The study focused on identifying certain indicators as possible markers of exposure to chronic heavy metal intoxication (Cd, Hg) at doses 500 times higher than MPC. Therefore, the monitoring of selected physiological, toxicological and reproductive parameters in rats were included in the set of monitored indicators.
4.1. Toxicological Parameters
During the experiment, no death of the experimental animals was recorded in any of the experimental groups (Cd, Hg or K). Thus, the survival was 100% and at the same time it was confirmed that the applied dose is low chronic dose. In a lifelong experiment [
9] evaluating the survival of animals exposed to cadmium, more individuals survived (one-year survival was also 100% as confirmed by the experiments) compared to other heavy metals. This may be because cadmium in low doses may function as an essential element [
10,
11,
12]. Hijová et al. [
13] also did not report any death of exposed animals during a chronic experiment in rats lasting 90 days, administering CdCl
2 in drinking water per os with a predicted daily dose of cadmium of 2.5 mg·kg
−1, similar to this study. A second possible explanation may be that rats received only 47.16% of LD
50 for a single administration per os throughout the experiment, which may also have resulted in no mortality. As reported by Kotsonis and Klaasen [
14], the LD
50 for single intoxication of Cd ingested as CdCl
2 per os in rats is 225 mg·kg
−1 of live weight. The fact that no death of experimental animals was recorded, even in the group exposed to mercury, may be related to the low total dose received, the LD
50 for Hg taken in the form of HgCl
2 per os for single intoxication is a dose of about 37 mg·kg
−1 of live weight [
15]. In the experiments, rats received only 55.51% of LD
50 for a single oral administration throughout the experiment.
At lifetime exposure to cadmium and mercury (156 days) in rats [
16], HgCl
2 was administered at a concentration of 1 µmol·L
−1, i.e., 0.2 mg of Hg per 1 L of drinking water, and CdCl
2 in a concentration of 20 µmol·L
−1, i.e., 2.0 mg of Cd per 1 L of drinking water. In the group exposed to Cd, 80% of animals survived; in the group exposed to Hg, only 40% of animals survived. Almášiová et al. [
17] stated that, at a concentration of 20 µmol·L
−1 CdCl
2 in drinking water, the survival of rats in the cadmium-exposed group was 10% lower than in the control group. In all the above works, however, no mortality was found in exposed animals for a period of one year, which is also confirmed by the experiments presented in this work. The mortality of animals in experiments with a similar focus can be significantly affected by breeding, handling conditions and overall welfare of the animals. For these reasons, in long-term experiments, it is necessary to adapt the conditions (experimental design) as much as possible to the natural method and the needs of the experimental animals. Ensuring adequate physical activity, daily physiological needs (e.g., shelter and cleaning) and well-being plays an important role and can fundamentally affect the results of an animal experiment. In the experiment, it is important to consider not only the duration of exposure but also other factors, e.g., concentration of toxic substance, light regime, handling and sampling time, heat stress, psychologically induced stress, etc. [
18].
4.2. Physiological Parameters
In Cd/female and Hg/female groups, the water intake in the first two stages was lower compared to the K group, which may indicate the so-called refuse effect. From the third stage, the water intake was increased in both the exposed groups, although statistically insignificant, but at the end of the experiment it was higher than in the K group, which can be attributed to the so-called taste habit phenomenon [
17,
19]. In the male groups, the situation in Hg group was similar to the female groups. However, in the Cd/male group, a significant difference was noticed, as in the first two stages no rejection effect was recorded, but, during third and fourth stages, at the end of the experiment and in the evaluation of the whole monitored period, the water intake was higher than in the Hg and K groups. These differences in water intake between females and males, especially in the cadmium-exposed group, require further experimental verification. A similar phenomenon between the sexes at the same concentration has not been reported in the literature so far.
When evaluating the water intake during the entire period of 52 weeks, in the Hg/female group, water consumption was higher, and, in the Hg/male group, it was lower when compared to the K group. This is consistent with another study [
20], suggesting that the higher intake of water after exposure was influenced by the kidney transport system and following the feeling of thirst, which is controlled by hypothalamus. A statistically significant difference (
p < 0.001) was observed between sexes in the Cd and K groups between males and females. In the male group, a statistical significance was recorded between the Cd and Hg groups (
p = 0.0145). In the Cd group, the intake of cadmium in the treated group was higher but statistically insignificant, as observed for both males and females. Ništiar et al. [
19] indicated that a relatively long time is needed for low doses of cadmium to be observed in food and water intake variance. The decrease in food and water intake occurs approximately after 91–100 weeks. In the Hg group, food intake by females was higher than by males compared to the K group. However, statistical significance was not observed. One of the reasons for different food intake can be attributed to the irritant effect of mercury on the mucosa of the digestive tract and the fact that mercury may impair appetite regulation [
21]. Some studies [
22,
23] have demonstrated that chronic exposure to low doses of mercury may have a difficult to predict course (but also consequence), and the response of the body to low (subtoxic) doses can vary. A statistical significance was observed between Cd/female and Cd/male groups (
p < 0.001), Hg/female and Hg/male groups (
p < 0.023) and K/females and Cd/female groups (
p < 0.021).
