Accumulation of Tritium in Aquatic Organisms Through a Food Chain

Accumulation of Tritium in Aquatic Organisms through

a Food Chain with Three Trophic Levels

KENSHI KOMATSU1), MASATAKA HIGUCHI2) AND MASATOSHI SAKKA1)

1) Department of Radiation Research, TohokuLaboratory University for School Radioisotopic of Medicine2)Research, Tohoku University School of Medicine, Seiryo-cho,Sendai 980, Japan (Received November25, 1980)

Tritium Accumulation/Food Chain/Aquatic Organism

Accumulation of tritium in aquatic organisms was estimated through a model food chain

such as;

tritiated water (THO) •¨ diatoms •¨ brine shrimps •¨ Japanese killifish.

Tritium accumulations in each organism as organic bound form are expressed as the R value which is defined as the ratio of tritium specific activity in lyophilized organisms (ƒÊCi/gH) to that in water (ƒÊCi/gH). The maximum R values were 0.5 in diatoms, Chaetoceros gracilis, 0.5

in brine shrimps, Artamia salina, and 0.32 in Japanese killifish, Oryzias latipes under the growing condition where tritium accumulation took place from tritiated water without tritiated diets. Brine shrimps and Japanese killifish, which grew from larvae to adult in tritiated sea water with

feeding on tritiated diets (model food chain), had the R value at 0.70 and 0.67 respectively,

indicating that more tritium accumulation in consumer populations with tritiated diets than those without tritiated diets. In addition, the R values of each organ of Japanese killifish, of DNA and the nucleotides purified rfom brine shrimps growing under the condition with or without our

model food chain were measured to estimate the tritium distribution in the body or various components of the organism. These results did not indicate the seeking characteristic of tritium to some specific organs of compounds.

INTRODUCTION

An expanding use of the nuclear power in recent years has resulted in a large

quantity of tritium release to the environment. Moreover, running of the nuclear fuel

reprocessing plant and development of the thermonuclear fusion reactor, in future, may

cause a large-scale release of tritium''. Consequently man will be increasingly exposed

to the environmental tritium released from nuclear power industries. The release of

tritum from those facities isexpected to deposit in aquatic ecosystem as tritiated water

and it will be accumulated in man predominantly through food chains, such as; tritiated

water , phytoplankton -p zooplankton edible animals (fish etc.) man.

1) 小松賢志,粟冠正利:東北大学医学部放射線基礎医学教室,仙台市星陵町2-1,〒980 2)樋口昌孝:東北大学医学部放射性同位元素中央実験室,仙台市星陵町2-1,〒980 There have been many studies on RBE of tritium'-'-, late effects of tritium"" or tritium turnover rate"-16' in organisms. Accumulation of tritium from tritiated water have been reviewed on various organisms"' and it has been shown that specific activ ity of tritium for each organism was less than that of tritiated water with only a few exception"'. Nevertheless there have been only a few reports pertinent to tritium transfer through aquatic food chains"-"). These reports suggest that a food chain plays an important part in tritium behavior in ecosystem. In fact, brine shrimps accumulate more tritium when fed on tritiated diet as described previously"). An experimental model ecosystem through food chain was designed in this investigation. The model consists of diatoms, brine shrimps and Japanese killifish, where brine shrimps were fed on diatoms that had been cultured in the medium containing tritiated water and the Japanese killifish were fed on thus labeled brine shrimps. The estimated R values (the ratio of specific activity of bound form tritium to that of tritiated water in the medium) under various conditions were compared to see whether the R value varied with feeding on tritiated diets in our model ecosystem.

MATERIALS AND METHODS

Organisms and Growing Conditions Diatoms, Chaetoceros gracilis (originally obtained from Laboratory of Oceanography, Tohoku University) were grown in glass flasks containing 2 liter of autoclaved medium at 28±2°C. The medium was a modified Allen-Nelson's one consisting of 0.4 g of KNO3i 0.05 g of Na2HPO4 ; 12H2O, 0.08 g of MgSO4 ; 7H2O, 0.026 g of CaC12 and 5 mg each of FeCl3 and Na2EDTA in 1 liter of filter-sterile natural sea water. The culture was bubbled with 5% CO2-95% air and always illuminated by long-wave fluorescent light. Brine shrimps, Artemia salina, dry eggs of brine shrinps (purchased from Cosmo Enterprize Co. Ltd., U.S.A.) were hatched by immersing in sea water for 3 days with vigorous air-bubbling and the nauplii were separated from the egg shells by phototaxis. New born brine shrimps were transfered into 3 liter of sterilized sea water and immediately fed on diatoms under a constant fluorescent illumination and with gentle air-bubbling. Japanese killifish, Oryzias latipes were kindly supplied from Zoological Institute, University of Tokyo and adapted to sea water. They were grown in 3 liter of sea water under an illumination of fluorescent light 14 hours/day and with gentle air bubbling. They were fed on brine shrimps only.

