- Email: [email protected]
Toxic Responses and Catalase Activity ofLemna minorL. Exposed to Folpet, Copper, and Their Combination
ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY
40, 194—200 (1998)
ENVIRONMENTAL RESEARCH, SECTION B ARTICLE NO.
ES981682
Toxic Responses and Catalase Activity of Lemna minor L. Exposed to Folpet, Copper, and Their Combination1 H. Teisseire, M. Couderchet, and G. Vernet Laboratoire de Zoologie et des Sciences de l+Environnement, BaL timent Europol+Agro, Faculte´ des Sciences, Universite´ de Reims Champagne-Ardenne, BP 1039, F-51687 Reims-02, France Received April 1, 1997
Toxicity of copper and folpet—two fungicides widely used on grape—was evaluated on Lemna minor L., a sensitive aquatic weed regularly used for (eco)toxicological studies. Toxicity assessments were based on inhibition of growth and chlorophyll content of L. minor cultures after 7 days. IC10 , IC50 , and IC90 were determined for both compounds alone and were respectively, 0.03, 0.16, and 0.95 mg liter21 for copper and 1.20, 7.50, and >40 mg liter21 for folpet. When both compounds were combined, the response of L. minor depended on the initial folpet concentration. Indeed, a slight synergy was observed for 5 mg liter21 folpet, while at folpet concentrations of 20 to 35 mg liter21, the two fungicides were antagonists. The antagonism was positively correlated with folpet concentration. Antagonism between Cu and folpet could not be explained by a reduced bioavailability of Cu since concentration of free copper in the mixture did not depend on the presence of folpet. One physiological defense response elicited by copper in plants is an increase in catalase activity. Copper and folpet stimulated catalase activity and changes in the activity of the enzyme could not account for the synergy but possibly for the antagonism. Nevertheless, catalase activity increased significantly after a 24-h exposure to 25 lg liter21 of copper. The use of this property as a rapid and sensitive biomarker to monitor the toxicity of xenobiotics alone or in combination and of environmental water is discussed. ( 1998 Academic Press Key Words: antagonism; catalase; copper; duckweed; folpet; fungicide; grapevine; Lemna minor; synergy.
INTRODUCTION
Runoff is an important phenomenon on the slopes of septentrional grape-growing areas, and pesticides and other chemicals may end up in surface waters, either dissolved in runoff water or adsorbed to soil particles. These polluted waters may then connect the hydrographic system downstream and generate a diffuse pollution that endangers plants and animals of the contaminated ecosystems.
Since copper and folpet (Fig. 1) control mildew and other fungal diseases in grape (Buchenauer, 1990), these two fungicides are generously sprayed on vineyards in Champagne, and they belong to the most widely used agrochemicals in those fields. As such, they are likely to be present in runoff water and present potential risks for the aquatic environment. The toxicological effects of copper are well documented for plants and animals (Filbin and Hough, 1979; Nemcso´k and Benedeczky, 1995), whereras those of folpet are rarely reported in the literature, especially for plants (Anonymous, 1995). Furthermore, the two fungicides, which are often applied together in the field, may interact on the plant surface and in the pathogenic fungi, but also in the environment. There, the presence of one of the pesticides may influence that of the other one, and their toxicology is more likely to be due to a combined action of the pesticides than to individual effects. Therefore, the toxicity of folpet and copper was examined and the potential risks resulting from their combination were investigated in duckweed (¸emna minor L.), a sensitive aquatic plant regularly used for ecotoxicological studies because of its small size, ease of handling and culture, and fast growth rate (Lewis, 1995). Furthermore, folpet seems to be a multitarget fungicide, the mode of action of which is not totally elucidated (Buchenauer, 1990). In contrast, understanding of the mode of action of copper in plants is rapidly expanding (Baro´n et al., 1995). One important effect of copper is an oxidative stress followed by elicitation of several defense enzymes such as catalase (Foyer et al., 1994). The possible involvement of oxidative stress in the toxicity of folpet is unknown. In this study the effect of both copper and folpet on catalase was therefore investigated. MATERIAL AND METHODS
Plant Material 1 Part of this work was presented at the 49th International Symposium on Crop Protection on May 6, 1997, in Gent, Belgium.
¸emna minor L. (duckweed) was collected from an artificial pond on the University campus. The plants were
194 0147-6513/98 $25.00 Copyright ( 1998 by Academic Press All rights of reproduction in any form reserved.
195
TOXICITY OF FOLPET AND COPPER
FIG. 1. Structure of folpet.
disinfected by immersing the fronds in NaOCl (0.01 M) for 20 s and rinsing with distilled water. The stock cultures were maintained in 2-liter plastic (PVC) aquariums containing 500 ml of inorganic growth medium (Chollet, 1993) that consisted of KNO , 202 mg liter~1; KH PO , 50.3 mg 3 2 4 liter~1; K HPO , 27.8 mg liter~1; K SO , 17.4 mg liter~1; 2 4 2 4 MgSO ) 7H O, 49.6 mg liter~1; CaCl , 11.1 mg liter~1; 4 2 2 Na EDTA, 10 mg liter~1; FeSO ) 7H O, 6 mg liter~1; 2 4 2 H BO , 5.72 mg liter~1; MnCl ) 4H O, 2.82 mg liter~1; 3 3 2 2 ZnSO , 0.6 mg liter~1; (NH )Mo O ) 4H O, 0.043 mg 4 4 7 24 2 liter~1; CuCl ) 2H O, 0.078 mg liter~1; CoCl ) 6H O, 2 2 2 2 0.054 mg liter~1. Before the medium was autoclaved its pH was adjusted to 6.5. The aquariums were placed in a controlled environment room at 25$1°C under continuous illumination provided by cool white fluorescent lamps (Sylvania Grolux F 36 W) with a light intensity of 2000$ 100 lx, and plants were subcultured twice a week. Agrochemicals Used A 100 mg liter~1 stock solution of copper (CuSO )5H O, 4 2 analytical grade, Sigma, Saint-Quentin-Fallavier, France) was prepared in distilled water. The commercial preparation of folpet, Folpan 500, containing 500 g liter~1 active ingredient (a.i.) was used and a 1% (v/v) stock solution was prepared just before the experiments were run. A blank formulation (without a.i.) was used for the controls. Both Folpan 500 and the blank formulation were generous gifts of Makhteshim Agan France. Cu and Folpet Treatments For toxicity experiments, six double-fronded ¸emna colonies of approximately equal size were transferred to 100-ml crystallizing dishes containing 50 ml of growth medium to which Cu and/or folpet had been added. The growth medium was the same as above for experiments with folpet alone but it did not contain EDTA when the experiments included Cu to avoid any interaction between the ion and the chelator (Wang, 1990). The experiments were conducted either under static or under semistatic conditions. Under static conditions, the
medium was not renewed and the concentration of active Cu ion or folpet (a.i.) was set at the beginning of the experiments. It was 0.05, 0.2, 0.4, 0.6, and 1 mg liter~1 for copper and 5, 20, 40, 70, and 100 mg liter~1 for folpet. Under semistatic conditions the fronds were transferred every day to a clean crystallizing dish containing fresh medium in which the contaminant concentration was readjusted to the initial values, which were 5, 10, 20, 30, and 40 mg liter~1 for folpet. No semistatic experiment was run with copper alone because the free copper concentration in the medium almost did not vary during the course of the experiments. In experiments including folpet, the quantity of blank formulation was the same in the controls and in the samples that received the highest dose of folpan (corresponding to 40 mg liter~1 a.i.). Formulation-free controls were also run. Fronds were counted daily and values of 0.25, 0.5, 0.75, and 1 were attributed to the fronds depending on their development. Relative growth was estimated after 7 days by integrating the area between the growth curve and the horizontal line y"n , where n is the initial number of 0 0 fronds in the colony (usually 12). Total chlorophyll (Chl) content was determined according to McKinney (1941) after 7 days of incubation. IC values were estimated graphically using relative 50 growth and Chl content inhibition curves. Combination of Cu and Folpet Experiments were conducted in EDTA-free media as described previously for individual contaminants. Sublethal concentrations of Cu and folpet were used, which can be seen under Results (Tables 3 and 4). In each sample, the quantity of blank formulation was adjusted to 0.07& (v/v). Experiments were run either under static conditions in which the media were not renewed (Table 3) or under semistatic conditions in which the media were renewed daily as above (Table 4). Possible interactions between copper and folpet were estimated using Abott’s formula (Gisi, 1996). In this widely used model, the expected inhibition of the mixture, expressed as percent C , can be predicted as %91 C
%91
"A#B!(AB/100),
where A and B are the inhibition levels given by the single chemicals (respectively copper and folpet). The ratio of inhibition (RI) was then calculated as follows for each pesticide combination: RI"C /experimentally observed efficacy. %91 Synergism or antagonism was evaluated by comparing the RI with 1. A RI (1 indicated synergism between the
196
TEISSEIRE, COUDERCHET, AND VERNET
formed for each concentration and all experiments were repeated twice. RESULTS
FIG. 2. Influence of folpet on growth and total chlorophyll content of ¸emna minor L. after a 7-day exposure to the fungicide. The plants were maintained under static conditions.
