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Effects of acute gamma irradiation on Folsomia candida (Collembola) in a standard test
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Ecotoxicology and Environmental Safety 71 (2008) 590–596 www.elsevier.com/locate/ecoenv
Effects of acute gamma irradiation on Folsomia candida (Collembola) in a standard test Taizo Nakamoria,, Satoshi Yoshidaa, Yoshihisa Kubotaa, Tadaaki Ban-naia, Nobuhiro Kanekob, Makiko Hasegawac, Ryosaku Itohc b
a Environmental Radiation Effects Research Group, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage, Chiba 263-8555, Japan Soil Ecology, Graduate School of Environment and Information Sciences, Yokohama National University, 79-7 Tokiwadai, Yokohama 240-8501, Japan c Biological Laboratory, Faculty of Arts and Sciences at Fujiyoshida, Showa University, 4562 Kamiyoshida, Fujiyoshida, Yamanashi 403-0005, Japan
Received 22 April 2007; received in revised form 19 September 2007; accepted 13 October 2007 Available online 21 December 2007
Abstract An understanding of the effects of ionizing radiation on non-human biota is required by the International Commission on Radiological Protection for the radiological protection of the environment. We examined dose–effect relationships for gamma radiation on survival, growth, and reproduction in the soil invertebrate Folsomia candida (Collembola) in a standard laboratory test. F. candida were acutely irradiated at increasing doses of gamma radiation, and subsequent survival, growth in body length, and number of neonates produced by irradiated specimens were examined. The 50% lethal dose was at 1356 Gy, and the 10% and 50% effective doses (ED10 and ED50) for growth were at 32 and 144 Gy, respectively. The ED10 and ED50 values for reproduction were at 7.1 and 21.9 Gy, respectively. These data establish important baselines for the radiological protection of terrestrial ecosystems based on scientific principles. r 2007 Elsevier Inc. All rights reserved. Keywords: Dose–effect relationship; Growth; Non-human biota; Radiological protection; Reproduction; Soil invertebrate; Springtails; Survival
1. Introduction The importance of radiological protection of the environment based on scientific principles is gaining international recognition as environmental issues garner more attention (International Commission on Radiological Protection (ICRP), 2003). The ICRP (2003) has suggested reference organisms for studying the impact of radiation on ecosystems. The Framework for Assessment of Environmental Impact (FASSET) project was launched under the European Commission Fifth Framework Programme to provide a framework for assessing the impact of ionizing radiation on non-human biota (Larsson, 2004). The FASSET project provided a set of reference organisms relevant to different exposure situations taking into account the environmental fate of radionuclide release and exposure pathways, and built an effects database for Corresponding author. Fax: +81 43 206 3267.
E-mail address: [email protected] (T. Nakamori). 0147-6513/$ - see front matter r 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2007.10.029
the reference organisms, including soil invertebrates, for terrestrial ecosystems (Larsson, 2004). However, available data for soil invertebrates were still limited. Single-species laboratory tests are used for ecological impact assessments for chemical substances. Because of their ecological importance, soil invertebrates are used for ecological impact assessments of terrestrial ecosystem pollutants (van Gestel et al., 1997; van Straalen, 2004). Standard laboratory tests using certain soil invertebrate species have been developed by international organizations such as the International Organization for Standardization (ISO) and the Organization for Economic Cooperation and Development (OECD; Ja¨nsch et al., 2005). Population and community level effects are extrapolated based on these tests. A similar methodology can be applied for assessing the environmental impact of ionizing radiation (Garnier-Laplace et al., 2004). Collembolans are part of the soil mesofauna and are the most numerous and widely distributed insects in soil ecosystems. They are thought to contribute to decomposition processes in the soil, and they serve as prey for several
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invertebrates and small vertebrates (Hopkin, 1997). Collembolans have been widely used for ecological impact assessment (Fountain and Hopkin, 2004; Hopkin, 1997; Timmermans et al., 2005). Among collembolans, the genus Folsomia is common and abundant in the soils of most biotopes. Folsomia candida Willem is frequently used in ecotoxicological testing as a representative collembolan species because of its cosmopolitan distribution and ease of rearing. A standard laboratory test was developed by the ISO (1999) using F. candida, which has since been suggested as a reference soil animal in ecotoxicology (Fountain and Hopkin, 2005; Ja¨nsch et al., 2005). Because of the accumulation of toxicity data for F. candida, a test using F. candida would allow us to access these data for comparative studies. In radioecology, effects of radiation or radioactive nuclides on collembolans have been studied both in the field (Krivolutzkii and Pokarzhevskii, 1992; Poinsot-Balaguer and Tabone, 1995; Reichele and Crossley, 1965; Styron and Dodson, 1973) and in the laboratory (Auerbach et al., 1957; Cavalloro and Delrio, 1971; Edwards, 1969; Styron, 1973). A standard test protocol has recently been used to evaluate the chemical toxicity of uranium in collembolans (Sheppard et al., 2004, 2005). Although Styron (1971) studied the lethal effects of acute gamma irradiation on the collembolan Sinella curviseta (Brook) using a method similar to the standard protocol, the effects of radiation on F. candida have not yet been studied in a standard laboratory test. To obtain basic data that may be used for the radiological protection of the environment, we examined the dose–effect relationships between external acute gamma irradiation and survival, growth, and reproduction in F. candida based on a standard laboratory test using a gamma cell. A plaster of Paris substrate was used in our exposure tests, although the standard test protocol recommends exposing animals to chemical substances via soil (ISO, 1999). Exposure via soil is important when considering the bioavailability of chemical substances, because soil properties affect this bioavailability, probably by altering chemical forms or absorbing chemical substances (Crommentuijn et al., 1997; Lock and Janssen, 2001; van Gestel and Mol, 2003). However, external exposure using a gamma cell has no connection to bioavailability, and emission doses from radionuclides are not affected by chemical forms. To study the effects of radiation, the doses absorbed by organisms can be estimated from the concentration of radionuclides in the environment and organisms (Go´mez-Ros et al., 2004). Furthermore, the use of a plaster substrate allows for continuous monitoring during the exposure period and is helpful to understand the biological effects of pollutants (Fountain and Hopkin, 2001). A plaster substrate was also used by Styron (1971). 2. Materials and methods 2.1. Animals F. candida is a soil-dwelling, unpigmented, eyeless, parthenogenic species belonging to the family Isotomidae. Adults reach 1.5–3 mm in
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body length. The biology of F. candida was reviewed by Fountain and Hopkin (2005). The test culture of F. candida used in the present study originated from a population in central Japan, and its laboratory life history data are available elsewhere (Itoh et al., 1995). Stock cultures were reared on baker’s yeast in glass vessels (50-mm diameter, 55-mm height) with a layer of moist culturing substrate (5-mm depth) on the bottom and polyethylene lids in a climate-controlled room (20 1C) and a light:dark regime of 12:12 h. The culturing substrate was plaster of Paris mixed with activated charcoal (8:1 by weight) and was consistent throughout the study. Synchronized collembolan specimens used in experiments were prepared according to ISO (1999). At the start of the experiments, the collembolan specimens were 10–12 days old.
