Comparative effects of four herbicides on non-photochemical fluorescence quenching in Lemna minor

Comparative effects of four herbicides on non-photochemical fluorescence quenching in Lemna minor

Environmental and Experimental Botany 49 (2003) 159 /168 Comparative effects of four herbicides on non-photochemic...

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Environmental and Experimental Botany 49 (2003) 159 /168

Comparative effects of four herbicides on non-photochemical fluorescence quenching in Lemna minor C. Frankart *, P. Eullaffroy, G. Vernet Laboratoire d’Eco-Toxicologie, Bat 18 Europol’agro, U.F.R Sciences Exactes et Naturelles, Universite´ de Reims Champagne-Ardenne, BP 1039, 51687 Reims Cedex 2, France Received 26 March 2002; received in revised form 20 September 2002; accepted 21 September 2002

Abstract Aquatic ecosystems are exposed to an increasing contamination of pesticides such as herbicides through water runoff. The pulse-amplitude-modulation (PAM) fluorometric method, as a sensitive and rapid method, was used to evaluate toxic effect of these pollutants in Lemna minor . Four herbicides (paraquat, norflurazon, flazasulfuron and atrazine) often found in outdoor water samples and inducing specific changes in the yield of the in vivo chlorophyll a fluorescence of PSII were selected. These herbicides affected photosynthesis via different ways by: accepting electron from PSI, inhibiting carotenoids and protein biosynthesis or blocking PSII electron transport. Data revealed that photosynthetic parameters based on fluorescence emission were modified with the increase of herbicide concentration. The toxicity of these compounds was as follows (from greatest to least): paraquat/norflurazon /atrazine / flazasulfuron. Growth rate and photosynthetic pigments analysis confirmed the results obtained with PAM fluorometry. We found that among the fluorescence parameters the non-photochemical quenching was the most appropriate indicator for the effects of herbicides. The components of non-photochemical quenching were then resolved by examination of relaxation kinetics of quenching upon DCMU addition and light saturation pulse in the entire plant. Three kinetically distinct phases were observed which have previously been identified in thylakoids (Horton and Hague, 1988) as being due to energy-state quenching (qE), state transition (qT) and photoinhibition (qI). These examined NPQ components showed different levels of sensitivity to the effect of herbicide. It was found that: (i) qE was the major NPQ component; (ii) qE was affected by all the selected herbicides; (iii) qT was significantly modified by paraquat and atrazine; (iv) qI was affected by norflurazon and flazasulfuron. We interpreted these results by the pesticide mode-ofaction. This study shows that the use of NPQ as a biomarker may be appropriate in laboratory and field herbicide bioassays. Moreover, application of non-photochemical quenching analysis may allow a better understanding of the mechanism of herbicide action. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Chlorophyll fluorescence; PAM fluorometry; Pesticides; Biomarker

1. Introduction * Corresponding author. Tel.: /33-3-2691-3410; fax: /33-32691-3347 E-mail address: [email protected] (C. Frankart).

Because of increasing agricultural and industrial productions, pesticides, also named phytosanitary

S0098-8472/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 9 8 - 8 4 7 2 ( 0 2 ) 0 0 0 6 7 - 9


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products, are became a major stress factor in the environment. Among these pesticides, herbicides are chemicals commonly used to control weeds in agricultural activities, and most of them are continuously discharged into aquatic environments through the surface runoff (Kloeppel et al., 1997). These herbicides are effective to control their targeted-weeds, however, it is of great concern to understand their effects on non-targeted organisms in aquatic environment. Numerous studies have been conducted to determine the harm of these pollutants on living organisms. Among these studies, growth rate, pigment contents and particularly chlorophyll fluorescence were used to elucidate the mode of action of herbicides on plant physiology (Conrad et al., 1993; Kim et al., 1999; Waldhoff et al., 2002). In the photosynthetic apparatus, light is absorbed by antenna pigments and excitation energy is transferred to the reaction centres of the two photosystems (PSI and PSII). There, the energy drives the primary photochemical reactions that initiate the photosynthetic energy conversion. Under optimal conditions, the primary photochemistry occurs with high efficiency and the dissipation of absorbed light energy by chlorophyll fluorescence is low and mainly emitted by PSIIassociated chlorophyll molecules (Krause and Weis, 1991; Lichtenthaler, 1996). This property of green plants is known to provide a simple and powerful non-intrusive means to determine the health state of plants (Lichtenthaler, 1988). In toxicity investigations, the saturation pulse method using pulse-amplitude-modulate (PAM) fluorometer is commonly used to study photosynthesis and is able to provide different fluorescence parameters giving reliable information of the effect of biotic and abiotic stress on plant physiology (Schreiber et al., 1994; Juneau et al., 2002). Among these parameters, the maximum quantum efficiency of PSII primary photochemistry (Fv/ Fm) and, photochemical and non-photochemical quenching (qP and NPQ) are very useful for laboratory and field studies (Conrad et al., 1993; Rascher et al., 2000). The two types of quenching occur in chloroplast: the photochemical quenching (qP) directly dependent to the electron transport, and the non-photochemical quenching (NPQ)

