Use of algal fluorescence for determination of phytotoxicity of heavy metals and pesticides as environmental pollutants

ECOTOXICOLOGY

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ENVIRONMENTAL

SAFETY

16,272-278

(1988)

Use of Algal Fluorescence for Determination of Phytotoxicity Heavy Metals and Pesticides as Environmental Pollutants GUY SAMSON AND RADOVAN

of

POPOVIC’

Centre de Recherche en Photobiophysique, Universitk du Q&bee ci Trois-Rivit?res, C.P. 500, Trois-Rivihes, Q&bec, Canada G9A 5H7 Received March 31, I988 The phytotoxicity of heavy metals and pesticides was studied by using the fluorescence induction from the alga Dunafiella tertiofecta. The complementary area calculated from the variable fluorescence induction was used as a direct parameter to estimate phytotoxicity. The value of this parameter was alfected when algae were treated with different concentrations of mercury, copper, atrazine, DCMU, Dutox, and Soilgard. The toxic effect of these pollutants was estimated by monitoring the decrease in the complementary area, which reflects photosystem II photochemistry. Further, the authors have demonstrated the advantage of using the complementary area as a parameter of phytotoxicity over using variable fluorescence yield. The complementary area of algal fluorescence can be used as a simple and sensitive parameter in the estimation of the phytotoxicity of polluted water. Q 1988 AC&&C PBS, Inc.

INTRODUCTION Deterioration of aquatic environment by numerous pollutants is a contemporary problem. The need for convenient methods and parameters to assesspollutant toxicity has become evident. Algae have already been used as biological indicators for this purpose (Wong and Beaver, 1980; Khan and Saifullah, 1986). The use of algae can be explained by their leading role at the primary step of the food chain (Reynolds, 1984). Deleterious effects on algae have serious consequences for the ecosystem equilibrium. Thus, the use of algae in bioassays has an acknowledged value in the estimation of the toxicity of pollutants (Couture et al., 1985). It has been confirmed that photosynthesis inhibition is a reliable indicator which rapidly reflects the toxic effects of pollutants (Overnell, 1976). Parameters closely associated with photosynthesis, such as ATF formation, radioactive carbon assimilation, and oxygen evolution, were used for this purpose (van Coillie et al., 1983). Their main disadvantages are the relative delay in obtaining a clear indication regarding the toxic effects and complexity of the methods. In addition to these methods, algal fluorescence induction phenomena were also used to study phytotoxicity (Moody et al., 1983). Upon illumination ofdark-adapted algae, the chlorophyll-a (Chl-a) fluorescence yield shows kinetics with characteristic transients usually termed 0, I, D, P, S, M, and T (Krause and Weis, 1984; Govindjee ’ To whom correspondence and reprinta should be addressed. * Abbreviations used: ATP, adenosine triphosphate; Atrazine, 2-chloro-4ethylamino-6-isopropylamino- 1,3,5&axine; C.A., complementary area; DCMU, 3-(3,4dichorophenyl)- 1, ldimethyl-mea; Hepes, N-2-hydroxylcthylpiperaxine-Y-2-ethanesu~onic acid Fc , constant fluorescence; FI , FD, and Fp, yields of fluorescence at I, D, and P transients; F-, maximal yield of fluorescence in the presence of DCMU; NADP, nicotinamide adenosine phosphate; PSII, photosystem Ik Q* and QB, primary and setondary electron acceptors of PSI& mspectively. 01476513/88

$3.00

copyright 0 1988 by Academic Rss Inc. Au rights of reproduction in any form rawnd.

