ISSN 00963925, Moscow University Biological Sciences Bulletin, 2013, Vol. 68, No. 2, pp. 75–82. © Allerton Press, Inc., 2013. Original Russian Text © I.V. Mosharova, V.V. Il’inskii, S.A. Mosharov, A.I. Azovskii, 2013, published in Vestnik Moskovskogo Universiteta. Biologiya, 2013, No. 1, pp. 27–34.

ECOLOGY

Distribution of Polychlorinated BiphenylsTransforming and Polychlorinated BiphenylsTolerant Bacteria in the Seas of the Temperate and Polar Latitudes with Different Levels of Polychlorinated Biphenyls I. V. Mosharovaa, V. V. Il’inskiia, S. A. Mosharovb, and A. I. Azovskiia a

b

Faculty of Biology, Moscow State University, Moscow, 119931 Russia Shirshov Institute of Oceanology, Nakhimovskii pr. 36, Moscow, 117218 Russia email: [email protected], [email protected] Received August 20, 2010

Abstract—The presence of PCBTRB and PCBTOB in the Baltic, Bering, and Chukchi seas was deter mined. It was found that most of these physiological groups of bacteria reach a chronically contaminated PCB marine ecosystem of the Baltic Sea. It was revealed that the number of PCBTRB and TCBTOB mainly depends on the concentration of PCBs in the suspended matter. The dependence manifests itself against the background of the impact of other abiotic environmental factors—temperature and salinity, as well as the content of various forms of phosphorus and nitrogen. Keywords: polychlorinated biphenylstransforming marine bacteria, polychlorinated biphenyls, tolerant marine bacteria. DOI: 10.3103/S0096392513020065

Contamination with chlorinated hydrocarbons is second after oil contamination with respect to the extent of danger for marine ecosystems [1]. Polychlo rinated biphenyls (PCBs) belong to xenobiotics, i.e., substances alien for the environment and enter the “dirty dozen” according to the UNEP classification [2]. These substances are weakly subjected to abiotic decomposition, and the main process of their transfor mation in the environment is microbial transforma tion that in many cases leads only to an incomplete oxidation and partial change of the structure of mole cules of PCB [3]. The possibility of development at contamination of their habitat with PCB exists for het erotrophic microorganisms of two groups: bacteria capable of transforming PCB (PCBtransforming bacteria or PCBTRB) and bacteria incapable of transforming PCB but tolerant to their presence (PCBtolerant bacteria or PCBTOB). The study of marine PCBTRB and PCBTOB began comparatively recently [4, 5], and the informa tion on the impact of environmental factors on the number of bacteria of these groups in marine ecosys tems is almost lacking altogether. The main task of our work was to study specific fea tures of the quantitative distribution of PCBTRB, as well as PCBTOB and saprotrophic bacteria (SB), in contaminated, weakly contaminated, and uncontam inated with PCB seas of temperate, subarctic, and arc tic latitudes, as well as to assess the impact of hydro

logical and hydrochemical factors of marine environ ment on the number of bacteria of these groups. MATERIAL This study is based on the data of integrated ecolog ical expeditions obtained in 2001–2004 in the Baltic and Chukot seas, as well as in Anadyr Bay of the Ber ing Sea. We used Niskin plastic bathometers sterilized with alcohol to take water samples from the surface (0.5 m) and nearbottom horizons, as well as from the ther mocline layer. To obtain temperature and salinity pro files at each station, CTDprobes (Sea Bird, United States) were used. The numbers of bacteria of different physiological groups were assessed using method of limiting dilu tions in liquid nutrient media. To assess the numbers of PCBTRB bacteria, we used marine mineral medium (MMM) [6, 7] of the following composition: 5.0 g NaCl, 1.0 g MgSO4 · 7H2O, 0.7 g Kcl, 2.0 g K2HPO4, 3.0 g Na2HPO4, 1.0 g NH4NO3 and 1.0 L distilled water. As a single source of carbon and energy, one to two drops of mother solution containing PCB (Chlophen A60) were introduced into each tube after inoculation. To obtain mother solution of PCB, a sample of 1 mg of substrate was dissolved in 1 mL of acetone, and the solution was introduced into 100 mL of MMM sterile medium. Mother solution was steril 75