representing all non-radiative mechanisms involved in dissipation of excess absorbed lightenergy (for review, see Horton et al., 1996; Ruban and Horton, 1995). NPQ is composed of three different components according to their relaxation kinetics in darkness following a period of illumination, as well as their response to specific inhibitors (Demming and Winter, 1988; Horton and Hague, 1988; Richter et al., 1999; Mu¨ller et al. 2001). The major component relaxing within seconds is the pH- or energy-dependent component (qE) related to a buildup of a thylakoid DpH generated by the photosynthetic electron transport. Simultaneously, the DpH activates the xanthophylls cycle involved in protection mechanisms of the photosynthetic apparatus (Ruban et al., 2001). A second NPQ component, so-called state-transition quenching (qT), relaxing within minutes involves the redistribution of the absorbed energy between PSII and PSI. This mechanism occurs via the reversible movement of a fraction of antenna from PSII to PSI (Finazzi et al., 1999). The third component of NPQ relaxing within tens of minutes, is caused by photoinhibition and called the photoinhibitory quenching (qI) (Oja and Laisk, 2000). Many works have shown that whatever the pollutant mode of action NPQ was one of the most appropriate indicator for inhibitory effect because of its high sensitivity and quickness of response (White and Critchley, 1999; Curviel and van Rensen, 1996; Tschiersch et al., 2002). However, no studies were achieved to determine which NPQ components (qE, qT, qI) were modified in plants exposed to xenobiotics. Because these components are related to different events of the photosynthetic process, it is expected that their susceptibility may vary with herbicides’ action sites. Four different herbicides chosen for their specific mode of action on the photosynthetic apparatus were selected: paraquat, an electron acceptor from PSI (Wong, 2000), atrazine, an inhibitor of electron transport (Nakajima et al., 1996), and flazasulfuron, and norflurazon, inhibitors of protein and carotenoids synthesis, respectively (Bo¨ger, 1996; Tomlin, 2000). It was expected that in regard to the mode of action of these herbicides, each NPQ component, as a physiological response, will

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be stimulated or inhibited in plant exposed to the contaminant showing specific effects of pesticides on photosynthetic process. This will give a new insight on the origin of the NPQ changes when used as a biomarker in toxicity test.

2. Material and methods 2.1. Plant material Fronds of duckweed (Lemna minor L.) were collected from pounds in the Ardennes area (France). Duckweed are assembled in colonies of 2 /5 fronds. They are widely distributed in the world especially in quiescent water bodies (Hillman and Culley, 1978). This plant is revelant to many aquatic environments, including lakes, streams and pounds, and it is often used in toxicity tests (Wang, 1990; Teisseire et al., 1999). Before experiments, L. minor were disinfected by immersion in NaOCl 1% (v/v) and Tween 0.01% (v/v) for 3 min and then rinsing with distilled water during 5 min. The stock cultures were maintained in PVC aquaria containing 400 ml of inorganic autoclaved growth medium (pH 6.5) adapted from Chollet (Chollet, 1993). This medium consisted of KNO3, 202 mg l 1; KH2PO4, 50.3 mg l1; K2HPO4, 27.8 mg l1; K2SO4, 17.4 mg l 1; MgSO4 ×/ 7H2O, 49.6 mg l1; CaCl2, 11.1 mg l1; FeSO4 ×/ 7H2O, 6 mg l 1; H3BO3, 5.72 mg l 1; MnCl2 ×/ 4H2O, 2.82 mg l 1; ZnSO4, 0.6 mg l 1; (NH4)Mo7O24 ×/ 4H2O, 0.043 mg l1; CuCl2 ×/ 2H2O, 0.078 mg l1; CoCl2 ×/ 6H2O, 0.054 mg l1. All aquaria were maintained in a growth chamber at 229/1 8C under continuous light (100 mmol photosynthetic active radiation [PAR] m 2 s 1) provided by cool white fluorescent lamps (Sylvania Gro Lux F30W, Germany). The plants were subcultured twice a week. 2.2. Exposition to agrochemicals Formulated herbicides were used: atrazine [6chloro-N -ethyl-N ?-(1-methylethyl)-1,3,5-triazine2,4-diamine] as Atraphyt† (Sipcam-Phyteurop, France), paraquat (1,1?-dimethyl-4,4?-bipyridinium dichloride) as Gramoxone† (Zeneca-Sopra,


