Bromophenol concentrations in fish from Salvador, BA, Brazil

Abstracts

The main objective of this work is to evaluate the occurrence of bromophenols (2bromophenol, 4bromophenol, 2,4dibromophenol, 2,6dibromophenol and 2,4,6tribromophenol), in the flesh and guts in two species of the LutjanidaeFamily: Lutjanus synagris and Ocyurus chrysurus. The bromophenols were extracted by steam distillation with pentaneether (7:3 v/v), identified by reverse phase High Performance Liquid Chromatography (HPLCUV), and quantified bythe externalstandard method. Total bromophenol concentrations were similar in the muscle of both species, rangingfrom 36 ng g¹ to 349 ng g¹. The total bromophenol concentrations in stomach (ranging from 12 ng g¹ to 586 ng g¹)were slightly higher than in muscle. The presence of bromophenol in the muscles of the species under study may occuras a result of their diet. The results of this work are therefore expected to contribute toward a better understanding ofthe path of bromophenol absorption from the fish's stomach to the rest of its body.

bromophenols; flavor; marine fishes; Lutjanus; Ocyurus

O principal objetivo do presente trabalho foi o estudo de bromofenóis (2bromofenol, 4bromofenol, 2,4dibromofenol,2,6dibromofenol and 2,4,6tribromofenol), no músculo e estômago de duas espécies de peixes da Familia Lutjanidae: Lutjanus synagris e Ocyurus chrysurus. Os bromofenóis foramextraídos através de destilação por arraste a vapor com pentanoéter (7:3 v/v), analisados por Cromatografia Líquida de AltaEficiência e quantificados por padronização externa. As concentrações totais de bromofenóis no músculo de ambas as espécies foram similares e estiveram na faixa de 36 ng g¹ a349 ng g¹. As concentrações totais de bromofenóis no estômago (na faixa de 12 ng g¹ a 586 ng g¹) foram mais altas queno músculo. A presença de bromofenóis no músculo das espécies estudadas pode ter origem na dieta. Os resultados destetrabalho contribuirão para o melhor entendimento das rotas deabsorção de bromofenóis nos peixes.

bromofenóis; flavor; peixes marinhos; Lutjanus; Ocyurus

CHEMICAL SCIENCES

Bromophenol concentrations in fish from Salvador, BA, Brazil

Aline S. Oliveira^III; Vilma M. Silva^I; Márcia C.C. Veloso^II; Gislaine V. Santos^III; Jailson B. de Andrade^I

^IInstituto de Química, Universidade Federal da Bahia, UFBA, Rua Barão de Geremoabo s/n ,Campus Universitário de Ondina, 40170-290 Salvador, BA, Brasil

IICentro Federal de Educação Tecnológica da Bahia, CEFET-BA, Rua Emídio dos Santos s/n, Barbalho, 40625-650 Salvador, BA, Brasil

IIIInstituto de Biologia, Universidade Federal da Bahia, UFBA, Rua Barão de Geremoabo s/n, Campus Universitário de Ondina, 40170-290 Salvador, BA, Brasil

^{Correspondence to}

ABSTRACT

The main objective of this work is to evaluate the occurrence of bromophenols (2-bromophenol, 4-bromophenol, 2,4-dibromophenol, 2,6-dibromophenol and 2,4,6-tribromophenol), in the flesh and guts in two species of the Lutjanidae Family: Lutjanus synagris and Ocyurus chrysurus. The bromophenols were extracted by steam distillation with pentaneether (7:3 v/v), identified by reverse phase High Performance Liquid Chromatography (HPLC-UV), and quantified bythe external-standard method. Total bromophenol concentrations were similar in the muscle of both species, rangingfrom 36 ng g^-1 to 349 ng . The total bromophenol concentrations in stomach (ranging from 12 ng g^-1 to 586 ng g¹)were slightly higher than in muscle. The presence of bromophenol in the muscles of the species under study may occuras a result of their diet. The results of this work are therefore expected to contribute toward a better understanding ofthe path of bromophenol absorption from the fish's stomach to the rest of its body.

