DOI: 10.2478/s11535-007-0014-1Research article

CEJB 2(2) 2007 169–181

Delayed fluorescence in algal growth inhibition tests

Maja Berden-Zrimec1∗, Luka Drinovec1, Alexis Zrimec1, Tatjana Tisler2

1 Institute of Physical Biology,SI – 1290 Grosuplje, Slovenia

2 National Institute of Chemistry,SI – 1000 Ljubljana, Slovenia

Received 30 January 2007; accepted 8 March 2007

Abstract: A series of 72-hour growth inhibition tests with green alga Desmodesmus (Scenedesmus)subspicatus (ISO 8692) has been performed to test the delayed fluorescence (DF) parameters as possibleendpoint measurements. Sensitivity to five toxicants with direct and indirect effects on photosynthesiswas tested, and the median effective concentration (EC50) values derived from the cell concentration,absorbance and DF were compared. The sensitivity of DF intensity (DFI) was comparable with thetwo endpoints suggested in ISO 8692 for all five toxicants: potassium dichromate, 3,5-dichlorophenol(3,5-DCP), DCMU, copper and cadmium. In the case of potassium dichromate and copper, DFI wasmore sensitive than the other endpoints in this study. The analysis of DF relaxation kinetics showeda specific response to the herbicide DCMU. Additionally, a 24-hour test was performed with the sametoxicant concentrations (except copper), where DFI was measured 30 minutes, one hour and 24 hoursafter the exposure. The influence of toxicants on DFI after a 24-hour exposure was comparable with theeffects after 72 hours. Only 3,5-DCP influenced DFI after 30 minutes. DF proved to be a simple, reliableand rapid measurement to assess toxicity in algal tests, which can to some extent differentiate amongvarious toxicants.c© Versita Warsaw and Springer-Verlag Berlin Heidelberg. All rights reserved.

Keywords: Delayed fluorescence intensity, relaxation kinetics, Desmodesmus (Scenedesmus) subspicatus,algal toxicity testing, short-term test

1 Introduction

Algae are the dominant primary producer in most aquatic habitats and have an impor-

tant role in food chains, indirectly affecting also humans. They are very sensitive to a

variety of potential toxicants [1, 2], thus being very applicable in to toxicity testing. The

∗ E-mail: maja@ifb.si

170 M. Berden-Zrimec et al. / Central European Journal of Biology 2(2) 2007 169–181

main holdback for an extensive use of algal growth inhibition tests are probably relatively

demanding protocols and measurements. At least two estimates of the biomass and/or

physiological response are recommended when monitoring algal cultures in toxicity tests

[3, 4]. Cell counting under a microscope is relatively simple, but can be less precise

than some other methods, although visual inspection provides important information on

the morphology of the cells [4]. On the other hand, electronic particle counting can be

time-consuming in many cases and the equipment costs are high [1]. Photometric deter-

mination of cell density is sensitive to interferences from suspended material and coloured

substances at low cell densities, which limits their use with environmental samples. Mea-

surements of extracted chlorophyll a are mostly preceded by a sample manipulation,

which represents an important source of errors [1]. These problems can be avoided by

simpler in vivo measurements, which do not demand an extensive handling of algae.

There are only a few means by which the abundance and physiological responses of

microalgae may be measured without destructive sampling [4]. These methods usually

require very sensitive measuring devices, but the procedures are simple with minimal

disturbance to the cells. In vivo chlorophyll fluorescence, which is a measure of prompt

fluorescence from the organisms, is already widely used in phyto-physiology studies [5, 6].

Flow cytometry is also a perspective method, offering many possibilities in toxicity testing

[7–9].

Delayed fluorescence (DF) is another in vivo measurement of the biomass and physi-

ological state of photosynthetic organisms, and can be observed as a long-lived low-level

photon emission after a short illumination pulse [10]. The emission spectrum resem-

bles the fluorescence emission spectrum of chlorophyll a [11–13]. The difference between

prompt fluorescence and DF is in the origin of the excited singlet state of the emitting

pigment molecule [13]. DF originates from the repopulation of excited states of chloro-

phyll from the stored energy after charge separation, whereas fluorescence reflects the

radiative de-excitation of excited chlorophyll molecules before the charge separation [13].