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ized by the method of tyndallization, i.e., by fractional boiling. To determine the numbers of PCBTOB, one to two drops of sterile 10% solution of glucose, parallel to PCB, were introduced into tubes with MMM medium [8]. To assess the numbers of saprotrophic bacteria, diluted sterile fish broth prepared on marine water from the site of sampling was used [8]. To determine the concentrations of PCB accumu lated in the suspended matter (suspended PCB), marine water samples were filtered through Dubna preliminarily weighed membrane filters (diameter of pores 0.45 μm). After drying filters with the suspended matter, they were weighed again and, according to the difference in weights and the volume of filtered water, we determined the concentration of the suspended matter. During the subsequent analysis of filters with the suspension, using method of higheffective gas liquid chromatography (HELC), the concentration of PCB in the suspended matter was determined, and the concentration of PCB suspended in water was calcu lated with regard to the volume of filtered water. To assess the combined effect on microbiological parameters of simultaneously several abiotic factors, the method of multiple stepbystep linear regression was used. To reduce the number of interacting factors, the method of principal components was used. All coefficients presented in the paper are significant at the level p ≤ 0.01. Statistical analyses were performed on personal computer using Statistica 6.0 and Excel 2002 software packages. RESULTS Microbiological Situation in the Open Part of the Baltic Sea in Autumn 2001 Since 1966, PCBs are stably found in all compo nents of the Baltic marine system [9]. Despite the fact that after the ban in 1977 on discharge of PCBs into the marine environment, a decrease in the concentra tion of these compounds in the surface water layer of the Baltic Sea is observed (from 4.8–6.1 to 0.05– 2.5 ng/L), PCBs continue to remain in it as rather widespread pollutants [10]. According to the data of the expedition performed in autumn 2001, it was established that heterotrophic bacteria of all three groups studied by us—PCBTRB, PCBTOB, and SB—are widespread in the open part of the Baltic Sea (Fig. 1). The average numbers of PCBTRB for the open part of the Baltic Sea were 590 cells/mL at variation from <10 cells/mL (at several stations) to 7500 cells/mL (at station BY1 in the Arkon basin of the sea). The numbers of PCBTOB in the same part of the Baltic Sea varied from <10 cells/mL to 25000 cells/mL (at an average value of 2360 cells/mL). Maximum numbers of bacteria of this group, as well as the highest abundance of PCBTRB, are observed in

the Arkon depression (the numbers of PCBTOB average for this area comprised 2800 cells/mL) (Fig. 1). The numbers of SB varied from less than 10 cells/mL to 250000 cells/mL (on average, 13200 cells/mL). Maximum abundance of bacteria of this group was revealed also in the area of the Arkon basin (Fig. 1). Maximum values of the numbers of bacteria of all three physiological groups (PCBTRB, PCBTOB, and SB) were established for the surface (0.5 m) layer of the water column (on average, for the section 1.57; 4900 and 29390 cells/mL (respectively), minimal, for the thermocline layer (on average 0.5; 2040 and 9400 cells/mL, respectively). In the nearbottom layer of the water column, the numbers of bacteria of all three studied groups again increased (on average 0.63; 3490 and 19800 cells/mL, respectively). Using pair correlation analysis for this horizon, we found close direct correlations between the amounts of PCBTRB and SB (R = 0.72), PCBTOB and SB (R = 0.69), as well as between PCBTRB and PCB TOB (R = 0.76). The correlations found between the numbers of bacteria of these two groups and SB allow us to suggest that PCBTRB and PCBTOB are a part of saprotrophic bacteriocenosis. Maximum values of the numbers of PCBTRB, as well as of PCBTOB and SB, were revealed in the area of the Arkon depres sion, against the background of higher water tempera tures, concentration of PCB in suspended matter (SM), and suspended PCB in water, as well as minimal values of salinity and concentrations of total and min eral phosphorus. Mass development of these groups of microorganisms in the composition of heterotrophic bacteriocenosis of the Arkon depression can be a result of the response of the ecosystem of the given area of the sea to its chronic contamination with PCB. The lowest average values of the numbers of PCBTRB, PCBTOB, and SB were found in the area of Western Gotland—they were present against the background of the low concentration of PCB in suspended matter (SM) and suspended PCB in water (Fig. 1). During analysis of pair relations between the microbiological and abiotic parameters, the closest relations of PCBTRB were revealed with concentra tions of PCB and SM (R = 0.81) and the amount of suspended PCB in water (R = 0.57), as well as with concentrations of total and mineral phosphorus (R = 0.62 and R = 0.63, respectively). To assess quantitatively these numerous relations, and, hence, the degree of impact of different abiotic factors on microbiological parameters, we used multi ple regression analysis. The calculations were per formed for all three studied water column horizons: surface horizon (0.5 m), thermocline layer, and near bottom horizon. A total of 11 abiotic factors were sub jected to regression analysis: SM content in water (mg/L), concentration of PCB and SM (ng/mg), con tent of suspended PCB in water (suspended PCB,

