Release of volatile halogenated organic compounds by unialgal cultures of polar macroalgae

Chemosphere, Vol. 31, No. 6, pp. 3387-3395, 1995

Pergamon

0045-6535(95)00190-5

Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0045-6535/95 $9.50+0.00

RELEASE OF VOLATILE HALOGENATED ORGANIC COMPOUNDS BY UNIALGAL CULTURES OF POLAR MACROALGAE

Frank Laturnus

Alfred Wegener Institute for Polar and Marine Research, Columbusstrasse, D-27568 Bremerhaven, Germany present address: Department of Chemistry, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium

(Received in Germany 20 April 1995; accepted 30 June 1995)

Abstract

The release of volatile haiogenated organic compounds (VHOC) by different cultivated polar brown, red and green macroalgae was monitored in laboratory experiments. Isolation and identification of VHOC was performed by a purge and trap technique and capillary gas chromatography with electron capture detection. By using a column with high stationary phase thickness, 11 compounds from bromomethane to diiodomethane were separated and their release rates determined. Of all compounds investigated, bromoform is released in highest quantities from all species studied. Significant linear correlations of bromoform with the halogenated methanes and ethanes investigated, suggest an enzymic formation for each single compound. No significant linear correlation is found for bromomethane with the other VHOC investigated. Therefore, a non-enzymic formation is assumed. The results are discussed with relation to a possible influence of VHOC on atmospheric ozone depletion.

Introduction

Volatile halogenated organic compounds (VHOC) are ubiquitous in the global environment and play an important role as reactants in atmospheric chemistry, such as, e.g. their reaction with ozone (Farman et al., 1985; Crutzen and Arnold, 1986; Solomon, 1990; Anderson et al., 1991). Anthropogenic VI-IOC, e.g. chlorofluorohydrocarbons, are well known and their global atmospheric input can be estimated from industrial production data. Only little knowledge exists about the atmospheric input of biogenic VHOC since the sources of these compounds have not been fully explored. Investigations of seawater and air samples suggest the oceans as a main source for biogenic VHOC (Singh et al., 1983; Class and Ballschmiter, 1987; Cicet:one et al., 1988; Krysell, 1991). The origin of these compounds in the oceans can be marine vulcanism or release by marine animals and plants (Singh et al., 1983; Gribble, 1992). Marine macro- and microalgae produce VHOC in large quantities and release them into seawater (Manley et al., 1992; Sturges et al., 1993). Investigations of near shore waters and the corresponding air show higher values ofbiogenic VHOC compared to the open ocean (Reifenhauser and Heumann, 1992a; Hdz and Hsu, 1978; Class and Ballschmiter, 1988).

3388

No data exists on the release of VHOC by polar macroalgae. Investigations on this field are necessary, especially with regards to increasing ozone depletion in the polar regions.

This paper reports the investigation of Arctic and Antarctic macroalgae for their release of VHOC. The measurements were done with unialgal cultures to minimize influences on the VHOC values by epiphytes and microorganisms.

Experimental Incubation of macroalgae Algae for production rate studies were placed without headspace in glass vessels with filtered natural seawater (Sartorious GF/C-filters, 0.2 lain). The glass vessels had a volume between 340-380 ml, containing a glass grating near the bottom and a septum-closed outlet in the middle of the vessel. They were sealed with polytetrafiuoroethylene (PTFE) collar surrounded glass plugs and metal clamps to prevent the loss of volatile compounds from the water by diffusion. A magnetic stirrer below the glass gratings effected a sufficient mixing of the medium, without injuring the algae. This avoided low nutrients and high VHOC levels near the alga during the incubation period, and effected a distribution of VHOC in the seawater. Glass guaranteed a sufficient lighting of the algae samples inside the vessels from all directions. It was necessary to use filtered natural seawater, as synthetic seawater (Kester et al., 1967) caused damage of the algal ceils immediately. Natural seawater had no obvious effect on cell activity, but a blank of VHOC had to be measured. The filtration of the water removed microorganisms and particular matter, which could influence the VHOC values. For laboratory experiments, small cultivated algae with complete thalli were used.

