Aquatic Toxicology 66 (2004) 319329

Review

Toxicological effects of malachite green

Shivaji Srivastava a , Ranjana Sinha a,b, , D. Roy a
a b

Department of Zoology, S.M.M. Town Post-Graduate College, Ballia 277001, India Central Inland Capture Fisheries Research Institute, Barrackpore 743101, WB, India Received 16 April 2002; accepted 29 September 2003

Abstract This review summarises the wide range of toxicological effects of malachite green (MG), a triarylmethane dye on various sh species and certain mammals. MG is widely used in aquaculture as a parasiticide and in food, health, textile and other industries for one or the other purposes. It controls fungal attacks, protozoan infections and some other diseases caused by helminths on a wide variety of sh and other aquatic organisms. However, the dye has generated much concern regarding its use, due to its reported toxic effects. The toxicity of this dye increases with exposure time, temperature and concentration. It has been reported to cause carcinogenesis, mutagenesis, chromosomal fractures, teratogenecity and respiratory toxicity. Histopathological effects of MG include multi-organ tissue injury. Signicant alterations occur in biochemical parameters of blood in MG exposed sh. Residues of MG and its reduced form, leucomalachite green have been reported from serum, liver, kidney, muscles and other tissues as also from eggs and fry. Toxicity occurs in some mammals, including organ damage, mutagenic, carcinogenic and developmental abnormalities. However, despite the large amount of data on its toxic effects, MG is still used as a parasiticide in aquaculture and other industries. It is concluded that the potential of alternative parasiticides, like humic acid, chlorine dioxide and Pyceze, should be explored to replace MG. Until then, MG should be used with extreme care at suitable concentrations and at times when the temperature is low. Removal of residual MG in treatment ponds should also be considered. 2003 Elsevier B.V. All rights reserved.
Keywords: Malachite green; Aquaculture; Toxicity; Parasiticide; Fish

1. Introduction Malachite green (MG) is an extensively used biocide in the aquaculture industry world-wide. It is highly effective against important protozoal and fungal infections (Hoffman and Meyer, 1974; Alderman, 1985; Schnick, 1988). Basically, it works as an ectoparasiticide: it has also been used to control skin ukes and gill ukes. Aquaculture industries have

Corresponding author. E-mail address: anu sinha1969@yahoo.co.uk (R. Sinha).

been using malachite green extensively as a topical treatment by bath or ush methods without paying any attention to the fact that topically applied therapeutants might also be absorbed systemically and produce signicant internal effects. On the other hand, it is also used as a food colouring agent, food additive, a medical disinfectant and anthelminthic as well as a dye in silk, wool, jute, leather, cotton, paper and acrylic industries (Culp and Beland, 1996). However, malachite green has now become a highly controversial compound due to the risks it poses to the consumers of treated sh (Alderman and

0166-445X/$ see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2003.09.008

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Clifton-Hadley, 1993) including its effects on the immune system, reproductive system and its genotoxic and carcinogenic properties (Fernandes et al., 1991; Rao, 1995; Gouranchat, 2000). Though the use of this dye has been banned in several countries and not approved by US Food and Drug Administration (Chang et al., 2001), it is still being used in many parts of the world due to its low cost, ready availability and efcacy (Schnick, 1988) and a considerable amount of research is being devoted to work out the wide spectrum of biological effects it exerts on different animals and mankind. The US Food and Drug Administration has nominated MG as a priority chemical for carcinogenicity testing (Culp and Beland, 1996). There is concern about the fate of MG and its reduced form, leucomalachite green in aquatic and terrestrial ecosystems since they occur as contaminants (Burchmore and Wilkinson, 1993; Nelson and Hites, 1980) and are potential human health hazards.

lachite green is structurally similar to classic aromatic amines and is a precursor of the dye during its production and could be present as a contaminant in the commercially prepared dye. While fungal metabolism of MG was rst reported by Bumpus and Brock (1988), its reduction by intestinal microora was shown by Henderson et al. (1997). Recently, MG is reported to be reduced and metabolised by lamentous fungus, Cunninghamella elegans (Chang-Jun et al., 2001).