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N SB, 6000 cells/mL 60° PCBTOB, 1000 cells/mL PCBTRB, 300 cells/mL BY 29 BY 31

BY 21

5 58° BY 20

6

BY 34 BY 15 BY 38

4

BY 10

56°

BY 9

BY 1

BY 3 BY 2

BY 4 BY 5

3

2

1 54°

12°

16°

E

20°

Fig. 1. Distribution of saprotrophic, polychlorinated biphenylstolerant, and polychlorinated biphenylstransforming bacteria in the open part of the Baltic Sea in summer 2001. Sea areas: (1) Arkon depression, (2) water area of Bornholm Island, (3) eastern area of the sea, (4) Eastern Gotland, and (5) Western Gotland.

ng/L), water temperature (t, °C), salinity (‰), water saturation with oxygen (O2, %), concentrations of mineral phosphorus (PO4, μgatoms/L), organic phosphorus (μgatoms, P/L), total phosphorus (μg atoms, P/L), nitrites (NO2μg/L), and ammonium +

The numbers of PCBTRB in the surface layer of the water column are also affected by the concentra tion of total phosphorus in marine water; this effect depended on the combination of factors, such as salin ity and temperature (R2 = 0.7):

( NH 4 , mg/L).

Y = 1.57 + 1.31PCA3 + 0.SPCA4 · (1 + 0.SPCA2),(1)

As a result of analysis, it was established that the numbers of PCBTRB in the surface and nearbottom layers of the water column, as well as in the ther mocline layer in the Baltic Sea, in the autumn period of 2001 were significantly related to the amount of SM, concentrations of PCB and SM, and the amount of PCB suspended in water.

where PCA2 is salinity and temperature; PCA3 is con tent of SM in water, concentrations of PCB and SM, and the content of suspended PCB in water; PCA4 is mineral and total phosphorus. The numbers of PCBTRB in the nearbottom water layer were significantly related to the concentra tions of PCB and SM and the content of suspended

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MOSHAROVA et al. N SB, 30000 cells/mL 60°

PCBTRB, 140 cells/mL 6 BY 29 BY 21

5

58°

BY 20 BY 34 BY 15 BY 38 BY 10

4 56°

BY 9 BY 8 BY 4 BY 3 BY 5 BY 1 BY 2

BY 7

3

2

1 54°

12°

16°

20°

E

Fig. 2. Distribution of saprotrophic, polychlorinated biphenylstolerant, and polychlorinated biphenylstransforming bacteria in the open part of the Baltic Sea in autumn 2004.

