# Reproductive Capacity, but not Food Consumption, is Reduced by Continuous Exposure to Typical Genotoxic Stressor γ-Rays in the sentinel species Gammarus fossarum

Sandrine Frelon, Rémi Recoura-Massaquant, Nicolas Dubourg, Laura Garnero, Jean-Marc Bonzom, Davide Degli-Esposti

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## ► To cite this version:

Sandrine Frelon, Rémi Recoura-Massaquant, Nicolas Dubourg, Laura Garnero, Jean-Marc Bonzom, et al.. Reproductive Capacity, but not Food Consumption, is Reduced by Continuous Exposure to Typical Genotoxic Stressor γ-Rays in the sentinel species Gammarus fossarum. Environmental Toxicology and Chemistry, 2024, 43 (9), pp.2071-2079. 10.1002/etc.5949. hal-04867566

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Reproductive capacity but not food consumption is reduced by continuous
exposure to the typical genotoxic stressor γ-rays, in the sentinel species
*Gammarus fossarum*.

Frelon, S.¹#, Recoura-Massaquant, R. ², Dubourg, N. ¹, Garnero L.², Bonzom, J.-M.¹, Degli-Esposti, D.²#

1 – IRSN / PSE-ENV / SERPEN Laboratoire d'écologie et d'écotoxicologie des radionucléides - Saint Paul lez Durance (13115) France

2 – INRAE, UR RiverLy, Ecotoxicology Team. Centre de Lyon-Grenoble Auvergne Rhône-Alpes, 5 rue de la Doua CS 20244, 69625 Villeurbanne, France.

# Corresponding authors

## HIGHLIGHTS

* The impact of chronic exposure to ionizing radiation was assessed in male and female gammarids.

* Ionizing radiation affects the reproduction of *Gammarus fossarum*, that is critical for population dynamics.

* Food consumption, which is linked to the general health status of gammarids, is not reduced by ionizing radiation.

* Radio-induced reprotoxicity is more severe in male than female gammarids after chronic exposure.

## ABSTRACT

The long-term impact of radiocontaminants (and the associated risks) for ecosystems are still subject to vast societal and scientific debate while wildlife is chronically exposed to various sources and levels of either environmental or anthropogenic ionizing radiation due to the use of nuclear energy.

This study aims to assess induced phenotypical responses in both male and female gammarids after short-term continuous γ-irradiation, acting as a typical well characterized genotoxic stressor that can interact directly with living matter. In particular, we started characterizing the effects using standardized measurements for biological effects on few biological functions for this species, especially feeding inhibition tests, molting and reproductive ability, which have already been proven for chemical substances and are likely to be disturbed by ionizing radiation. The results show no significant differences in terms of the survival of organisms (males and females), of their short-term food consumption which is linked to the general health status (males and females) and of the molting cycle (females). In contrast, exposure significantly affected fecundity (number of embryos produced) at the highest dose rates for irradiated females (51 mGyh⁻¹) and males (5 and 51 mGyh⁻¹). These
results showed that, in gammarids, reproduction, that is a critical endpoint for population dynamics, is the most radiosensitive phenotypic endpoint, with significant effects recorded on male reproductive capacity, which is more sensitive than in females.

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## INTRODUCTION

In the environment, wildlife is chronically exposed to various sources and levels of ionizing radiation, natural or anthropogenic due to the use of nuclear energy (civilian or military use, accidental release, and side activities) (UNSCEAR 2000). Natural ionizing radiation includes cosmic rays and terrestrial radiations coming from natural elements present in environments, such as uranium, thorium series and potassium (⁴⁰K). After accidental release (e.g. Chornobyl, Fukushima), the levels of ionizing radiation are mainly due to persistent radioisotopes such as ¹³⁷Cs, ⁹⁰Sr and plutonium isotopes, in terrestrial and aquatic habitats. Whatever their decay process, radiocontaminants are harmful to organisms in the environment due to their radioactivity, referred to herein as ionizing radiation (IR). Indeed, IR can interact directly (by the ionization of biomolecules) or indirectly (by water radiolysis and the production of reactive oxygen species) with DNA and other biomolecules, and cause damage at different biological levels (UNSCEAR 2008). Characterization of such chronically induced biological effects in different ecosystems and their representative non-human taxa, is crucial to define relevant levels of radiological protection, to assess and predict ecological risk and then minimize impacts.

