BULLETIN OF MARINE SCIENCE, 48(2): 517-523, 1991

BROMO PHENOLS IN CONCHILEGA (POL YCHAET A, ): THE INFLUENCE OF SEX, WEIGHT AND SEASON

Helmut Goerke and Kurt Weber

ABSTRACT The levels of four brominated compounds in 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 specific. Ten other terebellid species did not contain the compounds in significant concentrations. The levels in L. conchilega were not dependent on size, sex or seasons. Bromophenols were not detected in and only (3) was found in sea water (2 pg.g-I) near the sites of dense populations of the . 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 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. It could not be further investigated, since this population was of extremely low density and did not exist continuously. It can also be questioned whether the same pop- ulation was sampled at N Schliisseltonne on the different occasions. Also unusu- ally, L. conchilega lived in clay at this location, where it constructed very solitary tubes without fringes from the few coarse sand grains available. Therefore, it is difficult to generalize about the results from this station. It is striking that 2,6-dibromo-4-methylphenol was dominant among the four identified compounds at Dinard, St. Enogate, and N Schliisseltonne. At these two stations higher concentrations ofthe other compounds were also recorded. Goerke and Weber (1990) demonstrated that L. conchilega from other localities in Bri- tanny exhibited these characteristics, too; specimens from N Schliisseltonne were exceptional among those of the southern North Sea. The authors suggested that differences in biosynthetic capabilities or in availability of precursors may cause such variation. It may also be possible that the take up the bromo phenols as algal products. Brominated compounds are well known as natural constituents of brown and red algae (Higa, 1981). Fenical (1981) summarized evidence for liberation of dissolved halogenated organics and subsequent uptake by other organisms after transport over short distances. However, macro algae are of minor importance in the southern North Sea. Phytoplankton does not appear to be a source either, GOERKE AND WEBER: BROMO PHENOLS IN LANICE CONCHILEGA 521

1.0

Norderhever 10186

0.8 N = 2(100) N = 2(40) N =3(45) N = 3(24) N=4(12)

0.6 Compounds:

2,4-dibromophenol 0.4 III• 2,6-dibro mo-4- methylphenol [@ 2,4,6-tribromophenol 0.2 ~ 0 3,5-dibromo-4-hydroxybenz- .2!, aldehyde c .2 0.0 ~ 0.039 0.129 0.229 0.41g 1.15g 1:: 15 Q) (j c Dinard, St. Enogate 8188 0 () N ~ 1(60) N ~ 1(11) N = 1(10) N = 1(15)) N= 1(13) N = 1(12)

10

5

0.069 0.369 0.39g 0.599 1.03g 1.31g Figure 3. Concentration patterns (wet tissue basis) of bromo phenols in Lanice conchilega of various average wet weights. Bars represent means ± SE. See Figure 2 for description of notations.

since major phytoplanktonic species were found to be void of halogenated organics (Fenical, 1981). To obtain experimental information on the environmental influence on bro- mophenol levels in L. conchilega, water and plankton near the sites of dense populations of the species were analyzed for bromophenols. Only 2,4,6-tribromo- phenol was found in water at concentrations of2.3 ± 0.3 pg·ml-1 (N = 15). Ernst and Weber (1978) reported an exceptionally high capacity of L. conchilega to concentrate anthropogenic pentachlorophenol from polluted estuarine water. However, it is not concluded that 2,4,6-tribromophenol is taken up in a similar way, since the particular compound 2,6-dibromo-4-methylphenol of the worms was absent from the water (detection limit -0.1 pg'ml-I, N = 15). Phytoplankton of May, June and August contributing to the food of the microphagous did not contain bromo phenols at all (detection limit 5 ng·g-t, N = 9). Even if any brominated constituents of the natural diet should contribute to the compounds in the species, this incorporation would indicate a particular property in this polychaete. The microphagous actinia Sargartia troglodytes and the polychaete annelid Nereis virens living within the population of L. conchilega at Norderhever did not contain any of the bromophenols (detection limit 5 ng' g-t, N = 2 and 4). In order to test bioaccumulation pieces of L. conchilega were fed to N. virens (Annelida), Crangon crangon (Crustacea) and Ophiura texturata (Echinodermata) for five weeks in the laboratory. At the end of this period, the three species contained only 2, 0.4, 3% of the amount of the 2,4,6-tribromophenol applied with L. conchilega. Ophiura texturata accumulated 7% of 2,4-dibromo- 522 BULLETINOFMARINESCIENCE,VOL.48, NO.2. 199 J

