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Chlorotriazine Reactive Azo Red 120 Textile Dye Induces Micronuclei in Fish
Ecotoxicology and Environmental Safety 47, 149}155 (2000) Environmental Research, Section B doi:10.1006/eesa.2000.1931, available online at http://www.idealibrary.com on
Chlorotriazine Reactive Azo Red 120 Textile Dye Induces Micronuclei in Fish Kabil Al-Sabti Joz\ ef Stefan Institute, P.O. Box 3000, Jamova 39, 1001 Ljubljana, Slovenia Received September 27, 1999
The main objective of this work was to use micronucleus induction in 5sh erythrocytes to study the risk to aquatic ecosystems due to the genotoxicity of Chlorotriazine Reactive Azo Red 120 textile dye. The frequencies of micronuclei were studied for three low doses of 1, 5, and 10 mg/L and blood sampling was carried out on the same 5sh after 3, 6, and 9 days. It was found that micronuclei increased not only in a dose-dependent manner but also in a time-dependent way, compared with negative (tap water) and positive (10 ppm benzene) control groups. There was also a slight, time-dependent increase in erythrocyte micronuclei of the control 5sh specimens. This study proved the genotoxicity of this dye, and suggests that further studies should be made on other dyes and some other toxic industrial pollutant discharges in water ecosystems, using 5sh as an indicator to monitor pollutant genotoxicity. 2000 Acamedic Press Key Words : micronuclei; genotoxicity; 5sh erythrocytes; water; biomarker; Chlorotriazine Reactive Azo Red 120 dye.
INTRODUCTION
The increasing contamination of the aquatic environment in Slovenia by di!erent industrial waste products has proven to have direct and/or indirect e!ects on human health. It has been found that biological testing is a scienti"cally valid approach to controlling toxic substances in industrial wastewater discharges (Al-Sabti et al., 1994). The textile business started thousands of years ago, but the development of modern technology for textile production, including the use of many arti"cial chemicals to improve products, makes it a serious problem with respect to the discharge of waste products directly and indirectly to aquatic ecosystems. At present there are about 3000 di!erent dyes available on the commercial market, and more than half of these are azo compounds (Majcen}Le Marechal et al., 1997). The industrial discharge of chemical waste products of the textile industry directly into Slovenian rivers has increased the amounts of these substances in the aqueous environment, although this trend, as in most countries, is now being halted or reversed due to legislation that limits
the use of toxic chemicals. For that reason there is also a debate about the level of such textile waste products, particularly textile dyes, permitted to be discharged directly into Slovenian waters, according to various studies monitoring the e!ect of such chemicals on human health. Although occupational and accidental chemical exposure does still occur, food is now the main route of exposure to these chemicals for the general population. Fish may act as &&sentinel'' organisms, indicating the potential for exposure of human populations to genotoxic chemicals in drinking water. Food is the major route of human exposure to toxic chemicals, and "sh and shell"sh have been recognized as major vectors for contaminant transfer to humans. For example, marine "sh and shell"sh, which are a major source of protein in many countries, are often contaminated with high concentrations of methyl mercury (WHO, 1990). Induction of chromosomal damage has been found in the lymphocytes of persons exposed to methyl mercury through consumption of contaminated "sh (Skerfving et al., 1974), although the precise mechanism involved in this process is not yet understood. Several chemicals or chemical mixtures induce clastogenic e!ects in "sh. Fish may accumulate pollutants from direct contamination by chemicals present in water or indrectly by feeding on living organisms in the water that accumulate pollutants. Genotoxicological data are unavailable for thousands of toxic compounds that are routinely discharged into surface waters. It is important to attempt to detect the early genotoxic e!ects of wastewater pollutants on "sh, at the actual concentrations concerned. Within the last decade the micronucleus test in "sh has played an important role in assessing exposure to water pollutants, and has proved to provide an early warning of genotoxic threats to "sh, their ecosystem, and "nally humans. Induction of cytogenetic damage in "sh could serve for monitoring not only selected genotoxic agents in the laboratory, but also the presence of genotoxicants in surface waters and di!erent aqueous ecosystems. The convenience of the micronucleus (MN) test needs no emphasis since data can be scored at any time; moreover, data
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on micronuclei might not only be useful as evidence supporting chromosome aberration frequencies induced by genotoxic agents but may also be important for the study of the relative radiosensitivity and chemosensitivity of di!erent tissues. Such investigations are lacking in "sh genotoxicity testing and the study described here is an attempt to advance in this direction. Various studies have found that the peripheral erythrocytes of "sh have a high incidence of micronuclei after exposure to di!erent pollutants under "eld and laboratory conditions (Hooftman and Raat, 1982); Longwell et al., 1983; Al-Sabti, 1986a, b; Das and Nanda, 1986; Manna and Sadhukhan, 1986; Hose et al., 1987; Metcalfe, 1988; Smith, 1990; Al-Sabti and Hardig, 1990; Carrasco et al., 1990; Hughes and Hebert, 1991; Ueda et al., 1992; Kurihara et al., 1992; Schultz et al., 1993; De Flora et al., 1993; Al-Sabti et al., 1994; Bahari et al., 1994; Nepomuceno et al., 1997; Campana et al., 1998; Pacheco and Santos, 1998, 1999). Observations of micronuclei can be made very rapidly, because in most "sh species the erythrocytes are sizable and have a large nucleus, enabling their use in such investigations. Micronuclei that arise from chromosomes or chromosomal fragments that are not incorporated into daughter nuclei at mitosis due to the lack of a centromere have proven to be useful for studying "sh under laboratory conditions. In this study, the genotoxic e!ects of exposure of "sh to three doses of the textile dye Chlorotriazine Reactive Azo Red 120 (CRARD-120) are determined by using MN induction in Prussian carp (Carassius auratus gibelio) erythrocyte cells under laboratory conditions. The three concentrations of CRARD-120 used in the present investigation were below those which cause gross cytotoxicty to "sh. MATERIAL AND METHODS
Fish Specimens of the Prussian carp, Carassius auratus gibelio, were chosen for this study because this "sh is a common and relatively sedentary species, which is available year-round in most "sh farms and some rivers and ponds in Slovenia. Specimens of Prussian carp with a weight and length of about 100 g and 12 cm, respectively, were supplied for this study by the Ribe Maribor "sh farm, in Rac\ e near Maribor in Slovenia. Before the experiment, "sh were acclimated for 7 days at a population density of 100 specimens in a 400-L tank with well-aerated nonchlorinated water at 153C and pH 7. Fish were allowed no food during the last 2 days of the experimental period. Experimental Design Chlorotriazine Reactive Azo Red 120 (CRARD-120) (Fig. 1), obtained from the Dye Factory of Belinka in Ljubljana, was added at three concentrations (sublethal) of
FIG. 1. Structure of Chlorotriazine Reactive Azo Red 120 dye.
