail<EMAIL_ADDRESS>
T. Milojevic Space Biochemistry Group, Department of Biophysical Chemistry, University of Vienna, Vienna, Austria

e-mail<EMAIL_ADDRESS>
S. Miranda · W. E. Radstake Department of Molecular Biotechnology, Ghent University, Ghent, Belgium

Radiobiology Unit, Belgian Nuclear Research Center (SCK CEN), Mol, Belgium

e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>
V. Moris Research Unit in Environmental and Evolutionary Biology (URBE), University of Namur (UNamur-LEGE), Namur, Belgium

Research Unit in Molecular Biology and Evolution (MBE), Department of Biology, Université Libre de Bruxelles, Brussels, Belgium

S. Penninckx

Medical Physics Department, Hôpital Universitaire de Bruxelles (H.U.B.), Université Libre de Bruxelles, Brussels, Belgium e-mail<EMAIL_ADDRESS>
E. Rehnberg Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium

Department of Molecular Biotechnology, Ghent University, Ghent, Belgium e-mail<EMAIL_ADDRESS>
K. Tabury Department of Biomedical Engineering, University of South Carolina, Columbia, SC, United States of America

Radiobiology Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium e-mail<EMAIL_ADDRESS>
K. Van Doninck Research Unit in Environmental and Evolutionary Biology (URBE), University of Namur (UNamur-LEGE), Namur, Belgium

Université Libre de Bruxelles, Molecular Biology and Evolution, Brussels, Belgium

e-mail<EMAIL_ADDRESS>
O. Van Hoey Research in Dosimetric Applications Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium e-mail<EMAIL_ADDRESS>
#### G. Vogin

Centre Francois Baclesse, University of Luxembourg and Luxembourg Institute of Health, Luxembourg, Luxembourg e-mail<EMAIL_ADDRESS>
#### Y. Socol (\*)

Jerusalem College of Technology, Jerusalem, Israel e-mail<EMAIL_ADDRESS>
# **10.1 Introduction**

Experiments in space provided us with new insights into radiation biology. This chapter is organized as follows. First, we present a historical overview of the feld that can be traced to the frst experiments at the Eiffel tower. Then, we overview the space radiation environment and mathematical models used to describe it. Later in this chapter, we present a macroscopic picture of health effects in humans (observed or anticipated in the space environment). Afterward, we turn to a microscopic level and describe biomolecular changes introduced by space radiation. Then, we describe experimental evidence obtained from models—small animals, plants, eukaryotic cells, and extremophiles (organisms living under conditions extreme from a human point of view). Finally, we present an overview of ground-based facilities mimicking the space environment.

# **10.2 History of Space Radiation Studies and Space Radiobiology**

# **10.2.1 From Earth Ground to the Eifel Tower**

Human space travels were very early the concerns of a number of scientists like Johannes Kepler who warned that extraterrestrial trips would require ships ft to withstand the breezes of heaven [1]. The development of electroscopes manufactured by Pierre Curie made the assessment of local currents possible due to any particle crossing the two metallic plates [2]. The Italian physicist Domenico Pacini suggested in 1910 that the background noise measured with the Curie electroscopes was caused by radiation emitted from the Earth ground [3]. By performing some experiments with the Curie electroscope at the Eiffel tower, Theodor Wulf, a Jesuit priest, demonstrated that half of the radiation emitted by the Earth ground disappears at a height of 300 m. When the technology of balloons was safe, Victor Hess observed that the ionization density of the atmosphere progressively decreases up to 1000 m, but increases above 1800 m, suggesting the existence of two components of natural radiation: one from the Earth ground, the other from space, "cosmic rays" [4]. In 1936, Hess was awarded the Nobel Prize in physics for his discoveries [5].

# **10.2.2 From the Eifel Tower to the Balloon Experiments**

Between the 1930s and the 1940s, there were considerable advances in the technology of particle counters and in the knowledge of particle physics; thanks to balloon experiments, new clues were brought to support that cosmic rays consist of very energetic (108 –1020 eV) particles. Furthermore, data hinted an unexpectedly high proportion of the iron-associated elements in the galactic cosmic radiation (GCR). The latter observation led to the hypothesis of the nucleosynthesis origin of cosmic rays [3]. Figure 10.1 shows the advances in space radiation biology since that period.

