Interaction of radionuclide 131I and cadmium chloride in an alternative bioassay with Artemia franciscana evaluated by a digital record

The interaction of radionuclide 131I and cadmium chloride was investigated by an alternative bioassay using the crustaceans Artemia franciscana. Fifty individuals were placed in each Petri dish. Due to radiation protection, evaluation of the experiment was performed using digital recordings taken by a camera. In the group containing a cadmium solution with an added radionuclide with a volumetric activity of 32 MBq·l-1, the lethality was significantly lower than in the group containing only a cadmium solution of 0.250 mmol·l-1. In the cadmium solution group and higher volumetric activity of radionuclide 131I (370 MBq·l-1), the lethality was significantly higher than in the control group, which demonstrated a synergistic effect. It was found that lethality was lower in the group containing only radionuclide 131I with a volumetric activity of 138 MBq·l-1 than in the control group. This result supports the theory of radiation hormesis. Radioactive iodine, internal contamination, external irradiation, digital recording, FIJI The environment has been burdened by the impact of the adverse effects of an everincreasing global production. Undesirable products with varying degrees of toxicity enter the soil, surface water and air in each sector of production. This also applies to ionizing radiation, which is part of the natural background, but has also been increasingly used in healthcare, industry and other areas of human activities. The impact of these factors on living organisms needs to be investigated in order to prevent damage resulting from their possible undesirable combined effects. The level of cosmic radiation and the terrestrial part of the natural background in the early period of life on this planet were about three times higher than they are today (Karam and Leslie 1999). Recently, new sources of ionizing radiation have appeared as a result of human activities, particularly the use of ionizing radiation in medical diagnostics and therapy. It also includes the release of radioactive substances in nuclear power plant accidents, nuclear weapon testing, and the use of atomic bombs in Japan at the end of World War II. In the first phase of nuclear accidents following the release of radionuclides into the atmosphere, the main food contaminants are iodine radioisotopes (Pöschl and Nollet 2006). The effects of ionizing radiation on living organisms have been studied for a long time. They can be divided according to the type of emitted particles, the dose size (absorbed energy) and the exposure time. Constant low doses of natural background or episodically occurring higher doses during medical examinations are common. ACTA VET. BRNO 2020, 89: 413–420; https://doi.org/10.2754/avb202089040413 Address for correspondence: Jana Doležalová Palackého tř. 1946/1 612 42 Brno, Czech Republic Phone: +420 541 562 623 E-mail: dolezalovaj@vfu.cz http://actavet.vfu.cz/

The environment has been burdened by the impact of the adverse effects of an everincreasing global production. Undesirable products with varying degrees of toxicity enter the soil, surface water and air in each sector of production. This also applies to ionizing radiation, which is part of the natural background, but has also been increasingly used in healthcare, industry and other areas of human activities.
The impact of these factors on living organisms needs to be investigated in order to prevent damage resulting from their possible undesirable combined effects.
The level of cosmic radiation and the terrestrial part of the natural background in the early period of life on this planet were about three times higher than they are today (Karam and Leslie 1999). Recently, new sources of ionizing radiation have appeared as a result of human activities, particularly the use of ionizing radiation in medical diagnostics and therapy. It also includes the release of radioactive substances in nuclear power plant accidents, nuclear weapon testing, and the use of atomic bombs in Japan at the end of World War II. In the first phase of nuclear accidents following the release of radionuclides into the atmosphere, the main food contaminants are iodine radioisotopes (Pöschl and Nollet 2006).
The effects of ionizing radiation on living organisms have been studied for a long time. They can be divided according to the type of emitted particles, the dose size (absorbed energy) and the exposure time. Constant low doses of natural background or episodically occurring higher doses during medical examinations are common.
The effect of higher doses of ionizing radiation (i.e. doses higher than 500 mSv) has been thoroughly studied in vertebrates and described by several authors (e.g. Sesztáková et al. 1996;Falis et al. 2004;Beňová et al. 2006;Beňová et al. 2007), though it is less studied in invertebrates, e.g. Artemia salina (Dvořák and Beňová 2002).