1, 5, and 10 mg/L to 10-L aquaria containing seven "sh specimens each. Specimens were exposed to each of the di!erent CRARD-120 concentrations (1, 5, and 10 mg/L) for 3, 6, and 9 days. One or two drops of blood from each "sh specimen were sampled three times at 3, 6, and 9 days and analyzed. The concentration of CRARD-120 tended to decrease because of precipitation and adsorption, and so appropriate amounts of CRARD-120 stock solution were added after each spectrophotometric analysis of aquarium water. A negative tap water control group for all the experiments consisted of "sh specimens that were kept in a laboratory aquarium in clean tap water, while another seven specimens of Prussian carp were exposed for 3, 6, and 9 days to water polluted with 10 ppm of benzene. Micronucleus Test Before blood sampling, "sh were tagged by cutting one or a combination of two "ns to enable blood to be sampled from the same "sh specimen at 3, 6, and 9 days (time response) while exposed to the same concentration of CRARD-120 and from both negative and positive control groups. Blood samples for the smears were obtained by caudal vein puncture of the same "sh specimen at three intervals, and slides were prepared by the "sh micronucleated erythrocyte method (Al-Sabti, 1986a, b). From each "sh, two slides were prepared. After "xation in pure ethanol for 20 min, the prepared slides were left to air dry, and then the smears were stained with 5% Giemsa solution for 20 min. The slides were examined by light microscopy (Opton, Germany) under an oil immersion lens. Scoring of coded slides was done &&blind.'' Only erythrocyte cells that were isolated from surrounding cells were scored, and MNs were enumerated only if they were distinct from the two nuclei. One thousand erythrocytes per specimen for each slide (2000 per two slides) were analyzed in the subsequent treatments to determine the frequency of cells with one or two micronuclei. Statistical Analysis The standard deviation (SD) of the mean (X) of the number of micronuclei in mononucleated erythrocyte cells was calculated by the method of Becker et al. (1992), as a measure of variation in these parameters between duplicate erythrocyte cell samples exposed to the same treatment.
CHLOROTRIAZINE REACTIVE AZO RED 120 AND MICRONUCLEI
RESULTS
For clarity the results obtained for CRARD-120 for 3, 6, and 9 days are presented separately and for each individual "sh in Table 1, together with both the negative and positive control groups. Table 1 and Fig. 2 summarize MN induction per 1000 erythrocyte cells for the three di!erent CRARD-120 concentrations, compared with the negative tap water and positive 10 ppm benzene control groups. One "sh specimen exposed to 1 mg/CRARD-120, one to 10 mg/L CRARD-120, and two specimens from the control group were eliminated from the study, because after 7 to 8 days they were very weak and had physical injuries on their gills and skin that were probably the result of "shing and/or transport and handling. Each slide was found to contain at least 2000 to 3000 erythrocyte cells, and the number of mononucleated erythrocyte cells was greater (about 4000) in the control (untreated) groups than in the treated groups. Specimens of Prussian carp from the control treatments exhibited signi"cant di!erences in the frequency of micronuclei compared with those specimens exposed to CRARD-120. There was a minor increase in MN induction with time in the same negative control individuals analyzed at di!erent times of this study, while the positive control with 10 ppm of benzene demonstrated a signi"cant increase with time of exposure. Therefore, to evaluate the data, it was necessary to compare the genotoxic response in the chemical treatments with the response in the appropriate control treatment. DISCUSSION
The production and use of textile dyes make their fate in aquatic ecosystems of special interest. Evaluation of the toxicity of these dyes is hampered by the fact that there are many dyes with various chemical structures and di!ering e!ects on the environment. The biological activities of dyes also di!er greatly despite similarities in their structure, so their toxicological properties cannot be generalized by reference to one group only (Majcen}Le Marcehal et al., 1997). It has been found that biological testing is a scienti"cally valid approach to controlling toxic mixtures from various industrial wastewater discharges, including textile factories (Al-Sabti, 1995). A number of studies have provided evidence that "sh species possess metabolic mechanisms for the biotransformation of xenobiotics. Clastogenic activity may be a risk factor for genetic disease, teratogenesis, or carcinogenesis in "sh populations. However, testing of "sh for the clastogenic e!ects of chemicals has been hampered by operational problems with in vivo assays. Mutagenic chemicals have a high probability of inducing carcinogenic e!ects in various "sh species and the majority of these chemicals have been demo-
151
