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Photosynthetic responses of Lemna minor exposed to xenobiotics, copper, and their combinations
Ecotoxicology and Environmental Safety 53 (2002) 439–445
Photosynthetic responses of Lemna minor exposed to xenobiotics, copper, and their combinations C. Frankart,* P. Eullaffroy, and G. Vernet Laboratory of Eco-Toxicology, Europol’Agro, Faculty of Sciences, University of Reims Champagne-Ardenne, B.P. 1039, F-51687 Reims 02, France Received 18 September 2001; accepted 5 March 2002
Abstract The effects on the photosynthetic process of copper and pesticides, used in vineyards, and their combinations, were investigated by measuring different chlorophyll fluorescence parameters in Lemna minor. Cu and flumioxazin had a severe impact on duckweed since a decrease in their photosynthetic capacity was detected after 24 h of exposure to 200 and 1 mg L1 ; respectively. However, fungicides used to control Botrytis cinerea (procymidone, pyrimethanil, and fludioxonil) seem to have no marked effects on duckweed even at very high concentrations (50 mg L1 ). Analysis of the combinations between copper (200 mg L1 ) and pesticides revealed different patterns of response: a synergistic effect was observed when Cu2þ was added to flumioxazin (1mg L1 ). In contrast, an antagonism was detected when duckweed was exposed to a mixture of Cu2þ and fludioxonil or procymidone. However, these interactions always tended toward additivity when pesticide concentrations increased. Additivity was also observed for the Cu2þ –pyrimethanil mixture at each fungicide concentration. r 2002 Elsevier Science (USA). All rights reserved. Keywords: Chlorophyll fluorescence; Lemna minor; Copper; Pesticides; Mixture; Botrytis cinerea
1. Introduction The substantial use of copper sulfate over the past 100 years on vineyards has increased the input of the heavy metal copper into the aquatic environment (Courde et al., 2001). Copper sulfate is known to control mildew and other fungal diseases on grapes and therefore is generously sprayed on vineyards. Copper is an essential micronutrient for the plant life as a constituent of several enzymes and redox catalyst in a variety of metabolic pathways (Maksymiec, 1997). However, it becomes toxic to most aquatic lifeforms, particularly aquatic plants, at concentrations only slightly higher than the optimal requirement. In excessive quantities, copper interfaces with photosynthesis and respiratory processes, protein synthesis, or development of plant ! et al., 1995; Ralph and Burchett, organelles (Baron 1998; Raven et al., 1999; Yruela et al., 2000). Beside the use of copper sulfate, some of the most widely used agrochemicals on vineyards are antibotrytic *Corresponding author. Fax: +33-3-26-91-33-47. E-mail address: [email protected] (C. Frankart).
fungicides (such as procymidone, pyrimethanil, or fludioxonil). These fungicides are used to fight the development of Botrytis cinerea, a phytopathogenic fungus including a disease named ‘‘gray mold’’ or ‘‘Botrytis’’ (Tomlin, 1997). Procymidone, a dicarboximide fungicide inhibiting spore germination and growth, causes cellular leakage and lipid peroxidation on B. cinerea (Lee et al., 1998). Pyrimethanil, an analinopyrimidine fungicide, inhibits the secretion of cell-wall-degrading enzymes required for Botrytis infection (Leroux, 1996). Finally, fludioxonil, a phenylpyrrole pesticide, is able to uncouple oxidative phosphorylation and to inhibit electron transport in B. cinerea, inducing the formation of reactive oxygen species. As a consequence, it could cause peroxidation in fungi (Steel, 1996). Herbicides are also often sprayed on vineyards especially to control adventive plants. Flumioxazin, one of these herbicides, inhibits protoporphyrinogen IX oxidase, an enzyme that converts protoporphyrinogen IX to protoporphyrin IX, a precursor of chlorophyll molecules (Duke et al., 1991). All these pesticides may be introduced into natural aquatic systems through surface water runoff after their
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application. This phenomenon is particularly widespread in Champagne (France) (Desc#otes et al., 2001), since vineyards are established on hillsides. Then the polluted waters can get into the ecosystems and generate a diffuse pollution which could damage the aquatic organisms. To determine the effects of this pollution on aquatic plant photosynthesis, the authors used a chlorophyll fluorescence-based method often applied as an effective and nondamaging tool for the elucidation of various aspects of the physiological state of the photosynthetic apparatus of plants. So this method provides basic information on the performance of photosynthesis. It has become available to distinguish several fluorescence parameters such as photosynthetic capacity, and photochemical and nonphotochemical fluorescence quenching components (Schreiber et al., 1986; Lichtenthaler, 1988; Krause and Weis, 1991; Juneau and Popovic, 1999). Because copper is a ubiquitous element in Champagne aquatic ecosystems and then can be in contact with other phytosanitary products in contaminated water, these problems were investigated to determine the effects of pesticides and copper alone and in combination on the photosynthetic apparatus of an aquatic plant, Lemna minor, regularly used for ecotoxicological studies (Wang, 1990; Fairchild et al., 1997; Teisseire and Vernet, 2000).
