Environ. Sci. Technol. 1996, 30, 1238-1241
Significance of Bacteria in Marine Waters for the Distribution of Hydrophobic Organic Contaminants D A G B R O M A N , * , † , ‡ C A R I N A N A¨ F , † JOHAN AXELMAN,† CECILIA BANDH,† HARALD PETTERSEN,† RON JOHNSTONE,§ AND PETRA WALLBERG§ Aquatic Chemical Ecotoxicology, Department of Zoology, Stockholm University, S-106 91 Stockholm, Sweden, Institute of Applied Environmental Research, Stockholm University, S-10691 Stockholm, Sweden, and Department of Zoology, Stockholm University, S-106 91 Stockholm, Sweden
Pelagic bacteria represent the potentially most predominant biological, particulate sorptive surface for hydrophobic organic contaminant (HOC) and constitute a food source for the microheterotrophic food web, which conceivably continues up to pelagic fish. However, no data have been reported on HOCs in bacteria. Therefore, this fraction (0.2-2 µm) was isolated with a new technique in situ in the Baltic Sea followed by HOC analysis (HRGC-MS). Results show bacterial concentrations (PAHs and PCBs) in the same order as or higher than the larger particulate fraction (2-90 µm), which clearly illustrates the significance of heterotrophes for the distribution and dynamics of HOCs in marine waters.
Introduction In natural marine waters, particulate organic matter is of crucial importance for the dynamics and distribution of hydrophobic organic contaminants (HOCs). The carbon content in, or on the surface of, various particulate matrixes is of fundamental importance since the particulate organic matter/water partitioning process is governed by simple sorptive mechanisms. The particulate/water equilibrium of HOCs is attained within minutes to hours for surface particle sorption whereas the time for equilibrium between the water and the organic matrix within the particles is in the order of days to weeks (1, 2). Free-living marine bacteria, which are the ones most abundant of the total number of bacteria in seawater [50 L min-1) using a silicon tube with an inner diameter of 350 mm. Before entering the ultrafiltration system, the water was filtered through a 90-µm nylon mesh followed by a 2-µm prefilter (CP15-010, Millipore) for the removal of the bulk of flagellates, ciliates, and phytoplankton. The total volume sampled for the bacterial fractions during summer and autumn were 0.6 and 2 m3, respectively. Analysis. The concentrations of six ortho-substituted polychlorinated biphenyls (PCBs) (IUPAC Numbers 52, 101, 118, 138, 153 and 180) and 12 polycyclic aromatic hydrocarbons (PAHs) (flouranthene, pyrene, benzo[ghi]fluoranthene, benz[a]anthracene, chrysene/triphenylene, benzo[k]fluoranthene, benzo[e]pyrene, benzo[a]pyrene, perylene, indeno[1,2,3-c]pyrene, benzo[ghi]perylene, and coronene) all with log Kow of between approximately 5 and 7.5 were determined in both the bacterial fraction (0.2-2 µm) and in the larger particulate fraction (2-90 µm) by HRGC-MS analysis according to previously described techniques (12). The carbon content in aliquots of the two size fractions collected and in the seawater was determined on a Carlo Erba element analyzer, EA 1108 CHNS-O, connected to a Fisons Optima MS. The number of cyanobacteria and heterotrophic bacteria was determined in the two smaller size fractions and in the seawater. Cyanobacteria were quantified with an epiflourescence microscope using a green excitation filter (filterset: Zeiss 487714) with a 1200× magnification, after collecting 10 mL on a 25-mm, 0.6-µm polycarbonate filter. For enumeration of heterotrophic bacteria, 1 mL was stained with acridine orange (13) filtered onto 25-mm, 0.2-µm black polycarbonate filters (MSI) and counted in an epifluorescence microscope at 1200× magnification, using blue excitation light (filterset: Zeiss 487709). At least 300 cells per filter was counted.
FIGURE 1. Concentrations of the sum of six PCBs and 12 PAHs on a carbon weight basis for the larger size fraction (2-90 µm) and the bacterial (0.2-2 µm) fraction filtered at an open coastal location in the northern Baltic proper at two seasonal periods, summer (June) and autumn (November).
