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Year : 2021  |  Volume : 44  |  Issue : 3  |  Page : 131-134  

Studies on foliar uptake of tritiated water on Spinach sp. during light and dark simulated conditions using environmental chamber

1 Environmental Survey Laboratory (Environmental Studies Section, Environmental Monitoring and Assessment Division, Bhabha Atomic Research Center), Surat, Gujarat, India
2 Environmental Survey Laboratory (Environmental Studies Section, Environmental Monitoring and Assessment Division, Bhabha Atomic Research Center), Thane, Maharashtra, India
3 Environmental Monitoring and Assessment Division, Bhabha Atomic Research Center, Mumbai, Maharashtra, India

Date of Submission18-May-2021
Date of Decision30-Jul-2021
Date of Acceptance16-Aug-2021
Date of Web Publication04-Jan-2022

Correspondence Address:
Chetan P Joshi
Environmental Survey Laboratory (Environmental Studies Section, Environmental Monitoring and Assessment Division, Bhabha Atomic Research Center), Kakrapar Gujarat Site, P.O. Anumala, Surat - 394 651, Gujarat
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/rpe.rpe_15_21

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Studies on foliar uptake of tritiated water (HTO) on Spinach sp. during light and dark condition was simulated inside the environmental chamber, and an attempt was made to compute CTFWTHTO, CpfwHTO, CpcwOBT, CpfwOBT using basic equations available in IAEA TECDOC1616. The predicted activity was compared with the observed activity. The conversion of tissue-free water tritium (TFWT) to organically bound tritium (OBT) was more in the light condition. The ratio of OBT to TFWT varied from 0.006 to 0.082. The ratio of observed OBT activity (Bq/kg fresh weight) in light to dark conditions varied from 34% to 117%.

Keywords: Dry deposition, organically-bound tritium, tissue-free water tritium, tritium, wet deposition

How to cite this article:
Joshi CP, Patra A K, Jha M K, Sudheendran V, Baburajan A, Saradhi I V, VinodKumar A. Studies on foliar uptake of tritiated water on Spinach sp. during light and dark simulated conditions using environmental chamber. Radiat Prot Environ 2021;44:131-4

How to cite this URL:
Joshi CP, Patra A K, Jha M K, Sudheendran V, Baburajan A, Saradhi I V, VinodKumar A. Studies on foliar uptake of tritiated water on Spinach sp. during light and dark simulated conditions using environmental chamber. Radiat Prot Environ [serial online] 2021 [cited 2023 Jan 28];44:131-4. Available from: https://www.rpe.org.in/text.asp?2021/44/3/131/334776

  Introduction Top

Tritium (H3) is the only radioactive isotope of hydrogen having radioactive half-life of 12.3 years and is a pure low beta emitter of Emax 18.6 keV. In the atmosphere, H3 exists mainly in the form of tritiated water (HTO) and tritiated gas. H3 comes in the environment from three sources, natural production (cosmogenic interaction of N14 and O16 in the upper atmosphere), release from atmospheric tests, and routine release from nuclear plants.[1] H3 being isotope of hydrogen and since exists as HTO, follows the biogeological cycle of water. The atmospheric process of H3 removal is classified as a wet deposition and dry deposition. Wet deposition results from scrubbing of air by rain, snow or fog and dry deposition occur on contact with tritiated air via the process of collection through leaf epidermis or the ground. The deposition velocity of HTO to the plants depends upon plant characteristics such as leaf area index and stomatal resistance. For both soil and plant, the deposition velocities of HTO range from 0.001 to 0.1/ms as a function of atmospheric stability and surface properties.[2],[3] Foliar absorption and root absorption are the pathways of entry of H3 into vegetation. H3 is incorporated into plants primarily in the form of HTO via air moisture and soil water. Generally, H3 activity in plant leaves moisture to reach equilibrium quickly with the tritium activity in air moisture. By this mechanism, tissue-free water tritium (TFWT) forms in the plant and partly it is converted into organically bound tritium (OBT).[4] Tritium is believed to be incorporated into organic molecules in living systems predominantly through reactions or exchanges involving HTO molecules.[5] The fraction of OBT consisting of tritium atoms that are easily exchanged with hydrogen atoms in water molecules is referred to as “exchangeable OBT” and amounts to about 20% of the total OBT.[6] The remainder of the OBT is referred to as “nonexchangeable OBT.” The exchangeable OBT is a part of plant dry matter, but it behaves as tissue-free water both in the environment and in vegetation. OBT has a longer retention time as compared to TFWT; hence, it is very important to study the estimation of OBT in plants. Therefore, an attempt was made to study the foliar uptake of HTO in Spinach sp. and the formation of OBT by carrying out the HTO exposure experiment in light and dark conditions in the environmental chamber.