2.2. Gamma radiation effect test Two tests were performed, in which the effects on reproduction were assessed separately from the effects on growth and survival. The reproduction test was carried out as follows. One day before the irradiation, ten collembolan specimens from a synchronous culture were transferred into a glass test vessel (50-mm diameter, 55-mm height) with a layer of moist substrate on the bottom. Five and ten replicate vessels were used per dose radiation and non-irradiated control, respectively. The test vessels were sealed with parafilm, and the collembolan specimens were starved for 1 day prior to irradiation at 20 1C. Collembolan specimens were exposed to 137Cs gamma radiation in the test vessels at a constant dose rate of 8.3 Gy/min at doses of 4, 8, 12, 21, 36, 60, and 110 Gy. The gamma source was located at one side of the turntable (2 rpm) at a distance of 45 mm from the center of the test vessels. After irradiation, the test vessels were kept at 20 1C under a light:dark regime of 12:12 h. Immediately following the irradiation, and again 2 weeks later, the insects were supplied with food (2 mg of baker’s yeast) on the substrate in each vessel. Vessels were opened twice per week for aeration. Four weeks after irradiation, the collembolan specimens, excluding eggs, were transferred to other vessels, and the number of surviving adults and neonates were counted in each vessel. To examine radiation effects on egg production and egg hatchability, the remaining eggs were observed for an additional 4 weeks (8 weeks after irradiation). First, the food remaining in the test vessels was removed to avoid mold growth, then the neonates were counted and removed weekly, and finally the remaining unhatched eggs were counted. Dead collembolans were identified by the absence of activity, even after stimulation. The growth and survival test was carried out in a manner similar to the reproduction test, with the only differences described below. Five replicate vessels were used per dose radiation and non-irradiated control. Collembolan specimens were exposed to 60Co gamma radiation at a constant dose rate of 31 Gy/min at doses of 35, 45, 65, 90, 125, 230, 410, 740, 1320, 1460, 1610, and 1770 Gy in smaller glass vessels (20-mm diameter, 50-mm height) with a layer of moist substrate at the bottom. The gamma source was located 16 cm above the surface of the substrate. Specimens were then transferred to the test vessels, which were the same type as those used for the reproduction test. Growth was measured as an increment in body length (mean value for animals in each vessel) from 1 day prior to irradiation and then 4 weeks after. Body length (from the anterior margin of the head to the end of the posterior abdominal segment) was measured at 2 1C under a binocular microscope with a micrometer eyepiece at 40 magnification. All individuals and surviving adults were measured at the beginning and the end of the test, respectively.
2.3. Statistics The data were checked for normality and homogeneity of variance using a Kolmogorov–Smirnov test and Bartlett’s test, respectively. The logistic model of Haanstra et al. (1985), modified according to van Brummelen et al. (1996), was applied to estimate the effect parameters: the 50% lethal dose (LD50) and the 10% and 50% effective doses (ED10 and ED50, respectively). The treatment means were used for the regression analysis. Data were fitted to the model using the non-linear regression
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The tests fulfilled the ISO criteria for a validation test: in the growth and survival test controls, adult mortality was 0%, the average number of juveniles per vessel was 591.2, and the coefficient of variation (CV) of juveniles was 11.5%; in reproduction test controls, adult mortality was 0%, the average number of juveniles per vessel was 629.7, and the CV of juveniles was 6.5%. Among the studied parameters, radiation sensitivity increased in the following order: reproduction, growth, and survival (Fig. 1). Effect parameters are shown in Table 1. No mortality occurred at doses up to 740 Gy (Fig. 1a). Survival rates rapidly decreased in the first 10 days after irradiation and then gradually decreased until the end of the test (Fig. 2). All adults were eliminated at the dose of 1770 Gy after 4 weeks. The growth in body length was affected at non-lethal doses (Fig. 1b). The no observed effect dose and the lowest observed effect dose were 45 and 65 Gy, respectively (ANOVA, Po0.05). In the reproduction test, no mortality occurred up to the highest dose of 110 Gy, whereas reproduction was adversely affected by the gamma irradiation (Fig. 1c). The no observed effect and lowest observed effect doses were 4 and 8 Gy, respectively (ANOVA, Po0.05). The total number of eggs produced by irradiated collembolans and the hatch rates for eggs both decreased in a dose-dependent manner (Fig. 3). At each dose, the hatch rates were calculated as the total number of juveniles from the beginning of the experiment per total number of eggs produced during the experiment. The no observed effect and lowest observed effect doses for egg production (total number of hatched juveniles and unhatched eggs) at 4 weeks after irradiation were 36 and 60 Gy, respectively (ANOVA, Po0.05). Hatch rates of the eggs produced by irradiated animals increased with time and were saturated at 6 weeks after irradiation (Fig. 3b). The data for 6 and 7 weeks after irradiation did not differ more than 1% from those after 8 weeks. Differences in hatch rate between treatment levels and the controls became obscure with time. Significant differences from the control were observed at the lowest studied dose (4 Gy) at 4 weeks after irradiation, whereas the no observed effect dose and the lowest observed effect dose for hatch rate of these eggs at 8 weeks after irradiation were 8 and 12 Gy, respectively (ANOVA, Po0.05). Percentages of eggs hatched from 4 to 8 weeks after irradiation (distance between hatch rates at 8 and 4 weeks after irradiation; Fig. 3b) increased with exposure dose: 49.7%, 55.7%, 56.4%, 57.7%, 62.0%, 71.6%, and 70% for the control, 4, 8, 12, 21, 36, and
Survival rate of adults
3. Results
1 0.8 0.6 0.4 0.2 0
Growth in body length (mm)
module of JMP (Version 5.1, SAS Institute Inc., Chicago, IL). The data at lethal doses were not used for the calculation, because increases in body length at lethal doses are not due to growth, but to elongation of the body before death. One-way analysis of variance (ANOVA) and Dunnett’s comparison with a control at a 5% significance level were performed using JMP to determine the no observed effect and the lowest observed effect doses, respectively.
Number of juveniles per test vessel
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Dose of acute gamma-irradiation (Gy) Fig. 1. Effects of gamma irradiation on the survival (a), growth (b), and reproduction (c) in Folsomia candida 4 weeks after acute irradiation on a plaster of Paris substrate. The data points are the averages of replicates (n ¼ 10, non-irradiated control for reproduction test; n ¼ 5, others). Error bars indicate standard error. The lines are dose–response relationships fitted using a logistic model. The data at lethal doses for growth (b; open circles) were not used for the calculation, because these increases in body length are not a result of growth, but rather the elongation of the body before death.
Table 1 Effects of acute gamma irradiation on Folsomia candida after 4 weeks Endpoint
ED10 in Gy
ED50 in Gy
Survival Growth Reproduction
32 (13–52) 7.1 (4.0–11.3)
1356 (1343–1368) 144 (98–187) 21.9 (17.5–27.1)
Values in parentheses indicate 95% confidence intervals. ED, effective dose.
