ISSN 10630740, Russian Journal of Marine Biology, 2012, Vol. 38, No. 1, pp. 96–99. © Pleiades Publishing, Ltd., 2012. Original Russian Text © I.V. Ryzhik, 2012, published in Biologiya Morya.
ALGOLOGY
The Metabolic Activity of Cells of Fucus vesiculosus Linnaeus, 1753 (Phaeophyta: Fucales) from the Barents Sea under Conditions of Oil Pollution I. V. Ryzhik Murmansk Marine Biological Institute, Kola Scientific Center, Russian Academy of Sciences, 183010 Murmansk email:
[email protected] Received October 6, 2011
Abstract—The metabolic activity of cells of brown seaweed was studied. The metabolic activity of cells of F. vesiculosus increased in an oilpolluted environment. The period of formation of the response to the addi tion of a toxicant depends on conditions in the algal habitat. Metabolic activity increased during several min utes in samples that were taken from a clean area and on the third day of exposure in samples from an oil polluted area. The level of metabolic activity of cells of F. vesiculosus can be used as an indicator of the pres ence of stress factors in the algal habitat. Keywords: metabolic activity, Fucus vesiculosus, oil pollution. DOI: 10.1134/S1063074012010117
reparative or adaptation processes [4, 10] and the met abolic activity of cells of the plants increases. One of the methods for assessing the metabolic activity of cells is determination of the activity of dehydrogena ses, which significantly increases under stress [16, 20]. The purpose of this work was to assess the meta bolic activity of cells of Fucus vesiculosus from areas with different levels of oil pollution, as well as to esti mate changes in the metabolic activity of cells during the introduction of diesel fuel under experimental ambient conditions.
Oil pollution of waters in seas and oceans has become a global environmental problem. The planned development of oil and gas condensate fields in the Arctic region, urban infrastructure development and, as a consequence, an increase in the anthropogenic load can have a negative impact on the environmental situation in the coastal waters of the Barents Sea as well. Oil and petroleum products are nonspecific toxi cants that negatively affect the marine biota at differ ent levels of its organization, from the subcellular to the population level. Under high concentrations of oil and petroleum products in the water, the intensities of physiological processes of macrophytes (photosynthe sis and growth rate) decrease and their lifespan is reduced, inhibiting the development of spores and early ontogenetic stages and reducing species diversity and projective covering [2, 5, 8, 11–14, 18, 19]. For example, it has been shown that young plants die from the direct impacts of oil products, while the biomass of older algae may decline because of the loss of herbivo rous invertebrates and the mass development of epi phytic algae [21], as was observed in 1989 during the oil spill from the tanker Exxon Valdez. Macrophytic algae that inhabit the intertidal zone are among the first that are exposed to petroleum products; they become actively involved in the process of purification of coastal waters, absorbing the oil film and probably utilizing the products of its decomposi tion [1, 3]. In the presence of petroleum products or other toxicants in the water, the intensity of respiration in algae increases; this is related to the activation of
MATERIALS AND METHODS The study was conducted in July–August 2008 at the Dalnezelenetskaya Seasonal Biological Station of the Murmansk Marine Biological Institute (Kola Sci entific Center, RAS). Fucus vesiculosus plants (four to five dichotomous branches of the same age) were col lected in the Zelenetskaya and Yarnyshnaya bays at intertidal sites with similar hydrodynamic conditions. The concentrations of petroleum products in the water were determined in the chemical laboratory of the Murmansk Rosgidrometeocenter using a Fljuorat 02 3M fluorimeter (Lumex, Russia). Previously, the algae were acclimated over a 5day period to laboratory conditions (temperature of 8°C, constant illumination, PAR intensity of 100 W/m2). After the acclimation, intact F. vesiculosus thalli (with weights of 10–15 g) were placed in 5litre vessels with sea water. In the experiment, diesel fuel was added to the water at a concentration of 0.2 mg/l: this concen 96
Activity, A 570 nm/(g h)
THE METABOLIC ACTIVITY OF CELLS OF Fucus vesiculosus (a)
97
(b)
0.02
0.008
0.016
0.006
0.012
0.004
0.008
0.002
0.004 0
0 Zelenetskaya bay
Yarnyshnaya bay
Zelenetskaya bay
Yarnyshnaya bay
Fig. 1. Metabolic activity of Fucus vesiculosus cells from areas with different levels of pollution. a, natural conditions, b, after acclimation to laboratory conditions.
