Inhibition of photosystem II (PSII) electron transport as a convenient ...

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Jan F.H. Snel1, José H. Vos1, Ronald Gylstra2 and Theo C.M. Brock3 ... ment and Aquatic Ecology, Ritzema bosweg 32a, NL-6703 AZ Wageningen, The ...

Aquatic Ecology 32: 113–123, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

113

Inhibition of photosystem II (PSII) electron transport as a convenient endpoint to assess stress of the herbicide linuron on freshwater plants Jan F.H. Snel1 , Jos´e H. Vos1 , Ronald Gylstra2 and Theo C.M. Brock3 1 Wageningen

Agricultural University, Department of Biomolecular Sciences, Laboratory for Plant Physiology, Arboretumlaan 4, NL-6703 BD Wageningen, The Netherlands (Phone: +31 317 482825; Fax: +31 317 484740; E-mail: [email protected]); 2 Wageningen Agricultural University, Department of Water Quality Management and Aquatic Ecology, Ritzema bosweg 32a, NL-6703 AZ Wageningen, The Netherlands 3 DLO, Winand Staring Centre for Integrated Land, Soil and Water Research, P.O. Box 125, NL-6700 AC Wageningen, The Netherlands Accepted: 30 July 1998

Key words: chlorophyll fluorescence, microcosm, phenylurea herbicide, photosynthetic electron flow, single species test, toxicity

Abstract Effects of the herbicide linuron on photosynthesis of the freshwater macrophytes Elodea nuttallii (Planchon) St. John, Myriophyllum spicatum L., Potamogeton crispus L., Ranunculus circinatus Sibth., Ceratophyllum demersum L. and Chara globularis (Thuill.), and of the alga Scenedesmus acutus Meyen, were assessed by measuring the efficiency of photosystem II electron flow using chlorophyll fluorescence. In a series of single-species laboratory tests several plant species were exposed to linuron at concentrations ranging from 0 to 1000 µg l−1 . It was found that the primary effect of linuron, inhibition of photosystem II electron flow, occurred with a half-lifetime of about 0.1 to 1.9 h after addition of linuron to the growth medium. The direct effect of the herbicide on photosynthesis appeared to be reversible. Complete recovery from the inhibition occurred with a half-lifetime of 0.5 to 1.8 h after transfer of linuron treated plants to linuron free medium. The EC50,24h of the inhibition of photosystem II electron transport by linuron was about 9–13 µg l−1 for most of the macrophytes tested. For S. acutus the EC50,72h for inhibition of photosystem II electron flow was about 17 µg l−1 for the free suspension, and 22 µg l−1 for cells encapsulated in alginate beads. In a long-term indoor microcosm experiment, the photosystem II electron flow of the macrophytes E. nuttallii, C. demersum and the alga Spirogyra sp. was determined during 4 weeks of chronic exposure to linuron. The EC50,4weeks for the long-term exposure was 8.3, 8.7 and 25.1 µg l−1 for E. nuttallii, C. demersum and Spirogyra, respectively. These results are very similar to those calculated for the acute effects. The relative biomass increase of E. nuttallii in the microcosms was determined during 3 weeks of chronic exposure and was related to the efficiency of photosystem II electron transport as assessed in the different treatments. It is concluded that effects of the photosynthesis inhibiting herbicide on aquatic macrophytes, algae and algae encapsulated in alginate beads can be conveniently evaluated by measuring photosystem II electron transport by means of chlorophyll fluorescence. This method can be used as a rapid and non-destructive technique in aquatic ecological research.

Introduction An undesirable side-effect of the agricultural use of pesticides on land is that these chemicals may enter adjacent freshwater ecosystems such as ponds and drainage ditches. To protect aquatic ecosystems from

damage by these chemicals, national and international authorities have set criteria. Current procedures for hazard assessment of pesticides in freshwater ecosystems are based essentially on information about the concentrations of these chemicals in surface water (usually predicted with computer models), and on