60 Gy-exposure, respectively, suggesting that the number of late hatchings increased with dose. At 100 Gy, no eggs hatched.
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Survival rate
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Hatch rate of eggs produced by irradiated animals
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Fig. 2. Time course for the survival of Folsomia candida after gamma irradiation at doses of 37–1767 Gy in the survival test. Open circles are the non-irradiated control and doses up to 738 Gy; closed diamonds, 1323 Gy; closed squares, 1456 Gy; closed circles, 1607 Gy; closed triangles, 1767 Gy.
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Fig. 3. Effects of gamma irradiation on Folsomia candida egg production during 4 weeks post irradiation (a), and the hatch rate of eggs produced by irradiated animals (b). Hatch rates (total from the beginning) were observed until 8 weeks after irradiation. The data points are the mean of replicates (n ¼ 5, irradiated treatments; n ¼ 10, non-irradiated control). Error bars indicate standard deviation. (b) Triangles, 4 weeks after irradiation; open circles, 5 weeks after irradiation; closed circles, 8 weeks after irradiation (data for 6 and 7 weeks after irradiation are not shown because the data did not differ more than 1% from the 8-week data).
4. Discussion The present standard test results are important for establishing the basis for radiological protection of the environment. Because of its comparability, a standard test is also useful for characterization and comparison of risk trade-offs for different pollutants. Although the same total dose of radiation exposure can be delivered through both
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acute (high dose rate in a short period of time) and chronic (low dose rate over a long period of time) exposures, we addressed the effects of acute gamma irradiation. Although differences in the method of exposure (acute or chronic) exist between our data and data for chemical exposure, careful comparison allows us to identify the characteristics of radiation effects. On the other hand, our data for acute exposure can be used for ecological risk assessment after an accidental release of radiation, thus for risk trade-off. Among the parameters studied in F. candida, survival was the least sensitive to gamma radiation. Styron (1971) studied lethal effects of radiation on the collembolan S. curviseta using methods similar to the standard protocol. The value of LD50 for F. candida (1356 Gy) was higher than those for S. curviseta: the values of LD50 after 30 days of acute gamma irradiation for juveniles and adults were 128 and 149 Gy, respectively (Styron, 1971). These findings agree with those of Edwards (1969) that Entomobryidae, to which S. curviseta belongs, are more sensitive to gamma irradiation than Isotomidae, to which F. candida belongs. The family Isotomidae may have a higher radiation tolerance than other collembolan families, given that members of Isotomidae, Cryptopygus thermophilus (Axelson) and Desoria trispinata (MacGillivray), have been found in abundance at highly irradiated sites in field experiments (Poinsot-Balaguer, 1976; Loring, 1985). Radiation tolerance in invertebrates may be related to very efficient mechanisms of DNA repair, anhydrobiosis, or other unknown properties (Jo¨nsson et al., 2005; Watanabe et al., 2006). The ED10 value for F. candida reproduction (7.1 Gy) was within the range of effective doses for reproductive or related endpoints in other invertebrates, although there were differences in the test and evaluation methods. The ED10 ranges from 0.5 Gy in polychaetes (Harrison and Anderson, 1994) to 50 Gy in chironomids (Jo¨nsson et al., 2005) under acute gamma irradiation. This wide range of reproductive sensitivity to acute gamma irradiation may be explained by phylogenetic differences as well as the life stage during irradiation (Harrison and Anderson, 1994). Understanding why species and subgroups differ in their sensitivity to ionizing radiation allows cross-taxa extrapolation of radiation effects and creates a stronger scientific foundation for impact assessments for ecosystems where diverse taxa exist. Extrapolation across species is an important task for the radiological protection of the environment (Garnier-Laplace et al., 2004). Both a reduction in egg production and hatch rate appear to result in reduced reproductive success in F. candida. Reduced hatch rates have also been observed in another irradiated collembolan species (Styron, 1971). The outcome of reproductive success (the number of juveniles) involves both egg production and hatch rate. The lowest observed effect dose for reproductive success 4 weeks after irradiation was 8 Gy, lower than the 60 Gy for total egg production and higher than the 4 Gy for hatch rate 4 weeks after irradiation, suggesting that reduced
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reproductive success was the result of reduced hatch rate 4 weeks after irradiation. The additional time required for hatching increased with dose. Therefore, the reduced hatch rate after 4 weeks was not mainly attributable to egg mortality, but rather to delayed egg development before or after oviposition. This delayed egg development was thought to be the primary cause of reduced reproductive success in the 4-week-reproduction test. In contrast, egg production was reduced and hatching ceased at 110 Gy, a high dose. These results suggest that reduced egg production and increased egg mortality lead to reduced reproductive success at high doses. Among the parameters studied in F. candida, reproduction was the most sensitive to gamma radiation. The higher sensitivity of reproduction is consistent with findings in other invertebrates, including collembolans (Styron, 1971), earthworms (Suzuki and Egami, 1983), tardigrades (Jo¨nsson et al., 2005), chironomids (Watanabe et al., 2006), and polychaetes (Harrison and Anderson, 1994). In the polychaete Neanthes arenaceodentata Moore, reproduction is three orders of magnitude more sensitive than survival (Harrison and Anderson, 1994). Even in a radiation-tolerant tardigrade such as Richtersius coronifer (Richters), in which the LD50 is 30 days at 2.5 kGy, the hatch rate in acutely gammairradiated animals was dramatically reduced by the lowest studied dose (0.5 kGy). Developing tissues in reproductive organs and eggs require mitotic activity, and thus may be sensitive to radiation (Jo¨nsson et al., 2005). The sublethal sensitivity index (SSI), the ratio between the lethal effect concentration and the sublethal effect concentration, was proposed as a parameter expressing maintenance of sublethal functions under toxicant stress (Crommentuijn et al., 1995a). The SSI seems to be valuable for evaluating characteristics of pollutants, because a different pollutant may affect life-history functions in a pollutant-specific manner (Crommentuijn et al., 1995b). The SSI for gamma radiation, which was calculated at the ratio of LD50 to ED50, was 61.9 for reproduction. Based on the F. candida standard tests (4-week period at 20 1C), this value is higher than those for a number of chemicals (LC50/EC50 for reproduction), ranging from 1.2 for dimethoate (Krogh, 1995) to 6.6 for cadmium (Greenslade and Vaughan, 2003). Similarly, the SSI for growth was higher for gamma radiation (LD50/ED50 for growth in body length: 9.4) than for chemicals (zinc LC50/EC50 for growth in fresh or dry weight: 1.2–1.3; Smit and van Gestel, 1998; Smit et al., 1998). An increased SSI for reproduction and growth may be one of the characteristic effects of gamma irradiation, or at least for acute doses in a 4-week test. These findings suggest that energy allocation among reproduction, survival, and detoxification differs between acutely irradiated and chemically exposed animals. This increased sensitivity to acute gamma radiation in reproduction relative to survival may be explained by the uniform exposure of the whole body and by the higher sensitivity of reproductive tissues. Because acute gamma
