Environ. Sci. Technol. 2007, 41, 3607-3612
Natural Production of Organic Bromine Compounds in Berlin Lakes ALEXANDRA HU ¨ TTEROTH, ANKE PUTSCHEW,* AND MARTIN JEKEL Technical University Berlin, Department of Water Quality Control, Sekr. KF4, Strasse des 17. Juni 135, 10623 Berlin, Germany
Berlin surface waters are characterized by elevated concentrations of organic bound bromine (up to 35 µg/L) in late summer. Organic bromine compounds in lakes are of significant importance because human life is closely connected to fresh water. Apart from recreational use, fresh water is frequently used for the production of drinking water, e.g., after bank filtration. Therefore the source, particularly the mechanism responsible for the formation is studied. Field studies indicate that the organic bromine compounds, measured as adsorbable organic bromine (AOBr), are autochthonous. Staggered maxima concentrations of chlorophyll-a, DOC and AOBr indicate that phototrophic organisms might contribute to the AOBr after death. The involvement of phototrophic organisms was established in the laboratory using surface water and/or cultures of organisms. Light and the presence of phototrophic organisms are essential for an AOBr production. Phototrophic organisms incorporate bromide, which is released randomly and after cell death. A part of the incorporated bromide is used for the formation of organic bromine compounds in the cell. After death of the organisms the brominated compounds and the incorporated bromide are released into the water phase, and an extracellular AOBr production can lead to a further formation of AOBr, most probably due to the parallel release of haloperoxidases.
Introduction From the environmental point of view, organic halogen compounds are of special interest because they are, in general, characterized as toxic, persistent and/or carcinogenic and are often considered as anthropogenic pollutants. Newer studies have shown that organic halogen compounds are also produced by nature (1). The detection of haloacetates in deep firn cores from Antarctica show a deposition of organic halogen compounds in the 19th century, long before a large-scale industrial production of chlororganics began (2). The knowledge of the natural production of organohalogens is relatively recent. Up till now there have been more then 3800 identified natural organohalogen compounds detected in marine plants, animals, and bacteria and also in terrestrial organisms (1). It is also known that organohalogens are formed during natural abiogenic processes such as volcanoes, forest fires, and other geothermal processes. Other abiotic formations are known but not studied in detail (3, 4). Naturally produced organohalogen compounds are used for the purpose of communication and defense (5). Natural biotic halogenations are well-known in the marine environment. * Corresponding author phone: ++49(0)30 314 2 54 80; fax: ++49(0)30 314 2 38 50; e-mail:
[email protected]. 10.1021/es062384k CCC: $37.00 Published on Web 04/10/2007
2007 American Chemical Society
It has been proposed, that haloperoxidases are involved in the natural production of halogenated compounds (6). The haloperoxidase enzymes have been identified in an increasing number of marine algae and terrestrial lichen and fungi (7). Haloperoxidases are capable of catalyzing the peroxidative synthesis of the carbon-halogen bond in a large number of substrates in the presence of chloride, bromide, or iodide and hydrogen peroxide (8, 9). Our institute reported for the first time the occurrence of natural organic bromine compounds in a lake (10, 11). Organic bromine compounds were detected by differentiation of the group parameter AOX into adsorbable organic chlorine (AOCl), bromine (AOBr), and iodine (AOI) using ion chromatography (12). In the eutrophic and wastewaterinfluenced surface water, Tegeler See, an annual AOBr profile is recognized in grab samples of the upper few cm of the water body (10). This annual profile has been observed for several years (1998-2000) with high concentrations of up to 35 µg/L AOBr during August and September, whereas for other months, concentrations between 5 and 10 µg/L were detected. The AOBr detected in the lake is most probably produced within the lake because the inflow concentrations are always lower (11). In the mean time a similar AOBr profile is recognized in Wannsee, a surface water comparable to Tegeler See (13). By simulating the AOBr formation in laboratory using lake water and algae cultures (11), some factors influencing the AOBr formation were determined. Light and the presence of phototrophic organisms are of most importance for the AOBr formation. To establish whether the former results are reproducible and to gain further knowledge about the AOBr production in Berlin lakes, a field study and laboratory tests were conducted. The field study covered an AOBr monitoring in Berlin’s surface waters and the determination of depthprofiles of selected compounds in Tegeler See. The formation of AOBr in the laboratory is highly influenced by biology; for that reason, numerous laboratory tests were done to confirm former results and to examine the influence of phototrophic organisms, enzymes, and hydrogen peroxide. Knowledge about the origin of organic bromine compounds in surface waters/lakes is important because human life is closely connected to fresh water from lakes. In many countries lake water is used for the delivery of raw drinking water. In Berlin, purification of the lake water is performed only by natural bank filtration or artificial groundwater recharge. Additionally, the recreational use of lake water should not be underestimated, in particular with regard to small children drinking the water.
