Tolerance of High Oral Doses of Nonradioactive and Radioactive Caesium Chloride in the Pale Grass Blue Butterfly Zizeeria maha

Abstract

The biological effects of the Fukushima nuclear accident have been examined in the pale grass blue butterfly, Zizeeria maha (Lepidoptera: Lycaenidae). In previous internal exposure experiments, larvae were given field-collected contaminated host plant leaves that contained up to 43.5 kBq/kg (leaf) of radioactive caesium. Larvae ingested up to 480 kBq/kg (larva), resulting in high mortality and abnormality rates. However, these results need to be compared with the toxicological data of caesium. Here, we examined the toxicity of both nonradioactive and radioactive caesium chloride on the pale grass blue butterfly. Larvae were fed a caesium-containing artificial diet, ingesting up to 149 MBq/kg (larva) of radioactive caesium (¹³⁷Cs) or a much higher amount of nonradioactive caesium. We examined the pupation rate, eclosion rate, survival rate up to the adult stage, and the forewing size. In contrast to previous internal exposure experiments using field-collected contaminated leaves, we could not detect any effect. We conclude that the butterfly is tolerant to ionising radiation from ¹³⁷Cs in the range tested but is vulnerable to radioactive contamination in the field. These results suggest that the biological effects in the field may be mediated through ecological systems and cannot be estimated solely based on radiation doses.

Keywords: Fukushima nuclear accident; Zizeeria maha; caesium chloride; field effect; internal exposure; pale grass blue butterfly; radioactive caesium; toxicology.

Figure A1

Materials and methods used in this study. (a) Separated and washed Oxalis leaves. (b) Fresh Oxalis leaf paste. The dry weight of this paste is considered one part. (c) Soy powder (one part) (d) Insecta F-II (two parts). (e) Mixing the fresh Oxalis paste, soy powder, and Insecta F-II in a glass cup using a spoon. (f) Plated artificial diet in Petri dishes. Dishes were labelled and sealed. (g) Feeding larvae an artificial diet in a 90-mm Petri dish. This panel shows a preliminary experiment to test the quality of the diet. Only 35-mm Petri dishes were used for feeding in the toxicological experiments. (h) The larval rearing system. In a plastic container, 35-mm Petri dishes were confined. (i) Isolated pupae in 90-mm Petri dishes. (j) Original supply of ¹³⁷CsCl (3.7 MBq in 0.5 mL). (k) Image analysis for areas of the artificial diet consumed by the larvae. [A–C] Images before feeding; [D–F] Images after feeding. From the original pictures [A,D], the diet areas and their surroundings were cut out [B,E], and the diet areas were further isolated by applying a darkness threshold of 133 using Adobe Photoshop. Black squares are reference scale areas of 1.0 cm². Areas were then calculated using ImageJ. (l) Sequential washing of the prepupae (light green objects) with deionised water to remove the artificial diet from their bodies before measuring the radioactivity concentrations of ¹³⁷Cs. (m) Drying of the washed prepupae with silica gel (black arrowheads) for measuring the radioactivity concentrations of ¹³⁷Cs. Dried prepupae were black and hard (red arrowheads).

Figure 1

Evaluation of the artificial diet AD-FSI-112. (a) Ingredients of the artificial diets AD-F, according to Hiyama et al. (2010) [43], and AD-FSI-112. Ingredients are shown in weight proportions. (b) Survival rates (normalised). Data for AD-F and AD-D were obtained from Hiyama et al. [43]. (c) Forewing size. p-values are shown (Student’s t-test).

Figure 2

Caesium levels used in the present study. In a and b, numerical values were obtained from calculations based on the starting concentrations except for the last line in b. The amount of the artificial diet consumed was assumed to be 108.6 mg (see Materials and Methods). Larval weight was assumed to be similar to the weight and to be 0.033 g, from Nohara et al. [33]. (a) Nonradioactive (cold) caesium group. The radioactivity concentration equivalence was calculated based on the fact that 100 Bq of ¹³⁷Cs is 31.2 pg, according to Shozugawa [45]. C1, C2, C3, C8, and C10 were not used in the present study. (b) Radioactive (hot) caesium group. H4, H5, and H6 correspond to C4, C5, and C6, respectively. An asterisk indicates the levels of the previous internal exposure experiments. The bottom lines are not calculated but are measured values. na: Not applicable. (c) Radioactivity concentrations from the previous internal exposure experiments. Data were obtained from Nohara et al. [33]. The bottom concentration is not calculated but is the measured value.

Figure 3

Effects of nonradioactive caesium chloride. In a, c, and e, all individual samples in the 3 trials were summed together, and the differences among the groups were examined using a Kruskal–Wallis test. In b, d, and f, the three trials were independently plotted, and a Spearman correlation analysis was performed. N indicates the number of groups. (a,b) The pupation rate (%). (c,d) The eclosion rate (%). (e,f) The survival rate (%).

Figure 4

Forewing size of the individuals who consumed nonradioactive caesium chloride. In a and c, box plots were made, and the differences among groups were examined using a Kruskal–Wallis test. In b and d, individual sample data were plotted, and a Spearman correlation analysis was performed. (a,b) Male forewing size. (c,d) Female forewing size.

Figure 5

Caesium radioactivity in the prepupae. Spearman correlation coefficients and their associated P-values are reported. (a) Measured caesium radioactivity in the prepupae in proportion to that in the artificial diet. Both the x- and y-axes are on a logarithmic scale. (b) Percentage of the measured caesium radioactivity among the ingested caesium radioactivity plotted against the caesium radioactivity in the artificial diet. In this scatter plot, the x-axis is on a logarithmic scale.

Figure 6

Effects of radioactive caesium chloride. In a, c, and e, all individual samples in the three trials were summed together, and the differences among the groups were examined using a Kruskal–Wallis test. In b, d, and f, the three trials were independently plotted, and a Spearman correlation analysis was performed. N indicates the number of groups. (a,b) The pupation rate (%). (c,d) The eclosion rate (%). (e,f) The survival rate (%).

Figure 7

Forewing size of the individuals who consumed radioactive caesium chloride. In a and c, box plots were made, and the differences among groups were examined using a Kruskal–Wallis test. In b and d, individual sample data were plotted, and a Spearman correlation analysis was performed. (a,b) Male forewing size. (c,d) Female forewing size.

Figure 8

Comparisons of the effects of nonradioactive and radioactive caesium chloride. C0 and H0 were set at 100%, and the normalised values were compared between the cold (n = 3) and hot (n = 3) results. P-values were obtained using a Student’s t-tests (df = 4 in all combinations). (a) The pupation rate (%). (b) The eclosion rate (%). (c) The survival rate (%).