radiation can reach all cells unselectively, the most sensitive or non-replaceable cells or organs, such as reproductive tissues, may be affected more severely. In the tested animals, energy may have been diverted from reproduction to detoxification or survival, resulting in an increased radiation tolerance in terms of survival. In contrast, in chemically exposed animals, the most damaged organs are likely to depend on uptake routes and chemical substance behavior (Vijver et al., 2004) and may not always be restricted to the most sensitive tissues. In such animals, energy may be used for detoxification, survival, and reproduction. The SSIs for chemical exposures seem to depend on the organism’s strategy for energy allocation among reproduction, survival, and detoxification under stress conditions (Crommentuijn et al., 1995a; Eijsackers, 1994; van Straalen, 2004). Understanding the effects of chronic low-level exposure to ionizing radiation is required to safeguard ecosystems (Garnier-Laplace et al., 2004). However, such data are limited for most biota, including collembolans (Styron, 1973). Therefore, extrapolation from acute to chronic exposure is necessary for the radiological protection of the environment (Garnier-Laplace et al., 2004). Data on acute exposure may illustrate differences between acute and chronic irradiation effects, which are useful to develop methods of extrapolation. Animals are exposed to the same total dose of radiation at a lower dose rate in chronic versus acute exposures. Our results for acute exposure suggest that F. candida sustains irreversible damage to reproduction at high doses (4110 Gy), whereas reproductive damage at lower doses (o60 Gy) appears to be reversible. Therefore, at the same total dose, chronically irradiated animals may experience reversible reproductive effects, whereas acutely irradiated animals may become sterile. Because sterilized animals cannot use energy for reproduction, differences in the method of exposure (acute or chronic) may lead to changes in energy allocation among detoxification, survival, and reproduction. Additional data on chronic and acute exposures in standard tests are required to clarify this issue. 5. Conclusion Using a standard test, we obtained basic data that can be used for ecological impact assessments for radiation. Delayed egg development appears to be the primary cause of reduced reproductive success in response to acute gamma irradiation. Additional reproductive data for chronic low-level exposures, which is a realistic parameter from an ecological viewpoint, are needed to better protect the environment from radiological exposure. Funding sources This work was supported by management expenses grants from the National Institute of Radiological Sciences.
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Acknowledgments We thank Dr. Takafumi Kamitani at the National Institute of Advanced Industrial Science and Technology and Mr. Yoshikazu Fujii at Yokohama National University for helpful suggestions and technical support, and Dr. Shoichi Fuma and Dr. Yoshito Watanabe at the National Institute of Radiological Sciences for helpful suggestions. This work was supported by management expenses grants from the National Institute of Radiological Sciences. References Auerbach, S.I., Crossley Jr., D.A., Engelmann, M.D., 1957. Effects of gamma radiation on Collembola population growth. Science 126, 614. Cavalloro, R., Delrio, G., 1971. Population longevity in gamma-irradiated Collembola. Int. J. Appl. Radiat. Isot. 22, 216–218. Crommentuijn, T., Doodeman, C.J.A.M., van der Pol, J.J.C., Doornekamp, A., Rademaker, M.C.J., van Gestel, C.A.M., 1995a. Sublethal sensitivity index as an ecotoxicity parameter measuring energy allocation under toxicant stress: application to cadmium in soil arthropods. Ecotoxicol. Environ. Saf. 31, 192–200. Crommentuijn, T., Sta¨b, J.A., Doornekamp, A., Estoppey, O., van Gestel, C.A.M., 1995b. Comparative ecotoxicity of cadmium, chlorpyrifos and triphenyltin hydroxide for four clones of the parthenogenetic collembolan Folsomia candida in an artificial soil. Funct. Ecol. 9, 734–742. Crommentuijn, T., Doornekamp, A., van Gestel, C.A.M., 1997. Bioavailability and ecological effects of cadmium on Folsomia candida (Willem) in an artificial soil substrate as influenced by pH and organic matter. Appl. Soil Ecol. 5, 261–271. Edwards, C.A., 1969. Effects of gamma irradiation on populations of soil invertebrates. In: Nelson, D.J., Evans, F.C. (Eds.), Symposium on Radioecology. US Atomic Energy Commission Report CONF670503, pp. 68–77. Eijsackers, H., 1994. Ecotoxicology of soil organisms: seeking the way in a pitch-dark labyrinth. In: Donker, M.H., Eijsakers, H., Heimbach, F. (Eds.), Ecotoxicology of Soil Organisms. Lewis Publishers, Boca Raton, pp. 3–32. Fountain, M.T., Hopkin, S.P., 2001. Continuous monitoring of Folsomia candida (Insecta: Collembola) in a metal exposure test. Ecotoxicol. Environ. Saf. 48, 275–286. Fountain, M.T., Hopkin, S.P., 2004. A comparative study of the effects of metal contamination on Collembola in the field and in the laboratory. Ecotoxicology 13, 573–587. Fountain, M.T., Hopkin, S.P., 2005. Folsomia candida (Collembola): a ‘‘standard’’ soil arthropod. Annu. Rev. Entomol. 50, 201–222. Garnier-Laplace, J., Gilek, M., Sundell-Bergman, S., Larsson, C.-M., 2004. Assessing ecological effects of radionuclides: data gaps and extrapolation issues. J. Radiol. Prot. 24, A139–A155. Go´mez-Ros, J.M., Pro¨hl, G., Taranenko, V., 2004. Estimation of internal and external exposures of terrestrial reference organisms to natural radionuclides in the environment. J. Radiol. Prot. 24, A79–A88. Greenslade, P., Vaughan, G.T., 2003. A comparison of Collembola species for toxicity testing of Australian soils. Pedobiologia 47, 171–179. Haanstra, L., Doelman, P., Voshaar, J.H.O., 1985. The use of sigmoidal dose response curves in soil ecotoxicological research. Plant Soil 84, 293–297. Harrison, F.L., Anderson, S.L., 1994. Effects of acute irradiation on reproductive success of the polychaete worm, Neanthes arenaceodentata. Radiat. Res. 137, 59–66. Hopkin, S.P., 1997. Biology of the Springtails (Insecta: Collembola). Oxford University Press, Oxford. ICRP, 2003. A framework for assessing the impact of ionizing radiation on non-human species. Publication 91, Annals of the ICRP 33, Pergamon Press, Oxford.
