Journal of Applied Phycology 13: 509–515, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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A pulse-amplitude modulated fluorescence-based method for assessing the effects of photosystem II herbicides on freshwater periphyton Ursula Dorigo & Christophe Leboulanger∗ Station d’Hydrobiologie Lacustre, INRA, BP 511 74203, Thonon les Bains cedex, France (∗ Author for correspondence; phone +33-450-266-711; fax +33-450-260-760; e-mail [email protected]) Received 10 February 2001; revised 29 April 2001; accepted 7 May 2001

Key words: community tolerance, fluorescence, herbicides, periphyton, photosynthesis

Abstract A test was developed that measures in vivo chlorophyll a fluorescence variables to assess the apparent sensitivity of freshwater periphytic algae to photosystem II inhibitors. Natural periphytic communities from rivers were collected on artificial substrata, and the effects of short-term exposures to two PSII herbicides (atrazine and isoproturon) on the fluorescence parameters were measured with a pulse-amplitude modulated fluorometer. The EC50 for each herbicide were calculated from fluorescence yield indices, and these results were compared to 14 C-based primary production measurements on the same communities. The fluorescence-based method appears to give very reliable estimations of EC50 for each pesticide we tested, ranging from 0.46 to 5.18 µM and 0.07 to 6.77 µM for atrazine and isoproturon, respectively. This method could be used in ecotoxicology monitoring programs, to detect changes in natural periphyton populations sensitivity, following photosystem II herbicide contamination in rivers or lakes. Abbreviations: EC50 – Effective Concentration reducing by 50% a given parameter in test organism; PAM – Pulse-Amplitude Modulated fluorescence (fluorimeter)

Introduction Despite increasing restrictions on the use of pesticides, the freshwater systems of many countries are becoming more and more polluted by various chemicals. Among them, many herbicides, which are mainly used in intensive agriculture, and which are ending in rivers (e.g. Pereira & Rostad, 1990). Several metabolic processes of plants are inhibited by herbicides, and photosystem II (PSII) is a key target for most of them (Solomon et al., 1996). These bind to the plastoquinone binding protein of PSII, causing the disruption of photosynthetic electron flow, which leads to a herbicide concentration dependent rise in fluorescence yield and a decrease in photosynthetic efficiency. Hence, the herbicide effect results in a modification of the in vivo fluorescence pattern of photosynthetic organisms, which can be monitored us-

ing the pulse-amplitude modulated fluorometer (PAM) system, described by Schreiber et al. (1986) for phytoplankton. This system was previously employed in order to use microalgae as biosensors for herbicide determination in natural waters, after solid-phase concentration of the chemicals, and algal immobilization (Wilhelm et al., 1996). Microalgae, because of their trophic level, rapid growth and ubiquity, are good indicators of environmental changes and the health of aquatic ecosystems (McCormick & Cairns, 1994). Periphyton sampling is often sources of errors, because of the difficulty to collect organisms and because of the representativity of sampling (Cattaneo et al., 1997). We decided to use artificial substrates to collect periphytic algae in small streams. These are not supposed to give a realistic and complete estimation of the whole algal community, because the colonising al-

510 gae may be selected by the physico-chemical nature of the substratum (Cattaneo & Amireault, 1992), but they do allow the collection of homogenous samples. We used scraped glass discs to collect periphyton, and tested the sensitivity of these organisms to two PSII inhibitors, atrazine (2-chloro-4-ethylamino-6isopropylamine-s-triazine), and isoproturon (N, Ndimethyl-N’-[4-(1-methylethyl)phenyl]urea). The effects of semi-logarithmic increases in the concentrations of each herbicide on periphyton were monitored using a PAM fluorometer, and the fluorescence parameters were selected to draw dose-response curves. From these, we calculated EC50s in each experiment, comparing them to results from a 14 C-assimilation test of photosynthesis, which have already been proven useful for assessing toxic effects under both natural conditions (Kromkamp et al., 1998), and anthropogenic pollutions (Juneau & Popovic, 1999), and on periphyton (Blanck, 1985; Guasch et al., 1997; Herman et al., 1986). The aim of our study was to develop a method to reveal changes in periphyton sensitivity to a given set of toxicants (namely PSII inhibitors). This sensitivity is depending on the previous contamination level of the ecosystem the periphyton was taken from, and could finally be linked to changes in communities (Goldsborough & Robinson, 1986) under selective pressure of background pollution.