The macroalgae investigated in the laboratory are listed in Table 1. Himantothallus grandifolius, lridaea cordata,

Desmarestia anceps, Phaeurus antarcticus, Ascoseira mirabilis and Palmaria decipiens were cultivated at 0 °C, and Urosporapenicilliformis and Acrosophonia arcta at 5 °C. Most algae were grown as described by Wiencke (1990a) in 1-2 L beakers containing aerated membrane faltered North Sea water (Sartorius Sartobran II, 0.2 lam) enriched with nutrients (ES enrichment, Provasoli 1968). The media were changed weekly. The cultures were illuminated with coolwhite fluorescent neon tubes (Osram L58/W12) at different photon fluence rates and light : dark cycles. Iridaea

cordata, Phaeurus antarcticus, Himantothallus grandifolius and Palmaria decipiens at 25 ~tmol m"2sd and fluctuating daylengths mimicking the daylength conditions on King George Island. Ascoseira mirabilis and Desmarestia anceps at 10 ,mol m2s ~ and the same daylength conditions. Acrosiphonia arcta and Urosporapenicilliformis at 50 ~tmol m2 s~ and a constant light:dark cycle of 1 8 : 6 hours.

Isolation and analysis of VI-IOC Inert precleaned material and short distances of the gas tubes were important to avoid losses of compounds during stripping procedures. All gas tubes consisted of glass or stainless steel with swageloek connectors. For the glass tubes

3389 TABLE 1. Species of cultivated polar macroaigae used in VHOC release studiesKing George Island = Antarctic; Spitsbergen = Arctic; Disko Island = Greenland, Arctic. No

algalspecies

origin

PHAIgOPHYTA(browDalgae)

1

Himantothallus grandifofius (A. et E.S. Gepp)Zinova

2

Desmarestia anceps Montagne

3

Phaeurus antarcticus Skottsberg

4

Ascoseira mirabilis Skottsberg

King GeorgeIsland KingGeorgeIsland KingGeorgeIsland KingGeorgeIsland

RIIODOPHYTA(red algae) 5

Palmaria decipiens (Reinseh)Rieker

6

Iridaea cm:data Kotzing

KingGeorgeIsland KingGeorgeIsland

CHLOROPHYTA(green algae) 7

Urospora penicilliformis (Roth)Areschoug

8

Acroaiphonia arcta (Dillwyn)J. Agardh

9

Urospora penicilliformis (Roth)Areschoug

10

Acrosiphonia arcta (Dillwyn)J. Agardh

KingGeorgeIsland KingGeorgeIsland Spitsbergen Disko Island

swagelock connectors with TFE seals were used. All equipment was precleaned with n-hexane/acetone and heated 24 hours at 150 °C under ultra-pure helium current. The preconcentration trap consisted of a 20 cm stainless steel tube with an inner diameter of 1 ram. 2 cm of the tube were filled with dimethyldichiorosilane (DMS) treated glass balls (25 pm) and sealed with small plugs of DMS treated glass wool. This construction avoided loss of VHOC during the stripping procedure, which fi'equently was observed by using capillary column or unfilled stainless steel tubes (Graydon and Grob, 1983; Laturnus, 1990). Water was removed from the gas current by a glass tube filled with potassium carbonate, which did not affect the VHOC. This procedure prevented the input of moisture into the cold trap and avoided a rapid freeze-up of the trap. The separation of the different VHOC was performed with a gas chromatograph (Carlo Erba, type Fractovap series 4160) with an electron capture detector (ECD) and a peak measurement system (Perkin Elmer Nelson Systems Inc., Cupertino, USA). The capillary column from Chrompack was specified for the analysis of volatile compounds (PoraPLOT Q, length 25 m, inner diameter 0.53 ram, film thickness 20 I~m, with particle trap, length 2.5 m). Helium was used as carrier gas (14.5 cm s-1) and nitrogen as make-up gas (40 ml rain1); temperature program: 5 rain at 5 °C,