plants were exposed to copper at concentrations of 40, 200, and 400 mg L1 : The concentration of copper was fixed at 200 mg L1 when combined with other pesticides, in regard to its environmental representativity (Paris-Palacios, 1999). The authors used formulated flumioxazin, fludioxonil, procymidone, and pyrimethanil in this study. Their commercial names are, respectively, Pledge (Cyanimid Agro, France), Geoxe (Ciba, Switzerland), Sumisclex (Sopra/Zeneca, France), and Scala (Schering Agriculture, France). Each pesticide concentration given in this study is for the active ingredient. Flumioxazin and antibotrytic fungicides (fludioxonil, procymidone, and pyrimethanil) were tested, respectively, at concentrations of 1, 10, and 50 mg L1 ; and 1, 10, and 100 mg L1 in regard to their environmental relevance (Verdisson et al., 2000). All stock solutions of the contaminants were prepared just before the experiments. For toxicity experiments, 10 mature fronds of L. minor were transferred to 50-mL Erlenmeyer flasks containing 20 mL of growth medium where copper and/ or agrochemical (herbicide or fungicides) was added. The experiments were conducted under static conditions. Plants without added contaminants served as negative control. Triplicates of plant colonies were exposed to contaminants for 72 h:
2. Materials and methods
2.3. Combination of copper and pesticides
2.1. Plant material
Possible interaction between copper and other agrochemicals was estimated using Abott’s formula (Gisi, 1996). In this widely used model, the expected inhibition of the mixture (Cexp ), expressed as a percentage, can be predicted as
Fronds of duckweed (L. minor) were collected from pounds in the Ardennes area (France). These fronds were disinfected by immersing them in NaOCI 1% (v/v) and Tween 0.01% (v/v) for 3 min and then rinsing with distilled water for 5 min: The stock cultures were maintained in PVC aquariums containing 400 mL of inorganic autoclaved growth medium (pH 6.5) adapted from Chollet (Chollet, 1993). This medium consisted of KNO3 ; 202 mg L1 ; KH2 PO4 ; 50:3 mg L1 ; K2 HPO4 ; 27:8 mg L1 ; K2 SO4 ; 17:4 mg L1 ; MgSO4 7H2 O; 49:6 mg L1 ; CaCl2 ; 11:1 mg L1 ; FeSO4 7H2 O; 6 mg L1 ; H3 BO3 ; 5:72 mg L1 ; MnCl2 4H2 O; 2:82 mg L1 ; ZnSO4 ; 0:6 mg L1 ; ðNH4 ÞMo7 O24 4H2 O; 0:043 mg L1 ; CuCl2 2H2 O; 0:078 mg L1 ; CoCl2 6H2 O; 0:054 mg L1 : All aquariums were maintained in a growth chamber at 22711C under continuous light (100 mmol PFD m2 s1 ) provided by cool white fluorescence lamps (Sylvania Gro Lux F30W/Gro-T8, Germany). The plants were subcultured twice a week. 2.2. Exposure to agrochemicals A 50-mg Cu2þ L1 stock solution (CuSO4 5H2 O; Sigma, France) was prepared in cultural medium. The
Cexp ¼ A þ B ðA B=100Þ; where A and B are the inhibition levels given by the single chemicals. The ratio of inhibition (RI) was then calculated as follows for each contaminant combination: RI ¼ Observed inhibition=Cexp : Synergism or antagonism were evaluated by comparing RI to 1. A RI value > 1 indicates potential synergism between the two contaminants; RI ¼ 1; a simple additivity; and RIo1; an antagonism between the two chemicals. 2.4. Chlorophyll fluorescence measurements Measurements of chlorophyll fluorescence emission from the upper face of duckweed fronds were made using a pulse amplitude modulation chlorophyll fluorometer (PAM-FMS 1, Hansatech, England). Before each measurement, three fully developed fronds were taken and placed in darkness for 15 min at 22711C:
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After recording the dark signal level, the probing light beam was turned on and the steady F0 -level (ground fluorescence level) recorded. The intensity of this light was sufficiently low not to produce any significant variable fluorescence (1:6 kHz; 0:02 mmol PFD m2 s1 ). A single saturating flash (1 s; 4500 mmol m2 s1 ) was then applied to reach Fm (maximal fluorescence). Photosynthetic capacity (also named maximal quantum yield) was estimated by the ratio Fm Fo =Fm (also termed Fv =Fm ) for dark-adapted leaves (Genty et al., 1990). After the decline of the signal from Fm to its initial Fo -level, an induction kinetic was started by turning on the actinic light (200 mmol PFD m2 s1 ). During the induction with actinic light, the saturating light pulse was given every 10 s: When a steady state of the induction curve was reached (about 6 min later), the induction phase was completed by turning off the actinic light. The photochemical ðqP Þ and nonphotochemical
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ðqN Þ quenching were determined as in Schreiber et al. (1986) and Lichtenthaler and Rinderle (1988). Data presented here are the means 7 standard deviation (SD). Significant differences between controls and contaminated samples were determined by the Mann–Whitney test and P values o0:05 were considered significant (Mann and Whitney, 1952).
