Considerations on the Aquaculture Development and on the Use of Veterinary Drugs: Special Issue for Fluoroquinolones—A Review

Authors

  • Silvia Pilco Quesada,

    1. Dept. of Food Science, School of Food Engineering, Univ. of Campinas, Rua Monteiro Lobato, 80, 13083–862 Campinas, SP, Brazil
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  • Jonas Augusto Rizzato Paschoal,

    Corresponding author
    • Dept. of Food Science, School of Food Engineering, Univ. of Campinas, Rua Monteiro Lobato, 80, 13083–862 Campinas, SP, Brazil
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  • Felix Guillermo Reyes Reyes

    1. Dept. of Food Science, School of Food Engineering, Univ. of Campinas, Rua Monteiro Lobato, 80, 13083–862 Campinas, SP, Brazil
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Direct inquiries to author Paschoal (E-mail: jonaspaschoal@yahoo.com.br).

Abstract

Aquaculture has become an important source of fish available for human consumption. 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. Antimicrobial substances are compounds used in livestock production with the objectives of inhibiting the growth of microorganisms and treatment or prevention of diseases. It is well recognized that the issues of antimicrobial use in food animals are of global concern about its impact on food safety. This paper present an overview of the aquaculture production in the whole world, raising the particularities in Brazil, highlighting the importance of the use of veterinary drugs in this system of animal food production, and address the potential risks arising from their indiscriminate use and their impacts on aquaculture production as they affect human health and the environment. The manuscript also discusses the analytical methods commonly used in the determination of veterinary drug residues in fish, with special issue for fluroquinolones residues and with emphasis on employment of LC-MS/MS analytical technique.

Introduction

Aquaculture plays an important role in global food production. Due to changes in human dietary habits, fish consumption has been increasing, with more people changing to a healthier diet with an appropriate nutritional profile (Crepaldi and others 2006).

As in any animal production system, the emergence of diseases is unavoidable, leading to potential economic losses. In aquaculture, the aquatic environment favors the emergence and spread of infectious diseases. Any abrupt physicochemical changes in the aquatic environment, for example, a sharp drop in temperature, would have a direct effect on fish health, which leaves the population more vulnerable to the outbreak of infectious diseases. This scenario, coupled with possible inadequate management practices and poor environmental conditions, favors the appearance of parasitic, bacterial, viral, and fungal infections (Roberts and Bullock 1980; Schalch and others 2005).

The use of veterinary drugs is necessary for both the prevention and the treatment of infectious diseases. In general, veterinary drugs can be used for therapeutic, prophylactic, and metaphylactic purposes, or as growth promoters (Cabello 2004). Oxytetracycline, florfenicol, enrofloxacin, and erythromycin are some of the main antimicrobials used in aquaculture throughout the world (FAO 2005).

However, careful use of these substances in animals intended for human consumption is advisable. Their uncontrolled and inappropriate use carries potential risks related to microbial resistance, which can affect not only the production system itself but also human health and the environment. Regarding the impact on aquaculture production, once the antimicrobial resistance is established, the antimicrobial efficacy will be reduced. Regarding the impact on human health, the intake of veterinary pharmaceutical residues above the limit that is considered safe is an important public health issue because it can cause possible adverse effects, such as allergies and problems with the human intestinal flora. With respect to the environmental contamination caused by the use of veterinary drugs in aquaculture, emphasis is placed on the extensive length of time that drugs stay in the environment, thereby altering the chemical processes of the sediments (Lunestad 1992; FAO/OIE/WHO 2006; FAO 2010).

To protect against the possible presence of antimicrobial residues in products of animal origin, maximum residue limits (MRLs) are established to ensure the quality and safety of consumer products. Organizations such as the Codex Alimentarius and the European Medicines Agency (EMA) were created to regulate the use of these substances in animal production. In Brazil, the Ministry of Agriculture, Livestock and Supply (MAPA) and the Ministry of Health have set up the following regulatory programs: the National Control of Biological Residues in Products of Animal Origin (PNCRB) and the Program of Veterinary Drugs Residue Analysis in Foods of Animal Origin (PAMVet).

The use of high-performance liquid chromatography linked to mass spectrometry (LC-MS/MS) is among the modern analytical techniques most frequently mentioned in the literature for the determination of veterinary drug residues in fish. In addition to their high selectivity and detectability, these analytical techniques are also essential for analyte identity confirmation.

This paper aims to present a review of aquaculture production aspects, globally and in Brazil, highlighting the importance of the use of veterinary drugs and addresses the potential risks arising from their indiscriminate use and their impacts on aquaculture production as they affect human health and the environment. In addition, we will present the analytical methods commonly used in the determination of veterinary drug residues in fish, with an emphasis on the LC-MS/MS analytical technique.

Global Aquaculture

According to the United Nations Food and Agriculture Organization (FAO 2010), the world per capita consumption of fish as a result of aquaculture development increased from 0.7 kg in 1970 to 7.8 kg in 2008, with an average annual increase of 6.6%.

The global aquaculture production is intended primarily for fresh consumption. In 2009, according to the FAO (2010) data, fish are primarily intended for fresh consumption (46.8%), followed by freezing (26.8%), canning (14.4%), and curing (10.2%) (Figure 1). It should be noted that China and Japan are the largest consumers of aquaculture products where there is a greater availability of fresh fish. These statistics do not reflect the situation in other countries as Brazil, for example, where the consumption is primarily of frozen fish.

Figure 1.

Preference intended for global fish consumption (2009).

Source: FAO (2010).

In the past 60 yr, aquaculture has been growing and gaining ground relative to extractive fishing. In 2009, the total global fishing production reached the equivalent of 145 million tons, of which approximately 38% were produced through aquaculture and 62% were produced through extractive fishing. From 2004 to 2009, extractive fishing showed a slight decrease, while aquaculture increased by approximately 7% (OCDE-FAO 2011) (Table 1). There are more than 240 food species of animals and aquatic plants available for aquaculture (Crepaldi and others 2006).