Since the Rio conference in 1992 and the publication of the Convention on biological diversity, the International Atomic Energy Agency (IAEA) and the International Commission on Radiological Protection (ICRP) have started to propose methodologies and reference values for radiological protection of non-human species. Some reference levels have been proposed for different ecosystems, i.e. generic-, terrestrial-, aquatic- one, ranging from 10 µGy·h⁻¹ (Garnier-Laplace et al., 2010) to 400 µGy·h⁻¹ (UNSCEAR 2008; IAEA 1992) to assist in assessing the risk. In addition, 'Derived Consideration Reference Levels' (DCRLs) have been defined by International Commission of Radiological Protection (ICRP) for 'reference animals and plants' (RAPs) (ICRP 2008). However, the available RAPs do not consistently represent the diversity of the species in the ecosystems (Spurgeon, Lahive et al. 2020). In particular, crustaceans and their radio-sensitivity are currently considered as insufficiently described and characterized. For instance, while the radio-sensitivity of the parthenogenetic *Daphnia magna* has been quite well described, this is not true for crustaceans with a sexual reproductive strategy (Dallas, Keith-Roach et al. 2012; Fuller, Lerebours et al. 2015). Moreover, while both male and female reproductive functions are sensitive to exposure to ionizing radiation, the effects on males have been studied less (Fuller, Lerebours et al. 2015). Regarding the ICRP DCRLs, effects on crustaceans have been recorded from 0.42 mGy·h⁻¹ with few results on
reproductive capacities and mortality (ICRP 2008). These data deal with (i) the hatching success of the calanoid copepod Diaptomus clavipes, the fertility and fecundity of the females amphipod *Gammarus duebeni*; (ii) the proportion of total deaths at molting after the chronic exposure of the blue crab *Callinectes sapidus* at a dose rate of 70 mGyh⁻¹ for 70 days; and (iii) the mortality (LD50/40, corresponding to LD50 after 40 days) at 15 Gy for post-juvenile grass shrimp *Palaemonetes pugio*. Other data were mainly obtained under acute conditions and taking into account radiation other than gamma, e.g. X-rays (*Gammarus duebeni*).

Gammarids (Gammarus sp.) are amphipod crustaceans with sexual reproduction and often abundant in aquatic ecosystems (Europe: *G. fossarum* and *G. pulex*, Asia: *G. nipponensis*...). They play an important ecological role as detritivores, significantly contributing to the degradation of leaf litter in watercourses and therefore to the nutrient cycle (Kelly, Dick et al. 2002). They are used as sentinel organisms in ecotoxicology, and recognized for their sensitivity to the quality of the environment and characterized in terms of their biological responses to numerous chemical compounds (metallic and organic traces) (Kunz, Kienle et al. 2010; Chaumot, Geffard et al. 2015). In addition, some normalized bioassays, such as feeding behavior or reprotoxic response measurements are available to assess the response to chemical contaminants (Geffard, Xuereb et al. 2010; Coulaud, Geffard et al. 2011). Thus, *G. fossarum* is a freshwater species used (i) to assess the ecotoxicity of chemical substances in aquatic environments (caging in situ bioassays on rivers, effluents, etc.), or in laboratory (Geffard, Xuereb et al. 2010; Lopes, Chaumot et al. 2020), (ii) as a tool for biomonitoring chemical status and as a bioindicator for the ecological status of a wide variety of aquatic systems (standardized AFNOR XP T90-721 (Babut, Ferrari et al. 2020), AFNOR XP T90-722-1/2/3 according to the effect criteria), within the Water Framework Directive (WFD, EU 2013) (Alric, Geffard et al. 2019).

Gammarids have not been extensively documented in terms of IR, however they could offer a relevant tool for biomonitoring radio-contamination after the characterization of radio-induced biological effects. Thus, to increase our knowledge of radiation protection for a freshwater crustacean with a sexual reproductive strategy, we undertook a dose-response study in controlled laboratory conditions at five external gamma irradiation levels (0.0042, 0.047, 0.57, 5.01, 51mGyh⁻¹) and a control condition. This range of irradiation levels (dose rates) includes low levels (0.0042 and 0.047 mGyh⁻¹) framing the threshold value of 10 µGyh⁻¹ (Garnier-Laplace, Della-Vedova et al. 2010) and level of 51 mGyh⁻¹, close to those recorded in post-accident situations (e.g. immediately after the Chernobyl nuclear accident (Garnier-Laplace, Geras'kin et al. 2013)). This study aims to assess phenotypical responses after the continuous γ-irradiation of either males or females. In particular, life history traits, such as survival, feeding behavior, molting and reproductive ability, in male or female *Gammarus fossarum*, were assessed after exposure during a reproductive cycle (14 days). Beyond that, since ionizing radiation is a well-characterized genotoxic stressor that can interact
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directly with living matter, the knowledge of its ecotoxic profile from genotoxic effects to individual
adverse outcome, without interference from pollutant biokinetics, can be useful to understand
ecotoxicity of other chemical pollutants likely to exhibit genotoxicity.