two contaminants; a RI of 1, simple additivity; and a RI'1, antagonism between the chemicals. Catalase Assays Approximately 15 double-fronded colonies of ¸. minor were exposed to various concentrations of both chemicals (0 to 600 lg liter~1 copper and 0 to 40 mg liter~1 folpet) according to the procedure described above. After 24 h of incubation, catalase activity was determined according to Subhadra et al. (1991) with some modifications. The fronds were homogenized in cold 0.1 M sodium phosphate buffer (pH 7) with a Potter homogenizer (Servodyne mixer head, Cole Parmer, Niles, IL) and centrifuged 15 min at 2300g and 4°C. The supernatant was used as the enzyme extract. One milliliter of enzyme extract was added to the reaction mixture consisting of 0.01 M hydrogen peroxide (1 ml) and 0.1 M sodium phosphate buffer (3 ml, pH 7). After 5 min incubation in a water bath at 22°C, the reaction was stopped by addition of 10 ml of 2% H SO . The acidified 2 4 reaction mixture was titrated against 0.005 N KMnO to 4 determine the quantity of hydrogen peroxide consumed by the enzyme. The total protein content of each enzyme extract was determined according Bradford (1976) using bovine albumin (Sigma) for calibration. The catalase activity was expressed as millimoles of hydrogen peroxide consumed per milligram of protein and per minute.
Plants contaminated by copper sulfate were chlorotic and their fronds were smaller than those of controls. After 1 day of exposure to Cu the two fronds associated in colonies separated and lost their roots. Subsequently, after 2 to 3 days of incubation and for Cu concentrations of 0.2 and 0.4 mg liter~1, new fronds no longer separated from mother plants, forming colonies of six or seven fronds, whereas in controls the number of associated fronds per colony never exceeded 4. Under static conditions (when the medium was not renewed) the lowest folpet concentration (5 mg liter~1) stimulated growth. Above that concentration folpet inhibited growth of ¸emna (Fig. 2). The plants exposed to higher concentrations were chlorotic and had lost their roots. Under semistatic conditions all concentrations of folpet tested inhibited growth (Fig. 3). In contrast to what was observed under static conditions, 5 mg liter~1 folpet was enough to induce significant inhibition of growth and reduction of chlorophyll content. Concentrations of the two chemicals inhibiting 10, 50, and 90% (IC , IC , and IC ) of growth of ¸. minor are 10 50 90 listed in Table 1. Similar values were found when chlorophyll content was used as a marker of copper toxicity (Table 2). Correlation between growth and chlorophyll content was not as good with folpet (Figs. 2 and 3), for which chlorophyll appeared to be a more sensitive marker than growth (compare Tables 1 and 2).
Data Reliability and Presentation IC determination experiments were repeated at least twice and each sample was triplicated. When the combination of folpet and copper was studied, experiments were triplicated. For catalase assays, three replicates were per-
FIG. 3. Influence of folpet on growth and total chlorophyll content of ¸emna minor L. after a 7-day exposure to the fungicide. The plants were maintained under semistatic conditions; that is, the medium was changed daily and the folpet concentration in the medium was adjusted daily.
197
TOXICITY OF FOLPET AND COPPER
TABLE 1 Inhibition of Lemna minor Growth by Copper and Folpet Chemical
Conditions
Copper Folpet
Static Static Semistatic
IC
10
0.03$0.02 7.8$5.0 1.2$0.2
IC 50 0.16$0.05 50$15 7.5$2.5
IC
90
0.95$0.13 '100 '40
TABLE 3 Ratios (RI) of Theoretical to Observed Growth Inhibition of Lemna minor L. by Mixtures of Copper and Folpet under Static Conditions Copper (mg liter~1) Folpet (mg liter~1)
0.05
0.15
0.25
0.920$0.034 1.200$0.085 1.34$0.16
0.915$0.073 1.10$0.22 1.23$0.25
0.990$0.089 1.42$0.13 1.30$0.28
Note. The plants were cultivated 7 days in nonrenewed (static conditions) or renewed (semistatic conditions) medium. Inhibitory concentrations are expressed in mg liter~1.
5 20 35
When ¸. minor was exposed to mixtures of copper and folpet under static conditions, the comparison between the expected inhibition (C ) and experimentally observed %91 growth inhibition revealed two different responses (Table 3). For the lowest folpet concentration (5 mg liter~1), the experimentally observed growth inhibition values were superior to C and the RIs were slightly less than 1, %91 indicating a potential synergism between copper and folpet (Table 3). In contrast, for the highest folpet concentrations tested, some of the ratios were greater than 1, indicating antagonism between the two chemicals. Under semistatic conditions, the experimentally observed inhibition values were not significantly different from C %91 as indicated by the RI values presented in Table 4. In this case, the results suggested a simple additivity of the effects of the two chemicals. Catalase activity of control ¸. minor was variable and was approximately 20 mmol H O mg protein~1 min~1. The 2 2 presence of blank formulation in the controls of folpet experiments yielded somewhat superior activity (27.40$ 0.96 mmol H O mg protein~1 min~1). Catalase activity 2 2 was higher in the plants that had been exposed 24 h to Cu (Fig. 4) or folpet (Fig. 5). This increase in enzyme activity was significant for concentrations as low as 0.025 mg liter~1 copper and 2.5 mg liter~1 folpet, indicating the very high sensitivity of the enzyme to these pollutants. Enzyme activity increased with the concentration of pollutant up to 35 to
40 mmol H O mg protein~1 min~1, this activity being 2 2 reached with 0.10 mg liter~1 CuSO and 10 mg liter~1 fol4 pet. Further increasing the concentration of pollutant yielded a decrease in enzyme activity (Figs. 4 and 5).