114 concentration-effect relationships as studied in singlespecies toxicity tests. Recently, the member states of the European Union adopted the Uniform Principles (Council Directive 91/414/EEC concerning the placing on the market of plant protection products; EU, 1994). In this directive it is stated that the predicted environmental concentration of a pesticide in surface water should not exceed 0.01 times the acute EC50 1 or 0.1 times the chronic NOEC2 of the most susceptible standard test species (algae, Daphnia or fish). In 1994, a research project was initiated with the aim to validate the current hazard assessment procedures for herbicides in freshwater ecosystems. The phenylurea herbicide linuron was chosen as a benchmark compound in the following test systems, which showed increasing ecological complexity: (1) Single species toxicity tests with standard test species (e.g., the alga Scenedesmus) and aquatic macrophytes inhabiting the other test systems. (2) Indoor derived microcosms that simulate the community of Dutch drainage ditches (e.g., Brock et al., 1992). (3) Outdoor mesocosms that simulate drainage ditches (Leeuwangh et al., 1994). The present paper deals with the single species tests and the indoor microcosms only. The primary mode of action of linuron, a herbicide from the phenylurea class, is binding to PSII resulting in inhibition of PSII electron transport (e.g., Van Rensen, 1989). Environmental risk assessment studies involving photosystem II inhibiting herbicides should preferably be carried out using the most sensitive parameter, i.e., inhibition of PSII electron transport. Measurement of photosynthetic electron transport in situ has traditionally been a difficult matter. Advances in methodology and instrumentation in the field of chlorophyll fluorescence have made PSII electron transport a relatively easy accessible parameter (Schreiber & Bilger, 1993). The rate of PSII electron flow depends on the rate of photon absorption and the efficiency of PSII. The efficiency of PSII electron transport describes the probability of a photochemical event leading to PSII electron transport upon absorption of a photon by the antennae of PSII. The efficiency of PSII electron transport can be determined by means of chlorophyll fluorescence and is related to photosynthetic carbon uptake (Genty et al., 1989). The method 1 EC , EC , EC 90 50 10 – herbicide concentrations that cause 90%, 50%, 10% inhibition of PSII electron flow or growth; 2 NOEC – no observed effect concentration;

is based on measurement of the steady state modulated fluorescence in ambient light and the maximal fluorescence during a non-modulated saturating light pulse (Schreiber, 1986; Hofstraat et al., 1994). In this study we have used chlorophyll fluorescence to assess the acute linuron effects on the efficiency of PSII electron flow of aquatic macrophytes and algae in single species tests and both acute and long-term effects in macrophyte dominated indoor microcosms. The aims of the study: (1) To assess whether measurement of the efficiency of PSII electron transport by means of chlorophyll fluorescence is a convenient and sensitive endpoint to assess linuron stress on aquatic macrophytes and algae. (2) To compare the acute linuron effects on PSII efficiency in single species tests between aquatic macrophytes that inhabit the indoor microcosms and the outdoor experimental ditches. (3) To compare short-term and long-term effects of linuron on macrophytes in freshwater microcosms. (4) To compare the effects of linuron on PSII efficiency of the alga Scenedesmus acutus in free suspension and encapsulated in alginate beads (encapsulated algae can be conveniently used as bioassays for in situ monitoring programs). Materials and methods Acute toxicity experiments with macrophytes For the single species tests the aquatic plants Elodea nuttallii, Myriophyllum spicatum, Ranunculus circinatus, Chara globularis and Potamogeton crispus were collected at the experimental ditches at the ‘Sinderhoeve’ (Leeuwangh et al., 1994) in october 1994 and transferred to glass containers in a temperature controlled phytotron room (19 ◦ C) and grown at a PPFD of 180–200 µmol m−2 s−1 using a 14 h light/10 h dark cycle. The macrophytes were grown in medium consisting of nutrient-poor well-water supplemented with (in mg l−1 ): N (1.72), P (0.39), K (2.63), Mg (0.25), Ca (1.7), S (1.03), B (0.0035), Cu (0.00017), Fe (0.006), Mn (0.011), and Zn (0.0025). During the day the heat resulting from the lamps raised the air temperature to 21 ◦ C, but the water temperature remained close to 19 ◦ C. The single-species experiments described in this paper were carried out in the period December 1994-January 1995. The kinetics of the inhibitory effects of linuron on PSII electron flow were studied by selecting 5cm

115 young shoots from the macrophytes and placing them in 25 ml glass tubes with the top of the shoot faced upwards for preincubation in linuron free medium. The medium was replaced with medium containing 50 µg l−1 linuron and the efficiency of PSII electron flow was measured regularly during the following 24 h. After 24 h incubation with linuron, the linuron containing medium was replaced with fresh linuron free medium and the efficiency of PSII electron flow was regularly measured for another 24 h. After each measurement the medium was renewed to remove linuron leaking out of the plants into the medium. The steady-state inhibition of PSII electron flow of the macrophyte species by linuron was determined after 24 h incubation of the shoots with linuron containing medium. Young shoots (5 cm) were preincubated for 24 h in 25 ml glass tubes with the top of the shoot faced upwards in linuron free medium. The medium was replaced with medium containing linuron and the efficiency of PSII electron flow was measured after 24 h incubation. The nominal concentrations were 0, 0.1, 1, 10, 25, 100 and 150 µg l−1 . Water samples were taken in duplo from stock solutions before the start of the incubation and from each centrifuge tube at the end of the fluorescence measurements to determine the actual linuron concentration in the medium during incubation. Acute toxicity experiments with Scenedesmus The unicellular green alga Scenedesmus acutus was axenically grown in a chemostat in 100% Z8-medium under continuous illumination (100 ± 5 µmol m−2 s−1 ) at 18 ◦ C in a temperature controlled room and cells were immobilized in alginate beads as described in Van Donk et al. (1992). At day 0 the algal suspensions, or beads with immobilised cells, were transferred to sterile 250 ml Erlenmeijer flasks in 100% Z8 medium to which linuron was added (nominal concentrations 0, 0.1, 1, 10, 25, 100 and 150 µg l−1 ) and incubated under continuous illumination (100 ± 5 µmol m−2 s−1 ) at 18 ◦ C in a climatted room. The experiments with the free suspension of S. acutus started with a concentration of 104 cells ml−1 . The mean initial cell density chosen for the alginate beads was 5 × 104 cells bead−1 (volume of one bead: 0.06 cm3 ) and using 0.2 bead ml−1 the number of cells was similar to the free suspensions experiment. Samples for algal counts were taken after 24, 48 and 72 h for measurement of PSII efficiency and cell number. Algal cells were counted with a Coulter Multisizer. Before count-