3390 increasing temperature up to 220 °C with a rate of 5 °C min1, then hold at 220 °C for 41 min. The PLOT column showed a good separation for all compounds determined. Only iodomethane could not be separated from dichloromethane and trichlorofluoromethane. Identification and quantification of the compounds were achieved by external calibration standards and multipoint calibration. Standard solutions were prepared gravimetrically from authentic compounds (p.a., Merck and Aldrich) in methanol (LiChroSolv, Merck). As bromomethane is gaseous at room temperature, calibrations were done by preparing a gas standard every day. Evaluated purge efficiencies and calculated detection limits were in the range of 46-97 % and 0.02 - 2.6 ng 11, respectively. The highest ECD response, and, therefore, the lowest detection limit, was found for chloroiodomethane, and the lowest response for diiodomethane. Verification was done by coupling the purge and trap apparatus to a gas chromatographic/mass spectrometric system (Finnigan MAT 1020).

Results and Discussion

The method described, was developed to investigate polar macroalgae in laboratory experiments. The release rates of VHOC from different species of cultivated macroalgae were determined without stressing the algal samples. Microscopic investigations on the thalli after incubation showed no damage of thallus cells. Even algae which are difficult to cultivate, like the brown alga Laminaria saccharina (Wiencke, personal communication), showed no cell damage. With the described purge and trap/gas chromatographic method VHOC could be easily removed from the water sample and separated on the column. Thereby, it was possible to determine a wide range of VHOC from bromomethane to diiodomethane during one analysis step. Eight different Arctic and Antarctic brown, green and red macroalgae were investigated (Table 1) and their release rates of VHOC determined. Field-macroalgae and cultivated macroalgae show similar growth rates (Wiencke, 1990 a-b), so that the release of VHOC by cultivated macroalgae is comparable to the release by field-macroalgae. Contrary to field-species, unialgal cultures are very clean and not contaminated with epiphyts and microorganisms. Therefore, the detected release rates of VHOC can be very closed to the release rates of uncontaminated algae. During the investigation, the rates of release of following compounds were determined: bromomethane, dibromomethane, bromoform, bromoethane, 1,2-dibromoethane, bromochloromethane, bromodichloromethane, dibromochloromethane, diiodomethane, iodoethane and chloroiodomethane. The results of the detected release rates are given in Fig. 1. Bromoform was the dominant compound and had the highest rates of release from all algae investigated. Bromoethane, 1,2-dibromoethane,

dibromomethane and diiodomethane were also released in high rates.

Bromomethane, iodomethane and the mixed halogenated methanes showed only low rates of release. In general, high release rates of VHOC were detected from the brown macroaigae, whereas only low release rates were found from the red and green species. The Antarctic brown algae Himantothallus grandifolius and Desmarestia anceps showed the highest VHOC release rates'among all macroalgae investigated. As these species occur in high biomass (DeLaca and Lipps, 1976), they essentially contribute to the input of VHOC into the Antarctic environment. The formation ofhalogenated methanes by marine macroalgae is suggested to be an enzymic halogenation of organic compounds (Moore, 1978). Haloperoxidases, found in marine macroalgae (deBoer et al., 1986; Butler and Walker, 1993; Mehrtens, 1994), can catalyze, in the presence of hydrogen peroxide, the formation of many halogenated

3391

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Figure 1. Release rates [rig g'J wet alga weight d"~and nmol kg" wet alga weight d q] of volatile halogenated organic compounds from different cultivated species of Antarctic and Arctic brown, red and green macroalgae. The numbers correspond to the species numbers listed in Table 1 (nd = not detectable). CHBr3 = bromoform, CI-IBr2C1 = dibromochloromethane, CHBrCI2 = bromodichloromethane, CH2Br2 = dibromomethane, CH3Br = bromomethane, CH212 = diiodomethane, CH2CII = chloroiodomethane, C~Hsl = iodoethane, 1,2-C2I-LBr~= 1,2-dibromoethane, C2HsBr = bromoethane.

