*Babes-Bolyai University, Department of Biology, Cluj-Napoca, Romania Corresponding author: Laszlo Fodorpataki, Babes-Bolyai University, Department of Biology, 1 Kogalniceanu Str., 400084 Cluj-Napoca, Romania, tel.: 0040264431858, fax: 0040264431858, email@example.com
Analele Universitatii din Oradea, Fascicula Biologie [Tom. XVI / 1, 2009, pp. 51-56]
Keywords: algal biomass, antioxidants, environmental stress, lipid peroxidation, molecular indicators, water pollution
While a large number of papers deal with influence of herbicides on terrestrial vascular plants, there are only a few publications referring to their impact on aquatic organisms, even though these organic compounds usually exhibit a high solubility in water and they may easily accumulate in aquatic habitats, especially in lakes [5, 7, 19, 26]. Unwanted side effects of agricultural application of herbicides are to be expected on non-target organisms, for example on algae in the aquatic ecosystems adjacent to areas subject to agricultural activities. These side effects consist of toxicity, disturbance of developmental processes and the appearance of resistant ecotypes. On the basis of their site and mode of action, the different herbicides may act as specific inhibitors of the light reactions of photosynthesis, inhibitors of biosynthesis of carotenoid pigment, fatty acids and aromatic amino acids, growth regulators administrated in excessive amounts, and compounds that affect tetrapyrrole biosynthesis, causing damage by peroxidation in light. The non-selective herbicides usually impair the energy flux in plants, disturb vital metabolic processes and generate highly reactive oxygen species [18, 25, 28]. Microalgae have a certain ability to bioaccumulate, to immobilize, to sequester and to biotransform different herbicides and related organic compound, thus contributing to a substantial remediation of the aquatic habitats polluted with xenobiotics. This is why the knowledge of how they react to stress conditions imposed by water pollution is important in order to use them in bioindication and bioremediation of disturbed aquatic ecosystems [17, 24].
The aim of the present study is to investigate some of the physiological reactions of the highly adaptable green alga Scenedesmus opoliensis to long-term exposure to micromolar amounts of three largely used non-selective herbicides, and to reveal molecular indicators suitable for a good establishment of environmental stress condition imposed by water pollution with the applied herbicides.
The dynamics of cell divisions was evaluated cytometrically with a light microscope. The initial cell density of all the cultures was set to 52x104 cells per milliliter. The dry algal biomass of the cultures was determined after 10 days of development of the algal populations, when the control cultures were at the end of exponential growth phase. Net photosynthetic oxygen production of the algal cultures was measured with an Oxy-Lab oxymeter at a constant light intensity of 110 ìM photons m-2 s-1 and 20 °C [1, 12].
For determination of lipid peroxidation after 10 days of exposure to herbicides, algal suspensions were centrifuged at 2500g for 10 minutes and pellets were weighed. To each pellet 0.1% (w/v) trichloroacetic acid (TCA) was added in 1:3 (g/ml) ratio. Algae were disrupted in a Constant Systems cell disrupter, than the extracts were centrifuged at 6300g for 10 minutes at 10 °C. 0.5% (w/v) 2-thiobarbituric acid (TBA) solution in 20% (w/v) TCA was added to the harvested supernatants in the ratio 1:4 (v/v), in 10 ml thermoresistant glass tubes. The extracts were heated for 25 minutes at 96 °C and, after lowering temperature on ice, they were centrifuged at 6300g for 10 minutes at 10 °C. Determination of thiobarbituric acid-reactive substances (TBARS), consisting mostly in malondialdehyde, was performed photometrically on the supernatants, at A532 – A600 nm, using at extinction coefficient of 159.2 mM-1 cm-1 .