Key words: bromophenols, flavor, marine fishes, Lutjanus , Ocyurus .

RESUMO

O principal objetivo do presente trabalho foi o estudo de bromofenóis (2bromofenol, 4bromofenol, 2,4dibromofenol,2,6dibromofenol and 2,4,6tribromofenol), no músculo e estômago de duas espécies de peixes da Familia Lutjanidae: Lutjanus synagris e Ocyurus chrysurus. Os bromofenóis foramextraídos através de destilação por arraste a vapor com pentanoéter (7:3 v/v), analisados por Cromatografia Líquida de Alta Eficiência e quantificados por padronização externa. As concentrações totais de bromofenóis no músculo de ambas as espécies foram similares e estiveram na faixa de 36 ng g^-1 a349 ng g¹. As concentrações totais de bromofenóis no estômago (na faixa de 12 ng g^-1 a 586 ng g¹) foram mais altas queno músculo. A presença de bromofenóis no músculo das espécies estudadas pode ter origem na dieta. Os resultados destetrabalho contribuirão para o melhor entendimento das rotas deabsorção de bromofenóis nos peixes.

Palavras-chave: bromofenóis, flavor, peixes marinhos, Lutjanus, Ocyurus.

INTRODUCTION

Flavor, or the consumer's perception thereof, is an important attribute of the quality of marine fishes and other seafoods (Lindsay 1990, Stansby 1962), and is the first and principal discriminative factor in his evaluation, acceptance, rejection or preference for the product (Boyle et al. 1992a). This fact has led to extensive research in several areas, including agriculture and the food and beverage industry, aimed at putting on the market products of excellent nutritional quality and especially of pleasant flavor (Lindsay 1990).

The success of aquaculture products has been hampered by problems relating to the quality of their flavor, since many consumers can clearly distinguish the difference between the flavor of cultivated and wild harvest seafoods (Boyle et al. 1992a). Knowledge of the factors and chemical substances that determine flavor can contribute significantly to the improvement and expansion of aquaculture and to the preservation, storage, control and improved quality of seafoods.

However, there is still a paucity of information about the specific substances that give fishes and other seafoods their widely diverse flavors and other subtle differences. In the last few decades, a group of organic compounds called simple bromophenols has been considered the main component of the flavor of several seafoods (Boyle et al. 1992b, 1993, Silva et al. 2007, Whitfield et al. 1992a, Whitfield 1988). These compounds, including 2-bromophenol (2-BP), 4-bromophenol (4-BP), 2,4-dibromophenol (2,4-DBP), 2,6-dibromophenol (2,6-DBP) and 2,4,6-tribromophenol(2,4,6-TBP), (Fig. 1) in water, have very low sensorythreshold concentrations in the ng g^-1 range (Whitfield 1988).

Bromophenols, which have been found in marinefishes (Boyle et al. 1992a, Whitfield et al. 1998), crustaceans (Chung et al. 2003a, Whitfield et al. 1997, 2002)and mollusks (Boyle et al. 1992a), are strongly associated with pleasant (marine-or ocean-like) or unpleasant (plastic, medicinal, disinfectant, iodoform or iodinelike) flavors, alone or in different combinations and concentrations. Marine food gourmets describe the flavorof some fish species as mildly candylike and others as marine- or oceanic-like, which is characteristic of thepresence of bromophenols in different concentrations(Whitfield et al. 1998). Boyle et al. (1992a), who compared four Pacific salmon species (Oncorhynchus spp)from marine and freshwater environments, found thatthe marine species contained 6.1 to 34.8 ng.g^-1 of total bromophenols while the freshwater species not onlycontained none of the five bromophenols investigated but also had none of the characteristic oceanic-like flavor. Although 2,4DBP and especially 2,4,6-TBP havebeen considered important anthropic pollutants (playingan important role as industrially produced flame retardant and pesticides) (Polo et al. 2006), the presence of bromophenols in these marine organisms has been attributedto their natural diets; in other words, these compoundsmay come from other species in the food chain. Despite of their importance does not exists limit value tothe bromophenol content in marine species.