Table 1. Global fish production from 2004 to 2009
 (million tons)
Fishing production200420052006200720082009
  1. Source: FAO (2010).

Extractive fishing92.492.189.789.989.790.0
Aquaculture41.944.347.449.952.555.1
Total134.3 136.4 137.1 139.8 142.3 145.1 

In relation to fish consumption, the growth trend observed from 2008 to 2010 was not uniform for all regions of the world (Figure 2). The region surrounding Oceania showed the highest growth (24.6 kg/capita), followed by North America (21.1 kg/capita), Europe (19.6 kg/capita), Asia (17.5 kg/capita), Latin America (9.0 kg/capita), and Africa (8.1 kg/capita). The countries that led the development of aquaculture, such as France, Japan, and Spain, have registered a decrease in production in the last decade.

Figure 2.

Trends in fish consumption: growth by regions during the period of 2008–2010.

Source: OCDE-FAO (2011).

Despite the promising statistics, we must consider the existence of limiting factors for the global development of the aquaculture industry; among them are the high cost of land, environmental issues, energy costs, the shortage of skilled labor, and the lack of investment capital in developing countries.

However, there are incentives for the global development of aquaculture, that is, the production of quality products, which in turn will improve the quality of life of the human population. In addition, using systems aimed at optimizing the use of water resources, such as cages and water reuse systems, it is important to increase the possibilities of production in areas previously considered unsuitable for fish farming.

In 2008, aquaculture raised 76.4% of the world's freshwater fish, 68.2% of the fish that migrate between freshwater and saltwater, 64.1% of the mollusks, and 46.4% of the crustaceans (Figure 3). According to the FAO (2010), the world production of freshwater fish in 2008 was predominated by carp (Cyprinus carpio) with 20.4 million tons (71.1%).

Figure 3.

Aquaculture contribution for the global production: major species groups.

Source: FAO (2010).

Over the years, hybrid species, which consist of animals with intermediate characteristics due to the mixture of gene sets from different species, have been introduced into aquaculture. Thus, the creation of such species has become widespread because they have great adaptability to various conditions, with faster growth and increased resistance to disease ( Amaral Jr and others 2010). However, the actual levels of hybrid production through aquaculture throughout the world are unknown. Several countries use a considerable number of hybrids in their aquaculture production. From 1.1 million tons of Nile tilapia produced in China, approximately, one-fourth is hybrids between the Nile tilapia (Oreochromis nilotica) and the blue tilapia (O. aureus). In Brazil, the hybrid tambacu (♀ tambaqui and ♂ pacu) is widely farmed, and in recent years, its production exceeded 10000 tons (FAO 2010).

Aquaculture has fostered the global demand and consumption of species such as shrimp, salmon, clams, and tilapia, which previously originated mainly from extractive catching and are now being farmed, lowering their prices and increasing their commercialization (FAO 2010).

The increasing production of aquaculture species can also be observed when evaluating the consumption of the most important groups of fish. In Latin America, aquaculture has registered a remarkable breakthrough. Countries such as Chile, Brazil, Mexico, and Ecuador led the advance, with large productions of salmon, trout, tilapia, shrimp, and mollusks. The industrial scale of commercial aquaculture continues to dominate in Latin America. There are initiatives to develop this type of aquaculture in the Amazon, which is one of the largest aquatic environments in the world with significant aquaculture potential. In the Brazilian Amazon, 30% of household income comes from fishing (Almeida and others 2002).

Aquaculture in Brazil

In Brazil, aquaculture presents the highest growth among food producers of animal origin. In 2009, the total world production of fish reached approximately 146 million tons, of which the largest producers were China with 60.5 million tons, followed by Indonesia with 9.8 million tons, India with 7.9 million tons, and Peru with approximately 7 million tons. That same year, Brazil contributed 1.2 million tons, representing 0.86% of the world's production; thus occupying the 18th place in the overall ranking of the largest fish producers (FAO 2010; MPA 2012).

In Latin America, Brazil ranks second, behind Chile. According to the report on the State of World Fisheries and Aquaculture published by the Food and Agriculture Organization (FAO 2010), the growth of aquaculture in Latin America in recent years tripled the world's average.

Brazil has a significant natural potential for aquaculture development. It has 7367 km of coastline and 9 million hectares in water reservoirs and hydroelectric reservoirs; the climate is predominantly tropical, which favors the production of several aquatic species and concentrates 13.8% of the world's available surface freshwater. Brazil also has a significant extraction and industrial processing infrastructure, with fishing plants that are qualified according to the HACCP concept (Hazard Analysis and Critical Control Points). Considering the factors mentioned above, Brazil has the potential to become the largest fish producer in the world (Ostrensky and others 2008).

According to the Ministry of Fisheries and Aquaculture (MPA 2012), from 2008 to 2010, the Brazilian aquaculture production increased by 31.2% (Figure 4). In 2010, the continental pisciculture represented 82.3% of the total production, compared to 81.2% in 2009, and 77.2% in 2008. However, the marine aquaculture decreased compared with the previous years (Table 2).

Table 2. Annual production of continental and marine aquaculture from 2008 to 2010
 200820092010
Production(t)(%)(t)(%)(t)(%)
  1. Source: MPA (2012).

Continental28200877233735281.2394.34082.3
Marine833582287829618.885.05817.7
Total365366 415.648 479.398 
Figure 4.

Brazilian aquaculture production (marine and continental).

Source: MPA (2012).

Products of Brazilian aquaculture such as shrimp, tilapia (Oreochromis spp.), Chinese and common carp (C. carpio, Ctenopharyngodon idella and Hypophthalmichthys molitrix), pacu (Piaractus mesapotamicus), and tambaqui (Colossoma macropomum) are steadily increasing their market share in the international trade (FAO 2010).