3. Results 3.1. Effects of copper treatments To charactersize the influence of cupric ions on the photosynthetic activity of L. minor, measurements of in vivo chlorophyll fluorescence parameters were conducted. The values of the photosynthetic capacity ðFv =Fm Þ; and photochemical ðqP Þ and nonphotochemical quenching ðqN Þ are given in Fig. 1.
Fig. 1. Photosynthetic parameters in controls ð~Þ and copper-treated fronds ((\) 40; (m) 200; and () 400 mg Cu2þ L1 ) of Lemna minor after 72 h of exposure. Data are means 7SD: (A) Photosynthetic capacity; (B) Photochemical quenching; (C) nonphotochemical quenching.
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A significant decrease (10%) in Fv =Fm was observed in L. minor exposed for 24 h to 400 mg L1 or 48 h to 200 mg L1 of copper (9%). After 72 h of exposure, the percentage of Fv =Fm inhibition reached 14% and 42% in duckweed exposed to 200 to 400 mg L1 of Cu2þ ; respectively (Fig. 1A). During these 3 days of copper exposure, the photosynthetic capacity was not significantly inhibited by 40 mg Cu2þ L1 (Fig. 1A). Inhibition of photosynthetic capacity was concentration-dependant. The same pattern with photochemical quenching (qP ) was observed (Fig. 1B). A significant decrease of 12% and 28%, was noticed after 48 h when copper was present at 200 and 400 mg L1 ; respectively. After 3 days of exposure, the inhibition reached 23% and 41% for these two copper concentrations. A significant increase of 19%, 52%, and 32% of qN was observed within 24 h of exposure for samples containing 40, 200, and 400 mg Cu2þ L1 ; respectively (Fig. 1C). However, it was noted that after an increase during the first 24 h of exposure to 400 mg Cu2þ L1 ; qN was strongly decreased.
L. minor demonstrated an important sensitivity to copper since its photosynthetic capacity and the photochemical and nonphotochemical quenching mechanisms were affected by concentrations lower than 400 mg Cu2þ L1 : However the photosynthetic capacity seems to be damaged before the qP mechanisms (significant inhibition 24 h after exposure versus 48 h). Moreover, interpretation of the qN pattern could be difficult (see Discussion). The trends of these photosynthetic processes were confirmed when their related photosynthetic parameters with the selected pesticides and their combination with copper were measured. 3.2. Effects of pesticides alone and mixtures of copper and pesticides on Lemna minor The results presented in Fig. 2 are for the shortest time of exposure necessary for contaminants to induce a significant effect in duckweed. L. minor contaminated by flumioxazin herbicide had a strong decrease in photosynthetic capacity (Fig. 2A). This decrease was observed from the first day of exposure even for the lowest
Fig. 2. Photosynthetic capacity (Fv =Fm ) of Lemna minor treated for 24, 48, or 72 h with pesticides alone or in combination with copper. Data are means 7SD: (A) Flumioxazin (24-h exposure); (B) fludioxonil (72-h exposure); (C) procymidone (72-h exposure); (D) pyrimethanil (72-h exposure). (\) Pesticide alone; (&) pesticide þ200 mg Cu2þ L1 :
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herbicide concentration: the inhibition percentages were 23, 62, and 64 for 1, 10, and 50 mg L1 of flumioxazin, respectively. However, this inhibition was higher when plants were exposed to mixtures of copper (200 mg L1 Cu2þ ) and flumioxazin (Fig. 2A). The comparison between the expected inhibition (Cexp ) and the experimentally observed Fv =Fm inhibition revealed two different responses. For the lowest flumioxazin concentration (1 mg L1 ), the experimentally observed photosynthetic capacity inhibition values were superior to Cexp and the RI was greater than 1 (1.13 7 0.05), indicating a very slight synergism or an additional effect, between copper and flumioxazin. For the higher flumioxazin concentrations (10 and 50 mg L1 ), the same results were observed: RI values close to 1 (respectively, 1:0970:23 and 0:9970:12), indicating a simple additivity or a slight synergism between the two chemicals. L. Minor exposed to antibotrytic fungicides alone had a very slight inhibition of photosynthesis (lower than 5%) at all tested concentrations after 72 h of treatment (Figs. 2B–D). However, when L. minor was exposed to a mixture of copper and fungicides, measurements of the photosynthetic capacity revealed different responses in regard to the fungicide concentrations. When duckweed was in contact for 72 h with a combination of Cu2þ (200 mg L1 ) and the two lower fludioxonil concentrations (1 and 10 mg L1 ), RI values were lower than 1 ð0:7470:12 and 0:8670:10; respectively), indicating a slight antagonism between the two contaminants. However, for the highest fludioxonil concentration (100 mg L1 ), RI was greater than 1 ð1:7170:67), indicating a synergism between the two contaminants or a simple additivity in regard to SD. After 72 h of exposure, mixtures of Cu2þ (200 mg L1 ) and procymidone had the same results as mixtures of Cu and fludioxonil. The RIs were smaller than 1 for mixture with procymidone at the lower concentrations (1 and 10 mg L1 ): 0:5770:08 and 0:6770:13; respectively, indicating a strong antagonism between the two contaminants. As had already been observed in previous mixtures (copper with flumioxazin and fludioxonil), a simple additivity of copper and pesticide was noted with increasing fungicide concentrations (100 mg L1 ): 1:1270:22: For mixture between Cu2þ and pyrimethanil, RI values were always close to 1, regardless to fungicide concentration. In this case, only an additive effect between these two contaminants was observed.
