environments
Review
Tritium: Doses and Responses of Aquatic Living Organisms
(Model Experiments)
Lydia Bondareva 1, * , Nadezhda Kudryasheva 2,3






Citation: Bondareva, L.;
Kudryasheva, N.; Tananaev, I.
Tritium: Doses and Responses of
Aquatic Living Organisms (Model
Experiments). Environments 2022, 9,
51. https://doi.org/10.3390/
environments9040051
Academic Editor: Claude Fortin
Received: 4 February 2022
Accepted: 7 April 2022
Published: 14 April 2022
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Environments 2022, 9, 51. https://doi.org/10.3390/environments9040051
and Ivan Tananaev 4
1 F.F. Erisman Federal Scientific Center of Hygiene, 141014 Moscow, Russia
2 Institute of Biophysics, Federal Research Center Krasnoyarsk Science Center, Siberian Branch,
Russian Academy of Sciences, 660036 Krasnoyarsk, Russia; n-qdr@yandex.ru
3 Biophysics Department, Siberian Federal University, 660041 Krasnoyarsk, Russia
4 FRC Kola Science Center RAS, 184209 Apatity, Russia; geokhi@mail.ru
* Correspondence: lydiabondareva@gmail.com; Tel.: +7-(495)-5861144
Abstract: Tritium is a byproduct of many radiochemical reactions in the nuclear industry, and its
effects on aquatic organisms, particularly low-dose effects, deserve special attention. The low-dose
effects of tritium on aquatic microbiota have been intensively studied using luminous marine bacteria
as model microorganisms. Low-dose physiological activation has been demonstrated and explained
by the signaling role of reactive oxygen species through the “bystander effect” in bacterial suspensions.
The activation of microbial functions in natural reservoirs by low tritium concentrations can cause
unpredictable changes in food chains and imbalances in the natural equilibrium. The incorporation
of tritium from the free form into organically bound compounds mainly occurs in the dark and
at a temperature of 25 ◦ C. When tritium is ingested by marine animals, up to 56% of tritium is
accumulated in the muscle tissue and up to 36% in the liver. About 50% of tritium in the liver is
bound in non-exchangeable forms. Human ingestion of water and food products contaminated
with background levels of tritium does not significantly contribute to the total dose load on the
human body.
Keywords: tritium; living organisms; dose-forming effects
1. Introduction
Tritium, 3 H, is classified as a long-lived radionuclide and it can pollute the biosphere
on local, regional, and global scales [1]. Tritium is one of the most biologically significant
radionuclides. As a constituent of water molecules, tritium is an ideal label for studying the
exchange processes between the stratosphere and the troposphere when studying air-mass
transport and water cycles in nature. Tritium is present in many organic compounds,
including those which are biologically important.
The global air pollution by tritium is largely a result of nuclear and thermonuclear
weapons tests. The release of industry-related tritium into the environment began with
the launch of industrial reactors and radiochemical production facilities intended for the
development of weapons-grade plutonium in the United States. By the year 1945 (the
beginning of nuclear weapons tests), the situation dramatically changed, and the content of
3 H in rainwater increased by several orders of magnitude. By November 1952 (following the
first thermonuclear explosion), the amount of tritium released into the Earth’s atmosphere
exceeded its natural level by more than 60–190 times [2–6].
Currently, the main source of industry-related tritium is the nuclear industry. There
are more than 500 large reactors operating in 35 countries around the world. Tritium
is produced during the operation of nuclear reactors of all types. Its sources include
fission reactions of heavy nuclei and the interactions of fast neutrons with boron, lithium,
and deuterium nuclei [7]. According to [8,9], tritium discharges through liquid waste
during the operation of various types of reactors, amounting to 5–130 TBq·year−1 . Nuclear
https://www.mdpi.com/journal/environments
Environments 2022, 9, 51 2 of 17

fuel recycling plants annually release about 600 TBq of tritium in the form of gaseous waste
and about 15 PBq of tritium in the form of liquid waste into the environment. The rate of
tritium formation varies in different types of reactors. For example, the fast neutron reactor
at the Beloyarsk Nuclear Power Plant (NPP) releases 740 GBq of tritium per MWe per year.
After 10 years of operation of the heavy water nuclear reactor, an average of 700 GBq of
tritium per MWe per year was formed [10]. Tritium discharges from the VVER-1000 reactor
at the Novovoronezh NPP amount to 15 ± 1.9 GBq·MWe−1 per year [11]. According to the
data presented in [12,13], in other countries, the radionuclide release into the environment
from nuclear power plants is 1.9 to 38 GBq·MWe−1 per year.
Several studies have indicated a local increase in the tritium content in the areas where
large nuclear power centers are located. In the Russian Federation, within the Ural region,
the Beloyarsk and the Mayak NPPs are major sources of industry-related tritium pollution.
In addition to atmospheric radionuclide emissions, increased tritium concentrations have
been recorded in the aquatic ecosystems adjacent to these plants. Thus, in the Olkhovskaya
marsh–river ecosystem in the area of the Beloyarsk NPP, the tritium content in the water
varies from thousands to tens of thousands becquerels per liter [14,15]. The tritium activity
concentration of 1–4 Bq·L−1 in the water of industrial reservoirs of the Mayak NPP are not
at a high level. Low concentrations of tritium (2–3 Bq·L−1 ) can be found in precipitation in
the northern hemisphere during late spring and early summer (Global Network of Isotopes
in Precipitation, GNIP-IAEA) [16].
Periodic increases in tritium concentrations in waterways from the Krasnoyarsk Min-
ing and Chemical Combine, and in some aquatic ecosystems of the Chernobyl NPP area,
were recorded as compared with the technogenic background level, but did not reach the
level of intervention established for drinking water by the radiation safety standards NRB:
99–7700 Bq·L−1 [17–19]. It is worth noting that the Mining and Chemical Combine (MCC),
located by the Siberian Yenisei River, has engaged in the production of weapons-grade
plutonium for several decades.
Data analysis presented in the literature regarding the radiation–chemical state of the
Yenisei River identified several potential sources of tritium that contribute to the presence
of tritium in the water-collecting area of the Yenisei River, namely [20,21]:
(a) Nuclear weapons tests;
(b) MCC aerosol releases;
(c) Water discharge from reactor cooling systems;
(d) Tritium migrating from open reservoirs of sedimentation tanks at the MCP industrial site;
(e) Tritium migrating from the subsurface horizons of the Severny landfill sites. It is
known that up to 330,000 Bq·L−1 of tritium has been pumped into the subsurface
horizons of the landfill site over many years.
Therefore, during the operational period of MCP nuclear reactors, there have been
six potential sources of tritium entering the Yenisei River. This created a unique situation,
prompting the investigation of a variety of tritium migration routes in the environment.
The term ‘tritium problem’ has increasingly been used in the contemporary literature
on radiation exposure risk assessments. Radioactive beta decay of tritium leads to the
disruption of molecular structures and intermolecular bonds under the action of its own
beta radiation [22]. Due to the presence of tritium in surface and underground water, there
is a risk of 3 H migration in air and water flows. This may contribute to the entry of 3 H into
several trophic levels of the food chain.
This article assesses the level of the tritium impact on living organisms, including the
trophic chain of water–bacteria–aquatic plants–fish, and to model the processes of human
exposure to tritium-containing fish and water.

