Bromophenol concentrations in fish from Salvador , BA , Brazil

The main objective of this work is to evaluate the occurrence of bromophenols (2-bromophenol, 4-bromophenol, 2,4dibromophenol, 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 by the external-standard method. Total bromophenol concentrations were similar in the muscle of both species, ranging from 36 ng g–1 to 349 ng g–1. The total bromophenol concentrations in stomach (ranging from 12 ng g–1 to 586 ng g–1) were slightly higher than in muscle. The presence of bromophenol in the muscles of the species under study may occur as a result of their diet. The results of this work are therefore expected to contribute toward a better understanding of the path of bromophenol absorption from the fish’s stomach to the rest of its body.


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-bromo-phenol (4-BP), 2,4-dibromophenol (2,4-DBP), 2,6dibromophenol (2,6-DBP) and 2,4,6-tribromophenol (2,4,6-TBP), (Fig. 1) in water, have very low sensory threshold concentrations in the ng g -1 range (Whitfield 1988).Bromophenols, which have been found in marine fishes (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 flavor of some fish species as mildly candy-like and others as marine or oceanic-like, which is characteristic of the presence 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 that the marine species contained 6.1 to 34.8 ng.g -1 of total bromophenols while the freshwater species not only contained none of the five bromophenols investigated but also had none of the characteristic oceanic-like flavor.Although 2,4-DBP and especially 2,4,6-TBP have been considered important anthropic pollutants (playing an important role as industrially produced flame retardant and pesticides) (Polo et al. 2006), the presence of bromophenols in these marine organisms has been attributed to their natural diets; in other words, these compounds may come from other species in the food chain.Despite of their importance does not exists limit value to the bromophenol content in marine species.
The flavor of marine fishes varies depending on the location and time of year when they were caught (Whitfield et al. 1995), and the diet of certain fish species can vary considerably according to the availability of alimentary components, which depends on seasonal variations (Whitfield et al. 1998).It is believed that the bromophenols in marine fish come from their natural diet (Whitfield et al. 1998).This hypothesis is strongly supported by the fact that bromophenols have been detected in the stomach content and the flesh, with higher concentrations in the former (Chung et al. 2003a, Whitfield et al. 1995), allied to the fact that benthic carnivorous fishes feeding on polychaetes and herbivorous fishes feeding on macroalgae have a strong flavor while piscivorous fishes feeding primarily on other fish do not contain these bromophenols (Whitfield et al. 1994(Whitfield et al. , 1995(Whitfield et al. , 1996)).
However, the identification of specific organisms that may introduce bromophenols into the diet of marine fish requires a broader investigation (Whitfield et al. 1996(Whitfield et al. , 1998)).Some reports suggest that fish do not accumulate bromophenols, but gradually metabolize or excrete them (Whitfield et al. 1992b, Anthoni et al. 1990).Therefore, studies are needed to establish the route whereby bromophenols are transferred to different fish 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 and widely accepted by the consumer market of the city of Salvador.Nevertheless, few studies have focused on the chemistry of these species, particularly with regard to the volatile organic compounds (VOCs) that give these species their particular flavor (Santos et al. 2001, Veloso et al. 2001).The purpose of this work was therefore to evaluate the occurrence of bromophenols in the flesh and guts (stomach content and pyloric ceca or appendices) of two species of the Lutjanidae family, Lutjanus synagris and Ocyurus chrysurus, and to identify the probable alimentary source of these bromophenols in these species.

