International Journal of Salt Lake Research 8: 245–251, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.

Lake Shira, a Siberian salt lake: ecosystem structure and function. 3. The use of bioluminescent biotests to monitor ecological status N. KUDRYASHEVA, E. SHILOVA, E. KHENDOGINA and V. KRATASYUK Institute of Biophysics, Krasnoyarsk, 660036, Russia

Abstract. In vivo and in vitro bacterial bioluminescent test-systems were used to monitor the ecological status of Lake Shira (Khakassia, Russia) near a health resort. Three bacterial bioluminescent test-systems were used: (1) bioluminescent bacteria, (2) a water soluble coupled enzyme system [(NADH:FMN)-oxidoreductase-luciferase] and (3) an immobilized coupled enzyme system in a starch gel. The value of the in vivo bacterial bioluminescent test-system was established. The difference in the sensitivities of the in vivo- and in vitro-systems to organic compounds could form the basis of a technique for monitoring organic pollution in water of different salinity. The sensitivity of the in vitro enzymic system may be increased. Key words: bacterial bioluminescent bioassays, Lake Shira, Russia, Khakassia

Introduction Lake Shira is a salt lake in South Siberia, Khakassia Republic. It is about 9.5 km long and 5 km wide. Maximal depth is 22 m. The Son River contributes 80 per cent of annual inflows; 9 per cent of annual supply is from anthropogenic sources (Kuskovskii and Krivosheev, 1989). The physicochemical features, fauna, and algal flora of the lake are described elsewhere (Zotina et al., 1999). A map of Lake Shira and a map showing where Lake Shira is located in Russia are also provided by Zotina et al. (1999, this issue). Currently, Lake Shira has become a popular health resort of southern Siberia. As a result, its ecological state is a significant issue and needs to be monitored. An important element of such monitoring is provided by biological investigations. Amongst these, bioassays are commonly used for ecological monitoring since toxic substances affect the functions of test organisms. One advantage of bioassays is that they integrate toxic effects and can be used as preliminary tests in place of a large number of chemical analyses. Microorganisms are now widely used for ecological monitoring in bioassays e.g.

246 Bulish and Isenberg, 1981; Stom et al., 1992; Drzyzga et al., 1995; Guzzella et al., 1995; Cheung et al., 1996; Natecz-Jawecki et al., 1997; Wood and Gruber, 1996). Bioluminescent biotesting, as one form in which microorganisms are used, involves integral toxicity assays (e.g., Kratasyuk and Gitelson, 1986; Kratasyuk, 1990; Kudryasheva et al., 1997, 1998). Its main principle is a correlation between the toxicity of samples and changes in the kinetic parameters of bioluminescent reactions. Advantages of bioluminescent assays are high speed (10–60 sec.), sensitivity (up to 0.1 nM), analytical accuracy (the error is no more 10–15 per cent), a wide range in linear response, and the absence of reagents injurious to human health (e.g. radioisotopes). The luminescent bacteria test was first described in its current form using preserved luminescent bacteria as long ago as 1969 (Grabert and Kossler, 1986). In the late 1980s, the test was standardized in Germany as a method for detecting pollution. It was subsequently modified by others (e.g. Bulish and Isenberg, 1981; Stom et al., 1992). Applications of the method follow two directions: one involving bioluminescent bacteria (in vivo), and one involving bioluminescent enzyme systems (in vitro). Reagents are used which are based on the test organisms (e.g. lyophilized luminous bacteria) (Kuznetsov et al., 1996) or on enzyme systems (e.g. bacterial luciferase systems) (Tyulkova, 1990). The aim of the present work was to investigate the use of in vivo and in vitro bacterial bioluminescent biotests to monitor the ecological status of Lake Shira. Materials and methods Three bacterial bioluminescent test-systems were used: (1) bioluminescent bacteria, (2) a water soluble, coupled enzyme system (NADH:FMN)-oxidoreductase-luciferase, and (3) the coupled enzyme system of (2) immobilized into a starch gel. Lyophilized preparations of luciferase from Photobacterium leiognathi, and NADH:FMN-oxidoreductase from Photobacterium fisheri were supplied by the Biotechnology section of the Institute of Biophysics (Krasnoyarsk, Russia). The reaction mixtures of the test-systems have been described by Kuznetsov et al. (1996) and Kudryasheva et al. (1998). Test-system (1) was shown to be more sensitive to organic oxidants (quinones, for example) and earth metals; test-system (2) appeared to be more sensitive to phenols and bare metals; and the test-system (3) was sensitive to d-metals (Kudryasheva et al., 1996). Three or four replicates per sample were used in bioluminescent measurements.

247

Figure 1. Lake Shira showing sampling sites.

