Zinc and Cadmium Toxicity Using a Biotest with Artemia franciscana

Nováková J., D. Daňová, k. Strišková, R. Hromada, H. Mičková, M. Rabišková: Zinc and Cadmium Toxicity Using a Biotest with Artemia franciscana. acta vet. Brno 2007, 76: 635-642. of the various toxic elements heavy metals, particularly cadmium, lead, mercury and zinc, occur frequently in the environment due to their relatively high industrial use. While the toxicity of individual substances is usually well known, information about their mutual interactions is relatively scarce. in animal experiments the prevailing trend is to substitute higher vertebrates with biotests of the 2nd generation. our experiment focused on observation of the effect of combinations of ZnSo4.7H2o and CdCl2.2H2o on lethality to Artemia franciscana. The aim of the study was to observe the synergistic or antagonistic effects of these two metals. depending on concentration, cadmium may increase or decrease the toxicity of zinc. at higher concentrations of CdCl2.2H2o exceeding 100 mg·l -1 one can observe obvious synergistic toxic effects of both the substances. our observations allowed us to conclude that the use of optimum, relatively low concentrations of cadmium (up to 50 mg·l-1 CdCl2.2H2o) results in a significant decrease in lethality to Artemia franciscana caused by ZnSo4.7H2o at concentrations of 50, 100 a 250 mg·l-1. Artemia franciscana, crustacean, cadmium, lethality, nauplii, zinc modern civilization together with increased industrial activities brought gradual redistribution of a number of toxic elements from the terrestrial crust into the environment and thus increased the potential exposure of humans and animals. With increasing pollution also the interest arises in consequences of the action of xenobiotics, including hazardous chemical elements, on live organisms (kovalkovičová et al. 2000; Eliášová et al. 2003; Beyer et al. 2005; obi et al. 2006; Žáková et al. 2006). of the various toxic elements heavy metals, particularly cadmium, lead, mercury and zinc occur frequently in the environment due to their relatively high industrial use. The presence of these elements in tissues reflects the contact of organisms with their environment (Pechová et al. 1998). They belong to cumulative poisons that are toxic at low doses. The metabolism and toxicity of these elements depend to a considerable degree on their interactions with essential elements that are necessary for the nutrition of live organisms, such as calcium, zinc, iron, selenium, copper, chromium and manganese. From the viewpoint of water pollution observation it is suitable to focus on the examination of water plankton. This may include observation of direct pollution of aqueous environment, i.e. the effect of pollutants on plankton and its organisms, or observation of accumulation of pollutants in the food chain, as plankton constitutes its lower links. Cadmium belongs to heavy metals widely distributed in the environment. it is present ACTA VET. BRNO 2007, 76: 635-642; doi:10.2754/avb200776040635 Address for correspondence: mvdr. Jaroslava Nováková University of veterinary medicine in košice department of radiobiology komenského 73 041 81 košice, Slovak Republic Phone: +421 903 934 543 E-mail: novaks@internetkosice.sk http://www.vfu.cz/acta-vet/actavet.htm in trace amounts in the oceans and in a wide range of plant and animal species. in nature, it is found together with zinc at a ratio of 1 : 100, or even 1 : 1000. it is obtained as a by-product of refining. Cadmium compounds are used in the electroplating of metals, alkaline batteries or in compounds with other metals. relatively high quantities of cadmium are present in phosphate fertilisers (from some locations), which increase the concentration of cadmium in soil and plants. Cadmium is an element highly toxic to organisms living in the aqueous environment (koréneková et al. 2002; Drastichová et al. 2004). This is only one of the reasons why the crustacean Artemia franciscana is a suitable subject of these studies. The current trend in toxicology is the reduction of experiments on higher vertebrates. one of the possibilities is the use of alternative biotest of the 2nd generation performed on A. franciscana (Dvořák 1995; Dvořák and Beňová 2002). For conducting detailed observations the prolonged 10-day biotest is a more suitable alternative (Dvořák et al. 2005). With regard to the taxonomic revisions in nomenclature of the order Artemia, the majority of older studies referred to this species as Artemia salina. Zinc is one of the elements that can decrease the toxicity of orally administered cadmium and its effect can be observed in competition with cadmium in certain transportation systems as well as at binding sites on metallothionein (McDowell 1992; Barata et al. 2002; Seebaugh and Wallace 2004). Zinc and cadmium do not occur in the organism separately but they are bound to metallothionein an intracellular, low-molecular protein rich in cysteine. only after blocking all binding sites on this protein, the metals pass to the blood and tissues as free ions and cause intoxication. Under the action of medium and low concentrations of zinc and low concentrations of cadmium the metallothionein synthesis increases within several hours, which in turn increases the binding capacity of heavy metals (Barata et al. 2002; Trinchella et al. 2006). However, if the time for metallothionein synthesis is insufficient due to parallel administration of zinc and cadmium, statistically significant changes in the influence of zinc on the prevention of cadmium toxicity can not be observed (Hua-Luo et al. 2002; Martinez et al. 1999). The aim of the present study was to observe synergistic and antagonistic effects of cadmium and zinc in the form of water-soluble salts of zinc sulphate and cadmium chloride on lethality to artemia franciscana. Materials and Methods The experiment was carried out employing a 10-day biotest (Dvořák et al. 2005). We used Artemia franciscana hatched in sea-water (Dvořák 1995). Ten freshly hatched nauplii were placed into polystyrene Petri dishes, 60 mm in diameter, and the total content of sea-water was 10 ml including the sample. A. franciscana were provided glucose as an additional feed (Dvořák et al. 2005). During the experiment we used solutions of cadmium chloride (CdCl2.2H2o) at concentrations of 5, 10, 15, 25, 50, 100 and 250 mg and of zinc sulphate (ZnSo4.7H2o) at concentrations of 50 mg, 100 mg and 250 mg. all solutions used in our experiments were prepared in sea-water. The study was conducted on 27 experimental groups and one control group (pure sea-water). in every experiment we used 50 A. franciscana divided into 5 individual groups (dishes), 10 A. franciscana in each. Therefore 1,400 individual A. franciscana were used in the study. Petri dishes were placed into a thermostat set to the temperature of 20 ± 1 °C. during the subsequent 10 days the live A. franciscana were counted once in 24 h. as we were interested in the synergistic or antagonistic effects of the selected chemical substances, we compared the results obtained in the experimental groups (marked in our case Zn 50, Zn 100 and Zn 250) and evaluated them statistically. in order to eliminate the distant values we used the Dean-Dixon test. The significance of differences between individual A. franciscana groups was tested (Wayland and Hayes 1991).