Catalase activity was evaluated spectrophotometrically by determining the consumption of H2O2 associated with a change in absorbance at 240 nm. Algal cultures were centrifuged at 2500g for 10 minutes. The testing medium contained 750 ìl of sodium phosphate buffer (50 mM, pH 7.5), 100 ìl H2O2 (200 mM), and 150 ìl of algal extract with enzyme (5 ìg of protein) in a final volume of 1 ml. Proteins were extracted with 1.5 ml of 0.1 M sodium phosphate buffer (pH 7) and the extract was centrifuged at 2300g for 20 minutes at 5 °C . Ascorbic acid content of the algal cells was determined titrimetrically. 25 ml algal suspension was centrifuged at 2500g for 10 minutes, the pellet was resuspended in 10 ml of 5% (v/v) metaphosphoric acid, filtered and completed with 20 ml of 5% metaphosphoric acid. This extract was titrated with 0.025% (w/v) 2,6-dichlorophenol indophenol until a persistent light pink color appeared. Ascorbic acid content was determined with the help of a standard curve obtained with titration of known concentrations of ascorbic acid .
Every measurement was repeated 5 times. The experimental data were evaluated statistically, the significance of the differences among the control and the treated cultures was tested using one-way ANOVA (after verification of variance homogeneity with the Levene test), followed by the multiple comparison Tukey test (P < 0.05) .
The ascorbic acid (vitamin C) content of the algal cells, expressed on a dry weight basis, showed a statistically significant increase only when the cultures were exposed to 10 ìM methylviologen. No important change in the quantity of ascorbate was detectable in the algae treated with diuron and with glufosinate. As the most frequent non-enzymatic organic reducing agent in plant cells, vitamin C plays an important role in the antioxidative defense of algae and a higher ascorbate content is a prerequisite for a more efficient protection. It is worth mentioning that vitamin C occurs in plant cells in three interchangeable forms: as reduced ascorbic acid, as partly oxidized monodehydroascorbate and as fully oxidized dehydroascorbate. The method used in the present experiments allows only the determination of total vitamin C amount, without distinction between its different forms (Fig. 2).
From among the enzymatic components of the antioxidative defense system, catalase exhibited the most prominent changes under the influences of the three herbicides. Its catalytic activity in decomposing the highly toxic hydrogen-peroxide was not significantly modified by diuron, but it was obviously decreased by methylviologen, while glufosinate induced a pronounced rise in its enzymatic activity. The changes in the intensity of the detoxification reaction of hydrogen peroxide may be due to changes in both the amount of the catalase protein molecules and the catalytic activity of the enzyme modulated by different regulatory factors (Fig. 3).
The herbicides used in the experiments exerted a negative effect on the division rate of algal cells, resulting in changes of the dynamics of cell density in the populations during the first five days of exposure. Glufosinate caused only a delay in reaching the maximal cell density in the cultures, but did not lower significantly the final cell number in a unit of culture media volume. Diuron inhibited the growth of algal populations and forced the establishment of the steadystate growth phase at a much lower cell density than in the case of control. Methylviologen exerted a very pronounced inhibition of cell divisions, maintaining the cell density of the cultures close to the initial low values (Fig. 4).
The final dry biomass of the algal populations, reflecting the efficiency of net photosynthetic primary production, was impaired by all the three herbicides used in the experiments. The most pronounced decline of the algal biomass was registered in the presence of methylviologen, while the mildest, but still significant effect was exerted by glufosinate. The adverse influence of diuron on net biomass production was more moderate than that of methylviologen but more intense than the decrease caused by glufosinate (Fig. 5).
The net photosynthetic oxygen production of the constantly illuminated algal cells also changed under the influence of micromolar concentrations of herbicides. Oxygen production was mostly inhibited by diuron, and less (but still significantly) impaired by methylviologen. In contrast with the other two herbicides, glufosinate enhanced the net oxygen evolution of the algal cells, increasing it with almost 50% of the values measured in the control cultures (Fig. 6).