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ISO, 1999. Soil quality—inhibition of reproduction of Collembola (Folsomia candida) by soil pollutants, no. 11267, ISO, Geneva. Itoh, R., Hisamatsu, M., Matsunaga, M., Hishida, F., 1995. Growth and reproduction of a collembolan species, Folsomia candida (Willem), under experimental conditions. J. Coll. Arts. Sci. Showa Univ. 26, 23–30. Ja¨nsch, S., Amorim, M.J., Ro¨mbke, J., 2005. Identification of the ecological requirements of important terrestrial ecotoxicological test species. Environ. Rev. 13, 51–83. Jo¨nsson, K.I., Harma-Ringdahl, M., Torudd, J., 2005. Radiation tolerance in the eutardigrade Richtersius coronifer. Int. J. Radiat. Biol. 81, 649–656. Krivolutzkii, D.A., Pokarzhevskii, A.D., 1992. Effects of radioactive fallout on soil animal populations in the 30 km zone of the Chernobyl atomic power station. Sci. Tot. Environ. 112, 69–77. Krogh, P.H., 1995. Does a heterogeneous distribution of food or pesticide affect the outcome of toxicity tests with Collembola? Ecotoxicol. Environ. Saf. 30, 158–163. Larsson, C.-M., 2004. The FASSET framework for assessment of environmental impact of ionising radiation in European ecosystems—an overview. J. Radiol. Prot. 24, A1–A12. Lock, K., Janssen, C., 2001. Cadmium toxicity for terrestrial invertebrates: taking soil parameters affecting bioavailability into account. Ecotoxicology 10, 315–322. Loring, S.J., 1985. g Radiation effects on Collembola. Environ. Entomol. 14, 805–809. Poinsot-Balaguer, N., 1976. Re´ponse des communaute´s de Collemboles a` l’irradiation gamma chronique d’une foreˆt me´diterrane´enne. Rev. Ecol. Biol. Sol 13, 365–379. Poinsot-Balaguer, N., Tabone, E., 1995. Impact of chronic gamma irradiation on the litter decay of a mixed mediterranean forest in Cadarache, France: microarthoropod’s response. Pedobiologia 39, 344–350. Reichele, D.E., Crossley Jr., D.A., 1965. Radiocesium dispersion in a cryptozoan food web. Health Phys. 11, 1375–1384. Sheppard, S.C., Sheppard, M.I., Sanipelli, B., Dowsley, B., Stephenson, G., Feisthauer, N., Rowland, R., Gilbertson, M.-K., 2004. Uranium concentrations in Port Hope soils and vegetation and toxicological effect on soil organisms. Canadian Nuclear Safety Commission Contract Report 87055-01-0266-R161.1, 2004. Sheppard, S.C., Sheppard, M.I., Gallerand, M.-O., Sanipelli, B., 2005. Derivation of ecotoxicity thresholds for uranium. J. Environ. Radioact. 79, 55–83. Smit, C.E., van Gestel, C.A.M., 1998. Effects of soil type, prepercolation, and aging on bioaccumulation and toxicity of zinc for Folsomia candida. Environ. Toxicol. Chem. 17, 1132–1141. Smit, C.E., van Overbeek, I., van Gestel, C.A.M., 1998. The influence of food supply on the toxicity of zinc for Folsomia candida (Collembola). Pedobiologia 42, 154–164. Styron, C.E., 1971. Effects of beta and gamma radiation on a population of springtails, Sinella curviseta (Collembola). Radiat. Res. 48, 53–62. Styron, C.E., 1973. Effects of chronic beta radiation from simulated fallout on springtails, Folsomia sp. (Collembola). Health Phys. 25, 281–284. Styron, C.E., Dodson, G.J., 1973. Response of grassland community to simulated radioactive fallout. Radiat. Res. 55, 487–500. Suzuki, J., Egami, N., 1983. Mortality of the earthworm, Eisenia foetida, after g-irradiation at different stages of their life history. J. Radiat. Res. 24, 209–220. Timmermans, M.J.T.N., Ellers, J., Roelofs, D., van Straalen, N.M., 2005. Metallothionein mRNA expression and cadmium tolerance in metalstressed and reference populations of the springtail Orchesella cincta. Ecotoxicology 14, 727–739. van Brummelen, T.C., van Gestel, C.A.M., Verweij, R.A., 1996. Longterm toxicity of five polycyclic aromatic hydrocarbons for the terrestrial isopods Oniscus asellus and Porcellio scaber. Environ. Toxicol. Chem. 15, 1199–1210. van Gestel, C.A.M., Mol, S., 2003. The influence of soil characteristics on cadmium toxicity for Folsomia candida (Collembola: Isotomidae). Pedobiologia 47, 387–395.
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Ecotoxicology and Environmental Safety 71 (2008) 590–596 www.elsevier.com/locate/ecoenv
Effects of acute gamma irradiation on Folsomia candida (Collembola) in a standard test Taizo Nakamoria,, Satoshi Yoshidaa, Yoshihisa Kubotaa, Tadaaki Ban-naia, Nobuhiro Kanekob, Makiko Hasegawac, Ryosaku Itohc b
a Environmental Radiation Effects Research Group, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage, Chiba 263-8555, Japan Soil Ecology, Graduate School of Environment and Information Sciences, Yokohama National University, 79-7 Tokiwadai, Yokohama 240-8501, Japan c Biological Laboratory, Faculty of Arts and Sciences at Fujiyoshida, Showa University, 4562 Kamiyoshida, Fujiyoshida, Yamanashi 403-0005, Japan
Received 22 April 2007; received in revised form 19 September 2007; accepted 13 October 2007 Available online 21 December 2007
Abstract An understanding of the effects of ionizing radiation on non-human biota is required by the International Commission on Radiological Protection for the radiological protection of the environment. We examined dose–effect relationships for gamma radiation on survival, growth, and reproduction in the soil invertebrate Folsomia candida (Collembola) in a standard laboratory test. F. candida were acutely irradiated at increasing doses of gamma radiation, and subsequent survival, growth in body length, and number of neonates produced by irradiated specimens were examined. The 50% lethal dose was at 1356 Gy, and the 10% and 50% effective doses (ED10 and ED50) for growth were at 32 and 144 Gy, respectively. The ED10 and ED50 values for reproduction were at 7.1 and 21.9 Gy, respectively. These data establish important baselines for the radiological protection of terrestrial ecosystems based on scientific principles. r 2007 Elsevier Inc. All rights reserved. Keywords: Dose–effect relationship; Growth; Non-human biota; Radiological protection; Reproduction; Soil invertebrate; Springtails; Survival
1. Introduction The importance of radiological protection of the environment based on scientific principles is gaining international recognition as environmental issues garner more attention (International Commission on Radiological Protection (ICRP), 2003). The ICRP (2003) has suggested reference organisms for studying the impact of radiation on ecosystems. The Framework for Assessment of Environmental Impact (FASSET) project was launched under the European Commission Fifth Framework Programme to provide a framework for assessing the impact of ionizing radiation on non-human biota (Larsson, 2004). The FASSET project provided a set of reference organisms relevant to different exposure situations taking into account the environmental fate of radionuclide release and exposure pathways, and built an effects database for Corresponding author. Fax: +81 43 206 3267.