The effect of intermediate doses and doses comparable to the natural background, i.e. up to about 250 mSv (in vertebrates), is relatively difficult to prove statistically. This area of the impact of ionizing radiation on organisms is at the stage of intense investigation (Špalková et al. 2015). The course of dose-risk dependence has been described by several hypotheses. The most common are the linear threshold-free model (LNT), the threshold model, the linear quadratic model and the hormesis model (Luckey 1991;Luckey 1994;Sanders 2010). The combination of internal contamination and external irradiation with 131 I was used in the experiment that best describes a model of the real situation after nuclear accidents.
Cadmium and other heavy metals are among the most important environmental pollutants. They are usually present at very low concentrations but those concentrations are gradually increasing locally due to industrial and agricultural production. Cadmium is released as a by-product in ore mining and processing in industry, and in power engineering when fossil fuels are burned. It enters the soil as part of fertilizers such as phosphates. When dissolved in water, along with other substances, it may get through the root system into plants that feed livestock and other animals and thereby enter the food chain and represent an important source of contamination for humans as well (Jinadasa et al. 1997).
Cadmium and its compounds have been classified as human carcinogens. It may cause various oncological diseases, such as lung, kidney and prostate cancer. The harmfulness of cadmium results from its very slow excretion by the organism. The reported half-life is between 10 and 30 years (Ercal et al. 2001). For this reason, its use is regulated and controlled by the applicable laws of the Czech Republic and the EU, specifically by the EP and Council Directive ( /95/EC 2003. The effect of cadmium and other heavy metals on animals has long been studied (Lyn 2003;Falis et al. 2014).
In line with the 3R concept (Russell and Burch 1959), i.e. Replacement, Reduction, Refinement, an alternative second-generation bioassay was chosen for this experiment as a substitute for higher organisms. This is also subject to the EP and Council Directive on the protection of animals used for scientific purposes (2010( /63/EC 2010. Freshly hatched nauplionic stages of Artemia franciscana were used as a model organism (Dvořák et al. 2005).
The aim of this paper was to investigate the effects of cadmium chloride at a concentration lower than LC 50 in 216 h (Dvořák et al. 2005) on the nauplionic stages of A. franciscana in interaction with various volumetric activities of radionuclide 131 I. In this study, the counting of A. franciscana individuals was performed by digital record evaluation in order to avoid irradiation when reading the results.

Materials and Methods
In the prolonged A. franciscana toxicity bioassay, a 3% pure glucose solution was used as an energy source. Sea water (4.7% salinity) was prepared from chemicals of p.a. purity (Dvořák et al. 2005).
This experiment was prolonged to 456 h over and above the standard time of 264 h (Dvořák et al. 2005). Clear square polystyrene Petri dishes (120 × 120 mm) were used in this experiment. Each dish contained 45 ml of sea water with 3% glucose. Fifty randomly selected freshly hatched A. franciscana nauplium stages were placed in each dish. The dishes were shielded against each other by a 110-mm layer of copper plates of a layer thickness higher than the level of solution in the dish. This prevented the irradiation of nearby dishes (including the control).
The CdCl 2 2.5 H 2 O (Ing. Petr Švec -Penta s.r.o., Prague, Czech Republic) stock solution was prepared at a 10-fold concentration to be added to the dishes at one tenth of the total dish volume (5 ml). The concentration of this solution used in the Petri dishes was 0.25 mmol·l -1 Cd 2+ .

Results
The results of the experiment were divided into two graphs due to the validation of lethality of the control group being exceeded (20%). Data were interpolated with grade 4-6 polynomials. Correlation indices for each group were as follows: IC = 0.996, II138 = 0.983, ICd = 0.996, II32Cd = 0.995, II132Cd = 0.989, II370Cd = 0.997. Figure 1 shows the observed dependence of lethality on exposure time and the activity of added radionuclide 131 I from the start of the experiment to 264 h when the lethality in the control group did not exceed 20%.
