Potassium Dichromate as a Reference Substance for Embryonic Tests of Toxicity in the Common Carp ( Cyprinus carpio L . )

Krejãí R. , M. Pal íková: Potassium Dichromate as a Reference Substance for Embryonic Tests of Toxicity in the Common Carp (Cyprinus carpio L.). Acta Vet. Brno, 75: 259-263. Potassium dichromate (K2Cr2O7) has already been used as a reference substance in tests of toxicity with aquatic animals. The aim of this study was to determine and compare values of LC50 for potassium dichromate during the whole period of embryonic development (i.e., 120 h) and 48 h after hatching of embryos in the common carp (Cyprinus carpio L.). Fish eggs and embryos were exposed to 5 different concentrations of potassium dichromate (i.e., 372, 409, 450, 495, 545 mg⋅l-1) during two experiments. Such characteristics as the cumulative mortality, the start and the end of hatching, the number of deformities, body length, and body mass of surviving individuals were studied during the tests. The highest mortality was found in the hatched embryos. Mortality and frequency of deformities increased with the growing concentration of potassium dichromate. The value of 120 LC50 for potassium dichromate was 464.91 ± 23.83 mg⋅l-1 and the value of 48 LC50 was 458.94 ± 4.14 mg⋅l-1 (mean ± SD). No statistically significant difference between values 120 LC50 a 48 LC50 was found. This is why reduction of the exposure period to only 48 h after hatching seems a reasonable method to study the control of susceptibility using potassium dichromate in embryonic tests of toxicity. Lethal concentration, embryonic toxicity, mortality, deformations, hatching The toxicity of chemical substances for aquatic organisms may be influenced by a variety of factors such as the physical and chemical characteristics of water (e.g., temperature, pH, oxygen concentration, concentrations of salts dissolved in water, etc.) and the state of health of the animals tested. Although it is possible to create standard laboratory conditions, maintaining indicators of the state of health as well as susceptibility may be more problematic. That is why toxicity tests employing reference substances are used in ecotoxicology. Results of these tests enable comparisons with those obtained by testing various substances on fish of different origin. Potassium dichromate (K2Cr2O7) is the most commonly used and recommended reference substance for aquatic organisms (âSN EN ISO 6341, 1996 and âSN EN ISO 7346, 1996). The effect of chromium on the fish organism has already been studied in detail. Studies were particularly focused on the hexavalent chromium that is more toxic to aquatic organisms than chromium with the valency of III (Van der Put te et al. 1981ab). Effects of chromium on fish are reported to include damage of gills, impairment of osmoregulation, blood count changes, etc. (Al-Akel et al. 1996; Nath et al. 1988; Van der Put te et al. 1982). Chromium CrVI is more toxic at a lower pH, like other metals (Stouthar t et al.1996). Significantly higher levels of chromium were found in the epithelium of gills in the rainbow trout kept at the pH of 6.5 as compared to the pH of 7.8 (Van der Put te et al. 1981ab). In eggs and larvae of the common carp, Stouthar t et al. (1995) also found the mortality as well as the accumulation of chromium to be the significantly highest at the pH of 6.3. These ACTA VET. BRNO 2006, 75: 259–263 Address for correspondence: Ing. Roman Krejãí, Ph.D. Department of Zoology, Fisheries, Hydrobiology and Apiculture Mendel’s Agricultural and Forestry University in Brno Zemûdûlská 1, 613 00 Brno, Czech Republic Phone: +420 545 133 267 E-mail: krejca@mendelu.cz http://www.vfu.cz/acta-vet/actavet.htm authors also found that the chorion and the perivitelline membrane of eggs acts like an effective barrier against CrVI and that after hatching the accumulation of CrVI in embryos and larvae starts to grow. In spite of that, there are no published data on the medium lethal dose for fish eggs and sac-fry of the common carp. This paper is aimed at finding and comparing the LC50 values for potassium dichromate and the whole embryonic development (120 h) as well as the period of 48 h after hatching. Materials and Methods Two experiments were performed in order to evaluate the influence of different concentrations of potassium dichromate during the embryonic development in the common carp. Experiments were carried out to determine the influence of potassium dichromate during the whole embryonic development, i.e., from fertilization to the end of the embryonic period, and during 48 h after hatching, i.e., from hatching to the end of the embryonic development. Experiments were finished when control individuals had the gas bladders filled and were able to take feed. Fish eggs and embryos were subjected to the same concentrations of potassium dichromate. Values of LC50 (120 LC50 and 48 LC50) were computed for individual periods of development. Both experiments were performed under the same laboratory conditions and in accordance with the OECD 212 (1998) guideline. Eggs of the common carp were obtained using artificial spawning and unstuck for one hour by whole milk diluted with water in the ratio of 1:9. Production lines of the common carp were used for stripping (spawner female PL, spawner male M72). Fish eggs were used for the experiment 8 h after fertilization. Embryos were used just after hatching. Until hatching, fish eggs were incubated in Zugs vials in a re-circulating unit. Fish eggs and embryos were stocked 15 individuals per 15 ml of water for dilution prepared according to the Czech State Norm âSN EN ISO 7346 (1996). Water exchange was made using a semi-static method every 12 hours. Fish eggs and embryos were transferred using a fine sieve. Temperature of the medium during the experiment was kept at 23.5 ± 0.3 °C, pH values varied in the range of 6.0 ± 0.1, and the O2 concentration ranged from 7.5 to 8.0 mg⋅l-1 (90 95% saturation). Photoperiod consisted of 14 h light and 10 h dark. The experiments included controls with water for dilution and 5 different concentrations of potassium dichromate (i.e., 372, 409, 450, 495, 545 mg⋅l-1) in three repeated measurements determined by calculation on the basis of LC50 found in preliminary tests. Characteristics such as cumulative mortality, the onset and the end of hatching, the number of deformities, total body length (TL) and body mass (w) of surviving individuals were determined during the experiments. Body length and mass values of the surviving individuals were determined after 3 months of the specimens being fixed in 4% formalin. A computer probit analysis program was used to determine concentrations of potassium dichromate in the experiments as well as the LC50 values. Values of LC50 after 120 and 48h were compared using t-test in STATplus (Matou‰ková et al. 1992). As the tests were performed in accordance with the OECD 212 guidelines and under identical conditions, we obtained statistical evaluation of six individual tests of toxicity.