Methods Study site The River Ozanne, located in the centre of France, which drained an area of intensive colza crops and was thus influenced by agricultural runoff containing herbicides, mainly atrazine and isoproturon, was sampled five times during one season (May 2000 to January 2001). Four stations were chosen from upto downstream (Oz#6, Oz#4, Oz#3.1 and Oz#1.1). The Ozanne watershed was subject of an institutional monitoring program, giving a data background regarding the water contamination by herbicides: atrazine was measured using gas-chromatography coupled to mass-spectrometry, whereas isoproturon was measured using high performance liquid chromatography, with diode array detection. Both molecules were simultaneously assayed using a commercially available ELISA kit (Rhône Diagnostic, France). Water samples were collected during each periphyton sampling, in order to determine the herbicide contamination.

Periphyton sampling Small glass discs of 1.5 cm2 area were glued onto 18 × 22 cm pieces of 4 mm thick Plexiglas, using aquarium silicon sealant, assumed to be free of toxic chemicals. The upper surface of the glass discs was roughened to favour periphyton installation. The plates (bearing about 120 glass disks) were then fixed to concrete blocks and placed in the running part of the stream to avoid sediment covering the disks, and exposed to sunlight (width varying from 0.5 to 5 m), to minimize the side effects due to differences in light exposition. Glass discs were covered by 10 to 30 cm of running water at the beginning of colonization. Periphytic communities were allowed to grow for 2–3 weeks, to obtain sufficient biomass for a measurable fluorescent signal. The colonized plates were removed, placed in plastic bags filled with river water, put in an insulated box, together with water samples for basic physico-chemical measurements (nutrients, pH and conductivity), and transported to the laboratory. The samples were placed in a thermostated chamber, within small plastic aquaria containing river water, and acclimated to laboratory conditions for 24 h under a light cycle 14:8, with an intensity of about 80 µMol photon m−2 s−1 at the periphyton level (measured with a LiCor 1400 equipped with a LiCor 192 PAR sensor). Three to five glass disks were taken from the same plates, fixed with 5% (final concentration) formaldehyde, and used to identify the periphyton under the light microscope. Another set of colonized glass disks was sonicated in methanol / 0.5 m ammonium acetate (98/2 v/v), and the extracted matter was used to determine lipophilic pigments by reverse-phase HPLC (Wright & Jeffrey, 1997). The periphyton biomass was then estimated as µg chl a per unit of substrate surface (Bonin & Travers, 1992), and percentage of each dominant algal group estimated according to Wilhelm et al. (1991). Ratio of each diagnostic pigment identified (fucoxanthine, lutein, and zeaxanthin) versus total chl a in the sample were used to estimate abundance of diatoms, chlorophyceae, and cyanobacteria, respectively. Preparation of herbicide solutions Herbicides were dissolved in acetone prior to dilution in the test vessels. Stock solutions of 20 mM atrazine (MW 215.69), and isoproturon (MW 206.29) were prepared, and stored at –30 ◦ C. Atrazine and isoproturon were high grade pesticide standards (Cluzeau

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Figure 1. Typical fluorescence recording using a PAM 101-103 fluorometer. A: Control without herbicide; B: Sample with atrazine (32 µM). 1: background level of fluorescence signal (no light applied). 2: ground fluorescence yield, F0 (inactinic modulated light). 3: saturating light pulse, maximal fluorescence yield Fmax . 4: Herbicide addition (100 µL), followed by a series of non-saturating pulses (∗ ), with F’max determinations. 5: End of the fluorescence recording.

Info Labo, Paris, France). A semi-logarithmic concentration series was freshly prepared for each herbicide, with a multiplication factor of 100.5 , by serial dilution of the stock solution. The final test solution was prepared by diluting in river water filtered on Whatman GF/F glass fibre filter. A solvent blank made with acetone without herbicide was used for the controls, acetone concentration for controls and contaminated samples in all the tests was equal, i.e. 0.5% of total volume. Final test concentrations ranged from 0.01 to 100 µM for atrazine and isoproturon. Fluorescence testing conditions The glass disks bearing periphytic communities were removed from the plates, and all visible debris or living animals were removed. We used the wells of 24-wells polystyrene microplates as test vessel, whose diameter was appropriate for the glass disks and the armed fiberoptics from the fluorescence system.