Lethality (individuals) was monitored up to 456 h due to continuous irradiation and activity decrease, although the lethality of the control group had already risen above the recommended value of 20% due to poor nutrition. Figure 2 shows the lethality dependence at this extended experiment time from 264 h up to 456 h.
The lethality of control group C increased only slightly to 12% in 240 h (Fig. 1). From 240 h it increased approximately linearly to 72% at 456 h (Fig. 2).  The shape of the lethality/exposure curves varied significantly between groups. The difference was mainly caused by the higher volumetric activity of 131 I in combination with cadmium.
The lethality of the I138 group (self-acting radioiodide) was increased only very slightly up to 168 h. There was an increase to 14% between 192 h and 264 h (Fig. 1). The curve differs from that of the control group between 264 h and 384 h (Fig. 2). The addition of radionuclide 131 I of a volumetric activity of 138 MBq·l -1 to the test environment therefore led to a decrease in lethality compared to the control group throughout the experiment, with the exception of 456 h.
As can be seen in both graphs, the lethality of the I32Cd group was very similar to that of the Cd group, although it contained additional 131 I with a low volumetric activity of 32 MBq·l -1 . Between 144 h and 264 h, the lethality in both groups was significantly higher compared to control C. Comparing the two groups (Figs 1 and 2), the lethality of I32Cd up to 408 h was non-significantly decreased compared to the Cd group.
In the I132Cd group, the lethality at the start of the experiment was insignificantly reduced compared to the lethality in the control group. Up to 144 h, it was insignificantly reduced compared to the lethality of the Cd group. Up to 120 h, it was also insignificantly reduced compared to the lethality of the I32Cd group. At 168 h there was a significant increase in lethality relative to all groups. After 360 h, the lethality reached 100%.
In the I370Cd group the lethality was significantly increased from 96 h and its growth was faster than in the I132Cd group. Lethality reached 100% at 408 h. Although the volumetric activity of 131 I in this group was almost three times that of the I132Cd group, the course of lethality of both groups from 216 h was practically identical. The difference between activities was, therefore, only seen in the early stages of the experiment.

Discussion
In this study, the counting of A. franciscana individuals was performed using a digital recorder which allows evaluation of experiments with a higher overall activity of all dishes. The method of visual reading was not appropriate due to the risk of significant exposure of the observer to ionizing radiation, e.g. when handling the dishes. Photos were taken instead of video because the resolution of the pictures taken is more than 10 × higher. For instance, images taken by this device had a resolution of 24 megapixels, while the resolution of an HD video record may be approximately 2 megapixels.
Use of this high-resolution imaging also makes it possible to perform this bioassay employing 50 nauplia in one Petri dish. This provided a homogeneous environment for all individuals, in contrast to alternative bioassays employing this number as the total number of individuals.
It is also necessary to consider whether the subjects were mechanically initiated during the recording when evaluating photographs. Non-moving subjects were considered dead. The lethality at a given time might, therefore, be slightly higher than the lethality at another time. This effect can be reduced by repeating the records at short intervals and averaging the values obtained.
The specificity of the experiment using the 131 I radionuclide was that the observed lethality of A. franciscana was also dependent on its continuously decreasing activity. The radionuclide used has a relatively short half-life of only 8.025 days (192.6 h). On the other hand, this isotope is one of the most abundant in environmental contamination immediately after nuclear accidents and nuclear weapon explosions. The subjects were exposed to ionizing radiation continuously from the beginning till the end of this experiment. In a different experiment, irradiation (e.g. 60 Co) occurred at a single dose (Beňová et al. 2007) or continuously at the same dose rate (Ahlers et al. 1992). However, the total effect of ionizing radiation was a combination of internal contamination and external irradiation with radionuclide 131 I. Therefore, when assessing the lethality values obtained during the experiment, it ought to be considered that the decrease in the activity of 131 I (a half-life of 192.6 h) is half, and after 385.2 h equals one quarter of the baseline at the start of the experiment.
