2.1 Plants culture

The soybean seeds were sown in plastic pots（30 cm×30 cm）placed outdoors. The pots were filled with a medium loam soil. A few days before sowing, 5 g of composite fertilizer (N: P₂O₅:K₂O=14:17:14) were supplied to each pot and fully mixed with the soil.

A total of 12 pots were prepared for exposure to HTO, the soybean seeds were randomly sown into each pot, and the final number of seedlings was 3.The pots were watered one or two times per week to ensure a wet soil for plant growth. Appropriate amounts of the composite fertilizer were supplied twice during plant growth.

2.2 Exposure to HTO vapor

Potted soybean plants were exposed to evaporated HTO in a transparent plexiglas box (1.2m (length) ×1 m (width) ×1.2m (height)) at two different growth stages (the flowering and podding stage, and the filling stages) at night and the following day. The exposure box was composed of a bottom plate and an upper part. An electric furnace, a fan, and a hygrothermograph were placed on the inside, base of the box. There was a lid on the top of the upper part.

For each exposure, three pots with plants were mounted on the bottom plate and then the upper part was fitted onto the plate. A glass beaker containing 20 mL of HTO solution (7.65×10³ Bq mL^-1 in distilled water) was placed into the electric furnace. The exposure experiment was carried out for 1 h exposure in daytime and nighttime (from 9:00 to 10:00 for daytime and from 21:00 to 22:00 for nighttime). All the openings were sealed with silicon rubber. After heating for about 20 min, almost the entire volume of the HTO solution was evaporated. The surface soil of the pots was covered by vinyl paper during the exposure in order to prevent tritium deposition into the soil.

Temperature and relative humidity were recorded every 5 min. The fan was operated at the beginning of the heating to prevent the build-up of an HTO concentration gradient inside box. The lid was opened and fresh air was blown into the box for 15 min immediately after the end of the exposure duration and directly followed by the removal of the upper part of the box. Following the exposure, the plants were removed from the box and cultivated outdoors as usual.

2.3 Sampling and measurement

The air moisture in the exposure box was collected with dry silica gel. The soybean plants exposed were sampled twice at the end of each exposure and at harvest. At each sampling, three exposed plants were randomly selected from three pots. The samples were immediately divided into leaves and seeds, which were then cut into small pieces. The three samples were pooled together to form a compound sample put into zip-lock bags before storage in a freezer at -20°C.

The air moisture contained in the silica gel can be obtained through a distillation system, and the amount of water was collected and recorded. The tissue free water was extracted by a freeze-drying method and trapped using liquid nitrogen for the frozen samples. The plant samples were oven-dried at 60°C for 2-3 days after being freeze-dried. The dry samples were pulverized with mortar and pestle, and the powdered samples were then combusted using a combustion apparatus. Then the water generated from the combustion was collected in 20 ml polyethylene vials. The combustion apparatus consisted of a furnace composed of two heating regions and a quartz tube. The sample was held in a quartz boat enclosed in the quartz tube in the first heating region which was movable. The second heating region was fixed and the quartz tube inside the second heating region contains a catalyst. The temperatures of both regions were independent and adjustable. The samples pyrolysis combustion performed in the first region and the combustion gases were oxidized by the catalyst in the second one. The water vapor was trapped in a U-tube immersed in a cold trap. Finally, the OBT concentration could be determined by measuring the tritium concentration in the water generated from the combustion.

All prepared water samples were mixed with the scintillation cocktail (Ultima Gold LLT) and tritium concentration in the water samples were measured by liquid scintillation counter (TRI-CARB 3170TR/SL). The water samples were mixed in the proportion of 5 mL of water and 15 mL of cocktail for TFWT measurement and 1mL of water and 19 mL of cocktail for OBT measurement. The detection limit was 1.2 Bq L^-1 with a background of 2~3 cpm and a count efficiency of about 33%. 20 ml glass vials (low potassium borosilicate glass) were used in the experiments for tritium measure.

3.1 Conditions in the exposure box during exposures

The meteorological conditions in the exposure box during each exposure are shown in Table 1.

As shown in Table 1, at the end of the exposure, the temperature rose by about 4-5°C in the nighttime, and was about 6°C higher than at the beginning in the daytime. The temperatures ranged from 30.7°C to 35.6°C in the nighttime and from 31.6°C to 39.1°C in the daytime. The increase in temperature of the box resulted mainly from the heat generated by the electric furnace.

The changes of relative humidity were in the range of about 8%～17% in the night and about 8%～11% in the day. The mean relative humidity of air ranged from 51.4% to 54.4% during the night and from 56.7% to 58.9% during the day. At the beginning of the exposure, the relative humidity increased and then gradually decreased about 30 minute later.

Table 2 shows the mean HTO concentrations in air moisture inside the exposure box during each exposure. There were some differences in the mean HTO concentration in the air moisture in the exposure box which mainly depended on the HTO evaporation rate, the content of moisture in the air, the degree of air moisture condensation on the inner walls, and the amount of the HTO uptake by plant and the absolute humidity in the box.