Experimental Section A rough description of the experimental work is given below, for more details see the Supporting Information. Field Study. On September, 22nd 2005, the water system of Berlin was sampled (1 L, grab samples) at different sampling points (marked as a dot, Figure 1; results). From JuneOctober, 2005, Tegeler See was sampled approximately every second week. The sample site was located close to the deepest point (52°35′46′′N, 013°15′53′′E) and samples (2 L) were taken out at different water depths (upper few cm and 0.5, 1, 2, 4, 5, 10, and 12 m). In all samples the AOBr, DOC, chlorophyll-a, bromide, and hydrogen peroxide concentrations were determined. Chlorophyll-a and hydrogen peroxide measurements were done on the sampling day. Laboratory Studies. All laboratory batch tests were carried out using open glass vessels (20 L or 5 L) irradiated with VOL. 41, NO. 10, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Map of the Berlin water system and AOBr concentrations determined at September, 22nd 2005 and AOBr profiles of Tegeler See (1998; (10, 11)) and Wannsee (2004, 2005; (13)). artificial sunlight (12 h each day, six tubes, each 18 W) and aerated with filtered air (0.45 µm). To all tests 100 µg/L of bromide was added. All experiments were performed in an air conditioned chamber at 25 °C, except surface water batch tests which were done at room temperature. Surface Water Batch Tests. For lake water batch tests, 20 L Tegeler See and Wannsee water were used unfiltered. At the beginning, samples were taken monthly and later on every third month, for AOBr analysis. Phototrophic Organisms Batch Tests. Phototrophic organism batch tests (BT 1-5) were carried out with the cyanobacteria Microcystis aeruginosa (Culture Collection of Algae at the University of Go¨ttingen, SAG) in a nutrient solution (20 L) or using surface water as nutrient source and with the addition of bromide (100 µg/L). All batch tests are summarized in Table 1. BT 1a-d: The batch tests were carried out in filtered and autoclaved Tegeler See water as nutrient solution. One test was cultivated in darkness and the other three were irradiated with artificial sunlight. One test was spiked with the herbicide Diuron (10 µM), and a blank test was carried out without cyanobacteria addition. BT 2a-b: Cyanobacteria were cultivated in a nutrient solution of optimal concentration, and with a diminished nutrient supply (optimal nutrient solution 10 times diluted). BT 3: The test was carried out using an optimal nutrient supply. In contrast to all other batch tests, the volume was just 5 L, and for irradiation, new light tubes were used and a new purchased Microcystis aeruginosa culture (SAG). BT 4a-b: Batch tests with addition of hydrogen peroxide (10 µg/L) were carried out in optimal nutrient solution. A blank test was done without the addition of hydrogen peroxide. BT 5a-d: The tests were as described for BT 4 but here different amounts of hydrogen peroxide were added (1000; 100; 10 µg/L), and again, a blank test was set up without addition of hydrogen peroxide. Bromoperoxidase Batch Tests. BT 6a-d: Batch experiments with bromoperoxidase were carried out without the addition of phototrophic organisms. One test was carried out in autoclaved and filtered (0.45 µm) Tegeler See water 3608
