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Abstract

Aquaculture in Brazil has shown expressive development since the 1990s with growth rates superior to those of cattle and poultry. In order to achieve greater productivity, intensive fish cultivation systems are employed, which can cause greater susceptibility to diseases caused by viruses, bacteria, fungi, and parasites. The reduced availability of veterinary medications approved for use in aquaculture in Brazil has lead fish farmers to the indiscriminate use of several chemical substances with antimicrobial activity, such as the dye malachite green (MG). As a result of this use, residues of MG and its main biotransformation product, leucomalachite green (LMG), may be present in fish available for consumption. The presence of residues of these compounds represents a risk to human health due to their toxicity, as well as a potential impact on the environment, and could also raise barriers for commercialization in the country and for exportation. The objective of this review is to provide the context and evidence of the use of MG in aquaculture and of its toxicological and legislative aspects. A review of the analytical methods used to determine MG residues in fish, with emphasis on mass spectrometry, is also presented.
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Considerations on the Use of Malachite
Green in Aquaculture and Analytical
Aspects of Determining the Residues in
Fish: A Review
Juliana Campos Hashimoto
a
, Jonas Augusto Rizzato Paschoal
a
,
Júlio Ferraz de Queiroz
b
& Felix Guillermo Reyes Reyes
a
a
Department of Food Science, School of Food Engineering,
University of Campinas, Campinas, SP, Brazil
b
Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA),
Jaguariúna, SP, Brazil
Available online: 12 Jul 2011
To cite this article: Juliana Campos Hashimoto, Jonas Augusto Rizzato Paschoal, Júlio Ferraz de
Queiroz & Felix Guillermo Reyes Reyes (2011): Considerations on the Use of Malachite Green in
Aquaculture and Analytical Aspects of Determining the Residues in Fish: A Review, Journal of Aquatic
Food Product Technology, 20:3, 273-294
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Journal of Aquatic Food Product Technology, 20:273–294, 2011
Copyright © Taylor & Francis Group, LLC
ISSN: 1049-8850 print / 1547-0636 online
DOI: 10.1080/10498850.2011.569643
Considerations on the Use of Malachite Green
in Aquaculture and Analytical Aspects
of Determining the Residues in Fish: A Review
JULIANA CAMPOS HASHIMOTO,
1
JONAS AUGUSTO RIZZATO PASCHOAL,
1
JÚLIO FERRAZ DE QUEIROZ,
2
AND
FELIX GUILLERMO REYES REYES
1
1
Department of Food Science, School of Food Engineering, University
of Campinas, Campinas, SP, Brazil
2
Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), Jaguariúna,
SP, Brazil
Aquaculture in Brazil has shown expressive development since the 1990s with growth
rates superior to those of cattle and poultry. In order to achieve greater productivity,
intensive fish cultivation systems are employed, which can cause greater susceptibility
to diseases caused by viruses, bacteria, fungi, and parasites. The reduced availability
of veterinary medications approved for use in aquaculture in Brazil has lead fish farm-
ers to the indiscriminate use of several chemical substances with antimicrobial activity,
such as the dye malachite green (MG). As a result of this use, residues of MG and its
main biotransformation product, leucomalachite green (LMG), may be present in fish
available for consumption. The presence of residues of these compounds represents a
risk to human health due to their toxicity, as well as a potential impact on the envi-
ronment, and could also raise barriers for commercialization in the country and for
exportation. The objective of this review is to provide the context and evidence of the
use of MG in aquaculture and of its toxicological and legislative aspects. A review of
the analytical methods used to determine MG residues in fish, with emphasis on mass
spectrometry, is also presented.
Keywords malachite green (MG), fish, aquaculture, high performance liquid
chromatography (HPLC), mass spectrometry
Introduction
Fish is easily digested and is also a source of protein of high biological value with sat-
isfactory levels of unsaturated fats, vitamins A and D, and minerals. The consumption
of fish in Brazil is still unimpressive, approximately 6 kg/person/year, well below the
36 kg/person/year for beef. However, over the last few years many consumers have been
The authors gratefully acknowledge the financial support of FAPESP (Proc. 09/54622-7),
CAPES, and CNPq, Brazil, and thank Professor Hilary C. de Menezes for language assistance.
Address correspondence to Felix Guillermo Reyes Reyes, Department of Food Science, School
of Food Engineering, University of Campinas, P.O. Box 6121, 13083-970 Campinas, SP, Brazil.
E-mail: reyesfgr@fea.unicamp.br
273
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274 J. C. Hashimoto et al.
seeking healthier foods, encouraged by governmental programs to increase fish consump-
tion and fish production under appropriate and safe conditions (Ostrensky et al., 2008;
Souza, 2003).
Although Brazil has a great potential to significantly increase aquaculture production,
this will only be achieved if the production system is managed in a sustainable manner
with the quality required by the internal and external markets. With respect to other animal
breeding activities, the use of antimicrobials in aquaculture is an important tool to ensure
high productivity and healthy products. However, only one veterinary medication has been
regulated for use in the Brazilian aquaculture industry, contributing to the use of illicit
substances as alternatives. Amongst these substances, antimicrobials indicated for other
animal species, pesticides, and dyes such as malachite green (MG), are the most commonly
used.
After being absorbed by fish, MG is bio-transformed into leucomalachite green
(LMG). The residues of these substances are stable in both the environment and the fish
meat, even after heat treatment (Mitrowska et al., 2007; Srivastava et al., 2004). The pres-
ence of such residues in fish represents a risk to human health due to their toxicity, as
well as their potential to cause an i mpact on the environment. They could also be a reason
for export barriers, since their use is prohibited in many countries on the global market—
especially in the United States, Japan, and the European Union ( Lem and Karunasagar,
2007). Despite this, MG is persistently used in aquaculture, probably due to its low cost
and recognized antimicrobial and antiparasitic efficiency.
Concern about the magnitude of exposure to residues of these compounds is of fun-
damental importance to guide and to stimulate health-monitoring actions to protect the
consumer. It is, therefore, important to monitor the residues of these substances in fish by
properly selective analytical methods with good detection ability. MG residues in fish are
mostly determined by a liquid chromatography technique coupled to a mass spectrometer
(LC-MS). The objective of this study was to provide the context and the evidence of the
use of MG by aquaculture, as well as presenting the toxicological and legislative aspects
involved. A review of the analytical methods used to determine MG residues in fish is also
provided, with an emphasis on LC-MS, as described in the literature.
Economic Aspects of Aquaculture
Aquaculture is one of the fastest growing food production industries in the world. Data
from the Food and Agriculture Organization (FAO) of the United Nations for the period
between 1984 and 1995 indicated that the global production increased at a r ate of approx-
imately 10% a year during that period, from 10.4 to 27.8 million t per year. In terms of
the generation of financial resources, agricultural production tripled from US$13.1 billion
in 1984 to US$39.8 billion in 1994. Considering the period from 1995 to 2005, the total
aquaculture production presented an average annual growth of 7.3%, with a production
of 48.1 million t in 2005, equivalent to US$70.9 billion. Of the total amount produced,
89.7% was produced in the Asian-Pacific region (67.3% from China), 4.2% came f rom
Western Europe, and 2.9% was produced in Latin America and the Caribbean (1.5% from
Chile; Diegues, 2006; Paschoal, 2007). Figure 1 shows the production values and the profits
generated by global aquaculture during the period from 1984 to 2004.