The FAO recommends the consumption of fish due to its outstanding quality as a source of animal protein. In 2008, the per capita world consumption was 17.1 kg/yr, with a projection of 22.5 kg/yr by 2030, which represents more than 100 million tons/yr. Thus, Brazil has an excellent opportunity for the expansion of its aquaculture.

In 2010, according to data provided by the MPA (2012), the per capita consumption of fish in Brazil was 9.75 kg/yr, an increase of 8% over the previous year (Figure 5).

Figure 5.

Brazilian per capita consumption from 1996 to 2010.

Source: MPA (2012).

According to the MPA, tilapia (42.61%) is the most cultivated species in Brazilian continental aquaculture, followed by carp (25.93%); both species are considered exotic. Moreover, among the native species, tambaqui (14.89%), pacu (5.82%), and tambacu (5.93%) stand out (Figure 6).

Figure 6.

Percentage of fish species in the Brazilian continental aquaculture in 2010.

Source: Adapted from the 2012 MPA Statistical Bulletin.

Pisciculture is a branch of aquaculture dedicated to raising fish in captivity, and currently, it is a growing and economically feasible activity (Oliveira 2009). Among the exotic species introduced in Brazil are tilapia, carp, trout, and catfish, which present some advantages over the native species regarding the biology and availability of technical information. Undoubtedly, of all species raised through pisciculture, tilapia has the highest profile, as it is considered the most important species of the 21st century, being farm-raised in more than 100 countries (Fitzsimmons 2000).

Importance of the Use of Veterinary Drugs in Aquaculture

Inevitably, diseases are present in all animal operations, and aquaculture is no exception. The intensification of this activity contributes to the spread of disease in fish, which is linked to inadequate management and poor environmental conditions (Roberts and Bullock 1980; Schalch and others 2005).

Some of the issues related to inadequate management are the following: high stocking, feeding levels, removal and restocking, inadequate nutrition. According to Pavanelli and others (2002), high concentrations of fish facilitate the spread of pathogens, thus producing high-mortality levels. Among the environmental issues, it is important to consider the following factors: the decrease in dissolved oxygen levels; increasing levels of carbon dioxide (CO2), ammonia (NH3), and nitrite (NO2); sudden changes in temperature; the excessive accumulation of organic material; and other physical–chemical changes in the water. Another important factor to consider is the quick transportation of the fishes, which facilitates the spread of disease when purchasing care and quarantine guidelines are not respected (Pavanelli and others 2002).

The result of an imbalance between pathogen, host, and environment is disease in the fish (Toranzo and others 2004).

Several pathogens cause major economic losses in pisciculture. As an example, Kubitza (2005a) refers to the case involving the Acqua Corporation, the main tilapia producer in Costa Rica, which incurred a $2.5 million loss resulting from a chronic infection caused by the Piscirickettsia salmonis bacteria and possibly exacerbated by quality changes in the water supply.

Table 3 shows the main pathogens isolated during tilapia culture, demonstrating that the diseases can be parasitic, bacterial, viral, or fungal. Bacterial diseases are the main factor limiting productivity. Among the most common are columnariose (or gill rot), septicemia mobile, vibrio infections, streptococcosis disease (spiral swimming), edwardsiellosis, and visceral granuloma, which may be caused by different pathogenic bacteria (Ranzani-Paiva and others 1997; Kubitza 2005b).

Table 3. Pathogens reported in tilapia
PathogensFreshwaterSaltwater
  1. Source: Adapted from the Kubitza (2005a).

ProtozoaIchthyophonus sp. 
 Epistylis sp. 
 Chilodonella 
 Trichodiniasis 
FlagellatesIchthyobodo necatorAmyloodinium ocellatum
 Piscinoodinium pillulare 
MonogenicsDactylogyrus, GyrodactylusNeobenedenia melleni,
 Cleidodiscus, Cichilogyrus sp.Cichilidogynus sp.
CopepodsErgasilus spp. Branchiurans, Argulus spp.Caligus spp.
 Dolop spp. and Lemaea spp. 
VirusesIridovirus 
BacteriaFlavobacterium columnaris, Aeromonas hydrophila, Vibrio spp., Streptococcus iniae, Streptococcus agalactiae, Edwardsiella tarda, Francisella sp., Pseudomonas fluorescens, Piscirickettsia salmonis, Plesiomonas shiguelloidesStreptococcus iniae, Vibrio spp., Flexibacter maritimus (fin rot).
FungiSaprotegnia parasitic, Branchyomyceae spp. 

Given the above, the use of veterinary drugs such as antimicrobial agents, which help in the treatment and prevention of infectious diseases, becomes a necessity. Antimicrobials are generally used to inhibit the microorganisms’ growth. In the last century, with the development of new antimicrobial agents, the treatments for infectious diseases have improved, thus reducing the animal mortality (Jiménez and Sánchez 1997; Mcewen and Fedorka-Cray 2002; FAO 2005; Rodríguez 2011).

Antimicrobials are used in animals intended for human consumption for the following purposes:

  • Prophylactic: The administration of medication to all animals in the lot to prevent diseases before they occur (Rodríguez 2011).
  • Therapeutic: The administration of medication to treat sick animals (Rodríguez 2011).
  • Metaphylactic: The use of mass medication to eliminate or minimize an expected outbreak of a disease (Jiménez and Sánchez 1997).
  • Growth promoters: Administered to animals to improve the growth rate and the food conversion (Torres and Zarazaga 2002).

Among the commonly used antimicrobial agents in aquaculture are the penicillins (amoxicillin, ampicillin), the phenicols (florfenicol), the macrolides (erythromycin), the tetracyclines (oxytetracycline), the aminoglycosides (streptomycin), the sulfonamides, and the fluoroquinolones, among others (Lim and Webster 2001; FAO 2005).