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(DOC ) 6.9 mg/L) and for the second test, natural organic matter (NOM) isolated from Suwanee River (U.S.) was dissolved in ultrapure water (DOC ) 5.1 mg/L). Bromoperoxidase (0.2 units/mL) from Corallina officinal (SigmaAldrich) was added to each test. Each experiment was accompanied by a blank test without addition of bromoperoxidase. All tests were spiked with bromide (100 µg/L), hydrogen peroxide (10 mM) and MES buffer (0.5 mM; 2-morpholinoethanesulfonic acid, Sigma-Aldrich) to maintain a pH of 6.4. The end of the experiment was indicated by a low enzyme activity. BT 7a-c: A second bromoperoxidase batch test was carried out in autoclaved, filtered (0.45 µm), and 10 times concentrated Tegeler See water. Two blank tests without bromoperoxidase addition were carried out, of which one contained the same autoclaved and concentrated Tegeler See water and the other one contained ultrapure water. Additions of enzyme and reagents were as described for BT 6. Due to the high bromide concentration, sulfite was added before AOBr analysis to exclude interferences (see the Supporting Information). Analysis Procedures. For the determination of adsorbable organic bromine (AOBr), the filtered (0.45 µm) and acidified water samples (conc. HNO3, pH 2; if necessary stored at 4 °C) were enriched on activated carbon. In case of samples with a high bromide concentration, a second set of samples was analyzed with sulfite addition before acidification and enrichment. After combustion of the loaded activated carbon and trapping the combustion gas in deionized water with a trace of sodium sulfide, the halides were analyzed by ion chromatography (IC (12)). Chlorophyll-a analysis of the biomass was carried out according to German standard method DIN 38412-16. Dissolved organic carbon (DOC) was determined with a HighTOC analyzer (Elementar, Hanau, Germany). Hydrogen peroxide was measured by the N,N-diethyl-p-phenylenediamine (DPD) method (14) with small modifications. In batch tests BT 2 and 3 with Microcystis aeruginosa, the dissolved and intracellular bromine species were determined. The dissolved bromine covers bromide and the AOBr. The intracellular bromine consists of organic bound and inorganic
TABLE 1. Laboratory Batch Experiments with Phototrophic Organisms and Bromoperoxidase conditions batch
test
organism
duration (d)
BT 1
a b c d
M. aeruginosa M. aeruginosa M. aeruginosa none
32
a b
M. aeruginosa M. aeruginosa
BT 2
BT 3
nutrient solution
bromide
TS water TS water TS water TS water
100 µg/L 100 µg/L 100 µg/L 100 µg/L
80 84
optimal reduced
M. aeruginosab
77
other additions
sampling
parameters
interval (d)
determined
3-7
AOBr
100 µg/L 100 µg/L
7-14
AOBr intra bromine extra Br-
optimal
100 µg/L
14-21
AOBr extra Brintra.bromine intraOBr
darkness 10 µM Diuron
BT 4
a b
M. aeruginosa M. aeruginosa
60
optimal optimal
100 µg/L 100 µg/L
10 µg/L H2O2
7-14
H2O2 AOBr
BT 5
a b c d
M. aeruginosa M. aeruginosa M. aeruginosa M. aeruginosa
60
optimal optimal optimal optimal
100 µg/L 100 µg/L 100 µg/L 100 µg/L
1000 µg/L H2O2 100 µg/L H2O2 10 µg/L H2O2
7
H2O2 AOBr
BT 6
a
none
26
Suwannee River NOM
100 µg/L
7
H2O2
b
none
TS water
100 µg/L
c
none
100 µg/L
end (26 d)
AOBr
d
none
Suwannee River NOM TS water
340 mg/L H2O2; MES buffer pH 6.4; 0.2 units/mL BrPOD 340 mg/L H2O2; MES buffer pH 6.4; 0.2 units/mL BrPOD 340 mg/L H2O2; MES buffer pH 6.4 340 mg/L H2O2; MES buffer pH 6.4
a
none
1:10 conz. TS water
100 µg/L
7
H2O2
b
none
100 µg/L
c
none
1:10 conz. TS water u.p. water
340 mg/L H2O2; MES buffer pH 6.4; 0.2 units/mL BrPOD 340 mg/L H2O2; MES buffer pH 6.4 340 mg/L H2O2; MES buffer pH 6.4
BT 7
a
TS is Tegeler See
49
b
100 µg/L
100 µg/L
enzyme activity
enzyme activity end (49 d)
AOBr
New culture
bromine species. The dissolved bromide was determined by IC in the filtered (0.45 µm) samples. The intracellular bromine concentrations were determined by combustion of the separated biomass (1.2 µm filtration) followed by IC of the trapped combustion gas. For the determination of intracellular organic-bound bromine the biomass was separated by filtration (1.0 µm) and hydrolyzed under alkaline conditions (5% potassium hydroxide in methanol/ultrapure water 8:2 for 2 h at 85 °C). The hydrolyzed biomass was extracted with dichloromethane. The dichloromethane extract was