Labeling Conditions Without food chains; Diatoms in early stationary phase were collected and then inoculated (either 0.004 mg dry weight/ml or 0.92 mg dry weight/ml) in fresh medium containing 7.6 uCi/ml of tritiated water, THO (from New England Nuclear Corp.,). They were grown for 11 days for 0.004 mg of inoculum (rapid growth ; cell doubling time of 0.25 days) and 4 days for 0.92 mg of inoculum (slow growth; cell doubling time of 8.9 days). New born brine shrimps were grown for 3 days after their hatching in non-tritiated sea water and then for 21 days in 3 liter of sea water containing 2.9,uCi/ml of tritiated sea water with feeding on non-tritiated diatoms ad libitum. Japanese killifish when they are about 2 weeks old were grown in sea water containing 0.89 pCi/ml of tritiated water for 38 days with feeding on non-tritiated brine shrimps once every day. With food chains; Brine shrimps were grown in tritiated sea water and fed on tritiated diatoms. Diatoms were tritiated by growing in the presence of tritiatd water described above and were used when the specific activity reached a maximum value. Japanese killifish were also grown in tritiated water and fed on tritiated brine shrimps. Brine shrimps were used as a diet to killifish when the specific activity reached a maximum value during their growth in tritiated sea water with feeding on tritiated diatoms. DNA labeling of brine shrimps; Brine shrimps were grown in sea water containing 10.4 pCi/ml of tritiated water for 6 days immediately after hatching either with or without tritiated diatoms. For labeling of DNA nucleotides, they were in the presence of 60.8 pCi/ml of tritiated water for 2 days of larval period with tritiated diatoms as a diet.

Isolation of DNA and Nucleotides DNA of brine shrimps was isolated according to the method described by Ueno23' with a slight modification. Namely, 200 mg of freeze-dried samples were homogenized in 3 ml of solution containing 6% sodium 4-aminosalicylate, 1% sodium lauryl sulfate and 1%' sodium chloride. 3 ml of phenol-cresol mixture (phenol 500 ml, m-cresol 700 ml, water 55 ml and 8-hydroxyquinoline 0.5g) was added to the homogenate and the mixture was stirred for 20°C. The mixture was centrifuged at 6,000g for 20 min. This treatment was repeated with sodium acetate and the phenol-cresol mixture. The upper aqueous phase was carefully removed and added with sodium acetate and three volumes of cold ethanol. The precipitates of the nucleic acids were collected by a glass rod and were washed with 70%, 80% and 90% ethanol. The resulting DNA contami nated with RNA was treated with ribonuclease (20 pg/ml, treated for few min. at 100'C) in 0.05M Tris-HCl buffer, pH 7.4, for 60 min at 30'C. The DNA was finally purified by CsCl buoyant density gradient centrifugation at 38,000 rpm. The DNA content was measured by the spectrophotometer at the wavelength of 260 Pm. For measurement of tritium incorporated into the nucleotides, DNA of brine shrimps was purified as described above. An aliquot of DNA solution was counted for deter mining the specific activity. DNA of residual solution was hydrolyzed with DNAse 1 (200 pg/ml, Miles Co.,) in 0.05 M Tris-HCl solution, pH 7.0 at 37°C for 2 hours and then with Phosphodiesterase (200 pg/ml, Miles Co.,) in the above buffer solution, pH 9.0 at 37'C for 2 hours. The hydrolysate residue was chromatographed with a 20 x 1 cm Dowex-1 formate column and eluted by gradient elution of 1.0 M at pH 4.2 according to the method by Canellakis24'. Four nucleotides fractions eluted were separately collected and were absorbed at active charcoal of 50 mg for 30 min. The charcoal was combusted in a sample oxidizer (Intertechnique Coop.,) to measure the specific activity. Adequate resolution and purity of the four bases were shown by the matching of the mole fractions of the watson-Crick base pairs.