tration does not restrict the viability of F. vesiculosus [12]. Plants contained in vessels with clean sea water were used as the control. The duration of the experi ments was 5 days. The investigation was conducted in two replicates. The metabolic activity of the cells of the apical part of the thalus was assessed using the tetrazolium method [22] modified for the fucoid algae [9]. The method is based on assaying the intensity of restora tion of tetrazolic compounds by NADdependent dehydrogenases. The statistical processing of the data was performed with Excel software. The Student’s test was used to determine the significance of the differences. The mean arithmetic values and standard deviations are presented in the figures. RESULTS AND DISCUSSION In nature, Fucus vesiculosus cellular metabolic activity increases with an increase in the concentra tion of petroleum products in the water. Thus, plants from a clean area (Yarnyshnaya bay, with a concentra tion of petroleum products of 0.004 mg/l) showed a 4 fold lower metabolic activity of cells than that of plants from a region with considerable pollution (near the pier in Zelenetskaya bay, with a concentration of petroleum products of 0.26 mg/l, which is 5 times over the maximum permissible concentration (MPC)) (Fig. 1a). After acclimation of F. vesiculosus from Zelenetskaya bay to laboratory conditions (clean water), the metabolic activity of cells of the alga decreased and became comparable to the metabolic activity of cells of a sample from the clean area (Fig. 1b). Our experiments showed that the period of forma tion of the F. vesiculosus response to the introduction of diesel fuel into the water of the environment depends on the presence of petroleum products in the original habitat. In seaweeds from the clean area, the metabolic activity of cells increased by 2 times within the first 30 min; the cell activity was then reduced and became comparable to the control value of the cellular RUSSIAN JOURNAL OF MARINE BIOLOGY
Vol. 38
metabolic activity on the second day of the experiment (Fig. 2a). The metabolic activity of the plant cells that were sampled in the oilcontaminated area was, in contrast, similar to the level of cellular metabolic activity in the control for a long period of time and increased only on day 3 (Fig. 2b). Changes in the metabolic activity of cells may be associated with the activation of reparative processes directed at the restoration of the integrity of cellular structures. Earlier, it was shown that the introduction of petroleum products into the environment increases the permeability of cell membranes in Fucus serratus and Laminaria digitata. As well, oil hydrocarbons can damage the membrane complex and structure of the cell matrix [8]. In the process of restoration, the inten sity of respiration increases; this is confirmed by the active reduction of tetrazolic compounds, which occurs not only in mitochondria (the electrontrans port chain), but also in the cytoplasm [15]. It is known that some macroalgae are able to sur vive for a long time in an oilpolluted environment and accumulate (up to a certain level) oil hydrocarbons within their cells without visible damage [6, 7]. Under a prolonged stress exposure (through several genera tions) algae become obviously resistant to oil products, as it has been shown that, despite a decline in growth rate, F. vesiculosus is able to grow under constant and severe water pollution [2, 12, 17]. In our study, the increased cellular metabolic activ ity level in F. vesiculosus samples from the contami nated area under natural conditions and the retarded response of algal cells to the introduction of toxicants in experiments can be explained by the adaptation of the samples to oil pollution. Since the signs of con tamination in Zelenetskaya bay began to appear only in recent years, we assume that the plants growing in the area have not yet formed a genetically fixed adap tation; this is shown by reduction in the cellular meta bolic activity in the absence of stress factors. A rapid increase in cellular metabolic activity in the F. vesicu losus samples from a clean area shows a high rate of reparative processes in the cells upon introduction of the toxicant. No. 1
2012
98
RYZHIK (a)
0.014 0.012
pure water diesel fuel added
0.01
Activity, A 570 nm/(g h)
0.002 0.006 0.004 0.002 0
0.5 h
1h
3h
12 h (b)
0.012
24 h
2 days
3 days
0.010 0.008 0.006 0.004 0.002 0
0.5 h
1.5 h
12 h
24 h 2 days Duration of exposure
3 days
4 days
5 days
Fig. 2. The dynamics of cellular metabolic activity in Fucus vesiculosus from Yarnyshnaya (a) and Zelenetskaya (b) bays under the impact of diesel fuel (A570 nm/g wet weight per hour).
This study has shown that oil pollution of the envi ronment causes an increase in the metabolic activity of cells of F. vesiculosus. Obviously, this is also true for other algal species. Thus, the level of cellular meta bolic activity in algae may serve as an indicator of the presence of stress factors (oil products or other toxi cants) in the area of their habitat. ACKNOWLEDGMENTS The work was supported by Russian Foundation for Basic Research (project no. 100498804 northa). REFERENCES 1. Biologicheskie aspekty zagryazneniya morskoi sredy (Biological Aspects of Contamination of Marine Envi ronment), Kiev: Naukova dumka Publishing House, 1988. 2. Voskoboinikov, G.M., Matishov, G.G., Bykov, O.D., et al., Sustainability of Marine Macrophytes to Oil Pol lution, Doklady RAS, Obshch. Biol., 2004, vol. 397, no. 6, pp. 842–844. 3. Voskoboinikov, G.M., Il’insky, V.V., Lopushanskaya, E.M., and Pugovkin, D.V., On the Possible Role of Marine Macroalgae in Cleaning Water Surface from Oil Pollu tion, Neft i gas Arkticheskogo shelfa2008: Materialy Mezhdunarod. konf. Murmansk, 12–14 noyabrya (Oil and Gas of Arctic Shelf2008: Proceedings of the Interna
4.
5.
6.
7.
8.
9.