France), flazasulfuron (N -[[(4,6-dimethoxy-2-pyrimidinyl-2-yl)-3-(3-trifluoromethyl-2-pyridylsulfonyl)urea]]) as Katana† (Zeneca-Sopra, France) and norflurazon [4-chloro-5-methylamino-2-(3-trifluoromethylphenyl)-pyridazinon-3(2H)one] as Zorial† (Novartis, France). Each pesticide concentration given in this study is for the active ingredient. Duckweed were exposed to 1, 10 and 100 mg l1 of each chemical in regard to their environmental relevance (Garmouna et al., 1998). All stock solutions were prepared just before the experiments. Six colonies of duckweed were contaminated with herbicides under static conditions (same as growth conditions) during 48 h. Plant without added contaminant served as negative control. Each experiments were conducted twice. The stability of herbicides after the 48 h of experiment was analysed by HPLC (Ginkotech, Germany) with a reverse-phase C18 column (Kromazil C18 5 ml 250 /3 mm, Cluzeau, France). The recoveries of the four herbicides were at least 989/ 3% (n/3) after 2 days. 2.3. In vivo chlorophyll fluorescence measurement Measurements of chlorophyll fluorescence emission from the upper face of duckweed fronds were made using a pulse amplitude modulated chlorophyll fluorometer (PAM-FMS 1, Hansatech† , UK). Before each measurement, three fully developed fronds were taken and placed in darkness for 15 min at 229/1 8C. A background far-red light was applied to increase electron transfer speed through photosynthetic apparatus. After recording the dark signal level, the probing light beam was turned on and the steady Fo-level (ground fluorescence level) recorded. The intensity of the modulated light (ML) was sufficiently low not to produce any significant variable fluorescence (1.6 kHz, 0.02 mmol PAR m2 s 1). A single saturating flash (SP) (1 s, 4500 mmol PAR m 2 s 1) was then applied to reach Fm (maximal fluorescence). The maximum quantum efficiency of PSII primary photochemistry was estimated by the ratio Fv/ Fm/Fm/Fo/Fm for dark adapted leaves (Genty et al., 1990). After the decline of the signal from Fm to its initial Fo-level, an induction kinetics was


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started by turning the actinic light on (200 mmol PAR m 2 s 1). During the induction with actinic light (AL), saturating light pulses (SP) were given to determine photochemical (qP) and non-photochemical (NPQ) quenching as in Schreiber et al. (1986), and Mu¨ller et al. (2001). The quantum efficiency of PSII (also called the Genty parameter, FPSII) and the quantum efficiency of open reaction centres (Fv?/Fm?) were calculated as in Genty et al. (1989) and Oxborough and Baker (1997), respectively.

2.4. Resolution of NPQ components We quantified the contribution of the three NPQ components following the method described by Horton and Hague (1988). After reaching the steady state-Fs level, 10 ml of DCMU [3-(3,4dichlorophenyl)-1,1-dimethylurea] at 10 mg l1, was directly applied on the lower face of lacerated frond. After reaching FE level (Fig. 1), the actinic light was turned off and saturating pulses were applied inducing further relaxation phase with a t1/2 of approximately 5 min allowing the determination of the FT level. Contribution of each NPQ components were determined as follow: qE / (FE/F?m)/F?m; qT /(FT/FE)/F?m, and qI/ (Fm/FT)/F?m (Horton and Hague, 1988).

2.5. Photosynthetic pigments analysis and growth rate determination Duckweed’s pigments were extracted in 100% methanol and their concentrations were determined as in Lichtenthaler (1987). Growth rate of L. minor was calculated in counting number of fronds with a coefficient applied in function of their development state. 2.6. Statistical analysis Data presented in this study are the means9/ standard deviation (S.D.). Significant differences between controls and contaminated samples were determined by the Mann and Whitney test and P values B/0.05 were considered significant (a ,b ,c in Tables 1 and 2) (Mann and Whitney, 1952). In this study, all statistical analysis were performed with SigmaStat 2.03 (SSCP Inc.† ) for Windows.