TABLE 2 Inhibition of Chlorophyll Content of Lemna minor L. Colonies by Copper and Folpet Chemical
Conditions
Copper Folpet
Static Static Semi-static
IC
10
0.03$0.015 6.6$3.1 0.90$0.02
IC 50 0.16$0.04 22.8$6 4.4$0.6
IC
DISCUSSION
The IC values obtained for copper (Tables 1 and 2) were 50 in agreement with the 0.12 mg liter~1 or 0.14—0.32 mg liter~1 reported earlier for ¸emna (Walbridge, 1977; Jenner and Janssen-Mommen, 1993); they indicate that this metal is highly toxic to ¸. minor. The toxicity of the metal is confirmed by the very low IC values. These IC values 10 10 corresponded to those measured in decantation basins that collect runoff waters from the vineyards (unpublished data). Symptoms observed (chlorosis, colony breakup, root loss, etc.) in ¸. minor after exposure to CuSO reflect the intensity 4 and diversity of the disorders generated by Cu2` ions in cell metabolism. Chlorosis is a common response of plants to stress (heat, diseases, pollution) and has been observed after copper treatment in various aquatic plants such as Eichhornia crassipes and Hydrilla verticillata (Lewis, 1993), Chlorella pyrenoidosa (Vavilin et al., 1995), and ¸emna sp. (Filbin and Hough, 1979). Among the variety of targets reported for copper in plants, the photosynthetic apparatus seems the most sensitive (Baro´n et al., 1995). This effect of copper entrains an oxidative stress followed by peroxida-
TABLE 4 Ratios (RI) of Theoretical to Observed Gowth Inhibition of Lemna minor L. by Mixtures of Copper and Folpet under Semistatic Conditions
90
0.88$0.15 '100 '40
Note. The plants were cultivated 7 days in nonrenewed (static conditions) or renewed (semistatic conditions) medium. Inhibitory concentrations are expressed in mg liter~1.
Copper (mg liter~1) Folpet (mg liter~1) 1.6 3.2 4.8
0.016
0.032
0.048
1.37$0.74 1.03$0.20 0.99$0.13
1.12$0.13 1.34$0.32 1.11$0.27
1.07$0.28 1.20$0.16 1.29$0.25
198
TEISSEIRE, COUDERCHET, AND VERNET
FIG. 4. Catalase activity of ¸emna minor L. after the fronds were cultivated 24 h in nutrient medium contaminated with CuSO . 4
tion of membranes and pigments (Mattoo et al., 1986). This oxidative stress explains the induction of catalase activity observed in response to copper contamination (Fig. 4) because catalase induction is part of the defense mechanism in response to increasing concentrations of reactive oxygen species responsible for lipid peroxidation (Fridovich, 1978; Foyer et al., 1994). The stimulation of catalase activity in response to copper (and also to folpet) occurred at very low contaminant concentration; at higher concentration it reached a maximum and decreased again. This decrease can be interpreted as a classical stress response in which the intensity of the stress is too high and the stage of exhaustion is reached (Lichtenthaler, 1996). The stimulation of growth in response to the lowest folpet concentration tested under static conditions (Fig. 1) had already been reported in plants (Buchenauer, 1990). It is even common practice among farmers to use this property of the fungicide to heal grape leaves after a storm. Folpet IC values (Table 1) demonstrated that this mol50 ecule can be considered as poorly toxic to ¸. minor under static conditions. It should be kept in mind though that the inhibitory concentrations (Table 1) were estimated using the initial folpet concentrations in the medium. Since folpet readily hydrolyzes in water and a half-life of 0.8 h was reported for the pure active ingredient of the fungicide (Parilla et al., 1996), the actual toxicity of the fungicide may be substantially higher. Under the present experimental conditions folpet (used in its commercial preparation) had a half-life of 24 h (data not provided). Therefore, a better estimation of the phytotoxic potential of folpet was obtained from the experiments conducted under semistatic conditions in which the concentrations of folpet were readjusted daily (Fig. 3). IC values obtained with this method were lower (Table 2). According to European Directive L180/63 relative to the aquatic environment (EEC, 1991),
with such IC values associated with its log K '3 50 08 (Tomblin, 1994), folpet could be classified as a substance that is ‘‘toxic for the aquatic organisms.’’ In decantation basins collecting vineyard runoff water, folpet could not be detected (data not provided) reducing its toxicity to the environment. Folpet could not be recovered in the environment because (1) it is rapidly degraded in water (punctual contamination of the environment by folpet was thus best described by static experiments, in which the initial quantity of folpet rapidly decreased leading to higher IC values), and (2) its low water solubility (1 mg liter~1 at 20°C) and strong adsorption to soils (Tomblin, 1994) suggested that after it is released in the environment, in case it is not degraded, folpet is expected to absorb onto soil and sediment rather than dissolve in water. The toxicity of folpet is not completely explained (Buchenauer, 1990). Like other N-trichloromethylthio fungicides, the molecule is supposed to react with thiol groups in the cells, with thiophosgene and carbonyl sulfide as intermediary products. The binding of sulfur-containing fragments of the fungicide to protein was evoked to explain the toxicity of folpet (Siegel and Sisler, 1968; Dalvi and Mutinga, 1990), these adducts being responsible for conformational changes in the proteins that became inactivated. The stimulation of catalase activity by folpet (Fig. 5) suggests that the fungicide generated oxidative stress. Similar to copper, folpet would then exert its toxicity (at least partially) by possible peroxidation of the lipids. Further research needs to be conducted in this direction to define the extent of this pathway in the toxicity of folpet. Since pesticides never occur alone in the environment but rather in combination, experiments were conducted in which the plants were exposed simultaneously to both folpet and copper. Under static conditions, which best illustrated punctual environmental pollution, a slight synergism
FIG. 5. Catalase activity of ¸emna minor L. after the fronds were cultivated 24 h in nutrient medium contaminated with folpet.