Of the antimicrobials mentioned above, the fluoroquinolones are particularly important because they exhibit a broad spectrum of action (Gram-positive and Gram-negative), in addition to other features that will be presented in the next section.

Consequences for the Use of Veterinary Drugs in Aquaculture

As in all animal production, in aquaculture, veterinary drugs are widely used during production, primarily for the treatment of bacterial diseases (therapeutic use). Therefore, their use should be conducted in compliance with good veterinary practices. However, the producers, in an attempt to avoid economic losses, often make improper and uncontrolled use of these products, increasing the risk of antimicrobial residues affecting the aquaculture production, the environment, and consequently, human health (FAO 1997; WHO 1998; FAO/SEAFDEC/CIDA 2000; OIE 2003).

The use of veterinary drugs in aquaculture has a wide range of implications and offers many advantages, such as increased productivity. Thus, the use of these drugs has been easily extended, improving both the sanitation and the bio-safety. However, as the use of antimicrobials expands, there is growing concern about irresponsible use by producers, which consequently leads to antimicrobial residues in the products. These residues are directly related to the issue of antimicrobial resistance.

The development of microbial resistance is dependent on various circumstances. It is a proven fact that high concentrations of Mg2+ (54 mM) and Ca2+ (10 mM) in brackish waters cause a reduction (>90%) of the biological activity in antimicrobials such as the oxytetracycline and the quinolones (flumequine and oxolinic acid). Thus, the bacterial strain that colonizes fish intestines may be more susceptible or resistant to these products, depending on the aquatic environment (fresh or salt water) (Smith and others 1994; FAO/RCAAP/OMS 1999).

Microbial resistance has been identified through the use of plasmids (circular molecules of double-stranded DNA that are able to reproduce independently from the chromosomal DNA), making the bacteria resistant to 2 or more antibiotics, resulting in the presence of genes that confer resistance to several antimicrobials in a single plasmid. Currently, there are numerous plasmids that have been identified as resistant to many antimicrobial agents used in aquaculture, such as sulfonamides, streptomycin, and sulfamethoxazole, among others (FAO/RCAAP/OMS 1999).

The aquaculture activity produces environmental contamination (Figure 7) because of the accumulation of organic matter, comprising of food scraps (feed), and the mixture of fecal material. This accumulation of organic matter depends on several factors, for example , the species farmed, the feed quality, among others. The fecal and feed waste contains higher contents of carbon, nitrogen, and phosphorus compared with the natural sediments (Holby and Hall 1994; Sowles and others 1994).

Figure 7.

Flowchart of the processes involved in the aquaculture production cycle.

Source: Adapted from Bruschmann (2001).

The accumulated organic matter stimulates microbial growth, changing the chemical composition, the structure, and the functions of the sediments. Some of the effects caused by the increase in organic matter are reflected in the decreased concentrations of oxygen; thus, producing changes in the normal cycling of nutrients and increasing the inflow of nitrogen and phosphorus in the sediments (Kaspar and others 1988).

However, there is another type of contamination that must be taken into account, which is consequence of the veterinary drugs used in aquaculture. Veterinary drugs, once incorporated into the diet, are frequently administrated orally. A portion of the pharmaceuticals reach the aquatic environment due to leaching from uneaten food and feces, which later may be consumed by detritivorous organisms (heterotrophic animal that feeds on dead organic matter) or by wild fish that feed around the culture systems (Alderman and others 1994; Beveridge 1996).

The fish usually do not absorb many of the antimicrobials that are mixed with the feed, and they excrete these compounds instead. Depending on the antimicrobial used, 60% to 85% of the pharmaceuticals can be excreted via the feces. It is known that the persistence of antimicrobials in sediments can vary from 1 d to months. For example, Weston (1996) reports that oxytetracycline and oxolinic acid in the sediments can persist for 10 and 6 mo, respectively (Lunestad 1992; Coyne and others 1994).

Currently, it is known that antimicrobials may be present at hundreds of meters from the breeding systems (through the hydrologic cycle—the continuous exchange of water in the hydrosphere, soil water, among others—would be the quickest way of spreading agents), and they may remain in the environment for years (Lunestad 1992; Samuelsen 1992). Sturini and others (2012) studied the role of sunlight in degrading fluoroquinolones present in environmental waters, and pointed out that the byproducts possessed residual antibacterial activity.

Studies were performed to verify whether the presence of antimicrobials in the sediments could induce changes in the environment. It has been shown that these compounds can inhibit the processes of reduction of sulfate as well as the nitrification (conversion of ammonia into nitrate, which is performed by different soil bacteria such as nitrosomonas, nitrobacter, and nitrosococcus). However, there is little evidence of changes in the microflora or in the decomposition rates of organic matter because of the antimicrobial presence. Several studies have demonstrated an increased resistance of both harmless and pathogenic bacteria in aquaculture sites that used antimicrobials, even years after the activity had been interrupted (Hastings and Mckay 1987; Aoki 1992; Hansen and others 1992; Richards and others 1992; Kerry and others 1994; Klaver and Mathews 1994).

The presence of antimicrobial residues in foods with animal origin can cause many problems for human health. Brito and Portugal (2003) reported that chloramphenicol residues cause changes in the intestine bacterial flora, leading to reduced absorption of vitamins and creating problems related to bone marrow aplasia and the newborn gray syndrome. Furthermore, Nascimento and others (2001) indicated that the presence of penicillin residues in milk can cause allergies in sensitized persons (approximately 10% of the population), which is associated with asthma episodes, digestive disorders, and even the possibility of anaphylactic shock.