# **10.2.3 From the Balloon Experiments to Artifcial Satellites**

The pioneer works of William Gilbert, Carl Friedrich Gauss, and Henri Poincaré about magnetism suggested that charged particles may be infuenced by the Earth's magnetism and that a ring current should exist around the Earth. At the end of the 1950s and overall in the 1960s, the number of artifcial satellites increased drastically and permitted to verify these hypotheses. In 1958, James Van Allen and Louis A. Frank

**Fig. 10.1** Synopsis of advances in space radiation biology. The continuous increase of space mission duration up to 400 days is illustrated by the grey line on the left. For Mercury, Gemini, Apollo, Salyut, Skylab, and Mir missions, the maximum dose values are given as red dots [6]. *ALTEA* anomalous long-term effects on astronauts, *FISH* fuo-

rescence in situ hybridization, *HPRT* hypoxanthine guanine phosphoribosyl transferase, *ISS* International Space Station, *LNT* linear no-threshold, *NLT* nonlinear threshold, *PCC* premature chromosome condensation, *SilEye* silicon eye. (Reprinted with permission from Maalouf et al. [6])

pointed out the existence of the Earth's radiation belt, based on data collected by the Explorer I and Pioneer IV satellites. Protons and electrons were found to be the major constituents of the Van Allen belt [7, 8]. In the same decade, Mariner II provided important data about the solar wind that permitted to document our knowledge on the radiation component from our Sun [9].

# **10.2.4 From Artifcial Satellites to Manned Missions**

During short-term (less than 2 weeks) missions in the 1960s at low Earth orbit (LEO), astronauts were exposed to several mGy at an average dose rate of about 0.17 μGy/min (245 μGy/mission day). This dose was delivered discontinuously as (1) the inner and outer zones of the Van Allen radiation belt contain protons and electrons of differing energy spectra that result in different secondary particles at dose rates different and energies; (2) the South Atlantic Anomaly (SAA), the area where the inner Van Allen radiation belt is the closest to the Earth surface, leading to an impressive fux of protons and electrons is passed about 15 times a day; here the dose rate can increase sixfold resulting in a signifcant contribution to the radiation exposure; and (3) unpredicted solar particle events (SPE) can increase the total dose, while the protection in LEO is still suffcient to prevent life-threatening acute radiation syndrome (see Sect. <IP_ADDRESS>).

The characterization of individual heavy cosmic particles of high-energy and high atomic number—*Z*—(HZE) was performed with different physical radiation detectors (nuclear emulsions, plastics, silver chloride (AgCl) crystals, and lithium fuoride (LiF) thermoluminescence dosimeters) for the frst time in space in the Biostack experiments fown aboard Apollo 16 and 17 (see Sect. 10.5). In parallel, their biological effects were examined in different biological objects such as bacterial spores, protozoa cysts, plant seeds, shrimp eggs, and insect eggs investigating various radiobiological end-points [10].

Examples of short-term experiments of up to 2 weeks in LEO combining radiation dosimetry and biological investigations were loaded on Space Shuttles (e.g., STS 9, 42, 45, 65), on free-fying satellites (e.g., LDEF, EURECA, BIOPAN 1–6) and on the MIR space station (Perseus mission). Later on, similar long-term experiments were performed on the International Space Station (ISS) (EXPOSE-E, -R, -R2) [11, 12].

"Cytogenetics observations revealed for the frst time the major biological consequences of an exposure to space radiation: the yield of chromosome breaks seemed to increase after fight, but statistical signifcance was still needed (see Sect. <IP_ADDRESS>). Data from eye fashes and helmets (see Sect. <IP_ADDRESS>) suggested the existence of a certain "hidden part" of the heavy ions' component, probably due to secondary particles generated by the interaction of very high-energy particles with metallic materials. The contribution of these heavy ions to the total dose of radiation remained unknown at the end of the 1960s" [6].

# **10.2.5 From One Space Station to Another**

Space experiments in combination with ground-based research (see Sect. 10.10) enabled a better understanding of the effects of space radiation and microgravity on human cells, microbes, and other biological models such as the roles of different complementary DNA repair mechanisms, the reactive oxygen species detoxifcation system and the intracellular accumulation of compatible solutes summarized, e.g., in Senatore et al. [13]. The modern picture of the space radiation dosimetry and its effects on human cells may be summarized as following [6]:

# **10.3 Space Radiation Environment**

# **10.3.1 Origin and Nature of Space Radiation**

Space is permeated with radiation, both electromagnetic radiation and particles with mass. Electromagnetic radiation in space spans many wavelengths, from long wavelength radio waves to very short-wavelength gamma rays. Gamma rays, X-rays, and some far/extreme ultraviolet (UV) waves, which can be generated for example during some transient events on the Sun [14], are actually ionizing radiation. The wavelengths of UV, X-, and gamma rays are all shorter than those of visible light. The majority of these rays are absorbed by the Earth's atmosphere. The extraterrestrial solar UV radiation ranges from vacuum UV (wavelength <200 nm) to UVA (320–400 nm). The ozone layer absorbs some, but not all, of these types of UV radiation: UVA is not absorbed by the ozone layer, UVB (wavelength: 290–320 nm) is mostly absorbed by the ozone layer, but some does reach the Earth's surface, while UVC (wavelength: 100–290 nm) is completely absorbed by the ozone layer and atmosphere. Overall, the electromagnetic radiation reaching the Earth's surface encompasses radio waves, some microwaves, some infrared light, UVB and UVA radiation, and visible light. Of the light that reaches Earth's surface, infrared radiation makes up 49.4% while visible light provides 42.3%. UV radiation makes up just over 8% of the total solar radiation. UVA and UVB radiation contribute not only to premature aging of the skin but also to some serious health effects such as skin cancer, cataracts, and suppression of the immune system.

Generally however the expression "space radiation" mainly refers to radiation consisting of particles with a mass. There are three main radiation populations in space: galactic cosmic rays (intra- and extragalactic; GCR), solar radiation (including both the Solar Energetic Particles, SEP, and solar wind), and trapped radiation. A schematic representation of these radiation types and the environment which they can infuence is given in Fig. 10.2.

# **<IP_ADDRESS> Galactic Cosmic Rays (GCRs)**

GCRs are constantly present highly energetic radiation in space. Their intensity is slowly varying and low with a few particles per second traversing an area of a cm2 to a m2 or more. They are nearly isotropic, meaning that they impinge from all directions. They originate from outside the heliosphere, most likely from deep space high-energy phenomena [16], such as supernova shock waves throughout the Galaxy, and also possibly from stellar wind termination shocks, pulsars, or other more exotic objects. They are composed of 98% baryons, of which 87% protons (hydrogen ions), 12% helium ions (α-particles), and 1–2% high-energy and highly charged ions, called High charge *Z* and Energy (HZE) particles, and 1% electrons and positrons [17]. HZE comprises ions from *Z* = 3 (Li) to *Z* = 28 (Ni). The most common elements are C, O, Mg, Si, and Fe ions (Fig. 10.3). Ions heavier than Ni can be encountered, yet these are very rare.

The spectrum of the GCRs is infuenced by periodical, long-term, and short-term effects. Also, the Sun's behavior is periodical and follows an 11-year cycle which affects the interplanetary medium. The increased solar and heliospheric magnetic felds during the maximum phase of the solar cycle partially shield the solar system and decrease the low-energy portion of the GCRs fux, by preventing it from entering the inner heliosphere [18], while at solar minimum the reduced interplanetary magnetic feld strength implies a more intense GCRs population [19]. The GCR fux is thus inversely proportional to the solar activity and decreases by a factor of 2–4 when moving from solar minimum to solar maximum,

**Fig. 10.2** Radiation environment during a space mission. (Image courtesy by ESA and reprinted from Chancellor et al. [15] with permission under Creative Commons Attribution-NonCommercial-NoDerivatives License: http://creativecommons.org/licenses/ by-nc-nd/4.0/)

depending on the depth of the solar minimum and the intensity of the solar maximum [20, 21].

The modulation of the GCR fux for different ions is reported for the period of solar maximum and minimum. In the short term, GCRs can also be reduced by intense release of high-energy particles (mostly protons) during transient solar eruptions [22] (see Sect. <IP_ADDRESS>). The energy spectrum of GCRs covers a huge range of energies: it commences at about 107 –108 electron volt (eV) (10–100 MeV), and the most energetic *cosmic rays* reach up to 1020 eV (Fig. 10.4). A prominent feature is a so-called knee, with an energy of about 2.7–3.1 PeV (PeV = 1015 eV). This energy originated

**Fig. 10.3** GCR composition, as based on data from NASA's Advanced Composition Explorer (ACE) spacecraft. (Reprinted with permission from http://www.srl.caltech.edu/ACE/ACENews/ACENews83.html)

from the diffusive shock acceleration from the Galactic supernova remnants. The so-called anide or ankle, with an energy of about 5 × 1018 eV, is another characteristic of the energy spectrum. It is believed to mark the lower end of the energy of ultra-high energy GCRs, those that originate from extragalactic sources [24].