Bacterial Indices (BI) for system (1) and Luciferase Indices (LI) for systems (2) and (3) were evaluated to monitor water quality using the bacterial test-system and the coupled enzyme ones, respectively. BI (LI) = I/Icontr 100 (per cent), where I is the bioluminescent light intensity in the sample under study, and Icontr is the bioluminescent light intensity of the standard (distilled) water sample in systems (1), (2) or (3). Measurements were undertaken using a BLM 8801 Bioluminometer (Special Design Bureau “Nauka”, Krasnoyarsk). Data were analyzed using the least squares method. Experimental error did not exceed 15 per cent. Figure 1 shows part of the lake near the health resort (see also Zotina et al., 1999). Sample sites are marked by numbers (1–10). Samples were obtained in the afternoon in July 1996.

Results and discussion LI values for samples from sites 1–10 are shown in Figure 2. Site 1 is a beach, sites 3 and 4 are places where municipal wastes sometimes occur. Site 9 is where municipal waste constantly occurs. The composition of the wastes is unknown. As can be seen, LI values for all ten samples in the water soluble enzymic system, and the immobilized system, are similar (within 90 per cent of each other). On the basis of previous data on the ecological status of freshwater lakes and rivers of the Krasnoyarsk and Altay regions (Kratasyuk et al.,

248

Figure 2. The luciferase indices (LI) of the water samples. (2) water-soluble coupled enzyme system: NADH:FMN-oxidoreductase-luciferase, (1) immobilized into starch gel coupled enzymic system: NADH:FMN-oxidoreductase-luciferase. Samples collected July 9, 1996.

Figure 3. Bacterial indices (BI) of water samples. (1) July 6, 1996; (2) July 12, 1996.

1996), in vitro biotests in their current form indicate a low toxicity of the water in Lake Shira. Taking into account the sensitivity of these systems to particular groups of substances (Kudryasheva et al., 1998), it appears that the concentrations of phenolic compounds and some inorganic salts are low and do not differ significantly between sites in Lake Shira.

249

Figure 4. Bacterial (1) and luciferase (2) indices (LI and BI) at different concentrations of salt (g kg−1 ).

BI values for the sample are given in Figure 3. As can be seen, BI values differ: values for July 6 are generally lower than those for July 12. Given the inhibition activity of organic compounds (oxidants) in the bacterial bioluminescent test-system (Kudryasheva et al., 1998), their greater concentration at higher temperatures on July 6 (than July 12) may reflect this. The decrease of BI in the beach sample site (N1, Figure 3) on July 6 may reflect processes connected with the presence of many holiday-makers. The BI values clearly show the variation in littoral samples of water of Lake Shira on July 6. Minimum values occur near the wastes (samples 4, 9, 10) and also sample 7. These differences in the response of the bacterial bioluminescent test-system could have resulted not only from the presence of organic impurities which inhibit bacterial light emission, but also from the lower salinities of the samples (Kudryasheva et al., 1998). To examine the latter possibility, LI- and BI-dependency on salinity was studied. A salt solution similar in composition to Lake Shira water was prepared (Kuskovskii and Krivosheev, 1989). The solution had an overall salt concentration of 40 g L−1 and contained the chloride and sulfate salts of potassium, sodium, magnesium and calcium. The ratio of ions in solution by weight was K:Na:Ca:Cl:SO4 = 4.139:1.471:0.080:2.411:9.779. The salinity of Lake Shira has varied between 15 and 20 g kg−1 (Kuskovskii and Krivosheev, 1989). Figure 4 shows LI and BI values according to the salt concentration. As can be seen, both LI and BI decrease as salinity falls below 15 g kg−1 . Hence, both in vivo and in vitro test-systems should behave in the same way as salinity decreases. This was not observed in the experiment. This

250 suggests that in Lake Shira the test-system involving bioluminescent bacteria is more sensitive to waste containing organic impurities than is the enzymic test-system. Note that the bacterial test-system has already been shown to be more sensitive than the enzymic system to organic compounds such as quinones and to municipal wastes (Kratasyuk et al., 1996; Kudryasheva et al., 1998). It may be concluded that the results of the present investigation indicate the value of an in vivo bacterial bioluminescent test-system as a means of monitoring the ecological status of Lake Shira. The difference in the sensitivities of the in vivo- and in vitro- systems to organic compounds can form the basis of a technique for monitoring organic pollution in water of different salinity. Additionally, the sensitivity of the in vitro enzymic system may be increased. In summary, in vivo and in vitro bioluminescent test-systems can be used to obtain data on the ecological status of Lake Shira. Since the time to undertake in vivo and in vitro bioluminescent bioassays is small (1–2 minutes), it is possible to use these tests to map rapidly pollution in the lake.

Acknowledgements This work was supported by the Federal Purpose-Oriented program “State Support for Integration of Higher Education and Fundamental Science” N 73, the Grant INTAS-97-open-0519, Expedition Grant of Siberian Branch of Russian Academy of Sciences, and a Grant from Russian Humane Foundation 96-03-04406. The authors are grateful to O.I. Egorova for help in experiments.