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and Satoh, 1986). These kinetics are dependent on the light and dark photosynthetic reactions. During the first seconds, the fluorescence time course reflects the events of water splitting and subsequent electron transport (Papageorgiou, 1975; Lavorel and Etienne, 1977). It is accepted that excitation energy at antenna Chl-a is transferred to the reaction center of photosystem II (PSII), which subsequently reduces the PSI1 primary electron acceptor QA . Photosynthetic electron transport then proceeds via membrane electron carriers, finally resulting in NADP reduction and ATP formation. A portion of the energy trapped by the PSI1 antenna which is not able to transfer beyond QA via electron transport is reemitted as variable fluorescence. Consequently, variable fluorescence increases as the PSI1 primary acceptor QA is reduced or is quenched as QA is oxidized. The redox state of QA is thus determined by photosynthetic electron transport (Malkin, 1971; Melis and Schreiber, 1979). The environmental factors, including herbicides and pollutants which alfect either the water-splitting system or electron transport, will influence the variable fluorescence yield or its time course (Biihme et al., 198 1; Govindjee et al., 198 1). For this reason, chlorophyllu fluorescence induction in vivo is a highly sensitive and direct indicator of the physiological state of algae. In this study, these investigators used the “complementary area” calculated from fluorescence induction to assess the phytotoxicity of some pollutants. The complementary area (C.A.) corresponds geometrically to the surface above the fluorescence curve between’the O-P transients. The CA. is proportional to the photochemical capacity of PSI1 (Lavorel et al., 1986). Its value is decreased when PSI1 photochemistry is lowered by the effect of inhibitors such as copper (Samson et al., 1988). This investigation supports the idea that the C.A. represents a valid indicator for quickly estimating the phytotoxicity of pollutants. MATERIALS

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METHODS

The green alga Dunaliella tertiolecta Butcher (North East Pacific Culture Collection, University of British Columbia, Vancouver) was grown in the culture medium described by Harrison et al. (1980). Hepes (10 mA4) was added to the medium in order to maintain pH at 7.5. The growth conditions were 18°C with a light intensity of 10 mW cm-* provided by a combination of cool white fluorescent and incandescent bulbs, with a 16-hr l&t/8&r dark cycle. For experiments, cells were harvested by centrifugation for 3 min at 5OOOgin their exponential growth from 96-hr-old cultures. Algal cells were washed twice with a resuspending medium which consisted of their culture medium, less vitamins and microelements in order to avoid chemical interactions with heavy metals. Chlorophyll-a concentration was estimated in 80% acetone as described by Strain et al. ( 1972). For fluorescence measurements, cells (5 pg Chl-a ml-‘) in their resuspending medium were dark-adapted for 45 min and then were incubated for 15 min in the dark with varying concentrations of inhibitom prior to the measurements. Mercury and copper were added in the form of HgCI, and CuS04 (Sigma, St. Louis, MO). The herbicides Atrazine and DCMU and the insecticides Dutox (containing trichlorfon 18.0% and oxydemeton-methyl 6%, CIL Inc., Laval, Quebec) and Soilgard (chlorpyrifos lo%, Wilson Lab. Inc., Dundas, Ontario) were dissolved in methanol prior the use. The final concentration of methanol did not exceed 1.O% in the samples. Chlorophyll fluorescence induction curves were recorded with an integrating sphere as previously described (Morissette et al., 1988). The constant fluorescence 0 level (FO) was calculated by the method described by Morissette

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TIME

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FIG. 1. Schematic representation of the chlorophyll-a fluorescence induction rise in a subsecond time scale. & F,, FD, and Fp: fluorescence intensity levels corresponding to 0, I, D, and P transients. The hatched surface.represents the complementary area.