Measurements of Radioactivity and the Expression In this experiment, only tritium incorporation in organic bound form was measured. Namely, the samples were freeze-dried at 10-3 Torr for 3 days and catalytically com busted in a sample oxidizer. The tritium activity in the water was measured by liquid scintillation counter (Intertechnique Coop.,). When the lyophilization was continued for more than 3 days, the residual tritium activity was unchanged. The results were expressed as the ratio of the tritium specific activity in freeze dried tissues of each organism to that of water in which they were grown, i. e.,

R value= tritiumt specific activity of organic materials (,uCi/gH) ritium specific activity of medium or sea water (,uCi/gH)

The R value was also calculated from the growth rate, provided that ; (1) all newly formed organic materials incorporate tritium and protium in the same ratio as that present in medium or sea water, (2) tritium does not replace hydrogen in the organic compounds which were already in the inoculum.

Rcal=1 MO (1) mt

where in, was the dried cell weight or the dried body weight at time t after inocula tion and mo is dry weight at t=0.

RESULTS

Tritium Accumulation in Diatoms The results obtained on diatoms under rapid growth conditions (inoculum of 0.004 mg dry weight/ml) are shown in Figure 1-B. Tritium accumulation in diatoms as organic bound form increased with incubation time and saturated at 4 days, where the R value was about 0.5. Compared with the R calculated from the dry weight, the experimental R value increased in parallel with the calculated one but always 0.5 times of the calculated value. An experiment on tritium accumlation in diatoms under the slow growing conditions (inoculum of 0.92 mg/ml) was also performed. The tritium accumulation in this case was shown in Figure 1-A, in which the experimental R increased with the calculated R and gradually became 0.5 times of the calculated one. Figure 1-A also shows that the experimental R was almost same with the calculated R at an initial growth stage,

; R value calculated from the growth curve, (Rcal). -0-0-; experimental R value , (Rexp) -O-O ; R value due to replacement which calculated from the experimental R value and growth curve, (Rrep) .

Fig. 1. Tritium Accumulation in Diatoms, Chaetoceoos gracilis. Diatoms were cultured in the modified Allen-Nelson'smedium containing 7.61iCi/ml of tritiated water at the following inoculum size. Figure 1-A (upper figure) ; at 0.004 mg of inoculum (rapid growth). Figure 1-B (lower figure) ; at 0.92 mg of inoculum (slow growth). -40-0-; growth in dried cell weight per volume of the medium . which may be due to rapid replacement of protium in the organic compounds of inoculum by tritium. To estimate these contributions, the following assumptions were made for convenience. The amount of tritium incorporated into organic compounds at time t is a product of the tritium specific activity of hydrogen in organic compounds of the organism (SA0), amount of the hydrogen per unit weight (H) and dry weight of the organism (in,); SAt •H• mt. This can be considered to consist of the sum of the amount incorporated by tritium-protium exchange reaction into organic compounds which had been existed in inoculum (SA1ep• H• mo) and the amount incorporated into newly synthesized compounds (SA0Yn•H•(mt-mo)). Namely,

SAt •H• mt=SArep •H• mo+SA0Yn • H• (rnt-mo) . (2)

Equation (2) was divided by SAenv • H• nit, where SAenv is a specific activity of tritium in the environment. Then it becomes,

1?2a mt-mo Rt=Rfep nit +RsYn• m (3) t Where RreP and Rsyn represent the R value due to tritium incorporation into organic compounds in inculum and into newly synthesized compounds, respectively. When the incubation time is sufficiently long (t is infinite), m,)/m, tends to be zero. This makes it possible to determine Rryn as Rmax• As shown in Figure 1-B, the maximum R value becomes 0.5. Substituting Rmax of 0.5, mo/mt and Rt from Figure 1-A to equation (3), Rrep can be calculated. The result showed that the R fep rapidly increased up to 0.02 at 0.5 day and showed little change thereafter.

Tritium Accumulation in Brine Shrimps Tritium acumulation in brine shrimps from tritiated water was tested in two ways in separated experiments. Namely, brine shrimps were reared in sea water containing 2.9 iCi/ml and fed on either non-labeled diatoms (Figure 2-A) or tritiated diatoms (Figure 2-B). The experimental R value with non-labeled diatoms increased and satu rated at 0.5 on 21 days, while dry weight of a brine shrimp increased 50 times during the experiment. On the other hand, the R value through a food chain with tritiated diatoms saturated at 0.7.