3. Results 3.1. Control Fig. 1 shows the fluorescence induction curve for control obtained with a method described in materials and methods. The photosynthetic capacity (Fv/Fm) of the dark adapted Lemna was 0.78 (Table 1). Upon subsequent application of constant actinic light illumination (200 mmol PAR m2 s 1), a transient rise the fluorescence yield to a maximum (FP) and the quenching processes took place decreasing the fluorescence intensity to a steady state level (Fs). The photochemical quenching (qP) and non-photochemical quenching (NPQ) were 0.83 and 0.35, respectively. Addition of DCMU allowed further resolution of the NPQ components. The results showed that qE was the major component (64%) of the total NPQ, compared to qT and qI (11 and 25%, respectively) (Table 2; Fig. 1). 3.2. Paraquat

Fig. 1. Chlorophyll fluorescence induction kinetics measured in control plant.

After 2 days of exposure to the lowest paraquat concentration (1 mg l 1), no significant effect on

Pesticide concentration (mg l 1) Growth rate (%) Pigment concentrations (mg g fresh weight 1)

Fluorescence parameters

Chl a

Chl b







0.839/0.02 0.839/0.03 0.899/0.04 0a

0.359/0.04 0.679/0.07a 1.609/0.30b 0c

0.679/0.04 0.579/0.03a 0.359/0.04b 0c

0.739/0.03 0.699/0.02 0.509/0.05a 0b

Control Paraquat

0 1 10 100

1009/2.3 90.19/3.7a 70.29/4.5b 47.29/2.0c

5529/23 5269/28 5119/26a 1259/25b

1969/8 1479/12a 1299/12a 339/14b

2489/11 2209/12a 2069/11a 119/19b

0.789/0.02 0.779/0.02 0.679/0.02a 0b


1 10 100

87.49/2.6a 86.59/3.7a 85.69/3.7a

5539/31 5539/33 4029/8a

1739/11a 1799/13 1209/6a

2479/11 2439/12 1539/5a

0.709/0.05a 0.669/0.06a 0.279/0.03a 0.369/0.05a 0.579/0.05a 0.579/0.04b 0.599/0.06a 0.439/0.05 0.359/0.06a 0.509/0.05a 0.549/0.10b / 0.689/0.10b 0.329/0.06a 0.469/0.06a


1 10 100

94.99/3.5 85.89/5.2a 74.19/4.0b

5439/21 5419/14 4719/23a

1859/11 1849/8 1579/12a

2459/10 2449/7 2229/8a

0.789/0.01 0.779/0.02 0.739/0.08


1 10 100

96.79/3.0 94.99/1.5a 81.79/3.0b

5099/26a 5079/22a 4379/34b

1709/9a 1739/9a 1399/12b

2269/10a 2299/6a 1839/14b

0.779/0.03 0.829/0.04 0.389/0.13 0.559/0.07a 0.689/0.06 0.779/0.01 0.779/0.02a 0.359/0.03 0.529/0.03a 0.679/0.03 0.699/0.02a 0.119/0.05b 0.159/0.08a 0.159/0.02b 0.489/0.01a

0.879/0.01 0.419/0.03 0.709/0.02 0.869/0.01 0.429/0.05 0.679/0.02 0.669/0.08a 0.779/0.09a 0.619/0.04

0.739/0.03 0.719/0.04 0.709/0.01

a means significantly different (P B/0.05) from the control; a-c means with the same letter in each category do not significant differ (P B/0.05), according to Mann and Whitney test.