  Materials and Methods Top

Experimental set up

Experimental set up of 13 pots were made in Environmental Survey Laboratory, Kakrapar Gujarat Site. Spinach (Spinacia olerece) seeds were sown in 13 pots. Spinach was chosen because it is an edible leafy vegetable species having a comparatively higher surface area and grows very fast during the winter. The organic manure was thoroughly mixed in the soil in each pot before the seeds were sown for better growth of Spinach sp. Spinach sp. was harvested at 39–62 days. At different time intervals, the pots were irrigated with normal water. The exposure of plants was carried out in the environmental chamber. Environmental chamber is an artificially designed chamber having a size of 2000 mm (L) × 2000 mm (W) × 2000 mm (H). In the chamber, different conditions for relative humidity (RH), light intensity, and rainfall rate can be simulated. On every occasion, a known amount of tritium activity (23,600 Bq) was diluted to 20 ml and was evaporated in a round bottom flask using a heating mantle at 60°C for 1.5 h. After complete evaporation of HTO, the heating mantle was switched off. During the experiment, the wet-bulb temperature and dry-bulb temperature were recorded, and it was found 25°C and 33°C, respectively. RH in the chamber was maintained at 52.7% during the exposure period. Simultaneously air moisture samples were also collected for 12 different occasions (6 light conditions and 6 dark conditions) from the chamber during the exposure period. Pot number 1, 3, 5, 7, 9, and 11 were exposed for 4 h in light conditions after 39, 42, 48, 49, 60, and 62 days harvesting period and pot number 2, 4, 6, 8, 10, and 12 were exposed for 4 h in dark conditions after 39, 42, 48, 49, 60, and 62 days harvesting period, respectively. During the exposure of all samples, the soil was covered by plastic.

Experimental estimation of tissue-free water tritium in spinach leaf samples

About 100 g of leaf samples from each pot were sampled. Leaf water was extracted using freeze dryer. 10 ml of the extracted water sample was mixed with a 10 ml Ultima Gold (LLT) cocktail and counted for 3H using ultra low-level liquid scintillation spectrometer (Model: QUANTULUS-1220). The counting system was calibrated with an H3 standard supplied by Amersham International. The system background count rate was 0.5–1.0 CPM, and counting efficiency of LSC system for detection of H3 was about 21%.

Estimation of biomass growth

Five plants were removed from each pot, and its root length, stem length, number of branches, total height, and the total weight of the plant was measured. By using the harvesting period and the total weight of the plants, biomass growth was estimated.

Modeling method for tissue-free water tritium and organically-bound tritium in spinach leaf samples

For the estimation of TFWT and OBT, different equations were used from IAEA TECDOC-1616[7] and are mentioned below.

Tritium activity in Leaf

Plants take up tritium from both air and soil. The HTO concentration in the free water of the leaf (TFWT) is calculated using:

where CTFWTHTO is the HTO concentration in the leaf free water (Bq/l), RH is the RH and γ =0.909 is the ratio of the HTO vapor pressure to that of H2O.

The HTO concentration in the fresh weight (FW) plant (Bq/kg FW) is given by,

where, WCp is the fractional water content of the plant.