Neuharlingersiel Weser,Spieka

N ~ 5(33) N ~3(18) N = 3(20) N" 2(15) N.5(48) N.4(32) N ~ 3(25) N ~ 2(29) W.0,57 W~ 0,59 W.0,47 We 0,96 W" 0,41 We 0,99 W. 0,36 W. 0,57

:E>1 Ol ..=;, c: .Q 0 10/86 ~ 10 "E o

3/85 2/86

Figure 4. Concentration patterns (wet tissue basis) of brom 0phenols in Lanice conchilega from various seasons. Bars represent means ± SE, See Figure 2 for description of notations and for assignment of compounds. phenol and 1% of 2,6-dibromo-4-methylphenol in addition. Bromophenols were not accumulated in these representatives of three different phyla, which indicates that bromophenols are generally not accumulated by marine invertebrates. Since no environmental influence has been shown by these investigations, it is suggested that bromophenols are synthesized by L. conchilega. Aromatic compounds from phytoplankton and benthic algae may contribute as precursors.

ACKNOWLEDGMENTS

Research was carried out with the skillful technical assistance of Mrs, 1. St5lting. 3,5-Dibromo- 4-hydroxybenzaldehyde was synthesized and kindly provided as authentic compound by Dr. R. Em- rich. Advice and hospitality of Dr. C. Retiere, Laboratoire Maritime, Dinard, of Dr. L. Cabioch and Dr. F. Oenti!, Station biologique de Roscoff, of Mr. F. Pleijel, Tjlirno marinbiologiska laboratorium, of Prof. J. O. Stromberg, Kristinebergs marinbiologiska station, and the efforts of the crew of RV VICTORHENSENare gratefully acknowledged, This is Contribution No. 305 of the Alfred-Wegener- Institut fUr Polar- und Meeresforschung.

LITERATURE CITED

Ernst, W. and K. Weber. 1978. Chlorinated phenols in selected estuarine bottom fauna. Chemosphere 7: 867-872. Fenical, W. 1981. Natural halogenated organics. Pages 375-393 in E. K. Duursma and R. Dawson, eds. Marine organic chemistry. Evolution, composition and chemistry of organic matter in sea- water. Elsevier Scientific Publishing Company, Amsterdam. GOERKE AND WEBER: BROMOPHENOLS IN LAN/CE CONCH/LEGA 523

Goerke, H. and K. Weber. 1990. Locality-dependent concentrations of bromophenols in Lanice conchi/ega (Polychaeta, Terebellidae). Compo Biochem. Physio!. 97B: 741-744. Higa, T. 1981. Phenolic substances: Pages 93-145 in P. J. Scheuer, ed. Marine natural products, Vo!. 4. Academic Press. London. --- and P. J. Scheuer. 1975a. 3-Chloroindole, principal odorous constituent of the hemichordate Ptychodera !lava laysanica. Naturwissenschaften 62: 395-396. --- and ---. 1975b. Constituents of the marine annelid Thelepus setosus. Tetrahedron 31: 2379-2381. ---, T. Fujiyama and P. J. Scheuer. 1980. Halogenated phenol and indole constituents of acorn worms. Compo Biochem. Physio!. 65B: 525-530. King, G. M. 1986. Inhibition of microbial activity in marine sediments by a bromophenol from a hemichordate. Nature, London 323: 257-259. Sheikh, Y. M. and C. Djerassi. 1975. 2,6-Dibromophenol and 2,4,6-tribromophenols-antiseptic secondary metabolites of Phoronopsis viridis. Experientia 31: 265-266. Weber, K. and W. Ernst. 1978. Occurrence ofbrominated phenols in the marine polychaete Lanice conchi/ega. Naturwissenschaften 65: 262. Woodin, S. A., M. D. Walla and D. E. Lincoln. 1987. Occurrence ofbrominated compounds in soft- bottom benthic organisms. J. Exp. Mar. Bio!. Eco!' 107: 209-217.

DATEACCEPTED:September 19, 1990.

ADDRESS: Chemistry Section. Alfred- Wegener-InstitutjUr Polar- und Meeresforschung, Am Handel- shafen 12, D-2850 Bremerhaven. Federal Republic of Germany.