Tritium accumulation in Japanese Killifish Tritium accumulation in Japanese killifish was also measured in two ways. The first was tritium incorporation into Japanese killifish through tritiated sea water alone with feeding on non-labeled brine shrimps. The second was tritium incorporation into Japanese killifish not only tritiated sea water but also labeled brine shrimps grown in a model food chain. In the experiment with non-labeled brine shrimps (without food chain), the R value of whole body was 0.32 at 38 days as compared to 0.97 of the calculated one. The dried body weight of a fish increased 32 times of that at the Fig. 2. Tritium Accumulation in Brine Shrimps, Artemia salina. Brine shrimps were reared in 2.9 ,pCi/ml of tritiated sea water under the following feeding condition. Figure 2-A (upper figure) ; Brine shrimps fed on diatoms which had been cultured in tritium free medium. Figure 2-B (lower figure) ; Brine shrimps were reared in the above medium, but fed on diatoms which had been cultured in tritiated water medium, (mcdel food chain). -A-A-; growth in lyophilized body weight of brine shrimps . ; R value calculated from the growth curve, (Rcal). -0-0-; experimental R value , (Rexp). beginning during the experiment. In the experiment with tritiated brine shrimps (with food chain), the experimental R value of whole body was 0.67 at 38 days. The corre sponding R value calculated from the dried body weight change (21.2 times) was 0.95. Then Rmax was 0.71 (from 0.67/0.95). It is apparent from these findings that the R value of Japanese killifish was higher with food chain model than that without food chain. Table 1 also shows R values for various organs; gills, viscera and muscle of Japanese killifish with or without the food chain. The R value of each organ was similar to that of whole body and no specificity of tritium accumulation in each organ was detected.

Table 1 Accumulation of Tritium in Various Organs and Whole Body of Japanese Killifish, Orizyas latipes (R value).

Japanese killifish (2 weeks old) were grown in 0.89pCi/ml of tritiated water for 38 days under the condition with or without model food chain as described in text.

Tritium Incorporation into DNA and the Nucleotides of Brine Shrimps Table 2 shows the specific activity of tritium incorporated into DNA purified from brine shrimps reared in tritiated sea water during 6 days under two conditions; that is, either with feeding on non-labeled diatoms or labeled diatoms. Assuming that all tritium in the DNA fraction were covalently bound to carbon atoms and that there are 0.0303 moles of carbon-bound hydrogens per gram of DNA, the R values were also calculated according to method used in the case of diatoms. The results repre sented slightly higher R values of DNA than that of whole body in both case with and without food chain but the difference was not significant. Analysis of tritium in DNA nucleotides of brine of shrimps fed on labeled diatoms for 2 day, after hydrolysis of DNA and isolation by column chromatography are also listed in Table 2. The R values of nucleotides were also calculated as described above. Table 2 shows that all four bases are labeled but the R values are always lower, except 5-TMP, than that of purified DNA before hydrolysis.

Tritium Accumulation through a Model of Extended Food Chain To determine the relationship between tritium accumulation and number of steps in trophic level, a simplified compartment model system was assumed. Assuming that the rate of tritium uptake in an organsm is proportional to the concentration N f (tCi/gH) in foods, and that the rate of excretion of the incorporated tritium is f

Table 2 Tritium Incorporation into DNA and the Nucleotides of Brine Shrimps, Artemia salina.

Brine shrimps were grown in 10.41iCi/ml of tritiated water for 6 days immediately after hatching with or without tritiated foods (diatoms grown in tritiated water) to examine tritium incorporation into DNA. To examine tritium incorporation into DNA nucleotides, brine shrimps were kept in 60.8,uCi/ml for 2 days of larval period with tritiated foods. Each R value was calculated from the content of hydrogen covalently bound to carbon of DNA (as hydrogen content of 3.03%) and the nucleotides.

proportinal to the concentration N,, (pCi/gH) in the organism. The uptake coefficient and the excretion coefficient are expressed as k,,-,,. [day-1] (from n-1 to n compartment) and k, o [day-1] (from n to 0 compartment), respectively. dN,,/d t= k._,, .. Nf kn, o• Nn.. Since tissue-free-water tritium will rapidly equilibrate to the level of the environment25,2s,, 0 compartment is considered as the environment. The R value of the organism through n steps of a food chain with the same uptake and excretion coefficient will be expressed as, Rn=(kn-l.fllkk,o)n=R". This is, however, an oversimplified model, since tritiums is incorporated not only from tritiated food stuff but also from environmental tritiated water and there is an incorporation process of replacement of protium by tritium (rapid compartment). Further the R value of producer organisms is not 1 as in diatoms in the present experiment. Therefore, we assumed a model as shown in Figure 3. S compartment represents a slow compartment which incorporates tritium from tritiated food stuff and from environmental tritiated water. These uptake coefficients were expressed as ko,,,,, respectively. R compartment represents a rapid compartment which incorporates tritium from environmental tritiated water and the uptake coefficient was expressed as k(r)o,. The R value through n steps of trophic level with the same size of each compartment and the same uptake and excretion coefficient will be