Brazil is considered to be one of the countries presenting the greatest potential in
the world for aquaculture production due to the extent of its territory, with 2/3 located
in tropical regions, in addition to the privileged amount of water resources with emphasis
on the Amazon basin, which is responsible for 20% of the fresh water available in the
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Use of Malachite Green in Aquaculture 275
(US$ 1,000)
80,000,000
70,000,000
60,000,000
50,000,000
40,000,000
30,000,000
20,000,000
10,000,000
0
(t)
70,000,000
60,000,000
50,000,000
40,000,000
30,000,000
20,000,000
10,000,000
0
1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004
Production Values
Figure 1. Evolution of the production and income generated by global aquaculture during the period
from 1984 to 2004 (Source: Ostrensky et al., 2008).
world. It also has 5 million ha of reservoirs in the Southeast and Northeast regions; and the
Brazilian coastline is over 8,000 km long, which has a huge potential for use in a variety
of aquaculture activities for the production of marine species (Paschoal, 2007). Currently,
Brazil is the second most important country in South America for aquaculture, with growth
rates superior to the global average since 1995. Aquaculture presents a growth rate higher
than that of extractive fishing, and it is highlighted in relation to poultry, pig, and cattle.
Data regarding 2004 indicated that the Southeastern region contributed 7.8% of the national
aquaculture production; and of this, São Paulo State was responsible for 66.3%, principally
due to the culture of tilapia and carp (Ostrensky et al., 2008). Figure 2 shows the evolution
(t)
300,000
250,000
200,000
150,000
100,000
50,000
0
(US$ 1,000)
1,200,000
1,000,000
800,000
600,000
400,000
200,000
0
1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004
(t)
Values
Figure 2. Evolution of the production and income generated by Brazilian aquaculture during the
period from 1984 to 2004 (Source: Ostrensky et al., 2008).
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276 J. C. Hashimoto et al.
of this production and the income generated by Brazilian aquaculture during the period
from 1984 to 2004.
Over the last few years, aquaculture has played an important role in the commer-
cial trade balance in Brazil due to the production of fish, crustaceans, and mollusks. This
increase in the contribution of aquaculture in the production of aquatic animals can be
attributed mainly to the fact that the extractive fishing capture is stagnated, while the fish
farmers are investing in intensive production structures, especially for the cultivation of
shrimp for exportation (Diegues, 2006).
The Use of Malachite Green in Aquaculture
Malachite green is a diamine triphenyl methylene dye, originally used in the textile industry
and also widely used in fish culture since the early 1930s (Rushing and Thompson, 1997).
Such popularity is related to its broad spectrum of antimicrobial action and its efficiency
in preventing and treating certain types of fish diseases (Halme et al., 2007).
MG is capable of inhibiting ectoparasites and the development of fungi in fish eggs,
larvae, and adult fish (Wu et al., 2007). It is immediately absorbed by fish and metabolically
reduced to a non-polar and colorless compound known as leucomalachite green. Most of
the MG residues in fish tissue are found in the form of LMG, which accumulates in the
adipose tissues, thus this substance is used as a marker to control the illegal use of MG
(Arroyo et al., 2008; Hernando et al., 2006; Srivastava et al., 2004). The chemical structures
of MG and its biotransformation product are demonstrated in Figure 3.
Brazilian aquaculture is based mainly on semi-intensive production regimes, and most
fish culture is done in excavated ponds. The larvae and fingerlings are usually stocked in
the ponds and feed during the entire cultivation period. It is also common t o use fish cages
installed in reservoirs and big ponds. The main species cultivated in Brazil are tilapia and
tambaqui in the Northern region (Diegues, 2006). Nile tilapia (Oreochromis niloticus)is
becoming the most cultured species in cages (Figure 4) installed in big reservoirs located
in the Northeastern and Southeastern regions, such as the São Francisco and Tietê Rivers
(Ostrensky et al., 2008).
The most common diseases affecting fish culture are caused by facultative pathogens.
These diseases are especially common in fish submitted to chronic stress. The major causes
of stress are directly related to inadequate handling practices, poor water quality, and low
quality feeds that do not meet the nutritional requirements of the different species of fish.
Thus, low quality feeds increase the chances of diseases occurring and high mortality rates.
The use of high fish densities in cages is another factor of stress in such systems (Ostrensky
et al., 2008). The degree of stress resulting from t hese conditions is characterized by mod-
ifications in the biochemical, physiological, and behavioral mechanisms, with a possible
Figure 3. Chemical structures of malachite green (A) and leucomalachite green (B).
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Use of Malachite Green in Aquaculture 277
Figure 4. Photograph illustrating fish culture in cages.
increase in the susceptibility to infectious processes caused by opportunistic pathogens like
viruses, bacteria, fungi, and parasites (Santos, 2007).
Parasites are the pathogenic agents most studied in Brazilian fish culture. The gills are
the main targets where blood losses and necroses frequently occur causing reduction in
the respiratory rate and impairment of the regulatory capacity. Infestations by metazoary
ectoparasites can represent great losses for fish farmers, because they reduce fish growth
rate, promote hematological alterations, behavioral changes, and mortality—especially of
larvae and juveniles. Besides this, at the spot where the parasite penetrates the fish host,
blood losses and inflammation normally occur, with necrosis of the tissues facilitating the
attack by secondary infectious agents (Santos, 2007).
The Lerneaidae family of the subclass Copedoda is the most important group of par-
asites in fresh water fishes, especially the Lernaea cyprinacea species (Ostrensky et al.,
2008). The importance of this group is due to its occurrence in practically all states in
Brazil, and also because it affects several species of fish in closed culture systems, as
well as in their natural environments. Amongst several fish species sensitive to Lernaea
(Figure 5), Cyprinus carpio (common carp) and Oreochromis niloticus (tilapia) are the
most affected; they are also the two major species cultivated in the Southeastern region of
Brazil. Infestation by these parasites causes huge damage to fish culture. The copepods are
considered real parasites because small numbers of these organisms always cause anoma-
lies and clinical signs in the host. A single specimen can be lethal if the fish is small and the
cephalic hooks penetrate the brain or some other vital organ. In places where the parasite
attaches itself to the fish, an increase i n the secretion of mucus (skin, fin, and branchiae),
inflammation, blood losses, and ulcerated zones can be seen. A loss of scales around the
parasite and losses in appetite and weight are also observed (Pizzolatti, 2000; Figure 6).
Besides the Lernaea sp., other crustaceans threaten fish cultures in Brazil, including
the copepod Eragasilus sp. and the branchiura Dolops sp. and Argulus sp (Santos, 2007).
One of the difficulties related to preventing and treating fish diseases in Brazil is the
lack of specific legislation and approved veterinary medication for use in aquaculture. In
2008, the first and only medication based on florfenicol was approved to control bacterial
infections in fish (Schering-Plough Animal Health, 2007). These factors, associated with
the lack of scientific information about alternative treatments to control fish diseases, have
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278 J. C. Hashimoto et al.