E-mail address: [email protected] (T. Nakamori). 0147-6513/$ - see front matter r 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2007.10.029
the reference organisms, including soil invertebrates, for terrestrial ecosystems (Larsson, 2004). However, available data for soil invertebrates were still limited. Single-species laboratory tests are used for ecological impact assessments for chemical substances. Because of their ecological importance, soil invertebrates are used for ecological impact assessments of terrestrial ecosystem pollutants (van Gestel et al., 1997; van Straalen, 2004). Standard laboratory tests using certain soil invertebrate species have been developed by international organizations such as the International Organization for Standardization (ISO) and the Organization for Economic Cooperation and Development (OECD; Ja¨nsch et al., 2005). Population and community level effects are extrapolated based on these tests. A similar methodology can be applied for assessing the environmental impact of ionizing radiation (Garnier-Laplace et al., 2004). Collembolans are part of the soil mesofauna and are the most numerous and widely distributed insects in soil ecosystems. They are thought to contribute to decomposition processes in the soil, and they serve as prey for several
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invertebrates and small vertebrates (Hopkin, 1997). Collembolans have been widely used for ecological impact assessment (Fountain and Hopkin, 2004; Hopkin, 1997; Timmermans et al., 2005). Among collembolans, the genus Folsomia is common and abundant in the soils of most biotopes. Folsomia candida Willem is frequently used in ecotoxicological testing as a representative collembolan species because of its cosmopolitan distribution and ease of rearing. A standard laboratory test was developed by the ISO (1999) using F. candida, which has since been suggested as a reference soil animal in ecotoxicology (Fountain and Hopkin, 2005; Ja¨nsch et al., 2005). Because of the accumulation of toxicity data for F. candida, a test using F. candida would allow us to access these data for comparative studies. In radioecology, effects of radiation or radioactive nuclides on collembolans have been studied both in the field (Krivolutzkii and Pokarzhevskii, 1992; Poinsot-Balaguer and Tabone, 1995; Reichele and Crossley, 1965; Styron and Dodson, 1973) and in the laboratory (Auerbach et al., 1957; Cavalloro and Delrio, 1971; Edwards, 1969; Styron, 1973). A standard test protocol has recently been used to evaluate the chemical toxicity of uranium in collembolans (Sheppard et al., 2004, 2005). Although Styron (1971) studied the lethal effects of acute gamma irradiation on the collembolan Sinella curviseta (Brook) using a method similar to the standard protocol, the effects of radiation on F. candida have not yet been studied in a standard laboratory test. To obtain basic data that may be used for the radiological protection of the environment, we examined the dose–effect relationships between external acute gamma irradiation and survival, growth, and reproduction in F. candida based on a standard laboratory test using a gamma cell. A plaster of Paris substrate was used in our exposure tests, although the standard test protocol recommends exposing animals to chemical substances via soil (ISO, 1999). Exposure via soil is important when considering the bioavailability of chemical substances, because soil properties affect this bioavailability, probably by altering chemical forms or absorbing chemical substances (Crommentuijn et al., 1997; Lock and Janssen, 2001; van Gestel and Mol, 2003). However, external exposure using a gamma cell has no connection to bioavailability, and emission doses from radionuclides are not affected by chemical forms. To study the effects of radiation, the doses absorbed by organisms can be estimated from the concentration of radionuclides in the environment and organisms (Go´mez-Ros et al., 2004). Furthermore, the use of a plaster substrate allows for continuous monitoring during the exposure period and is helpful to understand the biological effects of pollutants (Fountain and Hopkin, 2001). A plaster substrate was also used by Styron (1971). 2. Materials and methods 2.1. Animals F. candida is a soil-dwelling, unpigmented, eyeless, parthenogenic species belonging to the family Isotomidae. Adults reach 1.5–3 mm in
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body length. The biology of F. candida was reviewed by Fountain and Hopkin (2005). The test culture of F. candida used in the present study originated from a population in central Japan, and its laboratory life history data are available elsewhere (Itoh et al., 1995). Stock cultures were reared on baker’s yeast in glass vessels (50-mm diameter, 55-mm height) with a layer of moist culturing substrate (5-mm depth) on the bottom and polyethylene lids in a climate-controlled room (20 1C) and a light:dark regime of 12:12 h. The culturing substrate was plaster of Paris mixed with activated charcoal (8:1 by weight) and was consistent throughout the study. Synchronized collembolan specimens used in experiments were prepared according to ISO (1999). At the start of the experiments, the collembolan specimens were 10–12 days old.
2.2. Gamma radiation effect test Two tests were performed, in which the effects on reproduction were assessed separately from the effects on growth and survival. The reproduction test was carried out as follows. One day before the irradiation, ten collembolan specimens from a synchronous culture were transferred into a glass test vessel (50-mm diameter, 55-mm height) with a layer of moist substrate on the bottom. Five and ten replicate vessels were used per dose radiation and non-irradiated control, respectively. The test vessels were sealed with parafilm, and the collembolan specimens were starved for 1 day prior to irradiation at 20 1C. Collembolan specimens were exposed to 137Cs gamma radiation in the test vessels at a constant dose rate of 8.3 Gy/min at doses of 4, 8, 12, 21, 36, 60, and 110 Gy. The gamma source was located at one side of the turntable (2 rpm) at a distance of 45 mm from the center of the test vessels. After irradiation, the test vessels were kept at 20 1C under a light:dark regime of 12:12 h. Immediately following the irradiation, and again 2 weeks later, the insects were supplied with food (2 mg of baker’s yeast) on the substrate in each vessel. Vessels were opened twice per week for aeration. Four weeks after irradiation, the collembolan specimens, excluding eggs, were transferred to other vessels, and the number of surviving adults and neonates were counted in each vessel. To examine radiation effects on egg production and egg hatchability, the remaining eggs were observed for an additional 4 weeks (8 weeks after irradiation). First, the food remaining in the test vessels was removed to avoid mold growth, then the neonates were counted and removed weekly, and finally the remaining unhatched eggs were counted. Dead collembolans were identified by the absence of activity, even after stimulation. The growth and survival test was carried out in a manner similar to the reproduction test, with the only differences described below. Five replicate vessels were used per dose radiation and non-irradiated control. Collembolan specimens were exposed to 60Co gamma radiation at a constant dose rate of 31 Gy/min at doses of 35, 45, 65, 90, 125, 230, 410, 740, 1320, 1460, 1610, and 1770 Gy in smaller glass vessels (20-mm diameter, 50-mm height) with a layer of moist substrate at the bottom. The gamma source was located 16 cm above the surface of the substrate. Specimens were then transferred to the test vessels, which were the same type as those used for the reproduction test. Growth was measured as an increment in body length (mean value for animals in each vessel) from 1 day prior to irradiation and then 4 weeks after. Body length (from the anterior margin of the head to the end of the posterior abdominal segment) was measured at 2 1C under a binocular microscope with a micrometer eyepiece at 40 magnification. All individuals and surviving adults were measured at the beginning and the end of the test, respectively.