~-1 R,=2-+s(Kw11--KK +Kfn-1R1) (4) Fig. 3. A Compartment Model of a Food Chain with n Successive Trophic Levels n=0 ; environmental sea water. n=1 ; producer organisms (for example, diatoms). n? 2 ; consumer organisms (for example, brine shrimps, Japanese killifish). r and s ; rapid and slow turn-over compartment of tritium. k,, and k ; coefficient of tritium transfer into or from r and s compartment, respec tively. that is, kn,0 excretion coefficient from organisms to sea water. ko, n ; uptake coefficient from sea water to organisms. kn-1 .n ; uptake coefficient from prey organisms to predator organisms.

where K,,;=ko, n/kn, o, Kf=kn_1 , n/kn, o, R1=0.5 (R value of diatoms) r and s represent fraction of hydrogen content in r and s compartment, respectively and r+s=1. In the equation, first term represents R value due to r compartment second term represents the summation of R value from environmental tritiated water in s compartment and R value from tritiated food stuff in s compartment. In the case of incorporation from only titiated food stuff, the equation (4) becomes a simple form, R fn=s(K f)n-1R1 . (5)

Parameters of s and r can be determined from the kinetics of tritium relese (unpub lished). The r values were 0.183 for brine shrimps and 0.035 for Japanese killifish. The r compartment consists of easily exchangeable tritium and probably tritium bound to bound water, 0-H and N-H groups. The R value of r compartment can be consid ered as 1 which is decribed in discussion. Kf and K,,, for brine shrimps and Japanese killifish were calculated from Rmax with or without food chain model. Number of the trophic levels in the natural environment can be 5 at the maximum. Therefore R5 value was calculated by equations (4) and (5). The results were shown in Table 3.

Table 3 Estimation of tritium accumulation through model extended food chain (R value).

Based on the maximum R values observed in brine shrimps and Japanese killifish, the R values of each trophic level in a food chain were calculated using a compartment model shown in Figure 3. The fourth consumer, R5, is the highest level usually seen in natural food chains. R f2 or R f3 represent the R value of first or second consumer organisms which were accumulated only through tritiated foods. a) R value measured in this experiment, b) R value calculated from equation (4) or (5).

Radiation Doses to Organisms Exposed to Tritiated Water with Food Chain We calculated the dose rate of organisms exposed to tritium through food chain. Assuming the R value of dry tissues in organ of reference is 0.71 as described in our experiment using fish, dose can be estimated by means of the following well known formula"); Dose Rate= - of2~ [rem/week] 2.8x 10-332 where q=body burden of tritium [,uCi] f2=the fraction of the body burden in organ of reference s=effective absorbed energy per disintegration of a radionuclide [MeV/disintegra tion] m=mass of the organ of reference [g]

The dose rate when only exposed to tissue water contaminated tritiated water was compared with that exposed to total tritium including tissue bound tritium. The result indicated that the increase of dose rate by tissue bound tritium depended on water content of the organ of reference and the values were 12% for testis (water content is 80%) and 69% for bone and fat (water content is 20%).

DISCUSSION Tritiation of Organisms from Tritiated Water Alone It is very difficult to give a comparison of our result of tritium accumulation in organisms as dry tissue form with that by other researchers, because of following two reasons. One is caused by difference in sample preparation, for example, promotion of tritiation by heating2S', loss of exchangeable tritium by washing29' and change of bound water content by degree of drying. Another is caused by various growing state of organisms during the period in tritiated water. In this experiment, we adopted freeze dry technique for gentle preparation of the sample and determined the R value together with Real which is calculated from growth curve of organisms. Our results were close to maximum R value reported by other researchers who made their experiments of tritium accumulation using chlorella29', fish 20' and rodents"' under similar sample preparation technique and growing condition as that in our experiment. On the other hand, Bruner171 reviewed on the R values in various orga nisms which varied from 0.2 to 1.0 under the various sample preparations and growing conditions. Untill now, fair agreement is obtained by various researchers for the R value less than 1, showing no biological concentration of tritium. We suppose that, in addition to dilution effect of protium by inhalation and unlabeled foods, mass isotope effect on molecular reaction contributes to lowering R value. For example, for reaction RH= H2O, equilibrium R value for N-H, O-H bond ranges from 0.9 to 1.0 and, for C-H bound, from 0.64 to 0.8531)32) As tritiated water is a reactant, in general, the R value of products in chemical reaction eventualy becomes less than 1. Therefore, discrimina tion by mass isotope effect of tritium will result in lower R value than 1 in organisms. In fact, it has been reported that R value for non-exchangeable C-H bond in many amino acids and sucrose of chlorella is about 0.633)