nstrated to cause tumors at speci"c or multiple sites in "sh (Harsbarger and Clark, 1990). As far as is known, only two studies have been published on the e!ect of some dyes in inducing micronuclei in "sh erythrocytes. Specimens of rainbow trout (Oncorhynchus mykiss) exposed to sublethal doses of wool shrink-proo"ng e%uent, Acid Violet 66 and Acid Red 217 (Marlasca et al., 1992, 1998), exhibited a signi"cant increase in erythrocyte MNs following 30 days of exposure. The present study was conducted on a di!erent "sh species and a di!erent dye, which could explain the di!erence between the present results and results of the above studies. The clastogenicity of the azo dye Direct Red 2 (DR2) has been investigated using the mouse murine bone marrow micronucleus assay; a potent dose-dependent response followed oral gavage of up to 4 mg/kg R2D after which signi"cant toxicity to the erythroid compartment was observed (Rajaguru et al., 1999). Table 1 and Fig. 2 illustrate the increased number of micronuclei in erythrocyte cells exposed to CRARD-120, not only as a dose response but also as a function of time. After 9 days the number of MNs resulting from exposure was found to increase when compared with that after 6 days. There might be a slight bias in the results probably related to the increase in organic substances in the water because of excretion and food additions. Another explanation could be related to the delay in mitosis and death of cells (Al-Sabti, 1994, Al-Sabti et al., 1994). Benzene was used as a positive control, because it represents a typical hydrocarbon in polluted waters. The positive control group exposed to 10 ppm of benzene and the group exposed to 10 mg/L of CRARD-120 exhibited similar induction of MNs after 9 days, whereas MN induction with the same dose of CRARD-120 was higher after exposure for 3 and 6 days compared with 10 ppm of benzene. The number of MNs induced in erythrocyte cells from the control group was also found to increase slightly with time, which can be related to shock, excrement, food addition, blood sampling, etc. But the results from the negative tap water control group are signi"cantly lower than those from the exposed groups, while the case is di!erent with the positive control after exposure to 10 ppm of benzene. When comparing e!ects of dose (1, 5, and 10 mg/L) (Figs. 2, 3 and Table 1), it can be seen that after 7 and 9 days, the values for MN induction with CRARD-120 di!er signi"cantly from those obtained after 3 days of exposure for all doses compared with the control. Except for CRARD-120 after 9 days of exposure, this observation is valid at the 95% con"dence level or better. The concentrations of CRARD-120 in the present investigation were lower than the threshold limit levels for natural waters. In Slovenia this limit is about 100 mg/L for CRARD-120, depending on the river quality category (I and II, or III and IV) (O.cial Gazette) of Republic of Slovenia, No. 18/85, 1985, p. 1073).
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KABIL AL-SABTI
TABLE 1 Frequencies of Micronuclei per 1000 Fish Erythrocytes from the Exposure to 1 (A), 5 (B), and 10 (C) mg/L CRARD-120, Compared with the Negative Tap Water (K) and Positive 10 ppm of Benzene (D) Controls for 3, 6, and 9 Days Exposure Specimen No.
Concentration (mg/L)
Days
MNs/specimen/ concn.
K1 K2 K3 K4 K5
Control Control Control Control Control
3 3 3 3 3
4.5 5.0 4.0 4.5 5.5
D1 D2 D3 D4 D5 D6 D7
Benzene Benzene Benzene Benzene Benzene Benzene Benzene
3 3 3 3 3 3 3
8.5 8.0 9.0 8.5 8.0 8.5 8.0
A1 A2 A3 A4 A5 A6
1 1 1 1 1 1
3 3 3 3 3 3
8.0 11.5 8.0 8.0 12.5 11.0
B1 B2 B3 B4 B5 B6 B7
5 5 5 5 5 5 5
3 3 3 3 3 3 3
16.0 17.0 16.5 14.0 16.5 18.0 14.5
C1 C2 C3 C4 C5 C6
10 10 10 10 10 10
3 3 3 3 3 3
20.0 18.5 20.5 18.0 19.0 20.0
K1 K2 K3 K4 K5
Control Control Control Control Control
6 6 6 6 6
5.0 5.0 5.5 5.0 5.5
D1 D2 D3 D4 D5 D6 D7
Benzene Benzene Benzene Benzene Benzene Benzene Benzene
6 6 6 6 6 6 6
14.5 14.5 14.5 15.5 16.0 15.5 15.0
A1 A2 A3 A4 A5 A6
1 1 1 1 1 1
6 6 6 6 6 6
11.5 13.5 11.5 11.5 14.0 12.0
B1 B2
5 5
6 6
20.0 23.0
Total No. of specimens
Total MN/all specimens/concn.
5
4.7$0.6
7
8.36$0.35
6
9.8$2.1
7
16.1$2.1
6
19.3$1.0
5
5.2$0.3
7
15.07$0.56
6
12.3$1.1
153
CHLOROTRIAZINE REACTIVE AZO RED 120 AND MICRONUCLEI
TABLE 1=Continued Exposure Specimen No.
Concentration (mg/L)
Days
MNs/specimen/ concn.
B3 B4 B5 B6 B7
5 5 5 5 5
6 6 6 6 6
18.5 18.5 22.5 18.0 22.0
C1 C2 C3 C4 C5 C6
10 10 10 10 10 10
6 6 6 6 6 6
24.5 23.5 22.5 23.0 21.5 25.5
K1 K2 K3 K4 K5
Control Control Control Control Control
9 9 9 9 9
6.6 6.0 5.7 6.4 6.1
D1 D2 D3 D4 D5 D6 D7
Benzene Benzene Benzene Benzene Benzene Benzene Benzene
9 9 9 9 9 9 9
24.5 23.5 23.5 23.5 24.5 24.5 25.5
A1 A2 A3 A4 A5 A6
1 1 1 1 1 1
9 9 9 9 9 9
16.5 16.0 14.5 14.0 17.0 15.0
B1 B2 B3 B4 B5 B6 B7
5 5 5 5 5 5 5
9 9 9 9 9 9 9
23.0 22.5 18.0 19.5 20.5 20.0 20.5
C1 C2 C3 C4 C5 C6
10 10 10 10 10 10
9 9 9 9 9 9
24.0 23.0 26.5 24.5 22.5 24.5
The present results for di!erent doses of CRARD120 could be used to interpret the data obtained from "eld monitoring. The mononucleated erythrocyte micronucleus assay may give a positive response in "sh exposed to both direct-acting and indirect-acting genotoxic chemicals. It is noted that some other factors, such as route of entry, duration and frequency of exposure, variations between di!erent species (interspecies), and variations among mem-
Total No. of specimens
Total MN/all specimens/concn.