In the study, the dynamics of food intake was the same in all groups in both males and females during the individual stages. However, an interesting result was observed in Cd/female group in the third quarter: a statistically significant reduction in food intake compared to the K group (
p = 0.037) was observed. At the end of the experiment, food intake was higher in both sexes in the Cd group than in the K group, but not statistically significant. Wirth and Nijal [
22] and Calabrese [
3] stated that chronic exposure to low doses of mercury can have an unpredictable course (but also consequences) and the body’s response to low (subtoxic) doses may be different. Ništiar et al. [
9] and Shibutani et al. [
24] stated that the determining indicator of food intake (but also changes in weight and heavy metal intake) is water intake, which only partially corresponds to the results in this study. The opposite situation may also be the fact that food intake is limiting in this respect and water intake is derived from it. In male groups, the dynamics of food intake for the entire experimental period in all groups was similar to that of water intake. On the contrary, in female groups, the food intake in the Cd and Hg groups was lower than in the K group, which in the Hg group does not correspond to water intake, and this is an interesting finding that requires further experimental verification.
According to water and food intake, an increase in weight during the individual stages was expected. This fact was confirmed by the experiment. However, at the end of the experiment as well as when evaluating the whole experimental period, the weight of females and males in both exposed groups was lower than in the K group. In the Cd group, this may indicate possible liver cell damage. An additional possible explanation for low body weight of rats after the exposure to a low dose of Cd is that Cd is bound to the mucous membrane of the gastrointestinal tract after per os intake and stomach and intestinal epithelial cells can be impaired. A long-term intake of a low dose of Cd may induce lipid peroxidation that can result in the damage of the mucosal barrier, ultimately resulting in ulcers. Further increase of the permeability through the intestine wall and its subsequent impairment may cause diarrhea in rats. Some studies [
25,
26,
27] have pointed to a possible link between low body weight and the weight of some organs (especially the hollow organs of the digestive tract). The overall toxic effect of the low dose of Cd administered to rats per os is signified by changes in weight of the organs, which consequently reflects in the changes of the overall body weight [
25]. Damage to the organs can be confirmed or rejected with follow-up histopathological examination. Santana et al. [
26] and Predes et al. [
27] reported that the weight also depends on the duration of the experiment and the total exposure (dose) of the toxic substance. Toman et al. [
28] reported that, already after nine weeks of cadmium administration, signs of liver cell damage were observed in the microscopic structure of the liver.
In the Cd/female group, the average daily dose was 0.29 mg·kg
−1·day
−1, receiving only 47.16% of LD
50 per animal per os. In the Cd/male group, the average daily dose was 0.28 mg·kg
−1·day
−1, receiving only 45.97% of LD
50 per os per animal. Based on these facts, the average daily dose of Cd for both males and females was the same over the entire experimental period. As reported by Kotsonis and Klaasen [
14], the LD
50 for a single Cd intoxication received in the form of CdCl
2 per os in rats is a dose of 225 mg·kg
−1 of live weight. Based on that, the average daily dose of cadmium in both females and males can be considered as a low dose for chronic exposures. The dose of Lowest-Observed-Adverse-Effect (LOAEL) for cadmium is 1.5–17.5 mg·kg
−1·day
−1 [
29], which was not exceeded in conducted experiments. If the Minimal Risk Level (MRL) is considered for per os chronic exposure whose value is 0.1 µg·kg
−1·day
−1 for Cd [
29], then the value was exceeded approximately 3000 times. With long-term intake of low doses cadmium, this toxic element is accumulated in the peripheral and central nervous system [
30], contributes to osteoporosis and is carcinogenic (causing prostate and lung cancer) [
31]. In the Hg/female group, the average daily dose was 0.02 mg·kg
−1·day
−1, receiving only 57.59% of LD
50 per animal per os. In the Hg/male group, the average daily dose was 0.03 mg·kg
−1·day
−1, receiving only 55.51% of LD
50 per os per animal. LD
50 for Hg received in the form of HgCl
2 per os for single intoxication is approximately 37 mg·kg
−1 of live weight [
15]. Almášiová et al. [
17] found that intoxication did not result in death of animals in the period of over one year. In both female and male groups, this value can be considered as a low dose for chronic exposures.The LOAEL dose for mercury is 1.9–3.9 mg·kg
−1·day
−1. In the experiments in both sexes, this dose was not exceeded. If MRL is considered for per os chronic exposure whose value is 0.007 µg·kg
−1·day
−1 for Hg [
31], then the value was exceeded approximately 9000 times.