3. Malachite green as a parasiticide MG has been extensively used as a topical fungicide (Hussein et al., 1999; Qureshi et al., 1998) and ectoparasiticide in sh farming throughout the world since 1936 (Foster and Woodbury, 1936). In African aquaculture, it has been used against infection by bacteria, protozoans, cestodes, trematodes, nematodes, crustaceans, etc. (Hecht and Endemann, 1998).

2. Chemistry Malachite green, a triarylmethane dye (C23 H26 N2 O, CI 42,000) is a dark green and crystalline solid prepared by condensing one part of benzaldehyde with two parts of diemethylaniline in the presence of concentrated sulphuric acid or zinc chloride. Malachite green is available in a number of forms, mainly as the oxalate or hydrochloride salt in a minimal 50% solution as a mixture of acetate and hydrochloride salts. Malachite green hydrochloride is an industrial grade variety which, during its manufacture, is precipitated by the addition of zinc chloride and is, therefore, produced as a double zinc salt. This dye, like other triphenylemethanes, can exist in two ionic forms- as the dye salt and as the carbinol or pseudobase. According to Albert (1979), it is probably as the pseudobase that these ions enter cells due to their much greater lipid solubility. The ionisation constant (pK) of MG is 6.90. It is 100% ionised at pH 4.0, 50% at pH 6.9, 25% at 7.4 and 0% at pH 10.1 (Goldacre and Philips, 1949). In animals, MG is reduced through biotransformation to its colourless form, leucomalachite green and persists in tissues (Werth and Boiteaux, 1967; Poe and Wilson, 1983; Michaels and Lewis, 1986; Ollikka et al., 1993; Azmi et al., 1998; Pointing and Vrijmoed, 2000). Leucoma4. Malachite green as a fungicide MG has been largely used to prevent outgrowth of oomycete fungi on sh and sh eggs, both as a post-infection therapy and prophylaxis (Alderman, 1985, 2002; Gerundo et al., 1991). It was found to be the most effective fungicide among 49 compounds tested against an oomycete fungus (Campbell et al., 2001). It has prevented the growth of Haliphthoros on rock lobster (Diggles, 2001) and Ful-2 on salmon (Huang et al., 1996). Saprolegniasis has also been effectively controlled by MG in salmons (Willoughby and Roberts, 1992), channel catsh (Bly et al., 1996) and rainbow trout (Valia and Fabian, 1998). Aphanomyces invadis (Lilley and Inglis, 1997) and Aspergillus avus (Bhattacharya, 1995) infection in Channa and some other sh have also been treated effectively with MG. Eggs of Cyprinus carpio and tench have been treated prophylactically to prevent fungal infection (Jaehnichen, 1976; Kouril et al., 1998).

5. Malachite green as an antiprotozoan MG has been used effectively to control protozoans (Rintamaki-Kinnunen and Valtonen, 1997), e.g.,

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Paranophrgs on Milten handed crab (Yunjiang, 1997); Ichthyophthirius on Ictarulus punctatus (Leteux and Meyer, 1972; Schachte, 1974; Moore, 1998; Tieman and Goodwin, 2001) and ornamental sh (Rodriguez and Fernandez, 2001); Trichodina on eel (Madsen et al., 2000), Epinephalus (Susanti et al., 1996) and Turbot (Diggles, 2000); Trichodinella epizootica on gill laments of grass carp (Abdel-Meguid, 1995); Dinoagellate ectoparasite on ornamental sh (Steinhagen et al., 1999) and Tetrahymena on guppy (Rie et al., 1999). MG has also been used against Uronema nigricans, the ciliate pathogen (Crosbie and Munday, 1999), Amylodinium ocellatum (Chang et al., 2001) and another scuticociliatid ciliate causing scuticociliatosis (Zhou et al., 1997).