7
18.9$1.4
6
22.8$0.8
5
6.2$0.4
7
24.2$0.7
6
15.5$1.2
7
20.6$1.7
6
24.6$1.4
bers of the same species (intraspecies), may also in#uence the toxicity of CRARD-120 and some other textile azo dye compounds. Such a study on the genotoxicity of CRARD120 can be used to establish exposure guidelines, and can help to establish restrictions on the waste discharges of such chemicals. The present results cannot be used alone as "nal data to assess the risk of industrial wastes, but they can form a foundation for decisions on further studies on other textile dyes and other water pollutants.
154
KABIL AL-SABTI
FIG. 2. Comparison of the frequency of MN induction ($SD) after three di!erent CRARD-120 exposure treatments (1, 5, 10 mg/L), compared with the negative (tap water) and positive (benzene/10 ppm) controls after 3, 6, and 9 days. For further explanation, see Table 1 and the text.
CONCLUSIONS
Using the micronucleus test, Chlorotriazine Reactive Azo Red 120 (CRARD-120) textile dye proved to be genotoxic to erythrocytes of Prussian carp (Carassius auratus gibelio) under laboratory conditions. Micronuclei were elevated not only in a dose-dependent (1, 5, and 10 mg/L) but also in a time-dependent (3, 6, and 9 days) manner, compared with the negative (tap water) and positive (10 ppm benzene) control groups. It should be noted that the concentrations of dye tested in this study were more than an order of magnitude below the threshold limit set by national regulations. Such a study on CRARD-120 genotoxicity can be used to establish exposure guidelines, and can help to establish restrictions on the waste discharge of textile dyes into the water ecosystem in Slovenia and elsewhere. ACKNOWLEDGMENT This work is part of a project supported by the Ministry of Science and Technology, Republic of Slovenia.
Bahari, I. B., Noor, F. M., and Daud, N. M. (1994). Micronucleated erythrocytes as an assay to assess actions by physical and chemical genotoxic agents in Clarias gariepinus. Mutat. Res. 313, 1}5. Becker, D., Christensen, R., Currie, L., Diamondstone, B., Eberhardt, K., Gills, T., Hertz, H.,Klouda, G., Moody, J., Parris, R., Scha!er, R., Steel, E., Taylor, J., Watters, R., and Zeiler, R. (1992). ;se of NIS¹ Standard Reference Materials for Decisions on Performance of Analytical Chemical Methods and ¸aboratories, National Institute of Standards and Technology Special Pub. 829, 1}27. Campana, M. A., Panzeri, A. M., Moreno, V. J., and Dulout, F.N. (1998). Genotoxic evaluation of thepyrethroid lambada-cyhalothrin using the micronucleus test in erythrocytes of the "sh Cheirodon interruptus interruptus. Mutat. Res. 438, 155}161. Carrasco, K. R., Tilbury, K. L., and Myers, M. S. (1990). Assessment of the piscine micronuclei test as an in situ biological indicator of chemical contaminant e!ects. Can. J. Fish. Aquat. Sci. 47, 2123}2136. Das, R. K., and Nanda, N. K. (1986). Induction of micronuclei in peripheral erythrocyte of "sh Heteropneustes fossilis by mitomycin C and paper mill e%uent. Mutat. Res. 175, 67}71. De Flora, S., Vigano, L., D'Agostini, F., Camoirano, A., M. Bagnasco, M., Bennicelli, C., Melodia, F., and Arillo, A. (1993). Multiple genotoxicity biomarkers in "sh exposed in situ to polluted river water. Mutat. Res. 319, 167}177. Harshbarger, M. A., and Clark, J. B. (1990). Epizootiology of neoplasms in bony "sh of North America. Sci. ¹otal Environ. 94, 1}32. Hooftman, R. N., and Raat, W. K. (1982). Induction of nuclear anomalies (micronuclei) in peripheral blood erythrocytes of Eastern mudminnow ;mbra pygmaea by ethyl methanesulphonate. Mutat. Res. 104, 147}152. Hose, J. E., Cross, J. N., Smith, S. G., and Diehl, D. (1987). Elevated circulating erythrocyte micronuclei in "shes from contaminated sites o! Southern California. Mar. Environ. Res. 22, 167}176. Hughes, J. B., and Hebert, A. T. (1991). Erythrocyte micronuclei in winter #ounder (Pseudopleuronectes americanus): Results of "eld surveys during 1980}1988 from Virginia to Nova Scotia and in Long Island Sound. Arch. Environ. Contam. ¹oxicol. 20, 474}479. Kurihara, Y., Rienkjkarn, M., and Etoh, H. (1992). Cytogenetic adaptive response of cultured "sh cells to low doses of X-rays. J. Radiat. Res. 33, 267}274. Longwell, A. C., Perry, D., Hughes, J. B., and Hebert, A. (1983). Frequencies of Micronuclei in Mature and Immature Erythrocytes of Fish as an Estimate of Chromosome Mutation Rates: Results of Field Surveys on =indowpane Flounder, =inter Flounder, and Atlantic Mackerel, ICES, C.M. 1983/E: 55, M.E.Q., Demersal and Pelagic Fish Committees. Majcen-Le Marechal, A., Slokar, Y. M., and Taufer, T. (1997). Decoloration of Chlorotriazine Reactive Azo Dyes with H O /UV. Dyes Pigments 33, 281}298. Manna, G. K., and Sadhukhan, A. (1986). Use of cells of gill and kidney of ¹ilapia "sh in micronucleus test (MNT). Curr. Sci. 55, 498}501.
REFERENCES Al-Sabti, K. (1986a). Clastogenic e!ects of "ve carcinogenic}mutagenic chemicals on the cells of the common carp Cyprinus carpio L. Comp. Biochem. Physiol. C 85, 5}9. Al-Sabti, K. (1986b). Comparative micronucleated erythrocyte cell induction in three cyprinids by "ve carcinogenic}mutagenic chemicals. Cytobios 47, 147}154. Al-Sabti, K. (1994). Micronuclei induced by selenium, mercury, methylmercury and theirmixture in binucleated blocked "sh erythrocyte cells. Mutat. Res. 320, 157}163. Al-Sabti, K., Franko, M., Andrijanic, B., Knez, S., and Stegnar, P. (1994). Chromium induced micronuclei in "sh. J. Appl. ¹oxicol. 14, 333}336.