Fluorescence variables were monitored using a PAM 101–103 (Walz, Effeltrich, Germany), equipped with a halogen lamp (Schott model KL1500) to provide actinic light. Two protocols were tested; the first consisted in simply exposing periphyton for at least 5 hours to the herbicides. PAM measurements started after 20 minutes of dark – adaptation. According to several authors, such as Samson and Popovic (1988), who reported the impact of pollutants on the potentially photosynthetic efficiency (Fv /Fmax ) of unialgal strains, a single saturation pulse with the halogen lamp (ca. 1200 µMol photon m−2 s−1 ) was applied to estimate Fv /Fmax for each concentration. The second protocol consisted in placing the periphyton sample in a well, adding 900 µL of Whatman GF/F filtered water from the algae sample site, and allowing the system to dark-adapt for 20 minutes. The fluorescence measurements started with the measurement of Fo , followed by a 600 ms saturating pulse to determine Fmax 80 seconds later. A series of non-saturating pulses (ca. 160 µE.m−2 .s−1 ) every 60 s was then programmed, and the herbicide (100 µL) was injected into the vessel just after the first non saturating pulse. The recording ran for twenty minutes, and data were transferred to a spreadsheet (Excel 97 software). A typical recorded fluorescence kinetic curve is given in Figure 1, for one control and one highly contaminated sample (32 µM atrazine). Calculation of fluorescence parameters and dose-response curves We used the method of Conrad et al. (1993), initially designed for unialgal phytoplankton studies, to calculate the herbicide sensitive fluorescence yield, shortly named y. This parameter was then transformed to give relative values comprised between 1 (control, healthy periphyton sample) and 0 (photosynthesis totally inhibited, maximal fluorescence yield, with no electron flows through the photosynthetic pathway): R =1−

y ymax

where R (relative yield) is the value used to draw the dose-response curve, and ymax the value obtained when the herbicide effect is maximal, and therefore background fluorescence is maximal. Data were fitted to a logistic equation (Seguin et al., 2001), using least square method (Nyholm, 1990), in order to determine the EC50 values for each PSII inhibitor.

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Figure 2. Compared dose-response curves obtained on samples from the same station (Oz#6 on June 2000) with atrazine. Parameters were relative yield R (closed circles) for y measured according to Conrad et al. (1993) (see text for details), and fluorescence yield Fv /Fm (open squares), linked to photosynthetic efficiency, measured after preincubation with the herbicide in the dark. Data are expressed as percent of the control value (R = 1 with y = 61.2; Fv /Fm = 0.46) obtained on a sample without atrazine. Only the R data permitted calculation of an EC50 (0.67 µM).

Figure 3. Example of dose-response curve fitted to a logistic model, allowing to calculate the EC50 value for a given PSII herbicide. Periphyton samples from station 6 in September 2000, contaminated with increasing amounts of isoproturon (open squares: PAM measurements of R, closed squares: 14 C measurements, dashed and bold lines: modelled dose-response curve for PAM and 14 C experiments, respectively). Data are expressed as% control value (R = 1 with y = 84; 14 C activity = 6141 dpm) obtained on a sample without isoproturon.

Measurements of photosynthetic activities by 14 C incorporation

Results

The periphyton disks were directly placed onto 20-mL scintillation vials, containing 2 mL Whatman GF/F filtered water collected from the same sampling site. The samples were contaminated with the same concentration range of herbicide as those used for the PAM test. After one hour of preincubation (temperature limits 8–23 ◦ C depending on the in situ water temperature of the sampling station, PAR ca. 70 µMol photon m−2 s−1 ), 20 µL NaH14 CO3 (0.4 µCi, CFA3, Amersham Pharmacia Biotech) was added to each vial, and photosynthesis was allowed to run for two hours under the same conditions. The reaction was stopped by adding formaldehyde (3% final concentration), followed by 200 µL glacial acetic acid (in a fume hood) to remove inorganic carbon (Nyström et al., 2000). Supernatant water was removed after one hour, and the disks dried for 6 h at 60 ◦ C under a stream of air. Labelled organic matter was dissolved in 1 mL dimethylsulfoxide (one hour at 45 ◦ C) and 15 mL of scintillation cocktail (Ultima Gold LLT, Packard Instruments) was added. The samples were counted after quenching attenuation on a 2100-TR (Packard Instruments). Dose-response curves were traced using gross radioactivity values, as percent of the radioactivity of the control at each herbicide concentration.