ing algal cells in the beads, the beads were dissolved separately in 5% sodium hexametaphosphate. Chronic exposure experiments in microcosms Twelve microcosms (1.1 × 1.1 × 0.7 m l × w × h, watervolume 600 l, 10 cm lake sediment, 50 cm watercolumn) were situated in a climatted room (19 ± 2 ◦ C) equipped with high pressure metal halide lamps (Philips HPI-T, 400 W) providing a PPFD3 of approximately 120 µmol m−2 s−1 at the watersurface with a daily illumination period of 14 h. See Brock et al. (1992) for details on the construction of the cosms. In the preparatory phase of the microcosm experiment, micro-organisms became introduced into the cosms, together with the natural sediment and well water. In addition, the macrophytes Elodea nuttallii and Ceratophyllum demersum and several populations of macroinvertebrates, typical for Dutch ditches, were deliberately introduced. A nutrient addition of P (nominal concentration: 0.05 mg l−1 ) and N (0.30 mg l−1 ) was also applied in this period. During an acclimation period of three months a biocoenosis was allowed to develop in the cosms. The aquatic macrophyte community became dominated by Elodea nuttallii. On February 15, 1994 linuron was added as the commercial herbicide Afalonr to 10 cosms in 5 duplicate doses with the remaining 2 cosms serving as a control. The nominal linuron concentrations were: 0.5, 5, 15, 50 and 150 µg l−1 . In the following 4 weeks additional herbicide was applied twice a week to keep the concentration constant. After 4 weeks the herbicide addition was stopped. See Van den Brink et al. (1997) for more experimental details. Direct effects of linuron on growth of the macrophyte E. nuttallii were determined by means of a bioassay. In each microcosm E. nuttallii shoots (total wet weight 4 g) were allowed to settle in a plastic beaker filled with sediment. Before the shoots were weighed, they were gently blotted with tissue paper. The beaker was transferred to a transparent cage (10 × 10 × 50 cm, l × w × h) with one side consisting of gauze (mesh size: 55 µm). Relative growth data were taken from Van den Brink et al. (1997). The caged plants were placed in the microcosm at a depth of 45 cm below the watersurface. Sufficient water exchange between the cage and the microcosm was ensured by regularly raising the cage. The initial Elodea dry weight was estimated from four extra portions of 4 g wet-weight dried at 105 ◦ C for 48 h. 3 PPFD – photosynthetic photon flux density.

116 The mean dry weight of these portions was 0.35 g. The bioassays started at the beginning of the linuron application and lasted 3 weeks when the dry weight of the Elodea shoots was determined. These data were used to calculate the relative biomass increase to be calculated. Chlorophyll fluorescence measurements In the single species tests the efficiency of photosystem II electron flow of the macrophytes was measured by means of chlorophyll fluorescence with a PAM fluorometer (Walz Mess- und Regeltechnik, Effeltrich, Germany) as described in Hofstraat et al. (1994). The fluorescence excitation light, the ambient light and the saturating light were guided to the sample via three arms of the four-armed optical fiber resulting in a PPFD at the watersurface of 0.03, 194 and about 10000 µmol m−2 s−1 , respectively. The duration of the saturating light pulses was 500 ms and the pulses were given every 30 s. The measurements on macrophytes were carried out using young shoots of about 5 cm length. The shoots were placed in a 25 ml glass tube with the top of the shoot faced to the common end of the optical fiber of the fluorometer. The top of the shoot was 5 mm below the water surface and the optical fiber was positioned 5 mm above the water surface. In the microcosm experiment and in the single species S. acutus tests the efficiency of PSII electron transport was determined from fluorescence measurements with a slightly modified version of the Xe-PAM fluorometer (Schreiber et al., 1993) using broadband excitation from 400–560 nm (Xenon flashlamp plus 4 mm Schott BG39) and emission detected above 650 nm (Balzer R65 plus Schott RG645). The XePAM fluorometer is equipped with two halogen lamps for actinic and saturating light. The saturating light was filtered through a 650 nm short-pass filter (Balzers DT Cyan special) and was used at an intensity of 6000 µmol m−2 s−1 , the actinic irradiance was adjusted with neutral density filters. The fluorescence measurements were corrected for background signals not related to chlorophyll fluorescence. Macrophyte shoots, algal suspensions and beads were measured in 1 cm glass cuvettes. The algal suspensions and beads were continuously stirred during measurements. The efficiency of PSII electron flow was determined from the steady state fluorescence and the maximal fluorescence of samples that had reached a