4. Discussion In this study, photosynthetic capacity and photochemical and nonphotochemical quenching were reliable to evaluate the toxicity of phytosanitary products used
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in vineyards. First, as had been observed in many cases of toxicity test, (qN ) increased with contaminant concentrations (Demming and Winter, 1988; Caja! nek et al., 1998). In this study, this increase induced by 200 and 400 mg Cu2þ L1 was followed by a decrease after 72 h: It is known that plants exposed to a long-term stress pass through different physiological states from resistance to exhaustion (Lichtenthaler, 1996). In this study, the qN decrease after 72 h of exposure to 200 and 400 mg Cu2þ L1 could indicate that the duckweed was entering the exhaustion phase. This reaction was confirmed by morphological observation revealing a separation of the fronds and large areas of chlorosis. Therefore, despite its sensitivity, the value of this parameter at a given time of exposure may not give the true degree of contaminant toxicity on the photosynthetic apparatus. In L. minor, 40 mg L1 of Cu2þ was sufficient to induce a decrease in the photosynthetic capacity, indicating that, although copper is an essential micronutrient for plant life, it can become a strong inhibitor of photosynthesis when in excess. Numerous sites were identified as targets of copper action in chloroplasts. The photosynthetic apparatus is particularly sensitive to this heavy metal, which is known to decrease the electron transfer rate, a disruption responsible for the decrease in photosynthetic capacity and photochemical quenching (Ruban and Horton, 1995; Pogson et al., 1998), or to catalyze the formation of reactive free radicals inducing oxidative damage (Navari-Izzo et al., 1998). The results clearly provided evidence that flumioxazin, as an herbicide, was very toxic to L. minor. It was a strong inhibitor of photosynthetic capacity even at a very low concentration (1 mg L1 ). This herbicide blocked the protoporphyrinogen oxidase activity, inhibiting the accumulation of protoporphyrinogen IX. This precursor of chlorophyll molecules is a potent photosensitizer that generates high levels of singulet oxygen in the presence of molecular oxygen and light (Duke et al., 1991). This metabolic disruption caused oxidative damages leading to plant death within 24 h of contamination (50 mg L1 ). Therefore, the inhibition of chlorophyll synthesis by flumioxazin may be the indirect cause of the photosynthetic capacity inhibition. A mixture of the smallest concentration of flumioxazin (1 mg L1 ) and Cu2þ (200 mg L1 ) induced a slight synergism of both contaminants. This herbicide, like copper, is known to generate oxidative stress (NavariIzzo et al., 1998); therefore, the combination of these two contaminants could increase the production of reactive oxygen species in the cell, causing a saturation or a destruction of the antioxidative systems of duckweed, resulting in more important damage to the ! photosynthetic apparatus (Duke et al., 1991; Baron et al., 1995).
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On the contrary, antibotrytic fungicides did not have any effects on the photosynthetic apparatus of L. minor at concentration between 1 and 100 mg L1 : Higher concentration (5–50 mg L1 ) of these fungicides were also tested, and even at these concentrations, no marked effects were observed (data not shown). The same results have been observed in duckweed, exposed to folpet, an antimildew fungicide (Teisseire et al., 1998). A mixture of these fungicides and copper seemed to induce different effects on L. minor. All kinds of interactions were observed in this study: synergism, additivity, and antagonism. To explain the slight synergism observed under static conditions, the influence of the blank formulation of pesticides may be evoked. The formulation may cause a dissolution of leave cuticles (Griffith, 1993; Zabkiewicz, 2000), which will make the copper penetration into plant cell easier, and so induce higher copper phytotoxicity. The antagonism between the lower concentrations of fludioxonil and procymidone (1–10 mg L1 ) and copper may be explained by a complexation between both contaminants. This complexation, already observed in other ecotoxicological studies, could reduce the bioavailability of copper and pesticide and lower their toxicity (Duda et al., 1996; Undabeytia et al., 1996). This hypothesis could also be advanced to explain the change from synergism to additivity observed when the higher concentrations of herbicide (10–50 mg L1 ) were added to copper.
5. Conclusion In this study, the pesticides affected duckweed photosynthesis as follows: flumioxazin> Cu2þ > antibotrytic fungicides (from greatest to least inhibition). The different results (antagonism, additivity, and synergism) obtained on the toxicity of pesticide mixtures in L. minor suggested that complex interactions between these types of contaminants could exist in the natural environment. Since the phytosanitary products rarely occur alone in the environment, but rather in combination, a better understanding of these interactions will be of major importance to assess the real potential effects of these contaminants in the natural environment.
Acknowledgments This work was cofinanced by the French Ministry of Environment as part of the ‘‘Vignes et Vins de Champagne’’ topic and by Europol’ Agro through the ‘‘Lutte Contre le Botrytis: Biomarqueurs pour une Conduite Inte! gre! e de la Vigne’’ program.