2. Effects of Tritium on Living Organisms

The radiological impact of tritium is a product of the characteristics and behavior of the
radionuclide. On one hand, tritium absorption is similar to that of other hydrogen isotopes
and has considerable biological significance [23]. On the other hand, some features of
Environments 2022, 9, 51 3 of 17

tritium impart low-level radiotoxicity. Indeed, low-energy beta particles emitted by tritium
have a maximum free path in water or tissue, ranging within 6 microns, which reduces
risks of external exposure. Therefore, tritium is associated with radiological risk only if the
human body absorbs it, especially after the ingestion of tritiated organic molecules [1,22,23].
The behavior of tritium in plants is of particular interest, because photosynthesis
is a necessary step in the production of organic matter, which moves throughout the
environment along food chains to its potential human consumers [3,24–27]. The radiological
effects of tritium can be evaluated from observations of irradiated cells or animals. On the
other hand, there are convincing data on the biological effects of tritium on plants [28–30].
No noticeable effect on biomass production was observed in experiments performed to
assess the impact of tritium on various vegetables [31–35]. However, it can be assumed
that DNA mutations and plant cell death may occur at very high levels of tritium impact,
as seen in animals.
The results of animal experiments cannot be directly transferred to humans. However,
the biological effectiveness of weak beta emissions under different irradiation conditions
can also be considered in order to assess the radiotoxicity of tritium in relation to humans.

3. Effects of Tritium on Marine Bacteria

Microorganisms are the basic and the simplest organisms of aquatic ecosystems;
they contribute significantly to the ecosystem equilibrium. Metabolic products of aquatic
microorganisms may influence all water inhabitants.
Luminous marine bacteria constitute a suitable bioassay system for radiotoxicity
monitoring in different multicomponent media. This bioassay is widely utilized in ecotoxi-
cological monitoring [36–38]. It applies bioluminescence intensity as a physiological test
parameter, which can be measured with simple devices, easily and quickly. The advantages
of the bioassay are its simplicity and the high rates of assay procedure (1–10 min) [39,40].
This enables the possibility of numerous sample analyses and proper statistical process-
ing. The high rates ensure a large number of measurements for comparable conditions,
and hence, enable robust statistical processing, which is extremely important for low-dose
exposures, usually described in terms of “stochastic effects” [41]. Furthermore, the rapid
luminescence response is supposed to indicate the non-genetic mechanism of low-intensity
exposures [42,43].
Previous studies [44–48] have demonstrated both the activation and inhibition effects
of tritium on marine bacteria (Figure 1A), as well as the absence of a monotonic depen-
dence of the luminescence response on tritium concentration at chronic low-dose exposure
(<0.03 Gy), in a wide range of tritium radioactivity from 0.0001 to 200 MBq·L−1 (Figure 1B).
Intact and lyophilized bacterial suspensions were studied and exhibited similar results.
The results obtained were explained in terms of the ability of the bacterial cells to adapt
to the low-dose radiation, based on the “hormesis” model. The term “hormesis” was
introduced by H. Schulz and R. Arndt at the end of the 19th century [49]. The current devel-
opment of the hormesis concept is attributed to E. Calabrese [50–54]. The term “radiation
hormesis” was suggested by Luckey [55]. The phenomenon of radiation hormesis was
intensively studied in [52,56,57].
An increase in the bacterial luminescence intensity in the presence of tritiated water,
HTO, was demonstrated in a series of experiments where bi-phasic dependence (activation
+ inhibition) was found in [44,46], whereas mono-phasic dependence (activation only) was
shown in [42,58,59]. The results were analyzed in reviews [47,48].
The mechanism of the activation effect of tritium is of special interest. The first
hypothetical mechanism is based on DNA damage repair [60–62]. The involvement of non-
genetic mechanisms in low-dose chronic radioactive effects in bacteria was demonstrated
in [42,59].
In addition, it was shown that tritium activated bacterial growth at the activity levels
of 10–104 kBq·L−1 and suppressed this growth at higher activities (>105 kBq·L−1 ) [45].
Environments 2022, 9, x
51FOR PEER REVIEW 4 4ofof 18
17

Figure 1. Effect of tritiated water on the bioluminescence of bacteria [47,48]. (A) Bioluminescence
kinetics of bacteria in tritiated
tritiated water,
water, 22 MBq ·L−−11; ;the
MBq·L thered
redarrows
arrowsdenote
denotethethetimes
times of
of exposure
exposure (20
(20 and
and
50 h); (B) bioluminescence intensity vs. activity
activity concentration
concentration of
of tritiated
tritiated water
water at
at 20 h (black) and
50
50 h
h (green)
(green) exposures.
exposures.

