Philippe Eullaffroy

The F684/F735 chlorophyll fluorescence ratio: a potential tool for rapid detection and determination of herbicide phytotoxicity in algae.

The use of herbicides constitutes the principal method of weed control but the introduction of these compounds into the aquatic environment (primarily through runoff) may have severe consequences for non-target plants. In this study, we describe a sensitive and inexpensive method for detection of photosynthesis-inhibiting herbicides, based on chlorophyll (Chl) fluorescence emission. Algae exhibited a Chl fluorescence signature with two maxima around 684 and 735 nm, correlated with the total Chl content of the algal suspension. The ratio of these two maxima (i.e. F684/F735) can be used as an indicator of stress in the photosynthetic apparatus, and thus represents a very simple method for in vivo evaluation of the health status of algae. Determination of the F684/F735 fluorescence ratio revealed the presence and phytotoxicity of atrazine, metribuzin, terbuthylazine, diuron, DCPMU, DCPU and paraquat. The toxic effect of these pollutants was estimated by monitoring the increase in the F684/F735 value, which reflects photosystem II and photosystem I photochemistry. We observed a drastic increase in the magnitude of this ratio, correlating quantitatively with herbicide concentration and corresponding to a decline in algal photosynthetic activity. For the tested herbicides affecting photosynthetic electron transport, the magnitude of the effect was as follows: diuron= DCPMU > metribuzin > atrazine > terbuthylazine > paraquat > DCPU. The F684/F735 Chl fluorescence ratio thus gives toxicity responses which compare favourably with tests such as the algal growth inhibition test, and could therefore be used to detect the presence and phytotoxicity of herbicides in aquatic environments.

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

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-of-action. 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.

Photosynthetic responses of Lemna minor exposed to xenobiotics, copper, and their combinations.

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 24h of exposure to 200 and 1 microg.L(-1), 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.L(-1)). Analysis of the combinations between copper (200 microg.L(-1)) and pesticides revealed different patterns of response: a synergistic effect was observed when Cu(2+) was added to flumioxazin (1 microg.L(-1)). In contrast, an antagonism was detected when duckweed was exposed to a mixture of Cu(2+) and fludioxonil or procymidone. However, these interactions always tended toward additivity when pesticide concentrations increased. Additivity was also observed for the Cu(2+)-pyrimethanil mixture at each fungicide concentration.

Toxicity of PAMAM dendrimers to Chlamydomonas reinhardtii

In recent decades, a new class of polymeric materials, PAMAM dendrimers, has attracted marked interest owing to their unique nanoscopic architecture and their hopeful perspectives in nanomedicine and therapeutics. However, the potential release of dendrimers into the aquatic environment raises the issue about their toxicity on aquatic organisms. Our investigation sought to estimate the toxicity of cationic PAMAM dendrimers on the green alga, Chlamydomonas reinhardtii. Algal cultures were exposed to different concentrations (0.3-10 mgL(-1)) of low dendrimer generations (G2, G4 and G5) for 72 h. Potential adverse effects on Chlamydomonas were assessed using esterase activity (cell viability), photosynthetic O2 evolution, pigments content and chlorophyll a fluorescence transient. According to the median inhibitory concentration (IC50) appraised from esterase activity, toxicity on cell viability decreased with dendrimer generation number (2, 3 and 5 mgL(-1) for G2, G4 and G5 dendrimers, respectively). Moreover, the three generations of dendrimers did not induce the same changes in the photosynthetic metabolism of the green alga. O2 evolution was stimulated in cultures exposed to the lowest generations tested (i.e. G2 and G4) whereas no significant effects were observed with G5. In addition, total chlorophyll content was increased after G2 treatment at 2.5 mgL(-1). Finally, G2 and G4 had positive effects on photosystem II (PSII): the amount of active PSII reaction centers, the primary charge separation and the electron transport between Q(A) and Q(B) were all increased inducing activation of the photosynthetic electron transport chain. These changes resulted in stimulation of full photosynthetic performance.