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## MATERIAL AND METHODS

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### Organism sampling and maintenance

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The gammarids came from the River Le Pollon, located in Saint-Maurice-de-Rements, France (45°57′25.8″N 5°15′43.6″E), a source population characterized by very low pollutant concentration levels (Alric, Geffard et al. 2019). They were collected using a hand-held net in March and June 2021. Adult organisms were recovered through 2 and 2.5 mm sieves and then quickly transferred to the laboratory. The organisms were acclimatized for a period of two weeks in 30 L tanks continuously supplied with drilling groundwater (300 µS/cm) and constantly aerated. A 10/14 h light/dark photoperiod was maintained, and the temperature was kept at 12 ± 1 °C. Organisms were fed *ad libitum* with alder leaves (*Alnus glutinosa*), previously conditioned for at least four days in water. Freeze-dried *Tubifex* worms were added as a dietary supplement twice a week.

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### Gammarid irradiation and experimental design

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The gammarids were irradiated in incubators (Panasonic MIR 54 without light, at 15.5°C controlled with Tinytag Aquatic 2® data loggers) for 14 days. The gammarids were placed in 500 mL glass beakers filled with drilling water from our laboratory, previously tempered at 15.5°C and oxygenated for 24 hours. This temperature, widely used for ecotoxicological experiments, was chosen as an intermediate between the holding temperature and a temperature which accelerates the reproductive cycle in order to perform the chronic exposure over the development period of females, avoiding a too important cumulative dose. Water was renewed every two days and dead organisms were removed. Beakers were placed according to a specific layout obtained based on Monte Carlo simulations to achieve the required dose rate for each experimental unit. Two Radio Photo Luminescent dosimeters (RPL, GD-301 type, Chiyoda Technol Corporation, Japan) were placed on beakers (one at the front, one at the rear) in order to calculate the average dose rate in the sample units, i.e. with the ratio between dose measured by dosimeters and the irradiated period. Pieces of plastic mesh were placed in each beaker as shelters to avoid cannibalism.

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Gamma-rays were emitted from external <sup>137</sup>Cs sources using the MICADO-Lab facility (*Moyen d'Irradiation Chronique pour l'Acquisition de relations DOse-effet en Laboratoire*) (370 GBq) (Cadarache, FRANCE). Five dose rates and a control, 0, 0.005, 0.054, 0.57, 5.01 and 51.5 mGyh<sup>-1</sup>, corresponding to six cumulative doses after 14 days (0, 1.7, 18.1, 191.7, 1 683, 17 312 mGy), were

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tested. The control condition was kept at a distance in the same room. In addition, it was offset from the source and separated with a concrete wall to maintain a low dose rate (130-140 nGyh⁻¹, reaching 0.03 mGy after 14 days).

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## Biological parameter measurements

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The following parameters were chosen on the basis of proven protocols used for chemical pollutants (Geffard, Xuereb et al. 2010; Coulaud, Geffard et al. 2011; Coulaud, Geffard et al. 2014), with regard to the existing literature on the effects of ionizing radiation (Fuller, Lerebours et al. 2015) and the known radiosensitivity of reproductive capacity (Garnier-Laplace, Della-Vedova et al. 2010).

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**Survival:** gammarids were counted for survival rate assessment for each assay after 7 or 14 days of irradiation.

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**Feeding assay:** The feeding rate during irradiation was assessed according to the procedure described in Coulaud et al (2011) (Coulaud, Geffard et al. 2011). Two series of experiments were carried out in March and June 2021. Three replicates of 20 male gammarids of homogenous size (11 ± 0.7 mm) per condition were considered in the first series and four replicates of 10 synchronized (i.e. at the same molting stage) female gammarids (10.3 ± 0.9 mm) per condition were considered in the second series. After 7 days, leaf discs were collected and numerically scanned to estimate leaf consumption. Feeding rate (FR) is expressed as a consumed surface per day per gammarid (mm² d⁻¹ organism⁻¹).