concentrated, dried with nitrogen gas, and then combusted, followed by IC of the trapped combustion gas. For counting the organisms, the samples were treated with Lugol’s solution for conservation and kept dark until individual microscopic count. Individual count was performed according to the Utermo¨hl procedure (15) with a Zeiss IM 35 microscope (Germany). The dry weight of the biomass was determined by separating the biomass on previously weighed membrane filters (1.2 µm), drying it in a desiccator overnight and reweighing the filter. The activity of the bromoperoxidase used in batch tests BT 6 and 7 was measured by monitoring the bromination of monochlorodimedone (16).
Results and Discussion Field Study. Figure 1 shows the Berlin water system, characteristic annual AOBr profiles with maximum concentrations in late summer of two surface waters, and the AOBr concentrations of different surface waters sampled at the
same day in September 2005. During the 2005 field study the AOBr reached only a maximum of 9.3 µg/L in Tegeler See. In 2001 a pipeline, pumping water from the Oberhavel into Tegeler See, was reactivated. Due to the pipeline the water is diluted and the retention time and the growth of phototrophic organisms are decreased. This is the most probable the reason why the AOBr in late summer 2005 is lower compared to the years before reactivating the pipeline. The improved situation at Tegeler See, concerning the AOBr, does not influence the AOBr formation downstream at Wannsee (20.3 µg/L). Even in Mu ¨ggelsee an elevated AOBr concentration (16.8 µg/L) was determined. The 2005 field study shows again that the AOBr of the lake inflows is always lower than in the lake (see Figure 1), indicating that AOBr must be produced within the lake and is carried downstream with the outflows. It is supposed that phototrophic organisms are involved in the AOBr formation, and for that reason it was checked if such a correlation could be seen in nature. Tegeler See was sampled from June to September, 2005, and different concentrations were determined of which chlorophyll-a, DOC and AOBr are the most significant (Figure 2). At the end of June, when the chlorophyll-a concentration increases, the phototrophic organism bloom starts. The death of the phototrophic organisms is indicated by decreasing chlorophyll-a and increasing DOC (August) concentration. An AOBr increase is observed during the DOC increase and continues after DOC decrease. The observed staggered maxima development of chlorophyll-a, DOC, and AOBr indicate that phototrophic organisms are probably involved in the AOBr formation and that elevated AOBr concentrations are deVOL. 41, NO. 10, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. AOBr, DOC, and chlorophyll-a (Chl-a) concentration in the top few cm of the water body of Tegeler See (grab samples); summer 2005. tectable after death of the organisms. Depth-profiles (not shown here) of chlorophyll-a and AOBr show that the AOBr is produced/released within the surface layer (1-5 m water depth). It is known that a biotic formation of brominated organic compounds requires, beside bromide and organic compounds, hydrogen peroxide, if haloperoxidases are involved. For that reason, the bromide and the hydrogen peroxide concentrations were determined as well (June/July 2005, n ) 7). Over the depth profile the bromide concentration was nearly constant with 120 ( 12 µg/L. Hydrogen peroxide was only detectable on sunny and windless days in the upper few cm of the water body. The highest hydrogen peroxide concentration was 11 µg/L on June, 21st and 10 µg/L on August 30th (limit of detection 0.2 µg/L (14)). We concluded that, in surface waters, the prerequisites for enzymatic formation of AOBr exists in the surface layers. Laboratory Studies. Putschew et al. (11) studied the