6. Malachite green in other diseases MG has also been used successfully against helminth infection, such as Dactylogyrus vastator in Cyprinus carpio (Molnar, 1995) and against Cichliodogyriasis (Flores et al., 1995). Dermocystidium koi in skin of carp (Wildgoose, 1995), proliferative kidney disease (PKD) in rainbow trout (Clifton-Hadley and Alderman, 1987; Alderman, 1992; Gouvello et al., 1999) and atlantic salmon (Quigley and Mc Ardle, 1998) and an ulcerative dermal necrosis in salmon (Murphy, 1973) have also been effectively controlled by MG. Another ulceratrive disease in juvenile turbot, caused by some protozoan and myxobacteria was also cured by MG/formalin treatment (Devesa et al., 1989).

7. Toxicological effects of malachite green on sh Several workers have estimated LC50 values of many commercial dyes at different time intervals on sh (Clarke and Anliker, 1980). It has been suggested that toxicity of individual toxicants to different species of sh are difcult to compare because they are inuenced by various factors such as temperature, pH, hardness and dissolved oxygen of test water (Schoettger, 1970; Smith and Heath, 1979; Gluth and Hanke, 1983). Bills et al. (1977) made a detailed study on LC50 values of MG on adults and nger-

lings of various sh species and observed the effects of pH, temperature and exposure time on the toxicity of this dye (Table 1). Their study indicates that toxicity of MG increases with rise in temperature. Similar observations have also been made by Alderman and Polglase (1984). Srivastava et al. (1995a) also observed changes in LC 50 values of MG in a freshwater catsh, Heteropneustes fossilis at different exposure times (Table 1) and stated that toxicity increases with exposure time. Wright (1976) evaluated the mortality rate of MG exposed eggs and fry of large mouth bass, Micropterus salmonides. A two-fold increase in MG concentration resulted in more than 20 times increase in mortality rate of eggs and fries. This observation led him to conclude that MG is extremely toxic and should not be used for any purpose involving large mouth bass eggs or fry. Several studies have shown this dye to be highly toxic to freshwater sh, in both acute and chronic exposures (Steffens et al., 1961; Werth and Boiteaux, 1967; Meyer and Jorgensen, 1983; Klein et al., 1991; Hormazabal et al., 1992; Alderman and Clifton-Hadley, 1993). Carcinogenesis, mutagenesis, chromosomal fractures, teratogenicity and reduced fertility have also been reported in rainbow trout following treatment with malachite green (Amlacher, 1961; Lieder, 1961; Steffens et al., 1961; Nelson, 1974; Bills et al., 1977; Schnick and Meyer, 1978; Meyer and Jorgensen, 1983). A considerable amount of research has been done to determine the teratogenic effects of MG on sh. Signicant developmental abnormalities in eggs, predominantly chromosome breaks, have been reported in rainbow trout, Oncorhynchus mykiss after long-term intoxication of MG (Keyl and Werth, 1959; Steffens et al., 1961; Mayer and Jorgensen, 1983). Chromosomal aberrations in eggs of MG treated freshwater sh have also been reported by Worle (1995). A marked decline occurs in survival of embryos within after 38 h fertilisation following long-term exposure to high doses of malachite green; and there is evidence for delayed hatching time and spinal, head, n and tail abnormalities in rainbow trout fry hatched from eggs (Meyer and Jorgensen, 1983). Malachite green also acts as a respiratory enzyme poison (Werth, 1958; Werth and Boiteaux, 1967) and causes respiratory distress in rainbow trout (Ross