The fluorescence emission ceases within several nanoseconds after the illumination, which

populates the excited singlet state, is switched off [13]. DF lifetimes are much longer and

the measurement usually lasts from milliseconds to seconds after the illumination [13].

The main source of DF are back reactions in the photosystem II (PSII) [14], whereas the

photosystem I contributes much less to the DF emission [13]. In PSII, charge pairs are

generated during the illumination with positive charges located on the oxygen evolving

complex (OEC) and negative charges on quinone acceptors (QA and QB). The slow com-

ponents of DF originate in back reactions between the S2 and S3 states of the OEC and

quinones QA and QB [15]. The half-times of these reactions in isolated chloroplasts are

1.5 s for QA+S2/3 and 25 s for QB+S2/3 [14].

DF has a monotonic decay kinetics in the first seconds, which is sometimes followed

by a transient peak [16–18]. DF is affected by chemical and physical parameters, such

as different chemicals [19–21] or temperature [22, 23]. This variety of influences has in

common that they affect the reduction state of the plastoquinone pool or its coupling

with PSII by inhibiting the reversed electron flow [24]. Most important, DF is a feature

M. Berden-Zrimec et al. / Central European Journal of Biology 2(2) 2007 169–181 171

of living cells with active photosynthesis and DF intensity (DFI) can be used as a measure

of the living algal biomass [25].

DF has been used in research of metals’ effects on photosynthesis of algae and higher

plants [19, 25, 26]. Simple, reliable and rapid measurements as well as minimal sample

manipulation, e.g. incubating tested organisms in measuring cuvettes, lead to the stud-

ies of possible DF use in ecotoxicological tests. Drinovec and co-workers [20] tested DF

applicability as an endpoint of a 24-hour test with duckweed Lemna minor and showed

comparable sensitivity and reproducibility for testing metals to the standard 7-day test

and prompt fluorescence measurements. Sensitivity of DF of green alga Pseudokirchner-

iella subcapitata to Simazine (CAT) and 3,5-DCP has recently been studied by Katsumata

and co-workers [21] and DF proposed as a rapid ecotoxicological bioassay.

Many research groups have suggested that low algal densities and short duration of

the test improve the environmental relevance and reproducibility of laboratory-derived

toxicity data [1, 7, 8, 27]. High algal densities change the physico-chemical properties

of the medium and can also directly affect the bioavailability and toxicity of the tested

substances [1, 8]. On the other hand, low algal densities require highly sensitive methods

for the measurement such as the flow cytometry [7]. Photosynthesis parameters have

been proposed as a good endpoint of short tests with low algal densities [28]. Because

DF enables detection of changes at low algal densities, we tested a 24-hour procedure as

a possible short and simple method for algal toxicity testing.

The aim of our research was to evaluate DF as a possible endpoint of algal growth

inhibition test in a standard 72-hour test and 24-hour exposure. DF intensity and relax-

ation kinetics were analysed. We compared the proposed endpoint to the two methods

recommended by the ISO standard [3]. Two standard reference toxicants, potassium

dichromate and 3,5-dichlorophenol (3,5-DCP), as well as chemicals specifically influenc-

ing photosynthesis were used. Additionally, DF was measured after 30 minutes and 1 hour

of exposure to the toxicants.

2 Experimental procedures

2.1 Culturing of Desmodesmus (Scenedesmus) subspicatus

Green unicellular alga Desmodesmus (Scenedesmus) subspicatus Chodat 1926 (CCAP

276/20) was cultured in a nutrient solution prepared according to Jaworski [29] on an

orbital shaker (Tehtnica Zelezniki, EV 403), alternating 15 min agitation at 150 rpm

and 15 min resting, at the constant room temperature of 23±1◦C and under continuous

illumination (60±5 µmol m−2 s−1 PAR) with cool-white fluorescent lights.

2.2 72-hour growth inhibition test (ISO 8692)

The growth inhibition tests were performed following the protocol suggested in interna-

tional standard ISO 8692 [3]. A pre-culture of algae was started three days before the

172 M. Berden-Zrimec et al. / Central European Journal of Biology 2(2) 2007 169–181

beginning of the test with the growth medium recommended in the ISO 8692. For the

main experiment, algae were harvested from the batch culture during their exponential

growth phase. 40 ml cultures were prepared in 100 ml Erlenmeyer flasks, with the initial

algal concentration of 104 cells ml−1. The flasks were continuously shaken at 150 rpm

under a permanent cool-white fluorescent light (80±8 µmol m−2 s−1 Photosynthetically

Active Radiation (PAR)) and temperature 23±1◦C. At least two preliminary experiments

were conducted for each tested toxicant to select the appropriate concentration range for

toxicity tests [3] (ISO 8692). For each toxicant, two or more definitive tests with three

replicates were performed.