modern civilization together with increased industrial activities brought gradual redistribution of a number of toxic elements from the terrestrial crust into the environment and thus increased the potential exposure of humans and animals.With increasing pollution also the interest arises in consequences of the action of xenobiotics, including hazardous chemical elements, on live organisms (kovalkovičová et al. 2000;Eliášová et al. 2003;Beyer et al. 2005;obi et al. 2006;Žáková et al. 2006). of the various toxic elements heavy metals, particularly cadmium, lead, mercury and zinc occur frequently in the environment due to their relatively high industrial use.The presence of these elements in tissues reflects the contact of organisms with their environment (Pechová et al. 1998).They belong to cumulative poisons that are toxic at low doses.The metabolism and toxicity of these elements depend to a considerable degree on their interactions with essential elements that are necessary for the nutrition of live organisms, such as calcium, zinc, iron, selenium, copper, chromium and manganese.
From the viewpoint of water pollution observation it is suitable to focus on the examination of water plankton.This may include observation of direct pollution of aqueous environment, i.e. the effect of pollutants on plankton and its organisms, or observation of accumulation of pollutants in the food chain, as plankton constitutes its lower links.
Cadmium belongs to heavy metals widely distributed in the environment.it is present in trace amounts in the oceans and in a wide range of plant and animal species. in nature, it is found together with zinc at a ratio of 1 : 100, or even 1 : 1000. it is obtained as a by-product of refining.Cadmium compounds are used in the electroplating of metals, alkaline batteries or in compounds with other metals.relatively high quantities of cadmium are present in phosphate fertilisers (from some locations), which increase the concentration of cadmium in soil and plants.Cadmium is an element highly toxic to organisms living in the aqueous environment (koréneková et al. 2002;Drastichová et al. 2004).This is only one of the reasons why the crustacean Artemia franciscana is a suitable subject of these studies.The current trend in toxicology is the reduction of experiments on higher vertebrates.one of the possibilities is the use of alternative biotest of the 2 nd generation performed on A. franciscana (Dvořák 1995;Dvořák and Beňová 2002).For conducting detailed observations the prolonged 10-day biotest is a more suitable alternative (Dvořák et al. 2005).With regard to the taxonomic revisions in nomenclature of the order Artemia, the majority of older studies referred to this species as Artemia salina.
Zinc is one of the elements that can decrease the toxicity of orally administered cadmium and its effect can be observed in competition with cadmium in certain transportation systems as well as at binding sites on metallothionein (McDowell 1992;Barata et al. 2002;Seebaugh and Wallace 2004).
Zinc and cadmium do not occur in the organism separately but they are bound to metallothionein -an intracellular, low-molecular protein rich in cysteine.only after blocking all binding sites on this protein, the metals pass to the blood and tissues as free ions and cause intoxication.Under the action of medium and low concentrations of zinc and low concentrations of cadmium the metallothionein synthesis increases within several hours, which in turn increases the binding capacity of heavy metals (Barata et al. 2002;Trinchella et al. 2006).
However, if the time for metallothionein synthesis is insufficient due to parallel administration of zinc and cadmium, statistically significant changes in the influence of zinc on the prevention of cadmium toxicity can not be observed (Hua-Luo et al. 2002;Martinez et al. 1999).
The aim of the present study was to observe synergistic and antagonistic effects of cadmium and zinc in the form of water-soluble salts of zinc sulphate and cadmium chloride on lethality to artemia franciscana.