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Table 1 LC50 Values of malachite green for various sh species Fish From Bills et al. (1977) Blue gill Sun sh (adult) (Lepormis macrochirus) LC50 (mg/l) 2.0 7.430 2.19 0.238 0.960 0.4 0.519 1.72 1.3 0.286 1.4 2.35 6.8 0.154 0.0453 0.282 0.0728 3.0 0.569 0.383 3.560 1.090 0.497 0.283 1.730 1.270 0.352 0.237 9.1 1.9 pH 8.0 6.5 7.5 7.5 7.5 7.5 9.5 8.0 8.0 8.0 7.5 8.0 8.0 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 Temperature ( C) 12 12 12 22 12 22 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 16 16 Time (h) 3 3 6 6 6 6 6 6 6 24 3 3 6 24 96 24 96 6 24 96 3 6 24 96 3 6 24 96 24 96

Fingerlings Channel catsh (Ictarulus punctatus)

Rainbow trout (Oncorhynchus mykiss)

Smallmouth bass (Micropterus dolomieui) Micropterus salmoides Coho Salmon (Oncorhynchus kisutch)

Atlantic salmon (Salmo salar)

Brown trout (Salmo trutta)

Freshwater prawn (Palaemonetes kadiakensis) From Srivastava et al. (1995a) Freshwater catsh (Heteropneustes fossilis)

5.60 1.40 1.25 1.0

7.7 7.7 7.7 7.7

22 22 22 22

24 48 72 96

et al., 1985) and Nile tilapia (Omoregie et al., 1998). Alderman and Clifton-Hadley (1988, 1993) studied the effects of malachite green under sh farm conditions and have made pharmacokinetic studies on malachite green in rainbow trout. Numerous other reports have also appeared dealing with both clinical and experimental aspects of malachite green

(Clemmensen et al., 1984; Gerundo et al., 1991; Khanna and Das, 1991; Klein et al., 1991; Marlasca et al., 1992; Hormazabal et al., 1992; Allen et al., 1994). Recent research, on the other hand, has attempted to characterise the effects of malachite green in various tissues and also on biochemical parameters of blood.

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8. Histopathological studies Histopathology has revealed that malachite green causes detrimental effects in liver, gill, kidney, intestine, gonads and pituitary gonadotropic cells. It causes sinusoidal congestion and focal necrosis in liver, damages mitochondria and also causes nuclear alterations (Gerundo et al., 1991). Hypertrophy and vacuolisation followed by necrosis and cirrhosis have been observed in hepatocytes of Heteropneustes fossilis following treatment with malachite green (Srivastava et al., 1998a). Exposure to this dye also causes severe damage to gills, resulting in necrosis of lamellar cells and gill epithelium, as well as leucocyte inltration in rainbow trout (Gerundo et al., 1991) and Heteropneustes fossilis (Srivastava et al., 1998b). The dye caused hyperplasia of epithelial cells in the proximal convoluted tubules and shrinkage of glomeruli, forming gaps between capsule and tuft, necrotic changes like karyorrhexis, karyolysis, pyknosis and desquamation of epithelial lining cells and vacuolisation in the kidney of Heteropneustes fossilis (Srivastava et al., 1998b). The effects of the dye on the intestine of the sh also included necrosis, desquamation and degeneration of epithelial cell lining, cytolysis and increase in goblet cell population, rupture of tip of intestinal villi, breakage of mucosal folds, necrosis and disorganisation of muscularis and serosa (Srivastava et al., 1998b). Inhibition of the activity of gonadotropic cells in pituitary gland and degenerative changes in the gonads have also been reported following acute and chronic exposures to subacute and sublethal concentrations of the dye in the catsh (Srivastava et al., 1998c).