Formation of organically bound tritium

Tritium is incorporated into the organic matter of plants during photosynthesis in the presence of light and through metabolic processes in the dark. Accordingly, the OBT concentration in water produced from the complete oxidation of organically bound hydrogen in plant tissues (Bq/l) is equal to the TFWT concentration in the leaf water modified by a partition factor (Rp)[7]

The partition factor accounts for the reduction in dry weight concentration due to the presence of exchangeable hydrogen in combustion water, as well as for isotopic discrimination.

The OBT concentration in the FW plant is given by:

Where WEQp is the water equivalent factor (kg of water produced per kg dry matter combusted).

  Results and Discussion Top

Tritium activity in Spinach sp. (CTFWTHTO and CpfwHTO)

[Table 1] shows the biomass growth rate observed in the experimental spinach samples during the study period. The biomass growth rate was found in the range of 0.069–0.127 g/day during the study period. [Table 2] shows the air moisture H3 activity (Bq/m3 or Bq/l) in the chamber, observed and predicted TFWT (Bq/l) in the plant, observed and predicted TFWT (Bq/kg) FW in the plant, observed and predicted OBT (Bq/l) in plant, observed and predicted OBT (Bq/kg) FW in plant, respectively. The range of air moisture H3 varied from 101 to 125 (Bq/m3) or 1848–2288 (Bq/l). The observed and predicted TFWT activity in spinach samples varied from 137 to 629 Bq/l and 1072–1326 Bq/l, respectively. The observed and predicted TFWT activity (Bq/kg) FW in spinach samples ranged from 123 to 567 (Bq/kg) FW and 964–1194 (Bq/kg) FW, respectively. There was a wide variation of experimental and predicted leaf HTO activity was observed. Observed TFWT activity is much lower than the predicted activity. The reason may be due to the nonuniform 3H activity inside the chamber and the deposition of HTO vapor on the inner walls of the environmental chamber. Keum et al.,[8] reported the HTO content of leaves decreases by several orders of magnitude in the space of a few hours.
Table 1: Biomass growth rate of Spinach sp. in different pots

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Conversion of CpfwHTO to CpfwOBT in Spinach sp.

The range of observed and predicted OBT was found to be 59–294 Bq/l and 579–716 Bq/l, respectively, whereas the observed and predicted OBT varied from 3 to 15 (Bq/kg FW) and 30–37 (Bq/kg FW), respectively [Table 2]. The estimation of biomass growth may be useful in the estimation/prediction of OBT in the plant. It was observed that [Table 2], the conversion of TFWT to OBT was more in the presence of light. In this study, the ratio of observed OBT activity (Bq/kg FW) in light to dark conditions varied from 34% to 117%. This wide variation may be due to natural conditions such as plant adaptability to the variability of light, temperature, water vapor deficit, as well as ambient CO2 concentration. In the simulated experiment, it is not possible to fully generate all the natural conditions for carrying out the experiment exactly. According to Diabate and Strack[9] the OBT formation at night suggests that tritium can be incorporated into organic matter not only by photosynthesis, but also by metabolic pathways independent of light, for instance by reaction of the tricarbonic acid cycle or other metabolic conversions. Therefore, the OBT formation in the nighttime pattern is more complex. According to Galeriu et al.[10], in the controlled study experiment, OBT/TFWT ratio is about 5 but not lower than 1. This may be because of illumination was too low compared to that outdoor. In the daytime, atmospheric HTO can enter the plant directly through the stomata and easily be incorporated into biological organisms as a TFWT.[11] After 1 h exposure in wheat and rice, the OBT concentration in water of combustion is up to 1.5% from the HTO concentration at the end of exposure for the day cases and about 0.1%–0.4% for the night cases.[12] Moses and Calvin[13] who exposed the chlorella algae to HTO in nutrient solution in light and dark conditions for 3 min, found that the incorporation of H3 in the nonexchangeable position of organic matter in the dark was one-third of that in the light. Thomson and Nelson[14] exposed the young leaves of Soybeans to HTO in the atmospheric humidity in light and dark conditions for 1–30 min. Related to the same exposure time, the assimilation of H3 in the dark was only 10% of that in light. According to Diabate and Strack[9], the concentration ratio in the night conditions is reduced to 23% in leaves, 25% in stems, and 59% in ears compared to those observed in high light conditions. Therefore the formation mechanism of organic matter is different in day and night conditions.
Table 2: 3H in air moisture predicted and observed tissue-free water tritium and organically bound tritium in Spinach sp.