Whenever oxidative stress occurs by generation of reactive oxygen species under adverse environmental conditions, a crucial component of tolerance is the induction of an efficient antioxidative protection system with both enzymatic and non-enzymatic components. Many external stress factors cause the formation and accumulation of reactive oxygen species (singlet oxygen, superoxide radical, hydrogen-peroxide and hydroxyl radical), triggering the synthesis of reducing compound (mainly ascorbate, tocopherol, glutathione and specific xanthophylls), and the enhancement of catalytic activity of antioxidative enzymes (such as catalase, ascorbate peroxidase, superoxide dismutase, peroxiredoxins, glutathione reductase etc.). 10 μM methylviologen increased the amount of ascorbate and decreased the catalase activity in the alga Scenedesmus opoliensis. These results indicate that the algal cells react by accumulation of the protective molecules of ascorbate in order to counteract the oxidative stress imposed by this herbicide, but the synthesis and/or the activity of catalase is inhibited by methylviologen, in consequence the enzyme cannot perform an efficient scavenging of the accumulated hydrogen peroxide. This reduced enzymatic protection is partly compensated by the higher amount of ascorbic acid, which is also involved in decomposition of hydrogen peroxide through the Halliwell-Asada-Foyer redox chain, with participation of glutathione reductase and ascorbate peroxidase [14, 20]. Similar antioxidative responses were reported for green algae exposed to water pollution with chromium ions, and significant differences between tolerant and sensitive algae could be established concerning changes in the antioxidative protective components . In contrast with methylviologen, glufosinate triggered a different antistress reponse, without any significant increase in the ascorbate content of the algal cells, but with an obvious enhancement of catalase activity. In the case of this herbicide, the enzymatic component of antioxidative system seems to play a more important protective role than the non-enzymatic one. This may be considered another new finding of our experiments, suggesting that more components of the antioxidative system have to be investigated in order to get a more accurate image of how the same organism reacts to similar oxidative stress situations imposed by different chemical pollutants.
Considering the influence of herbicides on growth and reproduction of the alga Scenedesmus opoliensis, one can observe that methylviologen exerted the strongest inhibitory effect both on cell division rate and on dry biomass production. Glufosinate had the mildest influence on these parameters, indicating that this is the mostly tolerated herbicide among the three types used in the experiments. Growth of both cell number and cell size was significantly impaired by the long-term exposure of the algal populations to 10 μM of herbicides, indicating that mainly all the physiological processes that ensure survival of individuals and of the species are endangered by the presence of these organic pollutants in the aquatic habitats.
The rate of net photosynthetic oxygen production is also a good functional indicator of algal condition in the presence of herbicides, and it is a suitable parameter for distinguishing between the actions of different herbicides. The net oxygen production of algae in the presence of a constant photon flux density is the result of photosynthetic water-splitting process in the thylakoids, of chlororespiratory oxygen consumption in the chloroplasts, of photorespiratory oxygen consumption in the chloroplast stroma and in the peroxisomes, and of the oxygen demand of mitochondrial respiration, the latter being very reduced due to the Kok-effect in the photosynthesizing algal cells [8, 16, 21]. Different herbicides may interact directly or indirectly with all of these processes involved in the overall oxygen budget of the algal cells. Diuron decreases oxygen production mainly by inhibiting water-splitting related to the photochemical processes in photosystem II. Methylviologen induces the consumption of extra amounts of oxygen for the generation of superoxide radicals and hydrogen peroxide, and this may be the main cause of its inhibitory effect on net oxygen release from algal cells. Glufosinate impairs the metabolic pathway of photorespiration because of disturbances in the endogenic nitrogen cycle of the algal cells. This reduces photorespiratory oxygen consumption and results in an increased net oxygen accumulation. Changes in oxygen production of algae were also observed under the influence of other polluting agents [27, 29, 31].
As a conclusion of the above presented results, one can notice that: 1. methylviologen increases lipid peroxidation in algal cells, inhibits catalase activity, induces accumulation of ascorbate, impairs cell divisions, reduces dry biomass production and lowers net oxygen production; 2. diuron inhibits photosynthetic oxygen production, growth and development of algal populations; 3. glufosinate increases catalase activity, lipid peroxidation and net oxygen production in Scenedesmus opoliensis, but it moderately reduces cell density and biomass production.