An increase the
Previously, in the bacterial
authors luminescence
demonstrated intensity
that in the presence
low-intensity of tritiateddid
tritium exposure water,
not
HTO, was increase
noticeably demonstrated in a series
the content of experiments
of reactive oxygen species where bi-phasic
(ROS) dependence
in bacteria-free media(activa-
[46];
tion
hence,+ inhibition) was foundproduction
the non-biological in [44,46], whereas
of ROS couldmono-phasic
hardly dependence
be responsible (activation only)
for this effect.
Subsequently,
was shown in it was confirmed
[42,58,59]. that the
The results wererates of ROS in
analyzed production were low in highly diluted
reviews [47,48].
tritiated
Thewater in the absence
mechanism of bacteriaeffect
of the activation [58]. ofThis resultiscan
tritium be explained
of special interest.by The
the low-energy
first hypo-
thetical mechanism is based on DNA damage repair [60–62]. The involvementlow-dose
radioactive decay of tritium. However, the exposure of marine bacteria to chronic of non-
tritium mechanisms
genetic irradiation ininhighly low-dosediluted tritiated
chronic water (<0.08
radioactive effectsGy) considerably
in bacteria increased the
was demonstrated
ROS
in content in the bacterial environment (up to 300%). A comparison of the low-dose
[42,59].
effects
In of tritium it
addition, (0.03,
was 4.0shownand that
500 MBq
tritium·L−activated
1 ) on the luminescence of marine bacteria and
bacterial growth at the activity levels
ROS
of content
10–10 in a−1 cellular
4 kBq·L water suspension
and suppressed this growth wasatperformed in [58]. (>10
higher activities It was demonstrated
5 kBq·L −1) [45]. that
an increase in the ROS concentration correlated with the intensification
Previously, the authors demonstrated that low-intensity tritium exposure did not no- of the physiological
bioluminescence
ticeably increase the process
contentin the bacteria oxygen
of reactive during the bacterial
species (ROS) lifetime.
in bacteria-free media [46];
Marine luminous bacteria are appropriate
hence, the non-biological production of ROS could hardly be responsiblebio-objects for studying thefor biological
this effect.ef-
fects of radiation,
Subsequently, andconfirmed
it was particular thatattention should
the rates be paid
of ROS to their metabolites,
production were low inparticularly
highly di-
ROS, because
luted tritiated theywater can insuppress
the absence or activate physiological
of bacteria [58]. Thisfunctions
result canofbe marine microorgan-
explained by the
isms. It is known that marine bacteria form ROS endogenously
low-energy radioactive decay of tritium. However, the exposure of marine bacteria to in aerobic environments
throughlow-dose
chronic the reaction between
tritium O2 andin
irradiation electron
highly donors
dilutedsuch as metal
tritiated water centers,
(<0.08 dihydroflavin,
Gy) consider-
etc. [63,64]. It is known that peroxide derivatives
ably increased the ROS content in the bacterial environment (up to 300%). of flavin molecules are native interme-
A comparison
diate compounds of the bacterial bioluminescence reaction
of the low-dose effects of tritium (0.03, 4.0 and 500 MBq·L−1) on the luminescence of marine[65,66]. It is suggested that
the oxygen-detoxifying function directed the emergence of
bacteria and ROS content in a cellular water suspension was performed in [58]. It was many bioluminescent systems,
including bacterial bioluminescence [67]. This suggestion provides an additional reason to
demonstrated that an increase in the ROS concentration correlated with the intensification
study the ROS balance in radioactive bacterial environments
of the physiological bioluminescence process in the bacteria during the bacterial lifetime.
HTO molecules can easily penetrate a cell membrane. Therefore, products of tritium
Marine luminous bacteria are appropriate bio-objects for studying the biological ef-
radioactive decay, electrons and ions of helium-3, may affect the bacterial structure, from the
fects of radiation, and particular attention should be paid to their metabolites, particularly
outer membrane to the enzymes and their substrates inside the cells. Thus, the stimulating
ROS, because they can suppress or activate physiological functions of marine microorgan-
effect of 3 H can be associated with the process of electron/ion transfer in intracellular
isms. It is known that marine bacteria form ROS endogenously in aerobic environments
bacterial structures, leading to increased rates of bioluminescent enzymatic reactions.
through the reaction between O2 and electron donors such as metal centers, dihydroflavin,
The same processes inhibit the luminescence function of bacteria at longer exposure times.
etc. [63,64]. It is known that peroxide derivatives of flavin molecules are native interme-
An important issue is the interaction of tritium decay products with bacterial cells.
diate compounds of the bacterial bioluminescence reaction [65,66]. It is suggested that the
Considering the concentration of cells and the number of ion pairs formed during the decay
oxygen-detoxifying function directed the emergence of many bioluminescent systems, in-
of tritium (up to 200 ions and/or excited molecules), the number of ion pairs per cell was
cluding
calculated: bacterial bioluminescence
3.33, 0.0266, and 0.0002 ion [67]. This
pairs suggestion
·(cell ·s)−1 for theprovides
tritiuman additional
activity reason to
concentrations
study the ROS balance in radioactive
− 1 bacterial environments
of 500, 4, and 0.03 kBq·mL , respectively [58]. This low value of ion pairs per cell suggests
HTO mechanism
a specific molecules can easily penetrate
underlying the effecta cell membrane.
of tritium on the Therefore,
cells. It isproducts
likely that of tritium
tritium
radioactive decay, electrons and ions of helium-3, may affect
decay products can serve as “triggers” for enhancing the metabolic oxygen-dependent the bacterial structure, from
the outer membrane
processes in cells, which to theresult
enzymes
in ROS andincrease
their substrates
within the inside
cell.theOncells. Thus,hand,
the other the stimu-
ROS
lating effect of 3H can be associated with the process of electron/ion transfer in intracellular
Environments 2022, 9, 51 5 of 17

released into the environment can serve as specific signaling molecules for other cells (the
so-called “bystander effect”).
It was shown [68–70] that for human cells, the bystander effect can be induced even by
one cell in a thousand-cell population, and it does not depend on the number of the initially
induced cells (one cell or 50% of cells). Additionally, it should be noted that the absence
of dose–response dependence is in accordance with the concept of the “stochasticity” of
low-dose radiobiological effects [71,72]. This concept assumes the involvement of free
radicals and ROS in radiobiological responses.
The results of studying tritium effects on luminous marine bacteria present a physico-
chemical approach to understanding the metabolic functioning of bacteria and their inter-
actions with aqueous environments, which can affect other water inhabitants [42–48,58,59].
Based on the rapid luminescence bioassay, the studies considered only short-time exposures
and did not reveal mutagenic effects or the cellular accumulation of tritium. The results
of another study [71] show the accumulation of tritium in phytoplankton populations
of Dunaliella tertiolecta and Nodularia spumigena and mussels Mytilus edulis. The paper
considers the scenarios where tritium may be concentrated or transferred in biota relevant
to Baltic coastal communities.
Hence, the activation of microbial functions and accumulation of tritium in marine
organisms introduced into food chains are highlighted in the scientific literature. The pro-
cesses mentioned may result in an imbalance in the natural equilibrium in water ecosystems.
It is important that molecular surroundings in natural aquatic ecosystems might
change the effects of tritium on microorganisms. It was reported that humic substances,
products of natural transformation of organic matter in water sediments, mitigate acti-
vating and inhibiting effects of tritiated water on marine bacteria [73,74], whereas gold
nanoparticles can additionally suppress physiological functions of the microorganism (so
called, radio-sensitizing effect) [75].

4. Effects of Tritium on Aquatic Plants

Aquatic organisms incorporate tritium as a fraction of TFWT (tissue-free water tritium)
contained in organic compounds as a result of isotopic exchange or enzyme-catalytic reac-
tions [28]. In exchange reactions, tritium binds to oxygen, sulfur, phosphorus, and nitrogen
atoms to form hydroxides, thiols, phosphides, and amines, respectively. Usually, this
produces exchangeable, organically bound tritium (OBT), which is in equilibrium with
TFWT in the studied plants or animals and behaves similarly to HTO. Tritium binds to the
carbon skeleton of organic molecules during enzymatically catalyzed reactions. In this case,
non-exchangeable tritium is formed. This is a form of tritium that remains in dry biological
matter even following repeated washes with light water.
It is necessary to have a clear definition of OBT formed from TFWT in living systems
through natural ecological or biological processes. This has been accomplished within
the framework of the International Atomic Energy Agency (IAEA) in the Environmental
Modelling of Radiation Safety (EMRAS) programm [28,71,76–78].
Previously, a study was carried out on the assimilation and transformation of tritium
in the aquatic plants Elodea canadensis and Lemna minor [79].
Branched shoots of Elodea canadensis can reach lengths of up to 100 cm long. They
were sorted out according to the biomass quality. Apical shoots from 3 to 6 cm in length
from the entire biomass were used for the study.
The Elodea biomass washed was used for accumulating tritium and estimating TFWT
and OBT in the control point (the initial point). For this purpose, the initial biomass was
divided into two parts. In one of them, tritium (in HTO form) was extracted from an aliquot
of 50 g taken from one part of the biomass. TFWT can be measured by azeotrope distillation
of the fresh sample, then liquid scintillation spectrometry–total OBT (exchangeable and non-
exchangeable) combustion in oxygen atmosphere of dry sample, purification of combustion
water, liquid scintillation spectrometry [25].
Environments 2022, 9, 51 6 of 17