Results and Discussion The results of the HOC analyses on the two size fractions for the two seasons are presented in Figure 1, which shows the concentrations of the sum of six polychlorinated biphenyls (PCBs) and the sum of 12 polycyclic aromatic hydrocarbons (PAHs) on a carbon weight basis. For the larger size fraction (comprised of phytoplankton, protists, and detritus), the concentrations of the sum of PCBs and PAHs were highest in summer [500 and 12000 ng (g of C)-1, respectively] and lowest during autumn [10 and 700 ng (g of C)-1, respectively]. The opposite pattern was found for the bacterial fraction, however, where the highest concentrations of the sum of PCBs and PAHs were found during autumn [2000 and 11000 ng (g of C)-1, respectively], while the lowest concentrations were found in summer [300 and 6000 ng (g of C)-1, respectively]. The relative distributions of the different PAH compounds in each of the samples (except the 0.2-2-µm size fraction from the summer sampling) were fairly similar both within the samples and between the samples. Factors of approximately 6-7 describe the differences between the PAHs found in highest and lowest concentrations, respectively, in each of the samples. The smaller size fraction from the summer sampling showed a higher relative contribution of lower molecular weight compounds, and a factor of more than 100 differed between the PAH found in highest concentration and the high molecular weight PAHs that were found close to the detection limit. The relative contribution of the different PCB congeners in each of the samples was more evenly distributed. The differences between the PCBs occurring in highest and lowest concentrations, respectively, ranged between approximately 3 and 6 for all samples except the smaller size fraction from the autumn sampling where this difference was a factor of approximately 9 due to a slightly higher contribution of the highest molecular weight PCBs. The particulate carbon content in the water, corresponding to the two fractions, was found to be approximately 120 and 180 µg L-1 for the larger size fractions for the summer and autumn sampling occasions, respectively, and approximately 10 and 8 µg L-1 for the bacterial fractions on the same occasions. The results of bacterial counts in the two smaller size fractions, in combination with the carbon analyses, indicated that the carbon content per cell was approximately 5 fg of C cell-1, which is about
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one-fourth of the commonly used literature value of 20 fg of C cell-1 (6). The results obtained show that the PCB and PAH concentrations in the bacterial fraction were in the same order of magnitude as the concentrations in the larger size fraction both on a carbon weight basis and on a volume basis. This clearly illustrates that bacteria can be very significant for the particulate distribution and dynamics of HOCs in the Baltic marine environment. The larger size fractions collected are likely to contain detritus in unknown amounts. It has, for instance, been shown that the detritus contribution to total the POC in nearshore Baltic during both summer and autumn can be as large as 80% (14). The ability of ‘living carbon’ to adsorb HOCs can be expected to be the same as or higher than the ability of detrital carbon to do so. A higher HOC sorption of living carbon (e.g., phytoplankton) can be expected if we assume a greater portion of ‘high quality’ carbon, such as lipids, in the living carbon than the detrital carbon (see further discussion below). Additionally, protists in the larger size fraction can take up HOCs actively through the ingestion of particles, which can give them a relatively larger HOC content than what would be the result of only surface adsorption. Also the smaller size fractions can be expected to contain detritus in unknown amounts, but since the carbon content of the bacterial cells was determined to only 5 fg of C cell-1, it is not likely that a detrital portion of the smaller size fraction is of any greater significance since the carbon content in this fraction is not high enough to make up for a detrital fraction as well. This reasoning is of course valid only on the condition that detritus particles were not counted as bacteria when using the epiflourescent microscopy technique where only sharp-edged spherical or rod-like particles were counted. In any case, the HOC adsorption ability of bacterial carbon should be the same as or greater than that of detritus for the same reasons as mentioned above for the larger size fraction. In partly the same size fraction as bacteria, Isao et al. (15) have found submicrometer particles in numbers far more than bacteria in oceanic waters. These nonliving submicrometer particles (size range 0.38-1 µm) were suggested to be fragile and flexible, to have a high water content, and to consist largely of organic material. In case these submicrometer particles are present also in Baltic waters, they are likely to be found in the smaller size fractions collected. It is uncertain though how large a portion of these submicrometer particles are collected in our smaller size faction since it was shown by Isao et al. (15) that almost half of the total submicrometer particles passed through a 0.1-µm Nuclepore filter due to their assumed flexible character. With the tangential cross-flow technique used in the present study for collection of the smaller size fractions, particles smaller than 0.2 µm are convected through the ceramic membranes under an applied pressure, and particles larger than the membrane pores are consequently concentrated during the total time of filtration (approximately 2-4 h) within the filtration system. It seems possible that during this process an even larger amount of the flexible submicrometer particles should pass through the ceramic membranes than through the Nuclepore filters due to both a larger pore size and a longer filtration time of the tangential filtration technique. The adsorption ability for HOCs of the nonliving submicrometer particles is unknown, but it seems likely that the organic material in these presumed high water content particles