Content of Exchangeable Tritium When organisms are exposed to tritiated water, two ways of incorporation into their organic compounds are assumed. One is the so-called T-H exchange reaction and another is the incorporation mediated through biochemical synthetic processes. The direct experimental estimation of a fraction of tritium incorporation, not elimination, into organic materials of growing organisms by exchange reactions is difficult at present time. However, it is possible to calculate the contribution of exchange reaction to the R value as described in the experiments with diatoms in slow growing and in rapidly growing condition. The result showed that tritium was incorporated with Rrep=0.02 for the first 0.5 day (Figure 1-A) and thereafter the value was almost constant. This value was similar to those obtained by others. Inomata34' reported that the R value was 0.05, when bacterial metabolism was inhibited by NaCN and HgC12. On the other hand, Weinberger29> also reported a similar value which was estimated from the amount of tritim removed after cell washing by tritim free water. These results indicate that the fraction of tritium incorporation by exchange reactions appears small in proliferating cells in contrast to that anticipated from high content of hydrogen bound to oxygen and nitrogen.

Uniformity of Tritiation in Organisms It has been observed by means of autoradiograms of the brine shrimps") and the tritium incorpotation into various organs of mice" that the tissue bound tritium from tritiated water is not distributed uniformly. These, however, are not dealing with the seeking characteristic of tritium to some specific organs compared with protium. Because the variation of hydrogen contents in each organ, too short exposure periods or dilution effect by protium from unlabeled food and metabolic H2O can affect the results easily. Therefore long period of tritium labeling was used in our experiment from larva to adult, leading Rca, near 1 and hydrogen content was measured in each organ to estimate each R value. Further, the possibility of dilution effect by inhalation can be excluded in our aquatic ecosystem. The results indicated no significant difference on the distribution among gills, viscera and muscle of Japanese killifish with or without food chain and the values are similar to that of whole body. Range of a j3-ray from tritium is short, about 1 nm, in tissue and may cause differential effects depending on its binding site, especially DNA. And radiobiological significance can be attributed to the difference in position of bound tritium in DNA base"'. Tritium incorporation into DNA and the base, therefore, is important for evaluating radiation effect on organisms. We investigated the accurate R values of DNA, using the purified DNA from brine shrimps grown in tritiated water and obtained similar R values of DNA to that of whole body of brine shrimps in both cases with and without food chain. Since the maximum R values for whole body are 0.5 and 0.7 with and without food chain, respectively, those for brine shrimps should be similar. These values are slightly lower than that in various tissues of mice previously reported by Commerford38'. This difference may be due to a species difference. The R values of nucleotides shown in Table 2 are smaller than that of purified DNA. This indicates the loss of carbon-bound tritium from nucleotides during hydrolysis although we used enzymatic hydrolysis instead of alkaline treatment. The R value of 5-TMP was the highest. A similar finding has been reported on DNA bases of Japanese killifish eggs in tritiated water alone.This is mainly because the hydrogen of thymine methyl group behaves in a different way from that of other bases during isolation of nucleotides. Higher R values of 5-dAMP and 5-dGMP than 5-dCMP may be due to too short exposure period. The incorporation route from water into the purines via folic acid coenzyme intermediate is rather direct, so that purines have a greater specific activity than pyrimidines as discussed by Hatch"'. Although the exact discussion of tritium accumulation among DNA bases can not be made, it is concluded from these evidences that the specificity of tritium accumulation was neither found among various organs, nor between DNA and organic compounds in whole body.