When traversing Earth's atmosphere, GCRs induce nuclear-electromagnetic-muon cascade reactions resulting in ionization of atmospheric molecules and generation of secondary particles [25, 26]. A small fraction of the initial primary particles, together with secondary particles of suffcient energy, reaches the ground. The maximum in secondary particle energy release (Pfötzer maximum) occurs at altitudes of 15–26 km depending on latitude and solar activity level. The radiation reaching the Earth's surface has levels similar to other low levels of radiation that humans are frequently exposed to. The average yearly exposure of a person is around 3.5 millisieverts (mSv). About half of this dose can be attributed to artifcial sources (X-ray, computer tomography (CT) scan, mammography), while the other half originates from natural sources, including around 10% from cosmic radiation.

#### **<IP_ADDRESS> Solar Energetic Particles (SEPs)**

SEPs originate from transient events on the Sun and come as massive injections of mostly protons and electrons (and to lesser extent helium (~4%) and heavier ions), with typical energies from ten to hundreds of MeV [27]. These transient events are Sun eruptions such as fares and Coronal Mass Ejections (CMEs). Characteristically, a fare lasts only minutes to hours and is the result of an explosive energy release from the Sun's coronal magnetic feld. Also, the electromag-

netic fux increases, particularly in the short-wavelength (Extreme ultraviolet—XUV, gamma ray) range, and also in the radio regime. Usually originating in active regions, CMEs are large-scale plasma-magnetic structures with high speeds (up to thousands of km/s) associated with prominence eruptions and fares.

The likelihood of CME events increases with the power and size of the related fare event, although not all CMEs are associated with fares. Such events extend from several hours to a few days, and they have a higher likelihood of occurring during solar maximum. The level of the Sun's activity is fairly described by the number of sunspots, which provides an indication of the phases of the cycle. The number of spots increases toward the solar maximum, while during solar minimum the Sun's surface is almost spotless. Nevertheless, such SEPs events are hardly predictable and can also occur during solar minimum. Examples of an active region, an initial fare, and then a prominent eruption initiating a CME is shown for the 28/10/2003 event as part of the "Halloween Storms of 2003" in Fig. 10.5, with the related sudden increase in proton fux as detected by the Geostationary Operational Environmental Satellite (GOES) satellite (Fig. 10.6a). Examples of fuences (integral of the fux over the period of the event) related to major SEP events are shown in Fig. 10.6b.

A classifcation exists between Impulsive SEP events, which are short (≤1 day), numerous (~1000/year in periods of high activity), and of low intensity, and gradual events, which are long (several days at energies of a few MeV/ nucleon), rather rare (a few tens per year), characterized by orders of magnitude higher protons fuences than impulsive events and ascribed to acceleration by CME-driven shocks as they propagate through the heliosphere. There is however some debate about the role played by "fare acceleration" in these events [29, 30].

Contrary to GCRs, SEP events can be considered mostly inducing deterministic effects (Sects. 10.4.2 and 10.6.2). Deterministic effects are those certainly occurring once a specifc threshold dose has been overpassed. The high-intensity SEP fux can signifcantly increase the absorbed dose to astronauts, for example, during extravehicular activities (EVA) at the ISS, or eventually, if the event is characterized by a "hard" spectrum with a strong high-energy component, also during both interplanetary mission or missions on thin atmosphere such as Mars. Acute radiation syndrome (ARS), sickness, or, in extreme cases, death after a lethal dose can occur [31].

A comparison between GCR and SPE can be found in Table 10.1 (adapted from NASA Space Flight Human System Standards—NASA Standard 3001).

**Fig. 10.5** The active regions (upper left), solar fare (upper right), and coronal mass ejections (CME, lower left and right) of the 28/10/2003 event captured by the Solar and Heliospheric Observatory (SOHO) satellite. The CME was imaged by the Large Angle and Spectrometric COronagraph (LASCO) instrument by blocking the light from the solar disk. (Courtesy of SOHO/EIT and SOHO/LASCO consortium. SOHO is a project of international cooperation between ESA and NASA)

**Fig. 10.6** (**a**) Proton fux between 28 and 31 October 2003. The 5-min averaged integral proton fux for energy thresholds of >10 (red line), >50 (blue line), and >100 MeV (green line) was measured by the primary Geostationary Operational Environmental Satellite (GOES) satellite of the Space Weather Prediction Center (SWPC). *CO* Colorado, *MeV* Mega electron volt, *NOAA* National Oceanic and Atmospheric Administration, *s* second, *sr* steradian, *UTC* Coordinated