The Lemna samples were grown in Climatostat apparatus for one week using Stein-
berg medium (ISO/DIS 20079, 2005). The algae cultures were grown in a cultivator,
KB-05, designed at the Siberian State University (Krasnoyarsk, Russia). An algae suspen-
sion, 125 ± 10 cm3 in volume, was poured into the reactor. To provide carbon dioxide,
the container with the suspension was rotated around its longitudinal axis. The constant
temperature of the medium was equal to 36.0 ± 0.5 ◦ C.
For the experiment on the tritium accumulation by Elodea, the plant shoots (~5 cm)
were placed in an aquarium and completely submerged in tritium-containing water (250 g
of the plants were placed into 2500 mL HTO). A photoperiod of ~12 h was set. The duration
of the experiment was 11 days, followed by measurement of the shoot length. Water from
the Yenisei River with the background tritium levels of 4 ± 1 kBq·L−1 was used as a control.
For the experiment on tritium accumulation by Lemna, culture samples were taken
by picking similar three-leaf rosettes. For each of the solutions being tested, 4 mL of 100%
Steinberg medium was poured into a measuring cylinder and filled to 200 mL with distilled
water or with water from the Yenisei River. Thus, near 20 Bq (100 kBq·L−1 ) of tritium
were introduced into the first tested solution, 60 Bq (300 kBq·L−1 ) into the second, 100 Bq
(500 kBq·L−1 ) into the third, and 200 Bq (1000 kBq·L−1 ) into the fourth. Four rosettes of
Lemna were placed into each flask with the prepared solutions. The flasks were placed
into the cassette of the Climatostat chamber, where conditions of constant light and a
temperature of 27–28 ◦ C were maintained.
The processes of the TFWT transformation into OBT in the plant biomass were studied
upon changing the ambient temperature and light regime. For this purpose, the apical
shoots of the green plants (3–4 cm) were used, which were preliminarily washed with
running water; the remaining water was removed using absorbent paper. The prepared
plants were placed in cylinders containing the same amount of tritium. The shoot weight
was equal to 200 g. The water volume was 1600 mL. The content of the tritium intro-
duced amounted to 1 kBq·L−1 . The ambient temperature was changed using a thermostat.
The light regime was provided by special chambers equipped with lamps. The experiment
duration was 14 days. At the end of the experiment, the content of tritium in the form of
TFWT and OBT was estimated.
To estimate the total tritium in the samples, it was necessary to eliminate all the liquid.
An aliquot of about 50 g dry weight was taken from the previously prepared sample. This
aliquot was placed into a round-bottomed flask, where it was mixed with toluene, chosen
for stripping the azeotropic mixture. The mixture obtained was kept in a corked flask for
12 h. Then, the flask was placed into a flask heater. A special device was put onto the flask
neck to strip the azeotropic mixture and separate aqueous and organic phases. To separate
OBT, an aliquot of 100–150 g dry weight was used. The aliquot of the prepared Elodea
samples was placed into a round-bottomed flask to be mixed with toluene [77,78].
The tritium content in each of the biological samples (plants or fish) under study was
measured using a liquid scintillation spectrometer, Quantuluse-1220, USA (The Joint Center
of the Krasnoyarsk Science Center, SB RAS), using a scintillation cocktail, UltimaGold
AB, where the sample under study was dissolved. The background determined for the
prepared tritium-free water samples ranged between 0.926 CPM and 1.002 CPM, and the
counting efficiency, using the internal standard method (ISO 9698:2010), was between 25.3%
and 26.1% for the maximum figure of merit.
Table 1 shows data on the content and distribution of tritium according to the occur-
rence forms (tissue-free water tritium (TFWT) and organically bound tritium (OBT)) in the
initial biomass and after the experiment with Elodea.
These results confirm the fact that during the chronic interaction of tritium with aquatic
plants, processes occur which are associated with the intensive accumulation and retention
of tritium in the biological structures of living organisms.
When conducting model experiments to study the influence of environmental param-
eters on tritium transformation, a dependence of OBT content on ambient temperature was
obtained. It was found that the proportion of tritium in the form of OBT in the Elodea shoots
 counting efficiency, using the internal standard method (ISO 9698:2010), was between
25.3% and 26.1% for the maximum figure of merit.
Environments 2022, 9, 51 Table 1 shows data on the content and distribution of tritium according to the occur-
7 of 17
rence forms (tissue-free water tritium (TFWT) and organically bound tritium (OBT)) in
the initial biomass and after the experiment with Elodea.
strongly depended on the ambient temperature, optimal temperature (~25 ◦ C; Figure 2),
Table 1. Results of tritium content estimation and its distribution with regard to the forms of bind-
and
ing toillumination. A large
the Elodea biomass (n =proportion
10, p = 0.95).of TFWT transformation into OBT was obtained in
the light/shadow mode—6/18 h (Table 2).
Tritium Concentration, Bq·kg−1 (%
Average
Table 1. Results of tritium content estimation Average of Tritium
and its distribution Accumulated
with regard to the forms in the Bi-
of binding
Plants
to the Elodea biomass (n = 10, p
Length, cm = 0.95). Weight, g omass)
TFWT OBT
Tritium Concentration, Bq·kg−1 (% of
Average Average 5.5 ± 1.1 0.20 ± 0.05
Plants
Initial 8.0 cm
Length, ± 0.5 0.59g± 0.3 Tritium Accumulated in the Biomass)
Weight,
(97 ± 1) (3 ± 1)
TFWT OBT
Following 141 ± 2 23 ± 2
15 ± 1 0.75 ± 0.9 5.5 ± 1.1 0.20 ± 0.05
Initial
exposure 8.0 ± 0.5 0.59 ± 0.3
(97 ± (85
1) ± 1) (3(15 ± 1)
± 1)
Following 141 ± 2 23 ± 2
These results 15
confirm
±1 the fact that
0.75 during the chronic interaction of tritium with
± 0.9
exposure (85 ± 1) (15 ± 1)
aquatic plants, processes occur which are associated with the intensive accumulation and
retention of tritium in the biological structures of living organisms.
TableWhen
2. OBTconducting
content (% model experiments
of the total to study
tritium content) the plant
in the influence of environmental
biomass as a function of pa-
the
rameters onmode
illumination tritium
(n =transformation,
5, p = 0.95). a dependence of OBT content on ambient tempera-
ture was obtained. It was found that the proportion of tritium in the form of OBT in the
Light/Dark,
Elodea shoots Hours 24/0
strongly depended 18/6 ambient16/8
on the temperature,12/12optimal6/18 0/24(~25
temperature
°C; Figure 2),%and illumination.
OBT, 3 A large6proportion9 of TFWT transformation
15 35 into OBT 22 was
obtained in the light/shadow mode—6/18 h (Table 2).
OBT, %

T, °С

Figure 2. Dependence of the proportion of the

Figure 2. the OBT content
content (percentage
(percentage from the
the tritium
tritium activity
activity
accumulated in the biomass) in the plant biomass on the ambient temperature.
accumulated in the biomass) in the plant biomass on the ambient temperature.