and Popovic (1987). The C.A. was calculated by the method of Melis and Homann (1975). Oxygen evolution was measured with a Clark-type electrode as described previously by Popovic et al. (1979). Chlorophyll a concentration for oxygen measurements was 20 pg ml-‘. Treatment conditions with inhibitors were the same as those for fluorescence measurements. RESULTS Figure 1 shows a typical fluorescence induction curve from a dark-adapted photosynthetic organism recorded in a subsecond time scale. The total fluorescence is composed of constant and variable components. The constant fluorescence denoted 0 (&,) is independent of PSI1 photochemistry. The fluorescence kinetics from 0 to P levels, via Fi and FD transients, have been explained by variations of the rates of QA reduction and reoxidation by the electron transport chain (Papageorgiou, 1975). The relative height of Fp transient depends on exciting light intensity (Fraser et al., 1987; Krause and Weis, 1984). The hatched area over the fluorescence induction curve represents the complementary area. The surface of this area is proportional to the PSI1 electron acceptor pool, which determines the PSI1 photochemical activity (Lavorel et al., 1986). Melis and Schreiber (1979) reported that the C.A. was a direct indicator of PSI1 photochemistry. Supporting this conclusion, Ma&in et al. (198 1) obtained a good quantitative correlation between the C.A. and photosynthetic capacity estimated by CO2 fixation. Figure 2 shows the fluorescence induction curves from D. tertiolecta incubated 15 min in the dark in the presence of varying concentrations of heavy metals, herbicides, and insecticides. Mercury and copper decreased the fluorescence intensity at the Fp transient, without significant change of the curve shape. Similar copper effects of algal fluorescence were obtained earlier (Shioi et al., 1978; Samson et al., 1988). These effects are attributed to an inhibition of photosynthetic electron transport on the oxidizing side of PSII. In this case, the electron flow from the water-splitting system cannot reduce the PSI1 primary acceptor QA (Vierke and Struckmeier, 1978; Samson et al., 1988). In the second class of inhibitors, the herbicides Atrazine and DCMU, well-known as,inhibitors of PSI1 electron transport, did not alter the lip level, but

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FIG. 2. Fluorescence induction transients from Dunaliella tertiolecta treated with different concentrations of mercury, copper, atrazine, DCMU, Dutox, and Soilgard. For details of experimental conditions. see Materials and Methods.

gradually increased the FD and FI levels. These herbicides block electron transport at the secondary electron acceptors QB (van Rensen, 1982; Oettmeier et al., 1984). In this case, QA cannot be oxidized by the plastoquinone pool via the QB electron carrier, and consequently it induces maximum fluorescence yield (Krause and Weis, 1984). The insecticides Dutox and Soilgard decreased the Fp level as observed with heavy metals. However, at high concentrations, they induced a rise in FI and FD levels as observed with the herbicides Atrazine and DCMU. Figure 3 shows the values in percentages of the fluorescence intensity at Fp transient and of the C.A. when different pollutant concentrations are used. It was evident that the value of Fp (or maximum fluorescence) was not always affected by pollutants. The inhibitory effects of the herbicides Atrazine and DCMU, which will maintain high fluorescence yield, are located at PSI1 secondary electron acceptor (Oettmeier et al., 1984). However, when the water-splitting system was inhibited as in the case with Cu (Vierke and Struckmeier, 1978; Samson et al., 1988) of Hg (DeFilippis et al., 198 1), fluorescence yield was decreased. The insecticides Dutox and Soilgard consistently decreased the C.A., which was not the case with Fp. Therefore, the fluorescence yield at Fp (or maximum fluorescence, F,, , in the case of herbicides) cannot always be a useful indicator of pollutant phytotoxicity. However, in Fig. 3, we showed that the C.A., as indicator of PSI1 photochemistry, always decreased when the concentration of pollutants was increased. Using this parameter, it is possible to indicate the starting precipitation point for some polIutants, as seen for copper at concentrations higher than 60 PM. A further increase in the Cu concentration induced a slow increase in the PSI1 photochemistry

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FIG. 3. Percentage offluorescence intensity and the complementary area a&ted mental conditions are the same as those in Fig. 2.

