Effects of litter feeders on the transfer of 137Cs to plants

The effects of the Japanese horned beetle larvae on the transfer of 137Cs from a contaminated leaf litter to the leaf vegetable, komatsuna (Brassica rapa var. perviridis) was studied. Feces of the larvae which were fed 137Cs-contaminated leaf litter were added to a potting mix in which komatsuna plants were cultivated. The presence of feces increased the harvest yield of komatsuna, suggesting that feces provided nutrients for the plant growth. In addition, the amount of exchangeable 137Cs in leaf litter was experimentally confirmed to be enhanced by the presence of feces which were excreted by larvae feeding. However, there was no difference in the soil-to-plant transfer factor of 137Cs for the presence and absence of feces. Interactions between clay minerals and exchangeable 137Cs in the soil beneath the litter layer may diminish the root uptake of 137Cs. From these results, it was concluded that the effect of exchangeable 137Cs released from feces was limited for the transfer of 137Cs to plants if plant roots were not present in litter layers.


Results and Discussion
Decomposition of leaf litter. Larvae were maintained on the 137 Cs-intact-litter bed (radiocesium contaminated uncrushable litter bed) to obtain decomposed leaf litter. No larvae mortality was observed, and they did not reach the prepupal state during the rearing period. Culture conditions, therefore, were suitable for larvae survival.
The 137 Cs-intact-litter was physically broken down into small pieces by larval feeding (Fig. 1). Some of the litter was chemically decomposed through digestive processes and then excreted as feces. Feces characteristically had a black columnar shape, and were present in the rearing vessels at the end of rearing period. Therefore, the decomposed-litter-mixture contained physically and chemically decomposed leaf litter.
Harvest yield. Komatsuna was successfully grown in the three types of potting mixes, but the yield at harvest differed among them (Fig. 2). ANOVA of the data confirmed that the amount of komatsuna grown in the PM-F soil was significantly larger than that in the other two mixes (p < 0.05 for vs. PM-L and p < 0.01 for vs. PM). No difference in the yield was found between komatsuna plants grown in PM and on PM-L. Because PM-L is a blended soil including PM and the 137 Cs-intact-litter, the results implied that the addition of 44 g of the 137 Cs-intact-litter had no effect on the amount of yield under the present experimental conditions. On the other hand, the decomposed-litter-mixture increased the harvest yield, and that suggested the decomposed-litter-mixture had plant growth-promoting abilities. The increase in the yield could be caused by the release of nutrients resulting from the decomposition of the 137 Cs-intact-litter by the beetle larvae; such release of nutrients like nitrogen, phosphorus, and potassium from leaf litter through decomposition has been demonstrated 8,9 . It was also possible that release of 137 Cs occurred, as leaching of 137 Cs from leaf litter after being submerged in water has been reported 10,11 . Extraction of 137 Cs from the 137 Cs-intact-litter and the decomposed-litter-mixture samples.
An extraction experiment was conducted to confirm the effect of biological decomposition on the release of 137 Cs  from the 137 Cs-intact-litter samples. In the experiment, eluents (deionized water and KCl solution) were passed through a syringe containing the 137 Cs-intact-litter or the decomposed-litter-mixture, so there was a contact-time between eluents and both litter samples of several minutes. Despite such a short contact-time, 137 Cs was detected in the dissolved fraction for all samples tested (Fig. 3). Sakai et al. 11 showed that the amount of extractable 137 Cs from litter increased with soaking time. The amount of released 137 Cs from 137 Cs-intact-litter may be enhanced under natural conditions, because the litter layer in the natural environment generally holds moisture allowing a prolonged contact-time of the 137 Cs-intact-litter with water. The contaminated leaf litter, therefore, becomes a potential source of 137 Cs for plants even if that litter is not biologically decomposed.
Availability of radiocesium for plant is affected by the chemical form of the radiocesium. Although the physical size of 137 Cs in the dissolved fraction was less than 0.45 µm, its chemical form was not evaluated. Tagami and Uchida 12 demonstrated that radiocesium in the <0.45 µm fraction was mostly in the Cs + form. In their study 137 Cs was extracted from leaf litter by deionized water and filtered through a membrane filter with 0.45-µm-pores. This treatment procedure was similar to that of the present experiment, so the extracted 137 Cs in the study was probably in a cationic form which is available for uptake by plants.