Environments 2022, 9, x FOR PEER REVIEW TableFigure

2. OBT3content
shows (% theofinitial duckweed
the total samplesin(a)
tritium content) theand samples
plant atas
biomass the end8ofofthe
a function experi-
of the
18 illu-
mination
ments: mode (n
control = 5,
(b), p = 0.95).with the maximum tritium activity in the media with distilled
samples
water (c) and those with the Yenisei River water (d).
Light/Dark,
24/0 18/6 16/8 12/12 6/18 0/24
Hours
OBT, % 3 6 9 15 35 22

Figure 3 shows the initial duckweed samples (a) and samples at the end of the exper-
iments: control (b), samples with the maximum tritium activity in the media with distilled
water (c) and those with the Yenisei River water (d).
(c) (d)
(a) (b)

Figure 3.Figure
Appearance of the duckweed:
3. Appearance initialinitial
of the duckweed: (a); at(a);
theat end of the
the end experiment:
of the experiment:control
control(b), 1000 Bq·L−1 :
(b),1000
Bq·L−1: distilled water (c); water of the Yenisei River (d).
distilled water (c); water of the Yenisei River (d).

No necrosis, chlorosis,
No necrosis, or other or
chlorosis, physiological changes were
other physiological found.
changes wereThere wasThere
found. an in-was an
crease in the number
increase in the of leaves:ofinstead
number leaves: of six leaves
instead of sixasleaves
in theascontrol, there were
in the control, thereeight
were eight
leaves in eachin
leaves system with the
each system maximum
with tritium
the maximum content,
tritium both both
content, with with
the water of the
the water of Ye-
the Yenisei
River
nisei River andand with
with distilled
distilled water.
water. An increase
An increase in theinsurface
the surface
area ofarea of leaves
leaves in the systems
in the systems
with the introduced tritium activities was observed as compared with the control systems,
especially in the systems with the water of the Yenisei River (tritium activity ~4 Bq·L−1).
Based on the experimental results, it was concluded that the OBT proportion de-
pended on the mode of illumination. The TFWT transformation into OBT occurred mainly
as a result of physiological processes associated both with photosynthesis and plant
Environments 2022, 9, 51 8 of 17

with the introduced tritium activities was observed as compared with the control systems,
especially in the systems with the water of the Yenisei River (tritium activity ~4 Bq·L−1 ).
Based on the experimental results, it was concluded that the OBT proportion depended
on the mode of illumination. The TFWT transformation into OBT occurred mainly as a result
of physiological processes associated both with photosynthesis and plant growth. During
the day, plants use energy from the sunlight to convert carbon dioxide into sugars. The latter
(including starch) are subsequently consumed by the plant itself, supplying energy for
cell division, the assembly of biological macromolecules, maintenance of physiological
processes, etc. This embeds the incoming tritium atom into the plant structures.

5. Studying the Effect of Tritium on the Physiological Functions of the Submerged

Aquatic Plant Elodea canadensis
To study the toxic effect (via phototaxis) of tritium on shoots of the submerged macro-
phyte Elodea canadensis [79], a sample of plant shoots was used. Before studying the photo-
taxis of chloroplasts, glasses containing Elodea shoots were kept in the dark for 15–30 min.
Phototaxis was observed on the upper surface cells of the leaves under the light microscope
“Mikmed-2” (LOMO, Russia) at 600× magnification. A leaf was separated from the shoot,
placed under a cover slip, and exposed to a lower illumination lamp for 3–5 min on the
microscope slide table. Under intense illumination, the chloroplasts adopted a parastrophic
position (perpendicular to the incident light rays), shifted to the cell edges, they encircled
the cell. The proportion of active chloroplasts was calculated as a ratio of the number of
chloroplasts in the parastrophic position shifted to the cell edges, to the total number of
chloroplasts in the cell. For each concentration, ten randomly selected cells from one leaf
were examined, in one or three replicates. To measure chlorophyll epifluorescence, an Elodea
leaf was placed under a cover slip and a drop of simazine herbicide (C7 H12 ClN5 , 10−5 M)
was introduced to block the non-cyclic electron transport between the two photosystems.
Epifluorescence was measured using a Lumam-I and FMEL microscope (LOMO, Russia),
excited by light with a wavelength of 410 nm in a circle with a diameter of 45 µm and with
an average cell size of 60 µm × 70 µm. On each leaf, ten fields were measured with random
movement of the preparation, in one or three replicates. The results are presented as mean
values or weighted mean values. The significance of the differences in the mean values was
calculated according to the Student’s t-test.
The following aqueous media were used: water from the Yenisei River, to which
tritium with different activity was introduced, and water collected from Atomic Lake
(Semipalatinsk test site (STS), Kazakhstan).
Reference: Atomic Lake. The power of all excavating explosions at STS with soil
displacement was 140 kt; 99% of the power of these explosions occurred in a single explosion
performed in Well 1004 on January 15, 1965, at the confluence of the Shagan and Aschisu
rivers, at a depth of 178 m. As a result of the explosion, long-lived radionuclides were
accumulated: 239,240 Pu—8.5 Ci, 137 Cs—800 Ci, 60 Co—80 Ci, 152 Eu—120 Ci, 90 Sr—400 Ci,
3 H—4·105 Ci [80]. Between 30% and 40% of the accumulated number of radionuclides

entered the bulk soil.