Figure 5. Photograph of the Lernaea sp. (Source: Pizzolatti, 2000).
Figure 6. Photograph of a fish infected by Lernaea cyprinacea (Source: Pizzolatti, 2000).
led to the indiscriminate use of substances such as MG dye by many fish farmers, despite
the danger and restrictions for its use in fish cultured for human consumption (Carneiro
et al., 2005).
Pavanelli (1998), as quoted by Pizzolatti (2000), emphasized that few studies had
been carried out in Brazil with the objective of testing the efficiency and secondary effects
of drugs used for the treatment of fish diseases, especially for fish produced in intensive
culture systems.
Souza (2003) affirmed that the indiscriminate use of chemical products to control
parasites in fish culture systems is becoming a cause for great concern. The author also
emphasized the easy access that everyone has to treatment prescriptions on the Internet;
for example, for organophosphorus compounds, MG, and formalin.
A review on therapeutic treatments to prevent crustacean parasites was published by
Pizzolatti (2000), including the use of substances such as organophosphorus pesticides,
sodium chloride, potassium permanganate, formalin, and MG oxalate. However, there
are doubts concerning the efficiency and safety of using these substances to treat fish.
Regarding the use of MG oxalate, the author explained that despite the fact that this sub-
stance is being used intensely to treat fish diseases, its use is prohibited by the United
States Food and Drug Administration (USFDA) and is not recommended for fish cultured
for human consumption, due to its carcinogenic potential and teratogenic action.
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Use of Malachite Green in Aquaculture 279
Evidence of the Use of Malachite Green in Aquaculture
Despite prohibition of the use of malachite green for fish cultured for human consump-
tion, it continues to be used by aquaculture on a global scale; this has been related
and attributed to its low cost, availability, and recognized antimicrobial and antiparasitic
efficiency (Halme et al., 2007; Hernando et al., 2006; Srivastava et al., 2004).
Monitoring and inspection programs in action in several countries have already issued
notifications concerning barriers to imports, due to the presence of MG residues in aquacul-
ture products. Recently, England (2002), Holland (2003), and Taiwan (2007) detected MG
residues in Chilean salmon. In 2003, the Rapid Alert System for Food and Feed (RASFF)
of the European Commission reported 11 notifications of the presence of MG in Chilean
salmon, representing 3% of the total of notifications for prohibited substances in food
of animal origins for that year (European Commission Health & Consumer Protection
Directorate, 2003; Lem and Karunasagar, 2007). The same occurred in 2006, when 17
occurrences of MG and LMG were found in aquaculture products; and in 2007, 9 cases
were reported, with 4 from Vietnam, 2 from Thailand, 2 from China, and 1 from Spain
(European Commission Health & Consumer Protection Directorate, 2007).
In Japan, 29 imported fish and fish products were refused in 2005, due to the presence
of veterinary medication and prohibited substances; the majority of the cases involved the
use of tetracycline, fluoroquinolones, and MG (Lem and Karunasagar, 2007).
In June 2007, the USFDA blocked the sale of five species of fish and seafood cultivated
in China, due to the repeated number of cases related to the presence of non- approved vet-
erinary medication such as nitrofurans, MG, gentian violet, and fluoroquinolones (Martin,
2007).
The notifications issued by the European Union, Japan, and the United States are con-
sidered of great relevance, since they represent 72% of the global market of fish imports
(Lem and Karunasagar, 2007).
In Brazil, little data is available on the monitoring of MG residues in fish, but there is
evidence of the use of this substance in fish culture. In 2006, a study was conducted based
on the application of questionnaires to fish farmers in the Mogi-Guaçu (SP) hydrographic
basin. The results obtained showed that 13 out of 84 fish farmers used MG on their prop-
erties for prophylaxis and/or disease treatment. Amongst the other compounds mentioned,
only calcium oxide (lime), pesticides (organophosphates, benzoylphenylurea, carbamates,
and pyrethroids), and common salt presented a higher frequency of use when compared to
MG (Santos, 2007).
Toxicological Aspects
Malachite green residues persist in the environment and cause acute toxicity to a wide
group of aquatic and terrestrial animals, resulting in serious risks to public health and
a potential environmental problem. Several effects have been reported in fish—such as
carcinogenesis, teratogenesis, reduction in fertility, and respiratory toxicity. Significant
alterations in some biochemical parameters of the blood also occur, as well as an increase
in total cholesterol and a reduction i n the phosphorous and calcium levels in the plasma
(Srivastava et al., 1995, 2004).
Clinical and experimental studies in mammals indicate that MG acts as a multi-organ
toxic compound. Amongst other effects, the following were reported: renal alterations in
rabbits, reduction in growth and fertility in rats; liver, spleen, kidney, and heart damage;
skin, eye, lung, and bone lesions; and teratogenic effects in rats and mice. Carcinogenic
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280 J. C. Hashimoto et al.
effects were verified in experimental animals involving liver, lung, mammary, ovary, and
thyroid tumors (Culp et al., 2006; Srivastava et al., 2004).
Besides the toxic effects presented, a few bacteria isolated from sick carp and trout
presented resistance to MG, representing evidence of an environmental problem, especially
in aquatic ecosystems (Srivastava et al., 2004).
The presence of MG residues in food becomes of greater concern when the stability
of these substances to heat treatments commonly used in food preparation is considered.
Mitrowska et al. (2007) studied the persistence of MG and LMG in carp fillets submitted to
different cooking processes. No reduction was observed in the initial concentration of LMG
after cooking the fish for 15 min, which indicates an elevated stability of this substance
under the heat processing conditions of foods.
In 2007, MG was included in the list of priorities for toxicological evaluation by
the FAO/WHO expert committee on food additives (JECFA), with the purpose of pro-
viding orientation f or the Codex Committee on Veterinary Medication Residues in Food
(CCRVDF) as to the use of this substance in animals used for human consumption
(USFDA, 2007). In the report of the 18th session of the CCRVDF and after reviewing
the literature available, JECFA reaffirmed that MG could not be used in animals utilized
for human consumption due to the toxicity of its main biotransformation product, LMG
(Codex Alimentarius Commission, 2009).
Legislation and Inspection
The definition of a residue by the European Union legislation includes pharmacologically
active substances, their metabolites, and other substances transmitted t o animal products
that can be hazardous to human health.
Regulation No 2377/90 of the European Community Council, which foresees a com-
munity process for the establishment of maximum limits for veterinary medication residues
in animal products used for human consumption, requires that only approved pharmaco-
logically active substances be used for veterinary treatment. The annexes of the Regulation
determine which substances are allowed, based on the different veterinary medications
used in animal products and the maximum limits of residues allowed in the edible parts.
Four categories of pharmacologically active substances are defined; MG is included in
Annex I V, which lists the pharmacologically active substances that have no maximum
limits determined because they are prohibited for application in animals used for human
consumption (European Commission, 1990).