199
TOXICITY OF FOLPET AND COPPER
(Table 3) was observed for the lowest folpet concentrations, whereas at higher concentrations (25—30 mg liter~1) the two contaminants were antagonistic. Under semistatic conditions (Table 4), in which the concentrations tested were lower, no synergism or antagonism could be found. Rather, the results suggested a simple additivity of the effects of both compounds. To explain the antagonism observed under static conditions complexation of copper by folpet may be evoked, which would reduce the bioavailability of copper, thereby lowering its toxicity. Such chelation or complexation of copper was described with other pesticides such as glyphosate (Undabeytia et al., 1996) and imazapyr (Duda et al., 1996). However, determination of available Cu2` with a specific electrode (Elit 227, BPS Nico, London, UK) demonstrated no significant influence of the presence of folpet on the concentration of available copper in the medium. Induction of catalase activity by folpet (Fig. 5) could also be proposed to explain antagonism. Indeed, this induction may harden the cells against oxidative stress generated by copper. Catalase was very sensitive to both pollutants tested in this study since copper levels as low as 25 lg liter~1 or 2.5 mg liter~1 folpet were enough to significantly stimulate the activity of the enzyme after 24 h of incubation. This high sensitivity suggests the possible use of catalase as a biomarker of exposure. Such a test is already used for aquatic animals (Di Giulio et al., 1989; Doyotte et al., 1997) but it remains rarely used for plants (Subhadra et al., 1994). With ¸emna this test was very easy to realize, did not require any expensive equipment or chemicals, and gave results in a short time (24 h). It needs to be further developed and its use has to be generalized with other molecules and ecotoxicological studies. CONCLUSIONS
Among the two chemicals tested, copper was highly toxic to ¸. minor. However, although folpet could be classified as ‘‘toxic for the aquatic organisms’’ according to the European directive (EEC, 1991), it did not appear to present any ecotoxicological risks for this species in nature. The main hazard generated by folpet was its potential synergism with copper observed at low concentrations (which are more likely in the environment), reinforcing the ecotoxicological risk of both pollutants. This synergism might even be more important when more pollutants interact. So synergism and also simple additivity of effects should be kept in mind when planning experiments to assess the ecotoxicological risk of a compound or industrial and agricultural wastewaters. Catalase activity of ¸. minor was found to be very sensitive and its use as a biomarker of exposure to oxidative stress-generating substances may be envisioned.
ACKNOWLEDGMENTS This work was financed in part by Europol’ Agro through the program ‘‘Toxicologie—e´cotoxicologie des pesticides et des me´taux lourds.’’ Thanks are due to Dr. J. Roederer, Makhteshim-Agan, France, for the gift of Folpan and blank formulation.
REFERENCES Anonymous (1995). Chemical review: Folpet. Dangerous Properties Ind. Mater. Rep. 15, 2—32. Baro´n, M., Arellano, J. B., and Gorge´, J. L. (1995). Copper and photosystem II: A controversial relationship. Physiol. Plant. 94, 174—180. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248—254. Buchenauer, H. (1990) Physiological reactions in the inhibition of plant pathogenic fungi. In Chemistry of Plant Protection (W. S. Bowers, W. Ebing, D. Martin, and R. Wegler, Eds.), Vol. 6, pp. 217—292. SpringerVerlag, Berlin. Chollet, R. (1993). Screening of inhibitors (antimetabolites) of the biosynthesis or function of amino acids or vitamins with a Lemna assay. In Target Assays for Modern Herbicides and Related Phytotoxic Compounds (P. Bo¨ger and G. Sandmann, Eds.), pp. 143—149. Lewis, Boca Raton, FL. Dalvi, R. R., and Mutinga, M. L. (1990). Comparative studies on the effects on liver and liver microsomal drug-metabolising enzyme system by the fungicides captan, captafol and folpet in rats. Pharmacol. Toxicol. 66, 231—233. Di Giulio, R. T., Washburn, P. C., Wenning, R. J., Winston, G. W., and Jewell, C. S. (1989). Biochemical responses in aquatic animals: A review of determinant oxidative stress. Environ. Toxicol. Chem. 8, 1103—1123. Doyotte, A., Cossu, C., Jacquin, M. C., Babut, M., and Vasseur, P. (1997). Antioxidant enzymes, glutathione and lipid peroxidation as relevant biomarkers of experimental or field exposure in the gills and the digestive glands of the freshwater bivalve ºnı& o tumidus. Aquat. Toxicol. 39, 93—110. Duda, A. M., Dyba, M., Kozlowski, H., Micera, G., and Pusino, A. (1996). Copper(II) complexes of imidazolinone herbicide imazapyr. J. Agr. Food Chem. 44, 3698—3702. EEC (1991). European directive No. L 180/63, 8 July 1991. Off. J. Eur. Communities. Filbin, G. J., and Hough, R. A. (1979). The effects of excess copper sulphate on the metabolism of the duckweed Lemna minor. Aquat. Bot. 7, 79—86. Foyer, C. H., Lelandais M., and Kunert K. J. (1994). Photooxidative stress in plants. Physiol. Plant. 92, 696—717. Fridovich, I. (1978). The biology of oxygen radicals. Science 201, 875—880. Gisi, U. (1996). Synergistic interaction of fungicides in mixtures. Phytopathology 86, 1273—1279. Jenner, H. A., and Janssen-Mommen, J. P. M. (1993). Duckweed Lemna minor as a tool for testing toxicity of coal residues and polluted sediments. Arch. Environ. Contam. Toxicol. 25, 3—11. Lewis, M. A. (1993). Freshwater primary producers. In Handbook of Ecotoxicology, 1 (P. Calow, Ed.), pp. 28—50. Blackwell Scientific, Oxford. Lewis, M. A. (1995). Use of freshwater plants for phytotoxicity testing: A review. Environ. Pollut. 87, 319—336. Lichtenthaler H. K. (1996). Vegetation stress: An introduction to the stress concept in plants. J. Plant. Physiol. 148, 4—14.
200
TEISSEIRE, COUDERCHET, AND VERNET
Mattoo, A. K., Baker, J. E., and Moline, H. E. (1986). Induction by copper ions of ethylene production in Spirodela oligorhiza: Evidence for a pathway independent of 1-aminocyclopropane-1-carboxylic acid. J. Plant Physiol. 123, 193—202. McKinney, G. (1941). Absorption of light by chlorophyll solutions. J. Biol. Chem. 140, 315—322. Nemcso´k J., and Benedeczky I. (1995). Pesticide metabolism and the adverse effects of metabolites on fishes. In Biochemistry and Molecular Biology of Fishes (Hochachka and Mommsen, Eds.), Vol. 5, pp. 313—348. Elsevier Science, Amsterdam. Parilla, P., Martinez-Vidal, J. L., Martinez-Galera, M., and GarridoFrenich, A. (1996). Degradation of fenamiphos and folpet in water. Int. J. Environ. Anal. Chem. 63, 137—145. Siegel, M. R., and Sisler, H. D. (1968). Reactions of folpet with purified enzymes, nucleic acids and subcellular components of Saccharomyces pastorianus. Phytopathology 58, 1129—1133.
Subhadra, A. V., Nanda, A. K., Behera, P. K., and Panda, B. B. (1991). Acceleration of catalase and peroxidase activities in Lemna minor L. and Allium cepa L. in response to low levels of aquatic mercury. Environ. Pollut. 69, 169—179. Tomblin C. (1994). ¹he Pesticide Manual (C. Tomblin Ed.), 10th ed., pp. 518—519. Crop Protection Publ., Farnham, UK. Undabeytia, T., Ceshire, M. V., and McPhail, D. (1996). Interaction of the herbicide glyphosate with copper in humic complexes. Chemosphere 32, 1245—1250. Vavilin, D. V., Polynov, V. A., Matorin, D. N., and Venediktov, P. S. (1995). Sublethal concentrations of copper stimulate photosystem II photoinhibition in Chlorella pyrenoidosa. J. Plant Physiol. 146, 609—614. Walbridge, C. T. (1977). A Flow Testing Procedure with Duckweed (Lemna minor). U.S. Technical Report No. EPA-600/3-77-108, USEPA, Duluth, MN. Wang, W. (1990). Literature review on duckweed toxicity testing. Environ. Res. 52, 7—22.