Experiments performed on periphytic communities confirmed that the measurement of Fv /Fmax itself is not sensitive enough to assess the effects of PSII herbicides. No important changes in Fv /Fmax were revealed with the first protocol (Figure 2), even when incubation with herbicide had been made for more than 24 h. In contrast, the modified version of the Conradmethod successfully generated signals that were dependent on the short-term effects of the PSII herbicide on photosynthesis. The dose-response effects (Figure 3), based on the estimation of y, of each PSII inhibitor allowed the calculation of EC50 in each case. When all the toxicity data (Table 1) obtained with the fluorescence method are considered, where isoproturon and atrazine were tested simultaneously, the former was more toxic than the latter, with mean EC50P AM values of 1.01 µM and 2.5 µM, respectively. Comparisons with 14 C assimilation were made for most of the tests with isoproturon (mean EC5014C of 0.25 µM) and atrazine (mean EC5014C of 1.14 µM). The EC5014C values were not correlated with the EC50P AM results for the same periphyton communities, and were lower than those calculated with the fluorescence method. The EC50 values show a gradient of sensitivity, which is broadly related to the contamination at each sampling site (Table 1). The downstream stations

513 Table 1. Minimal and maximal values, and mean (between brackets) for known concentrations (k.c.) of atrazine and isoproturon (italics) in the different sampling sites of R. Ozanne, and average chlorophyll a content, EC50 obtained from PAM measurements (‘Conrad’) and 14 C incorporation. The first line refers to the results with atrazine, the second with isoproturon (italics). More data were collected for PAM analysis, explaining part of the difference in range of apparent sensitivities Station

Chl a (µg cm−2 )

k.c. (nM)

EC50 (µM) PAM

14 C

Oz#1.1

1.3–53.8 (20.5)

0.19–21.32 (6.48) 0–2.33 (1.02)

1.39–4.48 (3.55) 0.43–6.77 (3.06)

0.16–1.0 (0.5) 0.34–0.56 (0.45)

Oz#3

9.3–20.4 (12.6)

0.23–78.8 (19.14) 0.24–6.79 (0.73)

1.92–5.18 (3.16) 0.19–0.7 (0.48)

0.24–2.65 (1.45) 0.1–0.23 (0.17)

Oz#4

1.2–8.4 (4.6)

0.33–19.94 (5.33) 0.24–0.53 (0.19)

1.0–3.34 (1.61) 0.07–0.64 (0.39)

0.62–1.53 (1.06) 0.9

Oz#5/6

0.3–9.7 (3.9)

0.14–0.46 (0.23) 0–1.07 (0.44)

0.26–1.36 (0.84) 0.46–1.43 (0.86)

0.42–0.97 (0.75) 0.21–0.26 (0.24)

be adapted to natural phytoplankton samples, provided that enough biomass can be concentrated on a small surface to give a readable fluorescence signal.

Discussion

Figure 4. Relative abundance (percentage of total biomass) of the three major groups of periphytic species, obtained by HPLC analysis of diagnostic pigments signatures, during the whole survey. Black: cyanobacteria; white: chlorophyta; striped: diatoms. Bars 1–3: sampling in May 2000; 4–8: June 2000; 9–12: September 2000; 13–17: January 2001.

are more contaminated than the upstream ones, and periphyton coming from the former ones was more resistant in our short-term tests. Oz#6 is the least contaminated station. All the stations show a seasonal trend in the change of the taxonomic composition (Figure 4), but diatoms are the dominant group, which was confirmed by microscopy (data not shown). Finally a downstream increasing gradient of biomass, expressed in terms of chl a, can be observed (Table 1). The fact that once we were able to use detached algae on a glass fibre filter indicates that this method can