In 1996, the European Commission published the Directives 96/22/EC and
96/23/EC, which constitute the current legal framework for the control of veterinary
medications in food of animal origin. The Directive 96/22/EC forbids the use of certain
substances with hormonal or thyrostatic actions in livestock, while Directive 96/23/EC
establishes measures for monitoring residues in live animals and their respective prod-
ucts. In addition, the Directive 97/747/EC establishes the levels and frequency of samples
necessary to monitor such substances and their residues in certain animal products. This
directive included the control of residues in poultry and rabbit meat, eggs, milk, honey,
and fish (Serratosa et al., 2006). In 2002, the Directive 2002/657/EC was published,
which deals with the performance of analytical methods and interpretation of the results,
and defines the maximum residue limits (MRL) and minimum required performance limit
(MRPL) applicable to the determination of contaminants in food. Directive 2004/25/CE
(European Commission, 2004) adds an MRPL equal to 2 μgkg
1
for the sum of MG and
LMG in aquaculture products to the previous directive.
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Use of Malachite Green in Aquaculture 281
FAO Directive 07/2005/QD-BTS also includes MG in the list of banished chemical
compounds used in aquaculture (FAO, 2005).
In Brazil, the National Plan for the Control of Residues and Contaminants in Animal
Products (Plano Nacional de Controle de Resíduos e Contaminantes em Produtos de
Origem Animal—PNCRC) was instituted by the Ministerial Ordinance No. 51 of May 6,
1986, and adapted by the Ministerial Ordinance No. 527 of August 15, 1995, of the
Ministry of Agriculture, Livestock and Food Supply (MAPA). The plan has specific pro-
grams for the control of residues in meat, milk, honey, and fish, resulting from the use
of pesticides and veterinary medications or due to accidents involving environmental
contaminants (Brasil, 1995, 1999).
The absolute necessity to meet the sanitary requirements of important international
markets (United States, European Union, and Japan), as well as concern at a national level,
has determined the establishment of a policy for the protection of consumer health and
for the presence of residues in fish products. In view of such concerns, the Program for
the Control of Residues in Fish (Programa de Controle de Resíduos em Pescados—PCRP)
was established along with the PNCRC with the objective of ensuring the integrity and
safety of fish within the national territory and for exports, with respect to contamination by
residues of hazardous substances originating from the application of agrochemicals, veteri-
nary medications, and environmental contaminants. However, the planning of the activities
and presentation of t he results of the PCRP for the period from 1999 to 2007 did not refer to
MG, giving greater emphasis to the determinations of mercury, nitrofurans, and chloram-
phenicol (Brasil, 1999, 2006, 2007). Nevertheless, determination of MG in cultivated fish
and shrimp has been considered since the PNCRC 2008 activities (Brasil, 2008). In 2008,
via the aforementioned program, 56 samples of cultivated fish were analyzed to verify the
presence of MG residues, and all samples presented negative results. Due to the fact that it
is a forbidden substance for use in aquaculture, the Reference Limit adopted for regulatory
action is equal to the established MRPL, in this case, equal to 2 μgkg
1
for the sum of
MG and LMG (Brasil, 2009).
In 2003, the National Health Surveillance Agency (Agência Nacional de Vigilância
Sanitária—ANVISA) created the National Program for the Analysis of Veterinary Drug
Residues in Foods of Animal Origin (Programa de Análise de Resíduos de Medicamentos
Veterinários em Alimentos de Origem Animal—PAMVet). This program contemplates sam-
ple collection actions on the retail market and the analysis of residues, with the objective
of evaluating t he exposure of the consumer to veterinary drug residues through the con-
sumption of animal products. In the initial schedule of the program, the analyses of fish
samples were planned for the fourth year of activity after its implementation (Brasil, 2003).
However, up to the present moment, only the results for the analysis of milk have been
reported (Brasil, 2006).
Determination of Malachite Green in Fish
Screening Methods
Immunoenzymatic methods such as ELISA (Enzyme Linked Immunosorbent Assay) are
based on the principle of immunospecific interaction between an antigen and its antibody.
Due to the speed in obtaining results, the ELISA method has been widely used for the
screening of contaminants in food products on account of its high detectability, speci-
ficity, simplicity, and the possibility of the simultaneous analyses of many samples (Jin
et al., 2006). Nevertheless, although commercial ELISA kits for the determination of MG
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282 J. C. Hashimoto et al.
in water and fish meat are available, there are no reports in the scientific literature on the
application of such kits. It must be mentioned that due to the cross-reactions with com-
ponents of the sample, these tests are generally susceptible to false positives. Therefore,
a more specific method for confirmation is necessary, such as chromatographic methods
coupled to mass spectrometry (MS).
Some kits used for the detection of malachite green have the disadvantage of not
presenting a specific or cross-reaction for leucomalachite green, which is the main bio-
transformation product of MG, and thus both substances can not be detected by the same
ELISA kit. According to Yang et al. (2007), the difference in reactivity against antibodies
is attributed to the “geometry of the molecule’s” central carbon atom, which is planar for
MG and presents a tetrahedron form for LMG. As an advantage, the sample preparation
is simple, consisting of homogenization and extraction steps with organic solvents such as
acetonitrile, methanol, or dichloromethane. In certain cases, a clean-up step of the extract
with cartridges of neutral alumina is necessary (Abraxis, n.d.; Bioo Scientific, 2009; Euro
Proxima, n.d.; Glyconex, 2009).
Quantification and Identification of Confirmation Methods
Several analytical methods have been developed to determine the presence of MG in
aquatic animals for human consumption (trout, carp, salmon, catfish, eels, shrimp, and
shellfish, among others), and the majority of these methods use high performance liquid
chromatography as the separation technique.
The chromatographic separation of MG and LMG is mostly carried out using r everse
phase octadecyl analytical columns (C
18
; Bergwerff and Scherpenisse, 2003; Halme et al.,
2007; Hernando et al., 2006; Lee et al., 2006; Scherpenisse and Bergwerff, 2005). Phenyl-
hexyl columns have been used less frequently (Bergwerff et al., 2004; Mitrowska et al.,
2005). The mobile phases used are generally constituted of mixtures of acetonitrile and
acidified aqueous buffer solutions, with isocratic or gradient elution. In terms of the ion-
izable analytes, the pH of the mobile phase must be adjusted in order to ensure that the
compound is present in a single format, generally adopting one or two units around the
pKa value of the compound of interest for the pH of the buffer. For basic compounds,
buffer solutions with a pH one unit lower than the pKa of the compound produce posi-
tively ionized species (Jardim et al., 2006). The composition and flow rate of the mobile
phase, temperature, and mode of elution are adjusted to optimize the separation of the ana-
lytes from interfering compounds. Due to the difference in polarity between MG and its
metabolite, the gradients and temperature are also adjusted to allow for chromatographic
separation in the shortest possible time.
The presence of malachite green and leucomalachite green can be determined by
means of high performance liquid chromatography with a visible range absorption detector
(HPLC-Vis) or fluorescence detector (HPLC-FLD), respectively. Liquid chromatography
coupled to mass spectrometry is more frequently employed.