Tilapia (Yildiz and Pulatsu, 1999). Disturbances in carbohydrate metabolism and osmoregulation have been reported in catsh after exposure to malachite green (Srivastava et al., 1995b). MG causes hepatic and muscle glycogenolysis with concomitant hyperglycemia and hyperchloraemia (Srivastava et al., 1995b); increases sensitivity to hypoxia and impairs protein synthesis in certain sh (Svobodova et al., 1997). Malachite green also affects haematological parameters: decreases in haematocrit values and anaemic responses have been reported in rainbow trout and Clarias gariepinus (Tanck et al., 1995; Musa and Omoregie, 1999). H. fossilis also exhibited a decrease in RBC count (dyscrasia), Hb (anaemia) and HTC (%); increase in WBC count (leucocytosis) and delay in blood coagulation post-exposure to malachite green (Srivastava et al., 1996). Decreases in monocyte count, haematocrit value and mean corpuscular volume and increase in mean corpuscular haemoglobin concentration have also been noticed after malachite green exposure (Svobodova et al., 1997). However, elevated packed cell volume and haemoglobin values have been observed in malachite green exposed rainbow trout (Alderman and Clifton-Hadley, 1993). Erythrocyte counts and haemoglobin values increased after 3 days, but erythrocytosis and leukopenia were found after 7 and 21 days respectively in MG treated channel sh (Grizzle, 1977).

10. Malachite green residues Malachite green used to treat and prevent fungal and parasitic infections is reduced to leucomalachite green and accumulates in the tissues of exposed sh (Roybal et al., 1995; Doerge et al., 1998a). Poe and Wilson (1983) for the rst time reported malachite green residue in sh tissue. It is stored primarily in serum, liver, kidney, muscle, skin and viscera of various experimental animals including sh (Edelhauser and Klein, 1986; Clifton-Hadley and Alderman, 1987; Kelin and Edelhauser, 1988; Alderman and Clifton-Hadley, 1993; Fink and Auch, 1993; Turnipseed et al., 1995; Machova et al., 1996; Rushing and Hansen, 1997; Alborali et al., 1997; Nowak and De Guingand, 1997; Doerge et al., 1998a). Plakas et al. (1996) analysed uptake, tissue

9. Biochemical and haematological studies Though Bills and Hunn (1976) denied any adverse effect of MG on blood chemistry of coho salmon, acute, subacute and sublethal concentrations of malachite green have been found to cause signicant alterations in biochemical parameters in the blood of Heteropneustes fossilis (Srivastava et al., 1995a,b). It causes depletion of serum calcium and protein levels; and also increases the total cholesterol level of blood in catsh (Srivastava et al., 1995a) and also decreases plasma phosphorus and calcium levels in

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distribution and metabolism of MG. It was rapidly absorbed and concentrated in the tissue during water borne exposure. Malachite green residues have also been detected in eggs, fry and adult muscle tissue of rainbow trout Oncorhynchus mykiss (John et al., 1994; Meinertz et al., 1995) and Atlantic salmon (Allen, 1990). Alderman (1992) reported that malachite green is absorbed by sh and that all sh tissues accumulate signicant and fairly persistent residues of the dye. The use of malachite green on sh destined for human consumption is, therefore, contra-indicated unless a long withdrawal period is employed. Different methods have been developed for residue analysis of MG. While Tarbin et al. (1998) and Swarbrick et al. (1997) have measured MG in trout muscle by HPLC; isotope dilution and ion pairing liquid chromatography methods have been used by other workers (Doerge et al., 1998a; Edder et al., 1997; Plakas et al., 1995). Meinelt et al. (2001) have reported that the toxicity of MG is greatly affected by presence of Ca2+ ions and humic substances. This dye being positively charged, competes with Ca2+ ions for the negatively charged binding sites of humic substances. Survival of MG exposed sh is highest in low Ca2+ /humic substance solution.