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Photosynthetic responses of Lemna minor exposed to xenobiotics, copper, and their combinations C. Frankart,* P. Eullaffroy, and G. Vernet Laboratory of Eco-Toxicology, Europol’Agro, Faculty of Sciences, University of Reims Champagne-Ardenne, B.P. 1039, F-51687 Reims 02, France Received 18 September 2001; accepted 5 March 2002
Abstract The effects on the photosynthetic process of copper and pesticides, used in vineyards, and their combinations, were investigated by measuring different chlorophyll fluorescence parameters in Lemna minor. Cu and flumioxazin had a severe impact on duckweed since a decrease in their photosynthetic capacity was detected after 24 h of exposure to 200 and 1 mg L1 ; respectively. However, fungicides used to control Botrytis cinerea (procymidone, pyrimethanil, and fludioxonil) seem to have no marked effects on duckweed even at very high concentrations (50 mg L1 ). Analysis of the combinations between copper (200 mg L1 ) and pesticides revealed different patterns of response: a synergistic effect was observed when Cu2þ was added to flumioxazin (1mg L1 ). In contrast, an antagonism was detected when duckweed was exposed to a mixture of Cu2þ and fludioxonil or procymidone. However, these interactions always tended toward additivity when pesticide concentrations increased. Additivity was also observed for the Cu2þ –pyrimethanil mixture at each fungicide concentration. r 2002 Elsevier Science (USA). All rights reserved. Keywords: Chlorophyll fluorescence; Lemna minor; Copper; Pesticides; Mixture; Botrytis cinerea
1. Introduction The substantial use of copper sulfate over the past 100 years on vineyards has increased the input of the heavy metal copper into the aquatic environment (Courde et al., 2001). Copper sulfate is known to control mildew and other fungal diseases on grapes and therefore is generously sprayed on vineyards. Copper is an essential micronutrient for the plant life as a constituent of several enzymes and redox catalyst in a variety of metabolic pathways (Maksymiec, 1997). However, it becomes toxic to most aquatic lifeforms, particularly aquatic plants, at concentrations only slightly higher than the optimal requirement. In excessive quantities, copper interfaces with photosynthesis and respiratory processes, protein synthesis, or development of plant ! et al., 1995; Ralph and Burchett, organelles (Baron 1998; Raven et al., 1999; Yruela et al., 2000). Beside the use of copper sulfate, some of the most widely used agrochemicals on vineyards are antibotrytic *Corresponding author. Fax: +33-3-26-91-33-47. E-mail address: [email protected] (C. Frankart).
fungicides (such as procymidone, pyrimethanil, or fludioxonil). These fungicides are used to fight the development of Botrytis cinerea, a phytopathogenic fungus including a disease named ‘‘gray mold’’ or ‘‘Botrytis’’ (Tomlin, 1997). Procymidone, a dicarboximide fungicide inhibiting spore germination and growth, causes cellular leakage and lipid peroxidation on B. cinerea (Lee et al., 1998). Pyrimethanil, an analinopyrimidine fungicide, inhibits the secretion of cell-wall-degrading enzymes required for Botrytis infection (Leroux, 1996). Finally, fludioxonil, a phenylpyrrole pesticide, is able to uncouple oxidative phosphorylation and to inhibit electron transport in B. cinerea, inducing the formation of reactive oxygen species. As a consequence, it could cause peroxidation in fungi (Steel, 1996). Herbicides are also often sprayed on vineyards especially to control adventive plants. Flumioxazin, one of these herbicides, inhibits protoporphyrinogen IX oxidase, an enzyme that converts protoporphyrinogen IX to protoporphyrin IX, a precursor of chlorophyll molecules (Duke et al., 1991). All these pesticides may be introduced into natural aquatic systems through surface water runoff after their
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application. This phenomenon is particularly widespread in Champagne (France) (Desc#otes et al., 2001), since vineyards are established on hillsides. Then the polluted waters can get into the ecosystems and generate a diffuse pollution which could damage the aquatic organisms. To determine the effects of this pollution on aquatic plant photosynthesis, the authors used a chlorophyll fluorescence-based method often applied as an effective and nondamaging tool for the elucidation of various aspects of the physiological state of the photosynthetic apparatus of plants. So this method provides basic information on the performance of photosynthesis. It has become available to distinguish several fluorescence parameters such as photosynthetic capacity, and photochemical and nonphotochemical fluorescence quenching components (Schreiber et al., 1986; Lichtenthaler, 1988; Krause and Weis, 1991; Juneau and Popovic, 1999). Because copper is a ubiquitous element in Champagne aquatic ecosystems and then can be in contact with other phytosanitary products in contaminated water, these problems were investigated to determine the effects of pesticides and copper alone and in combination on the photosynthetic apparatus of an aquatic plant, Lemna minor, regularly used for ecotoxicological studies (Wang, 1990; Fairchild et al., 1997; Teisseire and Vernet, 2000).