Cells for the measurements were harvested at the beginning of the test and subse-

quently every 24 hours. The cell concentration was determined in Burker counting cell

(Brand). Delayed fluorescence was measured in 1 ml samples in the luminometer [20].

Optical density of the same samples was measured in a spectrophotometer (Ocean Optics

USB2000-VIS-NIR). Absorbance in 400-700 nm range was measured in standard cuvettes

with 10 mm optical path (Sarstedt 67.740).

2.3 Tested toxicants

Algae were exposed to concentration series of two toxicants, which are used as a refer-

ence toxicants in many standardized toxicity tests: 0.18, 0.32, 0.58, 1.05 and 1.89 mg l−1

(0.61–6.42 µmol l−1) of potassium dichromate (K2Cr2O7) and 1, 2, 3, 4 and 5 mg l−1

(6.14–30.68 µmol l−1) of 3,5-DCP (3,5-dichlorophenol, Cl2C6H3OH). Cr(VI) has many

toxic effects on growth and physiological processes in photosynthetic organisms [30].

3,5-DCP is an inhibitor of respiration [21], of electron transport in thylakoids and of

photophosphorylation [31].

Copper and cadmium can influence photosynthesis by inhibiting processes in pho-

tosynthetic electron chain [32, 33]. The tested concentrations were 0.03, 0.045, 0.0675,

0.101, 0.152 mg l−1 (0.27–1.35 µmol l−1) of Cd2+ (as CdCl2), and 0.02, 0.06, 0.18, 0.54,

1.62 mg l−1 (0.32 – 25.49 µmol l−1) of Cu2+ (as CuCl2).

The herbicide DCMU (diuron, 3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a PSII-

specific inhibitor, blocking electron transport and is routinely used in photosynthesis

research [31, 34]. It was prepared in acetone, which was non-toxic to D. subspicatus in

the final tested concentration (0.0027 v/v%). Toxicity of different concentrations of ace-

tone to D. subspicatus was preliminary tested to select appropriate solvent for DCMU.

The algae were exposed to 0.0033, 0.01, 0.03, 0.09, 0.27 mg l−1 (0.014–1.158 µmol l−1)

DCMU with the same concentration of acetone, which was also used in controls for DCMU

testing.

2.4 24-hour delayed fluorescence test

The cultures for the tests were prepared following the same protocol as in the growth

inhibition tests with the initial algal concentration of 104 cells ml−1 [3]. The toxicants used

M. Berden-Zrimec et al. / Central European Journal of Biology 2(2) 2007 169–181 173

were potassium dichromate, 3,5-DCP, DCMU and Cd2+. After the toxicants addition

in the same final concentrations as for the growth inhibition tests, 1 ml samples were

transferred to 6 ml round cuvettes (Sarstedt 58.485), which were used for measurements

in the luminometer [20]. The cuvettes were closed with a transparent plastic foil and

incubated under continuous cool white fluorescent light 100±10 µmol m−2 s−1 PAR at

23±0.5◦C. Higher light intensity than in the 72-hour test was used to promote culture

growth. DFI of the samples was measured immediately after toxicant addition and after

24 hours. In one series of experiments the influence of exposure to toxicants on DFI was

measured also after 30 minutes and after one hour.

2.5 Delayed fluorescence measurements

DF was measured in a custom-made photon-counting luminometer [20]. The samples

were illuminated by a 20 W halogen lamp equipped with a heat-absorbing filter, which

provided 400-800 nm light with a maximum at 650 nm. The illumination intensity at

sample position was 60 µmol m−2 s−1 PAR. The sample was illuminated from above for

3 s in a temperature-controlled holder adjusted to 23±0.2◦C. DF was measured in the

interval 1-60 s after the illumination pulse. Before each DF measurement, the background

DF signal and dark count from the photomultiplier tube were measured and subtracted

from the DF values in the analysis. The detector was a red-light-sensitive photomultiplier

tube (Hamamatsu R1104) with a Hamamatsu C3866 Photon Counting Unit for signal

conditioning and amplification. The integration time was 100 ms.