40, 194—200 (1998)
ENVIRONMENTAL RESEARCH, SECTION B ARTICLE NO.
ES981682
Toxic Responses and Catalase Activity of Lemna minor L. Exposed to Folpet, Copper, and Their Combination1 H. Teisseire, M. Couderchet, and G. Vernet Laboratoire de Zoologie et des Sciences de l+Environnement, BaL timent Europol+Agro, Faculte´ des Sciences, Universite´ de Reims Champagne-Ardenne, BP 1039, F-51687 Reims-02, France Received April 1, 1997
Toxicity of copper and folpet—two fungicides widely used on grape—was evaluated on Lemna minor L., a sensitive aquatic weed regularly used for (eco)toxicological studies. Toxicity assessments were based on inhibition of growth and chlorophyll content of L. minor cultures after 7 days. IC10 , IC50 , and IC90 were determined for both compounds alone and were respectively, 0.03, 0.16, and 0.95 mg liter21 for copper and 1.20, 7.50, and >40 mg liter21 for folpet. When both compounds were combined, the response of L. minor depended on the initial folpet concentration. Indeed, a slight synergy was observed for 5 mg liter21 folpet, while at folpet concentrations of 20 to 35 mg liter21, the two fungicides were antagonists. The antagonism was positively correlated with folpet concentration. Antagonism between Cu and folpet could not be explained by a reduced bioavailability of Cu since concentration of free copper in the mixture did not depend on the presence of folpet. One physiological defense response elicited by copper in plants is an increase in catalase activity. Copper and folpet stimulated catalase activity and changes in the activity of the enzyme could not account for the synergy but possibly for the antagonism. Nevertheless, catalase activity increased significantly after a 24-h exposure to 25 lg liter21 of copper. The use of this property as a rapid and sensitive biomarker to monitor the toxicity of xenobiotics alone or in combination and of environmental water is discussed. ( 1998 Academic Press Key Words: antagonism; catalase; copper; duckweed; folpet; fungicide; grapevine; Lemna minor; synergy.
INTRODUCTION
Runoff is an important phenomenon on the slopes of septentrional grape-growing areas, and pesticides and other chemicals may end up in surface waters, either dissolved in runoff water or adsorbed to soil particles. These polluted waters may then connect the hydrographic system downstream and generate a diffuse pollution that endangers plants and animals of the contaminated ecosystems.
Since copper and folpet (Fig. 1) control mildew and other fungal diseases in grape (Buchenauer, 1990), these two fungicides are generously sprayed on vineyards in Champagne, and they belong to the most widely used agrochemicals in those fields. As such, they are likely to be present in runoff water and present potential risks for the aquatic environment. The toxicological effects of copper are well documented for plants and animals (Filbin and Hough, 1979; Nemcso´k and Benedeczky, 1995), whereras those of folpet are rarely reported in the literature, especially for plants (Anonymous, 1995). Furthermore, the two fungicides, which are often applied together in the field, may interact on the plant surface and in the pathogenic fungi, but also in the environment. There, the presence of one of the pesticides may influence that of the other one, and their toxicology is more likely to be due to a combined action of the pesticides than to individual effects. Therefore, the toxicity of folpet and copper was examined and the potential risks resulting from their combination were investigated in duckweed (¸emna minor L.), a sensitive aquatic plant regularly used for ecotoxicological studies because of its small size, ease of handling and culture, and fast growth rate (Lewis, 1995). Furthermore, folpet seems to be a multitarget fungicide, the mode of action of which is not totally elucidated (Buchenauer, 1990). In contrast, understanding of the mode of action of copper in plants is rapidly expanding (Baro´n et al., 1995). One important effect of copper is an oxidative stress followed by elicitation of several defense enzymes such as catalase (Foyer et al., 1994). The possible involvement of oxidative stress in the toxicity of folpet is unknown. In this study the effect of both copper and folpet on catalase was therefore investigated. MATERIAL AND METHODS
Plant Material 1 Part of this work was presented at the 49th International Symposium on Crop Protection on May 6, 1997, in Gent, Belgium.
¸emna minor L. (duckweed) was collected from an artificial pond on the University campus. The plants were
194 0147-6513/98 $25.00 Copyright ( 1998 by Academic Press All rights of reproduction in any form reserved.
195
TOXICITY OF FOLPET AND COPPER
FIG. 1. Structure of folpet.
disinfected by immersing the fronds in NaOCl (0.01 M) for 20 s and rinsing with distilled water. The stock cultures were maintained in 2-liter plastic (PVC) aquariums containing 500 ml of inorganic growth medium (Chollet, 1993) that consisted of KNO , 202 mg liter~1; KH PO , 50.3 mg 3 2 4 liter~1; K HPO , 27.8 mg liter~1; K SO , 17.4 mg liter~1; 2 4 2 4 MgSO ) 7H O, 49.6 mg liter~1; CaCl , 11.1 mg liter~1; 4 2 2 Na EDTA, 10 mg liter~1; FeSO ) 7H O, 6 mg liter~1; 2 4 2 H BO , 5.72 mg liter~1; MnCl ) 4H O, 2.82 mg liter~1; 3 3 2 2 ZnSO , 0.6 mg liter~1; (NH )Mo O ) 4H O, 0.043 mg 4 4 7 24 2 liter~1; CuCl ) 2H O, 0.078 mg liter~1; CoCl ) 6H O, 2 2 2 2 0.054 mg liter~1. Before the medium was autoclaved its pH was adjusted to 6.5. The aquariums were placed in a controlled environment room at 25$1°C under continuous illumination provided by cool white fluorescent lamps (Sylvania Grolux F 36 W) with a light intensity of 2000$ 100 lx, and plants were subcultured twice a week. Agrochemicals Used A 100 mg liter~1 stock solution of copper (CuSO )5H O, 4 2 analytical grade, Sigma, Saint-Quentin-Fallavier, France) was prepared in distilled water. The commercial preparation of folpet, Folpan 500, containing 500 g liter~1 active ingredient (a.i.) was used and a 1% (v/v) stock solution was prepared just before the experiments were run. A blank formulation (without a.i.) was used for the controls. Both Folpan 500 and the blank formulation were generous gifts of Makhteshim Agan France. Cu and Folpet Treatments For toxicity experiments, six double-fronded ¸emna colonies of approximately equal size were transferred to 100-ml crystallizing dishes containing 50 ml of growth medium to which Cu and/or folpet had been added. The growth medium was the same as above for experiments with folpet alone but it did not contain EDTA when the experiments included Cu to avoid any interaction between the ion and the chelator (Wang, 1990). The experiments were conducted either under static or under semistatic conditions. Under static conditions, the
medium was not renewed and the concentration of active Cu ion or folpet (a.i.) was set at the beginning of the experiments. It was 0.05, 0.2, 0.4, 0.6, and 1 mg liter~1 for copper and 5, 20, 40, 70, and 100 mg liter~1 for folpet. Under semistatic conditions the fronds were transferred every day to a clean crystallizing dish containing fresh medium in which the contaminant concentration was readjusted to the initial values, which were 5, 10, 20, 30, and 40 mg liter~1 for folpet. No semistatic experiment was run with copper alone because the free copper concentration in the medium almost did not vary during the course of the experiments. In experiments including folpet, the quantity of blank formulation was the same in the controls and in the samples that received the highest dose of folpan (corresponding to 40 mg liter~1 a.i.). Formulation-free controls were also run. Fronds were counted daily and values of 0.25, 0.5, 0.75, and 1 were attributed to the fronds depending on their development. Relative growth was estimated after 7 days by integrating the area between the growth curve and the horizontal line y"n , where n is the initial number of 0 0 fronds in the colony (usually 12). Total chlorophyll (Chl) content was determined according to McKinney (1941) after 7 days of incubation. IC values were estimated graphically using relative 50 growth and Chl content inhibition curves. Combination of Cu and Folpet Experiments were conducted in EDTA-free media as described previously for individual contaminants. Sublethal concentrations of Cu and folpet were used, which can be seen under Results (Tables 3 and 4). In each sample, the quantity of blank formulation was adjusted to 0.07& (v/v). Experiments were run either under static conditions in which the media were not renewed (Table 3) or under semistatic conditions in which the media were renewed daily as above (Table 4). Possible interactions between copper and folpet were estimated using Abott’s formula (Gisi, 1996). In this widely used model, the expected inhibition of the mixture, expressed as percent C , can be predicted as %91 C
%91
"A#B!(AB/100),
where A and B are the inhibition levels given by the single chemicals (respectively copper and folpet). The ratio of inhibition (RI) was then calculated as follows for each pesticide combination: RI"C /experimentally observed efficacy. %91 Synergism or antagonism was evaluated by comparing the RI with 1. A RI (1 indicated synergism between the
196
TEISSEIRE, COUDERCHET, AND VERNET
formed for each concentration and all experiments were repeated twice. RESULTS
FIG. 2. Influence of folpet on growth and total chlorophyll content of ¸emna minor L. after a 7-day exposure to the fungicide. The plants were maintained under static conditions.