The effect of biological decomposition on the extraction of 137 Cs was also confirmed. Decomposition of the 137 Cs-intact-litter by larvae significantly increased the extraction ratio of 137 Cs from 2.3 ± 0.4% to 3.0 ± 0.4% in the exchangeable fraction by KCl (Fig. 3). On the other hand, no difference was found for the extraction ratios of the soluble fraction (extracted with deionized water). In comparison to deionized water, the effect of KCl as extractant was evident. For example, extraction ratios by KCl for the 137 Cs-intact-litter samples were 2.8 times those by deionized water. Similarly, when 137 Cs was extracted from the decomposed-litter-mixture samples by KCl, 3.1 times higher amounts of 137 Cs were extracted in comparison with those extracted by deionized water. In addition, the difference in these extraction efficiencies (the ratio of 137 Cs extracted by KCl to 137 Cs extracted by deionized water) of 2.8 and 3.1 suggested that decomposition of the 137 Cs-intact-litter by larvae promoted the production of the exchangeable 137 Cs. Because simple physical decomposition affects chemical composition of the 137 Cs-intact-litter, digestive processes of larvae may cause a change in the chemical composition of the 137 Cs-intact-litter. Consequently, the amount of 137 Cs in the exchangeable fraction increased for the decomposed-litter-mixture samples. Some previous studies also observed changes in chemical composition of soil 13 and litter 14,15 by digestion. Additionally, Coeurdassier et al. 16 showed an increase of the water-soluble cadmium fraction in soil with earthworms. These examples suggest that digestive processes commonly play an important role in the change of chemical composition.  Table 1. The 137 Cs activity concentrations for the both potting mixes were similar. There were no differences in 137 Cs activity concentrations in komatsuna plants grown in both PM-L and PM-F. These results suggested that the 137 Cs-intact-litter was a source of 137 Cs regardless of decomposition.
TFs were determined using the activity concentrations of 137 Cs in potting mixes and plants (Table 1). A TF value of 2.6 × 10 −2 for the PM-F potting mix was similar to that for the PM-L potting mix (t-test, P > 0.05) in spite of the significant release of exchangeable 137 Cs from the decomposed-litter-mixture. This was in agreement with a previous result showing no increase in TF of 137 Cs from soil to lettuce even if saprophagous organisms (earthworms) were present in the soil 18 . The lack of difference in TF may be explained in two ways by the interaction between exchangeable 137 Cs and soil minerals. Firstly, the feeding activity by beetle larvae may have enhanced the release of the exchangeable 137 Cs from the 137 Cs-intact-litter (Fig. 3). Some of the exchangeable 137 Cs would be fixed by soil minerals immediately after its release, because 137 Cs is easily sorbed on the surface of soil minerals by ion exchange and chemical sorption 19 , and held in frayed edge sites 20 . These sorption and fixation processes could decrease the bioavailability of 137 Cs to plants. Secondly, the significant increase of exchangeable 137 Cs caused by the larvae in the present study was too small to significantly affect the transfer of 137 Cs.
The TFs of up to 0.026 obtained in this study were close to the minimum values reported in previous studies for plants belonging to the family Brassicaceae 21, 22 . The difference in the contact area between the 137 Cs source and plant roots may affect TF values. The previous studies used sand, loam, clay, and organic soils as the 137 Cs source. In such cases, most of the plant roots were probably in contact with the 137 Cs source. In the present study, 44 g of the decomposed-litter-mixture was mixed with 2.6 kg of soil. In this case, the volume of the decomposed-litter-mixture was small in comparison with that of the soil, and thus the contact area between plant roots and the 137 Cs source was also small. If the 137 Cs released from the decomposed-litter-mixture is fixed by the soil before the uptake of 137 Cs by the roots, TF value becomes low. The positional relationship between plant roots and the 137 Cs source is one of the factors controlling TF value. In the natural environment, plant and tree roots grow in humus and soil mineral layers beneath the litter layer 23 , so there is little direct contact between roots and a litter layer covering the soil.