Regarding the risk to human health through the development of microbial resistance in aquaculture, the FAO/OIE/WHO (2006) highlights these 2 important aspects: (1) the development of antimicrobial resistance acquired by aquatic bacteria, which can be regarded as a direct spread to humans; (2) the development of microbial resistance, acquired in aquatic bacteria through bacteria that act as a reservoir of resistance genes that are disseminated to the pathogenic bacteria and then humans. This is considered as indirect spread to humans, caused by horizontal gene transfer.

Currently, there is evidence to indicate that the genes that mediate this resistance can be transmitted from aquatic bacteria to bacteria capable of producing infections in humans and land animals. This shows that there are no boundaries with respect to the flow of microbial resistance genes and that this phenomenon is global because the use of antimicrobials in one environment will impact other environments that are seemingly distant (Sørum 1998; Rhodes and others 2000; Schmidt and others 2001; Cabello 2004).

The FAO/RCAAP/OMS (1999) also considers the possibility of the direct spread of antimicrobial resistance through the presence of antimicrobial residues in drinking water. In the case of developed countries, there is a remote possibility that antimicrobial residues may reach the consumer through the drinking water because the water treatment methods that are normally used ensure that viable pathogens do not reach the consumer. However, there is another reality for the undeveloped countries with low purchasing power and food shortages. In such countries, drinking water is not always attained through the appropriate water treatment processes. Additionally, in some undeveloped countries with tropical climate, a greater possibility exists that the fish pathogenic bacteria are acclimated to temperatures closer to the human body and are therefore able to survive in human intestines (FAO/RCAAP/OMS 1999).

According to the FAO/OIE/WHO (2006), the presence of antimicrobial residues in the human intestinal flora can have a direct effect on the health for the following reasons: (i) these residues can create a selective pressure on the dominant intestinal flora; (ii) they can promote the growth of microorganisms with natural or acquired resistance; (iii) they can directly or indirectly promote the development of acquired resistance in pathogenic enteric bacteria; (iv) they can impair the strength of the colonization, or (v) they may change the metabolic zymotic activity in the intestinal microflora (Serrano 2005). Although the available knowledge in this area is limited, some studies indicate that these effects on the human intestinal microflora may occur at a low level of exposure.

According to Cerniglia and Kotarski (2005), harmonized approach is needed in evaluating the veterinary antimicrobial drug residues in food based on their effects on the human intestinal microflora. The authors describes the background to current regulatory approaches used in applying in vitro and in vivo methods to set a microbiologic acceptable daily intake (ADI) for residues in food derived from animals treated with an antimicrobial agent.

Regulatory Aspects of Veterinary Drugs

FAO, WHO (World Health Organization), and OIE (Office International des Epizooties), in conjunction with governments from different countries, have focused attention on the issue of the irresponsible use of antimicrobials in all animal production systems. Thus, the regulations on the use of antimicrobials in aquaculture production have been strengthened (FAO 2002).

In this context, the establishment and application of MRLs for antimicrobials used in animal production is indispensable. The MRL is defined as the maximum acceptable concentration of a substance in the edible tissues of an animal (fat, kidney, liver, muscle, honey, milk, and eggs) that when ingested by humans, poses no health risks. MRLs are established for all antimicrobial agents approved for use in food animals, and they are differentiated between animal species and tissues. Special attention to the MRLs for antimicrobials is of great importance, to ensure that the available foods are safe for consumption. The establishment of MRLs aims to satisfy the ADI, which is defined as the amount of a substance ingested daily over a lifetime that should not be harmful to health and is expressed in mg/kg of body weight (JECFA 2007).

In the European Union, the body responsible for the establishment of MRLs is the Working Group on the Safety of Residues (WGSR) belonging to the Committee for Veterinary Medicinal Products (CVMP) from the EMA.

In cases of prohibited or unauthorized substances without MRL standards, the European Community, through the Commission's Decision 2002/657/EC from August 12, 2002, established the minimum required performance limits (MRPLs), which is recognized as the minimum amount of a substance(s) present in a sample to be detected and confirmed by a determined analytical method (EC 2002).

The FAO/WHO Experts Committee on Food Additives (JECFA); a body that advises the Codex Alimentarius, establishes IDA and MRLs standards for veterinary pharmaceuticals used in the production of foods of animal origin. The JECFA recommendations are adopted by the Codex Alimentarius to ensure food safety for consumers throughout the world. Table 4 shows MRLs standards established for quinolones that are globally used in aquaculture.

Table 4. Maximum residue limits established for quinolones use in aquaculture in the different regions
RegionQuinoloneMRL (μg/kg)Reference
  1. *Sum of enrofloxacin and its metabolite (ciprofloxacin).

BrasilEnrofloxacin100*Brazilian Ministry
 Ciprofloxacin100of Agriculture,
 Sarafloxacin30Livestock and
 Difloxacin300Supply—MAPA
 Oxolinic acid100 
 Flumequine600 
EuropeanSarafloxacin30European Medicines
 Oxolinic acid300 
 Flumequine600Agency—EMA
 Danofloxacin100 
 Difloxacin300 
 Enrofloxacin100* 
JapanOxolinic acid100, 60Japan Food Chemical
 Flumequine50Research Foundation—FFCR
 Danofloxacin500, 40, 600 
 Sarafloxacin100, 100 
  100, 30 
AsiaOxolinic acid50Asian Food Regulation
 Flumequine500Information
 Enrofloxacin100*Service—AFRIS
 Oxolinic acid100Agriculture and Livestock
ChileFlumequine600Service—SAG (Servicio
EcuadorEnrofloxacin100*Agrícola y Ganadero)
 Oxolinic acid100 
 Danofloxacin100National Institute for
 Flumequine200Fisheries—INP

Different countries adopt the Codex Alimentarius recommendations to establish sanitary monitoring measures. Currently, only a few countries have established MRL standards for veterinary pharmaceuticals used in aquaculture.