steady state in the light, which was usually attained after about 10 to 15 min illumination. Determination of linuron Linuron solutions and linuron samples were stored at 5–7 ◦ C. Linuron concentrations were determined by HPLC as described in Van den Brink et al. (1997). The 0.1 µg l−1 linuron solutions were concentrated by solid phase extraction before HPLC analysis. Plastipak cartridge columns (5 ml) were filled with 0.4 g octadecyl (C-18) between two polypropylene filters. The extraction columns were conditioned with 1×5 ml methanol and 2×5 ml deionized water, respectively. A known sample volume was extracted with the extraction column under vacuum. Elution of linuron from the extraction column was carried out with 3 portions of 0.5 ml acetonitrile. The eluate was collected in a 10 ml tube and diluted with distilled water to a total volume of 5.0 ml. The sample was sonicated and filtrated over a 0.45 µm filter before HPLC analysis. Data analysis The acute linuron effects on the efficiency of PSII electron flow were modelled assuming that linuron enters and leaves the plant by passive diffusion only and that the linuron concentration in the water is constant during the experiment. These assumptions imply that the inhibition of PSII electron flow by linuron binding is equivalent to the binding of a ligand to a macromolecule, a process with (pseudo) first-order kinetics (Snel & Van Rensen, 1983). In steady state, i.e., when there is an equilibrium between free and bound herbicide, the ligand-binding model results in a hyperbolic relation between PSII electron flow and the free herbicide concentration in isolated chloroplasts (Snel & van Rensen, 1983). The decrease in the steady-state efficiency of PSII electron flow is a direct measure for inhibited PSII centers containing linuron and the steady-state relation between the free herbicide concentration and the efficiency of PSII electron flow can be described by a hyperbolic function: φpi = φpc

1 E50 + [I ]

(1)

with φpc , φpi : steady state efficiency of PSII electron flow in control and linuron treated plants, respectively; EC50: herbicide concentration at 50% inhibition of φpc ; I: free herbicide concentration.

117 inhibition after transfer to linuron free medium this results in the following equation: φp (t) = φpi + (φpc − φpi )(1 − e−k2 t )

(2)

E

with φp (t): efficiency of PSII electron flow at time t; other abbreviations as in Equation (1). The kinetics of the inhibition after addition of linuron to the medium is described by the following equation: φp (t) = φpc + (φpc − φpi )(1 − e−k1 t ) I Figure 1. Kinetics of the inhibition of photosystem II electron flow in Elodea nuttallii by 50 µg l−1 linuron ( ) and the subsequent recovery of inhibition by washing with well water ( ). The curves represent exponential fits generated by the least squares procedure. The fit function was assumed to be an exponential function of time t containing the efficiency of PSII electron flow in the control (φPc ) and linuron treated (φPi ) shoots and the rate constant k as parameters: φP = φPi + (φPc − φPi )(1 − e−kt ). The photon flux density during the experiment was 194 µmol m−2 s−1 .

#

with φp (t): efficiency of PSII electron flow at time t; other abbreviations as Equation (1). The function parameters were fitted to the data in MathCad (MathSoft Inc.) using a method that minimizes the sum of the least squares of the deviations from the fit. In the long-term experiments the macrophytes may show adaptive responses. For practical reasons no proper control was available and the relation between the efficiency of PSII and the free herbicide concentration was not hyperbolic and a statistical model had to be applied. Assuming that distribution of the errors in the determination of the efficiency of photosynthesis are of the Poisson type, a logistic model proved to be useful and logistic regression fits were generated using the following function (McCullagh & Nelder, 1989):

E

φpi =

L Figure 2. Inhibition of photosystem II electron flow of Elodea nuttalli shoots by linuron. At each concentration 3 shoots were used. After 24 h incubation with linuron chlorophyll fluorescence was measured from randomly selected shoots. Water samples were taken in duplo from stock solutions before the start of the incubation and from each centrifuge tube at the end of the fluorescence measurements to determine the linuron concentration. The solid and dotted lines represent the fit of the data using the ligand-binding and logistic model, respectively.

