BULLETIN OF MARINE SCIENCE, 48(2): 517-523, 1991 BROMO PHENOLS IN LANICE CONCHILEGA (POL YCHAET A, TEREBELLIDAE): THE INFLUENCE OF SEX, WEIGHT AND SEASON Helmut Goerke and Kurt Weber ABSTRACT The levels of four brominated compounds in Lanice conchilega were measured: 2,4-di- bromophenol (I), 2,6-dibromo-4-methylphenol (2), 2,4,6-tribromophenol (3), 3,5-dibromo- 4-hydroxybenzaldehyde (4). The presence of all four of these secondary metabolites in the j.lg/grange was species specific. Ten other terebellid species did not contain the compounds in significant concentrations. The levels in L. conchilega were not dependent on animal size, sex or seasons. Bromophenols were not detected in plankton and only (3) was found in sea water (2 pg.g-I) near the sites of dense populations of the annelid. The compounds are generally not accumulated, which was tested by feeding pieces of L. conchi/ega to three marine invertebrate spllcies. Bromophenols are known as secondary metabolites in various Enteropneusta (Higa, 1981; King, 1986; Woodin et aI., 1987), in Phoronidea, Phoronopsis viridis (Sheikh and Djerassi, 1975), and in Polychaeta, Lanice conchilega and Arenicola cristata (Weber and Ernst, 1978; Woodin et aI., 1987). The compounds exhibit antimicrobial activity and are possibly of antiseptic importance for wound healing in bottom living species (Sheikh and Djerassi, 1975). King (1986) suggested that a dibromophenol inhibits the aerobic microbial degradation of the burrow-wall mucous lining. There is little information on the concentrations of the compounds in the taxa mentioned above, and it is not known whether the compounds are predominant during particular growth stages or under specific environmental conditions. METHODS Lanice conchilega (Pallas) of different sex, of various wet weights and from various seasons were collected at five stations of the German Bight and the English Channel: Neuharlingersiel-53°42.4'N, 7°38.4'E (intertidal); Weser, Spieka-53°48.8'N, 8°31.5'E (intertidal); Norderhever-54°24.5'N, 8°31.9'E (subtidal, 15 m); N SchliisseItonne-53°58.9'N, 7°54.TE (subtidal, 33 m); Dinard, S1. Enogate- 48°37. TN, 2°4.I'W (intertidal). These stations are part ofa comprehensive series between SyIt, North- ern Frisia, and Roscoff, Brittany (Goerke and Weber, 1990). Ten additional terebellid species were sampled in areas listed in Table I. Composites of generally 4-5 g (wet weight) from whole specimens were deepfrozen a few hours after collection. The frozen samples were ground with sodium sulfate and quartz sand to a dry tissue powder, from which the compounds were extracted by n-hexane/acetone. Acetone was removed using a rotary vacuum evaporator. Subsequently, acidic compounds were separated from neutral ones by partitioning into aqueous alkali. After buffering the alkaline solution with sodium borate, phenols were derivatized for gas chromatographic analysis by extractive acetylation using acetic anhydride/pyridine and n-hex- ane as solvent. Gas chromatographic analysis of phenol acetates was performed using fused silica capillary columns with SE-54 as stationary phase, applying appropriate temperature programs. Halogenated phenols were identified and quantified by electron capture detection using authentic compounds as standards. identifications were confirmed by combined gas chromatography and mass spectrometry. (Analytical details in Goerke and Weber, 1990.) Plankton samples of3-5 g (wet weight) collected with Apstein nets of 20-150 j.lmmesh and consisting mainly of phytoplankton were likewise treated. Phenols from 20 liter sea water samples taken by Niskin bottles were extracted by small volumes of n-hexane after acidification of the water. Bottom water was sampled down to 0.3 m above dense populations of L. conchilega. Extracts were processed as described above. 517 518 BULLETIN OF MARINE SCIENCE, VOL. 48, NO.2, 1991 (5) (2) (3) BrOOAe Br "0"CHO (1) (4) Figure 1. Example of gas chromatogram (ECD) of bromophenolacetates from Lanice conchi/ega. RESULTS AND DISCUSSION In all samples of L. conchilega five brominated compounds were detected, four of which were identified (Fig. 1): (1) 2,4-dibromophenol, (2) 2,6-dibromo-4-meth- ylphenol, (3) 2,4,6-tribromophenol, (4) 3,5-dibromo-4-hydroxybenzaldehyde. Mass spectra indicate that the fifth compound is probably a brominated indole. How- ever, a complete structural description cannot be given, since no authentic com- pound was available for comparison. This compound has therefore been excluded from further consideration. L. conchilega has a characteristic iodoform-like odor. Compounds 1, 2 and probably the indolic compound 5 contribute to the odor of this polychaete annelid (compare Higa and Scheuer, 1975a; Higa et al., 1980), while compounds 3 and 4 are odorless. To investigate whether the bromophenols in L. conchilega are commonly pres- ent in the Terebellidae, 10 additional species of the family were analyzed (Table 1). 