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**The reproductive capacities of females:** As described by Geffard et al. (2010) (Geffard, Xuereb et al. 2010), the reproductive cycle of *Gammarus* females is synchronized with their molting cycle. Synchronized females were selected at the start of their reproductive/molting cycle, between 0 and 48 hours after ecdysis (stage AB). Each molt stage is characterized by a specific marsupial embryonic development stage and the size of developing follicles visible on the ovarian membrane. Six molt stages were defined, A and B for postmolt, C1 and C2 for intermolt, and D1 and D2 for premolt. The mean follicle surface is highly dependent on the molting stage, with the highest follicle growth occurred during the C2 stage. In AB females, stage 1 embryos correspond to newly fertilized, undifferentiated eggs. Then embryos are characterized by a comma-like shape (stage 2), which appeared during the C1 molting stage. Stage 3 embryos appeared at the C2 molting stage and are characterized by the presence of cephalothorax and segmented appendages. At stage 4, the compound eye is fully visible and appendages are also fully developed. Stage 5 corresponds to newly hatched juveniles.

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Reproductive output was assessed by euthanizing 15 females for each condition at the end of exposure. When reproductive output is assessed, females are expected to be in the middle of the reproductive cycle, i.e. in stage C2 (Geffard et al (2010)).

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The molting stage, the number of oocytes, and the number of embryos per female were evaluated as described by Geffard et al (2010). The integumental morphogenesis of the third and fourth periopod pairs was observed by optical microscopy (x200) to identify the molting stage of the euthanized females, as described by Geffard et al. (2010).

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Embryos were manually recovered from the marsupium and counted under a binocular microscope. The number of oocytes per female was counted by in vivo observation of the gonads under a binocular microscope. Since the number of oocytes and embryos depends on female body size and molting stage, this number was standardized by dividing the total count of oocytes and embryos by female body size minus size at puberty (5 mm). Only females at molting stages C2 and D1 were considered, as described in Geffard et al 2010.

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Female gammarids were fed *ad libitum* with alder leaves. These measurements were repeated twice, in March and June 2021, with a different batch of females each time.

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**Post-exposure reproductive assessment of males and females:** After 14 days of irradiation, batches of irradiated male and female gammarids were reciprocally paired with non-irradiated organisms and were kept in the laboratory in control conditions for a new reproductive/molting cycle. Regarding the batch of irradiated males, three replicates of five organisms were paired with the same number of non-irradiated females, at the very end of the molting and reproduction cycle, just before ecdysis. Concerning the batch of irradiated females, at the start of irradiation, female gammarids were selected in the middle of their molting and reproductive cycle (i.e. in stage C1) so that the end of irradiation coincided with the day before ecdysis (Chaumot, Coulaud et al. 2020; Lopes, Chaumot et al. 2020). Thus, two replicates of ten organisms were paired with the same number of mature non-irradiated males. During this post-exposure period, organisms were placed in separate cages, with one cage per condition. The organisms were kept at 16°C, continuously supplied with drilled water and constantly aerated. A few days after pairing, the cages were monitored to ensure that the females laid eggs. Female gammarids that had not laid eggs after a few days were removed. The embryos produced were counted seventeen days after ecdysis and egg laying by the females.

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## Statistical analysis

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All statistical analyses were run using R software version 3.2.4. Control and irradiation or recovery exposures were compared. Differences in survival, consumption rates, post-irradiation fertility and the percentage of females without embryos between conditions were tested by a pairwise comparison adjusted for multiple comparisons by Bonferroni corrections. The nonparametric test (Kruskal-Wallis) was used to compare both fecundity and fertility between conditions. Proportion tests were used to identify differences in molting rates versus the control conditions. The significance level (alpha risk) was set at 0.05.
## 208 RESULTS AND DISCUSSION

### 209 - Survival assay

210 After 14 days of gamma irradiation, the gammarid survival rate remained high for both males and 211 females at all doses with mean survival systematically above 70% (Table 1). Our dose rate exposure 212 and maximum cumulative dose (17.3 Gy) results match all existing data for crustaceans after gamma 213 exposure (Fuller et al, 2015), meaning that no mortality is expected under 50 mGyh⁻¹ (~15 Gy). In 214 particular, data showed that 30d LD50 has been assessed at 15 Gy for grass shrimp, *Palaemonetes* 215 *pugio*. In addition, mortality is reached for dose rates as high as 212 mGyh⁻¹ for *Daphnia Pulex* when 216 irradiated after 385 days (~twelve “generations”, cumulative dose / generation ~ 152 Gy), or, 290 217 mGyh⁻¹ for *Callinectes sapidus* over a 70-day period (cumulative dose ~ 487 Gy).