Tritiation of Organisms through Food Chain The organisms grown in tritiated water with the model food chain have 40% or 110°1, higher R value in brine shrimps or Japanese killifish, respectively than those grown in tritiated water alone. Patzer20' also reported a similar result, using mosquitofish grown in tritiated water with artificial or natural feeding. Tritium accumulation through the food chain is generally expected to cause higher R value in predator organisms with feeding on tritiated foods than that without feeding on tritiated foods, because heterotrophic organism can synthesize their hydrogen of organic materials not only from water containing tritiated water, but also from components of their foods contaminated with tritium. Our results indicate that the extent of dilution by protium of unlabeled food stuff is 0.2 in brine shrimps or 0.35 in Japanese killifish of the R value. It is important to determine whether tritium concentrates in some of organisms through food chain or not, as reported for many radioactive nuclides. When the Japanese killifish were continuously fed on brine shrimps (R=0.7) tritiated in the first step of the model food chain system, we anticipated that the R value of Japanese killifish became higher than 0.7 if tritium in predator organisms accumulated with a same rate as in brine shrimps. However, as shown in Table 1, fish on the second step of the model food chain have a tendency to achieve a saturated R value. It is difficult to determine this tendency in a model ecosystem experiment where extended food chain system was introduced. Therefore, we calculated the R value of organisms on a simplified compartment model shown in Figure 3. The calculation based on the brine shrimp experiments resulted in an increase of tritium accumulation in organisms on a stepwise increase in food chain and gave R5 value of 0.79 even at fourth consumer organisms which are usually the last one in the natural environment. Whereas, calcu lated value from Japanese killifish was 0.71, suggesting the difference on trophic levels in a food chain. Lower R value of fish encouraged us to consider no concentration of tritium through food web, because the fish are higher consumer organisms in a food chain and because the experimental condition involved less dilution effects by unlabeled food stuff and inhalation as previously described. However, tritium accumulation probably depends not only on trophic level in a food chain, but also on accumulation complications in food web including saprophytic or parasitic chain. Other species may be further necessary to be examined an increased tritium accumulation through food chains by model ecosystem or by field work.

ACKNOWLEDGEMENTS The authors wish to thank Dr. Takehito Sasaki for his discussion and for his help in preparing the manuscript. REFERENCES