3,5-Dibromo-4-hydroxybenzaldehyde, which was detected as one of five bro- minated compounds in Thelepus setosus (Higa and Scheuer, 1975b), was present at a concentration of23 ~g'g-1 in this species. The other species contained no or only trace quantities of the compounds. It is apparent, therefore, that the occur- rence of bromophenols identified in L. conchilega is not characteristic for the whole family Terebellidae. Similarly, 2,6-dibromophenol reported by Woodin et al. (1987) from Arenicola cristata at the very high concentration of -6 mg·g-l or other bromophenols do not appear to be typical for the family Arenicolidae. The compounds are absent in Abarenicola pacifica (Woodin et al., 1987) and were not detected in A. marina at Neuharlingersiel and Weser, Spieka, during this inves- tigation. A study was undertaken to determine whether the different concentration pat- terns sporadically observed in L. conchilega could be attributed to variations in GOERKE AND WEBER: BROMOPHENOLS IN LANlCE CONCHILEGA 519 Table I. Concentrations of bromophenols in various species of Terebellidae. (I) 2,4-Dibromophenol, (2) 2,6-dibromo-4-methylphenol, (3) 2,4,6-tribromophenol, (4) 3,5-dibromo-4-hydroxybenzaldehyde. Dash: <0.005 Itg·g-' Number of specimens, Average Concentration of compounds number of wet ljlg.g-' wet weight) samples weight Species and area (in brackets) (g) (I) (2) (3) (4) Lanice conchilega* Channel, Brittany 270 (19) 0.80 0.61 10.3 3.22 1.30 Lanice conchilega* North Sea, Frisia 520 (29) 0.38 0.20 0.65 0.81 0.36 Pista cristata Swedish west coast, Gullmarfjord 13 (I) 0.13 0.01 0.32 Pista spinifera Weddell Sea 6 (2) 0.85 0.01 0.01 0.04 Amphitrite edwardsi Channel, St. Pol de Leon I (I) 8.40 Neoamphilrite affinis Swedish west coast, Gullmarfjord 3 (I) 1.11 0.02 0.06 Neoamphitrite figulus Swedish west coast, Tjiirno 4 (4) 1.08 Eupo/ymnia nebu/osa Swedish west coast, Tjiirno I (I) 1.01 Eupo/ymnia nebu/osa Channel, Rance 7 (I) 1.43 Eupo/ymnia nesidensis Channel, Rance 8 (I) 0.07 Nico/ea zosterico/a Swedish west coast, Tjiirno 25 (I) 0.02 Nicolea venustu/a Channel, Roscoff 14 (I) 0.03 The/epus setosus Channel, Roscoff 8 (I) 0.06 0.30 0.05 22.8 • Calculated from Goerke and Weber (1990). sex and weight. At five sampling times during the period of gamete maturation and at maturity, separate samples of males and females were prepared. Sexes were distinguished by determining gamete color through the body wall and/or by mi- croscopic inspection of the coelomic fluid. There was only very little variation in the concentrations of the four compounds measured in parallel samples of males and females (Fig. 2). These similarities in concentration patterns were particularly conspicuous, as significant differences in patterns and levels were observed if non- parallel samples were compared. It is concluded that concentrations and concen- tration patterns of bromo phenols in L. canchi/ega are not dependent on the sex of the species. The effect of size (age) on bromophenol synthesis and accumulation was tested: samples of L. canchi/ega of different average wet weights were analyzed, including the smallest tube living worms (mean 0.03 g) and the largest specimens (mean 1.3 g) available at two stations (Fig. 3). Bromophenol levels and concentration patterns at each station did not exhibit significant differences compared to dif- ferences between the two stations. Therefore, all samples could be composed regardless of sexual status and compared regardless of average wet weight. Seasonal variations in the various bromophenol compounds were investigated at four stations. The concentrations and patterns remained remarkably constant at Neuharlingersiel and Weser, Spieka (Fig. 4). Up to threefold concentration differences at Norderhever and up to elevenfold differences at N Schliisseltonne 520 BULLETIN OF MARINE SCIENCE, VOL. 48, NO.2, 1991 2.0 Neuharlingersiel 5/89 1.5 N= 1(10) N= 1(5) N = 1(12) N= 1(5) W= 0.82 W=1.30 W = 0.84 W= 0.99 Compounds: 1.0 • 2,4-dibromophenol ~ 2,6-dibromo-4-methylphenol 2,4.6-tribromophenol 0.5 WJ Oi C, o 3,5-dibromo-4-hydroxybenzaldehyde ~ ,9 0,0 III ~ 14 Q) o N Schliisseltonne G 12 o 3/85 2186 T 4/86 6/86 10 N = 2(20) N = 2(20) N = 4(4) N=5(5) N = 10(10) N = 15(15) N = 4(30) N = 3(30) W = 0.45 W = 0.42 W = 0.30 W=0.25 W = 0.42 W = 0.37 W = 0.30 W = 0.31 Figure 2. Concentration patterns (wet tissue basis) of bromo phenols in Lanice conchilega of different sex. Bars represent means ± SE, Notations: name of station, month and year, number N of samples and of total specimens, average wet weight W (g) of Lanice conchilega. occurred. The latter variation appears exceptional and is not understood.