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Table 1 Growth rate, photosynthetic pigments and fluorescence parameters (means value9/S.D.), after 48 h exposure of Lemna minor fronds to paraquat, norflurazon, flazasulfuron and atrazine



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Table 2 qE, qT and qI components in percentage (means value9/S.D.) of the total NPQ in Lemna minor fronds exposed 48 h to paraquat (10 mg l 1), norflurazon (100 mg l 1), flazasulfuron (100 mg l 1), and atrazine (100 mg l 1) Concentration (mg l 1)

Control Paraquat Norflurazon Flazasulfuron Atrazine

10 100 100 100


0.329/0.04 1.609/0.30* 0.689/0.05* 0.779/0.09* 0.159/0.08*

% total NPQ qE



649/5 729/3* 799/3* 509/3* 349/4*

119/3 49/2* 129/4 109/3 369/8*

259/5 249/3 99/6* 409/7* 309/6

* Significant difference at P B/0.05.

physiological parameters of duckweed was observed. Only NPQ showed a strong increase. At 10 mg l 1, we noticed important effects of paraquat since the growth of L. minor was inhibited by about 13% and Fv/Fm, FPSII and Fv?/Fm? were decreased by 14, 52 and 7%, respectively (Fig. 2A). The NPQ value was increased by a factor 4.5 (Table 1). Resolution of non-photochemical quenching showed that qE still the major NPQ component since its proportion was significantly increased, compared to the control (72%). This

increase was correlated to the decrease of qT. The photoinhibitory quenching (qI) seemed to be not affected by paraquat at 10 mg l 1 (Table 2). At the same time, a decrease of about 15% of chlorophyll and carotenoid contents were observed. After 48 h of exposure, the highest concentration tested (100 mg l1) seemed to lead the plant to death as it was not possible to determine any fluorescence parameters. We also noticed a large decrease of the pigment concentrations (Table 1). The presence of totally bleached fronds confirmed these results.

Fig. 2. Chlorophyll fluorescence induction kinetics of Lemna minor exposed 48 h to 10 mg l 1 paraquat (A), 100 mg l 1 norflurazon (B), 100 mg l 1 flazasulfuron (C) and 100 mg l 1 atrazine (D).

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3.3. Norflurazon

4. Discussion

We observed an inhibition of the growth rate and a decrease of Fv/Fm from 1 to 100 mg l1. The pigment contents were significantly decreased (38%) only at the highest norflurazon concentration (Table 1). At this concentration the NPQ value was 2-fold higher than the control (Fig. 2B). As seen in paraquat treated fronds, this increase was especially due to a qE enhancement, but at this time at the expense of qI whereas qT seemed unchanged (Table 2).

Herbicides such as paraquat, norflurazon, flazasulfuron and atrazine are commonly used in agriculture and their residues can be easily translocated into aquatic environments through the surface runoff (Garmouna et al., 1998; Kloeppel et al., 1997). The study of physiological effects of these pollutants on non-target organisms requires criteria to distinguish their lethal or sublethal effects on plants (Fairchild et al., 1997). The use of growth rate, pigment contents and PAMfluorometric methods, may be of great relevance to determine the toxicity of herbicides on L. minor . The impacts of the tested herbicides were located upon the electron acceptors around PSI and PSII (paraquat and atrazine) while the others affect protein or pigment synthesis (flazasulfuron and norflurazon). This variety of impact sites and whether the effects on the photosynthetic apparatus is direct or indirect, will induced the related change of the growth rate, pigment contents and fluorescence parameter values. From our study, it appeared that paraquat was more toxic than norflurazon, atrazine and flazasulfuron (from greatest to least toxicity) to L. minor. Among the fluorescence parameters, the change of non-photochemical quenching was very fast and sensitive since herbicide concentration as low as 1 mg l 1 induced its variation. NPQ involves very complex mechanisms which are still not well understood (Mu¨ller et al., 2001). Here, analysis of the relaxation of the non-photochemical chlorophyll fluorescence quenching in duckweed’s fronds has revealed three distinct phases of recovery, in agreement with previous studies using thylakoids (Horton and Hague, 1988; Richter et al., 1999). We found that these three NPQ components were affected differently in regard to the mode of action of the tested herbicides. Paraquat, also named methyl-viologen, disrupts photosynthetic electron transfer by accepting electrons from PSI and producing superoxide radicals (Preston et al., 1991; Bo¨ger and Sandmann, 1998; Eullaffroy et al., 2001). This property will enhance the linear photosynthetic electron transport rate generating a transthylakoid DpH. The formation of a pH gradient associated with an activation of

3.4. Flazasulfuron After 2 days of exposure, the two lowest flazasulfuron concentrations (1 and 10 mg l1) induced no marked effect on physiological parameters (Table 1). At 100 mg l1, we observed a strong inhibition (26%) of the growth rate and a significant (P B/0.05) decrease (21%) of qP (Fig. 2C). At this concentration, NPQ increased by two fold due to a qI enhancement at the expense of qE, whereas qT seemed to stay constant (Table 2). A significant inhibition of pigment contents was only seen at the highest herbicide concentration (Table 1).