As a result of soil displacement during the explosion, a funnel with a diameter of
430 m along the initial surface was formed; the height of the bulk ridge was 20–35 m,
and the bulk width from the funnel ridge was 400 m. The volume of the visible funnel
was more than 10 million m3 from the bulk ridge and 6 million m3 from the initial surface.
With spring flood waters filling the funnel, two large reservoirs were formed: the inner one
in the funnel with the volume of 7 million m3 and water surface area of 0.5 km2 , and the
outer one in the floodplains with the volume of 10 million m3 and water surface area of
3.5 km2 . The reservoir waterfront was formed by an earth-and-rockfill dam, a rockfill dam
with a bottom discharge and a surface flood spillway. The constructed reservoir was the
first hydroelectric facility of this kind, and it was considered to be a hydrotechnical test site.
 bulk width from the funnel ridge was 400 m. The volume of the visible funnel was more
than 10 million m3 from the bulk ridge and 6 million m3 from the initial surface. With
spring flood waters filling the funnel, two large reservoirs were formed: the inner one in
the funnel with the volume of 7 million m3 and water surface area of 0.5 km2, and the outer
one in the floodplains with the volume of 10 million m3 and water surface area of 3.5 km2.
Environments 2022, 9, 51 9 of 17
The reservoir waterfront was formed by an earth-and-rockfill dam, a rockfill dam with a
bottom discharge and a surface flood spillway. The constructed reservoir was the first
hydroelectric facility of this kind, and it was considered to be a hydrotechnical test site.
WeWe determined
determined thethe
metal
metalcontent
contentbybyICP-MS,
ICP-MS, and thethe
and anion content
anion contentbyby
ionic liquid
ionic liquid
chromatography.
chromatography. To To
estimate thethe
estimate tritium content
tritium in the
content samples
in the samplesunder study
under wewe
study used
used thethe
procedures described
procedures described above.above.
TheThe tritium
tritiumcontent in the
content water
in the waswas
water ~5.6~5.6 ·L−1 .−1In
kBqkBq·L addition,
. In thethe
addition, water contained
water contained
(mg · L − 1 ): Na—1100; Cl—1700; Mg—160; S—350; Ca—94; Sr—6.4; K—4; Br—1.7; Si—0.58;
(mg·L ): Na—1100; Cl—1700; Mg—160; S—350; Ca—94; Sr—6.4; K—4; Br—1.7;
−1 Si—0.58;
B—0.55,
B—0.55, and
and(µg/L):
(µg/L):Cu—87;
Cu—87;I—52;I—52;Ba—22;
Ba—22;U—16;
U—16; Al—12;
Al—12; Mo—9.2; P—7.6; Ti,
Mo—9.2; P—7.6; Ti, Ni,
Ni,As,
As,Se
Se ~3;
~3; R—1.1.
R—1.1. The The main −
·L 1 ):−1):
main anions
anions in inthe
thewater
waterfrom
fromAtomic
Atomic Lake were
Lake wereas follows
as follows(mg(mg·L
HCO − − 2 −
HCO 3 3—214;
−—214; ClCl−—1430;
—1430; SO —1290.
SO442−—1290.
Figure 4 shows the images obtained
Figure 4 shows the images obtained during the experiments
during the experimentson theoneffect of tritium
the effect on
of tritium
theonphotosynthesis of Elodea.
the photosynthesis of Elodea.

(a) (b) (c) (d)

Figure
Figure 4. Results
4. Results of the
of the tritium
tritium action
action on movement
on the the movement of chloroplasts
of chloroplasts of theofElodea
the Elodea
leavesleaves
in the in
ex-the
experiments with water from the Yenisei River—14 days, (a–c): (a) control (~4 Bq·L −1), (b) +100
periments with water from the Yenisei River—14 days, (a–c): (a) control (~4 Bq·L−1 ), (b) +100 Bq·L−1 ,
Bq·L−1, (c) +2900 Bq·L−1; and with water from Atomic Lake—5 days, (d): tritium content of 5600
(c) +2900 Bq·L−1 ; and with water from Atomic Lake—5 days, (d): tritium content of 5600 Bq·L−1 in
Bq·L−1 in the mixture with other radionuclides.
the mixture with other radionuclides.

It was
It was found
found that
that whenwhenthethe system
system with
with thethe water
water from
from thethe Yenisei
Yenisei River
River contained
contained
tritium, a lethal effect occurred at a tritium concentration of ~3000 Bq·L , 29 daysafter
tritium, a lethal effect occurred at a tritium concentration of ~3000 Bq·L −
−1 ,
1 29 days afterthe
the beginning of the experiment. In addition, significant damage to the integrity of the cellcell
beginning of the experiment. In addition, significant damage to the integrity of the
membranes
membranes waswas observed,
observed, followed
followed by leakage
by leakage of theofintracellular
the intracellular fluid (Figure
fluid (Figure 3). De-
3). Despite
thespite the external
external well-being
well-being of the used,
of the shoots shootsitused, it was observed
was observed under aunder a microscope
microscope that at that
a
at a tritium
tritium contentcontent
of ~100of ·L−1 ,Bq·L
Bq~100 the −1 , the movement
movement of chloroplasts
of chloroplasts decreaseddecreased
by 70% by 70%
in 29 in 29
days,
anddays, and at
by 25% bya25% at a of
content content
~10 Bqof·L~10−1 . Bq·L−1.
In the experiments with the water
In the experiments with the water samples samplesfromfrom Atomic
Atomic Lake,
Lake, the movement
the movement of chlo-
of chloro-
roplasts
plasts decreased
decreased by 40%
by 40% fromfromthe the initial
initial valuevalue
fivefive
daysdays after
after thethe start
start of of
thethe experiment.
experiment.
In In this
this case,
case, thethe lethal
lethal effect
effect wouldwould occur
occur muchmuch earlier
earlier because
because thethe radiation–chemical
radiation–chemical
composition
composition of of water
water is quite
is quite complex
complex andand diverse.
diverse.
Chlorophyll fluorescence is widely used for biotesting as an activity indicator of
the photosynthetic apparatus of aquatic plants [79,81–83]; epifluorescence microscopy has
previously been used for detecting chlorophyll complexes with heavy metals in vivo [82,83].
It is assumed that the decrease in chlorophyll fluorescence under the action of tritium
probably occurs as a result of the substitution of a fraction of the protium atoms in the
chlorophyll molecule “a” with tritium atoms.
In contrast to fluorescence, the phototaxis reaction of chloroplasts is rarely used for
biotesting, although it is often used for duckweed [81–84].
The movement of chloroplasts in duckweed cells in light was experimentally shown
to not only occur as a result of the light effect on photosynthesis and respiration [81].
The movement of chloroplasts in cells involves the participation of actin-myosin proteins,
ATP, photosynthesis, and other processes [82–84]. Therefore, as compared with epifluores-
cence, phototaxis is a more integral reaction. Our experiments show that a certain decrease
in the epifluorescence signal under the action of tritium occurs faster than the inhibition of
chloroplast phototaxis. One of the main advantages of the phototaxis reaction in terms of
biotesting appears to be the absence of the need for control. Under favorable conditions,
the phototaxis of chloroplasts should be 100%. In contrast, the lethal effect is accompanied
by the complete extinction of phototaxis.
Environments 2022, 9, 51 10 of 17

Consequently, the effect of tritium on the biological functions of Elodea with the chronic
radionuclide intake is observed at a tritium content of 2900 Bq·L−1 , which is several times
lower than the intervention level established by NRB-99 (7700 Bq·L−1 ).