(3)

1 1+

eb(ln(I )−a)

× φpc

(4)

with φpc , φpi : steady-state efficiency of PSII electron flow in control and linuron treated plants, respectively; a: logarithm of the herbicide concentration at 50% inhibition of φpc and b: first derivative of the first term of the function at 50% of maximal inhibition; I: free herbicide concentration. The logistic model was programmed in Genstat version 5.31 (Payne & Lane, 1987). No Observed Effect Concentration (NOEC) calculations were performed with the Williams test. This test is based on ANOVA and assumes an increasing effect with an increasing dose (Williams, 1972).

Results The kinetics of the effects of linuron on PSII efficiency is given by an exponential function with as additional parameters the rate constants k1 for the herbicide binding to PSII and k2 for the dissociation from the PSII-herbicide complex. For the recovery from

Acute linuron effects on PSII efficiency in Elodea nuttallii The kinetics of the inhibitory effects of linuron on PSII electron flow were studied by replacing the medium

E

118

R Figure 3. Relationship between the efficiency of photosystem II electron transport measured at day 1 from shoots of the standing stock in the microcosm and the relative biomass increase of Elodea nuttallii determined from the bioassays. The fit was generated from the logistic dose-response relationships of the efficiency of photosystem II electron transport (see Table 5, week 1) and the relative biomass increase (for original data see (Van den Brink et al., 1997)).

in which E. nuttallii plants were incubated for 24 h in the light in medium containing linuron (50 µg l−1 ) and the efficiency of PSII electron flow was measured regularly. Figure 1 shows at time t = 0 the steadystate efficiency of PSII electron flow is about 0.5. The binding of linuron and the resulting inhibition of PSII electron flow has reached a steady-state within about 4 h. The steady-state efficiency of PSII at constant PPFD depends on the concentration of linuron. Figure 1 shows that linuron inhibition is reversible and that recovery is complete after about 6 h. Exponential fits yield a half-time of 0.41 h for inhibition and 1.5 h for the recovery of PSII electron flow from inhibition. Figure 2 shows the acute effects of linuron on the efficiency of PSII electron flow in the steady-state at a PPFD of 194 µmol m−2 s−1 . A logistic fit of the experimental data of Figure 2 (solid line) yields an EC50,24h of 9.0 µg l−1 which is very similar to the 9.2 µg l−1 of the ligand binding model (dotted line). The 95% confidence intervals (c.i.) were also similar: 7.5–10.7 µg l−1 for the logistic model versus 8.0– 10.4 µg l−1 for the ligand binding model. We used the logistic approach to quantify the effects of linuron in both the single species tests and the microcosm experiments. Relation between PSII efficiency and relative growth Figure 3 shows the relationship between the efficiency of PSII electron flow measured after one day exposure to linuron and the relative biomass growth of

the E. nuttallii bioassay determined at the end of the 3 weeks constant chronic exposure in the microcosms. The efficiency of PSII electron transport at day 1 was lower than the control at all but the lowest dose (NOEC = 0.5 µg l−1 ) and the EC50,24h of the inhibition of PSII electron flow was 11.7 µg l−1 (95% c.i.: 9.1–15.0). At the end of the bioassay period the NOEC using the efficiency of PSII electron transport as an endpoint was 0.5 µg l−1 . The NOEC with relative growth in the bioassay as endpoint was 0.5 µg l−1 and the EC50,3weeks was 2.5 µg l−1 (95% c.i.: 1.1–6.0 µg l−1 ) (Van den Brink et al., 1997). Tables 1 and 2 show the effects of linuron on PSII efficiency and the relative growth rate of Scenedesmus acutus. The EC50 of the effect of linuron on PSII efficiency is about 18 µg l−1 in a suspension of algae and about 24 µg l−1 in beads. These EC50 ’s are not very dependent on the time between day 1 and day 3. In contrast with the effect of linuron on PSII efficiency, the effect on growth seems to be time-dependent. Notably on day 1 the EC50 with growth as effect endpoint is higher than on days 2 and 3. But even on day 3 growth is more sensitive to linuron than PSII electron flow. This might be caused by part of the photosynthetically fixed energy being not available for growth but being required for maintenance. This would imply a non-linear relationship between PSII electron flow and growth. Figure 4 shows the relationship between PSII electron flow and growth as derived from the data at day 3 in Tables 1 and 2. The relationship is nonlinear but very similar for the algae in free suspension and the algae in beads. Single species tests with macrophytes In a comparative study the kinetics and extent of the short-term effects of linuron on a number of macrophyte species were investigated. The kinetics of a number of macrophytes are summarized in Table 3. The data show that, except for P. crispus, inhibition is faster than recovery and that differences between species are relatively small. Not surprisingly, Chara, with its small distance between chloroplast and external medium, shows the most rapid kinetics. Table 4 shows the effect concentrations for 10%, 50% and 90% inhibition of PSII electron transport in the macrophytes. The lowest and highest EC10 , EC50 and EC90 are 0.7–2, 11.8–13.4 and 90–194 µg l−1 , respectively. Differences in effects between the macrophytes tested in this study were not statistically significant. The results are based on the nominal linuron