two contaminants; a RI of 1, simple additivity; and a RI'1, antagonism between the chemicals. Catalase Assays Approximately 15 double-fronded colonies of ¸. minor were exposed to various concentrations of both chemicals (0 to 600 lg liter~1 copper and 0 to 40 mg liter~1 folpet) according to the procedure described above. After 24 h of incubation, catalase activity was determined according to Subhadra et al. (1991) with some modifications. The fronds were homogenized in cold 0.1 M sodium phosphate buffer (pH 7) with a Potter homogenizer (Servodyne mixer head, Cole Parmer, Niles, IL) and centrifuged 15 min at 2300g and 4°C. The supernatant was used as the enzyme extract. One milliliter of enzyme extract was added to the reaction mixture consisting of 0.01 M hydrogen peroxide (1 ml) and 0.1 M sodium phosphate buffer (3 ml, pH 7). After 5 min incubation in a water bath at 22°C, the reaction was stopped by addition of 10 ml of 2% H SO . The acidified 2 4 reaction mixture was titrated against 0.005 N KMnO to 4 determine the quantity of hydrogen peroxide consumed by the enzyme. The total protein content of each enzyme extract was determined according Bradford (1976) using bovine albumin (Sigma) for calibration. The catalase activity was expressed as millimoles of hydrogen peroxide consumed per milligram of protein and per minute.
Plants contaminated by copper sulfate were chlorotic and their fronds were smaller than those of controls. After 1 day of exposure to Cu the two fronds associated in colonies separated and lost their roots. Subsequently, after 2 to 3 days of incubation and for Cu concentrations of 0.2 and 0.4 mg liter~1, new fronds no longer separated from mother plants, forming colonies of six or seven fronds, whereas in controls the number of associated fronds per colony never exceeded 4. Under static conditions (when the medium was not renewed) the lowest folpet concentration (5 mg liter~1) stimulated growth. Above that concentration folpet inhibited growth of ¸emna (Fig. 2). The plants exposed to higher concentrations were chlorotic and had lost their roots. Under semistatic conditions all concentrations of folpet tested inhibited growth (Fig. 3). In contrast to what was observed under static conditions, 5 mg liter~1 folpet was enough to induce significant inhibition of growth and reduction of chlorophyll content. Concentrations of the two chemicals inhibiting 10, 50, and 90% (IC , IC , and IC ) of growth of ¸. minor are 10 50 90 listed in Table 1. Similar values were found when chlorophyll content was used as a marker of copper toxicity (Table 2). Correlation between growth and chlorophyll content was not as good with folpet (Figs. 2 and 3), for which chlorophyll appeared to be a more sensitive marker than growth (compare Tables 1 and 2).
Data Reliability and Presentation IC determination experiments were repeated at least twice and each sample was triplicated. When the combination of folpet and copper was studied, experiments were triplicated. For catalase assays, three replicates were per-
FIG. 3. Influence of folpet on growth and total chlorophyll content of ¸emna minor L. after a 7-day exposure to the fungicide. The plants were maintained under semistatic conditions; that is, the medium was changed daily and the folpet concentration in the medium was adjusted daily.
197
TOXICITY OF FOLPET AND COPPER
TABLE 1 Inhibition of Lemna minor Growth by Copper and Folpet Chemical
Conditions
Copper Folpet
Static Static Semistatic
IC
10
0.03$0.02 7.8$5.0 1.2$0.2
IC 50 0.16$0.05 50$15 7.5$2.5
IC
90
0.95$0.13 '100 '40
TABLE 3 Ratios (RI) of Theoretical to Observed Growth Inhibition of Lemna minor L. by Mixtures of Copper and Folpet under Static Conditions Copper (mg liter~1) Folpet (mg liter~1)
0.05
0.15
0.25
0.920$0.034 1.200$0.085 1.34$0.16
0.915$0.073 1.10$0.22 1.23$0.25
0.990$0.089 1.42$0.13 1.30$0.28
Note. The plants were cultivated 7 days in nonrenewed (static conditions) or renewed (semistatic conditions) medium. Inhibitory concentrations are expressed in mg liter~1.
5 20 35
When ¸. minor was exposed to mixtures of copper and folpet under static conditions, the comparison between the expected inhibition (C ) and experimentally observed %91 growth inhibition revealed two different responses (Table 3). For the lowest folpet concentration (5 mg liter~1), the experimentally observed growth inhibition values were superior to C and the RIs were slightly less than 1, %91 indicating a potential synergism between copper and folpet (Table 3). In contrast, for the highest folpet concentrations tested, some of the ratios were greater than 1, indicating antagonism between the two chemicals. Under semistatic conditions, the experimentally observed inhibition values were not significantly different from C %91 as indicated by the RI values presented in Table 4. In this case, the results suggested a simple additivity of the effects of the two chemicals. Catalase activity of control ¸. minor was variable and was approximately 20 mmol H O mg protein~1 min~1. The 2 2 presence of blank formulation in the controls of folpet experiments yielded somewhat superior activity (27.40$ 0.96 mmol H O mg protein~1 min~1). Catalase activity 2 2 was higher in the plants that had been exposed 24 h to Cu (Fig. 4) or folpet (Fig. 5). This increase in enzyme activity was significant for concentrations as low as 0.025 mg liter~1 copper and 2.5 mg liter~1 folpet, indicating the very high sensitivity of the enzyme to these pollutants. Enzyme activity increased with the concentration of pollutant up to 35 to
40 mmol H O mg protein~1 min~1, this activity being 2 2 reached with 0.10 mg liter~1 CuSO and 10 mg liter~1 fol4 pet. Further increasing the concentration of pollutant yielded a decrease in enzyme activity (Figs. 4 and 5).