plants were exposed to copper at concentrations of 40, 200, and 400 mg L1 : The concentration of copper was fixed at 200 mg L1 when combined with other pesticides, in regard to its environmental representativity (Paris-Palacios, 1999). The authors used formulated flumioxazin, fludioxonil, procymidone, and pyrimethanil in this study. Their commercial names are, respectively, Pledge (Cyanimid Agro, France), Geoxe (Ciba, Switzerland), Sumisclex (Sopra/Zeneca, France), and Scala (Schering Agriculture, France). Each pesticide concentration given in this study is for the active ingredient. Flumioxazin and antibotrytic fungicides (fludioxonil, procymidone, and pyrimethanil) were tested, respectively, at concentrations of 1, 10, and 50 mg L1 ; and 1, 10, and 100 mg L1 in regard to their environmental relevance (Verdisson et al., 2000). All stock solutions of the contaminants were prepared just before the experiments. For toxicity experiments, 10 mature fronds of L. minor were transferred to 50-mL Erlenmeyer flasks containing 20 mL of growth medium where copper and/ or agrochemical (herbicide or fungicides) was added. The experiments were conducted under static conditions. Plants without added contaminants served as negative control. Triplicates of plant colonies were exposed to contaminants for 72 h:
2. Materials and methods
2.3. Combination of copper and pesticides
2.1. Plant material
Possible interaction between copper and other agrochemicals was estimated using Abott’s formula (Gisi, 1996). In this widely used model, the expected inhibition of the mixture (Cexp ), expressed as a percentage, can be predicted as
Fronds of duckweed (L. minor) were collected from pounds in the Ardennes area (France). These fronds were disinfected by immersing them in NaOCI 1% (v/v) and Tween 0.01% (v/v) for 3 min and then rinsing with distilled water for 5 min: The stock cultures were maintained in PVC aquariums containing 400 mL of inorganic autoclaved growth medium (pH 6.5) adapted from Chollet (Chollet, 1993). This medium consisted of KNO3 ; 202 mg L1 ; KH2 PO4 ; 50:3 mg L1 ; K2 HPO4 ; 27:8 mg L1 ; K2 SO4 ; 17:4 mg L1 ; MgSO4 7H2 O; 49:6 mg L1 ; CaCl2 ; 11:1 mg L1 ; FeSO4 7H2 O; 6 mg L1 ; H3 BO3 ; 5:72 mg L1 ; MnCl2 4H2 O; 2:82 mg L1 ; ZnSO4 ; 0:6 mg L1 ; ðNH4 ÞMo7 O24 4H2 O; 0:043 mg L1 ; CuCl2 2H2 O; 0:078 mg L1 ; CoCl2 6H2 O; 0:054 mg L1 : All aquariums were maintained in a growth chamber at 22711C under continuous light (100 mmol PFD m2 s1 ) provided by cool white fluorescence lamps (Sylvania Gro Lux F30W/Gro-T8, Germany). The plants were subcultured twice a week. 2.2. Exposure to agrochemicals A 50-mg Cu2þ L1 stock solution (CuSO4 5H2 O; Sigma, France) was prepared in cultural medium. The
Cexp ¼ A þ B ðA B=100Þ; where A and B are the inhibition levels given by the single chemicals. The ratio of inhibition (RI) was then calculated as follows for each contaminant combination: RI ¼ Observed inhibition=Cexp : Synergism or antagonism were evaluated by comparing RI to 1. A RI value > 1 indicates potential synergism between the two contaminants; RI ¼ 1; a simple additivity; and RIo1; an antagonism between the two chemicals. 2.4. Chlorophyll fluorescence measurements Measurements of chlorophyll fluorescence emission from the upper face of duckweed fronds were made using a pulse amplitude modulation chlorophyll fluorometer (PAM-FMS 1, Hansatech, England). Before each measurement, three fully developed fronds were taken and placed in darkness for 15 min at 22711C:
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After recording the dark signal level, the probing light beam was turned on and the steady F0 -level (ground fluorescence level) recorded. The intensity of this light was sufficiently low not to produce any significant variable fluorescence (1:6 kHz; 0:02 mmol PFD m2 s1 ). A single saturating flash (1 s; 4500 mmol m2 s1 ) was then applied to reach Fm (maximal fluorescence). Photosynthetic capacity (also named maximal quantum yield) was estimated by the ratio Fm Fo =Fm (also termed Fv =Fm ) for dark-adapted leaves (Genty et al., 1990). After the decline of the signal from Fm to its initial Fo -level, an induction kinetic was started by turning on the actinic light (200 mmol PFD m2 s1 ). During the induction with actinic light, the saturating light pulse was given every 10 s: When a steady state of the induction curve was reached (about 6 min later), the induction phase was completed by turning off the actinic light. The photochemical ðqP Þ and nonphotochemical
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ðqN Þ quenching were determined as in Schreiber et al. (1986) and Lichtenthaler and Rinderle (1988). Data presented here are the means 7 standard deviation (SD). Significant differences between controls and contaminated samples were determined by the Mann–Whitney test and P values o0:05 were considered significant (Mann and Whitney, 1952).