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 Aldrich and filtered through a 0.45μm membrane.The other reagents (pentane, diethyl ether, sodium chloride and sulfuric 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 fish caught in the coastal waters of Bahia, Brazil (13 • 01 S and 38 • 31 W), were purchased from commercial fishermen.Three specimens of each species were purchased, with an average weight of 1.0 kg and 30 cm length.In the laboratory, the fish were washed in distilled water, gutted, and the flesh was separated from the heads, tails, and backbone.
The flesh was washed in a saturated NaCl solution and then blended into a smooth purée in a food processor (Triton-Arno).Samples of puréed flesh (in portions of 250 g) were stored in sealed polyethylene bags at −15 • C prior to their analysis.
The guts (full stomach contents and pyloric ceca) were dissected, weighed and stored in a refrigerator (0 • C).Subsequently, an incision was made in each stomach to check the types of food items and classify them according to the highest taxon.This material and the pyloric ceca were then blended together into a purée.Stomachs that were empty, everted, or contained baitlike contents were rejected.The samples consisting of three blended stomachs, each weighing about 30 g, were stored in sealed polyethylene bags at −15 • C 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 in acetonitrile.The standard calibration solutions were prepared by diluting the bromophenol stock solutions in acetonitrile, in concentrations of 200 to 1000 ng mL -1 .The resulting solutions were stored at 4 • C in dark glass flasks.The standard solutions were prepared at least once a week.More detailed information is available elsewhere (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 pH 1 with 10 mol L -1 sulfuric acid, were left to stand at ambient temperature (26 ± 3 • C) for about 12 h.The volatile components were isolated by combined continuous hydrodistillation-solvent extraction with 2 mL of pentane/diethyl ether (6:4) using a modified Clevenger apparatus (Vidrosel Ltda, Brazil) adapted for this study (Silva et al. 2005).The hydrodistillation process was completed 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 in acetonitrile (500μL) and stored in 2 mL dark glass vials at −15 • C 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 LiChrospher 100 Rp-18 (244 mm × 4.4 mm I.D., 5μm; Merck) column coupled to a LiChrospher guard column with similar characteristics (14 mm × 4 mm I.D.; Merck).The mobile 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 min -1 at ambient tem-An Acad Bras Cienc (2009) 81 (2) perature.The detection was performed at 286nm, where the 2-BP, 4-BP, 2,4-and 2,6-DBP show significant absorptive values and at 297nm for 2,4,6-TBP.Analytical calibration curves were built by plotting the observed peak height against the amount of injected bromophenol (200 to 1000 ng mL -1 ).Quantification of the bromophenols was performed using an external standard (n = 5), by measuring the peak height at each retention time calculated from the calibration curve.Spikes of each bromophenol were produced in the samples to ascertain the exact retention times.The total bromophenol content (TBC), used in the literature to reflect the impact of flavor produced by all the bromophenols contained in a food (Whitfield et al. 1995(Whitfield et al. , 1996(Whitfield et al. , 1998)), was calculated by incrementing the concentration (ng g -1 ) 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 were Crustacea (Decapoda) and Teleostei, with a smaller proportion of representatives of the taxon Mollusca.When full, the pyloric appendices or pyloric ceca presented a 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 each species.
The highest bromophenol concentrations in the two fish species involved 2,4-DBP and 2,4,6-TBP, which were present in concentrations exceeding 110 ng g -1 in muscle and stomach (Tables II and III).2-BF and 4-BF showed the lowest concentrations.
Total bromophenol concentrations showed a similar predominance in the muscle of both species, ranging from 36 ng g -1 to 349 ng g -1 (Table II).The total concentrations in the stomach (ranging from 12 ng g -1 to 586 ng g -1 ) (Table III ) were slightly higher than in the muscle.
In the muscle of the species Lutjanus synagris, 2,4-DBF (110 ng g -1 ) and 2,4,6-TBF (171 ng g -1 ) stood out, 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., 2,4-DBF (158 ng g -1 ) and 2,4,6-TBF (119 ng g -1 ) (Table II).The analysis of bromophenol in the stomach also showed a predominance of 2,4-DBF and 2,4,6-TBF in both species, although the species O. chrysurus showed significant concentrations of 2,6-DBF as well (Table III).The higher bromophenol concentrations found in the specimens collected in winter were consistent with the greater abundance and weight of L. synagris and O. chrysurus (Costa et al. 2002) in autumn and winter.Low temperature seasons are associated with the growth cycle of marine species (Chung et 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 TBCs in the muscle (15 samples) and stomach (9 samples) of the species L. synagris and O. chrysurus during the period of this study.With the exception of 2,4-BF, 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 2,4-BF, 2,6-BF 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, Szpilman 1991, Filho 1994, La Morinière et al. 2003).With the 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 from their diets.
The route of bromophenol absorption from the fish'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 therefore necessary to clarify such questions, whose answers are of paramount importance for aquaculturists.

Fig. 2 -
Fig. 2 -Bromophenol concentrations and TBCs in the muscle and stomach of L. synagris collected in winter (A) and summer time (B).

Fig. 3 -
Fig. 3 -Average bromophenol concentrations and TBCs in the muscle and stomach of L. synagris.