10.
tional Conference, Murmansk, 12–14 November), Mur mansk: MMBI KSC RAN, 2008, pp. 63–65. Golovko, T.K., Dykhanie rastenii. Fiziologicheskie aspe kty (Respiration of Plants. Physiological Aspects), St. Petersburg: Nauka, 1999. Klochkova, N.G. and Berezovskaya, V.A., Makrofito bentos Avachinskoi Guby i ego antropogennaya destruktsiya (Macrophytobenthos of Avachinskaya Guba Bay and Its Anthropogenic Destruction), Vladi vostok: Dalnauka, 2001. Mironov, O.G., Vzaimodeistvie morskikh organizmov s neftyanymi uglevodorodami (Interaction of Marine Organisms with Oil Hydrocarbons), Leningrad: Gidrometeoizdat, 1985. Mironov, O.G. and Shchekaturina, T.L., Hydrocarbons in Marine Organisms, Gidrobiol. Zhurnal, 1976, vol. 12, no. 6, pp. 5–14. NelsonSmith, A., Neft i ekologiya morya (Oil and Sea Ecology), Leningrad: Gidrometeoizdat, 1977 (In Rus sian). Ryzhik, I.V., Tetrazolic Method as a Tool for Assess ment of Metabolic Activity of Fucoid Algae, Materialy XXVI konf. molodykh uchenykh MMBI (Materials of XXVI Conference of Young Scientists of MMBI), Apatity: Izdvo KSC RAN, 2008, pp. 124–127. Semikhatova, O.A. and Chirkova, T.V., Fiziologiya dykhaniya rastenii (Physiology of Respiration of Plants), SPb: Izdo SPbGU, 2001.
RUSSIAN JOURNAL OF MARINE BIOLOGY
Vol. 38
No. 1
2012
THE METABOLIC ACTIVITY OF CELLS OF Fucus vesiculosus 11. Stepan’yan O.V., Effect of Oil and Oil Products on the Development of Fucus vesiculosus (Phaeophycea, Fucales), Materialy conf. molodykh uchenikh MMBI, aprel’ 2002 (Materials of the Conference of Young Scien tists MMBI, April 2002), Murmansk: MMBI KSC RAN, 2002, pp. 153–160. 12. Stepan’yan O.V., Morphofunctional Reconstructions in Macrophytic Algae of the Barents Sea under the Effect of Oil and Oil Products, Extended Abstract of Cand. Sci. (Biol.) Dissertation MMBI KSC RAN, Mur mansk, 2003. 13. Stepan’yan, O.V. and Voskoboinikov, G.M., Effect of Oil and Oil Products on Morphofunctional Peculiari ties of Marine Macroalgae, Rus. J. Mar. Biol., 2006, vol. 32, Suppl. 1, pp. S32–S39. 14. Yartseva, I.A., Phyllophora and Oil Contamination, Tez. dokl. 3ego Vsesoyuz. soveshch. po algologii—mak rofitobentosu, Sevastopol, oktyabr’ 1979 (Abstracts of Reports at the 3rd AllUnion Conference on Algology Microphytobenthos, Sevastopol, October 1979), Kiev: Naukova dumka, 1979, pp. 130–131. 15. Apoptosis, Cell Death, and Cell Proliferation Manual, Roche Applied Science, 2004, pp. 1–7. 16. Chang WanChun, Chen MingHwa, and Lee Tse Min. 2.3.5Triphenyltetrasolium Reduction in the Via bility Assay of Ulva fasciata (Chlorophyta) in Response to Salinity Stress, Bot. Bull. Acad. Sin., 1999, vol. 40, pp. 207–212.
RUSSIAN JOURNAL OF MARINE BIOLOGY
Vol. 38
99
17. Coelho, S.M., Jan, W., Rijstenbil, M., and Brown, T., Impacts of Anthropogenic Stresses on the Early Devel opment Stages of Seaweeds, J. Aquat. Ecosyst. Stress Recovery, 2000, vol. 7, pp. 317–333. 18. Driskell, W.B., Ruesink, J.L., Lees, D.C., et al., Long Term Signal of Disturbance: Fucus gardneri After the Exxon Valdez oil spill, Ecol. Appl., 2001, vol. 11, pp. 815–827. 19. Kawai, H., Kamiya, M., Komatsu, T. et al., Ten Years’ Monitoring of Intertidal Macroalgal Vegetation of Hyogo Prefecture, Northwestern Coast of Honshu, Japan to Assess the Impact of the Nakhodka Oil Spill, Algae, 2007, vol. 22, no. 1, pp. 37–44. 20. Lu IFan, Sung MingShiuan, and Lee TseMin, Salin ity Stress and Hydrogen Peroxide Regulation of Anti oxidant Defense System in Ulva fasciata, Mar. Biol., 2006, vol. 150, no. 1, pp. 1–15. 21. Stekoll, M.S. and Deysher, L., Response of the Domi nant Alga Fucus gardneri (Silva) (Phaeophyceae) to the Exxon Valdez Oil Spill and CleanUp, Mar. Pollut. Bull., 2000, vol. 40, no. 11, pp. 1028–1041. 22. Vistica, D., Skehan, Ph., Scudiero, D. et al., Tetrazo liumBased Assays for Cellular Viability: a Critical Examination of Selected Parameters Affecting Forma zan Production, Cancer Res., 1991, vol. 51, pp. 2515– 2520.
No. 1
2012