Marlasca, M. J., Valles, B., Riva, M. C., and Crespo, S. (1992). Sublethal e!ects of synthetic dyes on rainbow trout Oncorhynchus mykiss: a light and electron microscope study. Dis. Aquat. Org. 12, 103}110. Marlasca, M. J., Sanpera, C., Riva, M. C., Sala, R., and Crespo, S. (1998). Hepatic alteration and induction of micronuclei in rainbow trout (Oncorhynchus mykiss) exposed to a textile industry e%uent. Histol. Histopathol. 13, 703}712. Metcalfe, C. D. (1988). Induction of micronuclei and nuclear abnormalities in the erythrocytes of mudminnows (;mbra limi) and brown bullheads (Ictalurus nebulosus). Bull. Environ. Contam. ¹oxicol. 40, 489}495. Nepomuceno, J. C., Ferrari, I., Spano, M. A., and Centeno, A. J. (1997). Detection of micronuclei in peripheral erythrocytes of Cyprinus carpio exposed to metallic mercury. Environ. Mol. Mutagen. 30, 293}297.
CHLOROTRIAZINE REACTIVE AZO RED 120 AND MICRONUCLEI Pacheco, M., and Santos, M. A. (1998). Induction of liver EROD and erythrocytic nuclear abnormalities by cyclophosphamide and PAHs in Anguilla anguilla L. Ecotoxicol. Environ. Saf. 40, 71}76. Pacheco, M., and Santos, M. A. (1999). Biochemical and genotoxic responses of adult eel (Anguilla anguilla L.) to resine acids and pulp mill e%uent: Laboratory and "eld experiments. Ecotoxicol. Environ. Saf. 42, 81}93. Rajaguru, P., Fairbairn, L. J., Ashby, J., Willington, M. A., Turner, S., Woolford, L. A., Chinnasamy, N., and Ra!erty, J. A. (1999). Genotoxicity studies on the azo dye Direct Red 2 using the in vivo mouse bone marrow micronucleus test. Mutat. Res. 444, 175}180. Schultz, N., Norrgren, L., Grawe, J., Johannisson, A., and Medhage, O. (1993). Micronuclei frequency in circulating erythrocytes from rainbow trout (Oncorhynchus mykiss) subjected to radiation, an image analysis and #ow cytometric study. Comp. Biochem. Physiol. 105, 207}211.
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Skerfving, S., Hansson, K., Mangs, C., Lindsten, J., and Ryman, N. (1974). Methylmercury-induced chromosomal damage in man. Environ. Res. 7, 83}89. Smith, I. R. (1990). Erythrocytic micronuclei in wild "sh from lakes Superior and Ontario that pollution-associated neoplasia. J. Great ¸akes Res. 16, 139}142. Ueda, T., Hayashi, M., Ohtsuka, Y., Nakamura, T., Kobayashi, J., and Sofuni, T. (1992). A preliminary study of the micronucleus test by acridine orange #uorescent staining compared with chromosomal aberration test using "sh erythropoietic and embryonic cells. =ater Sci. ¹echnol. 25, 235}240. WHO (World Health Organization) (1990). Environmental Health Criteria for Methylmercury. Environmental Health Criteria 101, p. 144, WHO, Geneva.
Chlorotriazine Reactive Azo Red 120 Textile Dye Induces Micronuclei in Fish Kabil Al-Sabti Joz\ ef Stefan Institute, P.O. Box 3000, Jamova 39, 1001 Ljubljana, Slovenia Received September 27, 1999
The main objective of this work was to use micronucleus induction in 5sh erythrocytes to study the risk to aquatic ecosystems due to the genotoxicity of Chlorotriazine Reactive Azo Red 120 textile dye. The frequencies of micronuclei were studied for three low doses of 1, 5, and 10 mg/L and blood sampling was carried out on the same 5sh after 3, 6, and 9 days. It was found that micronuclei increased not only in a dose-dependent manner but also in a time-dependent way, compared with negative (tap water) and positive (10 ppm benzene) control groups. There was also a slight, time-dependent increase in erythrocyte micronuclei of the control 5sh specimens. This study proved the genotoxicity of this dye, and suggests that further studies should be made on other dyes and some other toxic industrial pollutant discharges in water ecosystems, using 5sh as an indicator to monitor pollutant genotoxicity. 2000 Acamedic Press Key Words : micronuclei; genotoxicity; 5sh erythrocytes; water; biomarker; Chlorotriazine Reactive Azo Red 120 dye.