Phytoremediation of fungicides by aquatic macrophytes: Toxicity and removal rate

The rate of removal of two fungicides (dimethomorph and pyrimethanil) from water by five macrophyte species (L. minor, S. polyrhiza, C. aquatica, C. palustris and E. canadensis) was assessed in laboratory tests. In order to assure that these studies were performed with healthy plants the effects of the fungicides on chlorophyll fluorescence were studied as well. At exposure concentrations of 600microgL(-1) the effects of the fungicides on chlorophyll fluorescence were minor, so that this initial concentration level was selected for the fungicide removal rate tests. The removal yields during the 4-d test periods varied from 10% to 18% and 7% to 12% for dimethomorph and pyrimethanil, respectively. The maximum removal rate during the 4-d test period was 48microgg(-1) fresh weight (FW) for dimethomorph and 33microgg(-1) FW for pyrimethanil. L. minor and S. polyrhiza showed the highest removal efficiency for the two fungicides.

Influence of initial pesticide concentrations and plant population density on dimethomorph toxicity and removal by two duckweed species.

Aquatic plants take up, transform and sequester organic contaminants and may therefore be used in phytoremediation for the removal of pollutants from wastewaters. A better understanding of factors affecting the rate of contaminant uptake by aquatic plants is needed to improve engineered systems for removal of pollutants from wastewaters. This work focused on the influence of initial concentrations of pesticide and population density of plants on toxicity and uptake of the fungicide dimethomorph by two duckweed species. An increased sensitivity to dimethomorph was observed with increasing duckweed population density. Less light, due to crowding, may explain this higher sensitivity and reduced removal rate. A positive relationship was also found between toxicity or contaminant uptake and initial pesticide concentration with a maximal removal of 41 and 26 microg g(-1) fresh weight of dimethomorph (at 600 microg L(-1) of dimethomorph and an initial density of 0.10g E-flask(-1)) by Lemna minor and Spirodela polyrhiza, respectively. This research also indicated that these aquatic plants can efficiently eliminate organic contaminants and may ultimately serve as phytoremediation agents in the natural environment.

Potential Use of Lemna Minor for the Phytoremediation of Isoproturon and Glyphosate

Pesticides are being detected in water bodies on an increasingly frequent basis. The present study focused on toxicity and phytoremediation potential of aquatic plants to remove phytosanitary products from contaminated water. We investigated the capacity of Lemna minor (L. minor) to eliminate two herbicides isoproturon and glyphosate from their medium. Since phytoremediation relies on healthy plants, pesticide toxicity was evaluated by exposing plants to 5 concentrations (0–20 μg L−1 for isoproturon and 0–120 μg L−1 for glyphosate) in culture media for 4 d using growth rate and chlorophyll a fluorescence as endpoints. At exposure concentrations of 10 μg.L−1 for isoproturon and 80 μg.L−1 for glyphosate, effects on growth rate and chlorophyll fluorescence were minor (< 25%), so that this initial concentration was selected to study herbicide removal. After a 4-d incubation, removal yields were 25% and 8% for isoproturon and glyphosate, respectively.

Enzymatic basis for fungicide removal by Elodea canadensis

PurposePlants can absorb a diversity of natural and man-made toxic compounds for which they have developed diverse detoxification mechanisms. Plants are able to metabolize and detoxify a wide array of xenobiotics by oxidation, sugar conjugation, glutathione conjugation, and more complex reactions. In this study, detoxification mechanisms of dimethomorph, a fungicide currently found in aquatic media were investigated in Elodea canadensis.MethodsCytochrome P450 (P450) activity was measured by an oxygen biosensor system, glucosyltransferases (GTs) by HPLC, glutathione S-transferases (GSTs), and ascorbate peroxidase (APOX) were assayed spectrophotometrically.ResultsIncubation of Elodea with dimethomorph induced an increase of the P450 activity. GST activity was not stimulated by dimethomorph suggesting that GST does not participate in dimethomorph detoxification. In plants exposed to dimethomorph, comparable responses were observed for GST and APOX activities showing that the GST was more likely to play a role in response to oxidative stress. Preincubation with dimethomorph induced a high activity of O- and N-GT, it is therefore likely that both enzymes participate in the phase II (conjugation) of dimethomorph detoxification process.ConclusionsFor the first time in aquatic plants, P450 activity was shown to be induced by a fungicide suggesting a role in the metabolization of dimethomorph. Moreover, our finding is the first evidence of dimethomorph and isoproturon activation of cytochrome P450 multienzyme family in an aquatic plant, i.e., Elodea (isoproturon was taken here as a reference molecule). The detoxification of dimetomorph seems to proceed via hydroxylation, and subsequent glucosylation, and might yield soluble as well as cell wall bound residues.