The sum of MG and its biotransformation product can be determined by HPLC-
Vis, after conversion of the LMG into MG using oxidation pre-columns filled with lead
oxide (IV; PbO
2
). This is an adequate screening method, since the combined signs cause
a gain in sensitivity, allowing the use of simpler equipment (Valle et al., 2005). In addi-
tion, the MRPL established refers to the sum of the MG and LMG residues present in
fish meat, and it is not necessary to determine the separate analytes. Several methods
rely on PbO
2
post-column oxidation, which allows determining MG and LMG separately
(Rushing and Thompson, 1997; Bergwerff and Scherpenisse, 2003; Hajee and Haagsma,
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Use of Malachite Green in Aquaculture 283
1995). However, some disadvantages have been reported such as the formation of addi-
tional oxidation products, a short life cycle of the oxidation columns, and the possibility
of cross-contamination in multi-residual laboratories (Mitrowska et al., 2005). Valle et al.
(2005) reported the partial degradation of MG to a demethylated form using an oxidation
column filled with PbO
2
: celite, the extension of degradation variation according to the pro-
portion of PbO
2
and the time of permanence of the sample in the column. Such processes
can compromise the precision and selectivity of the method.
Several published methods do not require the post-column reaction. Andersen et al.
(2005, 2006), Tarbin et al. (2008), and Bueno et al. (2010) used the reagent 2,3-dichloro-
5,6-dicyano-1,4-benzoquinone for analysis by HPLC-Vis. Rushing and Hansen (1997)
performed the oxidation in an electrochemical cell, and Long (2008) used an iodine reactor
for post-column derivatization.
The liquid chromatographic technique coupled to a visible absorbance detector and a
fluorescence detector in series allow for the simultaneous determination of the presence
of both analytes. An advantage of this experimental array is that, unlike the equipment
coupled to a mass spectrometer, these detectors are available in many laboratories since
they are cheaper than other similar equipment and are also easier to operate (Mitrowska
et al., 2005; Rushing and Hansen, 1997). Nevertheless, methods based on the use of LC-MS
have recently become more common to verify the presence of MG residues, especially due
to their advantages related to selectivity and detectability; for this reason, this technique
will be discussed in more detail.
Mass Spectrometry
The analysis of residues and contaminants in food involves the determination of com-
pounds present in a complex sample at levels of µgkg
1
. Thus, adequate detectability and
selectivity of the analytical methods are necessary.
The complexity of food samples can be a problem in mass spectrometry, because sup-
pression of the ionization of the analytes can occur. Thus, certain steps must be followed
during sample preparation using extraction techniques to clean the extract and promote
an efficient chromatographic separation in order to minimize the presence of interfering
compounds (Careri et al., 2002). However, in many cases, mass spectrometry shows intrin-
sic characteristics that allow for elevated selectivity, simplifying the analytical methods
frequently used in spectrophotometric detection.
The mass spectrometer enables one to confirm the identity of compounds of interest
from the molecular mass data of the precursory ion and its fragmentation products, which
provides information about the molecular structure. In addition, in view of the characteris-
tics of the chromatogram of mass extracted, it is possible to solve problems in determining
compounds that do not present good resolution in chromatographic separations due to
structural similarities.
The appearance of ionization techniques using electronebulization (ESI) and atmo-
spheric pressure chemical ionization (APCI) have allowed for the analysis of almost all
types of molecules. Both techniques consist of mild ionization, which produces ions with
low energy, generally singly charged, facilitating the identification of compounds with-
out the obligation of using spectrum libraries and also allowing for high detectability and
selectivity for fast analyses.
ESI works by providing a source of electric charge to the eluent when it emerges from
the nebulizer. The result is an aerosol with electrically charged droplets, which suffers
a reduction in size until it reaches sufficient charge density to permit ejection from the
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284 J. C. Hashimoto et al.
surface of the droplet of ions of the sample (ionic evaporation). At the APCI source, the
sample is vaporized in a heated nebulizer before emerging to plasma constituted of ions
from the solvent, formed at the source at atmospheric pressure by a corona discharge.
The analytical methods available for the determination of MG commonly use ioniza-
tion sources compatible with liquid chromatography, such as APCI and especially ESI.
ESI provides greater sensitivity to determine the presence of MG (Halme et al., 2007; Wu
et al., 2007). In a comparative study, Turnipseed et al. (2006) found no-discharge APCI to
provide greater sensitivity for both MG and LMG when compared to ESI. Scherpenisse
and Bergwerff (2005) also describe APCI as being a more sensitive ionization technique
than ESI for LMG.
Various mass analyzers have been described in the literature—such as quadrupole
(Doerge, 1998; Sanders et al., 2005; Scherpenisse and Bergwerff, 2005; Turnipseed et al.,
2005; van de Riet et al., 2005; Valle et al., 2005), time of flight (ToF; Hernando et al.,
2006), and ion trap (IT; Wu et al., 2007; Lee et al., 2006). For quantitative purposes, the
quadrupole analyzers in tandem (triple quadrupole) provide lower limits of detection and
quantification, since they are continual analyzers that operate in a discriminatory man-
ner. For confirmation purposes, the ToF analyzers present advantages in comparison to the
quadrupole analyzers because of their higher resolution and mass accuracy.
Methods using MS are particularly adequate to confirm the identity of analytes by
means of exact mass measurements and information about the chemical structure. The
Directive 2002/657/EC defines confirmatory methods for organic residues in food as those
techniques that provide complete or complementary indications of the unequivocal iden-
tification of substances of interest (European Commission, 2002). In this context, the use
of mass spectrometry techniques with high and low resolution meets the criteria for the
identity confirmation of residues based on the identification points (IP) system, which is
related to the quantity of fragment ions necessary to confirm the identity of a substance.
Using low resolution techniques, for example, when a triple quadrupole analyzer is used,
three or four ions are necessary depending on the matrix or the analyte in order to con-
firm the identity of the residues of substances used as allowed or prohibited, respectively,
whereas high resolution techniques only need two or three ions. Thus, the use of the ToF
analyzer, which is classified as being of high resolution, easily reaches the IP necessary to
confirm the presence of contaminants. Despite the fact that the sensitivity of ToF analyzers
is lower than that of triple quadrupole analyzers, they can be accurate enough to detect
substances at levels below the MRL or MRPL, thus meeting the requirements established
by the national or regional regulatory agencies or by international organizations such as
Codex Alimentarius (Hernando et al., 2006).
Preparation of Fish Samples for Chromatographic Analysis
In a simplified manner, the sequence of steps for sample preparation consists of: homog-
enization of the frozen sample, extraction with buffer solution and/or organic solvents,
partition with dichloromethane for sample clean-up, residue isolation by solid phase extrac-
tion (SPE) or the addition of absorbents. Typically, the extract is defatted by partition with
hexane or by the addition of alumina prior to SPE clean-up to prevent fat from fouling the
SPE material. According to the technique used, the eluates or extracts can be evaporated
and the residue resuspended in the solvent itself.
The homogenization step is generally performed in an Ultra Turrax homogenizer with
acidified ammonium acetate, sodium acetate, or citrate buffer solutions (Halme et al.,
2007; Mitrowska et al., 2005; Rushing and Thompson, 1997; Tarbin et al., 1998), or using
Downloaded by [UNICAMP], [Mr Felix Guillermo Reyes Reyes] at 13:14 12 July 2011
Use of Malachite Green in Aquaculture 285
McIlvaine solution at pH 3.0 (Bergwerff and Scherpenisse, 2003; Valle et al., 2005; Wu
et al., 2007). The extracts are cleaned by solid phase extraction, employing alumina and
propylsulfonic acid cartridges (Halme et al., 2007; Rushing and Thompson, 1997), reverse
phase polymeric cartridges (Lee et al., 2006), or cation exchange cartridges (Bergwerff
and Scherpenisse, 2003; Mitrowska et al., 2005; Valle et al., 2005; Wu et al., 2007).