TABLE 2 Inhibition of Chlorophyll Content of Lemna minor L. Colonies by Copper and Folpet Chemical
Conditions
Copper Folpet
Static Static Semi-static
IC
10
0.03$0.015 6.6$3.1 0.90$0.02
IC 50 0.16$0.04 22.8$6 4.4$0.6
IC
DISCUSSION
The IC values obtained for copper (Tables 1 and 2) were 50 in agreement with the 0.12 mg liter~1 or 0.14—0.32 mg liter~1 reported earlier for ¸emna (Walbridge, 1977; Jenner and Janssen-Mommen, 1993); they indicate that this metal is highly toxic to ¸. minor. The toxicity of the metal is confirmed by the very low IC values. These IC values 10 10 corresponded to those measured in decantation basins that collect runoff waters from the vineyards (unpublished data). Symptoms observed (chlorosis, colony breakup, root loss, etc.) in ¸. minor after exposure to CuSO reflect the intensity 4 and diversity of the disorders generated by Cu2` ions in cell metabolism. Chlorosis is a common response of plants to stress (heat, diseases, pollution) and has been observed after copper treatment in various aquatic plants such as Eichhornia crassipes and Hydrilla verticillata (Lewis, 1993), Chlorella pyrenoidosa (Vavilin et al., 1995), and ¸emna sp. (Filbin and Hough, 1979). Among the variety of targets reported for copper in plants, the photosynthetic apparatus seems the most sensitive (Baro´n et al., 1995). This effect of copper entrains an oxidative stress followed by peroxida-
TABLE 4 Ratios (RI) of Theoretical to Observed Gowth Inhibition of Lemna minor L. by Mixtures of Copper and Folpet under Semistatic Conditions
90
0.88$0.15 '100 '40
Note. The plants were cultivated 7 days in nonrenewed (static conditions) or renewed (semistatic conditions) medium. Inhibitory concentrations are expressed in mg liter~1.
Copper (mg liter~1) Folpet (mg liter~1) 1.6 3.2 4.8
0.016
0.032
0.048
1.37$0.74 1.03$0.20 0.99$0.13
1.12$0.13 1.34$0.32 1.11$0.27
1.07$0.28 1.20$0.16 1.29$0.25
198
TEISSEIRE, COUDERCHET, AND VERNET
FIG. 4. Catalase activity of ¸emna minor L. after the fronds were cultivated 24 h in nutrient medium contaminated with CuSO . 4
tion of membranes and pigments (Mattoo et al., 1986). This oxidative stress explains the induction of catalase activity observed in response to copper contamination (Fig. 4) because catalase induction is part of the defense mechanism in response to increasing concentrations of reactive oxygen species responsible for lipid peroxidation (Fridovich, 1978; Foyer et al., 1994). The stimulation of catalase activity in response to copper (and also to folpet) occurred at very low contaminant concentration; at higher concentration it reached a maximum and decreased again. This decrease can be interpreted as a classical stress response in which the intensity of the stress is too high and the stage of exhaustion is reached (Lichtenthaler, 1996). The stimulation of growth in response to the lowest folpet concentration tested under static conditions (Fig. 1) had already been reported in plants (Buchenauer, 1990). It is even common practice among farmers to use this property of the fungicide to heal grape leaves after a storm. Folpet IC values (Table 1) demonstrated that this mol50 ecule can be considered as poorly toxic to ¸. minor under static conditions. It should be kept in mind though that the inhibitory concentrations (Table 1) were estimated using the initial folpet concentrations in the medium. Since folpet readily hydrolyzes in water and a half-life of 0.8 h was reported for the pure active ingredient of the fungicide (Parilla et al., 1996), the actual toxicity of the fungicide may be substantially higher. Under the present experimental conditions folpet (used in its commercial preparation) had a half-life of 24 h (data not provided). Therefore, a better estimation of the phytotoxic potential of folpet was obtained from the experiments conducted under semistatic conditions in which the concentrations of folpet were readjusted daily (Fig. 3). IC values obtained with this method were lower (Table 2). According to European Directive L180/63 relative to the aquatic environment (EEC, 1991),
with such IC values associated with its log K '3 50 08 (Tomblin, 1994), folpet could be classified as a substance that is ‘‘toxic for the aquatic organisms.’’ In decantation basins collecting vineyard runoff water, folpet could not be detected (data not provided) reducing its toxicity to the environment. Folpet could not be recovered in the environment because (1) it is rapidly degraded in water (punctual contamination of the environment by folpet was thus best described by static experiments, in which the initial quantity of folpet rapidly decreased leading to higher IC values), and (2) its low water solubility (1 mg liter~1 at 20°C) and strong adsorption to soils (Tomblin, 1994) suggested that after it is released in the environment, in case it is not degraded, folpet is expected to absorb onto soil and sediment rather than dissolve in water. The toxicity of folpet is not completely explained (Buchenauer, 1990). Like other N-trichloromethylthio fungicides, the molecule is supposed to react with thiol groups in the cells, with thiophosgene and carbonyl sulfide as intermediary products. The binding of sulfur-containing fragments of the fungicide to protein was evoked to explain the toxicity of folpet (Siegel and Sisler, 1968; Dalvi and Mutinga, 1990), these adducts being responsible for conformational changes in the proteins that became inactivated. The stimulation of catalase activity by folpet (Fig. 5) suggests that the fungicide generated oxidative stress. Similar to copper, folpet would then exert its toxicity (at least partially) by possible peroxidation of the lipids. Further research needs to be conducted in this direction to define the extent of this pathway in the toxicity of folpet. Since pesticides never occur alone in the environment but rather in combination, experiments were conducted in which the plants were exposed simultaneously to both folpet and copper. Under static conditions, which best illustrated punctual environmental pollution, a slight synergism
FIG. 5. Catalase activity of ¸emna minor L. after the fronds were cultivated 24 h in nutrient medium contaminated with folpet.