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### 219 - Feeding assay

220 No significant difference in feeding rate was observed between the control conditions and 221 gammarids irradiated to different doses, for both males and females (Figure 1 – Pairwise 222 comparisons, Bonferroni correction, adjusted p-value>0.05).

223 Feeding, linked to general health status of organisms, is a well-known marker in ecotoxicology, and is 224 sensitive to different types of chemical contamination, indicating general toxicity in various pollution 225 contexts (agricultural, mining, urban, industrial) (Coulaud, Geffard et al. 2015). While this experiment 226 is usually carried out on males, we targeted females in order to obtain the levels of food 227 consumption in the irradiated females and take this data into account regarding female reproductive 228 output as described by Coulaud et al. (2015). Only synchronized females were selected in the first 229 stage of their reproductive cycle to avoid varying leaf consumption depending on the stage of their 230 reproductive cycle. Gamma radiation did not alter the feeding behavior of gammarids at the tested 231 doses during the exposure time regardless of the sex. This result is consistent with the absence of 232 effect on *Daphnia magna* filtration rate after chronic external irradiation (Gilbin, Alonzo et al. 2008) 233 or alpha contamination (Alonzo, Gilbin et al. 2008). However, this results on 14j does not presume of 234 the results on its detritivore capacities usually assessed on longer period.

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### 236 - Molting cycle assessment in females

237 The proportions of females before or after the C2 stage of the reproductive cycle are shown in 238 Table 2. 15 females were used for each condition to determine their molting stage. For the two 239 series of experiments, after 14 days of exposure, around 70% of the control females were at molting 240 stage C2. At all doses, the proportions of females at stage C2 do not differ significantly (proportion 241 test, experiments 1 & 2; p-value >0.05). The molting cycle of females was not delayed by gamma
radiation exposure. Molting is a key physiological process for crustacean growth and reproduction (Gouveia, Bonneton et al. 2018; Street, Eytcheson et al. 2019). This process is controlled by hormones, and a number of chemicals, some of which act as endocrine disruptors, have been shown to affect molting, even inducing increased mortality for the organisms in some cases, such as for *Daphnia* (Song, Villeneuve et al. 2017; Chen, Gu et al. 2019; Wang, Yang et al. 2023). To our knowledge, only one study has tested the effects of gamma radiation on the molting of crustaceans (Han, Won et al. 2014). In this study, acute gamma radiation affected molting during the larval stage in the copepod *Tigriopus japonicas*. The lack of observed effects in the adult gammarids investigated in this study suggests that the susceptibility of the molting process to gamma ray radiation may depend on the species and life stage and warrant further studies to explore these differences across crustacean species.

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254 - Reproductive capacity of irradiated females

Figure 2 shows the numbers of oocytes and embryos (normalized per the female size) produced during the irradiation period for the two series of experiments. The number of oocytes produced was not significantly different whatever the dose rate; however, a non-significant trend to decrease in the production of the number of oocytes seems to be observed at the highest dose (Kruskal Wallis test, A1 p-value: 0.069, A2 p-value: 0.36). in addition, the number of embryos produced during irradiation was not impacted at any dose (Kruskal Wallis test, B1 p-value: 0.72, B2 p-value: 0.45). This result is consistent with an *in situ* study finding no evidence of impact on female *A. aquaticus* reproductive parameters of current dose rates in aquatic systems at Chernobyl (< 27.1 µGy/h) (Fuller, Ford et al. 2018). However, brood size is not fully integrative of reproductive impairment assessment since it considers a number of embryos produced but not the viability of progeny that can also depend on the quality of ovocytes. Another study found that the brood size of the parthenogenetic *Daphnia magna* was significantly reduced after chronic exposure to gamma rays (35mGyh⁻¹). While the authors did not consider whether it was due to inadequate oogenesis, embryogenesis or both (Parisot, Bourdineaud et al. 2015), other paper described a decrease in ovulation rate in *C. elegans* with no impact on hatching success after chronic irradiation exposure at the same dose rate (Dufourcq-Sekatcheff et al 2021).