119 Table 1. Short-term effect of linuron on the efficiency of photosystem II electron flow in a free suspension of Scenedesmus acutus and in encapsulated S. acutus cells (beads). Chlorophyll fluorescence was measured at days 1,2 and 3. The effect concentrations resulting in 10, 50 and 90% inhibition of PSII electron flow were calculated using a logistic fit model and the numbers in brackets indicate the 95% confidence limits Test conditions

Free suspension Free suspension Free suspension Beads Beads Beads

Day

1 2 3 1 2 3

Inhibition of efficiency PSII EC10 (µg l−1 ) EC50 (µg l−1 )

EC90 (µg l−1 )

1.3 (0.7–2.3) 1.9 (1.3–2.7) 2.5 (1.8–3.5) 1.7 (0.2–16.4) 1.6 (0.5–5.2) 1.3 (0.7–2.3)

117.3 (84.1–163.5) 189.2 (154.8–231.4) 120.4 (98.4–147.2) 295.2 (73.5–1186.3) 401.3 (198.0–813.4) 379.2 (271.0–530.7)

12.2 (9.4–16.0) 18.7 (16.0–21.9) 17.3 (14.8–20.2) 22.2 (8.6–57.5) 25.7 (16.2–40.7) 22.1 (17.7–27.7)

Table 2. Short-term effect of linuron on the relative volume growth rate of Scenedesmus acutus in free suspension and encapsulated in beads. Total algal volume was measured at days 0, 1, 2 and 3. The relative growth rate was calculated as the relative increase in volume with respect to day 0. The effect concentrations resulting in 10, 50 and 90% reduction of growth were calculated using a logistic fit model and the numbers in brackets indicate the 95% confidence limits Inhibition of relative volume growth rate EC10 (µg l−1 ) EC50 (µg l−1 ) EC90 (µg l−1 )

0–1 0–2 0–3 0–1 0–2 0–3

0.9 (0.0–22.7) 0.5 (0.3–0.7) 1.2 (0.7–2.0) 4.4 (1.4–13.6) 2.0 (1.0–3.9) 2.8 (1.8–4.3)

E

Free suspension Free suspension Free suspension Beads Beads Beads

Day

R Figure 4. Relationship between relative volume growth and efficiency of photosystem II electron transport of Scenedesmus acutus in free suspensions and encapsulated in alginate beads. The data and fit were taken from the toxicity experiment in Tables 1 and 2. The fit was generated from the logistic dose-response relationships computed from the growth and the photosystem II efficiency measurements.

23.6 (6.9–80.3) 5.4 (4.6–6.3) 6.0 (4.9–7.5) 24.8 (14.7–41.6) 10.9 (8.2–14.5) 13.2 (10.6–16.5)

639.8 (96.9–4270.8) 56.3 (46.4–68.3) 30.3 (22.7–40.5) 140.2 (69.3–283.6) 58.8 (34.8–99.4) 63.7 (43.4–93.5)

concentrations, not on the concentrations as measured in the first experiment with E. nuttallii shown in Figure 2. This was to avoid the time-consuming and costly procedure of measuring small amounts of linuron. It can be expected that the actual free linuron concentration in the water is somewhat lower than the nominal concentration as a result of breakdown and binding to the plant and to the surfaces of the glass tube. This could explain the fact that the apparent EC50 of E. nuttalli in the second experiment is higher than the EC50 as calculated for the first experiment (Figure 2). We therefore conclude that the EC50 of the macrophytes tested is similar to the value found for Elodea in Figure 2, i.e., about 9 µg l−1 .