The European Community, Canada, and Norway are among the regions that have already approved antimicrobials for specific use in aquaculture. Canada has approved the following antimicrobials for aquacultural use: oxytetracycline, florfenicol, sulfadiazine (Trimethoprim®) and sulfadimethoxine (Ormetoprim®) (HC-SC 2011). Regulations not only determine the types of antimicrobial agents that can be used, but they also specify the species for which it is intended, the diagnosis, the dosage, and duration of treatment, as well as the interruption period to be observed between the last dosing and the slaughter (grace period) when the antimicrobial is to be used as a therapeutic agent. Compliance with these conditions and regulations ensures that residues are below the established MRL standards, and the risk of pathogenic bacteria developing resistance is negligible or at least acceptable. In the developed countries (EC, United States, and Canada), the majority of approved antimicrobials are available by prescription only and under the supervision of a qualified professional (FAO 2002).

As a member of Codex Alimentarius, Brazil adheres to the MRL standards for pharmaceuticals used for the production of food of animal origin. To ensure food safety and for inspection purposes, the MAPA created the PNCRB, approved by the Normative Instruction No. 42, December 20, 1999. This plan is responsible for monitoring the presence of compounded veterinary residues and environmental contaminants in products of animal origin.

In 2003, the PAMVet was established, coordinated by the National Sanitary Surveillance Agency (ANVISA) under the Ministry of Health. This program aims to control the presence of residues from the use of veterinary drugs in foods of animal origin. However, it is still not as developed as the PNCRB, which was established by the MAPA to perform analysis in aquacultural products.

The Ministerial Decree No. 50 of February 20, 2006, which specifies the Sectoral Programs for meat (PNCRC), honey (PNCRM), milk (PNCRL), fish (PNCRP), and eggs (PNCRO), extended the PNCRB.

The Normative Instruction No. 9 of March 30, 2007 initiates the control of residues in fish, targeting the following substances: nitrofurantoin, nitrofurazone, furazolidone, furaltadone, and chloramphenicol. In 2011 the Normative Instruction No. 24 was published, dated August 9, for the control of antimicrobial residues in farmed fish, which included the quinolones (Table 4).

In Latin America, Brazil stands out as the largest producer of veterinary drugs and as the second largest aquaculture producer. However, there are currently only 3 veterinary drugs regulated for use in aquaculture: florfenicol, oxytetracycline, and a parasiticide based on trichlorfon. Due to the lack of alternative veterinary medicinal products prescribed for use in aquaculture and the high cost of the available drugs, there is the suspicion that fish farmers are resorting to the irregular use of veterinary drugs prescribed for other animal species and even the use of illicit substances, such as the malachite green dye (FAO 2010; Hashimoto and others 2011, 2012; MPA 2012).

Fluoroquinolones

While experimenting with alkaloid degradation in 1949, price described the first compound of the quinolone group. He presented a molecule with no biological activity and named it quinolone (1-methyl-4-quinolon-3-carboxylic acid) (Perez 1999). The first quinolone used, in the clinical form, for the treatment of urinary tract infections was nalidixic acid, discovered in 1962 by Lesher and Cols, which is useful due to its activity against certain Gram-negative bacteria (Tovar and Bremón 1998). More derivatives with broader spectrum antibacterial action, such as oxolinic acid, piromidic acid, flumequine, and others, were later developed (Moellering 2000). These are called first-generation quinolones. In the 1980s, a second generation of quinolones, the fluoroquinolones (FQs), was developed. The compounds belonging to this group are ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, norfloxacin, ofloxacin, and sarafloxacin.

The basic structure of quinolones (Figure 8) has a hydroxyl group in the 3′ position and a ketone group in the 4′ position, which is required to confer the antibacterial activity. Fluorine at the 6′ position differentiates the FQs from the quinolones and contributes to the improvement of activity against Gram-negative and Gram-positive bacteria. In the 1′ position, the addition of a cyclopropyl group (for example, enrofloxacin and ciprofloxacin), an ethyl group, or a fluorophenyl group improves the spectrum of activity against both Gram-positive and Gram-negative bacteria. The addition of a piperazinyl group at the 7′ position, as in the case of ciprofloxacin and enrofloxacin, improves the spectrum of activity against Pseudomonas aeruginosa (Riviere and Papich 2009). The molecular structures of some fluoroquinolones of interest in aquaculture are shown in Figure 9.

Figure 8.

Basic structures of quinolones.

Figure 9.

Molecular structures of some fluoroquinolones of interest in aquaculture.

The FQs have a high potential for the treatment of diseases, as they have a broad spectrum of activity against Gram-positive and Gram-negative organisms, with therapeutic action in infections caused by microorganisms that are resistant to other drugs (Souza 2005). The FQs have been used primarily to treat respiratory and gastrointestinal infections. In 1995, the FDA approved the therapeutic use of sarafloxacin in poultry, which thus became the first FQ approved for use in farm animals (FDA 1995).

The FQs are considered bactericides (that is, produces bacterial death) because the MIC (minimum inhibitory concentration, that is, the minimum concentration of antibiotic that prevents visible microbial growth) and an MBC (minimum bactericidal concentration, that is, the minimum concentration of antibiotic that kills 99.9% of the original number of bacteria) have similar magnitudes.

Another important FQs characteristic is the extended post-antibiotic effect with a slow release from the tissue, which allows the administration of a single daily dose (Buffe and others 2001). Among the most commonly used FQs in global pisciculture are flumequine, sarafloxacin, and enrofloxacin (Mella and others 2000; Woo and Bruno 2011).