Certain extract cleaning procedures use absorbents such as basic alumina (Rushing and
Thompson, 1997; Wu et al., 2007) or primary/secondary amines (PSA; Hernando et al.,
2006).
Certain chemical compounds are frequently added with the objective of increas-
ing the recovery of the analytes and/or minimizing their degradation. For this purpose,
hydroxylamine hydrochloride, p-toluenesulfonic acid, pentanesulfonic acid, and N,N,N,N-
tetramethyl-1,4-phenylenediamine dihydrochloride (TMPD) are used in different quanti-
ties and combinations (Bergwerff and Scherpenisse, 2003; Halme et al., 2007; Valle et al.,
2005; Wu et al., 2007).
Due to their renowned selectivity, methods that use mass spectrometry allow for sim-
pler sample preparation procedures. However, the majority of studies published described
laborious procedures with a high consumption of organic solvents in order to extract
and clean the extracted phases. In part, this can be attributed to the fact that several
methods resulted from adaptations of procedures previously developed for HPLC-Vis or
HPLC-FLD. In several cases, a single extract of the sample is used for quantification
by HPLC-Vis/FLD and also for the confirmation of its identity by mass spectrometry
(Andersen et al., 2006; Halme et al., 2004; Stoev and Stoyanov, 2007).
Bueno (2010) has developed an analytical procedure to determine MG residues in
salmon samples using molecularly imprinted polymers (MIPs) as the extraction and
clean-up material, followed by a hybrid triple quadrupole/linear ion trap mass spectrom-
etry system. The extraction method used is a modification of the procedure previously
described by Andersen et al. (2009) and included a solid–liquid extraction (SLE) method,
two clean-up steps, and one oxidation reaction with DDQ to convert leucometabolites
to the colored forms. This analytical procedure has demonstrated an optimal sensitivity
and high specificity, enabling the determination of target compounds at ng kg
1
lev-
els. In spite of the high selectivity and specificity showed by the LC–MS/MS systems,
only it has been possible to achieve these very low limits through the use of highly
selective extraction materials (MIPs), which significantly reduce the matrix effects, thus
facilitating the detection and quantification of the analytes. However, although promis-
ing, the procedure described is laborious and time consuming, inadequate for routine
analyses.
Thus, a reduced number of simple methods are available; some of which involve,
basically, extraction with acetonitrile followed by centrifugation (Hernando et al., 2006;
Sanders et al., 2005). Van de Riet (2005) published a simple method for LC-MS/MS analy-
sis that relies on an acetonitrile-perchlorate extraction with a minimal clean-up, employing
a chemically bonded octadecyl C
18
solid-phase extraction column. More recently, Chen
and Miao (2010) have developed a simple extraction method for HPLC-Vis-FLD analysis
that does not require liquid-liquid defatting. In this method, the extraction was performed
in pH 3 McIlvaine buffer and acetonitrile, followed by clean-up using a polymeric strong
cation-exchange column. However, it must be emphasized that these methods are highly
dependent on the detectability of the analytical instrument used, since they result in dilute
extracts obtained from a small sample mass.
Table 1 describes a few characteristics of the chromatographic methods published in
the literature to determine the presence of MG and LMG in fish meat.
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Table 1
Chromatographic methods used to determine malachite green and leucomalachite green in fish meat
Analytes Matrix LSE LLE or SPE Rec. (%) SP and MP Det.
CCα
(ng g
1
)
CCβ
(ng g
1
)Ref.
MG, LMG, GV,
LGV
Catfish pH 3.0
McIlvaine
buffer and
ACN
Cation exchange
(SPE)
44.5–49.2
(MG)
74.3–84.5
(LMG)
C
18
(150 × 4.6
mm, 3 µm)
Ammonium
acetate buffer
and ACN
Vis
FLD
0.38
(LOD MG)
0.10
(LOD LMG)
–Chen&
Miao
(2010)
C
18
(100 × 2.0
mm, 3 µm)
Ammonium
acetate buffer
and ACN
MS/MS
(QqQ)
<0.5 for MG
and LMG
MG, LMG, CV,
LCV, GV,
LGV
Catfish pH 4.5 acetate
buffer and
ACN
Dichloromethane
(LLE)
Alumina and cation
exchange (SPE)
90.3 C
18
(150 × 4.6
mm, 3 µm)
Ammonium
acetate buffer
and ACN
Vis 0.15
(LOD)
0.49
(LOQ)
Andersen
et al.
(2009)
82.3 Phenyl (50 × 4.0
mm, 3 µm)
Formic acid and
ACN
MS/MS
(IT)
0.24
(LOD)
0.75
(LOQ)
MG, LMG Bass, trout,
gilthead bream
and turbot
ACN 48.1
(MG)
92.1 (LMG)
RP (150 × 2.0
mm, 4 µm)
Ammonium
acetate buffer
and ACN
MS/MS
(QqQ)
0.45–0.55 0.76–0.92 Arroyo
et al.
(2009)
Triarylmethane
and
phenothiazine
dyes
Salmon pH 4.5 acetate
buffer and
ACN
Dichloromethane
(LLE)
Cation exchange
(SPE)
–C
18
(100 × 2.1
mm, 3.5 µm
Ammonium
acetate buffer
and ACN)
MS/MS
(QqQ)
1.2 2.0 Tarbin
et al.
(2008)
286
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MG, LMG Rainbow trout pH 4.5 acetate
buffer and
ACN
Dichloromethane
(LLE) Alumina
and cation
exchange (SPE)
58–65
(MG) 59–68
(LMG)
C
18
(150 × 2.1
mm, 3.5 µm)
Ammonium
acetate buffer
and ACN
MS/MS
(QqQ)
0.13 (MG)
0.16
(LMG)
0.22 (MG)
0.27
(LMG)
Halme
et al.
(2007)
MG, LMG, GV,
LGV
Grass carp, eel,
salmon,
shrimp, and
shellfish
pH 3.0
McIlvaine
buffer and
ACN
Alumina (dSPE)
Dichloromethane
(LLE) Cation
exchange (SPE)
80.8–112.1
(MG)
91.1–115.6
(LMG)
Column (150 ×
2.0 mm, 5 µm
Ammonium
acetate buffer
and ACN)
MS/MS
(IT)
0.04–0.08
(MG)
0.02–0.05
(LMG)
0.07–0.13
(MG)
0.04–0.09
(LMG)
Wu et al.
(2007)
MG, LMG, Trout, salmon,
and shrimp
pH 4.5 acetate
buffer and
ACN
Dichloromethane
(LLE) Alumina
and cation
exchange (SPE)
84.3–102.6 C
18
(150 × 4.6
mm, 3 µm)
Ammonium
acetate buffer
and ACN
Vis 1.0 (LOD) Andersen
et al.
