Aquatic Toxicology 66 (2004) 319–329
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 fish
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 fish 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. Significant alterations occur in biochemical parameters of blood in MG exposed fish.
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
flukes and gill flukes. 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 flush methods without paying
any attention to the fact that topically applied therapeutants might also be absorbed systemically and
produce significant 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 fish (Alderman and
0166-445X/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2003.09.008
320
S. Srivastava et al. / Aquatic Toxicology 66 (2004) 319–329
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
efficacy (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 first reported
by Bumpus and Brock (1988), its reduction by intestinal microflora was shown by Henderson et al.
(1997). Recently, MG is reported to be reduced and
metabolised by filamentous 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 fish 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). Leucoma-
4. Malachite green as a fungicide
MG has been largely used to prevent outgrowth
of oomycete fungi on fish and fish 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 catfish (Bly et al.,
1996) and rainbow trout (Valia and Fabian, 1998).
Aphanomyces invadis (Lilley and Inglis, 1997) and
Aspergillus flavus (Bhattacharya, 1995) infection in
Channa and some other fish 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.,
S. Srivastava et al. / Aquatic Toxicology 66 (2004) 319–329
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 fish (Rodriguez
and Fernandez, 2001); Trichodina on eel (Madsen
et al., 2000), Epinephalus (Susanti et al., 1996)
and Turbot (Diggles, 2000); Trichodinella epizootica on gill filaments of grass carp (Abdel-Meguid,
1995); Dinoflagellate ectoparasite on ornamental
fish (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 fish
Several workers have estimated LC50 values of
many commercial dyes at different time intervals
on fish (Clarke and Anliker, 1980). It has been suggested that toxicity of individual toxicants to different
species of fish are difficult to compare because they
are influenced 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 finger-
321
lings of various fish 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
catfish, 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 fish, 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 fish.
Significant 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 fish
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, fin 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 fish species
LC50 (mg/l)
pH
Temperature (◦ C)
2.0
7.430
2.19
8.0
6.5
7.5
12
12
12
3
3
6
0.238
0.960
0.4
0.519
1.72
1.3
0.286
7.5
7.5
7.5
9.5
8.0
8.0
8.0
22
12
22
12
12
12
12
6
6
6
6
6
6
24
Rainbow trout (Oncorhynchus mykiss)
1.4
2.35
6.8
7.5
8.0
8.0
12
12
12
3
3
6
Smallmouth bass (Micropterus dolomieui)
0.154
0.0453
7.5
7.5
12
12
24
96
Micropterus salmoides
0.282
0.0728
3.0
0.569
0.383
7.5
7.5
7.5
7.5
7.5
12
12
12
12
12
24
96
6
24
96
Atlantic salmon (Salmo salar)
3.560
1.090
0.497
0.283
7.5
7.5
7.5
7.5
12
12
12
12
3
6
24
96
Brown trout (Salmo trutta)
1.730
1.270
0.352
0.237
7.5
7.5
7.5
7.5
12
12
12
12
3
6
24
96
Freshwater prawn (Palaemonetes kadiakensis)
9.1
1.9
7.5
7.5
16
16
24
96
5.60
1.40
1.25
1.0
7.7
7.7
7.7
7.7
22
22
22
22
24
48
72
96
Fish
From Bills et al. (1977)
Blue gill Sun fish (adult) (Lepormis macrochirus)
Fingerlings
Channel catfish (Ictarulus punctatus)
Coho Salmon (Oncorhynchus kisutch)
Time (h)
From Srivastava et al. (1995a)
Freshwater catfish (Heteropneustes fossilis)
et al., 1985) and Nile tilapia (Omoregie et al., 1998).
Alderman and Clifton-Hadley (1988, 1993) studied
the effects of malachite green under fish 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.
S. Srivastava et al. / Aquatic Toxicology 66 (2004) 319–329
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 infiltration 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 fish 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 catfish (Srivastava et al., 1998c).
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 significant
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 catfish (Srivastava et al., 1995a) and also
decreases plasma phosphorus and calcium levels in
323
Tilapia (Yildiz and Pulatsu, 1999). Disturbances in
carbohydrate metabolism and osmoregulation have
been reported in catfish 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 fish (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 fish (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
fish (Roybal et al., 1995; Doerge et al., 1998a).
Poe and Wilson (1983) for the first time reported
malachite green residue in fish tissue. It is stored
primarily in serum, liver, kidney, muscle, skin and
viscera of various experimental animals including
fish (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
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S. Srivastava et al. / Aquatic Toxicology 66 (2004) 319–329
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 fish and that all fish tissues accumulate
significant and fairly persistent residues of the dye.
The use of malachite green on fish 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 fish is highest in low Ca2+ /humic substance
solution.
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; inflicts 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
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 significant 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
fly (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 fish 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 fish 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
S. Srivastava et al. / Aquatic Toxicology 66 (2004) 319–329
chlorine dioxide (Chen et al., 2001) and hydrogen
peroxide (Yamamoto et al., 2001), that have been
found to control fungal infections in fish and fish 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 fish, 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 fish
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 fish farms
(Aitcheson et al., 2000); and its use should be avoided
in all other industries.
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