6. Effects on the Development of Phytophagous Freshwater Prussian carp

Among living organisms in polluted reservoirs, fish are the most suitable objects
of study due to their biological peculiarities that make it possible to assess the medium-
term transformation processes of water reservoirs. Fish are sensitive to a wide variety of
direct impacts and integrate the adverse effects of the entire range of different impacts,
including impacts on other components of the aquatic ecosystem (habitat, macroinverte-
brates, primary products, etc.). The dose loads on fish bodies are formed due to external
radiation (from water and bottom sediments) and, internal radiation (from incorporated
radionuclides) [5,85–88].
The embryonic period of development does not end when the embryo exits the shell;
it includes a period of time after hatching, while the sac fry, with a number of embryonic
peculiarities related to the structure of respiratory, circulatory and digestive organs, goes
through the final stages of embryonic development.
To estimate the tritium content in the samples under study, we used the procedures
described above. In the experiments, we utilized water from the Yenisei River (Table 3),
which differs in terms of the level of added tritium activity, and the water from Atomic
Lake (Table 3). At the end of the study, the number of dead eggs, number of fries with
developmental abnormalities, and the number of fries with the approximately normal
physiological development were calculated in each experimental system (Table 3).

Table 3. Effects of the tritium water on the development of carp eggs 25 days after starting the
experiment (p = 0.95).

Tritium Concentration, Initial Eggs, Dead Eggs, Eggs with Abnormal

Bq·L−1 Units Units (%) Development Units (%)
Water from the Yenisei River
control 3500 ± 150 280 ± 20 (9) 250 ± 15 (7)
500 ± 10 7600 ± 200 850 ± 15 (11) 750 ± 20 (10)
5000 ± 18 3500 ± 100 700 ± 10 (20) 1100 ±100 (30)
50,000 ± 57 4400 ± 200 950 ± 30 (20) 1400 ± 50 (30)
Water from Atomic Lake
5600 ± 28 5800 ± 250 3900 ± 200 (67) 1500 ± 170 (26)

According to the assessment of external physical characteristics, at the level of the

tritium impact approximately equal to (5000 Bq·L−1 ) or exceeding (6.5 times) the level
of intervention (7700 Bq·L−1 ), a clear radiation effect was manifested only at the stage of
egg development. In this case, the proportion of the dead eggs and fry with abnormal
development in the systems with 5000 Bq·L−1 and 50,000 Bq·L−1 was comparable.
The system with the water from Atomic Lake stood out to a large extent. In this case,
the number of the dead eggs reached 67%, while 26% of the eggs showed abnormal development.
Most fry from the system with the addition of 50,000 Bq·L−1 were placed in a clean
medium (clean water, pure feed), where they remained for breeding and subsequent use.
The fry from the model systems with the additions of 500 Bq·L−1 and 5000 Bq·L−1 were
not used in further studies.
The remaining fry from the system with the water from Atomic Lake were also placed
in a clean system for nursery purposes. However, all the fry died within ten days.
Table 4 shows the results of changes in the length of the fries under nursery conditions
in clean water where pure feed was used as food. In such conditions, the fry were kept for
150 days before entering the third stage of the experiment.
Environments 2022, 9, 51 11 of 17

Table 4. Changes in the length of the fish fry in relation to the control system (p = 0.95).

Length, mm
Age of Fish,
System Units
Days Average (Difference in
Range, mm
Relation to Control, %)
17 ± 1
Control 800 16.8–17.4
18 ± 1
50,000 Bq·L−1 35 1050 17.6–18.1
(1.1%)
28.3 ± 2.8
Control 500 27.8–28.9
28.4 ± 2.4
50,000 Bq·L−1 76 500 28.0–28.9
(0.4%)
65.7 ± 6.6
Control 206 65.0–67.0
70.6 ± 6.7
50,000 Bq·L−1 125 203 69.6–77.5
(9.7%)
80.3 ± 8.6
Control 75 88.3–92.2
81.7 ± 7.9
50,000 Bq·L−1 150 97 70.1–93.3
(2.2%)
17 ± 1
Control 800 16.8–17.4
18 ± 1
50,000 Bq·L−1 35 1050 17.6–18.1
(1.1%)

The maximum difference in fry length was detected only on day 125 (9.7%); however,
on day 150, this difference diminished by several times.
After 150 days, the carp fry were placed in large aquariums, with clean filtered water
from the Yenisei River. At this stage of the experiment, the accumulation and distribution of
tritium that entered the body with food were studied. The pre-used feed had been enriched
with tritium during the course of the previous experiments on the accumulation of tritium
by aquatic plants [74,84]. Previously, the tritium-containing plants (~1.5 kBq·kg−1 ) were
dried at ~45 ◦ C in a nitrogen atmosphere to prevent the tritium exchange with the laboratory
atmosphere. The anhydrous concentrates obtained were mixed with dry fish fodder (1:1),
and the mixture was pressed into 0.3 g granules. The tritium amount in each granule
was ~50 Bq, and the total amount of radionuclide fed daily in each experimental system
was up to 600 Bq. The maximum amount of the introduced tritium was up 300 kBq per
individual [78,89].
Table 5 shows the data on the changes in the morphological parameters of the studied
fish during the last stage of the study.

Table 5. Changes in the morphological parameters of the studied carp specimens (n = 10, p = 0.95).

Parameters Initial 250 Days 550 Days

Mass, g 120 ± 17 500 ± 55 800 ± 110
Length, cm 17 ± 3 35 ± 5 60 ± 6

From the results presented, it is evident that the fish developed quite well. This is
indicated by the increase in the weight and length of the specimens.
Due to the fact that the welfare conditions for the fish were more favorable (sufficient
food, optimal lighting and temperature, and water circulation in the aquariums) compared
with natural conditions, a fairly rapid weight gain was observed [90,91].
The methods from [92] were used for the complete ashing and isolation of lipids and pro-
teins from the fish. The main muscle mass components (%, n = 10) were: proteins—16 ± 1%,
water—70 ± 2%, fat—13 ± 1%, ash—~1%.
Following the conclusion of experimentation, we obtained the following results:
the muscle tissue accumulated a large proportion of the tritium absorbed by the whole fish
(up to 56%), although the proportion of the accumulated tritium did not depend on the
feed consumed by the fish. In both cases, about 35% of the total tritium accumulated by the
fish was bound to the liver.
Environments 2022, 9, 51 12 of 17