INTRODUCTION
The increasing contamination of the aquatic environment in Slovenia by di!erent industrial waste products has proven to have direct and/or indirect e!ects on human health. It has been found that biological testing is a scienti"cally valid approach to controlling toxic substances in industrial wastewater discharges (Al-Sabti et al., 1994). The textile business started thousands of years ago, but the development of modern technology for textile production, including the use of many arti"cial chemicals to improve products, makes it a serious problem with respect to the discharge of waste products directly and indirectly to aquatic ecosystems. At present there are about 3000 di!erent dyes available on the commercial market, and more than half of these are azo compounds (Majcen}Le Marechal et al., 1997). The industrial discharge of chemical waste products of the textile industry directly into Slovenian rivers has increased the amounts of these substances in the aqueous environment, although this trend, as in most countries, is now being halted or reversed due to legislation that limits
the use of toxic chemicals. For that reason there is also a debate about the level of such textile waste products, particularly textile dyes, permitted to be discharged directly into Slovenian waters, according to various studies monitoring the e!ect of such chemicals on human health. Although occupational and accidental chemical exposure does still occur, food is now the main route of exposure to these chemicals for the general population. Fish may act as &&sentinel'' organisms, indicating the potential for exposure of human populations to genotoxic chemicals in drinking water. Food is the major route of human exposure to toxic chemicals, and "sh and shell"sh have been recognized as major vectors for contaminant transfer to humans. For example, marine "sh and shell"sh, which are a major source of protein in many countries, are often contaminated with high concentrations of methyl mercury (WHO, 1990). Induction of chromosomal damage has been found in the lymphocytes of persons exposed to methyl mercury through consumption of contaminated "sh (Skerfving et al., 1974), although the precise mechanism involved in this process is not yet understood. Several chemicals or chemical mixtures induce clastogenic e!ects in "sh. Fish may accumulate pollutants from direct contamination by chemicals present in water or indrectly by feeding on living organisms in the water that accumulate pollutants. Genotoxicological data are unavailable for thousands of toxic compounds that are routinely discharged into surface waters. It is important to attempt to detect the early genotoxic e!ects of wastewater pollutants on "sh, at the actual concentrations concerned. Within the last decade the micronucleus test in "sh has played an important role in assessing exposure to water pollutants, and has proved to provide an early warning of genotoxic threats to "sh, their ecosystem, and "nally humans. Induction of cytogenetic damage in "sh could serve for monitoring not only selected genotoxic agents in the laboratory, but also the presence of genotoxicants in surface waters and di!erent aqueous ecosystems. The convenience of the micronucleus (MN) test needs no emphasis since data can be scored at any time; moreover, data
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on micronuclei might not only be useful as evidence supporting chromosome aberration frequencies induced by genotoxic agents but may also be important for the study of the relative radiosensitivity and chemosensitivity of di!erent tissues. Such investigations are lacking in "sh genotoxicity testing and the study described here is an attempt to advance in this direction. Various studies have found that the peripheral erythrocytes of "sh have a high incidence of micronuclei after exposure to di!erent pollutants under "eld and laboratory conditions (Hooftman and Raat, 1982); Longwell et al., 1983; Al-Sabti, 1986a, b; Das and Nanda, 1986; Manna and Sadhukhan, 1986; Hose et al., 1987; Metcalfe, 1988; Smith, 1990; Al-Sabti and Hardig, 1990; Carrasco et al., 1990; Hughes and Hebert, 1991; Ueda et al., 1992; Kurihara et al., 1992; Schultz et al., 1993; De Flora et al., 1993; Al-Sabti et al., 1994; Bahari et al., 1994; Nepomuceno et al., 1997; Campana et al., 1998; Pacheco and Santos, 1998, 1999). Observations of micronuclei can be made very rapidly, because in most "sh species the erythrocytes are sizable and have a large nucleus, enabling their use in such investigations. Micronuclei that arise from chromosomes or chromosomal fragments that are not incorporated into daughter nuclei at mitosis due to the lack of a centromere have proven to be useful for studying "sh under laboratory conditions. In this study, the genotoxic e!ects of exposure of "sh to three doses of the textile dye Chlorotriazine Reactive Azo Red 120 (CRARD-120) are determined by using MN induction in Prussian carp (Carassius auratus gibelio) erythrocyte cells under laboratory conditions. The three concentrations of CRARD-120 used in the present investigation were below those which cause gross cytotoxicty to "sh. MATERIAL AND METHODS
Fish Specimens of the Prussian carp, Carassius auratus gibelio, were chosen for this study because this "sh is a common and relatively sedentary species, which is available year-round in most "sh farms and some rivers and ponds in Slovenia. Specimens of Prussian carp with a weight and length of about 100 g and 12 cm, respectively, were supplied for this study by the Ribe Maribor "sh farm, in Rac\ e near Maribor in Slovenia. Before the experiment, "sh were acclimated for 7 days at a population density of 100 specimens in a 400-L tank with well-aerated nonchlorinated water at 153C and pH 7. Fish were allowed no food during the last 2 days of the experimental period. Experimental Design Chlorotriazine Reactive Azo Red 120 (CRARD-120) (Fig. 1), obtained from the Dye Factory of Belinka in Ljubljana, was added at three concentrations (sublethal) of
FIG. 1. Structure of Chlorotriazine Reactive Azo Red 120 dye.
1, 5, and 10 mg/L to 10-L aquaria containing seven "sh specimens each. Specimens were exposed to each of the di!erent CRARD-120 concentrations (1, 5, and 10 mg/L) for 3, 6, and 9 days. One or two drops of blood from each "sh specimen were sampled three times at 3, 6, and 9 days and analyzed. The concentration of CRARD-120 tended to decrease because of precipitation and adsorption, and so appropriate amounts of CRARD-120 stock solution were added after each spectrophotometric analysis of aquarium water. A negative tap water control group for all the experiments consisted of "sh specimens that were kept in a laboratory aquarium in clean tap water, while another seven specimens of Prussian carp were exposed for 3, 6, and 9 days to water polluted with 10 ppm of benzene. Micronucleus Test Before blood sampling, "sh were tagged by cutting one or a combination of two "ns to enable blood to be sampled from the same "sh specimen at 3, 6, and 9 days (time response) while exposed to the same concentration of CRARD-120 and from both negative and positive control groups. Blood samples for the smears were obtained by caudal vein puncture of the same "sh specimen at three intervals, and slides were prepared by the "sh micronucleated erythrocyte method (Al-Sabti, 1986a, b). From each "sh, two slides were prepared. After "xation in pure ethanol for 20 min, the prepared slides were left to air dry, and then the smears were stained with 5% Giemsa solution for 20 min. The slides were examined by light microscopy (Opton, Germany) under an oil immersion lens. Scoring of coded slides was done &&blind.'' Only erythrocyte cells that were isolated from surrounding cells were scored, and MNs were enumerated only if they were distinct from the two nuclei. One thousand erythrocytes per specimen for each slide (2000 per two slides) were analyzed in the subsequent treatments to determine the frequency of cells with one or two micronuclei. Statistical Analysis The standard deviation (SD) of the mean (X) of the number of micronuclei in mononucleated erythrocyte cells was calculated by the method of Becker et al. (1992), as a measure of variation in these parameters between duplicate erythrocyte cell samples exposed to the same treatment.