3392 organic compounds (Neidleman and Geigert, 1986; Theiler et al., 1978). It has been shown that haloperoxidases are involved in the formation of VHOC (Walter and Balischmiter, 1992). Significant linear correlations (p<0.001) of dibromomethane with bromoform are in good agreement with the described formation mechanisms. Also a significant linear correlation (p<0.01) could be found for diiodomethane with bromoform. Therefore, it is probable that both compounds are formed by the same enzyme. Dibromochloromethane, bromodichloromethane, bromochioromethane and chloroiodomethane are possibly directly formed by macroalgae (Burreson et al., 1976) or by nuleophilic substitution of bromoform, dibromomethane and diiodomethane with chloride ions in seawater (Class and Ballschmiter, 1988). Investigations of Arctic macroalgae did not show any chloroperoxidase activities suggested for the enzymic formation of chlorobromo- and chloroiodomethanes (Mehrtens, 1994). However, Geigert et al. (1984) reported that chloroperoxidases are not absolute necessary for the formation of chlorinated metabolites. In the presence ofbromoperoxidase, additionally bromochloro-compounds were found. The detetcted halogenated ethanes are probably also formed by enzymic mechanisms, as significant linear correlations (p<0.001) with bromoform showed. A suggestion is a biogenic formation of c~,13-dibrominated products from alkenes (Geigert et al.; 1984). However, the exact mechanisms for their formation have not been fully explored,

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Figure 2. Linear correlation between the release rates of bromomethane, dibromomethane and bromoform from cultivated Antarctic and Arctic macroalgae, r = correlation factor, n = numbers of samples, p = significance Jail values in ng g'~ wet alga weight d'l].

3393 Polybromo- and polyiodomethanes/-ethanes are an important source for bromine and iodine radicals in the atmosphere. Because of their low stability in photochemical reactions, probably most of them are already decomposed in the troposphere and can influence the tropospheric ozone concentration. In addition to their function as a halogen source, brominated and iodinated hydrocarbons can take part in the global greenhouse effect, since IR radiation was adsorbed more strongly compared to other discussed greenhouse gases (Reifenhauser and Heumann, 1992a). These environmental aspects indicate that detailed information about the global atmospheric input of these gases are necessary. Compared to the polybrominated and -iodinated methanes, the monohalogenated methanes can be more important for stratospheric photochemical reactions. Whereas iodomethane mainly reacts in the troposphere (Zafirou, 1974), bromomethane is stable enough to reach the stratosphere, and, therefore, can be a direct source for reactive halogen radicals. Mass spectrometric investigation of VHOC released by cultivated polar macroalgae showed the occurrence ofbromomethane and iodomethane, and the release rates of bromomethane could be determined (Fig. 1) A non-correlation was found for the release rates ofbromomethane and dibromomethane/bromoform (Fig. 2). These results agree with investigations of Schall and Heumann (1993) and Reifenhauser and Heumann (1992b), who found no correlations for iodomethane with brominated methanes detected in Arctic and Antarctic seawater. Compared with the significant linear correlations of the release rates of the polybrominated hydrocarbons, a different mechanism for the formation ofbromomethane can be suggest. Manley and Dastoor (1988) assumed a formation inside the algae without support by haloperoxidase. White (1982) discussed a formation of monohalogenated methane by reaction of dimethylsulfoniopropionate with halide of seawater. This reaction is specific only for the monohalomethanes and can be an explanation for the missing correlation with the other compounds investigated. As dimethylsulfoniopropionate were detected in many marine macroalgae as a possible part of an 'antifreeze' system (Karsten et al., 1990), this mechanism seems to be more likely. Comparison of the VHOC rates released by cultivated macroalgae (this study) and by field-macroalgae (Laturnus et al., submitted), showed in general lower release rates from cultivated material. However, for some cultivated species higher rates of VHOC release were found compared to their field-species. Whether these different results were caused by growth of bacteria and microorganisms on the surfaces of the field-macroalgae, or by culture conditions, can not be answered at the moment. Therefore, to calculate the contribution of macroalgae to the input of VHOC into the polar environment, the release rates should be investigated direct from field-algae. Investigations of cultivated macroalgae are more suitable to obtain data about the dependence of the VHOC release rates on external factors such as light, temperature, salinity and nutrients. The variations of these factors are easier to control in laboratory experiments than in the field.

Acknowledgement I greatfully acknowledge W. Ernst for discussing the manuscript, C. Wiencke for the cultures of various macroalgae and helpful comments on the manuscript, and C. Langreder for fundamental help with macroalgae cultures. Contribution No. 807 of the Alfred Wegener Institute for Polar and Marine Research.

3394

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