prerequisites for the AOBr formation by performing batch tests using surface water (Tegeler See and Mu ¨ ggelsee) and the algae Desmodesmus subspicatus. It could be shown that light and phototrophic organisms are prerequisites for the AOBr formation. Additionally, it was found that stress induced by a lack of nutrients might favor the AOBr production. Several new batch tests with surface water and cultures of phototrophic organisms were performed to confirm the results and to receive additional knowledge concerning the AOBr formation. Batch Tests with Lake Water and Phototrophic Organisms. As in the cited study, it was possible to simulate an AOBr production with surface water (Figure S1, Supporting Information). Batch tests with Tegeler See water resulted in an AOBr concentration of 76 µg/L in 190 d; 62 µg/L in 200 d for Wannsee water. For batch tests with phototrophic organisms, Microcystis aeruginosa (cyanobacterium) which often proliferates in nutrient enriched lakes was used. Figure 3 shows the AOBr concentration of these tests (BT 1) under the influence of light, darkness, light and inhibition of photosynthesis by Diuron as well as a light test without phototrophic organisms (blank). In all tests with Microcystis aeruginosa, an AOBr increase was detected, whereas without Microcystis aeruginosa, the AOBr didn’t increase, showing that an abiotic AOBr formation can be excluded. In the light experiment the AOBr rises from 12 µg/L to 55 µg/L within 11 days. In darkness 28 µg/L AOBr was detectable after 11 days, respectively 20 µg/L if photosynthesis is disrupted. The AOBr increase in darkness is probably based on the fact that phototrophic organisms can survive temporarily in darkness and, for the Diuron test, on a delayed effect. The inhibition of AOBr production in darkness and by Diuron, as well as the absence of an AOBr production in the blank test, gives 3610
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FIGURE 3. AOBr concentration of four parallel batch tests at 25 °C using autoclaved water from Tegeler See inoculated with Microcystis aeruginosa, light: irradiated with artificial sunlight; dark: kept in darkness; Diuron ) spiked with Diuron (10 µM) and irradiated; blank ) without Microcystis aeruginosa and irradiated.
FIGURE 4. Mycrocystis aeruginosa cultivated with a diminshed nutrient supply at 25 °C, aerated and irradiated. A: Dissolved bromide, intracellular bromine species and sum ) dissolved bromide + intracellular bromine + AOBr. B: Individuals and AOBr. evidence for the necessity of phototrophic organisms, which is in agreement with the field observations. Bromine Mass Balance. To demonstrate the involvment of organisms and to sort out if the AOBr is produced intraor extracellularly, a bromine mass balance was generated. Besides the concentration of dissolved bromine species (inorganic bromide and AOBr), the intracellular bromine content was determined. Figure 4A shows the bromine mass balance of the batch test with a low nutrient supply (BT 2b). In case of a reduced nutrient supply, a higher AOBr production (4.3 µg/L in 77 d) was observed than under an optimal nutrient supply (2.5 µg/L in 62 d, BT2a) which is consistent with the former study (11), where Desmodesmus subspicatus was used. The data of these tests are given in the Supporting Information (Figure S2). The mass balance of all analyzed bromine species comes out to be the amount of
FIGURE 6. Autoclaved Tegeler See water (TS) tenfold concentrated and spiked with H2O2 (10 mM) and bromide (100 µg/L) in 0.5 mM MES buffer (pH 6.4) at 25 °C: Initial ) TS, TS+BrPODdTS with adition of BrPOD (20 units/mL), TS-BrPODdTS without addition of BrPOD. Samples were taken after 49 d, AOBr* initial not determined. AOBr* ) sulfite addition prior enrichment (see Supporting Information and Figure S3).