4.3. Reproduction Parameters
In the Cd group, the number of litters, the number of born pups and the number of raised pups for the entire period of the experiment were lower than in the K group. Lower numbers of born pups per litter and raised pups per litter compared to the K group were recorded. Cd acts preferentially through deprivation of testosterone secretion [
16], as well as through the hypothalamus—pituitary —testes axis [
11]. Predes et al. [
27] indicated that, even a very small difference in the administered dose of Cd in males causes a sudden increase in testicular damage. Thompson [
32] stated that cadmium can affect the female reproductive and endocrine systems and hormone levels change. It is assumed that lower values of some monitored reproductive parameters in the Cd group compared to the K group indicate a decrease in reproductive function in rats. In general, the number of born pups per litter and the number of raised pups per litter are among the most important indicators of reprotoxicity. A similar conclusion was achieved at a concentration of 20 mmol·L
−1, i.e., 2.0 mg Cd per liter [
16]. The body’s response to low doses may vary [
3]. One example of the unexpected hormonal effect of a toxic substance is the finding that cadmium acted as a hormonally active substance even in concentrations that were considered safe. In a multigenerational study in rats at doses up to 100 mg Cd per kg of live weight no adverse effects on reproduction have been reported. In a four-generation study at a dose of 1 mg·L
−1 in water and at a mean daily dose of 0.125 mg Cd·kg
−1·day
−1, no adverse effects on fertility were observed in rats and mice. However, at a dose of 50 mg Cd·kg
−1 of live weight, slight testicular changes were observed in rats during a 15-month period [
33]. In terms of the effect of cadmium on animals in chronic poisoning, the highest accumulation of cadmium occurred in rabbits (females) in the ovaries (0.47 mg·kg
−1), uterus (0.25 mg·kg
−1) and testes (0.10 mg·kg
−1) [
34]. Soukupová and Dostál [
35] described the embryotoxic effects of cadmium in rodents, i.e., fetal malformations (cleavage of the climate, oligodactylia, polydactylia, exencephalia and others).
In the Hg group, the numbers of litters born pups and raised pups for the entire period of the experiment were lower compared to the K group. The number of born pups per litter and the number of raised pups per litter in the Hg group were higher but insignificantly compared to the K group, which could be attributed to the adaptation process to a certain environment with a certain load factor. However, it is very similar to hormesis after exposure to a low dose of harmful substances. At low doses of mercury, there may be no signs of a toxic effect on the body. However, it can cause increased production of proteins from the group of matalothionines, which bind heavy metals and ensure their subsequent removal from the body. In addition, the role of metallothionein is to protect cells from further damage (e.g., from heritable information damage). Therefore, it is possible that the low dose of mercury was not only not dangerous for the organism, but also contributed to the organism becoming more resistant to the adverse effects to which it was exposed. A similar finding was made by Lukačínová et al. [
36] but at a concentration 200 times higher than the MPC in water containing HgCl
2 at a concentration of 1 mmol·L
−1 (0.2 mg Hg·L
−1). If the results in this study are attributed to the hormetic effect, there can be a logical explanation. The low doses of the toxic substance used in the cells did not have to cause much damage; on the contrary, they were able to trigger protective reactions in the cell which prevented the action of other adverse effects. However, this assumption, based on the results of this study, requires further experimental studies. The body’s response to different low doses of heavy metals may be different. It is known that high doses inhibit growth, shorten life expectancy and reduce fertility, while low doses can improve some parameters. A higher number of born pups (rats) with chronic exposure to heavy metals (significantly higher doses and significant mortality) than in the experiments in this study was described by Kotsonis et al. [
14]. Harmful fetal development and decreased fertility in rats of both sexes were also confirmed [
21]. Comparing the results and their effect on reproductive parameters between low and high dose studies is quite challenging because it is a confirmation of the exact mechanism of action and role of reactive oxygen species and metallothionein in chronic exposure to heavy metals. It is also necessary to take into account the effect of the components of innate and adaptive immunity, as well as the defense mechanisms that could be activated by gradually increasing the concentration of the toxic substance (albeit in the range of low doses) in the environment.