Universal Time. Reprinted with permission under terms of the Creative Commons Attribution License [28]. (**b**) Distribution in the energy of proton fuences for major past SEPs events (free space). (Reprinted with permission from: The space radiation environment: an introduction. Schimmerling W. https://three-jsc.nasa.gov/concepts/ SpaceRadiationEnviron.pdf. Date posted: 2-5-2011)

Adapted from https://msis.jsc.nasa.gov/sections/section05.htm#\_5.7\_RADIATION

# **<IP_ADDRESS> Solar Wind**

The solar wind is a continuous fow of plasma from the Sun's corona, mainly consisting of protons, electrons with a small percentage of He ions, with kinetic energies between 0.5 and 10 keV. There are also some trace amounts of heavy ions and atomic nuclei such as C, N, O, Ne, Mg, Si, S, and Fe. Their energy results from the high temperature of the Sun's corona and allows them to escape the Sun's gravity. The fux of the solar wind varies over time, solar longitude and latitude, together with its temperature, density, and speed. At distances of more than a few solar radii from the Sun, the solar wind reaches supersonic speeds of 250–750 km/s [32]. At much greater distances, about 75–90 astronomical units (1 au is the distance Sun-Earth), the so-called "termination shock," interactions of the local interstellar medium with the solar wind slow it down to subsonic speed.

There are different classes of solar wind [30]:

and their main properties differ signifcantly according to the location and magnetic properties at the source;

The Earth's magnetosphere defects the solar wind, causing most of the solar wind to fow around and beyond us. Nevertheless, a small number of particles from the solar wind reach the upper atmosphere and ionosphere. This may produce phenomena such as aurora and geomagnetic storms, the latter occurring when large infation of the magnetosphere, due to an increased pressure of the contained plasma, distorts the geomagnetic feld.

In space missions, the solar wind has no impact on astronauts, as it is effciently stopped by the spacecraft shielding and also by appropriate astronaut suits, because of the small range in a matter of the low speed-solar wind particles. However, if not appropriately shielded, the solar wind particles may affect the human body during eventual EVAs in deep space or on the surface of airless bodies, such as the Moon.

#### **<IP_ADDRESS> Trapped Radiation**

Trapped radiation particles are produced mainly by the interaction of GCRs and SEPs with the Earth's atmosphere and are trapped by its magnetic feld into the Van Allen radiation belts. These comprise:

In the radiation belts, the energetic particles move along Earth's magnetic feld lines, via the combination of three types of motion: a fast rotation (or "gyration") around magnetic feld lines, typically thousands of times each second; a back-and-forth bouncing along the stronger magnetic felds in the northern and southern hemispheres, typically lasting 1/10 s; a slow drift around the magnetic axis of the Earth (the drift is eastward for electrons and westwards for ions), such drift is from the current feld line to its neighbor, with the particle keeping roughly the same distance from the axis. A

**Fig. 10.7** Radiation belts of the Earth. (Figure from Van Allen radiation belt. Reprinted with permission from Wikipedia. Author Booyabazooka at English Wikipedia, https://commons.wikimedia.org/ wiki/File:Van\_Allen\_radiation\_belt.svg)

typical time to complete a full circle around the Earth is a few minutes.

In the area above the southeastern part of South America and the South Atlantic, the inner radiation belt approaches the surface of Earth down to a few hundreds of kilometers (South Atlantic Anomaly, SAA). This is caused by the tilt and shift of the axis of the dipole-like magnetic feld of the Earth with respect to its axis of rotation [33]. The dip of magnetic lines leads to an increased particle fux within this region.

The dose rate experienced by the astronauts on the ISS has a considerable contribution from trapped protons in the inner Van Allen belt because the ISS orbit with an altitude of about 400 km passes through this belt at the SAA (roughly 50% of the total dose rate) [34].

# **10.3.2 Radiation Environment in Low Earth Orbit (LEO)**

A low Earth orbit (LEO) is an Earth-centered orbit close to our planet with an altitude ranging from 160 to 2000 km. Thus, the ISS, which fies at an altitude of around 400 km, is also in such an orbit, with an orbital inclination (the tilt of the orbital plane with respect to the equatorial plane, which helps to understand an orbit's orientation with respect to the equator) of 51.6° and an orbiting period of 90–93 min. Consequently, in 24 h the ISS makes 16 orbits of Earth and travels through 16 sunrises and sunsets. The environment of these altitudes is extreme and characterized by microgravity, high vacuum, meteoroids, extremes of temperature, ionospheric plasma, space debris, and UV as well as ionizing radiations.