Journal of Applied Phycology 13: 509–515, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
509
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
511
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.
512
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
514
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.
515
References
Blanck H (1985) A simple, community level, ecotoxicological test
system using samples of periphyton. Hydrobiologia 124: 251–
261.
Bonin DJ, Travers M (1992) Examen critique des méthodes
d’estimation de la biomasse et de l’activité des microorganismes
dans les systèmes aquatiques. Mar. Life 2: 1–29.
Briggs BJF, Goring DG, Nikora VI (1998) Subsidy and stress responses of stream periphyton to gradients in water velocity as a
function of community growth form. J. Phycol. 34: 598–607.
Cattaneo A, Amireault MC (1992) How artificial are artificial substrata for periphyton ? J. North Am. benthol. Soc. 11: 244–256.
Cattaneo A, Kerimian T, Roberge M, Marty J (1997) Periphyton
distribution and abundance on substrata of different size along a
gradient of stream trophy. Hydrobiologia 354: 101–110.
Conrad R, Büchel C, Wilhelm C, Arsalane W, Berkaloff C, Duval
JC (1993) Changes in yield of in vivo fluorescence as a tool for
selective herbicide monitoring. J. appl. Phycol. 5: 505–516.
Goldsborough LG, Robinson GGC (1986) Changes in periphytic
algal community structure as a consequence of short herbicide
exposures. Hydrobiologia 139: 177–192.
Guasch H, Ivorra N, Lehmann V, Paulsson M, Real M, Sabater S
(1998) Community composition and sensitivity of periphyton to
atrazine in flowing waters: the role of environmental factors. J.
appl. Phycol. 10: 203–213.
Guasch H, Muñoz I, Rosés N, Sabater S (1997) Changes in atrazine
toxicity throughout succession of stream periphyton communities. J. appl. Phycol. 9: 137–146.
Guasch H, Sabater S (1998) Light history influences the sensitivity
to atrazine in periphytic algae. J. Phycol. 34: 233–241.
Herman D, Kaushik NK, Solomon KR (1986) Impact of atrazine
on periphyton in freshwater enclosures and some ecological
consequences. Can. J. Fish aquat. Sci. 43: 1917–1925.
Juneau P, Popovic R (1999) Evidence for the rapid phytotoxicity
and environmental stress evaluation using the PAM fluorometric
method: importance in future application. Ecotoxicology 8: 449–
455.
Kirby MF, Sheanhan DA (1994) Effects of atrazine, isoproturon,
and mecoprop on the macrophyte Lemna minor and the alga
Scenedesmus subspicatus. Bull. environ. Contam. Toxicol. 53:
120–126.
Kromkamp J, Barranguet C, Peene J (1998) Determination of microphytobenthos PSII quantum efficiency and photosynthetic
activity by means of variable chlorophyll fluorescence. Mar.
Ecol. Progr. Ser. 162: 45–55.
Maxwell K, Johnson GN (2000) Chlorophyll fluorescence – a
practical guide. J. exp. Bot. 51: 659–668.
McCormick PV, Cairns J, Jr (1994) Algae as indicators of environmental changes. J. appl. Phycol. 6: 509–526.
Nyholm N (1990) Expression of results from growth inhibition
toxicity tests with algae. Arch. environ. Contam. Toxicol. 19:
518–522.
Nyström B, Paulsson M, Almgren K, Blanck H (2000) Evaluation of
the capacity for development of atrazine tolerance in periphyton
from a Swedish freshwater site as determined by inhibition of
photosynthesis and sulfolipid synthesis. Environ. Toxicol. Chem.
19: 1324–1331.
Pereira WE, Rostad CE (1990) Occurrence, distribution and transport of herbicides and their degradation products in the lower
Mississippi River and its tributaries. Environ. Sci. Technol. 24:
1400–1406.
Petersen S, Kusk KO (2000) Photosynthesis as an alternative to
growth tests for hazard assessment of toxicant. Arch. environ.
Contam. Toxicol. 38: 152–157.
Samson G, Popovic R (1988) Use of algal fluorescence for determination of phytotoxicity of heavy metals and pesticides as
environmental pollutants. Ecotoxicol. environ. Safety 16: 272–
278.
Schreiber U, Schliwa U, Bilger W (1986) Continuous recording of
photochemical and non-photochemical fluorescence quenching
with a new type of modulation fluorometer. Photosynth. Res. 10:
51–62.
Seguin F, Leboulanger C, Rimet F, Druart JC, Bérard A (2001) Effects of atrazine and nicosulfuron on phytoplankton in systems
of increasing complexity. Arch. environ. Contam. Toxicol. 40:
198–208.
Solomon KR, Baker DB, Richards P, Dixon KR, Klaine SJ, LaPoint
TW, Kendall RJ, Weisskopf CP, Giddings JM, Giesy JP, Hall LW,
Jr, Williams WM (1996) Ecological risk assessment of atrazine
in North American surface waters. Environ. toxicol. Chem. 15:
31–76.
Wilhelm C, Conrad R, Meitzler L, Mühlenweg A (1996) Combination of solid phase extraction and a microalgal test system
based on pulse-amplitude modulated fluorescence to detect photosystem II herbicides up to 0.05 µeq l−1 . J. appl. Phycol. 8:
171–173.
Wilhelm C, Rudolph I, Renner W (1991) A quantitative method
based on HPLC-aided pigment analysis to monitor structure and
dynamics of the phytoplankton assemblage – A study from Lake
Meerfelder Mar (Eifel, Germany). Arch. Hydrobiol. 123: 21–35.
Wright SW, Jeffrey SW (1997) High-resolution HPLC system for
chlorophylls and carotenoids of marine phytoplankton. In Jeffrey
SW, Mantoura RFC, Wright SW (eds), Phytoplankton Pigments
in Oceanography, UNESCO-SCOR, Paris, pp. 327–341.