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271 - Post-exposure reproductive assessment of males and females

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As described by Geffard et al. 2010, oocytes produced during a molting cycle contribute to the formation of the embryos in the next brood. In order to assess the impact of gamma radiation on the quality of the gonads, we analyzed the ability of males and females to reproduce after 14 days of
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irradiation. Batches of irradiated organisms were maintained in the laboratory during a new molting cycle, without irradiation, and paired with non-irradiated organisms.

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**Figure 3** shows the amounts of embryos produced (A) in non-irradiated females after mating with irradiated males, (B) in irradiated females after mating with non-irradiated males as well as the results of females with (C) and without (D) embryos from irradiated females paired with non-irradiated males.

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The fertility of the females was significantly impacted when the males had been irradiated at the highest doses, with a threshold effect observed from 5 mGyh⁻¹ and a median embryo production of 0.2 embryos/female at the highest dose (Pairwise comparisons, Bonferroni correction, p<0.01). This result could be due to unrepaired DNA damage in male gametes as previously observed by (Lacaze, Geffard et al. 2011). In particular, reproduction defects in males have already been observed for levels of DNA damage exceeding a minimal threshold suggesting that gammarid spermatozoa are extremely sensitive to genotoxic insults and unable to repair this damage (Lacaze, Geffard et al. 2011). Only scarce data in the literature describes the effects on crustaceans after chronic exposure to ionizing radiation, especially on males (Fuller, Lerebours et al. 2015) but our results are consistent with the ones observed after exposure of male *E. marinus* to ³²P β-emitter at a lower dose rate (up to 0.4 mGyh⁻¹) (Fuller, Smith et al. 2019). In that paper, significant effects were observed on resultant female fecundity at all dose rates and in the frequency of embryo abnormalities per female for males exposed to 0.04 and 0.4 mGy.h⁻¹. *E. marinus* also exhibit a statistically significant reduction of sperm quality with significant differences in DNA damage for the higher dose rate but no significant effects recorded for sperm count. In the same paper no significant results were found on male *G. pulex* (for which high mortality was observed in all groups including controls) which can be consistent with our observed effects that seem starting to appear between 0.57 and 5 mGyh⁻¹.

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A decrease in the fertility of females was also observed when they were irradiated during the previous reproductive cycle (exposure between stages C1 and D2) and paired with non-irradiated males. While high inter-individual variability was observed, mainly due to the high percentage of females without embryos in the marsupium increasing rapidly in proportion to the exposure dose rate (figure 3D), a decreasing trend was observed starting from the lowest dose (0.5 mGyh⁻¹) (figure 3C). However, even at the highest dose, some females managed to produce embryos, which is not the case when the males were irradiated (only a tiny number of embryos were produced). This result can be linked to the oocyte production during irradiation. At this step no significant results were found on the number of oocytes (only a trend to decrease). On another invertebrate, ionizing radiation have been shown to slow down oocyte production, probably due to apoptosis and cell cycle arrest (Dufourcq Sekatcheff, Godon, et al. 2023). This hypothesis remains to be investigated in freshwater crustaceans.

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In terms of the differences in female fecundity observed after male or female irradiation, these results are consistent with both the genotoxic properties of ionizing radiation (Pereira, Bourrachot et al. 2011) and the conclusions of Lacaze, Geffard et al. 2011) for experiments on *Gammarids* with genotoxicants, i.e. that spermatozoa were considerably more sensitive to DNA damage than oocytes.
This has already been identified in other organisms and partly attributed to a loss of apoptotic capabilities during spermatogenesis (Aitken and De Iuliis 2007). For instance, in the soil invertebrate *C. elegans* after irradiation, cell cycle arrest to enable DNA repair and increasing apoptosis occurred in oocytes whereas spermatids were 40% less numerous in the same condition of irradiation, but without involvement of apoptosis (Dufourcq-Sekatcheff et al 2021; Dufourcq Sekatcheff, Godon, et al. 2023).

Other studies described in the literature are mainly performed in acute exposure conditions. For the dipteran *Drosophila suzukii*, full female sterility was achieved at 75 Gy, while 99.67% male sterility was observed at 200 Gy, suggesting that females are more sensitive than males for this species (Krüger, Schlesener et al. 2018), while male and female sterility was observed at similar doses for another dipteran, *Anastrepha fraterculus*, at 50 Gy for females and 70 Gy for males (Krüger, Ferreira et al. 2021). Interestingly, sexual dimorphism is not systematically evaluated in arthropods as an effect of chronic exposure to gamma radiation and may actually vary across species and type of exposure.