References Bulish, A.A. and Isenberg, D.L. 1981. Use of the luminescent bacterial system for rapid assessment of aquatic toxicity. ISA Transactions 20: 29–33. Cheung, Y.H., Neller, A., Chu, K.H., Tam, F.Y., Wong, C.K., Wong, Y.S. and Wong, M.H. 1996. Assessment of sediment toxicity using different tropic organisms. Arch. Environ. Contam. Toxicol. 32: 260–267. Drzyzga, O., Jannsen, S. and Blotevogel, K.H. 1995. Toxicity of diphenylamine and some of its nitrated and aminated derivatives to the luminescent bacterium Vibrio fisheri. Ecotoxicol. Environ. Saf. 31: 149–152. Grabert, E. and Kossler, F. 1986. About the effects of nutrients on the luminescent bacteria test. In: J.W. Hastings, L.J. Kricka, and P.E. Stanley (eds) Bioluminescence and Chemiluminescence, pp. 291–294. John Wiley & Sons, Chichester.

251 Guzzella, L., Bartone, C., Ross, P., Tartari,G. and Mutau, H. 1995. Toxicity identification evaluation of Lake Orta (Northern Italy) sediments using the microtox system. Ecotoxicol. Environ. Saf. 31: 231–235. Kratasyuk, V.A. 1990. Principle of luciferase biotesting. In: B. Jezowska-Trzebiatowska, B. Kochel, J. Stawinski and W. Strek (eds) Biological luminescence, Poland, 1989, pp. 550– 558. World Scient. Singapore. Kratasyuk, V.A. and Gitelson, I.I. 1986. Using bacterial bioluminescence and bioluminescent analyses. Uspekhi Mikrobiologii 21: 3–30 (In Russian). Kratasyuk, V.A., Kuznetsov, A.M., Rodicheva, E.K., Egorova, O.I., Abakumova, V.V., Gribovskaya, I.B. and Kalacheva, G.G. 1996. Problems and perspectives of bioluminescent testing in ecological monitoring. Siberian Ecological Journal 3: 397–403 (In Russian). Kudryasheva, N.S., Kratasyuk, V.A., Esimbekov, E.N., Vetrova, E.V., Nemtseva, E.N. and Kudinova, I.Y. 1998. Development of the bioluminescent bioindicators for analyses of environmental pollution. Field Anal. Chem. Tech. 2: 277–280. Kudryasheva, N.S., Shilova, E.B., Khendogina, E.V. and Kratasyuk, V.A. 1997. Bioluminescence biotest in monitoring Lake Shira. In: V.P. Parnaev, A.G. Degermengy, N.N. Nicolaeva, T.S. Krasnova and I.Y. Utkina (eds) Proceedings of the Conference “MedicalBiological and Ecological Problems Health-Resort Complex “Lake Shira”, Tomsk”, pp. 100–105. Tomsk State University, Tomsk (In Russian). Kudryasheva, N.S., Zuzikova, E.V. Gutnik, T.D. and Kuznetsov, A.V. 1996. Influence of metallic salts on bacterial bioluminescent systems of different complexity. Biofizika 2: 1264–1269 (In Russian). Kuskovskii, V.S. and Krivosheev, A.S. 1989. Mineral lakes in Siberia. Nauka, Novosibirk, Siberian branch, Novosibirk (In Russian). Kuznetsov, A.M., Rodicheva, E.K. and Shilova, E.B. 1996. Biotest based on lyophilized bacteria. Biotekhnologia: 57–61 (In Russian). Natecz-Jawecki, G., Rudz, B. and Sawicki, J. 1997. Evalution of toxicity of medical devices using Spirotox and Microtox tests: I. Toxicity of selected toxicants in various diluents. Biomedical Materials Research 35: 101–105. Stom, D.I., Geel, T.A., Balayan, A.E. and Kuznetsov, A.M. 1992. Bioluminescent method in studying the complex effect of sewage components. Arch. Environ. Contam. Toxicol. 22: 203–208. Tyulkova, N.A. 1990. Purification of bacterial luciferase from Photobacterium leiognathi with the use of FPLC-system. In: B. Jezowska-Trzebiatowska, B. Kochel, J. Stawinski and W. Strek (eds) Biological Luminescence, Poland, 1989, pp. 369–374. World Scient. Singapore. Wood, K.V. and Gruber, M.G. 1996. Transduction in microbial biosensors using multiplexed bioluminescence. Biosensors & Bioelectronics 11: 207–214. Zotina, T.A., Tolomeyev, A.P. and Degermengy, N.N. 1999. Lake Shira, a Siberian salt lake: ecosystem structure and function 1. Major physico-chemical and biological features. Int. J. Salt Lake Research 8: 211–232.