The toxicity of chemical substances for aquatic organisms may be influenced by a variety of factors such as the physical and chemical characteristics of water (e.g., temperature, pH, oxygen concentration, concentrations of salts dissolved in water, etc.) and the state of health of the animals tested.Although it is possible to create standard laboratory conditions, maintaining indicators of the state of health as well as susceptibility may be more problematic.That is why toxicity tests employing reference substances are used in ecotoxicology.Results of these tests enable comparisons with those obtained by testing various substances on fish of different origin.Potassium dichromate (K 2 Cr 2 O 7 ) is the most commonly used and recommended reference substance for aquatic organisms (âSN EN ISO 6341, 1996 andâSN EN ISO 7346, 1996).
The effect of chromium on the fish organism has already been studied in detail.Studies were particularly focused on the hexavalent chromium that is more toxic to aquatic organisms than chromium with the valency of III (Van der Putte et al. 1981ab).Effects of chromium on fish are reported to include damage of gills, impairment of osmoregulation, blood count changes, etc. (Al-Akel et al. 1996;N ath et al. 1988;Van der Putte et al. 1982).Chromium Cr VI is more toxic at a lower pH, like other metals (Stouthart et al.1996).Significantly higher levels of chromium were found in the epithelium of gills in the rainbow trout kept at the pH of 6.5 as compared to the pH of 7.8 ( Van der Putte et al. 1981ab).In eggs and larvae of the common carp, Stouthart et al. (1995) also found the mortality as well as the accumulation of chromium to be the significantly highest at the pH of 6.3.These authors also found that the chorion and the perivitelline membrane of eggs acts like an effective barrier against Cr VI and that after hatching the accumulation of Cr VI in embryos and larvae starts to grow.In spite of that, there are no published data on the medium lethal dose for fish eggs and sac-fry of the common carp.
This paper is aimed at finding and comparing the LC 50 values for potassium dichromate and the whole embryonic development (120 h) as well as the period of 48 h after hatching.