Materials and Methods
The experiment was carried out employing a 10-day biotest (Dvořák et al. 2005).We used Artemia franciscana hatched in sea-water (Dvořák 1995).Ten freshly hatched nauplii were placed into polystyrene Petri dishes, 60 mm in diameter, and the total content of sea-water was 10 ml including the sample.A. franciscana were provided glucose as an additional feed (Dvořák et al. 2005).During the experiment we used solutions of cadmium chloride (CdCl 2 .2H 2 o) at concentrations of 5, 10, 15, 25, 50, 100 and 250 mg and of zinc sulphate (ZnSo 4 .7H 2 o) at concentrations of 50 mg, 100 mg and 250 mg.all solutions used in our experiments were prepared in sea-water.The study was conducted on 27 experimental groups and one control group (pure sea-water).
in every experiment we used 50 A. franciscana divided into 5 individual groups (dishes), 10 A. franciscana in each.Therefore 1,400 individual A. franciscana were used in the study.Petri dishes were placed into a thermostat set to the temperature of 20 ± 1 °C.during the subsequent 10 days the live A. franciscana were counted once in 24 h.
as we were interested in the synergistic or antagonistic effects of the selected chemical substances, we compared the results obtained in the experimental groups (marked in our case Zn 50, Zn 100 and Zn 250) and evaluated them statistically. in order to eliminate the distant values we used the Dean-Dixon test.The significance of differences between individual A. franciscana groups was tested (Wayland and Hayes 1991).

Results
Fig. 1 and tables include the control group that was not exposed to any chemical substance added artificially to the aqueous environment because the lethality in this group even after 240 h was lower than 20%.This condition must be met in order to evaluate the test (Dvořák et al. 2005).all other results are presented in Tables 2 and 3 and in Fig.  1.For both separately acting zinc (Zn 50, Zn 100 and Zn 250) and cadmium (Cd 5 to Cd 250) the relationship between lethality and concentration is obvious.These groups served as comparison groups for the studies of mutual interactions.
a significant increase in lethality compared to the first zinc exposure (Zn 50) was observed on day 8 of observation with the combination Zn50Cd50, on days 6 to 8 with the combination Zn50Cd100 and on days 3 to 8 with the combination Zn50Cd250.
a significant decrease in lethality compared to the lowest zinc exposure group (Zn 50) was recorded on days 9 and 10 with combination Zn50Cd5 and on day 6 with combination Zn50Cd10.
a significant increase in lethality compared to the second zinc exposure group (Zn 100) was observed on day 4 with combinations Zn100Cd25 and Zn100Cd50, on days 3 to 6 from the beginning of the experiment with the combination Zn100Cd100 and on days 2 to 8 with the combination Zn100Cd250.
a significant decrease in lethality compared to the second zinc exposure group (Zn 100) was detected on days 5 to 10 with combinations Zn100Cd5 and Zn100Cd10 and on days 7 to 10 with combinations Zn100Cd25 and Zn100Cd50.a significant increase in lethality compared to the third zinc exposure group (Zn 250) was observed on days 3 to 6 with combination Zn250Cd100 and as early as on days 2 to 7 with combination Zn250Cd250.a significant decrease in lethality compared to the third zinc exposure group (Zn 250) was recorded on days 6 to 10 with the combination Zn250Cd5, on days 7 to 9 with the combination Zn250Cd10, 6 to 9 with the combination Zn250Cd25 and 7 to 8 with the combination Zn250Cd50 (Tables 2 and 3).
other differences were statistically non-significant.
The effect of interactions of low cadmium concentrations (Cd 5, Cd 10 and Cd 25) is illustrated in Fig. 1.This graph indicates that the toxicity of individual concentrations of zinc is decreased by low concentrations of cadmium used in optimum concentration combinations.637