PCB in water (PCAS5), as well as to salinity and tem perature (R2 = 0.8): Y = 0.5 + 0.6PCA5 + 0.4PCA2. (2) Microbiological Situation in the Open Part of the Baltic Sea in Summer 2004 Repeated microbiological studies of the open part of the Baltic were performed in summer 2004, the observations comprised the determination of the num bers of bacteria of two groups—PCBTRB and SB. The latter group of bacteria dominated at all stations. Its numbers varied from 0.25 to 600000 cells/mL and averaged 13 200 cells/mL. In autumn 2001, the num bers of SB were almost three times lower and averaged

13 200 cells/mL. In summer 2004, SB reached maxi mum quantitative development in the areas of South eastern Baltic and Eastern Gotland, whereas maxi mum numbers of SB were observed in water areas of Eastern Gotland and Arkon depression in autumn 2001. According to the distribution over the horizons of the water column, the highest average value of the numbers of SB were found for the nearbottom hori zon—113 400 cells/mL at a variation of 2500 to 600000 cells/mL, whereas maximum values of the numbers of SB were typical for the surface horizon in 2001 (0.5 m). In the surface and thermocline layers, the numbers of SB were approximately equal and aver aged 26 600 and 26 300 cells/mL, respectively.

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N Chukotka Peninsula

SB, 3000 cells/mL PCBTOB, 1500 cells/mL PCBTRB, 200 cells/mL

66°

46 43

48 52

64°

55 60

42 2

58

St. Lawrence Island 1 62° BERING 176°

180°

SEA 176°

172°

168°

E

Fig. 3. Distribution of saprotrophic, polychlorinated biphenylstolerant, and polychlorinated biphenylstransforming bacteria in Anadyr Bay of the Bering Sea.

The highest average value of the numbers of PCB TRB for all three studied groups in this period, unlike autumn 2001, was discovered in the nearbottom layer of the water column. It comprised 1200 cells/mL, the numbers of PCBTRB varied from <10 cells/mL to 2500 cells/mL. The lowest average value of the num bers of PCBTRB in summer 2004 was found in the surface water layer (0.5 m)—90 cells/mL; the range of fluctuations of amounts of PCBTRB in this layer was from <10 cells/mL to 250 cells/mL. The spatial distribution of PCBTRB over the areas of the Baltic Sea in summer 2004, as well as in autumn 2001, was nonuniform (Fig. 2). Maximum amounts of bacteria of this group were found in Eastern Gotland; their average value for this area was 510 cells/mL. The high value of this index was recorded also for the area of Bornholm Island: 480 cells/mL. The lowest average value of the numbers of PCBTRB was found for the area of Western Gotland: 60 cells/mL (Fig. 2). In 2001, maximum numbers of PCBTRB were found in the area of Arkon depression, and the lowest values of this parameters were found also in the area of Western Gotland. Thus, both horizontal and vertical distribu

tion of PCBTRB in 2004 differed from the distribu tion of these bacteria in 2001. In 2004, significant pair correlations between the numbers of PCBTRB and SB in the thermocline layer (R = 0.59), as well as between PCBTRB and SB and hydrologohydrochemical parameters, were found again. As in autumn 2001, in summer 2004, the numbers of PCBTRB in the surface layer of the water column (0.5 m) significantly depended on the concen trations of PCB and SB (R = 079), as well as on the concentrations of mineral (R = 0.530 and total phos phorus (R = 0.55). In addition, in summer 2004, the relationship between the numbers of PCBTRB and the concentrations of nitrites (R = 0.69) and ammo nium nitrogen (R = 0.75) was found. At the same time, no significant relations of the numbers of PCBTRB to water temperature and salinity in 2004, unlike 2001, were found. The assessment of the combined effect of abiotic factors on the numbers of PCBTRB, PCB TOB, and SB, using the method of multiple step linear regression, demonstrated that in summer 2004, as well as in autumn 2001, the numbers of PCBTRB in the surface layer (0.5 m) of the Baltic Sea in the first turn significantly depended on the concentrations of PCB

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N

20

19

Wrangel Island 16

22 70° 24

29

30

15

25 34

26 68°

35 SB 3000 cells/mL

37

13 11

38

39

PCBTOB, 200 cells/mL PCBTRB, 100 cells/mL

66° 176°

180°

8 Chukotka Peninsula 176°

172°

168°

W

Fig. 4. Distribution of saprotrophic, polychlorinated biphenylstolerant, and polychlorinated biphenylstransforming bacteria in the Chukot Sea.