FIGURE 5. Mycrocystis aeruginosa batch test at 25 °C, aerated and irradiated. A: Dissolved and intracellular bromide and sum of both concentrations. B: Dissolved AOBr, intracellular organic bound bromine (intraOBr), individual numbers and sum ) dissolved AOBr + intraOBr. added bromide (100 ( 10 µg/L), after a short period (22 d) of adaptation. A dependency between dissolved bromide and intracellular bromine is obvious. If the intracellular bromine reaches a maximum, the dissolved bromide concentration is at a minimum and vice versa. This result leads to the conclusion that Microcystis aeruginosa can incorporate bromide. At the end of the experiment the dissolved bromide concentration was just lower (98.4 µg/L) than the added bromide concentration and thus, only a low amount of brominated organic compounds can be produced within the cell, if any at all. Figure 4B shows the number of individuals and the dissolved AOBr. During the first 14 days of the experiment the cell number increased and then decreased thereafter. The AOBr is very low at the beginning and rises up to 4.3 µg/L after 35 days. The trend of the cell numbers and the AOBr indicates a correlation between the cell number and the AOBr concentration in the water phase, which is in agreement with the field observation. It is not clear if the AOBr is produced within the cell or after cell death. In a second set of cyanobacteria batch tests, the bromine content of the cells was differentiated into intracellular organic bound bromine (intraOBr) and intracellular inorganic bromide (intraBr-). The amount of intraBr- was calculated by subtracting the intraOBr from the intracellular bromine concentration. The devolution of the dissolved bromide and the intraBr- is of opposite direction (Figure 5A). With increasing time the sum of the determined bromide concentrations decreased, indicating that bromide is consumed. The concentration of intracellular and the dissolved organic bromine compounds increases with time (Figure 5B), whereby the organic-bound bromine content is always higher within the cell than in the water phase, pointing out the organic bromine compounds can be produced by Microcystis aeruginosa. With increasing time, the sum of both parameters increases which is consistent with the decrease of the inorganic bromide concentration. The individual number
was constant until day 60 and rises thereafter (Figure 5B). In contrast to the experiment discussed before, an intracellular production of organic bromine compounds could be shown which might be related to the different organism development and leads to the assumption that the bromide uptake is a fast process (BT 2a), whereas the bromination of organic compounds within the cell is slow. Batch Test with Hydrogen Peroxide and Peroxidase. The AOBr concentrations measured in surface water batch tests and tests done with phototrophic organisms attract attention. The maximum AOBr concentration in laboratory surface water batch tests (40-50 µg/L in 74 d, Figure S1 in the Supporting Information) is much higher than in batch tests with organisms (4 µg/L in 77 d; Figure 4B). Additional factors must influence the AOBr formation. Haloperoxidases are known to catalyze the bromination of organic compounds in the presence of bromide and hydrogen peroxide (17) which were detected in Tegeler See (see Field Study). We investigated the influence of H2O2 on the AOBr formation. The presence of H2O2 (10 µg/L) in a batch test using Microcystis aeruginosa (BT 4) turned out to increase the AOBr production significantly. In such a batch test 40 µg/L AOBr was produced within 60 d, which is about ten times the AOBr without H2O2. To examine the influence of the H2O2 concentration, four parallel batch tests with Microcystis aeruginosa were done, adding different amounts of H2O2 (BT 5) and a blank test without H2O2 addition. In all experiments 54 ( 9 µg/L (n ) 8) AOBr was produced in 60 d (results not shown). This result is surprising but explainable by the fact that the culture used was in contact with H2O2 before and thus, it seems that the presence of H2O2 enables Microcystis aeruginosa to produce brominated organic compounds. In