### - Radiological protection concerns

Our results on reproductive parameters showed that reproduction is the most radiosensitive phenotypic endpoint in gammarids. Indeed, neither mortality nor short-term food consumption, proxy of their general health status, are impacted in our controlled conditions of irradiation. This work is consistent with many other studies in invertebrates describing radiation-induced reprotoxicity as the most radiosensitive individual endpoint among the ones used to derive species radiosentitivity curves i.e. mortality, morbidity, reprotoxicity (Garnier-Laplace, Della-Vedova et al. 2010; Beresford, Horemans et al. 2020; Shuryak 2020).

In addition, our results showed that radio-induced reprotoxicity is more severe for male than female gammarids after chronic exposure. Here, we reported significant effects on male reproductive capacity at 5 mGy·h⁻¹ in the parental generation of a freshwater crustacean which is much lower than for other crustaceans such as *Daphnia magna* (35 mGy·h⁻¹) regarding a phenotypical endpoint (Parisot, Bourdineaud et al. 2015). Excepted the data on male marine crustaceans *E. marinus* (Fuller, Smith et al. 2019) most data on chronic exposure to gamma irradiation for other crustaceans only demonstrated molecular damage at such exposure dose rates (Fuller, Lerebours et al. 2015) that
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remain high dose rates only present after nuclear accidents. And, many data on freshwater crustaceans come from studies on the (female) parthenogenetic species *Daphnia Magna*.

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The dose rates found for the radio-induced phenotypical effects observed for the parent generation exposed for 14 days are consistent with the ICRP DCRLs for crustaceans defined as between 0.4 and 4 mGy h⁻¹. However, this observed No-Effect Dose Rate is likely to be decreased if results observed after multi-generational exposure on *Daphnids* (Parisot, Bourdineaud et al. 2015) can be extrapolated for *Gammarids*.

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Finally, these *Gammarus fossarum* data seem confirming the biological key events (KE) “oogenesis/spermatogenesis impairment” in females/males, leading to the adverse outcome “reproduction decrease” previously identified in an Adverse Outcome Pathway consensus (AOPc) set up with the results of many taxonomic group (Tollefsen, Alonzo et al. 2022). Further analysis of sub-individual endpoints will enable to decipher biological pathway involved in the observed effects and to fuel the up-stream KE of this previous AOPc scheme. This can also enable to link together events at different biological levels and get sub-individual key events likely to be sensitive at lower dose rates.
All of that requires further investigations but provides worthwhile prospects for ERA.

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## CONCLUSION

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Our results showed that short-term food consumption in *Gammarids* is not impacted in our conditions while reproduction is the most radiosensitive phenotypic endpoint of those tested. This is of main interest since reproduction is a key ecological parameter as the ability of a species to reproduce directly influences its population dynamics. In addition, our results suggest the greater sensitivity of males, as previously observed in other species (Lacaze, Geffard et al. 2010; Dufourcq-sekatcheff, Cuiné et al. 2021) but for which little data is available for crustacean taxa (Fuller, Lerebours et al. 2015). This observation of effects for the first generation, after continuous exposure over 14 days, also confirm the chance of observing earlier effects at lower dose levels if irradiated for longer, over several generations, as in the freshwater ecosystems, since the response of the population tends to change and worsen over generations (Parisot, Bourdineaud et al. 2015). This ecotoxic profile at the individual biological level for ionizing radiation, that is a known well-characterized genotoxic stressor, may be helpful to fuel a database of adverse outcomes likely to be induced by other chemical pollutants with genotoxic properties. This is a first step in the characterization of *Gammarus fossarum* facing ionizing radiations in order to fuel the existing AOPc on reproductive impairments and before its further use.

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## ACKNOWLEDGMENTS
377 The authors would like to thank the IRSN for their financial support. The authors benefitted from the
378 French GDR "Aquatic Ecotoxicology" framework which aims at fostering stimulating scientific
discussions and collaborations for integrative approaches. Moreover, the authors would like to thank
380 O. Simon for its careful review of the paper and valuable suggestions.

381

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498 **Table 1**: Percentage of gammarids survival<sup>a</sup> at different dose rates of *gamma*-rays after 14d of exposure – No
499 significant differences between conditions were observed.