The experiment was intentionally prolonged due to this decrease in volumetric activity and its effect on lethality, for which reason the experiment continued even though the lethality of the control group was higher than the recommended value from 288 h. It would be optimal for validation for the lethality of the control group not to exceed 20% of the prolonged 240 h test with A. franciscana (Dvořák et al. 2005). However, the duration of exposure to the test substance (especially ionizing radiation) is chosen to exhibit a maximum difference in values between the experimental and control groups, thereby allowing a less common test extension to 456 h. It was found, thanks to this extension, that the lethality of group I138, containing only the radionuclide, was reduced significantly from 336 h to 384 h compared to the control group. Low doses of ionizing radiation may, therefore, result in lower lethality of the subjects, in this case the I138 group, compared to the control group.
The results of this work support the theory of radiation hormesis (Hrnčíř 1999;Sanders 2010). As suggested in many other biological and medical studies (Clewell et al. 2019), it appears that the natural mechanisms controlling living organisms are much more complex and defy the generally accepted concept of a simple linear cause-effect. Our results also support the notion that low doses of ionizing radiation might not be unambiguously negative (Luckey 2008).
Higher doses of ionizing radiation in combination with the toxic effects of cadmium, the I370Cd and I132Cd groups, show a synergistic negative effect. Cadmium toxicity has been described by several authors, such as Ochi and Ohsawa (1985). Bencko et al. (1995) reported the relationship between a higher Cd content in soil and the mortality of the population in the monitored area. Statistics have shown that there is a small (but demonstrable) risk of an increased incidence of ovarian cancer. High concentrations of cadmium in the liver and kidneys have been linked to the ability of these organs to produce more melatonin. Lethal doses of cadmium may inhibit mitochondrial phosphorylation in the liver (Kottferová and Koréneková 1998). The mechanism of action is believed to be based on the blocking of the (SH-) groups contained in proteins. This effect is similar to ionizing radiation. Cadmium binds to metallothionein in tissues. It is bonded via sulphhydryl groups. It is reported that they can reach about 10% of the molecular weight of metallothionein (Frazier and Rayner 1982;Kottferová et al. 2002). Its toxicity becomes evident when metallothionein synthesis is low. The cadmium chloride concentration we used was low for A. franciscana.
Several studies have also demonstrated the radioprotective effect of cadmium, both at the cellular level and throughout the whole organism (Privezencev et al. 1996). In our experiment, however, the radioprotective effect of cadmium was not demonstrated. The low volume activity of 131 I (32 MBq·l -1 ) had no significant effect on the lethal cadmium chloride curve, although it was found to be somewhat lower almost throughout the experiment.
From the point of view of our experiment, the volume activity of 131 I 32 MBq·l -1 appeared to be non-significant; independent activity of 138 MBq·l -1 represented the radiation hormone; and activities of 132 and 370 MBq·l -1 already represented interactions that significantly increased cadmium chloride toxicity.
In comparison with the limiting activities for drinking water, which are 5 to 6 orders of magnitude lower for mammals than those used in this experiment, it should be noted that radiosensitivity of A. franciscana is also about 2 orders of magnitude lower (LC 50 for 96 h 600 Gy) than in mammals (LD 50 for 30 day 4-6 Gy) (Dvořák and Beňová 2002).
The group containing only the radionuclide showed lower lethality than the control group, which is in conflict with the linear threshold-free model, thereby supporting the theory of the hormesis effect. The observed cadmium concentrations and higher 131 I radioisotope activity showed a synergistic effect. Digital recording allows the evaluation of experimental results when working with high activities of open sources of ionizing radiation without a risk of exposing the experimenter to ionizing radiation.
The results of this work model a real situation after a nuclear accident, when the interaction of radioiodine and cadmium salts contaminating the environment occurs.