199
TOXICITY OF FOLPET AND COPPER
(Table 3) was observed for the lowest folpet concentrations, whereas at higher concentrations (25—30 mg liter~1) the two contaminants were antagonistic. Under semistatic conditions (Table 4), in which the concentrations tested were lower, no synergism or antagonism could be found. Rather, the results suggested a simple additivity of the effects of both compounds. To explain the antagonism observed under static conditions complexation of copper by folpet may be evoked, which would reduce the bioavailability of copper, thereby lowering its toxicity. Such chelation or complexation of copper was described with other pesticides such as glyphosate (Undabeytia et al., 1996) and imazapyr (Duda et al., 1996). However, determination of available Cu2` with a specific electrode (Elit 227, BPS Nico, London, UK) demonstrated no significant influence of the presence of folpet on the concentration of available copper in the medium. Induction of catalase activity by folpet (Fig. 5) could also be proposed to explain antagonism. Indeed, this induction may harden the cells against oxidative stress generated by copper. Catalase was very sensitive to both pollutants tested in this study since copper levels as low as 25 lg liter~1 or 2.5 mg liter~1 folpet were enough to significantly stimulate the activity of the enzyme after 24 h of incubation. This high sensitivity suggests the possible use of catalase as a biomarker of exposure. Such a test is already used for aquatic animals (Di Giulio et al., 1989; Doyotte et al., 1997) but it remains rarely used for plants (Subhadra et al., 1994). With ¸emna this test was very easy to realize, did not require any expensive equipment or chemicals, and gave results in a short time (24 h). It needs to be further developed and its use has to be generalized with other molecules and ecotoxicological studies. CONCLUSIONS
Among the two chemicals tested, copper was highly toxic to ¸. minor. However, although folpet could be classified as ‘‘toxic for the aquatic organisms’’ according to the European directive (EEC, 1991), it did not appear to present any ecotoxicological risks for this species in nature. The main hazard generated by folpet was its potential synergism with copper observed at low concentrations (which are more likely in the environment), reinforcing the ecotoxicological risk of both pollutants. This synergism might even be more important when more pollutants interact. So synergism and also simple additivity of effects should be kept in mind when planning experiments to assess the ecotoxicological risk of a compound or industrial and agricultural wastewaters. Catalase activity of ¸. minor was found to be very sensitive and its use as a biomarker of exposure to oxidative stress-generating substances may be envisioned.
ACKNOWLEDGMENTS This work was financed in part by Europol’ Agro through the program ‘‘Toxicologie—e´cotoxicologie des pesticides et des me´taux lourds.’’ Thanks are due to Dr. J. Roederer, Makhteshim-Agan, France, for the gift of Folpan and blank formulation.
REFERENCES Anonymous (1995). Chemical review: Folpet. Dangerous Properties Ind. Mater. Rep. 15, 2—32. Baro´n, M., Arellano, J. B., and Gorge´, J. L. (1995). Copper and photosystem II: A controversial relationship. Physiol. Plant. 94, 174—180. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248—254. Buchenauer, H. (1990) Physiological reactions in the inhibition of plant pathogenic fungi. In Chemistry of Plant Protection (W. S. Bowers, W. Ebing, D. Martin, and R. Wegler, Eds.), Vol. 6, pp. 217—292. SpringerVerlag, Berlin. Chollet, R. (1993). Screening of inhibitors (antimetabolites) of the biosynthesis or function of amino acids or vitamins with a Lemna assay. In Target Assays for Modern Herbicides and Related Phytotoxic Compounds (P. Bo¨ger and G. Sandmann, Eds.), pp. 143—149. Lewis, Boca Raton, FL. Dalvi, R. R., and Mutinga, M. L. (1990). Comparative studies on the effects on liver and liver microsomal drug-metabolising enzyme system by the fungicides captan, captafol and folpet in rats. Pharmacol. Toxicol. 66, 231—233. Di Giulio, R. T., Washburn, P. C., Wenning, R. J., Winston, G. W., and Jewell, C. S. (1989). Biochemical responses in aquatic animals: A review of determinant oxidative stress. Environ. Toxicol. Chem. 8, 1103—1123. Doyotte, A., Cossu, C., Jacquin, M. C., Babut, M., and Vasseur, P. (1997). Antioxidant enzymes, glutathione and lipid peroxidation as relevant biomarkers of experimental or field exposure in the gills and the digestive glands of the freshwater bivalve ºnı& o tumidus. Aquat. Toxicol. 39, 93—110. Duda, A. M., Dyba, M., Kozlowski, H., Micera, G., and Pusino, A. (1996). Copper(II) complexes of imidazolinone herbicide imazapyr. J. Agr. Food Chem. 44, 3698—3702. EEC (1991). European directive No. L 180/63, 8 July 1991. Off. J. Eur. Communities. Filbin, G. J., and Hough, R. A. (1979). The effects of excess copper sulphate on the metabolism of the duckweed Lemna minor. Aquat. Bot. 7, 79—86. Foyer, C. H., Lelandais M., and Kunert K. J. (1994). Photooxidative stress in plants. Physiol. Plant. 92, 696—717. Fridovich, I. (1978). The biology of oxygen radicals. Science 201, 875—880. Gisi, U. (1996). Synergistic interaction of fungicides in mixtures. Phytopathology 86, 1273—1279. Jenner, H. A., and Janssen-Mommen, J. P. M. (1993). Duckweed Lemna minor as a tool for testing toxicity of coal residues and polluted sediments. Arch. Environ. Contam. Toxicol. 25, 3—11. Lewis, M. A. (1993). Freshwater primary producers. In Handbook of Ecotoxicology, 1 (P. Calow, Ed.), pp. 28—50. Blackwell Scientific, Oxford. Lewis, M. A. (1995). Use of freshwater plants for phytotoxicity testing: A review. Environ. Pollut. 87, 319—336. Lichtenthaler H. K. (1996). Vegetation stress: An introduction to the stress concept in plants. J. Plant. Physiol. 148, 4—14.
200
TEISSEIRE, COUDERCHET, AND VERNET
Mattoo, A. K., Baker, J. E., and Moline, H. E. (1986). Induction by copper ions of ethylene production in Spirodela oligorhiza: Evidence for a pathway independent of 1-aminocyclopropane-1-carboxylic acid. J. Plant Physiol. 123, 193—202. McKinney, G. (1941). Absorption of light by chlorophyll solutions. J. Biol. Chem. 140, 315—322. Nemcso´k J., and Benedeczky I. (1995). Pesticide metabolism and the adverse effects of metabolites on fishes. In Biochemistry and Molecular Biology of Fishes (Hochachka and Mommsen, Eds.), Vol. 5, pp. 313—348. Elsevier Science, Amsterdam. Parilla, P., Martinez-Vidal, J. L., Martinez-Galera, M., and GarridoFrenich, A. (1996). Degradation of fenamiphos and folpet in water. Int. J. Environ. Anal. Chem. 63, 137—145. Siegel, M. R., and Sisler, H. D. (1968). Reactions of folpet with purified enzymes, nucleic acids and subcellular components of Saccharomyces pastorianus. Phytopathology 58, 1129—1133.
Subhadra, A. V., Nanda, A. K., Behera, P. K., and Panda, B. B. (1991). Acceleration of catalase and peroxidase activities in Lemna minor L. and Allium cepa L. in response to low levels of aquatic mercury. Environ. Pollut. 69, 169—179. Tomblin C. (1994). ¹he Pesticide Manual (C. Tomblin Ed.), 10th ed., pp. 518—519. Crop Protection Publ., Farnham, UK. Undabeytia, T., Ceshire, M. V., and McPhail, D. (1996). Interaction of the herbicide glyphosate with copper in humic complexes. Chemosphere 32, 1245—1250. Vavilin, D. V., Polynov, V. A., Matorin, D. N., and Venediktov, P. S. (1995). Sublethal concentrations of copper stimulate photosystem II photoinhibition in Chlorella pyrenoidosa. J. Plant Physiol. 146, 609—614. Walbridge, C. T. (1977). A Flow Testing Procedure with Duckweed (Lemna minor). U.S. Technical Report No. EPA-600/3-77-108, USEPA, Duluth, MN. Wang, W. (1990). Literature review on duckweed toxicity testing. Environ. Res. 52, 7—22.