with pollutants. Experi-

inhibition, as we reported earlier (Samson et al., 1988). A slow increase in free cupric ion concentration tier the precipitation point was detected with an Orion Model 94 29 cupric ion selective electrode (data not shown). DISCUSSION The biological significance of the C.A. is confirmed by the results presented in Table 1. Table 1 presents the concentrations of the above-mentioned inhibitors required to reduce by 50% (Iso) the rates of oxygen evolution, the fluorescence intensity at Fr transient and the CA. With the exception of mercury, it was not possible to obtain the Iso value for Fr intensity for other pollutants. However, IS0 of the C.A. for all pollutants had welldefined values, as seen for the Is0 of,oxygen evolution. The difference in concentration of the same pollutant required to induce I50 of Oz evolution or Iso of C.A. resulted from the fact that the oxygen evolution value does not represent all photosynthetic photochemistry because of oxygen-consuming processes present in the algal cells (Popovic d al., 1983). With these results, we demonstrated

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TABLE 1 CONCENTRATIONS OF SIXPOLLUTANTSREQUHWDTOREDUCE BY~O%(&) THERATESOFOXYGENEVOLTJTION,THE FLUORESCENCE~NTENSITYAT Fp TRANSIENT, AND THE COMPLEMENTARY AREA Isovalues Inhibitors

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140&f 175ph4 1.25&f

250 /AM n.0. n.0. n.0. n.0. n.0.

175&f loopikf 0.17@ 0.25 pit4 230 mg-liter-’ 60 mg.liter-’

l.oopM 250 mg-liter-’ 65 mg.liter-’

Note. no., not observed. Conditions are the same as those in Fig. 2. Rate of oxygen evolution for the control was 252 NO2 mg-i Chl hr-‘.

that the C.A. can be a reliable indicator of the phytotoxicity of different pollutants. An earlier study indicated that the C.A. is not dependent on light intensity (Lavorel et al., 1986), which is not the case for the FP fluorescence level (Fraser et al., 1987; Krause and Weis, 1984). This study provides the fluorescence method with a simple and sensitive parameter for the determination of the phytotoxicity of different pollutants. CONCLUSION This paper demonstrated the greater significance of the complementary area in the determination of the inhibitory effects of pollutants on the capacity of photosynthetic electron transport than that by using the total fluorescence yield. This study provides the fluorescence method with a reliable and sensitive parameter for assessing the phytotoxicity of a wide range of pollutants. ACKNOWLEDGMENT This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) through Grants G- 1929, G- 1930, and A-3047, awarded to R. Popovic.

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GOVINDJEE, DOWNTON, W. J. S., FORK, D. C., AND AR~~OND, P. A. (198 1). Chhrophyh fluorescence transient as an indicator of water potential in leaves. Plant Sci. Lett. 20,19 1- 194. HARRISON, P. J., WATERS, R. E., AND TAYLOR, F. J. R. (1980). A broad spectrum artificial seawater medium for coastal and open ocean phytoplankton. J. Phycol. 16,28-35. KHAN, S. H., AND SAIFULLAH, S. M. ( 1986). Bioassay studies of phytoplankton of coastal waters of Karachi Pakistan in relation to heavy metals pollution I. Effect of copper and lead on Slcefctonema costarrum. Pak. J. Bot. l&137-146. KRAUSE, G. H., AND WEIS, E. (1984). Chlorophyll fluorescence as a tool in plant physiology. Photo,syrrth. Rex 5, 139-157. LAVOREL, J., BRETON, J., AND LUTZ, M. (1986). Methodological principles of measurements of light emitted by photosynthetic systems. In Light Emission by Plants and Bacteria (Govindjee, J. Amesz, and D. C. Fork, Eds.), pp. 57-98. Academic Press, San Diego. LAVOREL, J., AND ETIENNE, A.-L. (1977). In vivo chlorophyll fluorescence. In Primary Processes ofPhotosynthesis (J. Barber, Ed.), pp. 206-268. Elsevier, Amsterdam. MALKIN, S., ARMOND, P. A., MOONEY, H. A., AND FORK, D. C. (1981). Photosystem II photosynthetic unit sizes from fluorescence induction in leaves. Correlation to photosynthetic capacity. Plant Physiol. 67,570-579.

MALKIN, S. (1971). Fluorescence induction studies in isolated chloroplasts. Biochim. Biophys. Actu 234, 415-427.