In Brazil, there is a great potential to FQs be regulated for use in aquaculture by at least 3 main reasons: Brazil presents an important demand for regulation of new veterinary drugs for use in aquaculture; some FQs (flumequine, oxolinic acid, sarafloxacin, enrofloxacin, norfloxacin, and danofloxacin) are already regulated for use in aquaculture in other countries (European Union, China, Japan, Thailand); some FQs (enrofloxacin, ciprofloxacin, norfloxacin, and danofloxacin) are already regulated for use in Brazil (CPVS-SINDAN 2013), nevertheless, prescribed for other animal species (bovine, chicken, porcine).

Analytical Methods for the Determination of Fluoroquinolone Residues

The development of an analytical method for the determination of veterinary pharmaceutical residues in food is essential to ensure that products are safe for consumer health. Thus, to guarantee the attainment of adequate results, the method must go through a procedure called analytical validation, which aims to evaluate and verify whether the results are suitable for the intended analysis (Paschoal and others 2008).

There are different agencies that provide validation parameters for analysis of veterinary drugs residues. The following are found at the international level: the European Community (EC 2002), the United Nations Food and Agriculture Organization (FAO 1998), the International Union of Pure and Applied Chemistry (IUPAC) (Thompson and others 2002), the International Organization for Standardization (ISO), and the International Conference on Harmonization (ICH). At the national levels are the Food and Drug Administration (FDA), the National Sanitary Surveillance Agency (ANVISA 2003), the National Institute of Metrology, the Standardization and Industrial Quality (INMETRO 2007), and the Ministry of Agriculture, Livestock and Supply (MAPA 2011).

MAPA (2011) published a “Guide for Validation of Analytical Methods and Quality Control of Analysis of Internal Monitoring of the National Plan for Biological Residues Management in Animal Products – PNCRB,” thus establishing the parameters to be considered in the objective analytical validation, including linearity, selectivity and matrix effect, recovery, accuracy, precision, decision limit (CCα), and detection capability (CCβ), in addition to stability studies, quality control, internal analysis, and inclusion of a validated new sample matrix. The detailed evaluation of each one of these parameters is described in the MAPA guide (MAPA 2011), which is similar to the EC guide (EC 2002).

The analysis of residues in fish flesh should consider the sample preparation, a step that anticipates the instrumental analysis. Food products in general are formed in complex matrices containing significant interferences, such as carbohydrates, lipids, proteins, among others.

For the development of sample preparation, one should take into consideration the work matrix and the physical and chemical characteristics of the analytes to be determined, to achieve the best possible selection of reagents involved in the analytical procedures. It is also essential to consider the MRL standards for the planning and to establish the method validation procedure.

The sample preparation generally involves the extraction(s) of the analyte(s) required by the matrix through solid–liquid and/or liquid–liquid extraction, the clean-up and the concentration of the analyte(s). Therefore, it is important to know the chemical and physical–chemical composition of the analyte(s) in question. In the case of FQs, these compounds show good solubility in polar organic solvents and are insoluble in apolar solvents (for example, hexane and toluene). Thus, extraction of the FQs from biological matrices can be accomplished by using organic solvents with medium to high polarity, an aqueous buffer solution, and an immiscible organic solvent, mixtures of aqueous-organic solutions as well as buffered aqueous solutions (Stolker and Brinkman 2005).

With reference to the determination of FQs in fish, the literature mentions methods for cleaning the extract. Among them are solid-phase extraction (SPE) (Johnston and others 2002; Paschoal and others 2009a,b; Zheng and others 2009; Evaggelopoulou and Samanidou 2013), dispersive solid-phase extraction (DSPE) like QuEChERS extraction (quick, easy, cheap, effective, rugged, and safe) (Stubbings and Bigwood 2009; Lopes and others 2012), among others. The concentration of the analyte(s) may be accomplished by the evaporation route, nitrogen stream evaporation (55 °C) and using other procedures to assist in the reduction of the extract's final volume, thereby enhancing the detectability of the method.

The literature describes different methods for determining FQs in fish, among which, the use of high-performance liquid chromatography (HPLC) combined with different detection systems stands out (Rubies and others 2007).

The HPLC technique primarily uses the reverse phase based on silica (C18 and C8) analytical columns for chromatographic separation. However, the presence of residual silanols and metallic impurities from the filling material can affect the symmetry of the chromatographic peaks. To solve the issue of the chromatographic peaks tailing effect, end-capped columns or high-purity silica are used instead (Inertsil, Kromasil, Zorbaz RX, XTERRA, and so on) (Stolker and Brinkman 2005).

Regarding the mobile phases recommended in the literature, when using a stationary reverse phase to determine the FQs, the use of ACN or MeOH, or a combination of both, with aqueous solutions has been proposed (Stubbings and Bigwood 2009).

The most frequently used detection techniques in combination with LC are molecular spectroscopy (by UV absorption and fluorescence) and MS. The earlier researchers employed UV absorption for the determination of FQs. However, compared to the fluorescence detection system, it presents lower selectivity and detectability.

The European Union (EC 2002) recommends the LC coupled with MS for the analysis, quantification, and confirmation of the analytes in antimicrobial residues in food samples of animal origin. An important parameter to consider when working with MS is the matrix effect because it can causes ion suppression, affecting the analyte ionization. Nevertheless, MS is highly sensitive and selective, and it is the method most commonly used in studies to confirm the identity of veterinary pharmaceuticals, whose residues are present in foods (Johnston and others 2002).

In MS, there are different types of mass analyzers, and the most commonly used in the analysis of residues from veterinary drugs are the triple quadrupole (QqQ) and time-of-flight MS. The time-of-flight (ToF) mass analyzer provides excellent results because of the accurate mass measurement of the analytes; thus, making it an excellent tool for confirming the identity of pharmaceutical agents (Pozo and others 2006).

Some of the published studies that determined FQs in fish are summarized in Table 5, which presents the main sample preparation procedures for the extraction of FQs in fish and the analytical methods used according to the chromatographic techniques.