According to previous studies, bromophenolshave been detected in a variety of other marine organisms such as macroalgae (Chung et al. 2003b, Lee et al.2007, Pedersén et al. 1974, Phillips and Towers 1981,Whitfield et al. 1992b, 1999a, Xu et al. 2003), polychaetes (Goerke and Weber 1990, 1991, Steward and Lovell 1997, Whitfield et al. 1999b), sponges (Hattori etal. 2001, Unson et al. 1994, Vetter and Janussen 2005)and bryozoans (Whitfield et al. 1999b), which are amajor dietary source for many marine organisms including fish (Whitfield et al. 1997, 1998, 1999a, 1999b, Ma et al. 2005).

The flavor of marine fishes varies depending onthe location and time of year when they were caught (Whitfield et al. 1995), and the diet of certain fish speciescan vary considerably according to the availability ofalimentary components, which depends on seasonalvariations (Whitfield et al. 1998). It is believed thatthe bromophenols in marine fish come from their naturaldiet (Whitfield et al. 1998). This hypothesis is stronglysupported by the fact that bromophenols have been detected in the stomach content and the flesh, with higherconcentrations in the former (Chung et al. 2003a, Whitfield et al. 1995), allied to the fact that benthic carnivorous fishes feeding on polychaetes and herbivorous fishesfeeding on macroalgae have a strong flavor while piscivorous fishes feeding primarily on other fish do not contain these bromophenols (Whitfield et al. 1994, 1995, 1996).

However, the identification of specific organismsthat may introduce bromophenols into the diet of marine fish requires a broader investigation (Whitfield etal. 1996, 1998). Some reports suggest that fish donot accumulate bromophenols, but gradually metabolizeor excrete them (Whitfield et al. 1992b, Anthoni et al.1990). Therefore, studies are needed to establish theroute whereby bromophenols are transferred to differentfish species.

Fish is a staple food among coastal populations in the state of Bahia. Standing out among the most popular fish species are the members of the Lutjanidae family(popularly known as "red"), which are highly valued andwidely accepted by the consumer market of the city ofSalvador. Nevertheless, few studies have focused on thechemistry of these species, particularly with regard tothe volatile organic compounds (VOCs) that give these species their particular flavor (Santos et al. 2001, Velosoet al. 2001). The purpose of this work was therefore toevaluate the occurrence of bromophenols in the flesh andguts (stomach content and pyloric ceca or appendices) oftwo species of the Lutjanidae family, Lutjanus synagris and Ocyurus chrysurus, and to identify the probable alimentary source of these bromophenols in these species.

MATERIALS AND METHODS

SOLVENTS, REAGENTS AND STANDARDS

The bromophenols standards were obtained from Aldrich(Milwaukee, WI), in purities ranging from 97 to 99%. Purified water was obtained by distillation and filtration through an E-pure Alltech system (Deerfield, IL).Acetonitrile (HPLC grade) was obtained from Aldrichand filtered through a 0.45µm membrane. The other reagents (pentane, diethyl ether, sodium chloride andsulfuric acid) were of analytical grade produced by Merck (Darmstadt, Germany).

COLLECTION AND PREPARATION OF SAMPLES

Two fish species of the Lutjanidae family were studied: Lutjanus synagris and Ocyurus chrysurus. Fresh fishcaught in the coastal waters of Bahia, Brazil (13º01'S and 38º31'W), were purchased from commercial fishermen. Three specimens of each species were purchased, withan average weight of 1.0 kg and 30 cm length. In thelaboratory, the fish were washed in distilled water, gutted,and the flesh was separated from the heads, tails, andbackbone.

The flesh was washed in a saturated NaCl solutionand then blended into a smooth purée in a food processor(TritonArno). Samples of puréed flesh (in portions of 250 g) were stored in sealed polyethylene bags at -15ºC prior to their analysis.