addition to the hydrogen peroxide involvement, we investigated the ability of bromoperoxidase (BrPOD) to produce AOBr in the presence of bromide (100 µg/L), H2O2 (10 mM) and natural organic matter (NOM) without addition of phototrophic organisms. Two different NOM sources were used: NOM from Suwannee River in ultrapure water (BT 6a, DOC ) 5.1 mg/L) and autoclaved and filtered Tegeler See water (BT 6b, DOC ) 6.9 mg/L, AOBr ) 2.9 µg/L). In both tests AOBr was produced within 29 d with the highest concentration in the Suwannee River NOM test (22 µg/L) and only a small amount in the Tegeler See water test (4 µg/L). Blank tests (BT 6c-d) using the different NOM without addition of bromoperoxidase showed no AOBr formation. A second test was carried out with autoclaved, filtered, and concentrated (tenfold) Tegeler See water (BT 7a and b, AOBr ) 53 µg/L) with and without addition of BrPOD plus VOL. 41, NO. 10, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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bromide and hydrogen peroxide addition as stated before. After 49 days, a high AOBr concentration was determined in the BrPOD test (391 µg/L) and a moderate value in the test without BrPOD addition (148 µg/L, Figure 6). In a blank test done with ultrapure water, BrPOD, hydrogen peroxide, and bromide addition, no AOBr was detectable (BT7c, data not shown). Thus, in the presence of BrPOD, hydrogen peroxide and organic material AOBr can be produced extracellular. The batch tests and field observations lead to the conclusion that AOBr can be produced extracellularly after the death of the phototrophic organisms (Figure 2 and 4B; BT 2b). The organisms do incorporate inorganic bromide and release it during growth stagnation or organism death (e.g., by cell lysis, Figure 4A and 5B; BT 2b and 3). The inorganic bromide release is accompanied by a starting AOBr production. It is presumed that the mechanism for the AOBr formation is among others an enzymatic bromination of dissolved organic matter catalyzed by haloperoxidases which are released, too, after cell death. The isolation of haloperoxidases from fresh water algae by Verdel et al. 2000 (18) supports this theory. The extracellular enzymatic reaction is very likely because it could be shown that with hydrogen peroxide contact (BT 4 and 5) the amount of produced/released AOBr in batch tests with Microcystis aeruginosa is comparable to lake water batch tests and the naturally produced amount. Additionally, an intracellular production of organic bromine compounds has been observed (Figure 5B; BT 3). It correlates better with the organism growth, indicated by higher production rates during periods of individual increase, then is does during growth stagnation.
Acknowledgments A special thanks goes to the three reviewers as many helpful comments were provided in preparing the paper. We thank Luisa Petri, David Kutzner, Ines Bewersdorff, and Katrin Noack for the AOBr analysis and various lab works. Karen Barry is thanked for editing the manuscript. We thank the Berlin Water Works for lending their boathouse to us. Thanks to Manuela Vogel for many new results extracted from her diploma thesis. The project was financially supported by the German Research Foundation (DFG; PU 199-2/1-2/2) and the “Berliner Programm zur Fo¨rderung der Chancengleichheit fu ¨ r Frauen in Forschung und Lehre”.
Supporting Information Available Detailed experimental procedures on sample sites, AOBr, intra- and extracellular bromine species, ion chromatography, chlorophyll-a, hydrogen peroxide, enzyme activity, nutrient solution for algae growth, figure S1: surface water batch tests with Tegeler See and Wannsee water, Figure S2: Microcystis aeruginosa batch test with optimal and reduced nutrient supply (BT 2a,b), figure S3: BrPOD batch test: AOBr with and without sulfite addition prior enrichment. This material
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is available free of charge via the Internet at http:// pubs.acs.org.
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Received for review October 5, 2006. Revised manuscript received February 27, 2007. Accepted March 9, 2007. ES062384K