<table><thead><tr><th>Dose rate in mGy.h<sup>-1</sup></th><th>0</th><th>0.005</th><th>0.054</th><th>0.57</th><th>5.01</th><th>51.5</th></tr></thead><tbody><tr><th>Total cumulated dose in mGy</th><td>0.03</td><td>1.70</td><td>18.1</td><td>191.7</td><td>1 683</td><td>17 312</td></tr></tbody></table><table><thead><tr><th rowspan="2"></th><th colspan="6">Males</th></tr><tr><th>n=3</th><th>n=3</th><th>n=2</th><th>n=2</th><th>n=3</th><th>n=3</th></tr></thead><tbody><tr><th>14 d</th><td>78.3<br>(70 - 85)</td><td>81.7<br>(75 - 85)</td><td>90<br>(85 - 95)</td><td>92.5<br>(85 - 100)</td><td>83.3<br>(75 - 95)</td><td>90<br>(90 - 90)</td></tr></tbody></table><table><thead><tr><th rowspan="2"></th><th colspan="6">Females</th></tr><tr><th>n=4</th><th>n=4</th><th>n=4</th><th>n=4</th><th>n=4</th><th>n=4</th></tr></thead><tbody><tr><th>14 d</th><td>75<br>(70 - 80)</td><td>80<br>(40 - 100)</td><td>90<br>(80 - 100)</td><td>72.5<br>(60 - 100)</td><td>87.5<br>(80 - 90)</td><td>77.5<br>(40 - 100)</td></tr></tbody></table>

<footnote><sup>a</sup>Survival is indicated by the mean and the min-max values</footnote>
<table><caption>**Table 2** : Percentage of gammarid females in the different molting stages after 14 days of gamma-rays irradiation for the two series of experiments</caption><thead><tr><th colspan="7">Percentage of Gammarids in the molt stage (%)</th></tr><tr><th>Dose rate in mGy.h<sup>-1</sup></th><th>0</th><th>0.005</th><th>0.054</th><th>0.57</th><th>5.01</th><th>51.5</th></tr></thead><tbody><tr><th colspan="7">Female - Experiment 1</th></tr><tr><th>n</th><td>15</td><td>15</td><td>15</td><td>15</td><td>15</td><td>15</td></tr><tr><th>&lt;C2</th><td>33.3</td><td>20.0</td><td>46.7</td><td>40.0</td><td>13.3</td><td>40.0</td></tr><tr><th>&gt; C2</th><td>66.7</td><td>80.0</td><td>53.3</td><td>60.0</td><td>86.7</td><td>60.0</td></tr><tr><th colspan="7">Female - Experiment 2</th></tr><tr><th>n</th><td>15</td><td>15</td><td>15</td><td>15</td><td>15</td><td>15</td></tr><tr><th>&lt;C2</th><td>26.7</td><td>46.7</td><td>20.0</td><td>26.7</td><td>20.0</td><td>20.0</td></tr><tr><th>&gt; C2</th><td>73.3</td><td>53.3</td><td>80.0</td><td>73.3</td><td>80.0</td><td>80.0</td></tr></tbody></table>

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<figure><img src="image_3.png" /></figure>

**Figure 1** : Measurement of feeding rates (FR) at different doses of *gamma*-rays after 7d of exposure (black points: mean of experimental replicates; grey points: replicate value per dose) for (A) male gammarids (number of replicates = 3 beakers of 20 males); and (B) molting synchronised female gammarids (number of replicates = 4 beakers of 10 females)
<figure><img src="image_4.png" /><figcaption>**Figure 2** : Individual responses to 14-days *gamma*-rays exposure at different doses during two experiments.

(A1) and (A2) Oocyte production (size-normalized oocyte number per female). (B1) and (B2) Embryo production (size-normalized embryo number per female). The median, first and third quartile are shown.</figcaption></figure>

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<figure><img src="image_5.png" /><figcaption>**Figure 3:** Effect of 14 days irradiation with gamma-rays at different doses on the production of embryos resulting from a new reproductive cycle without irradiation. (A) Results from irradiated males paired with non-irradiated females, (n=3 beakers of 5 irradiated males and 5 females). (B) Results from all irradiated females paired with non-irradiated males (n=2 beakers of 10 irradiated females and 10 males). The median, first and third quartile are shown. (C) Results from irradiated females with embryos paired with non-irradiated males. The median, first and third quartile are shown. (D) Percentage of females without embryos from irradiated females paired with non-irradiated males. *means significant difference compared to control (p.value < 0.05 – For 5.01 mGy/h, p.value = 0.0506)</figcaption></figure>

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