and BB. This dependence manifested itself against the background of the combined effect of the concentra tions in water of total phosphorus and ammonium nitrogen (R2 = 0.8): Y = 0.09 + 0.05(1.6PSA5 + PSA1), where PSA1 is total phosphorus, mineral phosphorus, and ammonium nitrogen and PCA5 is the concentra tion of PCB and SM. Thus, at stations with high numbers of PCBTRB, higher concentrations of PCB and BB, as compared to the remaining stations, were recorded. They were also characterized by increased concentrations of total phosphorus and ammonium nitrogen. On the basis of equations we obtained, we can state that the numbers of PCBTRB in waters of the Baltic Sea in the summer–autumn seasons are significantly

affected not only by the concentrations of PCB in water and the content of PCB and BB but also by other hydrologicohydrochemical factors (in particular, concentrations of total and mineral phosphorus, nitrite and ammonium nitrogen, temperature, water salinity), against the background of which the impact of PCB is exhibited. Microbiological Situation in Anadyr Bay of the Bering Sea According to published data, Anadyr Bay of the Bering Sea belongs to weakly contaminated marine ecosystems [1, 11]. We established that PCBTRB, PCBTOB, and SB are also widespread in Anadyr Bay of the Bering Sea (Fig. 3). The average numbers of PCBTRB for the bay were 540 cells/mL at a range of

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fluctuations from <10 cells/mL to 6000 cells/mL. The highest average for the water column (from surface to bottom) values of the numbers of PCBTRB were found for stn. 52 (1520 cells/mL) and stn. 55 (1070 cells/mL) exposed to the effect of the discharge of the Anadyr River, and minimal was 60 cells/mL, for stn. 1 located in the most out at sea part of the bay (Fig. 3). PCBTOB were found at all stations of Anadyr Bay. Their numbers varied from 25 cells/mL to 25000 cells/mL (on average, 1440 cells/mL). The highest for the water column values of the numbers of PCBTOB were found at stn. 55 (9860 cells/mL) and stn. 52 (3440 cells/mL) located in the zone of the impact of the Anadyr River discharge (Fig. 3). The numbers of SB varied from <10 cells/mL to 25000 cells/mL at an average value 4920 cells/mL. The highest average for the water column values of the numbers of SB were found at stn. 60 (18300 cells/mL) and for stn. 48 (10 400 cells/mL), and the minimal high was found at stns. 1 and 2 in the outatsea part of the bay (730 and 330 cells/mL, respectively). A direct correlation (R = 0.71) was found between the numbers of SB in the surface layer 0.5 m and the concentration of suspended PCB in water. In the nearbottom layer, a direct correlation was found between the numbers of SB and the concentration of PCB and SM (R = 0.60). Both for the surface (0.5 m) and the nearbottom horizons, correlation relations between the numbers of PCBTRB and PCBTOB (R = 0.53 and R = 0.62, respectively) and between concentrations of SM and the numbers of PCBTRB (R = 0.57 and R = 0.61, respectively) were revealed. Statistically significant dependencies of the numbers of PCBTRB on abiotic environmental factors were found in neither studied horizon in Anadyr Bay of the Bering Sea. For the numbers of PCBTOB, close correlation relations with the concentrations of SM and water temperature were found (R = 0.68 and R = 0.83, respectively). In the nearbottom layer, significant relation of the numbers of PCBTOB was found only with SM concentrations (R = 0.93). Microbiological Situation in the Chukot Sea in Autumn 2002 The Chukot Sea belongs to unpolluted marine eco systems [1, 11]. In the Chukot Sea, PCBTRB turned out to be the most scarce, they were found only in sin gle cases, and their numbers varied from <10 kl/mL to 2.5 thousand kl/mL (on the average, for the studied area of the sea–150 kl/mL). Maximum numbers of PCBTRB were found in the nearbottom layer of the Chukot Sea. The numbers of PCBTOB varied from < 10 kl/mL to 25 thousand kl.ml. The numbers of PCBTOB equal to 25 thousand kl/mL were found only in one sample at stn. 39 (horizon 25 m). Maximum numbers