120 Table 3. Kinetics of the effect of linuron on the efficiency of photosystem II electron flow in aquatic macrophytes. The half-life time t1/2 data were calculated from two independent measurements as described in Figure 1 Plant species

Inhibition t1/2 (h)

Recovery t1/2 (h)

E. nuttallii M. spicatum P. crispus R. circinatus C. globularis

0.41 0.23 1.9 0.16 0.1

1.5 1.8 0.57 1 0.45

Macrophytes and filamentous algae in freshwater cosms The long-term effects of chronic linuron exposure on aquatic macrophytes were studied in a series of macrophyte-dominated freshwater microcosms. The linuron concentrations in the water compartments were kept at a constant level during the first 4 weeks of the experiment. After 4 weeks linuron was no longer added and the concentration in the water compartment decreased linearly (see Van den Brink et al., 1997). At regular intervals young shoots of the macrophytes were taken to the laboratory and the efficiency of PSII electron transport was measured at a PPFD of 92 µmol m−2 s−1 . Table 5 shows that the EC50 of E. nuttallii is about 9 µg l−1 during the entire experiment. At 5 weeks incubation the EC50 of C. demersum is not much different from the EC50 of E. nuttalii. The filamentous alga Spyrogyra sp. is less sensitive to linuron as judged from the EC10 (11.1 µg l−1 ) and the EC50 (25.1 µg l−1 ), but the EC90 is not different from the EC90 of the vascular plants.

Discussion Acute linuron effects on PSII efficiency in Elodea nuttallii On ecological timescale, i.e., longer than during the experimental exposures, the effects of linuron on PSII electron flow are fast which makes the efficiency of PSII electron flow a convenient early warning method to detect stress of photosynthesis inhibiting herbicides in aquatic ecosystems. At an ecological timescale also the rate of recovery is fast when linuron is removed

from the medium. This suggests that a brief exposure of aquatic plants to linuron will not result in dramatic effects on ecosystem structure. The similarity in the sensitivity of macrophytes to linuron (see EC50,24h in Table 4 suggests that linuron will affect the tested macrophytes to the same extent on the short term. At longer exposures the larger energy reserves of macrophytes might allow the macrophytes to sustain growth in the presence of linuron, in contrast to planktonic algae. The EC50,24h of inhibition PSII electron flow in E. nuttallii by linuron (about 9 µg l−1 , Figure 2) is comparable to the 10 µg l−1 reported for inhibition of electron flow in isolated spinach chloroplasts (Arnaud et al., 1994) but lower than the 25–50 µg l−1 reported for its herbicide action (Fedtke, 1982). The discrepancy with the latter results might be related to differences in experimental conditions, notably the light intensity applied. At light-saturated rates of photosynthesis the rate of PSII electron flow is not determined by PPFD but by down-regulation of the efficiency of PSII electron flow. This down-regulation is reversible and is the result of conversion of absorbed light energy into heat. Under these conditions PSII does not operate at its maximum efficiency. Inhibition of a fraction of the PSII centers does not result in inhibition of steadystate photosynthesis as long as the remaining PSII are not yet operating at maximum efficiency. In contrast the efficiency of PSII electron flow is already high at limiting light and inhibition of a small fraction of PSII by linuron will result in a proportional inhibition of photosynthesis. It was shown that up to 85% of PSII could be blocked by diuron without significant inhibition of photosynthesis under high light where PSII is not the rate-limiting factor in photosynthesis (Heber et al., 1988). This has important consequences for the toxicity of linuron in aquatic ecosystems. The extent of the light-saturated region of photosynthesis of a species is modulated by a number of factors (e.g., availability of carbon dioxide, temperature, developmental stage, etc.) and this means that the EC50 of linuron is modulated by these factors as well. In other words the toxicity of linuron will be modulated by the microclimate. An individual plant in full sunlight might be nearly unaffected while another plant of the same species in the shade might be affected to a much greater extent. This phenomenon deserves further attention and is currently being investigated.

121 Table 4. Short-term effect of linuron on the efficiency of photosystem II electron flow in aquatic macrophytes. The EC10 , EC50 and EC90 data are based on nominal concentrations and were calculated using a logistic fit model. The numbers in brackets indicate the 95% confidence limits Plant species

EC10 (µg l−1 )

EC50 (µg l−1 )

EC90 (µg l−1 )

Elodea nuttallii1 Elodea nuttallii Myriophyllum spicatum Potamogetum crispus Ranunculus circinatus Chara globularis

0.9 (0.5–1.4) 2.0 (1.0–3.9) 0.7 (0.2–3.2) 1.0 (0.5–1.9) 1.7 (1.1–2.7) 1.0 (0.3–2.7)

9.0 (7.5–10.7) 13.4 (10.7–16.8) 11.8 (6.9–20.5) 12.9 (9.6–17.4) 13.2 (11.0–16.0) 12.1 (7.9–18.5)

91 (63–131) 90 (52–153) 194 (69–547) 173 (106–281) 101 (72–142) 152 (72–321)

1 Based on data from Figure 2 using measured linuron concentrations.

Table 5. Effect of linuron on the efficiency of photosystem II electron flow of aquatic macrophytes and filamentous algae in the microcosms. The EC10 , EC50 and EC90 data were calculated using a logistic fit model and the numbers in brackets indicate the 95% confidence limits Plant species Elodea nuttallii

Ceratophyllum demersum Spirogyra sp.