(2006)
75.0–94.0 Phenyl (50 × 4.0
mm, 3 µm)
Formic acid
and ACN
MS/MS
(IT)
0.25 (LOD)
Antibiotics,
MG, and
LMG
Salmon ACN (0.1%
HAc) and
NaCl
PSA (dSPE) 93–107
(MG)
91–109
(LMG)
C
18
(250 × 3.0
mm, 5 µm)
Formic acid and
ACN
MS (ToF) 8 (MG) 38
(LMG)
13 (MG) 65
(LMG)
Hernando
et al.
(2006)
MG, LMG Carp pH 4.5 acetate
buffer and
ACN
Dichloromethane
(LLE) Cation
exchange (SPE)
60.4–63.5
(MG)
89.0–91.5
(LMG)
Phenyl (150 ×
4.6 mm, 5 µm)
Ammonium
acetate buffer
and ACN
Vis and
FLD
0.15 (MG)
0.13 (LMG)
0.37 (MG)
0.32 (LMG)
Mitrowska
et al.
(2005)
(Continued)
287
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Table 1
(Continued)
Analytes Matrix LSE LLE or SPE Rec. (%) SP and MP Det.
CCα
(ng g
1
)
CCβ
(ng g
1
)Ref.
MG, LMG Salmon, trout,
tilapia, and
catfish
ACN 39–49 (MG)
82–118
(LMG)
Phenyl (150 ×
4.6 mm, 5 µm)
Ammonium
acetate buffer
and ACN
MS/MS
QqQ
< 0.3 < 0.5 Sanders
et al.
(2005)
LMG Salmon, trout,
pangasius,
tilapia, and
Victoria perch
pH 3.0
McIlvaine
buffer and
ACN
Dichloromethane
(LLE) Cation
exchange (SPE)
66–112
(LMG)
C
18
(150 × 2,0
mm, 3 µm)
Ammonium
acetate buffer
and ACN
MS/MS
QqQ
0.11 (LMG) 0.15 (LMG) Scherpenisse
& Bergwerff
(2005)
LMG Salmon pH 4.5 acetate
buffer and
ACN
Dichloromethane
(LLE) Alumina
and cation
exchange (SPE)
86–109
(LMG
measured
as MG)
Phenyl (50 × 4.0
mm, 3 µm)
Formic acid
and ACN
MS (IT) < 0.15
(LOD)
0.15
(LOQ)
Turnipseed
et al.
(2005)
MG, LMG Salmon pH 3.0
McIlvaine
buffer and
ACN
Dichloromethane
(LLE) Cation
exchange (SPE)
70 (MG)
85 (LMG)
C
18
(250 × 3.0
mm, 5 µm)
Formic acid
and ACN
MS (Q) 0.15 (LOD
for the
sum of
MG and
LMG)
-Valle
et al.
(2005)
MG, LMG Salmon Perchloric acid-
acetonitrile
solution
Octadecyl (C
18
)
(SPE)
98 (MG)
81 (LMG)
Column C
18
Ammonium
hydroxide-
formic acid
buffer in ACN
MS/MS
(QqQ)
0.1 - van de
Riet
et al.
(2005)
288
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MG, LMG Catfish, eel,
rainbow trout,
salmon,
tropical
prawns, and
turbot
pH 3.0
McIlvaine
buffer and
ACN
Dichloromethane
(LLE) Cation
exchange (SPE)
44–68 (MG)
86–105
(LMG)
C
18
(150 × 2,0
mm, 3 µm)
Ammonium
acetate buffer
and ACN
MS/MS
QqQ
0.2 (LOD) - Bergwerff
&
Scherpenisse
(2003)
C
18
(100 × 3,0
mm)
perchlorate
containing
sodium acetate
and 1-
pentanesulfonic
acid and ACN
Vis 1.0 (LOD) -
LSE: liquid-solid extraction; LLE: liquid-liquid extraction; SPE: solid phase extraction; Rec.: recovery; SP: stationary phase; MP: mobile phase; Det.: detection;
CCα: decision limit; CCβ: detection capacity; LOD: detection limit; LOQ: limit of quantification; Ref.: bibliographic reference; MG: malachite green; LMG:
leucomalachite green; CV: crystal violet; LCV: leucocrystal violet; BG: brilliant green; LBG: leucobrilliant green; ACN: acetonitrile; HAc: acetic acid; PSA:
primary/secondary amine; dSPE: dispersive solid-phase extraction; RP: reverse phase; MS/MS: sequential mass spectrometry; QqQ: triple quadrupole analyzer;
Vis: visible absorbance detector; IT: ion trap analyzer; ToF: time of flight analyzer; FLD: fluorescence detector; MS: mass spectrometry; Q: quadrupole analyzer;
GV: gentian violet; LGV: leucogentian violet.
289
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290 J. C. Hashimoto et al.
Final Considerations
The consolidation of aquaculture as an economic activity of great worldwide importance,
especially in Brazil, presents challenges in terms of the production of safe food. Like other
agribusiness activities, the use of veterinary medications is an important tool to ensure
high productivity in intensive production systems. In Brazil, due to the low availability
of regulated veterinary medication for use in agriculture, several problems have arisen
as a result of the use of illicit substances. Amongst these substances, the dye malachite
green, which presents elevated acute and chronic toxicity for a wide group of aquatic
and terrestrial animals, is still used by fish farmers. Thus, residues of MG and of its
main biotransformation products, leucomalachite green, may be present in fish offered
directly to the consumers. The presence of this residue represents a risk to human health,
as well as being a potential environmental threat and leading to exportation barriers. For
this reason, monitoring of the presence of MG residues in aquaculture products is of
fundamental importance to protect consumers and also for the development of the aqua-
culture industry. Since 2008, the Brazilian Ministry of Agriculture, Livestock and Food
Supply, by way of the National Program for the Control of Residues and Contaminants,
monitors the presence of MG in fish and shrimp samples. In order to maintain these
governmental actions, it is important to emphasize the need to develop analytical meth-
ods with proper performance to allow the determination of these residues by routine
analyses. The determination of MG residues in fish is mainly carried out using liquid
chromatography techniques coupled to mass spectrometry, due to its advantages with
respect to selectivity, detectability, and the possibility of confirming the identity of the
analytes.
References
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... Malachite green (MG) is a synthetic basic dye belonging to the triphenylmethane family which has been widely used in fish farming, particularly salmonids, since 1930 as a very effective antimicrobial against fungi and protozoan ectoparasites [1]. In aqueous solutions, the compound might be present as a green-colored free cation (cationic form) or as its metabolite, leucomalachite green (LMG), a colorless neutral form. ...
... Due to its carcinogenic, mutagenic and teratogenic potential to mammals, MG has never been registered as a veterinary drug and its use in aquaculture production is illegal. Furthermore, given its persistence in edible fish, the United States and the European Union established maximum legal residue limits of 2 µg/kg for the total MG and LMG in food [1]. Human exposure to these contaminants has been documented as mainly occurring through food and water ingestion [4,5] indicating the need to find effective, reliable and easy to implement analytical methods for controlling these toxic products in food and water. ...