Discussion
Cadmium is an element highly toxic to live organisms (koréneková et al. 2002).its toxicity was described also for organisms living in the aqueous environment (drastichová et al. 2004).For this reason it appeared advantageous to use the crustacean A. franciscana in our experiments (Dvořák 1999).The alternative 10-day biotest was used for the first time to examine the mutual interactions of toxic elements.its main advantage is the limited influence of biological variability, i.e. random results.our study was conducted on a large number of experimental crustaceans (28 groups, 50 in each, i.e. 1,400 A. franciscana).an experiment of similar extent hardly be conducted on higher vertebrates or it would require extraordinary expenses.

638
(+*) differences between the values marked with the same symbol were significant (α = 0.05) it has been recognised that zinc is one of the elements that can decrease the toxicity of orally administered cadmium.its effect has been observed in competition with cadmium in certain transport systems or for binding sites on metallothionein (McDowell 1992;Barata et al. 2002;Seebaugh and Wallace 2004).Zinc has a favourable effect on liver cells, protecting them against damage and helping to preserve membrane integrity through direct action on free radicals.When administered before or together with cadmium, it can protect kidneys against the nephrotoxic effect of cadmium without decreasing its level in this organ.
Zinc and cadmium do not occur alone in the organism but they are bound to metallothionein -an intracellular, low-molecular protein rich in cysteine.only after all binding sites on this protein become occupied, the metals pass to blood and tissues as free ions and cause intoxication.Under the action of medium and low concentrations of zinc and low concentrations of cadmium the metallothionein synthesis increases within several hours which results in the increase of binding capacity for heavy metals (Barata et al. 2002;Trinchella et al. 2006).However, if the time for metallothionein synthesis is insufficient due to parallel administration of zinc and cadmium, one may observe no significant preventive effect of the addition of zinc on toxicity of cadmium (Hua et al. 2002;Martinez et al. 1999).
Studies of many authors indicated that the relationship between cadmium and zinc is important for the toxicology of cadmium (Dvořák 1999).The protective role of higher uptake of zinc during cadmium intoxication and higher accumulation and toxicity of cadmium during zinc deficiency suggest that the metabolism and effect of cadmium on the organism may be modified by regulation of the zinc uptake.
additional multiple interactions can also play an important role.The study by Dvořák and Šucman (1996) reported interactions between cadmium chloride, potassium dichromate 90 Sr radionuclide and polychlorinated biphenyls.The paper mentioned predominantly only synergistic, toxicity-increasing effects.Contrary to the studies mentioned above, our experiments indicate that mutual combinations of individual concentrations of both elements are of decisive importance, i.e. suitably low concentrations of cadmium can also decrease toxicity of zinc which is an observation that has not been reported before.Toxicological hormesis can also be important with regard to the final effect (Beňová et al. 2007).
in conclusion, relatively high concentrations of zinc and cadmium resulted in a synergistic effect of these two metals.However, at suitable combinations of zinc and cadmium concentrations and elapse of sufficient time after the onset of action of these substances, a significant decrease in their lethality to A. franciscana was observed.

Table 1 .
Experimental A. franciscana were divided to 27 experimental groups as follows

Table 2 .
Lethality to Artemia franciscana (%) resulting from single action either of cadmium chloride (CdCl 2 .2H 2 o) or zinc sulphate (ZnSo 4 •7H 2 o) in dependence on the time of action (+*) differences between the values marked with the same symbol were significant (α = 0.

Table 3 .
Lethality to Artemia franciscana (%) resulting from single action either of cadmium chloride (CdCl 2 .2H 2 o) or zinc sulphate (ZnSo 4 .7H 2 o) in dependence on the time of action