High amounts of products of lipid peroxidation and of ascorbate are sensitive molecular indicators of oxidative stress conditions imposed by the presence of methylviologen in the aquatic environment, and these parameters may be suitable for detection of various pollutants that generate reactive oxygen species in algae. Net oxygen production, dry biomass accumulation and the rate of cell divisions are efficient functional indicators of the presence of all the three herbicides used in these experiments to study reactions of the alga to water pollution with organic xenobiotics. The green alga Scenedesmus opoliensis proves to be a promising test organism for bioindication of the effects of environmental stress factors on aquatic ecosystems. Studies on the impact of chemicals on aquatic organisms involve standardized single-species test systems evaluated under controlled laboratory conditions, but in order to achieve a more realistic knowledge of what happens in the complex natural ecosystems, these studies have to be continued with field experiments which take into account multilevel interactions among abiotic and biotic environmental factors [5, 15, 21].
Acknowledgements. This research was supported by grant MENER PC-D06-PT25-293.
 Barsanti, L., Gualtieri, P. (2006): Algae: anatomy, biochemistry and biotechnology, CRC Press, New York, 97-126.
 Becker, E. W. (2003): Microalgae: biotechnology and microbiology, Cambridge University Press, Cambridge, 113-138.
 Bigot, A., Fontaine, F., Clément, C., Vaillant-Gaveau, N. (2007): Effect of the herbicide flumioxazin on photosynthetic performance of grapevine, Chemosphere 67: 1243-1251.
 Bonilla, S., Conde, D., Blanck, H. (1998): The photosynthetic responses of marine phytoplankton, periphyton and epipsammon to the herbicides paraquat and simazine, Ecotoxicology 7: 99-105.
 Celekli, A., Balci, M., Bozkurt, H. (2008): Modelling of Scenedesmus obliquus; function of nutrients with modified Gompertz model, Bioresource Technology 99: 8742-8747.
 Daam, M. A., Rodrigues, A.M.F., Van den Brink, P.J., Nogueira, A.J.A. (2008): Ecological effects of the herbicide linuron in tropical freshwater microcosms, Ecotoxicology and Environmental Safety, doi: 10.1016/j.ecoenv.2008.07.009.
 Dewez, D., Didur, O., Vincent-Héroux, J., Popvic, R. (2008): Validation of photosynthetic fluorescence parameters as biomarkers for isoproturon toxic effects on alga Scenedesmus obliquus, Environmental Pollution, doi: 10.1016/j. envpol. 2008.03.002.
 Dragos, N., Péterfi, L.S., Momeu, L., Popescu, C. (1997): An introduction to the algae and the culture collection of algae at the Institute of Biological Research Cluj-Napoca, Cluj University Press, Cluj-Napoca, pp. 197.
 Fodorpataki, L., Bartha, Cs., (2004): Salt stress tolerance of a freshwater green alga under different photon flux densities, Studia Universitatis Babes-Bolyai, Biologia 49(2): 85-94.
 Fodorpataki, L., Bartha, L. (2008): Differential sensitivity of the photosynthetic apparatus of a freshwater green alga and of duckweed exposed to salinity and heavy metal stress. pp: 1451-1454. In: Allen, J.F., Gantt, E., Golbeck, J.H., Osmond, B. (eds.): Photosynthesis: energy from the Sun, Springer.
 Fodorpataki, L., Márton, A., Csorba, T., (2001): Stressphysiological investigation of algal cell cultures in polluted media, Contributii Botanice 36: 101-108.
 Geoffroy, L., Frankart, C., Eullaffroy, P., (2004): Comparison of different physiological parameters in Lemna minor and Scenedesmus obliquus in response to herbicide flumioxazin, Environmental Pollution 131: 233-241.
 Geoffroy, L., Teisseire, H., Couderchet, M., Vernet, G., (2002): Effect of oxyfluorfen and diuron alone and in mixture on antioxidative enzymes of Scenedesmus obliquus, Pesticide Biochemistry and Physiology, 72: 178-185.