3. Results 3.1. Effects of copper treatments To charactersize the influence of cupric ions on the photosynthetic activity of L. minor, measurements of in vivo chlorophyll fluorescence parameters were conducted. The values of the photosynthetic capacity ðFv =Fm Þ; and photochemical ðqP Þ and nonphotochemical quenching ðqN Þ are given in Fig. 1.
Fig. 1. Photosynthetic parameters in controls ð~Þ and copper-treated fronds ((\) 40; (m) 200; and () 400 mg Cu2þ L1 ) of Lemna minor after 72 h of exposure. Data are means 7SD: (A) Photosynthetic capacity; (B) Photochemical quenching; (C) nonphotochemical quenching.
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A significant decrease (10%) in Fv =Fm was observed in L. minor exposed for 24 h to 400 mg L1 or 48 h to 200 mg L1 of copper (9%). After 72 h of exposure, the percentage of Fv =Fm inhibition reached 14% and 42% in duckweed exposed to 200 to 400 mg L1 of Cu2þ ; respectively (Fig. 1A). During these 3 days of copper exposure, the photosynthetic capacity was not significantly inhibited by 40 mg Cu2þ L1 (Fig. 1A). Inhibition of photosynthetic capacity was concentration-dependant. The same pattern with photochemical quenching (qP ) was observed (Fig. 1B). A significant decrease of 12% and 28%, was noticed after 48 h when copper was present at 200 and 400 mg L1 ; respectively. After 3 days of exposure, the inhibition reached 23% and 41% for these two copper concentrations. A significant increase of 19%, 52%, and 32% of qN was observed within 24 h of exposure for samples containing 40, 200, and 400 mg Cu2þ L1 ; respectively (Fig. 1C). However, it was noted that after an increase during the first 24 h of exposure to 400 mg Cu2þ L1 ; qN was strongly decreased.
L. minor demonstrated an important sensitivity to copper since its photosynthetic capacity and the photochemical and nonphotochemical quenching mechanisms were affected by concentrations lower than 400 mg Cu2þ L1 : However the photosynthetic capacity seems to be damaged before the qP mechanisms (significant inhibition 24 h after exposure versus 48 h). Moreover, interpretation of the qN pattern could be difficult (see Discussion). The trends of these photosynthetic processes were confirmed when their related photosynthetic parameters with the selected pesticides and their combination with copper were measured. 3.2. Effects of pesticides alone and mixtures of copper and pesticides on Lemna minor The results presented in Fig. 2 are for the shortest time of exposure necessary for contaminants to induce a significant effect in duckweed. L. minor contaminated by flumioxazin herbicide had a strong decrease in photosynthetic capacity (Fig. 2A). This decrease was observed from the first day of exposure even for the lowest
Fig. 2. Photosynthetic capacity (Fv =Fm ) of Lemna minor treated for 24, 48, or 72 h with pesticides alone or in combination with copper. Data are means 7SD: (A) Flumioxazin (24-h exposure); (B) fludioxonil (72-h exposure); (C) procymidone (72-h exposure); (D) pyrimethanil (72-h exposure). (\) Pesticide alone; (&) pesticide þ200 mg Cu2þ L1 :
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herbicide concentration: the inhibition percentages were 23, 62, and 64 for 1, 10, and 50 mg L1 of flumioxazin, respectively. However, this inhibition was higher when plants were exposed to mixtures of copper (200 mg L1 Cu2þ ) and flumioxazin (Fig. 2A). The comparison between the expected inhibition (Cexp ) and the experimentally observed Fv =Fm inhibition revealed two different responses. For the lowest flumioxazin concentration (1 mg L1 ), the experimentally observed photosynthetic capacity inhibition values were superior to Cexp and the RI was greater than 1 (1.13 7 0.05), indicating a very slight synergism or an additional effect, between copper and flumioxazin. For the higher flumioxazin concentrations (10 and 50 mg L1 ), the same results were observed: RI values close to 1 (respectively, 1:0970:23 and 0:9970:12), indicating a simple additivity or a slight synergism between the two chemicals. L. Minor exposed to antibotrytic fungicides alone had a very slight inhibition of photosynthesis (lower than 5%) at all tested concentrations after 72 h of treatment (Figs. 2B–D). However, when L. minor was exposed to a mixture of copper and fungicides, measurements of the photosynthetic capacity revealed different responses in regard to the fungicide concentrations. When duckweed was in contact for 72 h with a combination of Cu2þ (200 mg L1 ) and the two lower fludioxonil concentrations (1 and 10 mg L1 ), RI values were lower than 1 ð0:7470:12 and 0:8670:10; respectively), indicating a slight antagonism between the two contaminants. However, for the highest fludioxonil concentration (100 mg L1 ), RI was greater than 1 ð1:7170:67), indicating a synergism between the two contaminants or a simple additivity in regard to SD. After 72 h of exposure, mixtures of Cu2þ (200 mg L1 ) and procymidone had the same results as mixtures of Cu and fludioxonil. The RIs were smaller than 1 for mixture with procymidone at the lower concentrations (1 and 10 mg L1 ): 0:5770:08 and 0:6770:13; respectively, indicating a strong antagonism between the two contaminants. As had already been observed in previous mixtures (copper with flumioxazin and fludioxonil), a simple additivity of copper and pesticide was noted with increasing fungicide concentrations (100 mg L1 ): 1:1270:22: For mixture between Cu2þ and pyrimethanil, RI values were always close to 1, regardless to fungicide concentration. In this case, only an additive effect between these two contaminants was observed.