Conclusions
The present study showed that biological decomposition of the 137 Cs-intact-litter by saprophagous beetle larvae enhanced the release of exchangeable 137 Cs which is available for uptake by plants. Involvement of the larval digestive processes in the change in the chemical composition was suggested. Although the amount of 137 Cs in the exchangeable fraction increased, no detectable effect on the transfer of 137 Cs to plants was observed under the experimental conditions. Therefore, the effect of litter feeders on the transfer of 137 Cs to plants was limited. In the present study, the decomposed-litter-mixture was blended with some soils, and the exchangeable 137 Cs was probably fixed by clay minerals. Therefore, further studies on interactions between soil minerals and exchangeable 137 Cs and the positional relationship between plant roots and litter will be important to understand the behavior of 137 Cs in actual forest ecosystems.

Methods
Sampling locations. Leaf litter which was contaminated with radiocesium (hereafter 137 Cs-intact-litter) was collected in a deciduous broadleaf forest (mainly Quercus serrata) of the Kami-Oguni river catchment (37°7′N, 140°6′E) in Fukushima Prefecture on 29 June 2012. This forest was located about 53 km north-west of the FDNPP. The concentration of 137 Cs was 3.2 × 10 5 Bq/kg-dry on the collection day.
Last instar larvae of Japanese horned beetle were collected in a mixed deciduous forest of Showa-no-Mori Park (35°31′N, 140°16′E), Chiba City, Japan in March 2013. The Ministry of Education, Culture, Sports, Science and Technology (MEXT) has reported 24 that the area surrounding this park was contaminated with radiocesium ( 137 Cs and 134 Cs) at less than 10 × 10 4 Bq/m 2 .
Rearing of larvae. Each larva was grown on 50 g-wet weight of 137 Cs-intact-litter in a polyethylene rearing vessel (500 cm 3 ) at 25 °C under light (16 hours)-dark (8 hours) conditions for 14 days; nine replicates were prepared. To retain the moisture conditions, deionized water was sprayed into the rearing vessels every 2 days during the cultivation period. All feces pellets excreted by the larva were removed at day 9 because these feces would have been affected by the mater ingested before the controlled rearing was started. The feces pellets present in the vessel at the end of the rearing were, therefore, excreted after day 9. Finally, after removing the larvae, the remaining 137 Cs-intact-litter, physically crushed litter, and feces pellets in the rearing vessel were well mixed (hereafter called the decomposed-litter-mixture). The decomposed-litter-mixture was used in the two types of experiments: extraction and planting. The 137 Cs-intact-litter alone was also prepared as a negative control culture with nine replicates.
Extraction experiment. Five grams-dry weight of the 137 Cs-intact-litter or the decomposed-litter-mixture were put into a syringe (volume of 50 mL). The concentration of 137 Cs was 3.2 × 10 2 Bq/g-dry for both litter samples. The bottom of the syringe was filled with quartz wool to keep the pieces of the litter samples in the syringe. Eluent (100 mL) was poured into the syringe to extract 137 Cs from the 37 Cs-intact-litter and the decomposed-litter-mixture samples. Two kinds of eluents were used: deionized water and 2 M KCl solution. Deionized water and KCl were used to obtain the water soluble 137 Cs and the exchangeable 137 Cs, respectively. The solution passing through the quartz wool was further filtered through a membrane filter having 0.45-µm-pores. The plant uptake is presumably limited to soluble chemical species. Even particles/colloids smaller than 0.45-µm may not be taken up by plant roots. The final filtrate samples were collected in U8 type polystyrene containers for the measurement of the activity concentrations of 137 Cs. The ratio of extraction was calculated as follows: Potting mix Treatment Activity concentration of 137 Cs (mean ± sd Bq/kg-dry) TF (mean ± sd) Soil Plant PM-L +Litter 2.2 × 10 3 ± 1.4 × 10 2 5.6 × 10 1 ± 6.6 × 10 1 2.5 × 10 −2 ± 2.9 × 10 −3 PM-F +decomposed litter and feces 2.6 × 10 3 ± 1.6 × 10 2 6.8 × 10 1 ± 6.2 × 10 1 2.6 × 10 −2 ± 2.0 × 10 −3 where C e is the total amount of 137 Cs in the dissolved fraction (<0.45 µm), and C 0 is the total amount of 137 Cs in 5 g of the 137 Cs-intact-litter or the decomposed-litter-mixture. These extraction experiment was repeated nine times.
Planting experiment. Leaf vegetable plants of the family Brassicaceae, komatsuna (Brassica rapa var. perviridis) were grown in three potting mixes: (1) a base potting mix (PM), (2) PM with the 137 Cs-intact-litter (PM-L) and (3) PM with the decomposed-litter-mixture (PM-F). The PM consisted of Akadama soil, leaf mold, Kuro-tsuchi, and a chemical fertilizer (8% nitrogen, 8% phosphorus, and 8% potassium by weight). First, the Akadama soil, leaf mold, and Kuro-tsuchi, were mixed with a volume ratio of 6:3:1, and then 10 g of the chemical fertilizer was added to 2.6 kg of the soil mixture. For the preparation of PM-L and PM-F, 44 g of the 137 Cs-intact-litter or the decomposed-litter-mixture were added to 2.6 kg of PM, respectively. Plastic containers (400 mm wide × 300 mm deep × 145 mm high) were filled with each of the potting mixes. Eight komatsuna plants were grown in each potting mix (PM, PM-L and PM-F). The containers were kept in a greenhouse under natural illumination for 30 days (growing period). The temperature was controlled at 25 ± 5 °C during the growing period. Planting was independently repeated three times at the same period.
The edible parts of komatsuna were harvested on day 30 after planting, and they were dried at 80 °C in a drying oven. After the harvest, the potting mixes were air dried, and then three subsamples were collected from each dried potting mix. The dried plants and soil samples were powdered with a grinder (Labo Milser LM-PLUS, Osaka Chemical Co., Ltd.), and each powdered sample was put into a U8 container for the analysis of 137 Cs.
Measurement of 137 Cs. The activity concentrations of 137 Cs in the samples were determined by a gamma spectrometry method using a high-purity germanium detector (GMX-type, ORTEC, Seiko EG&G, Tokyo, Japan). The detector was calibrated with volume radioactivity standard gamma sources (MX033U8PP, Japan Radioisotope Association). The standard reference material JSAC-0471 (the Japan Society for Analytical Chemistry) was used for an accuracy check. The activity concentrations were corrected for radioactive decay to the sample collection day.

Soil-to-plant transfer factors.
The soil-to-plant transfer factors (TFs) were calculated as the ratio of the activity concentration of 137 Cs in the plant (Bq/kg-dry weight) to its concentration in the potting mix (Bq/kg-dry weight).
Statistical analysis. Significant differences in the amount of harvested komatsuna were determined by one-way analysis of variance (ANOVA) with Tukey's HSD post hoc test. Student's t-test was carried out to confirm the effect of decomposition on the extraction of 137 Cs from leaf litter.