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of PCBTOB were confined to the nearbottom layer. On the average, for this layer they comprised 1.84 thousand kl/mL. Maximum average for the water column value of the numbers of PCBTOB was found for stn. 24— 6580 cells/mL (Fig. 4); comparatively high value of this index was established also for stn. 26—1490 cells/mL. The numbers of SB in the Chukot Sea in the autumn period of 2002 varied from <10 cells/mL to 25000 cells/mL at an average for the section value 4100 cells/mL. Maximum averages for the water col umn values of SB were found for stn. 24 (16860 cells/mL) and for stn. 34 (13 810 cells/mL) (both stations are located in the zone exposed to the effect of the river drainage from the coast (Fig. 4)). In the surface (0.5 m) and nearbottom horizons, pair correlations were revealed between the numbers of PCBTRB and PCBTOB (R = 0.78, R = 0.66, respectively). Statistically significant dependencies of the numbers of PCBTRB, PCBTOB, and SB on abiotic environmental factors were found for none of the studied horizons in the Chukot Sea. Published data available towards the beginning of the study [4, 5, 10] allowed us to suggest that PCB TRB and PCBTOB are not an independent group of heterotrophic bacteria but represent a part of saprotrophic bacterioplankton possessing abilities to transform PCB. Under conditions of the chronic pres ence of increased concentrations of PCB in marine ecosystems, these groups of bacteria possibly obtain advantages and, respectively, their numbers increase. As a result, the numbers of PCB, TRB, and PCBTOB in such seas can depend on PCB concentrations whose action will manifest itself against the background of other abiotic environmental factors. Our studies in the Baltic Sea as a whole confirmed these suggestions. The studies performed in less polluted with PCB arctic seas (Bering and Chukot) also demonstrated the presence in them of physiologically active bacteri oplankton. In the composition of heterotrophic bacte riocenoses of these seas, both PCBTRB and PCB TOB are present; however, their numbers here are small and are evidently within the natural background. CONCLUSIONS PCBTRB and PCBTOB bacteria are present in the composition of heterotrophic bacterioplanton of marine ecosystems of different latitudes (from tem perate to polar) despite different levels of their pollu tion with PCB. PCBTRB and PCBTOB reach maximum devel opment in the chronically contaminated with PCB ecosystem—the Baltic Sea. Here, numbers of PCB TRB and PCBTOB depend on the concentrations of PCB in the suspended matter; the relation to this fac tor manifests itself against the background of the effect on the numbers of bacteria of other abiotic environ

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mental parameters—salinity and water tempera ture—and concentrations of phosphorus and ammo nium nitrogen. The study was supported by the Ministry of Educa tion and Science of the Russian Federation. REFERENCES 1. AMAP. Arctic Pollution: Persistent Organic Pollutants, Heavy Metals, Radioactivity, Human Health, Chang ing Pathways. Arctic Monitoring and Assessment Pro gramme (AMAP), Oslo, 2002. 2. UNEP Dioxin and Furan Inventories (National and Regional Emissions of PCDD/PCDF), Genewa, 1999. 3. Abramowicz, D.A., Aerobic and Anaerobic PCB Bio degradation in the Environment, Environ. Health Per spect., 2005, vol. 103, suppl. 5, pp. 97–99. 4. Colwell, R., Walker, J., and Nelson, J., Microbial Ecol ogy and the Microbial Degradation of Oil Pollutants, in Microbiol. Degradation of Oil Pollutants. Workshop Held at Georgia State Univ., Atlanta, 1973, p. 322. 5. Luigi, M., Di, M.G., Bruni, V.L., and Guidice, An., Biodegradative Potential and Characterization of Psy chrotolerant Polychlorinated Biphenyl Degrading Marine Bacteria Isolated from a Coastal Station in the Terra Nova Bay (Ross Sea, Antarctica), Mar. Poll. Bull., 2007, vol. 54, pp. 1754–1761. 6. Coronelli, T.V. and Il’inskii, V.V, Determination of the Number of HydrocarbonOxidizing Bacteria in the Sea

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