4. Discussion In this study, photosynthetic capacity and photochemical and nonphotochemical quenching were reliable to evaluate the toxicity of phytosanitary products used
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in vineyards. First, as had been observed in many cases of toxicity test, (qN ) increased with contaminant concentrations (Demming and Winter, 1988; Caja! nek et al., 1998). In this study, this increase induced by 200 and 400 mg Cu2þ L1 was followed by a decrease after 72 h: It is known that plants exposed to a long-term stress pass through different physiological states from resistance to exhaustion (Lichtenthaler, 1996). In this study, the qN decrease after 72 h of exposure to 200 and 400 mg Cu2þ L1 could indicate that the duckweed was entering the exhaustion phase. This reaction was confirmed by morphological observation revealing a separation of the fronds and large areas of chlorosis. Therefore, despite its sensitivity, the value of this parameter at a given time of exposure may not give the true degree of contaminant toxicity on the photosynthetic apparatus. In L. minor, 40 mg L1 of Cu2þ was sufficient to induce a decrease in the photosynthetic capacity, indicating that, although copper is an essential micronutrient for plant life, it can become a strong inhibitor of photosynthesis when in excess. Numerous sites were identified as targets of copper action in chloroplasts. The photosynthetic apparatus is particularly sensitive to this heavy metal, which is known to decrease the electron transfer rate, a disruption responsible for the decrease in photosynthetic capacity and photochemical quenching (Ruban and Horton, 1995; Pogson et al., 1998), or to catalyze the formation of reactive free radicals inducing oxidative damage (Navari-Izzo et al., 1998). The results clearly provided evidence that flumioxazin, as an herbicide, was very toxic to L. minor. It was a strong inhibitor of photosynthetic capacity even at a very low concentration (1 mg L1 ). This herbicide blocked the protoporphyrinogen oxidase activity, inhibiting the accumulation of protoporphyrinogen IX. This precursor of chlorophyll molecules is a potent photosensitizer that generates high levels of singulet oxygen in the presence of molecular oxygen and light (Duke et al., 1991). This metabolic disruption caused oxidative damages leading to plant death within 24 h of contamination (50 mg L1 ). Therefore, the inhibition of chlorophyll synthesis by flumioxazin may be the indirect cause of the photosynthetic capacity inhibition. A mixture of the smallest concentration of flumioxazin (1 mg L1 ) and Cu2þ (200 mg L1 ) induced a slight synergism of both contaminants. This herbicide, like copper, is known to generate oxidative stress (NavariIzzo et al., 1998); therefore, the combination of these two contaminants could increase the production of reactive oxygen species in the cell, causing a saturation or a destruction of the antioxidative systems of duckweed, resulting in more important damage to the ! photosynthetic apparatus (Duke et al., 1991; Baron et al., 1995).
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On the contrary, antibotrytic fungicides did not have any effects on the photosynthetic apparatus of L. minor at concentration between 1 and 100 mg L1 : Higher concentration (5–50 mg L1 ) of these fungicides were also tested, and even at these concentrations, no marked effects were observed (data not shown). The same results have been observed in duckweed, exposed to folpet, an antimildew fungicide (Teisseire et al., 1998). A mixture of these fungicides and copper seemed to induce different effects on L. minor. All kinds of interactions were observed in this study: synergism, additivity, and antagonism. To explain the slight synergism observed under static conditions, the influence of the blank formulation of pesticides may be evoked. The formulation may cause a dissolution of leave cuticles (Griffith, 1993; Zabkiewicz, 2000), which will make the copper penetration into plant cell easier, and so induce higher copper phytotoxicity. The antagonism between the lower concentrations of fludioxonil and procymidone (1–10 mg L1 ) and copper may be explained by a complexation between both contaminants. This complexation, already observed in other ecotoxicological studies, could reduce the bioavailability of copper and pesticide and lower their toxicity (Duda et al., 1996; Undabeytia et al., 1996). This hypothesis could also be advanced to explain the change from synergism to additivity observed when the higher concentrations of herbicide (10–50 mg L1 ) were added to copper.
5. Conclusion In this study, the pesticides affected duckweed photosynthesis as follows: flumioxazin> Cu2þ > antibotrytic fungicides (from greatest to least inhibition). The different results (antagonism, additivity, and synergism) obtained on the toxicity of pesticide mixtures in L. minor suggested that complex interactions between these types of contaminants could exist in the natural environment. Since the phytosanitary products rarely occur alone in the environment, but rather in combination, a better understanding of these interactions will be of major importance to assess the real potential effects of these contaminants in the natural environment.
Acknowledgments This work was cofinanced by the French Ministry of Environment as part of the ‘‘Vignes et Vins de Champagne’’ topic and by Europol’ Agro through the ‘‘Lutte Contre le Botrytis: Biomarqueurs pour une Conduite Inte! gre! e de la Vigne’’ program.
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