The guts (full stomach contents and pyloric ceca)were dissected, weighed and stored in a refrigerator(0ºC). Subsequently, an incision was made in each stomach to check the types of food items and classify themaccording to the highest taxon. This material and thepyloric ceca were then blended together into a purée. Stomachs that were empty, everted, or contained baitlike contents were rejected. The samples consisting ofthree blended stomachs, each weighing about 30 g, were stored in sealed polyethylene bags at -15ºC until required for analysis.

PREPARATION OF BROMOPHENOL STANDARDS AND CALIBRATION SOLUTIONS

Stock solutions (100 mg mL^-1) were prepared by first weighing each bromophenol and then dissolving it inacetonitrile. The standard calibration solutions were prepared by diluting the bromophenol stock solutions inacetonitrile, in concentrations of 200 to 1000 ng mL^-1.The resulting solutions were stored at 4ºC in dark glass flasks. The standard solutions were prepared at leastonce a week. More detailed information is available else where (Silva et al. 2005).

EXTRACTION OF BROMOPHENOLS

Representative samples of flesh (250 g) or guts(30 g) were homogenized separately in purified water (1000 mL) and the homogenates, acidified to pH1 with 10 mol L¹ sulfuric acid, were left to stand at ambient temperature (26 ± 3ºC) for about 12 h. Thevolatile components were isolated by combined continuous hydrodistillation-solvent extraction with 2 mL ofpentane/diethyl ether (6:4) using a modified Clevengerapparatus (Vidrosel Ltda, Brazil) adapted for this study(Silva et al. 2005). The hydrodistillation process wascompleted after 4 hours, and the pH of the residues was measured. The collected extract was concentrated under a gentle stream of ultrahigh purity (99.999%) nitrogen. The concentrated extract was then dissolved inacetonitrile (500µL) and stored in 2 mL dark glass vials at -15ºC until it was analyzed.

SEPARATION OF COMPOUNDS

A PerkinElmer series 200 liquid chromatograph equipped with a Rheodyne (Cotati, California, USA) injector valve with a 20µL sample loop and a PerkinElmer UV-visible detector were used. Chromatographic separation of bromophenols was performed in a LiChrospher100 Rp18 (244 mm X 4.4 mm I.D., 5µm; Merck) column coupled to a LiChrospher guard column with similar characteristics (14 mm X 4 mm I.D.; Merck). Themobile phase vacuum-degassed in a sonicator was a mixture of water: acetonitrile pumped in gradient mode(Table I) at a flow rate of 1.0 mL min¹ at ambient temperature. The detection was performed at 286nm, where the 2-BP, 4-BP, 2,4and 2,6-DBP show significant absorptive values and at 297nm for 2,4,6-TBP.

ANALYTICAL CALIBRATION CURVE AND QUANTIFICATION

Analytical calibration curves were built by plotting theobserved peak height against the amount of injected bromophenol (200 to 1000 ng mL^-1). Quantification of thebromophenols was performed using an external standard(n = 5), by measuring the peak height at each retentiontime calculated from the calibration curve. Spikes ofeach bromophenol were produced in the samples to ascertain the exact retention times. The total bromophenolcontent (TBC), used in the literature to reflect the impactof flavor produced by all the bromophenols contained ina food (Whitfield et al. 1995, 1996, 1998), was calculated by incrementing the concentration (ng g¹) of all the simple bromophenols detected in a sample.

RESULTS AND DISCUSSION

The items found in the stomach contents of the specimens indicated that the main taxonomic categories wereCrustacea (Decapoda) and Teleostei, with a smaller proportion of representatives of the taxon Mollusca. Whenfull, the pyloric appendices or pyloric ceca presenteda stomach fluid, probably due to a previous digestion.The five simple bromophenols were found to occur in the muscle and stomach of the species under study. Tables II and ^III list the concentration of each of the five bromophenols and the total bromophenol content (TBC)found, respectively, in the muscle and stomach of eachspecies.

The highest bromophenol concentrations in the twofish species involved 2,4-DBP and 2,4,6-TBP, whichwere present in concentrations exceeding 110 ng g^-1 in muscle and stomach (Tables II and ^III). 2BF and4BF showed the lowest concentrations.