3.2 HTO uptake by the soybean leaf

The tissue free water tritium (TFWT) concentrations in the soybean leaves were measured and the results are given in Table 2.

The leaf TFWT concentration at equilibrium and steady condition can be described by the following equation (1) on the assumption that the soil HTO concentration is zero [12,13]:

where C_TFWT, the TFWT concentration in the plant leaf, is at equilibrium (Bq/L); A is the ratio of the specific activity of tritium (T/H) in the water to that in the vapor at equilibrium (1.1); R_CH is the relative humidity; C_air is the the HTO concentration in the air moisture (Bq/L). In order to calculate C_TFWT, an approximation was made by using the mean values for R_CH in the equation (1).

The percentages of the leaf TFWT concentrations at the end of exposure to the estimated equilibrium concentrations were 50% for nighttime conditions, and 80% for daytime ones at two different growth stages. The TFWT concentrations at the end of the exposure were considerably lower than the estimated equilibrium concentrations in the nighttime condition.

These results indicate that it usually takes longer than 1h for the TFWT concentrations in the soybean leaves to reach quasi-equilibrium with the HTO concentration in the ambient air moisture. This phenomenon is in agreement with the experimental results for wheat, Chinese cabbage and radish. Wheat plants were exposed to HTO vapor for 2 h in two different light conditions of about 120 and 550Wm^-2 and a relative humidity in the range of 85–90% during the early to middle phase of the rapid seed growth stage. The results showed that the leaf TFWT concentration reached 75–86% of the HTO concentration in air moisture at the end of exposure [11]. The potted Chinese cabbage and radish plants were exposed to HTO vapor under semi-outdoor conditions at daytime for 1 h in different meteorological conditions with mean temperatures in the range of 15.2～34.5°C, a mean light intensity in the range of 22.9～60.5klx, and a mean relative humidity in the range of 67.9%～89.0%. The results indicated that the percentages of the leaf TFWT concentration at the end of exposure to the estimated equilibrium concentration were in the range of 9.4～68.0% and 28.9～57.7% for Chinese cabbage and radish, respectively [13].

Experimental reports on grape leaves, potato, and rice leaves showed that the time of reach quasi-equilibrium were shorter than 1h. The TFWT concentration in the leaves reached a theoretical plateau level after about 30 min when the grape plants were exposed to HTO vapor under a controlled outdoors conditions during the daytime in June. A field experiment concerning potato and grape plants exposed to HTO vapor in a clear chamber for 4 h during the daytime resulted in the TFWT concentrations in the leaves of both plants reaching an apparent plateau 45 min after the start of the exposure. Potted rice plants were exposed to atmospheric HTO in a box outdoors for 1 h at 9 different times from booting to yellow-ripe stages. Inside the box during each exposure, the means of relative humidity, temperatures and solar radiations were in the range of 61.4～94.2%, 25.1～34.8°C and 234～678Wm^-2, respectively. It is indicated that the leaf TFWT concentration may reach equilibrium within 1 h at clear weather [12].

The discrepancy between these experimental results are mainly due to the difference of physiological characteristics of the leaves, the exposure times, the meteorological conditions during the exposure, and the leaves age during the exposure.

The experimental results show that the absorption of atmospheric HTO by soybean leaves is much slower at night than during the day. This is in agreement with the experimental results obtained by others [1, 11]. It is well known that plant uptake of HTO vapor is closely related to the stomatal movement. The stomata open when stimulated by sunlight in the day, and are more or less closed at night. Thus the plant leaf can easily absorb HTO vapor from the atmosphere under daylight, but uptake is slower in the dark for various plants. The closure of the stomata partly prevents HTO vapor uptake by soybean leaves.

3.3 TFWT concentration in the soybean plant at the end of the exposure in the nighttime condition and in the daytime conditions

The TFWT and OBT concentration in the leaf and beans of soybean at the end of exposure are summarized in Table 3.

To compare the difference of HTO transfer from air to leaves and from leaves to bean between nighttime and daytime, the TFWT concentrations in soybean leaves and beans at the end of the exposure were normalized to the relative values defined as the ratio betweenTFWT at the end of the exposure and the average HTO concentrations of the air moisture during the exposure as referenced by others [13].

Figure 1 (a) and (b) show the relative TFWT concentrations in the soybean leaves and beans at the end of the exposure to HTO vapor in nighttime and daytime at the flowering and podding stage and at the filling stage.

Fig. 1Full-screen View

Relative TFWT in soybean plants at the end of the exposure to HTO vapor under nighttime and daytime at the flowering and podding stage (a) and filling stage (b) .