Table 5. Some procedures for determination of (fluoro)quinolones in fish by chromatographic techniques
  RecoverySeparation LODLOQ 
AnalytesExtraction(%)SP—MPDet.(ng/g)(ng/g)Reference
  1. SP, stationary phase; MP, mobile phase; LOD, limit of detection; LOQ, limit of quantitation; NAL, nalidixic acid; FLU, flumequine; OXO, oxolinic acid; SAR, sarafloxacin; ENR, enrofloxacin; CIP, ciprofloxacin; DIF, difloxacin; MAR, marbofloxacin; ORBI, orbifloxacin; RIP, piromidic acid; Na2SO4, sodium sulfate; EtAc, ethyl acetate; H3PO4, phosphoric acid; NaOH, sodium hydroxide; KH2PO4, monopotassic phosphate; PSA, primary-secondary amine; QuEChERS, quick, easy, cheap, effective, rugged and safe; SDS, sodium dodecylsulfate; TEA, triethylamine; MeOH, methanol; ACN, acetonitrile; THF, tetrahydrofuran; TFA, trifluoroacetic acid; PAD, photodiode array detector; FLD, fluorescence detector; Det, detection; MLC, miscellar liquid chromatography; SPME, solid phase micro-extraction; IT, ion trap; QqQ, triple-quadrupole; QToF, quadupole—time-of-flight.

FLU, OXOHexane, Buffer pH 10, H3PO4, Na2SO4, NaOH73 to 86RP (150 × 4.6 mm, 5 μm)—KH2PO4/H3PO4/ACN/THFFLD25Malvisi and others (1997)
SAR, OXO, FLUMeOH, NaOH, Buffer pH 9.060 to 71RP (150 × 4.6 mm, 5 μm)—H3PO4/ACNFLD2 to 715 to 75Roudaut and Yorke (2002)
OXO, FLU,PIR, CIP, ENR, DAN, SAR, ORBIACN and SPE60 to 80C18 (150 × 2.1 mm, 5 μm)—ACN/formic acid/waterQqQ1 to 35Johnston and others (2002)
OXOEthyl Acetate70 to 78C18 (250 × 4, 6 mm, 5 μm)—TFA/MeOH/canFLD25Tyrpenou and Rigos (2004)
ENR, CIP, FLUBuffer pH 9.1, ACN62 to 78RP (150 × 4.6 mm, 5 μm)—H3PO4/ACN/THFFLD1Kirbis and others (2005)
CIP, DAN, SAR, ENR, OXO, NAL, FLUNaOH and NaCl90 to 116C18 (250 × 4.6 mm, 5 μm)—MeOH/ACN/formic acidQqQ2 to 36 to 8Samanidou and others (2008)
ENR, CIP, DAN, SAR, OXO, FLUATCA/MeOH and SPE73 to 110C18 (250 × 4.6 mm, 5 μm)—ACN/oxalic acidFLD2 to 810 to 27Paschoal and others (2009a)
ENR, CIP, DAN, SAR, OXO, FLUATCA/MeOH and SPE89 to 112C18 (150 × 2.1 mm, 5 μm)—ACN/formic acidQToF4 to 614 to 21Paschoal and others (2009b)
SAR, ENR, CIP, OXO, FLU, NALQuEChERS38 to 93C18 (100 × 2.1 mm, 2,5 μm)—formic acid/ACN/MeOHQqQ3Stubbings and Bigwood (2009)
CIP, DAN, ENR, SAR, DIF, OXO, FLUAcetone 5 mL (SPME)81 to 113C18 (250 × 2.0 mm, 5 μm)—ACN/formic acidQToF0.2 to 10.6 to 3Zheng and others (2009)
DAN, CIP, OXO and other drugsACN, hexane>50Phenyl (4 × 50 mm, 3 μm)—ACN/formic acid/NaOHIT10Smith and others (2009)
DIF, ENR, FLU, OXO, SARSDS, TEA pH 396 to 106C18 (150 × 4.6 mm, 5 μm)—SDS/propanol/TEAMLC5 to 3010 to 70Rambla-Alegre and others, (2010)
OXO, DAN, ENR, MAR, SAR, FLUModified QuEChERS69 to 125C18 (100 × 2.1 mm, 1.7 μm)—formic Acid/canQqQDAN: 15 Others: < 8DAN: 25 Others: < 50Lopes and others (2012)
CIP, DAN, ENR, SAR, OXO, NAL, FLUCitrate buffer and SPE88 to 99ODS–2 120 (250 × 5 mm, 5 μm)—TFA/ACN/MeOHPAD2 to 126 to 35Evaggelopoulou and Samanidou (2013)

Final Considerations

Aquaculture is the fastest growing food production system in the world. Brazil has a high potential for fish production based on both the exotic and the native species. The use of veterinary drugs is essential to increase the efficiency of the aquaculture production. However, despite the importance of these substances, Brazil still faces a shortage of veterinary drugs approved for the use in the various species and diseases that the aquaculture holds. Veterinary drugs should be administered in a responsible and prudent manner, always respecting the good veterinary practices to avoid future problems in the efficiency of the product. Thus, it will be possible to manage the issue of antimicrobial resistance, which is currently discussed with consideration of the impacts on aquaculture production, human health, and the environment. The analytical method is a vitally important tool to ensure that the products are accurately presented in legal determinations. Importantly, LC linked to MS is an analytical technique that is widely used and recommended for the determination of residues from veterinary drugs because it presents high selectivity and sensitivity.

Acknowledgments

The authors gratefully acknowledge the financial support received from São Paulo Research Foundation (FAPESP, Brazil), the National Council for Scientific and Technological Development (CNPq, Brazil) and Coordinator for the Improvement of Personnel in Higher Education Brazil (CAPES, Brazil). The authors also thank Elsevier Language Editing Services for language assistance. The authors declare that they do not have any interest conflict for this publication.

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