Total bromophenol concentrations showed a similar predominance in the muscle of both species, rangingfrom 36 ng g^-1 to 349 ng g^-1 (Table II). The total concentrations in the stomach (ranging from 12 ng g^-1 to586 ng g¹) (^{Table III} ) were slightly higher than in the muscle.

The results presented in Table II are consistentwith those found by Whitfield (1998, 1999), Chung etal. (2003a) and Silva et al. (2005) in marine fish. Thoseauthors reported high total bromophenol concentrations (2.72 to 462 ng g¹), especially for 2,4,6-TBP, which wasthe most frequent and abundant bromophenol (Chung etal. 2003a, Silva et al. 2005). The concentrations of2,6-DBP, 2,4-DBP and 2,4,6-TBP determined in sevenfish species (Branchiostegus wardi, Girella tricuspidata, Nemadactylus douglassi, Rhabdosargus sarba, Acanthopagrus australis, Meuschenia trachylepis, and Pseudorhombus jenynsii) (Parejo et al. 2004) fell within therange of 0.4-18 ng g¹, 112-150 ng g^-1 and 5.7-170 ng g¹, respectively. The concentrations reported here(Table II) show a similar predominance of these three bromophenols in the two fish species studied.

In the muscle of the species Lutjanus synagris, 2,4-DBF (110 ng g¹) and 2,4,6-TBF (171 ng g¹) stoodout, particularly in the specimens collected in winter,as indicated in Table II and showed in the Figure 2.The muscle of the species Ocyurus chrysurus showed similar results, i.e., 2,4DBF (158 ng g¹) and 2,4,6-TBF (119 ng g¹) (Table II). The analysis of bromophenol in the stomach also showed a predominance of 2,4DBF and 2,4,6-TBF in both species, although the species O. chrysurus showed significant concentrations of 2,6DBF as well (^{Table III}). The higher bromophenol concentrations found in the specimens collected in winterwere consistent with the greater abundance and weight of L. synagris and O. chrysurus (Costa et al. 2002) inautumn and winter. Low temperature seasons are associated with the growth cycle of marine species (Chunget al. 2003a, b). The abundance and weight, as well the bromophenol content, decrease in summer.

Figures 3 and 4 indicate, respectively, the variation in average bromophenol concentrations and TBCsin the muscle (15 samples) and stomach (9 samples) ofthe species L. synagris and O. chrysurus during the period of this study. With the exception of 2,4BF, the bromophenol pattern found for Lutjanus synagris was similar in the muscle and stomach (Fig. 3). In contrast, O. chrysurus showed substantial differences, especially in 2,4BF, 2,6BF and TBF (Fig. 4), which were found predominantly in the stomach.

The predominant items in the diet of L. synagris and O. chrysurus are crustaceans (mainly Decapoda)and fish, and a smaller proportion of mollusks and polychaetes (Costa et al. 2002, Druzhinin 1970, Szpilman1991, Filho 1994, La Morinière et al. 2003). Withthe exception of polychaetes, the stomach contents of the specimens collected were compatible with those reported in the literature. Thus, the bromophenol found in the muscle of the species under study may come fromtheir diets.

The route of bromophenol absorption from thefish's stomach to the rest of its body, as well as its physiological roles, is still unknown (Boyle et al. 1992b,Chung et al. 2003b). Additional research is thereforenecessary to clarify such questions, whose answers areof paramount importance for aquaculturists.

ACKNOWLEDGMENTS

The present work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq), Fundação de Apoio a Pesquisa do Estado da Bahia (FAPESB)-PRONEX/FAPESB/CNPq, Coordenação de Formação de Pessoal de Nível Superior (CAPES)and Financiadora de Estudos e Projetos (FINEP).

Manuscript received on July 14, 2008; accepted for publication on October 7, 2008; contributed by JAILSON B. DE ANDRADE^*

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*

Member Academia Brasileira de Ciências

Publication Dates

• Publication in this collection
  26 May 2009
• Date of issue
  June 2009

History

• Accepted
  07 Oct 2008
• Received
  14 July 2008