MELIS, A., AND !XHREIBER, U. (1979). The kinetic relationship between the absorbance change, the reduo tion of Q and the variable fluorescence yield change in chloroplasts at room temperature. B&him. Biophys. Acta 547,47-57. MELIS, A., AND HOMANN, P. H. ( 1975). Kinetic analysis of the fluorescence in 3-(3,4-dichlorophenyl)- 1,l dimethylurea poisoned chloroplasts. Photochem. Photobiol. 21,43 l-437. MOODY, R. P., WEINBERGER, P., AND GREENHALGH, R. (1983). Algal fluorometric determination of the potential phytotoxicity of environmental pollutants. In Aquatic Toxicology (J. 0. Nriagu, Ed.), Vol. 13, pp. 504-5 12. Wiley-Interscience, New York. MOFUSSETTE, J. C., AND POPOVIC, R. (1987). A new method for the separation of the constant and the variable fluorescence of chlorophyll-a in vivo. Biochem. Biophys. Res. Commun. 149,385-390. MORISSETTE, J. C., MEUNIER, P. C., AND POPOVIC, R. (1988). An automatic integrating fluorometer using an Apple-II and Scope-85. Rev. Sci. Instrum. 59,934-936. OETTMEIER, W., SOLL, H.-J., AND NEUMANN, E. (1984). Herbicide and plastoquinone binding to photosystem II. Z. Naturforsch. C39,393-396. OVERNELL, J. (1976). Inhibition of marine algal photosynthesis by heavy metals. Mar. Biol. 38,335-342. PAPAGEORGIOIJ,G. (1975). Chlorophyll fluorescence: An intrinsic probe of photosynthesis. In Bioenergetits OfPhotosynthesis (Govindjee, Ed.), pp. 3 19-37 1. Academic Press, New York. POPOVIC, R., COLBOW, K., VIDAVER, W., AND BRUCE, D. (1983). Evolution of OZ in brown algal chloroplasts. Plant Physiol. 73,889-892. POPOVIC, R., KYLE, D. J., COHEN, A. S., AND ZALIK, S. (1979). Stabilization of thylakoid membranes by spermine during stress-induced senescence of barley leaf discs. Plant Physiol. 64,72 l-726. REYNOLDS, C. S. (1984). The EcoIogy ofFreshwater Phytoplunkton. Cambridge Univ. Press, Cambridge. SAMSON, G., MORISSETTE, J. C., AND Popovrc, R. (1988). Copper quenching of the variable fluorescence in Dunaliella tertiolecta. New evidence for a copper inhibition on PSI1 photochemistry. Photochem. Photobiol. 48,329-332. SHIOI, Y., TAMAI, H., AND SASA, T. (1978). Inhibition of photosystem II in the green alga Ankistrodesmus falcatus by copper. Physiol. Plant. 44,434-438. STRAIN, H. H., COPE, B. T., AND SVEC, W. A. (1972). Analytical procedures for the isolation, identification, estimation, and investigation of the chlorophylls. In Methods in Enzymology (A. San Pietro, Ed.), Part B, Vol. 23, pp. 452-476. Academic Press, New York. VAN COILLIE, R., COUTURE, P., AND VISSER, S. A. (1983). Use of algae in aquatic ecotoxicology. In Aquatic ToxicoIogy (J. 0. Nriagu, Ed.), Vol. 13, pp. 488-502. Wiley-Intersciences, New York. VAN RENSEN, J. J. S. (1982). Molecular mechanisms of herbicide action near photosystem II. Physiol. Plant. 54,5 15-52 1. VIERKE, G., AND STRUCKMEIER, P. (1978). Inhibition of millisecond luminescence by copper (II) in spinach chloroplasts. Z. Natu&rsch. C33,266-270. WONG, S. L., AND BEAVER, J. L. (1980). Algal bioassays to determine toxicity of heavy metals mixtures. Hydrobioiogia 74, 199-208.