At the flowering and podding stages of the experiment, the relative HTO concentration in leaves and young pods during nighttime conditions were about 0.31 and 0.09, but the relative HTO concentration during daytime conditions were 0.48 in the leaves and 0.06 in the young pod. The relative TFWT concentration at the end of the exposure in soybean leaves under the nighttime conditions was 63.66% of the daytime conditions. Whereas the relative TFWT concentration in young beans during HTO vapor released at the nighttime was 1.37 times higher than that during the daytime exposure at the end of the exposure. The discrepancy between leaves and beans is probably due to physiological differences. The high relative TFWT concentration observed for young beans during the night at the flowering and podding stage can be attributed to the fact that the soybean were in the early stages of its development. The green bean could have also absorbed the HTO vapor, and the loss of TFWT via moisture exchange and transpiration might have taken place at a much lower rate in the nighttime than during daytime.

The filling stage is a crucial time for the determination of the future soybean yield. As shown in Fg.1 (b), in the night condition, a lower HTO vapor uptake and conversion to TFWT in soybean leaves and beans was observed compared to the day condition. The relative TFWT concentration under nighttime conditions are 0.28 in leaves, 0.14 in beans, 0.53 in leaves, and 0.23 in beans under daytime conditions. The relative TFWT concentration in leaves in dark conditions represented 52.3% of that under light conditions, and was of 57.8% in beans. The percent ratio of the relative TFWT concentration in leaves between night and day at the end of exposure was not as large as that for the beans. This is likely because at the filling stage the soybean pods gradually become drier and the ability to uptake HTO from the air is reduced. Therefore the HTO was mainly absorbed by leaves during the night and then transfered to the pods.Simultaneously, the water content was decreased and dry matter was increased in the beans.

The results indicate that during nighttime conditions, the ability of foliage to uptake HTO from the ambient environment during the flowering and podding stage was greater than that at the filling stage. This may be attributed to the leaves’ strength during the early-middle stage of growth. In this study, the amount of HTO uptake in the leaves during the nighttime conditions is higher than that reported in experiments carried out with D₂O releases in soybean and in experimental data obtained by wheat [1, 11, 19]. This phenomenon can be explained by the difference in the growth stage exposed, the environmental conditions, and the crop characteristics and so on.

3.4 OBT concentration in soybean plants at the end of the exposure during the nighttime and daytime conditions

In order to compare the difference of OBT formation in soybean leaves and beans at the end of the exposure for the nighttime conditions and daytime conditions, the OBT concentrations at the end of the exposure in soybean leaves and seeds were normalized on order to obtain relative values. These values were calculated by relating the OBT concentrations in plant in each of the plant’s organs to the leaves TFWT concentrations at the end of the exposure. This is based on the knowledge that the plant’s OBT is mainly produced in leaves and then translocated to other organs.

The relative OBT concentrations in leaves and beans of the soybean plant after its exposure to atmospheric HTO at the flowering and podding stage, and at the filling stage are given in Fig. 2(a) and (b).

Fig. 2Full-screen View

Relative OBT in soybean plants at the end of the exposure to HTO vapor under nighttime and daytime at the flowering and podding stage (a) and at the filling stage (b).

During the flowering and podding stage, the relative OBT concentration at the end of the exposure were 0.096 at nighttime and 0.18 at daytime in the leaves, whereas in beans the obtained values were 0.075 and 0.1, respectively. At the filling stages, the relative OBT concentrations under night conditions were 0.087 in leaves and 0.063 in beans at the end of the exposure; under day conditions the values were 0.21 and 0.13, respectively.

At the end of exposure, the relative OBT concentration levels in soybean leaves and beans are higher than those in rice leaves (0.7-1.2), and wheat leaves (0.34%-1.25%) during the day. However, these values are comparable to 5–25% in various crop plants from the experiments conducted by Guenot, Belot and by Indeka [12]. Under night conditions, the relative OBT concentrations observed in leaves and beans are higher than that observed in white leaves and grains. This discrepancy may be attributed to the difference of the time of exposure, the development stage, and the plant species.

As shown in Fig.2, whether exposure experiments are carried out at the flowering and podding stage or at the filling stage, the amount of OBT formation in soybean leaves and beans in the nighttime conditions was less than that in the daytime conditions. During the flowering and podding stage, the relative OBT concentration in leaves at the end of the exposure during the night represented1/2 of thedaytime’s value compared to the value in beans which represented 7/10 of the daytime values.During the filling stage, the corresponding values were 2/5 and 1/2, respectively. These values are slightly higher than those found in the experiments with wheat and rice [1, 11, 19].

Table 4 gives the ratios of OBT concentrations to TFWT concentrations in the soybean plants at the end of exposure.

The TFWT concentration were one to eleven times higher than the OBT concentration depending on the time of experiments and the plant part. The OBT-to-TFWT ratio in the leaf and bean for the daytime conditions was about 2～3 times higher than that for the nighttime conditions. In case of soybean plants acute exposure to HTO vapor, if the beans were immediately ingested at the end of exposure, most of the ingestion dose would be generally given by TFWT because of the low OBT-to-TFWT ratios in spite of the high dry matters contents in the beans. The values obtained from our experiment are much higher than the published ratios in the leaves of the mature cabbage plants exposed to HTO vapor for 1 h and were considerably different from the result following long-term exposure [21].