 Gorbi, G., Torricelli, E., Pawlik-Skowronska, B., Toppi, L.S., Zanni, C., Corradi, M.G. (2006): Differential responses to Cr(VI)-induced oxidative stress between Crtolerant and wild-type strains of Scenedesmus acutus (Chlorophyceae), Aquatic Toxicology 79: 132-139.
 Grover, J.P., Holt, R.D. (2005): Plants in trophic webs. pp. 556-565. In: Crawley, M. J. (ed.): Plant ecology, Blackwell, Oxford.
 Gurbuz, F., Ciftci, H., Akcil, A. (2008): Biodegradation of cyanide-containing effluents by Scenedesmus obliquus, Journal of Hazardous Materials, doi:10.1016/j. jhazmat. 2008.05.008.
 Horváth G., Droppa M., Fodorpataki L., Istokovics A., Garab Gy., Oettmeier, W. (1996): Acridones: a chemically new group of protonophores, Proceedings of the National Academy of Sciences of the USA, 96: 3876- 3880.
 Li, X., Ping, X., Xiumei, S., Zhenbin, W., Liqiang, X. (2005): Toxicity of cypermethrin on growth, pigments, and superoxide dismutase of Scenedesmus obliquus, Ecotoxicology and Environmental Safety, 60: 188-192.
 Madhava-Rao, K.V., Sresty, T.V.S., (2000): Antioxidative parameters in the seedlings of pigeonpea (Cajanus cajan Millspaugh) in response to Zn and Ni stresses, Plant Science, 157: 113-128.
 McCormick, P.V., Cairns, J., (1997): Algal indicators of aquatic ecosystem condition and change. pp. 177-208. In: Wang, W., Gorsuch, J.W., Hughes, J.S. (eds.): Plants for environmental studies, Lewis Publisher, Boca Raton.
 Nagy-Tóth, F., Barna, A., (1998): Alge verzi unicelulare, Cluj University Press, Cluj-Napoca, pp. 156-179.
 Perales-Vela, H.V., Gonzalez-Moreno, S., Montes- Horcasitas, C., Canizares-Villanueva, R.O., (2007): Growth, photosynthetic and respiratory responses to sublethal copper concentrations in Scenedesmus incrassulatus (Chlorophyceae), Chemosphere, 67: 2274- 2281.
 Rodriguez-Garcia, I., Guil-Guerrero, J.L., (2008): Evaluation of the antioxidant activity of three microalgal species for use as dietary supplements and in the preservation of foods, Food Chemistry, 108: 1023-1026.
 Sandermann, H., (2004): Molecular ecotoxicology of plants, Springer, Heidelberg, pp. 244-253.
 Stabenau, H., Winkler, U., (2005): Glycolate metabolism in green algae, Physiologia Plantarum, 123: 235-245.
 Tukaj, Z., Aksmann, A. (2007): Toxic effects of anthraquinone and phenanthrenequinone upon Scenedesmus strains (green algae) at low and elevated concentration of CO2, Chemosphere, 66: 480-487.
 Tukaj, Z., Pokora, W., (2006): Individiual and combined effect of anthracene, cadmium, and chloridazone on growth and activity of SOD izoforms in three Scenedesmus species, Ecotoxicology and Environmental Safety, 65: 323-331.
 Vallotton, N., Moser, D., Eggen, R.I.L., Junghans, M., Chevre, N. (2008): S-metolachlor pulse exposure on the alga Scenedesmus vacuolatus: effects during exposure and the subsequent recovery, Chemosphere, 73: 395-400.
 Zar, J.H., (2000): Biostatistical analysis, Prentice-Hall, New Jersey, pp. 178-214.
 Zhang, E., Wang, B., Wang, Q., Zhang, S., Zhao, B., (2008): Ammonia-nitrogen and orthophosphate removal by immobilized Scenedesmus sp. isolated from municipal wastewater for potential use in tertiary treatment, Bioresource Technology, 99: 3787-3793.
(Click image to enlarge)
(Click image to enlarge)
(Click image to enlarge)
(Click image to enlarge)
(Click image to enlarge)
(Click image to enlarge)