The results obtained with the two fluorescence protocols show the importance of taking the most sensitive parameter to measure the endpoint (Figure 2). Estimates of the toxic effects of PSII inhibitors done by measuring the fluorescence yield Fv /Fmax , calculated after herbicide pre-incubation in the dark, were insufficiently sensitive to ensure a satisfactory estimate of their effects in the systems tested, whereas estimation of the herbicide sensitive fluorescence yield did achieve this. The reason for this difference is still unclear. Other fluorescence parameters, such as photochemical quenching, non-photochemical quenching, and quenching of the quantum yield, are promising, and will be studied in the near future. The use of variable fluorescence appears to be an efficient noninvasive tool to assess PSII quantum efficiency, and therefore to detect changes in photosynthetic activity. The PAM system appears to be well-suited for this purpose, despite the need for a concentrated biomass in a small surface, to obtain reliable fluorescence measurements. The EC50s we calculated were far higher than the known maximal contamination in Ozanne (2.33

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Figure 5. Comparison of periphyton sensitivity to atrazine and to isoproturon. Each point represents atrazine and isoproturon EC50 obtained from the same sampling site and date, based on fluorescence and primary productivity measurements. The correlation parameters were: r = 0.638, p = 0.001, n = 43. Closed squares: samples from Oz#1.1; closed circles: samples from Oz#3.1; open circles: samples from Oz#4; open squares: samples from Oz#6.

nM and 78.8 nM for isoproturon and atrazine, respectively), but our results are comparable to those of published studies on the sensitivity of freshwater periphyton to atrazine (e.g. Guasch & Sabater, 1998, which gave EC50s for atrazine ranging from 0.21 to 3.32 µM using photosynthesis-irradiance curves). The EC50 values calculated for each station are generally in relation to the degree of contamination. Isoproturon displayed a greater toxicity to periphyton than atrazine and, the two were correlated (Figure 5). The slope of the regression curve, obtained with the fluorescence data, indicates that isoproturon was 2.5 times more toxic than atrazine, results were similar to those mentioned by Kirby & Sheanhan (1994). Co-tolerance would be possible, as the defence mechanism might be the same against atrazine and isoproturon, since both have the same mode of action. If this mechanism can be shown to be general, then it would allow the use of only one toxicant during short term testing to assess the sensitivity / tolerance of periphyton to any chemical with the same metabolic target. The relationship between periphyton biomass (expressed as chl a) and EC50 values were examined by linear regression analysis (data not shown). No correlation was found, indicating that in all cases the periphyton cells were saturated in toxics and that the measured effect corresponded to the maximal effect. Fluorescence modification due to PSII inhibitors can be used in a practical assay (Juneau & Popovic, 1999; Maxwell & Johnson, 2000). Fluorescence meas-

urements are less demanding in time and handling than radiolabelling of photosynthesis, they produce no radioactive wastes, and the laboratory does not require administrative authorisation. Furthermore, there is more specificity, assuming that fluorescence patterns are strictly linked to the PSII inhibition process, which is not the case for other toxicants which would affect other steps in photosynthesis. The differences in final EC50 values are not a significant caveat in such a case, and PAM based toxicity tests are thus as suitable as photosynthetic activity tests (Petersen & Kusk, 2000). PAM-based assessment on periphyton could be employed in the field, taking advantage that portable systems are commercially available. Accurate comparisons of the toxicity of PSII inhibitors to natural communities at different sites requires parameters sich as the trophic level, light climate (Guasch & Sabater, 1998; Guasch et al., 1998) and current velocity (Briggs et al., 1998) of the stations to be similar. This method is intended to detect the changes in the apparent short-term toxicity of a given compound under field conditions. It was applied successfully for a river with various levels of biomass and composition, under a varying level of contamination. This information could be most useful when examining how the background pollution may select freshwater periphytic communities, favouring the development of more resistant taxa, and needs coupling with fine analysis of community structure, either by classical taxonomy and pigment analysis, or by alternative methods using molecular probes for example.

Acknowledgements We thank Dr Annette Bérard for support and scientific help. Xavier Bourrain and the Agence de l’Eau LoireBretagne are acknowledged for technical and financial aid, and pesticide data for the River Ozanne. This work was made possible with grants from the French Ministry of the Environment. Nicolas Cauzzi and Isabelle Mercier, Audrey Duchaine, Alexandre Saint-Olive are acknowledged for their help in taxonomic determination, HPLC pigment analysis, and 14 C experiments, respectively. The authors wish to thank anonymous referees and the Editor, Prof. Brian A Whitton, for valuable improvements on this manuscript.

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