Limnol. Oceanogr., 44(5), 1999, 1348–1352

Volatile halocarbons released by 12 Falkenbergia phase isolates of Asparagopsis taxiformis and Asparagopsis armata collected from widely scattered geographical locations were identified using a purge and trap technique and gas chromatography/mass spectrometry. Production of the compounds under normal and high irradiance was quantified where possible. Bromoform (CHBr3), dibromomethane (CH2Br2), 1,2-dibromoethylene (C2H2Br2), dibromochloromethane (CHBr2Cl), and tribromoethylene (C2HBr3) were identified as algal products. C2H2Br2 and C2HBr3 have not previously been recorded as natural products. Considerable qualitative and quantitative differences existed between isolates in the halocarbons released. The rates of CHBr3 and C2H2Br2 production were negatively correlated. Increased irradiance invariably resulted in higher rates of release of halocarbons. Initial rates of production ranged between ,8 and 866 ng g21 fresh weight (FW) h21 for CHBr3, ,3 and 59 ng g21 FW h21 for CH2Br2, and ,0.4 and 17 ng g21 FW h21 for C2H2Br2. Volatile halocarbons are produced by many marine algae (Gschwend et al. 1985; Manley and Dastoor 1987; Manley et al. 1992; Gribble 1996; Goodwin et al. 1997b). The red alga Asparagopsis taxiformis (Delile) Trev., which is cosmopolitan in warm-temperate, subtropical, and tropical waters, is a particularly prolific source releasing over 100 such compounds, mainly brominated and iodinated methanes and acetones (Burreson et al. 1976; McConnell and Fenical 1977). This alga is a dioecious gametophytic stage that alternates in its life cycle with a heteromorphic sporophyte known as Falkenbergia hillebrandii (Born.) Falkenb. (Dixon and Irvine 1977). Surprisingly, Burreson et al. (1976) were unable to demonstrate production of any volatile halogenated compounds by the asexual form that they identified as Falkenbergia rufolanosa (Harvey) Schmitz. However, the latter attribution is open to question as F. rufolanosa is the tetrasporophyte form of the only other currently recognized Asparagopsis taxon, A. armata, a species endemic to cold-temperate waters. This confusion probably arises as the Falkenbergia stages of both Asparagopsis species are considered morphologically indistinguishable (Dixon 1964). Both form small dense fluffy balls of branched filaments in shallow sublittoral environments. We have studied volatile halocarbon production by laboratory-grown cultures of 12 Falkenbergia stages of Asparagopsis spp. from a wide range of geographical locations including both warmand cold-water environments and have demonstrated release of several bromocarbons, two previously unreported as natural products. Production of several of the bromocarbons has been quantified under both normal and high irradiance because increased irradiance can stimulate volatile halocarbon production by many marine algae (Mtolera et al. 1996). Isolates and culture conditions—Isolates of Falkenbergia stages were obtained from the collection of Professor Michael Guiry of the Department of Botany, National University of Ireland, Galway, Ireland. The collection accession numbers of each isolate, together with the geographical locations where they were collected are indicated in Table 1. Isolates were maintained in seawater enriched with 20% von Stosch medium (von Stosch 1964). Initially algal cultures were established in petri dishes (50 mm diameter 3 20 mm) containing culture medium (15 ml) and incubated at 208C illuminated from above with Thorn Grolux and Sylvana Lifeline fluorescent lights at an irradiance of 30 mmol photons m22 s21 (16 : 8 light : dark [LD] cycle). The algae were later subcultured into fresh medium (100 ml) in 250-ml conical flasks shaken at 100 rpm at 208C under a similar light regimen. For monitoring of volatile halocarbon release, alga (1 g fresh weight [FW]) was suspended in culture medium (10 ml) under an atmosphere of air in each of a series of screwcapped vials (25 ml) fitted with polytetrafluoroethylene (PTFE) septa and incubated at 208C at an irradiance of 50 mmol photons m22 s21 under fluorescent lights or an irradiance of 330 mmol photons m22 s21 from a quartz halogen lamp for periods from 6 to 48 h. Duplicate vials were removed from the incubation chamber after the appropriate time period, and medium (5 ml) was withdrawn from each for analysis as described below. The remaining medium was either discarded or stored at 2208C for future reference. Control experiments in which culture medium that had not been exposed to the alga was spiked with authentic samples of various halocarbons and incubated in the absence of algae were also conducted. Halocarbon and other analyses—Halocarbons present in the algal incubation medium were identified and quantitatively determined using a DANI 37.50 purge and trap sampler linked to a Hewlett Packard 5890 gas chromatograph equipped with a mass selective detector (MSD). Incubation medium (5 ml) was placed in a vial (20 ml) and 1,2-dibromoethane (1 ml of a 10 mg liter21 solution was employed as an internal standard), previous investigations in the laboratory having established that this compound was not released by any of the Falkenbergia isolates under the conditions of the experiments. Vials sealed with aluminum crimp caps fitted with PTFE-coated butyl rubber seals were equilibrated in the purge and trap sampler at 508C before purging with helium at a flow rate of 30 ml min21 for 30 s. For qualitative measurements the vials were purged for 5 min, but for quantitative determination a purge time of 30 s was used to ensure that analysis was conducted in the range where a linear relationship existed between concentration and response. Volatiles were trapped on Tenax TA and thermally desorbed at 2508C for 1 min onto a Chrompak Poroplot Q cap-