Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 1, pp. 87-89, 2005

AcceptedThe Formation of Insoluble Tc Depends on Bacterial Activity
Nobuyoshi Ishii,*, a Hiroyuki Koiso,b and Shigeo Uchidaa
aOffice of Biospheric Assessment for Waste Disposal, National Institute of Radiological Sciences, Anagawa 4-9-1,
Inage-ku, Chiba 263-8555, JapanbTokyo Nuclear Services Co., Ltd., 7-2-7 Ueno, Taito-ku, Tokyo 110-0005, Japan
Received: November 15, 2004; In Final Form: April 21, 2005
The formation of insoluble Tc (> 0.2 µm in size) in ponding water of paddy soil was investigated. The ponding
water prepared by flooding with deionized water was collected at a one-week intervals during the 120 days of theexperimental period. The collected sample was incubated with 3 kBq mL−1 TcO4− at 25 ˚C for 14 days. The meanvalue of relative amounts of insolubilized Tc was less than 0.6% of total Tc. This result suggested that floodingwith deionized water was not effective for the formation of insoluble Tc. When the ponding water sample wasincubated with TcO4− under anaerobic conditions, Tc was scarcely insolubilized. On the other hand, a significant
fraction of the Tc was insolubilized in the ponding water sample supplemented with nutrients that promote bacterial activity. Also, the amount of insoluble Tc increased with time. From these results, it was concluded that theformation of insoluble Tc in the ponding water depends on enhanced bacterial activity rather than just anaerobicconditions.

1. Introduction
Technetium-99 (99Tc) is produced by thermal fission of 235U
and 239Pu with a yield of 6%, making 99Tc relatively high inabundance among fission products. In addition, the half-life of99
Tc is very long (2.1 × 105 y). Under aerobic conditions, Tc ispresent in the heptavalent form as pertechnetate (TcO4−). Thischemical species is soluble and mobile in the environment andis readily taken up by plants.1 The uptake of Tc by rice is anespecially serious issue in the transfer of 99 Tc to humans.
Therefore, it is important to predict the behavior of 99Tc in
agricultural environments.
Recently, the authors found that soluble TcO4− was changed
to insoluble forms in ponding water in several types of paddy
soil2 and that the formation of insoluble Tc was mainly caused
by bacteria.3 The chemical species of the insoluble Tc was notidentified, but it was probably TcO2.4 In the previous study,
0.3% glucose water was used for flooding paddy soil todemonstrate the possibility of forming insoluble Tc. Paddy
fields in nature, however, are not flooded by glucose water
even though there are other sources of organic carbon such as
plant litter, added organic matter for fertilization, and remains
from previous crops. Therefore, the aims in this study were todetermine the amounts of insoluble Tc in ponding water
without addition of glucose and to clarify the main factors
causing Tc insolubilization.
2. Materials and Methods

The flooded soil was statically incubated at 25 ˚C for 120 days
under 12 h-12 h light-dark cycles. The initial conditions of thesample were aerobic because the sample had more than 40 mLof headspace air. Flooded soil samples were prepared in triplicate.
During the 120-day incubation period, 1.2 mL of the ponding
water was collected once a week from each flooded sample.
After collection, the flooded soil sample was supplemented
with autoclaved deionized water to maintain the initial volumeof flooding. Of the collected ponding water, 0.2 mL was used
to determine the concentration of ferrous iron, and the rest wasused for the following experiments.
Insolubilization of Tc in ponding water samples. The
experimental procedure is shown in Figure 1. The stock solution of NH 4 TcO 4 in H 2 O was filter-sterilized by passing it
through a 0.2 µm pore size membrane. In the aqueous solution, Tc is present as pertechnetate (TcO4−). The Tc solution
was added to each of the collected ponding water samples at afinal concentration of 3 kBq mL−1. These samples were thenstatically incubated at 25 ˚C for 14 days in the dark. The initialconditions of these samples were aerobic because the samples
had about 14 mL of headspace air. After incubating for 0, 4, 7,
and 14 days, a part of the sample was passed through a 0.2µm-pore size filter to separate insoluble Tc. The relative
amount of insoluble Tc was calculated by dividing “(the 99Tc
radioactivity at day 0) – (the radioactivity of 99 Tc at eachsampling time)” by “the 99Tc radioactivity at day 0”.
(a)

Preparation of ponding water samples. Paddy soil containing
Tc-insolubilizing bacteria3 was collected from Koriyama City,
Japan. Characteristics of the soil were described in a previous
report.2 The Tc-insolubilizing bacteria in the paddy soil havenot been isolated yet. The soil was air-dried and passed througha 2-mm-mesh sieve. This procedure eliminated plant litter andprevious crop residues from the soil. For flooding, 5 g of theair-dried soil were put in a polypropylene tube with a cap andthe tube was filled with 7.5 mL of autoclaved deionized water.
*Corresponding author. E-mail: nobu@nirs.go.jp. FAX: +81-43251-4853.

Flooding soil|
Incubation for 120 days(b)Collection of ponding waterat a one-week interval|
Addition of 99TcO4to the ponding water sample|
Determination of insoluble Tc amountafter incubating for 4, 7, 14 days

Flooding soil|
Collection of ponding water
after 120 days of incubation||

Anaerobic culture

Promoting culture||
Addition of TcO4 to both cultures|Determination of insoluble Tc amountafter incubating for 4, 7, 14 days

Figure 1. Diagrams showing the two different experimental procedures. (a) Experiment for Tc insolubilization, and (b) Experiment forfactors affecting Tc insolubilization.

© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 07/31/2005

IshiiJ. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

Determination of Factors affecting Tc insolubilization.
The experimental procedure is shown in Figure 1. To clarify
the factors affecting Tc insolubilization, two subsamples wereprepared using the ponding water from the 120-day flooding:1) a sample incubated under anaerobic conditions (anaerobicculture), and 2) a sample with organic substrates added topromote bacterial metabolic activity (promoting culture). For
the anaerobic culture, the ponding water sample was incubatedin the AnaeroPackTM System (MGC Co., Inc. Tokyo, Japan) for
14 days after addition of TcO4− at a final concentration of 3
kBq mL−1. For the promoting culture, the following reagentswere added to the ponding water sample (in milligrams permilliliter of culture): tryptone, 1.0; yeast extract, 0.5; and NaCl,1.0. This subsample was also incubated for 14 days after addition of 99TcO4− at a final concentration of 3 kBq mL-1. Relative
amounts of insoluble Tc were determined after incubating for
4, 7, and 14 days in the same way as mentioned above.
Analytical methods. The concentration of Fe(II) ions in
ponding water samples was colorimetrically determined by the1, 10-phenanthroline monohydrate method5 at every time ofsample collection.
The radioactivity of 99Tc was measured by liquid scintillation counting of 0.1 mL filtrate samples mixed with 4 mL ofliquid scintillation cocktail, Ultima gold LLT (PerKinElmer,Inc., Massachusetts, USA).

10Day 450-5Insoluble Tc (% of total)

88-10
10Day 750-5-101020Day 14
50-5-10
020

4060

80

100120140Time of flooding (days)

Figure 2. Formation of insoluble Tc in ponding water samples. The
number of days means the time after the addition of 99 Tc to theponding water. The means ± standard deviations (error bars) fromthree experiments are shown.

25

3. Results and Discussion

Anaerobic
Insoluble Tc (% of Total)

Formation of insoluble Tc. Variations in the relative amountsof insoluble Tc were small for 120 days of flooding (Figure 2).
For 4-day incubation samples with different flooding periods,
the mean value of the relative amounts of the insoluble Tc was
0.2 ± 1.7% of the total 99Tc. Similar results were observed forthe samples after incubating for 7 and 14 days (Figure 2), andmean values of the relative amounts of the insoluble Tc forboth samples were less than 0.6% of the total 99Tc. These data
indicated that Tc was hardly insolubilized in the ponding water
samples. However, the presence of Tc-insolubilizing bacteria
in the paddy soil studied was certain because, as reported
previously, a significant fraction of Tc was insolubilized in theponding water samples prepared by flooding the same paddy
soil with glucose water.3 The addition of glucose as a carbon
source promotes microbial metabolic activity, and the activated microbes rapidly exhaust molecular oxygen to decompose the organic carbon source. Both microbial activity6 andanaerobic conditions7 are responsible for removal of Tc fromsolution. The limited formation of insoluble Tc under thepresent conditions, therefore, was probably due to the low
metabolic activity of the bacteria or the aerobic conditions in
the ponding water samples. Among microorganisms, bacteria
are key for Tc insolubilization in ponding water. 3 In thefollowing experiment, the effects of the metabolic activity ofbacteria and the aerobic conditions on insolubilization of Tc
were investigated.
Factors affecting Tc insolubilization. Anaerobic conditionsin the ponding water samples were evaluated by analysis of theferrous ion because Fe(II) is generally released to ponding waterfrom paddy soil under anaerobic conditions. The concentrationsof Fe(II) ion in the ponding water samples were less than 0.45 µg
mL−1 during the experimental period, while the addition of glucose
to flooding water resulted in 143 µg of Fe(II) ion per milliliter
after 7 days of flooding.8 Therefore, the ponding water samples
without addition of glucose were under aerobic conditions.
To clarify the effect of anaerobic conditions on Tc insolubilization, the ponding water sample spiked with 99 Tc wasforcibly incubated under anaerobic conditions (anaerobicculture). Tc was hardly insolubilized at 14 days (Figure 3),
suggesting that anaerobic conditions were a not key factor for
Tc insolubilization in the ponding water.

20Promoting
151050-5-505Time (days)

1015

Figure 3. Formation of insoluble Tc in the anaerobic culture and thepromoting culture. The means ± standard deviations (error bars) fromthree experiments are shown.

The effect of bacterial activity on Tc insolubilization wasalso determined by the addition of organic substances to theponding water sample (promoting culture). Insoluble Tc wasformed and its relative amount increased with time (Figure 3),
while Tc was hardly insolubilized without addition of organic
substances (Figure 2). These results suggest that Tc-insolubilizing bacteria were present in the ponding water sample andtheir metabolic activity resulted in formation of insoluble Tc inthis sample. Organic substrates added to the cultures may
function as electron donors because the previously known Tcinsolubilizing bacterium, Escherichia coli, requires organic
substrates such as formate, pyruvate, glucose, or glycerol aselectron donors.9
4. Conclusion
Insoluble Tc was scarcely formed in ponding water samples
prepared by flooding of the air-dried paddy soil with deionizedwater. The amount of insoluble Tc was increased by the addition of organic substrates to the ponding water samples, but not
by incubation under anaerobic conditions without addition oforganic substrates. The formation of insoluble Tc in ponding
water is therefore due to bacterial activity.

The Formation of Insoluble Tc Depends on Bacterial Activity

Acknowledgement. This work has been partially supportedby the Agency for Natural Resources and Energy, the Ministryof Economy, Trade and Industry (METI) Japan.
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J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

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