1 day 4 weeks 5 weeks 8 weeks 5 weeks 5 weeks

EC10 (µg l−1 )

EC50 (µg l−1 )

EC90 (µg l−1 )

1.7 (0.9–3.0) 1.5 (1.2–1.8) 1.4 (0.9–2.0) 1.3 (0.6–2.7) 1.0 (0.6–1.7) 11.1 (6.7–18.4)

11.7 (9.1–15.0) 8.4 (7.7–9.2) 8.3 (7.1–9.7) 10.5 (7.6–14.4) 8.7 (6.8–11.1) 25.1 (19.2–32.8)

81.9 (52.1–128.7) 48.5 (41.2–57.0) 50.2 (37.4–67.2) 85.1 (48.1–150.6) 77.1 (50.4–117.7) 56.9 (38.5-84.2)

Relation between PSII efficiency and growth In the microcosms the EC50 of E. nuttallii for inhibition of PSII electron flow was higher during the 3 weeks of chronic exposure (EC50,24h = 11.7 µg l−1 , EC50,5weeks = 8.3 µg l−1 ) than the EC50 for inhibition of growth measured as biomass increase in the bioassay (EC50,3weeks = 2.5 µg l−1 ). A similar phenomenon is observed in S. acutus where the EC50,72h for inhibition of PSII electron flow (17.3 µg l−1 ) is higher than the EC50,72h for inhibition of biovolume growth (6.0 µg l−1 , see Tables 1 and 2). This difference in EC50 for PSII electron flow and growth results in a non-linear relationship between PSII electron flow and growth (Figures 3 and 4). The reason for this non-linearity is not well understood; possibly the organisms have adapted to the presence of linuron adapted by decreasing the amount of light absorbed by PSII or by the number of photosystems II. Regardless of the physiological mechanism underlying the nonlinear relationship, the data in Figures 3 and 4 show a positive correlation between both endpoints, i.e. inhibition of photosynthesis and reduction of growth. This means that measurement of PSII electron flow can be

used to assess toxic effects of herbicides on aquatic macrophytes. Moreover, since the EC50 does not seem to be dependent on the duration of the exposure (Table 5), chlorophyll fluorescence may be a useful tool to predict herbicide effects on biomass growth of aquatic macrophytes. Chlorophyll fluorescence as a tool in ecotoxicological studies Conrad et al. (1993) have used chlorophyll fluorescence from a test alga as a bioassay to monitor the presence of herbicides in water samples. The method presented here is also based on the effects of herbicides on chlorophyll fluorescence, but the measurement of the efficiency of PSII electron flow not only allows the detection of herbicides but allows estimation of the effects of linuron on photosynthesis and hence on primary production as well. Accurate estimation of macrophyte biomass production at the vegetation or the single plant level, however, requires more experimental parameters. The efficiency of PSII electron transport, i.e. the probability of a photochemical event leading to electron

122 transport once a photon is absorbed in the antennae of PSII, is a fundamental property of a single PSII which is modulated by external factors such as light, availability of inorganic carbon and water temperature. If the number of absorbed photons per unit leaf area can be measured, the number of electrons transported per PSII can be calculated and if the number of PSII centers per unit leaf area is known, the total number of electrons can be calculated. In the short term experiments it is not likely that physiological (the number of PSII centers per leaf area or the size of the PSII antennae) or morphological (leaf reflection, leaf area etc.) adaptations of the plants occur. As the photon flux density during our measurements is equal to the photon flux density during growth, the experimentally observed efficiency of PSII is a direct measure for photosynthetic electron flow per unit leaf area. During long term incubation with linuron, however, morphological and physiological differences may develop (see, e.g., Kemp et al., 1985) which make assessment of differences in photosynthesis or primary production, as estimated from PSII efficiency measurements, more complex in long term microcosm or field experiments.

Conclusions This study demonstrates that effects of the herbicide linuron on photosynthesis of aquatic macrophytes and algae in single species tests and in microcosms can be conveniently evaluated by means of the efficiency of PSII electron flow. In the tested macrophytes the EC50,24h for inhibition of PSII electron flow by linuron was about 9 µg l−1 . The effects were reached within a few hours and were not much different after 4 weeks of chronic treatment. The EC50,72h for Scenedesmus cells in free suspension was lower than for cells encapsulated in beads and in ecotoxicological studies using beads as a model system one should account for this difference in EC50 .

Acknowledgements This study was carried out in the framework of the graduate school Experimental Plant Sciences. The authors acknowledge the National Institute for Coastal and Marine Management (RIKZ) for kindly making the Xe-PAM chlorophyll fluorometer available for some of the experiments. We would like to thank Drs.

Jacques van Rensen and M.J.M. Hootsmans for critically reading the manuscript and Paul van den Brink for statistical analyses of the data.

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