The distribution of OBT in the parts of the fish body was considered in a number of
studies [93–96]. However, our research deals with the total tritium content, i.e., the to-
tal sum of TFWT and OBT [74,86]. These experiments are presented in detail in prior
research [77,78,89]. The consumption of fish products is increasing all over the world;
the quantity and quality of this product depends on the type of fish and on its origin
(growing in natural or artificial conditions). Quantitatively, the content of the muscle tissue
of each specimen varies within 40–50% of the total weight of the fish [92]. Therefore, when
fish is consumed by humans, the main dose-forming part is the muscle tissue.
The obtained results did not reveal any differences in the radionuclide accumulation
in the systems with duckweed and hornweed. Hence, it was concluded that both of these
plants were equally digested in the digestive organs of carp intentionally fed with pellets
containing plants with tritium.
In the course of the studies, it was revealed that tritium accumulated in the liver,
mainly binding to adipose tissues which included lipids (~39%). A slightly lower amount
of tritium was bound to proteins (~19%). The smallest amount of tritium was found in
the form of free water (~3% of the total tritium content in the liver) [85]. The unrecorded
tritium content accumulated by the liver was bound to unidentified substances in the liver.
The liver has a very high level of metabolic activity. Food lipids are re-esterified in
the liver and are carried to other tissues and fat depots by the blood. The lipids that are
mobilized from the depot are again transferred to the liver, which is the main site of their
oxidation and synthesis [30]. An increase in the level of the lipid-bound tritium in the
muscle mass of fish, up to 39% of the tritium content in the whole fish, and a greater
contribution of lipids included in the liver due to the accumulation of tritium (~39% of the
total tritium content in the liver), were found.
The quantity of phospholipids in the tissues, as compared with other fractions, was
lower as related to the physiological state of the fish. It did not depend on feeding habits.
The welfare conditions for all the fish were the same, and the content of phospholipids had
the least variability, which also affected the proportion of tritium binding by this fraction,
the maximum being 7.9%.
The use of cholesterol in biosynthesis processes and in the regulation of membranes
motivates assessing the proportion of tritium binding. In the experiments conducted,
cholesterol had the greatest contribution to the accumulation of tritium (~30%). Considering
cholesterol esters, the proportion of tritium binding reached 50% (in terms of the dry weight)
of all the tritium accumulated by the liver.
During the period of intensive weight uptake, lipids are accumulated in the depot
in the form of triacylglycerols [32,92]. The feeding and welfare conditions for the fish
specimens did not change for the entire duration of the experiment; therefore, the content of
triacylglycerols did not decrease, in contrast to the fish in natural conditions, and continued
to accumulate. This was indicated by the overall increase in the mass of the fish muscle
tissue, especially by the increase in lipid content. This pattern also manifested in the liver.
Therefore, the increase in the tritium content in triacylglycerols has a direct relationship
with the content of lipids in the liver.
Thus, the accumulation of tritium in the liver occurs due to the formation of non-
exchangeable organically bound forms (more than 50% of the total tritium accumulated by
the liver).
An increase in the proportion of non-exchangeable organically bound tritium, both in
the liver and in the whole body, increases the radiotoxicity of tritium as compared with
tritium water (TFWT).
Another important parameter is the determination of the biological half-life of tritium
for carp, including the consumption of food enriched with tritium. For this purpose, three
fish specimens were placed in clean water after 250 days from the tritium accumulation
experiment. For 300 days, the fish were fed with pure tritium-free food. At the end of
the experiment, the fish were also dissected. The tritium content was determined at each
Environments 2022, 9, 51 13 of 17

stage. According to the recommendations given in the paper by Melintescu et al. [26],
the half-lives of tritium were calculated for fish organs.
The results obtained showed that the decrease in tritium varied from 27% to 65%,
depending on the organ under study (Table 6). The average value for the half-life for the
whole fish can be assumed to be 175 days, whereas the half-life for the liver is 550 days.

Table 6. Determination of the half-life period of tritium from the model fish (n = 3, p = 0.95).

Organ 250 Days 550 Days

Gills 1.0 ± 0.1 <MDA
Bones + head <MDA <MDA
Skin + fins + scale 2.0 ± 0.4 <MDA
Liver 6.0 ± 0.4 4.4 ± 0.6
Intestine with contents 8.0 ± 0.6 2.8 ± 1.0
Stomach with contents 9.0 ± 1.0 3.2 ± 1.0
Muscle tissue 74.0 ± 3.0 33.0 ± 4.0

Thus, the accumulation of tritium in the liver occurs due to the formation of non-
exchangeable forms strongly bound to organic compounds (more than 50% of the to-
tal amount of tritium accumulated by the liver). An increase in the proportion of non-
exchangeable organically bound tritium, both in the liver and in the whole body, increases
the radiotoxicity of tritium as compared with tritium water (TFWT).
It is known that the effects of tritium and its compounds on the human body are
characterized by a number of peculiarities, one of which is its ability to be incorporated in
the composition of biologically active molecules. Tritium can replace hydrogen in DNA
molecules, which may lead to an increase in its biological half-life and, accordingly, to an
increase in the risk of the long-term effects of radiation including carcinogenic risk [97–100].
To calculate the radiation doses, we considered the following: eggs with the introduc-
tion of the maximum tritium content (50,000 Bq·L−1 ) and fish with the longest exposure
interval (550 days). Both external and internal radiation was calculated, according to
the IAEA recommendations [85–87] (Table 7). Specifically, we used the values of the to-
tal tritium content in the eggs and fish according to the recommendations provided in
the literature.

Table 7. Radiation doses for the eggs and fish.

Types of Eggs Fish

Irradiation µGy·hour−1 µGy·25 Days−1 µGy·hour−1 µGy·550−1
Internal 5.36·10−7 3.2·10−4 1.89·10−6 2.50·10−2
External 4.82·10−6 2.9·10−3 2.36·10−10 3.12·10−7

7. Conclusions
Tritium is a by-product of many radiochemical reactions in the nuclear industry,
and its effects on water organisms, particularly low-dose effects, deserve special attention.
In the past few decades, the low-dose effects of tritium on aquatic biota have been studied
in detail using luminous marine bacteria as model microorganisms, as well as during
the transformation of fresh tritium water to organically bound tritium. The efficiency of
activation and inhibition processes has been demonstrated and studied using cellular and
enzymatic systems. Low-dose activation can be explained by the “trigger” function of
the products of tritium decay for bacterial metabolic oxygen-dependent processes and by
the signaling role of reactive oxygen species (ROS) in the “bystander effect” in bacterial
suspensions. ROS are supposed to serve as intercellular messengers in the latter process.
Briefly, despite the low energy of tritium decay, its influence on living organisms in
water environments may be considerable. The activation of microbial functions in natural
Environments 2022, 9, 51 14 of 17

reservoirs, due to the low content of tritium, may result in unpredictable changes in food
chains and an imbalance of natural equilibria.
The accumulation of tritium in the liver occurs mainly due to the formation of organi-
cally bound tritium, and, for the most part, these compounds are non-exchangeable. This
leads to the fact that the half-life of tritium in the liver is the longest and reaches 550 days.

Author Contributions: Conceptualization, L.B., I.T. and N.K.; methodology, L.B.; validation, N.K.;
formal analysis, I.T.; investigation, L.B. and N.K.; resources, L.B. and N.K.; data curation, N.K.;
writing—original draft preparation, L.B. and I.T.; writing—review and editing, I.T. and N.K.; visual-
ization, L.B. and N.K.; supervision, N.K.; project administration, L.B. and I.T.; funding acquisition,
L.B. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing not applicable.
Acknowledgments: This review was prepared with the partial financial support of the Program of
the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Russian
Federation) 2020–2025 and of the Sevastopol State University, project identifier 42-01-09/169/2021-7.
Conflicts of Interest: The authors declare no conflict of interest.

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