3.5. Atrazine No significant effect was observed after 48 h of exposure at the lowest concentrations of atrazine (1 and 10 mg l1) (Table 1). At 100 mg l 1, the fluorescence kinetics was largely modified since the steady-state level (Fs) was higher than in control plants (Fig. 2D). The growth rate was inhibited (18%), and all the fluorescence parameters were diminished. NPQ was also decreased. The lower value of NPQ was due to a decrease of qE which still not the major NPQ component. This decrease of qE was correlated to the increase of qT. Here, the three NPQ components represented the same proportion (Table 2). The pigment concentrations were significantly diminished in 100 mg l 1 treated fronds (around 25% compared to the control) (Table 1).


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the xanthophyll carotenoids cycle (Thiele and Krause, 1994) may explain the increase of the qE component. Horton and Hague (1988) showed that a rise in qE is correlated to a decline in qT due to an alteration of the distribution of excitation energy arriving at the two photosystems. Flazasulfuron provoked the switching on of the protective mechanisms as shown by the enhancement of NPQ. As an inhibitor of protein synthesis, this herbicide may disrupt the assembly of chlorophyll /protein complexes included in light harvesting complex of PSII (LHCII). The accumulation of free chlorophyll (not bound to protein) could allowed an additional quenching of excess absorbed light energy, by increasing energy dissipation (Gilmore and Govindjee, 1999; Mu¨ller et al., 2001). The qI and qE components rose with chemical treatment. The photoinhibitory quenching (qI) can reflect an organisation change in antenna’s protein structure that enables more effective transfer of energy to these quenching centres. Beside the accumulation of free chlorophyll, this might allow an additional quenching of excited energy. As flazasulfuron, norflurazon acts indirectly on the photochemistry of photosynthesis. Norflurazon inhibits phytoene desaturase which catalyses a rate-limiting step in carotenoid biosynthesis and then decreases carotenoid contents (Bo¨ger, 1996; Kim et al., 1999). These pigments are known to be important quenchers of the triplet state of chlorophyll (3Chl) which can generate singlet oxygen. Norflurazon, by eliminating these protecting compounds (i.e. carotenoids), triggers a photo-oxidative process inducing the photodestruction of plastid components such as photosynthetic pigments and membrane lipids (Kim et al., 1999). This may lead to the substantial inhibition of the photochemical efficiency of photosynthesis. As it had been shown in previous works (Jung et al., 2000; Tschiersch et al., 2002), the NPQ parameter was clearly increased by norflurazon. However, contrarily to the results obtained in paraquat- and flazasulfuron-treated fronds and in previous studies (Ruban et al., 1993; Curviel and van Rensen, 1996), we noticed an enhanced participation of the qE component at the expense of qI. This data is difficult to discuss since the mechanisms involved

in qI are not well understood, and may contribute to the debate on its origin. In this study, atrazine is the only herbicide inducing a decrease of the NPQ value. Atrazine affects photosynthesis by binding to the second electron acceptor (QB) then strongly inhibiting the electron transport shortly after PSII. This will subsequently induce an over-excitation of PSII (Conrad et al., 1993; Ralph, 2000) and cause an increase of the steady state fluorescence level (Fs) (Fig. 2D). The energy-dependent quenching (qE) was the most inhibited NPQ component since it is directly related to the electron transport necessary for the occurrence of the transthylakoid DpH (Ruban et al., 1992). The decrease of qE was compensated by a significant increase of qT likely due to a lateral redistribution of excitation rates in favour of PSI to reduce the damaging-excitation energy reaching the PSII.

5. Conclusion Our results show that all tested physiological parameters are sensitive and give information on the toxicity of herbicides on duckweed: paraquat was more toxic than norflurazon, atrazine, and flazasulfuron. Among the physiological parameters, NPQ is the first to respond when plants were exposed to herbicides, and therefore may be recommended to be used as a biomarker. The resolution of NPQ made in an entire plant seems to be appropriate to determine specific effect of pesticides on photosynthetic process and could provide a foundation for further detailed investigations into the physiological effects of herbicide on aquatic plants. Moreover, this can contribute to a better understanding of the complex mechanisms of the three NPQ components as well as their relationship.

Acknowledgements This work was supported by the French Ministry of Education and Research, and by the Europol’Agro Foundation.

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