2.3. Statistics The data were checked for normality and homogeneity of variance using a Kolmogorov–Smirnov test and Bartlett’s test, respectively. The logistic model of Haanstra et al. (1985), modified according to van Brummelen et al. (1996), was applied to estimate the effect parameters: the 50% lethal dose (LD50) and the 10% and 50% effective doses (ED10 and ED50, respectively). The treatment means were used for the regression analysis. Data were fitted to the model using the non-linear regression
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The tests fulfilled the ISO criteria for a validation test: in the growth and survival test controls, adult mortality was 0%, the average number of juveniles per vessel was 591.2, and the coefficient of variation (CV) of juveniles was 11.5%; in reproduction test controls, adult mortality was 0%, the average number of juveniles per vessel was 629.7, and the CV of juveniles was 6.5%. Among the studied parameters, radiation sensitivity increased in the following order: reproduction, growth, and survival (Fig. 1). Effect parameters are shown in Table 1. No mortality occurred at doses up to 740 Gy (Fig. 1a). Survival rates rapidly decreased in the first 10 days after irradiation and then gradually decreased until the end of the test (Fig. 2). All adults were eliminated at the dose of 1770 Gy after 4 weeks. The growth in body length was affected at non-lethal doses (Fig. 1b). The no observed effect dose and the lowest observed effect dose were 45 and 65 Gy, respectively (ANOVA, Po0.05). In the reproduction test, no mortality occurred up to the highest dose of 110 Gy, whereas reproduction was adversely affected by the gamma irradiation (Fig. 1c). The no observed effect and lowest observed effect doses were 4 and 8 Gy, respectively (ANOVA, Po0.05). The total number of eggs produced by irradiated collembolans and the hatch rates for eggs both decreased in a dose-dependent manner (Fig. 3). At each dose, the hatch rates were calculated as the total number of juveniles from the beginning of the experiment per total number of eggs produced during the experiment. The no observed effect and lowest observed effect doses for egg production (total number of hatched juveniles and unhatched eggs) at 4 weeks after irradiation were 36 and 60 Gy, respectively (ANOVA, Po0.05). Hatch rates of the eggs produced by irradiated animals increased with time and were saturated at 6 weeks after irradiation (Fig. 3b). The data for 6 and 7 weeks after irradiation did not differ more than 1% from those after 8 weeks. Differences in hatch rate between treatment levels and the controls became obscure with time. Significant differences from the control were observed at the lowest studied dose (4 Gy) at 4 weeks after irradiation, whereas the no observed effect dose and the lowest observed effect dose for hatch rate of these eggs at 8 weeks after irradiation were 8 and 12 Gy, respectively (ANOVA, Po0.05). Percentages of eggs hatched from 4 to 8 weeks after irradiation (distance between hatch rates at 8 and 4 weeks after irradiation; Fig. 3b) increased with exposure dose: 49.7%, 55.7%, 56.4%, 57.7%, 62.0%, 71.6%, and 70% for the control, 4, 8, 12, 21, 36, and
Survival rate of adults
3. Results
1 0.8 0.6 0.4 0.2 0
Growth in body length (mm)
module of JMP (Version 5.1, SAS Institute Inc., Chicago, IL). The data at lethal doses were not used for the calculation, because increases in body length at lethal doses are not due to growth, but to elongation of the body before death. One-way analysis of variance (ANOVA) and Dunnett’s comparison with a control at a 5% significance level were performed using JMP to determine the no observed effect and the lowest observed effect doses, respectively.
Number of juveniles per test vessel
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1.2 1 0.8 0.6 0.4 0.2 0
700 600 500 400 300 200 100 0 0
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Dose of acute gamma-irradiation (Gy) Fig. 1. Effects of gamma irradiation on the survival (a), growth (b), and reproduction (c) in Folsomia candida 4 weeks after acute irradiation on a plaster of Paris substrate. The data points are the averages of replicates (n ¼ 10, non-irradiated control for reproduction test; n ¼ 5, others). Error bars indicate standard error. The lines are dose–response relationships fitted using a logistic model. The data at lethal doses for growth (b; open circles) were not used for the calculation, because these increases in body length are not a result of growth, but rather the elongation of the body before death.
Table 1 Effects of acute gamma irradiation on Folsomia candida after 4 weeks Endpoint
ED10 in Gy
ED50 in Gy
Survival Growth Reproduction
32 (13–52) 7.1 (4.0–11.3)
1356 (1343–1368) 144 (98–187) 21.9 (17.5–27.1)
Values in parentheses indicate 95% confidence intervals. ED, effective dose.
60 Gy-exposure, respectively, suggesting that the number of late hatchings increased with dose. At 100 Gy, no eggs hatched.
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Survival rate
1 0.8 0.6 0.4 0.2 0 0
10
20
30
Time after irradiation (days)
Hatch rate of eggs produced by irradiated animals
Number of eggs produced
Fig. 2. Time course for the survival of Folsomia candida after gamma irradiation at doses of 37–1767 Gy in the survival test. Open circles are the non-irradiated control and doses up to 738 Gy; closed diamonds, 1323 Gy; closed squares, 1456 Gy; closed circles, 1607 Gy; closed triangles, 1767 Gy.
1600 1400 1200 1000 800 600 400 200 0 0
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0
20 40 60 80 100 Dose of acute gamma irradiation (Gy)
120
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1 0.8 0.6 0.4 0.2 0
Fig. 3. Effects of gamma irradiation on Folsomia candida egg production during 4 weeks post irradiation (a), and the hatch rate of eggs produced by irradiated animals (b). Hatch rates (total from the beginning) were observed until 8 weeks after irradiation. The data points are the mean of replicates (n ¼ 5, irradiated treatments; n ¼ 10, non-irradiated control). Error bars indicate standard deviation. (b) Triangles, 4 weeks after irradiation; open circles, 5 weeks after irradiation; closed circles, 8 weeks after irradiation (data for 6 and 7 weeks after irradiation are not shown because the data did not differ more than 1% from the 8-week data).