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should not posses any higher ability for HOC sorption than the organic material-rich (16) bacteria. Hence, the HOC concentration of the bacteria collected in the smaller size fraction ought not in any case be lower than what is reported on in the present study. If we assume a lower carbon content of the submicrometer than bacteria, and if these particles are present in the smaller size fractions and presumably also counted concomitantly as bacteria, the carbon content of the individual bacteria cells is underestimated. It has recently been shown that a large fraction of traditional total counts of bacteria consists of non-nucleoidcontaining “ghosts” (17). The implication of this is that bacterial cell residues with the same shape and size as bacteria are included in both the counting of bacteria and the content of the smaller size fraction. It can only be speculated on what the HOC content of these nonactive bacterial ghosts can be compared with active bacteria, but it seems reasonable to assume that it should be the same or somewhat lower, which is an indication that the suggested HOC concentrations of the active bacteria in the smaller size fractions are not in any case overestimated. Zweifel and Hagstro¨m (17) concluded, however, that their results do not change the common concept of carbon transfer in the microbial food web merely that the production and uptake rates per cell may be substantially higher than previously estimated. Elements of inorganic nature as possible contributors to the total matter content in both size fractions probably do not have any importance for the HOC concentrations (as given on a carbon weight basis) since the HOC adsorption ability for inorganic matter is much less than that of organic matter (i.e., organic carbon) (18). Free-living bacteria have no settling velocity, and studies have shown that bacterivore grazing (by mainly nanoflagellates) is generally the most significant fate of the bacterial production (19, 20). The turnover times within the microheterotrophic food web can be very fast. For example, Fenchel (21) estimated that, in temperate coastal waters during the summer, flagellates filter between 10 and >100% of the water column for bacterial cells/day. Nanoflagellates are in turn preyed upon by larger protists and zooplankton. For phytoplankton (collected in the 2-90-µm size fraction), on the other hand, a significant proportion can sink out rapidly from the trophogenic layer especially during periods of bloom, making a significant proportion of the associated HOCs unavailable for direct pelagic food transfer. Also, apart from in acute seasonal periods of high phytoplankton production, the standing stock of bacteria remains relatively even in comparison to phytoplankton. This might, at least seasonally, lead to a relatively larger proportion of the HOCs found in the bacterial fraction being transferred into the marine food chains (via the microheterotrophic food web) than those HOCs found in phytoplankton in the larger size fraction. During seasons of low autotrophic production, the bacterial generation times becomes longer, which in turn makes the time for equilibrium between HOCs and the carbon in bacteria concomitantly longer. This would, in turn, lead to an increase in the concentrations of HOCs in bacteria in terms of total carbon. The results obtained in the present study can be interpreted as a support to this idea since the highest concentrations of PCBs and PAHs were found in the bacterial fraction during autumn (Figure 1). It can be added that the number of bacteria present in
the seawater that was filtered during the two occasions was approximately the same (5-6 × 106 cells mL-1 of which approximately 0.1% consisted of cyanobacteria). Notably, however, the differences in HOC concentrations in the bacterial fractions were not as great as the concentration differences found for the larger size fractions. This can probably be interpreted as an effect of the generally higher heterogeneity of the larger particulate fraction. Furthermore, the HOC concentrations in the larger size fraction were much higher for the summer period than for the autumn period, which can be a result of the fact that during this season the carbon in this size fraction has a large lipid component (22, 23) which, in turn, has a high absorption efficiency for HOCs (23). The particulate material in the larger size fraction during the autumn sampling probably also contains a greater fraction of older resuspended material with a poorer absorption capacity. In summary, it can be concluded that the carbon represented by pelagic bacteria constitutes a significant part of the living, particulate carbon available for HOC sorption in the pelagic system, and this is shown by a similar magnitude of both PCB and PAH concentrations in the bacterial and larger size fractions analyzed here. Furthermore, as also indicated in the present study, the significance of the bacterial fraction for the dynamics of HOCs is of even greater importance during periods of low phytoplankton production.
Acknowledgments
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The authors would like to thank Bertil Thulin and Kåre Larsson at Millipore, Sweden, for assistance in the development and construction of the filtration setup unit. Comments and suggestions from anonymous reviewers helped improve this manuscript. This study was financially supported by the Swedish Environmental Protection Agency.
Received for review June 27, 1995. Revised manuscript received December 11, 1995. Accepted December 11, 1995.X
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Abstract published in Advance ACS Abstracts, February 15, 1996.
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