CHLOROTRIAZINE REACTIVE AZO RED 120 AND MICRONUCLEI
RESULTS
For clarity the results obtained for CRARD-120 for 3, 6, and 9 days are presented separately and for each individual "sh in Table 1, together with both the negative and positive control groups. Table 1 and Fig. 2 summarize MN induction per 1000 erythrocyte cells for the three di!erent CRARD-120 concentrations, compared with the negative tap water and positive 10 ppm benzene control groups. One "sh specimen exposed to 1 mg/CRARD-120, one to 10 mg/L CRARD-120, and two specimens from the control group were eliminated from the study, because after 7 to 8 days they were very weak and had physical injuries on their gills and skin that were probably the result of "shing and/or transport and handling. Each slide was found to contain at least 2000 to 3000 erythrocyte cells, and the number of mononucleated erythrocyte cells was greater (about 4000) in the control (untreated) groups than in the treated groups. Specimens of Prussian carp from the control treatments exhibited signi"cant di!erences in the frequency of micronuclei compared with those specimens exposed to CRARD-120. There was a minor increase in MN induction with time in the same negative control individuals analyzed at di!erent times of this study, while the positive control with 10 ppm of benzene demonstrated a signi"cant increase with time of exposure. Therefore, to evaluate the data, it was necessary to compare the genotoxic response in the chemical treatments with the response in the appropriate control treatment. DISCUSSION
The production and use of textile dyes make their fate in aquatic ecosystems of special interest. Evaluation of the toxicity of these dyes is hampered by the fact that there are many dyes with various chemical structures and di!ering e!ects on the environment. The biological activities of dyes also di!er greatly despite similarities in their structure, so their toxicological properties cannot be generalized by reference to one group only (Majcen}Le Marcehal et al., 1997). It has been found that biological testing is a scienti"cally valid approach to controlling toxic mixtures from various industrial wastewater discharges, including textile factories (Al-Sabti, 1995). A number of studies have provided evidence that "sh species possess metabolic mechanisms for the biotransformation of xenobiotics. Clastogenic activity may be a risk factor for genetic disease, teratogenesis, or carcinogenesis in "sh populations. However, testing of "sh for the clastogenic e!ects of chemicals has been hampered by operational problems with in vivo assays. Mutagenic chemicals have a high probability of inducing carcinogenic e!ects in various "sh species and the majority of these chemicals have been demo-
151
nstrated to cause tumors at speci"c or multiple sites in "sh (Harsbarger and Clark, 1990). As far as is known, only two studies have been published on the e!ect of some dyes in inducing micronuclei in "sh erythrocytes. Specimens of rainbow trout (Oncorhynchus mykiss) exposed to sublethal doses of wool shrink-proo"ng e%uent, Acid Violet 66 and Acid Red 217 (Marlasca et al., 1992, 1998), exhibited a signi"cant increase in erythrocyte MNs following 30 days of exposure. The present study was conducted on a di!erent "sh species and a di!erent dye, which could explain the di!erence between the present results and results of the above studies. The clastogenicity of the azo dye Direct Red 2 (DR2) has been investigated using the mouse murine bone marrow micronucleus assay; a potent dose-dependent response followed oral gavage of up to 4 mg/kg R2D after which signi"cant toxicity to the erythroid compartment was observed (Rajaguru et al., 1999). Table 1 and Fig. 2 illustrate the increased number of micronuclei in erythrocyte cells exposed to CRARD-120, not only as a dose response but also as a function of time. After 9 days the number of MNs resulting from exposure was found to increase when compared with that after 6 days. There might be a slight bias in the results probably related to the increase in organic substances in the water because of excretion and food additions. Another explanation could be related to the delay in mitosis and death of cells (Al-Sabti, 1994, Al-Sabti et al., 1994). Benzene was used as a positive control, because it represents a typical hydrocarbon in polluted waters. The positive control group exposed to 10 ppm of benzene and the group exposed to 10 mg/L of CRARD-120 exhibited similar induction of MNs after 9 days, whereas MN induction with the same dose of CRARD-120 was higher after exposure for 3 and 6 days compared with 10 ppm of benzene. The number of MNs induced in erythrocyte cells from the control group was also found to increase slightly with time, which can be related to shock, excrement, food addition, blood sampling, etc. But the results from the negative tap water control group are signi"cantly lower than those from the exposed groups, while the case is di!erent with the positive control after exposure to 10 ppm of benzene. When comparing e!ects of dose (1, 5, and 10 mg/L) (Figs. 2, 3 and Table 1), it can be seen that after 7 and 9 days, the values for MN induction with CRARD-120 di!er signi"cantly from those obtained after 3 days of exposure for all doses compared with the control. Except for CRARD-120 after 9 days of exposure, this observation is valid at the 95% con"dence level or better. The concentrations of CRARD-120 in the present investigation were lower than the threshold limit levels for natural waters. In Slovenia this limit is about 100 mg/L for CRARD-120, depending on the river quality category (I and II, or III and IV) (O.cial Gazette) of Republic of Slovenia, No. 18/85, 1985, p. 1073).
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KABIL AL-SABTI
TABLE 1 Frequencies of Micronuclei per 1000 Fish Erythrocytes from the Exposure to 1 (A), 5 (B), and 10 (C) mg/L CRARD-120, Compared with the Negative Tap Water (K) and Positive 10 ppm of Benzene (D) Controls for 3, 6, and 9 Days Exposure Specimen No.
Concentration (mg/L)
Days
MNs/specimen/ concn.