with malachite green results in prominent weight loss and change in serum urea and aspartate aminotransferase levels in rats (Clemmensen et al., 1984). Malachite green has been found to be mutagenic in rats and mice; and it causes signicant developmental abnormalities in pregnant New Zealand white rabbits (Oryctolagus cuniculus) (Meyer and Jorgensen, 1983). It also produces chromosomal derangement in chironomid larvae (Keyl and Werth, 1959) and fruit y (Drosophila melanogaster) (Pfeiffer, 1961). Malachite green is highly cytotoxic to mammalian cells (Fessard et al., 1999) and carcinogenic to liver, thyroid and other organs of experimental animals (Rao, 1995; Rao and Fernandes, 1996; Doerge et al., 1998b; Mahudawala et al., 1999; Sundarrajan et al., 2000). Incidences of tumours in lungs, breast and ovary have also been reported from rats exposed to malachite green (Werth, 1958). In the thyroid gland, leucomalachite green results in blockade of hormone synthesis, decreases T4 and increases TSH concentrations and causes tumours in thyroid follicle cells of rats (Doerge et al., 1998b).

12. Conclusion The preceding account of MG reveals that this dye has now become one of the most debated and controversial compounds used in aquaculture, due to the risks it poses to the consumers, including its effects on the immune system and reproductive system as well as its genotoxic and carcinogenic potentials. In Germany, malachite green is not allowed to be used as an animal drug because of the possible carcinogenic, mutagenic and teratogenic risks for human health. A zero tolerance of 0.01 mg/kg for the sum of malachite green and leucomalachite green in edible sh has been established (Klein et al., 1991). Despite being banned in several countries, the dye is still being used in many parts of the world due to lack of a proper alternative. Recently, a pharmaceutical alternative to MG, Pyceze with bronopol as its active ingredient, has been developed in UK. It is being used for the treatment of sh and their ova; and appears to be a safe and effective replacement for MG in prevention of fungal infections (Cawley, 1998; Pottinger and Day, 1999; Hardwick, 2000; Kaijser et al., 2001). There are some other compounds, such as stable

11. Toxicological effects of malachite green on mammals and other animals Malachite green is environmentally persistent and acutely toxic to a wide range of aquatic and terrestrial animals. It causes serious public health hazards and also poses potential environmental problem. Both clinical and experimental observations reported so far reveal that malachite green is a multi-organ toxin. Desciens and Bablet (1994) found renal changes in rabbit following repeated oral dosing of this dye. It decreases food intake, growth and fertility rates; causes damage to liver, spleen, kidney and heart; inicts lesions on skin, eyes, lungs and bones; and produces teratogenic effects in rats and mice (Werth and Boiteaux, 1967, 1968; Culp et al., 1999). Apoptosis in the transitional epithelium of the urinary bladder and thyroid follicles was also observed in MG fed mice (Culp et al., 1999). Clemmensen et al. (1984) observed no systemic effects in rats following dermal application of the dye. However, long-term (28 days) treatment

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chlorine dioxide (Chen et al., 2001) and hydrogen peroxide (Yamamoto et al., 2001), that have been found to control fungal infections in sh and sh eggs as effectively as MG. Humic acid has also been evaluated as an alternative disinfectant (Heidrich et al., 1999). Moreover, some bacterial isolates from diseased carp and trouts have been found to be resistant to MG (Prasenjit et al., 2001). It is, therefore, timely to explore further the potential of these alternatives for the eventual complete replacement of MG. Until then, its use in aquaculture should be limited to when it is an absolute necessity; and then only with extreme care within the safe concentration. Its use should be restricted to eggs and fry (Guandalini et al., 1998). In tropical countries, the most suitable time for applying MG to a pond should also be carefully considered. Early morning treatment, when water temperature is not too high, is recommended. During peak summer months, the exposure time for MG treatment should be decreased (Chinabut, 1995). The use of MG should also be avoided with scaleless sh, since they are more vulnerable to this dye (Chinabut, 1995). It has been observed that stress modulates the response of an animal to toxic substances (Pottinger and Calder, 1995). It is, therefore, suggested that sh should be subjected to minimum disturbance and handling while being exposed to MG. We should also explore ways to remove excess MG left in large ponds after treatment. Activated carbon may facilitate the removal of MG from sh farms (Aitcheson et al., 2000); and its use should be avoided in all other industries.

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