Materials and Methods
Two experiments were performed in order to evaluate the influence of different concentrations of potassium dichromate during the embryonic development in the common carp.Experiments were carried out to determine the influence of potassium dichromate during the whole embryonic development, i.e., from fertilization to the end of the embryonic period, and during 48 h after hatching, i.e., from hatching to the end of the embryonic development.Experiments were finished when control individuals had the gas bladders filled and were able to take feed.Fish eggs and embryos were subjected to the same concentrations of potassium dichromate.Values of LC 50 (120 LC 50 and 48 LC 50 ) were computed for individual periods of development.Both experiments were performed under the same laboratory conditions and in accordance with the OECD 212 (1998) guideline.
Eggs of the common carp were obtained using artificial spawning and unstuck for one hour by whole milk diluted with water in the ratio of 1:9.Production lines of the common carp were used for stripping (spawner female PL, spawner male M72).Fish eggs were used for the experiment 8 h after fertilization.Embryos were used just after hatching.Until hatching, fish eggs were incubated in Zugs vials in a re-circulating unit.
Fish eggs and embryos were stocked 15 individuals per 15 ml of water for dilution prepared according to the Czech State Norm âSN EN ISO 7346 (1996).Water exchange was made using a semi-static method every 12 hours.Fish eggs and embryos were transferred using a fine sieve.Temperature of the medium during the experiment was kept at 23.5 ± 0.3 °C, pH values varied in the range of 6.0 ± 0.1, and the O 2 concentration ranged from 7.5 to 8.0 mg⋅l -1 (90 -95% saturation).Photoperiod consisted of 14 h light and 10 h dark.The experiments included controls with water for dilution and 5 different concentrations of potassium dichromate (i.e., 372, 409, 450, 495, 545 mg⋅l -1 ) in three repeated measurements determined by calculation on the basis of LC 50 found in preliminary tests.
Characteristics such as cumulative mortality, the onset and the end of hatching, the number of deformities, total body length (TL) and body mass (w) of surviving individuals were determined during the experiments.Body length and mass values of the surviving individuals were determined after 3 months of the specimens being fixed in 4% formalin.
A computer probit analysis program was used to determine concentrations of potassium dichromate in the experiments as well as the LC 50 values.Values of LC 50 after 120 and 48h were compared using t-test in STATplus (Matou‰ková et al. 1992).As the tests were performed in accordance with the OECD 212 guidelines and under identical conditions, we obtained statistical evaluation of six individual tests of toxicity.

Cumulative mortality
Mortality during 120 h of exposure (Table 1) Mortality during the first 12 h of exposure to potassium dichromate varied from 1.1 to 4.4%.There was no mortality during the period from 12 h until hatching (except for the control).The highest mortality was found in the period after hatching.It was growing with the increasing concentration of potassium dichromate.No increase in mortality was observed in control individuals after hatching.Mortality during 48 h of exposure (Table 1) There was no mortality in the control groups.Growing concentrations of potassium dichromate resulted in growing mortality of individuals.

Hatching
Table 2 presents the start and end of hatching in both experiments.In groups exposed to potassium dichromate hatching started from 57.3 to 61.5 h, and was finished from 69.2 to 74.2 h.Hatching started at 62.8 h, and ended at 73 h in the control group.

Deformities
The number of deformities varied from 6.7 to 6.8 % in control animals (Table 2).A control animal without deformities is presented in Fig. 1 (Plate IX).Deformities of the vertebral column and yolk sac were most frequent in control groups.The number of deformities was growing with the increasing concentration of potassium dichromate.Deformities of embryos exposed for 120 h to potassium dichromate included bowing of the vertebral column (Fig. 2), deformities of the head, missing fin margins (Fig. 2), strangulation and enlargement of the yolk sac (Fig. 2 and 3).Swelling of the heart area was very frequent at higher concentrations of potassium dichromate (Fig. 3, Plate X, Fig. 4, 5).Individuals exposed to potassium dichromate for 48 h showed bowing of the vertebral column less frequently.This duration of exposure resulted most frequently in deformities of the head, yolk sac and swelling of the heart area.