... Common methods to determine MG in water involve a pre-concentration step followed by liquid chromatography [1,[6][7][8][9] and spectrophotometric (UV-Vis) analyses [10][11][12]. These methods are time-consuming and their need for chromatographic separations produce toxic organic waste. ...
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... It is used for dyeing fabrics like leather, silk, and cotton (Daneshvar et al., 2007). It is also applied as an antifungal agent in aquaculture to control infections in fish (Hashimoto et al., 2011). ...
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Relationship Between Microbes and Environment for Sustainable Ecosystem Services, Volume Two: Microbial Mitigation of Waste for Sustainable Ecosystem Services promotes advances in sustainable solutions, value-added products, and fundamental research in microbes and the environment. Topics include advanced and recent discoveries in the use of microbes for sustainable development. Volume Two describes the successful application of microbes and their derivatives for waste management of potentially toxic and relatively novel compounds. This proposed book will be helpful to environmental scientists, experts and policymakers working in the field of microbe- based mitigation of environmental wastes. The book provides reference information ranging from the description of various microbial applications for the sustainability in different aspects of food, energy, environment industry and social development.
... Malachite green (MG) dye is a synthetic cationic dye. MG dye is very popular in treating fungus, antibacterial, ectoparasites and Ichthyophthirius multifiliis in freshwater aquaria 21,39 . This dye is used extensively in the textile industries for dying wool, jute, leather, silk and paper 7,40 . ...
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... Malachite green (MG), a toxic cationic triphenylmethane dye, has been used as a topical antiseptic to treat protozoan, fungal and bacterial infections in aquaculture (Srivastava et al., 2004). However, it is always present in the wastewater and will become a highly lethal compound due to their acute and chronic toxicity (Hashimoto et al., 2011). In addition, MG has broadly been used as a disinfectant, food additive, anthelmintic, and as a dyeing agent for the textile industry. ...
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An attempt has been made to collect upto date information of the toxicity of a dye malachite green on H. fossilis which are frequently used in fish farming. Exposure to malachite green to a concentration of 0.20 mg/l () for 96 h caused significant depletion of serum calcium and protein levels. Short-term (10–20 days) exposures to sub-acute 0.10 mg/l () and sub-lethal 0.05 mg/l () levels of the dye also affect significant decrease in the serum calcium and protein levels; however, long-term (30–60 days) exposure did not show any changes in comparison to control fish. The total cholesterol level of blood is increased significantly at all the concentrations of malachite green in respect to all the time intervals.
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Liquid chromatography with time-of-flight mass spectrometry (LC–TOF-MS) method has been developed for simultaneous confirmation by accurate mass measurement and quantitative determination of antibiotics (enrofloxacin, oxolinic acid, flumequine, erythromycin), fungicides (malachite green MG, leucomalachite green LMG) and parasiticide (emamectin benzoate) residues in edible portion of salmon. Confirmation of chemotherapeutant residues has been based on the system of identification points (IPs) established in the Commission Decision 2002/657/EC concerning the use of mass spectrometry (MS) techniques. A validation study on matrix is presented evaluating accuracy in terms of precision (λppm 0.83–1.15) and trueness (0.22–0.70 Da). Limits of detection (LODs) and limits of quantification (LOQs) were in ranges of 1–3 and 3–9 μg/kg, below the maximum residue limits (MRLs) established in current EU legislation (100–200 μg/kg) for these chemotherapeutants. Considering the EU guidelines, decision limits (CCα) and detection capabilities (CCβ) were determined (ranges of 103–218 and 107–234 μg/kg, respectively) for authorised substances. For no authorised compounds (MG and LMG), LODs were 2 and 1 μg/kg, respectively, but exceed the MRPL (minimum required performance limit) established in the legislation which corresponds to the sum of MG and LMG (2 μg/kg). Acceptable intra-day and inter-day variability, in terms of relative standard deviation (R.S.D.) of the analytical method, were obtained (2–15%). Linearity was demonstrated from the LOQs of the analytes to 600 μg/kg (r > 0.9991). The method has involved an extraction procedure based on solid–liquid extraction (SLE) with recoveries higher than 80% for most target chemotherapeutants, with exception of enrofloxacin (40%).
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A sensitive method for the determination and confirmation of the sum of malachite green (MG) and leucomalachite green (LMG) in salmon muscle has been developed. It is based on the use of an oxidative pre-column reaction which converts LMG into MG previous to liquid chromatography–atmospheric pressure chemical ionisation–mass spectrometry (LC–APCI–MS) analysis. The determination of both compounds together constitutes a good screening method to confirm the presence of this kind of residue, taking into account that the combined signals will provide a gain of sensitivity. The detection limit, determined for spiked salmon samples using the confirmatory ion m/z 313, was 0.15 μg/kg. The recoveries determined at a spiking level of 2 μg/kg were 85 and 70% for LMG and MG, respectively, with respective relative standard deviations of 1.3 and 3.1%.
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With a new oxidant for post-column chemical derivation, a novel approach was developed for the determination of Malachite Green (MG) and Leucomalachite Green (LMG) in fish by high-performance liquid chromatography (HPLC). A C(8) column was used for separation, and elution was performed with a pH 2.5 phosphate buffer (0.02 mol L(-1)) containing 40% acetonitrile. When the eluate was combined with 3.0 x 10(-4) mol L(-1) iodine solution, LMG was converted to MG and detected at 618 nm after post-column derivatization. The recoveries of MG and LMG were ranged from 67.3% to 73.9% and 84.7% to 92.1%, respectively, which were obtained by measuring the amount of MG and LMG in the samples with solvent calibration curve. The decision limit (CCalpha) and the detection capability (CCbeta) obtained for MG and LMG were in the range of 0.10-0.17 and 0.13-0.23 microg kg(-1) in grass carp, shrimp and shellfish. This method appeared suitable for the control of MG and LMG residues in aquatic products.
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A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of leuco-malachite green (LMG) in various fish tissues is described. LMG, which is the primary metabolite of the parasiticide and fungicide malachite green (MG), is the targeted analyte to reveal abuse of this veterinary drug in fish. After extraction using McIlvaine buffer and acetonitrile, the extract was purified on an aromatic sulphonic acid solid-phase extraction column. After conversion of LMG into MG by post-column oxidation with PbO2, the effluent was analysed by LC-MS/MS in the multiple reaction monitoring (MRM) mode. The MS–MS trace m/z 329 →m/z 313 was used for quantification of LMG and, for salmon, gave an averaged decision limits (CCα; α 1%) and detection capability (CCβ; β 5%) of 0.11 and 0.18μgkg−1, respectively, with a measurement each of three consecutive days. The last values were comparable for those for MG. Other traces were used to collect sufficient identification points to establish the identity of this prohibited veterinary drug, which was achieved at CCβ and higher. These values were comparable for other tested species, including pangasius, tilapia, trout and Victoria perch. Recoveries ranged from 66% in trout at 0.4μgkg−1 to 112% in pangasius at 0.1μgkg−1. Three out of nineteen samples including pangasius, salmon, shrimps and trout bought in local shops, revealed detectable amounts of residues, i.e. in excess of CCα, and were considered non-compliant. The findings demonstrate the suitability of the presented analytical method to detect residues of malachite green in various aquatic species at relatively low residue levels.