K1 K2 K3 K4 K5
Control Control Control Control Control
3 3 3 3 3
4.5 5.0 4.0 4.5 5.5
D1 D2 D3 D4 D5 D6 D7
Benzene Benzene Benzene Benzene Benzene Benzene Benzene
3 3 3 3 3 3 3
8.5 8.0 9.0 8.5 8.0 8.5 8.0
A1 A2 A3 A4 A5 A6
1 1 1 1 1 1
3 3 3 3 3 3
8.0 11.5 8.0 8.0 12.5 11.0
B1 B2 B3 B4 B5 B6 B7
5 5 5 5 5 5 5
3 3 3 3 3 3 3
16.0 17.0 16.5 14.0 16.5 18.0 14.5
C1 C2 C3 C4 C5 C6
10 10 10 10 10 10
3 3 3 3 3 3
20.0 18.5 20.5 18.0 19.0 20.0
K1 K2 K3 K4 K5
Control Control Control Control Control
6 6 6 6 6
5.0 5.0 5.5 5.0 5.5
D1 D2 D3 D4 D5 D6 D7
Benzene Benzene Benzene Benzene Benzene Benzene Benzene
6 6 6 6 6 6 6
14.5 14.5 14.5 15.5 16.0 15.5 15.0
A1 A2 A3 A4 A5 A6
1 1 1 1 1 1
6 6 6 6 6 6
11.5 13.5 11.5 11.5 14.0 12.0
B1 B2
5 5
6 6
20.0 23.0
Total No. of specimens
Total MN/all specimens/concn.
5
4.7$0.6
7
8.36$0.35
6
9.8$2.1
7
16.1$2.1
6
19.3$1.0
5
5.2$0.3
7
15.07$0.56
6
12.3$1.1
153
CHLOROTRIAZINE REACTIVE AZO RED 120 AND MICRONUCLEI
TABLE 1=Continued Exposure Specimen No.
Concentration (mg/L)
Days
MNs/specimen/ concn.
B3 B4 B5 B6 B7
5 5 5 5 5
6 6 6 6 6
18.5 18.5 22.5 18.0 22.0
C1 C2 C3 C4 C5 C6
10 10 10 10 10 10
6 6 6 6 6 6
24.5 23.5 22.5 23.0 21.5 25.5
K1 K2 K3 K4 K5
Control Control Control Control Control
9 9 9 9 9
6.6 6.0 5.7 6.4 6.1
D1 D2 D3 D4 D5 D6 D7
Benzene Benzene Benzene Benzene Benzene Benzene Benzene
9 9 9 9 9 9 9
24.5 23.5 23.5 23.5 24.5 24.5 25.5
A1 A2 A3 A4 A5 A6
1 1 1 1 1 1
9 9 9 9 9 9
16.5 16.0 14.5 14.0 17.0 15.0
B1 B2 B3 B4 B5 B6 B7
5 5 5 5 5 5 5
9 9 9 9 9 9 9
23.0 22.5 18.0 19.5 20.5 20.0 20.5
C1 C2 C3 C4 C5 C6
10 10 10 10 10 10
9 9 9 9 9 9
24.0 23.0 26.5 24.5 22.5 24.5
The present results for di!erent doses of CRARD120 could be used to interpret the data obtained from "eld monitoring. The mononucleated erythrocyte micronucleus assay may give a positive response in "sh exposed to both direct-acting and indirect-acting genotoxic chemicals. It is noted that some other factors, such as route of entry, duration and frequency of exposure, variations between di!erent species (interspecies), and variations among mem-
Total No. of specimens
Total MN/all specimens/concn.
7
18.9$1.4
6
22.8$0.8
5
6.2$0.4
7
24.2$0.7
6
15.5$1.2
7
20.6$1.7
6
24.6$1.4
bers of the same species (intraspecies), may also in#uence the toxicity of CRARD-120 and some other textile azo dye compounds. Such a study on the genotoxicity of CRARD120 can be used to establish exposure guidelines, and can help to establish restrictions on the waste discharges of such chemicals. The present results cannot be used alone as "nal data to assess the risk of industrial wastes, but they can form a foundation for decisions on further studies on other textile dyes and other water pollutants.
154
KABIL AL-SABTI
FIG. 2. Comparison of the frequency of MN induction ($SD) after three di!erent CRARD-120 exposure treatments (1, 5, 10 mg/L), compared with the negative (tap water) and positive (benzene/10 ppm) controls after 3, 6, and 9 days. For further explanation, see Table 1 and the text.
CONCLUSIONS
Using the micronucleus test, Chlorotriazine Reactive Azo Red 120 (CRARD-120) textile dye proved to be genotoxic to erythrocytes of Prussian carp (Carassius auratus gibelio) under laboratory conditions. Micronuclei were elevated not only in a dose-dependent (1, 5, and 10 mg/L) but also in a time-dependent (3, 6, and 9 days) manner, compared with the negative (tap water) and positive (10 ppm benzene) control groups. It should be noted that the concentrations of dye tested in this study were more than an order of magnitude below the threshold limit set by national regulations. Such a study on CRARD-120 genotoxicity can be used to establish exposure guidelines, and can help to establish restrictions on the waste discharge of textile dyes into the water ecosystem in Slovenia and elsewhere. ACKNOWLEDGMENT This work is part of a project supported by the Ministry of Science and Technology, Republic of Slovenia.
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Skerfving, S., Hansson, K., Mangs, C., Lindsten, J., and Ryman, N. (1974). Methylmercury-induced chromosomal damage in man. Environ. Res. 7, 83}89. Smith, I. R. (1990). Erythrocytic micronuclei in wild "sh from lakes Superior and Ontario that pollution-associated neoplasia. J. Great ¸akes Res. 16, 139}142. Ueda, T., Hayashi, M., Ohtsuka, Y., Nakamura, T., Kobayashi, J., and Sofuni, T. (1992). A preliminary study of the micronucleus test by acridine orange #uorescent staining compared with chromosomal aberration test using "sh erythropoietic and embryonic cells. =ater Sci. ¹echnol. 25, 235}240. WHO (World Health Organization) (1990). Environmental Health Criteria for Methylmercury. Environmental Health Criteria 101, p. 144, WHO, Geneva.