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Foliar uptake of HTO on Spinach sp. during light and dark conditions was simulated in environmental chamber to understand the conversion process from TFWT to OBT. Spinach sp, which was exposed in the presence of the light condition, shows more conversion of TFWT to OBT. The basic equations given in IAEA TECDOC-1616 were used for the computation of TFWT and OBT. The ratio of OBT to TFWT varied from 0.006 to 0.082. Further study is in progress for the conversion of TFWT to OBT for longer time exposure in light and dark conditions in the environmental chamber.


The authors would like to thank Site Director, Kakrapar Site, Station Director, KAPS-1 and 2, and ACE (E and US) for their keen interest and encouragement. The assistance rendered by Smt. Padma Chaudhary, Shri M. K. Chaudhary, Shri. J. J. Chaudhary and all other ESL staff are thankfully acknowledged.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Okada S, Momoshima N. Overview of tritium: Characteristics, sources, and problems. Health Phys 1993;65:595-609.  Back to cited text no. 1
Eisenbud M, Bennett B, Blanco R, Compere E, Goldberg E, Jacobs D, et al. Tritium in the environment. In: National Council on Radiation Protection and Measurements, NCRP Report 62; 1978.  Back to cited text no. 2
Galeriu D, Davis P, Raskob W, Melintescu A. Recent progresses in tritium radioecology and dosimetry. Fusion Technol 2008;54:237-42.  Back to cited text no. 3
Choi YH, Lim KM, Lee WY, Park HG, Choi GS, Keum DK, et al. Tritium levels in Chinese cabbage and radish plants acutely exposed to HTO vapor at different growth stages. J Environ Radioact 2005;84:79-94.  Back to cited text no. 4
Diabaté S, Strack S. Organically bound tritium. Health Phys 1993;65:698-712.  Back to cited text no. 5
Belot Y. Tritium in plants: A review. Radiat Prot Dosimetry 1986;16:101-5.  Back to cited text no. 6
Quantification of Radionuclide Transfer in Terrestrial and Freshwater Environments for Radiological Assessments, IAEA TECDOC-1616; 2009.  Back to cited text no. 7
Keum DK, Lee HS, Kang HS, Jun I, Choi YH, Lee CW. Prediction of tritium level in agricultural plants after short term exposure to HTO vapor and its comparison with experimental results. Health Phys 2006;90:42-55.  Back to cited text no. 8
Diabate S, Strack S. Organically bound tritium in wheat after short-term exposure to atmospheric tritium under laboratory conditions. J Environ Radioact 1997;36:157-75.  Back to cited text no. 9
Galeriu D, Melintescu A, Strack S, Atarashi-Andoh M, Kim SB. An overview of organically bound tritium experiments in plants following a short atmospheric HTO exposure. J Environ Radioact 2013;118:40-56.  Back to cited text no. 10
Boyer C, Vichot L, Fromm M, Losset Y, Tatin-Froux F, Guetat P, and Badot PM, Tritium in plants: A review of current knowledge. Environ Exp Bot 2009;67:34-51.  Back to cited text no. 11
Galeriu D, Melintescu A, Diabate S, Strack S, Mariko AA, Kim SB. Overview on Tritium Transfer from Air to Plants and Conversion to OBT with Focus on Night Cases. IAEA TECDOC EMRAS II, WG7; 2014.  Back to cited text no. 12
Moses V, Calvin M. Photosynthesis studies with tritiated water. Biochim Biophys Acta 1959;33:297-312.  Back to cited text no. 13
Thomson RG, Nelson CD. Photosynthetic assimilation and translocation of 3H and 14C–organic compounds after 3HHO and 14CO2 were simultaneously offered to a primary leaf of soybean. Can J Bot 1971;49:757-66.  Back to cited text no. 14


  [Table 1], [Table 2]

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