4. Discussion The present standard test results are important for establishing the basis for radiological protection of the environment. Because of its comparability, a standard test is also useful for characterization and comparison of risk trade-offs for different pollutants. Although the same total dose of radiation exposure can be delivered through both
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acute (high dose rate in a short period of time) and chronic (low dose rate over a long period of time) exposures, we addressed the effects of acute gamma irradiation. Although differences in the method of exposure (acute or chronic) exist between our data and data for chemical exposure, careful comparison allows us to identify the characteristics of radiation effects. On the other hand, our data for acute exposure can be used for ecological risk assessment after an accidental release of radiation, thus for risk trade-off. Among the parameters studied in F. candida, survival was the least sensitive to gamma radiation. Styron (1971) studied lethal effects of radiation on the collembolan S. curviseta using methods similar to the standard protocol. The value of LD50 for F. candida (1356 Gy) was higher than those for S. curviseta: the values of LD50 after 30 days of acute gamma irradiation for juveniles and adults were 128 and 149 Gy, respectively (Styron, 1971). These findings agree with those of Edwards (1969) that Entomobryidae, to which S. curviseta belongs, are more sensitive to gamma irradiation than Isotomidae, to which F. candida belongs. The family Isotomidae may have a higher radiation tolerance than other collembolan families, given that members of Isotomidae, Cryptopygus thermophilus (Axelson) and Desoria trispinata (MacGillivray), have been found in abundance at highly irradiated sites in field experiments (Poinsot-Balaguer, 1976; Loring, 1985). Radiation tolerance in invertebrates may be related to very efficient mechanisms of DNA repair, anhydrobiosis, or other unknown properties (Jo¨nsson et al., 2005; Watanabe et al., 2006). The ED10 value for F. candida reproduction (7.1 Gy) was within the range of effective doses for reproductive or related endpoints in other invertebrates, although there were differences in the test and evaluation methods. The ED10 ranges from 0.5 Gy in polychaetes (Harrison and Anderson, 1994) to 50 Gy in chironomids (Jo¨nsson et al., 2005) under acute gamma irradiation. This wide range of reproductive sensitivity to acute gamma irradiation may be explained by phylogenetic differences as well as the life stage during irradiation (Harrison and Anderson, 1994). Understanding why species and subgroups differ in their sensitivity to ionizing radiation allows cross-taxa extrapolation of radiation effects and creates a stronger scientific foundation for impact assessments for ecosystems where diverse taxa exist. Extrapolation across species is an important task for the radiological protection of the environment (Garnier-Laplace et al., 2004). Both a reduction in egg production and hatch rate appear to result in reduced reproductive success in F. candida. Reduced hatch rates have also been observed in another irradiated collembolan species (Styron, 1971). The outcome of reproductive success (the number of juveniles) involves both egg production and hatch rate. The lowest observed effect dose for reproductive success 4 weeks after irradiation was 8 Gy, lower than the 60 Gy for total egg production and higher than the 4 Gy for hatch rate 4 weeks after irradiation, suggesting that reduced
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reproductive success was the result of reduced hatch rate 4 weeks after irradiation. The additional time required for hatching increased with dose. Therefore, the reduced hatch rate after 4 weeks was not mainly attributable to egg mortality, but rather to delayed egg development before or after oviposition. This delayed egg development was thought to be the primary cause of reduced reproductive success in the 4-week-reproduction test. In contrast, egg production was reduced and hatching ceased at 110 Gy, a high dose. These results suggest that reduced egg production and increased egg mortality lead to reduced reproductive success at high doses. Among the parameters studied in F. candida, reproduction was the most sensitive to gamma radiation. The higher sensitivity of reproduction is consistent with findings in other invertebrates, including collembolans (Styron, 1971), earthworms (Suzuki and Egami, 1983), tardigrades (Jo¨nsson et al., 2005), chironomids (Watanabe et al., 2006), and polychaetes (Harrison and Anderson, 1994). In the polychaete Neanthes arenaceodentata Moore, reproduction is three orders of magnitude more sensitive than survival (Harrison and Anderson, 1994). Even in a radiation-tolerant tardigrade such as Richtersius coronifer (Richters), in which the LD50 is 30 days at 2.5 kGy, the hatch rate in acutely gammairradiated animals was dramatically reduced by the lowest studied dose (0.5 kGy). Developing tissues in reproductive organs and eggs require mitotic activity, and thus may be sensitive to radiation (Jo¨nsson et al., 2005). The sublethal sensitivity index (SSI), the ratio between the lethal effect concentration and the sublethal effect concentration, was proposed as a parameter expressing maintenance of sublethal functions under toxicant stress (Crommentuijn et al., 1995a). The SSI seems to be valuable for evaluating characteristics of pollutants, because a different pollutant may affect life-history functions in a pollutant-specific manner (Crommentuijn et al., 1995b). The SSI for gamma radiation, which was calculated at the ratio of LD50 to ED50, was 61.9 for reproduction. Based on the F. candida standard tests (4-week period at 20 1C), this value is higher than those for a number of chemicals (LC50/EC50 for reproduction), ranging from 1.2 for dimethoate (Krogh, 1995) to 6.6 for cadmium (Greenslade and Vaughan, 2003). Similarly, the SSI for growth was higher for gamma radiation (LD50/ED50 for growth in body length: 9.4) than for chemicals (zinc LC50/EC50 for growth in fresh or dry weight: 1.2–1.3; Smit and van Gestel, 1998; Smit et al., 1998). An increased SSI for reproduction and growth may be one of the characteristic effects of gamma irradiation, or at least for acute doses in a 4-week test. These findings suggest that energy allocation among reproduction, survival, and detoxification differs between acutely irradiated and chemically exposed animals. This increased sensitivity to acute gamma radiation in reproduction relative to survival may be explained by the uniform exposure of the whole body and by the higher sensitivity of reproductive tissues. Because acute gamma
radiation can reach all cells unselectively, the most sensitive or non-replaceable cells or organs, such as reproductive tissues, may be affected more severely. In the tested animals, energy may have been diverted from reproduction to detoxification or survival, resulting in an increased radiation tolerance in terms of survival. In contrast, in chemically exposed animals, the most damaged organs are likely to depend on uptake routes and chemical substance behavior (Vijver et al., 2004) and may not always be restricted to the most sensitive tissues. In such animals, energy may be used for detoxification, survival, and reproduction. The SSIs for chemical exposures seem to depend on the organism’s strategy for energy allocation among reproduction, survival, and detoxification under stress conditions (Crommentuijn et al., 1995a; Eijsackers, 1994; van Straalen, 2004). Understanding the effects of chronic low-level exposure to ionizing radiation is required to safeguard ecosystems (Garnier-Laplace et al., 2004). However, such data are limited for most biota, including collembolans (Styron, 1973). Therefore, extrapolation from acute to chronic exposure is necessary for the radiological protection of the environment (Garnier-Laplace et al., 2004). Data on acute exposure may illustrate differences between acute and chronic irradiation effects, which are useful to develop methods of extrapolation. Animals are exposed to the same total dose of radiation at a lower dose rate in chronic versus acute exposures. Our results for acute exposure suggest that F. candida sustains irreversible damage to reproduction at high doses (4110 Gy), whereas reproductive damage at lower doses (o60 Gy) appears to be reversible. Therefore, at the same total dose, chronically irradiated animals may experience reversible reproductive effects, whereas acutely irradiated animals may become sterile. Because sterilized animals cannot use energy for reproduction, differences in the method of exposure (acute or chronic) may lead to changes in energy allocation among detoxification, survival, and reproduction. Additional data on chronic and acute exposures in standard tests are required to clarify this issue. 5. Conclusion Using a standard test, we obtained basic data that can be used for ecological impact assessments for radiation. Delayed egg development appears to be the primary cause of reduced reproductive success in response to acute gamma irradiation. Additional reproductive data for chronic low-level exposures, which is a realistic parameter from an ecological viewpoint, are needed to better protect the environment from radiological exposure. Funding sources This work was supported by management expenses grants from the National Institute of Radiological Sciences.
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