Body length and mass
Table 3 presents such characteristics as the total body length (TL) and body mass (w) of individuals surviving until the end of the experiment.The total body length and body mass in individuals exposed to different concentrations of potassium dichromate were even lower than in the control animals.LC 50 estimate for potassium dichromate The value of 120 LC 50 for potassium dichromate was 464.91 ± 23.83 mg⋅l -1 (mean ± SD) and the value of 48 LC 50 was 458.94 ± 4.14 mg⋅l -1 .No significant difference was found between these values.

Discussion
Mortality during the first 12 h of the experiment was probably caused by unfertilized fish eggs or damage through handling during transport.
It is mostly reported that the LC 50 value for the hexavalent chromium amounts to 96 h in fish.According to B e n o u t (1976), the 96 LC 50 values for the rainbow trout and the brook trout equal to 69 and 59 mg Cr⋅l -1 , respectively.A l -A k e l (1996) mentions the 96 LC 50 of hexavalent chromium in the common carp to be 93.6 mg⋅l -1 .These authors, however, were not experimenting with embryos, but older age categories.Buhl (1997) reports the 96 LC 50 value of hexavalent chromium of three species of fish (i.e., colorado squawfish -Ptychocheilus lucius, bonytail -Gila elegans and razorback sucker -Xyrauchen texanus) in the range of 32 -123 mg⋅l -1 .This author reports the same or higher susceptibility of larvae than juveniles of these species of fish.All these values are many times lower than those found in our study, but they concern a longer time period, i.e., 96 h as compared to 48 h of our experiments.It should hold good that the longer the test period, the lower the lethal concentration of the substance.Such results are reported, e.g., by M u r t i et al. (1983) for chromium and the freshwater prawn -Macrobrachium lamarreia.The LC 50 values are 24, 48, 72, and 96 h and show a decreasing tendency of 5.44, 3.69, 2.47, and 1.84 mg⋅l -1 , respectively.Comparing the 96 LC 50 and 48 LC 50 values, we see that the first one is twice as high as the second.V y k u s o v á and S v o b o d o v á (1990) report the range of the 48 LC 50 values for chromium in the common carp to be 100 -350 mg⋅l -1 .Again, these authors were not engaged in embryonic tests, but tests using small fish.
From our results it is clear that the highest mortality in all concentrations of potassium dichromate can be found after hatching, i.e. in the life period when embryos are away from the egg envelopes.This statement has also been confirmed by Stouthart (1995) finding the highest mortality in hatched embryos.The content of Cr VI in fish eggs drops nearly to zero levels during hatching and starts to grow in the embryos again after hatching.The higher mortality of the period after hatching can be explained by a barrier formed by the perivitelline membrane of fish eggs, preventing the accumulation of Cr VI in embryos.This fact is reflected in values of LC 50 determined after 120 and 48 h, which are very similar.Considering the fact that chromium accumulates in the embryos only after hatching, the duration of action of potassium dichromate lasts de facto 48 h from the total of 120 h of exposure.The difference between 120 LC 50 and 48 LC 50 values amounted to 4.43 % (A) and 6.42 % (B).The LC 50 values differing by up to 10 %, however, are considered to be rather the same.
The 120 LC 50 value may be, due to the susceptibility of embryos to potassium dichromate only after hatching, influenced by this process.The time of hatching may influence the mortality of individuals due to differences in the action of potassium dichromate on hatched embryos.An unusually early or late start of hatching, on the other hand, may also influence mortality and thus the LC 50 value.
No statistically significant deference between values 120 LC 50 and 48 LC 50 was found.That is why the reduction of the exposure period to only 48 h after hatching seems a reasonable method to study the control of susceptibility using potassium dichromate in embryonic tests of toxicity.A similar reduction of the exposure to a reference substance (from 96 to 24 h) has already been accepted in the Czech State Norm âSN EN ISO 7346.

Fig. 4 .
Fig. 4. Embryo (120 h old) exposed to the action of K 2 Cr 2 O 7 at the concentration of 495 mg⋅l -1 .Deformities of yolk sac and head, heart area oedema.Magnification × 20