1. Kouts, H. and Long, H. (1973) Chap. II. Tritium Production in Nuclesr Reactors. Tritium. Messenger Graphycs. Phoenix, pp. 38. 2. Brues. A. M., Stroud, A. N. and Riets, L. (1952) Toxicity of tritium oxide to mice. Pro. Soc. Exptl. Biol. Med., 70: 174. 3. Furchner, J. E. (1957) Relative biological effectiveness of tritium beta particles and 60Co gamma rays measured by lethality in CFl mice. Radiation Res., 6: 483. 4. Hall, E. J., Oliver R. and Bedford, J. S. (1967) The relative biological effectiveness of tritium beta particles compared to gamma radiation-its dependence on dose rate. Br. J. Radiol., 40: 704. 5. Storer, J. B., Harris, P. S., Furchner, J. E. and Lanham, W. H. (1957) The relative biological effecttiveness of various ionizing radiations in mammalian systems. Radiation Res., 6: 188. 6. Lambert, B. E. (1969) Cytological damage produced in the mouse testes by tritiated thymid ine, tritiated water, and X-rays. Health Phys., 17: 547: 547. 7. Dobson, R. L. and Kwan, T. C. (1975) The tritium RBE at low level exposure variation with dose, dose rate, and exposure duration. Curr. Top. Radiat. Res. Q., 12: 44. 8. Baserga, R., Lisco, H. and Kisieleski, W. E. (1966) Tumor induction in mice by radioactive thymidine. Radiation Res., 29: 583. 9. Cahill, D. F., Wright, J. F., Godbold, J. H., Ward, J. M., Laskey, J. W. and Tompkins, E. A. (1975) Neoplastic and lifespan effects of chronic exposure to tritium. I. Effects on adult rats exposed during pregnancy. J. Natl. Cancer Inst., 55: 371. 10. Richmond, C.R., Langham, W.H. and Trujillo, T.T. (1962) Comparative metabolism of tritiated water by mammals. J. Cell. Comp. Physiol., 59: 45. 11. MacFarlane, W. V. (1964) Terrestrial animals in dry heat: Ungulates. Handbook of Physiol ogy. Adaptation to the Environment. Amer. Physiol Soc., Washington, D.C. 12. Yousef, M. K. (1973) Chap. V. Tritiated Water Turnover Rate in Desert Mammals. Tritium. Messenger Graphics. Phoenix. pp. 333. 13. Sanders, S. M. Jr. and Reinig, W. C. (1968) Assessment of tritium in man. In Nucl. Med. Biol., 534. 14. Lehman, J. W. (1973) Tritium cycling in a Lake Erie marsh ecosystem. Proc. 16th Conf. Great Lakes Res., 65. 15. Harrison, F. L., Koranda, J. J. and Tncker, J. S. (1973) Chap. V. Tritiation of Aquatic Animals in an Experimental Marine Pool. Tritium. Messenger Graphics, Phoenix. pp. 363. 16. Takeda, H. and Kasida, Y. (1979) Biological benavior of tritium after administration of tritiated water in the rat. J. Radial. Res., 20: 174. 17. Bruner, H. D. (1973) Distribution of Tritium between the Hydrosphere and Invertebrates. Tritium. Messenger Graphics, Phoenix. pp. 303. 18. Koranda, J. J. (1965) Preliminary studies of the persistence of tritium and 14C in the Pacific Proving Ground. Health Phys., 11 : 1445. 19. Kirchmann, R., Charles, P., van Bruwaene, R., Remy, J., Koch, G. and van den Hoek, J. (1977) Distribution of tritium in the different organs of calves and pigs after ingestion of various tritiated feeds. Curr. Top. Radiat. Res. Q., 12: 291. 20. Patzer, R. G., Moghissi, A. A. and McNelis, D. N. (1973) Accumulation of Tritium in Various Species of Fish Reared in Tritiated water. Environmental Behaviour of Radionuclides Re leased in the Nuclear Industry. IAEA. -SM-172/29: 403. 21. Bittle, R., Kirchmann, R., van Gelder.Bonnihns, G. and Koch, G. (1974) Etude d'un ecosys teme aquatique naturel contamene in situ per des effluents liquides trities, ev vue de 1'evalu ation de la seseibilite des paramtres des niveaux d'exposition du public. IAEA. -SM-184 /27: 613. 22. Higuchi, M., Komatsu, K. and Mukade, A. (1980) Assessment of tritium effect on brine shrimp, Artemia salina, reared in a model ecosystem contaminated with tritiated water. Radiation effects on aquatic organisms. Japan Scientific Societies Press, Tokyo/Uuiversity Park Press, Baltimore. pp. 13. 23. Ueno, A. M. (1974) Incorporation of tritium from tritiated water into nucleic acids of oryzias latipes eggs. Radiation Res., 59: 629. 24. Canellakis, E. S. and Mantsavinos, R. (1958) Conversion of deoxynucleoside 5'-monophosphates labeled with carbon-14 to the corresponding di and triphosphates by soluble mammalian enzymes. Biochim. Biophys. Acta., 27: 643. 25. Mukade. A. and Higuchi, M. (1974) Tritium incorporation into the cellular materials and its biological effects to Artemia salina reared in tritiated sea water. Bull. Plankton Soc. Japan, 21 : 32. 26. Kirchmann, R., Bonotto, S., Soman, S. D., Krishnamoorthy, T. M., Iyengar, T. S. and Moghissi, A.A. (1979) Transfer and incorporation of tritium in aquatic organisms. Behaviour of tritium in the environment. IAEA.-SM-232/96: 187. 27. ICRP, (1959) Report of committee II on permissible dose for internal radiation. ICPR Publi cation 2. Pergamon Press, New York. 28. Doppler-Bernardi, F. and Felsenfeld, G. (1969) In vitro incorporation of tritium into native DNA. Biopolymers, 8: 733. 29. Weinberger, D. and Porter, J. W. (1953) Incorporation of tritium oxide into growing Chlo rella pyrenoidosa cells.Science, 117: 636. 30. Hatch, E. T. and Mazrimas, J. A. (1972) Tritiation of animals from tritiated water. Radiation Res., 50: 339. 31. Weston, R. E. Jr. (1973) Kinetic and equilibrium isotope effects on tritium substitution. Tritium. Messenger Graphics, Phoenix. pp. 289. 32. Meloche, H. P., Monti, C. and Cleland, W.W. (1977) Magnitude of the equilibrium isotope effect on carbontritium bond synthesis. Biochim. Biophys. Acta., 480: 517. 33. Rambeck, W. A. and Bassham, J. A. (1973) Tritium incorporation and retention in photo synthesizing algae. Biochim. Biophys. Acta., 304: 725. 34. Inomata, T. and Higuchi, M. (1906) Incorporation of tritium into cell materials of Rhodo pseudomonas spheroides from tritiated water in the medium under aerobic condition. J. Biochem., 80: 569. 35. Higuchi, M. and Mukade, A. (1976) Distribution of the bound form tritium in the whole body of brine shrimps, Artemia salina, reared in the sea water containing tritiated water. Radioisotopes, 25: 814. 36. Thompson, R. C. (1853) Studies of metabolic turnover with tritium as a tracer, II. Gross studies on the rat. J. Biol. Chem., 200: 731. 37. Person, S. and Backrath, R. C. Jr. (1965) Mutagenic local effect accompanying the decay of incorporated T in Escherichia coli. J. Molec. Biol. 13: 600. 38. Commerford, A. L., Carsten, A. L. and Cronkite, E. P. (1977) The distribution of tritium in the glycogen, Hemoglobin, and chromatin of mice receiving tritium in their drinking water. Radiation Res., 72: 333.