2.6 Data analyses

The median effective concentration (EC50) was defined as a toxicant concentration that

resulted in the 50% reduction of growth rate compared to the control (cultures without

added toxicants). Values were obtained by the linear regression analysis on the growth

rates in different toxicant concentrations. Data were statistically analysed by means of the

Student’s t-test using a significance level of 0.05 [35]. DF intensity (DFI) was calculated

from the relaxation curve as an average DF in the time interval from 1 to 60 s after the

illumination.

3 Results

3.1 72-hour growth inhibition test - comparison of EC50 values of different

endpoints derived from growth rates

EC50 values for the measured endpoints were calculated from the growth rates and are

presented in the Table 1. In general, DFI sensitivity to the tested toxicants was compara-

ble to the sensitivity of the cell concentration and absorbance. DFI was significantly more

sensitive to potassium dichromate, 3,5-DCP and copper than the cell concentration. DFI

174 M. Berden-Zrimec et al. / Central European Journal of Biology 2(2) 2007 169–181

was significantly more sensitive than absorbance only to potassium dichromate, mainly

due to high variation of the absorbance results.

Endpoint Potassium 3,5-DCP DCMU Cd2+ Cu2+

dichromate

Cell concentration 0.71±0.19 3.39±0.52 0.03±0.07 0.08±0.01 0.97±0.06Absorbance 1.10±0.50 3.00±1.30 0.03±0.11 0.10±0.02 1.07±0.36DFI 0.44±0.18 2.51±0.22 0.02±0.02 0.08±0.01 0.24±0.32DFI (24-hour) 0.35±0.06 1.38±0.57 0.01±0.002 0.07±0.03 –

Table 1 EC50 values (mg l−1) of 72-hour and 24-hour tests calculated from growth rates

of tested endpoints with 95 % confidence intervals.

3.2 72-hour growth inhibition test - comparison of dose-responses of

different endpoints calculated from growth inhibition

For a further comparison of DFI results with the cell concentration and absorbance, dose-

responses are presented in Figures 1a-e. The inhibition of growth due to the exposure

to toxicants was used in the dose-response Figures. It should be noted that EC50 values

presented in the Table 1, which were calculated from the growth rates, can’t be obtained

from these Figures. The results of the growth inhibition tests from all three endpoints

were normalized for comparison.

Figures 1a-e show that DFI had a similar dose-response as other measured endpoint

parameters in the growth inhibition test. The stimulating action of 0.02 mg Cu l−1

detected by the cell concentration and absorbance measurements was also observed with

DFI.

3.3 72-hour growth inhibition test - changes of DF relaxation kinetics

DF relaxation kinetics is a 60 s measurement of DF after the 3 s illumination pulse that

reflects the physiological state of the whole photosynthetic electron chain, including the

PQ pool, PS I and dark reactions. Effects on DF relaxation kinetics of D. subspicatus

were observed only during the exposure to the herbicide DCMU.

DF kinetics were markedly changed with different concentrations of DCMU (Figure 2).

However, other tested toxicants did not influence DF relaxation kinetics outside their

natural variation (data not shown).

M. Berden-Zrimec et al. / Central European Journal of Biology 2(2) 2007 169–181 175

a) b)

c) d)

e)

Fig. 1 Normalized dose-response of different endpoint measurements, presented as

inhibition of growth due to exposure to toxicants: (a) Potassium dichromate;

(b) 3,5-dichlorophenol; (c) DCMU; (d) Cadmium; (e) Copper. Error bars represent stan-

dard deviation. The concentrations indicated as “0” represent non-exposed algae and are

all normalized to value “1” for easier comparison.

176 M. Berden-Zrimec et al. / Central European Journal of Biology 2(2) 2007 169–181

Fig. 2 DF relaxation kinetics of D. subspicatus after 72-hour exposure to five DCMU

concentrations, log-log scale.

3.4 24-hour test - effects of toxicants on DFI

The 24-hour tests were conducted simultaneously with 72-hour growth inhibition tests.

Only DFI was measured in these tests and EC50 values were calculated from the growth

rates. DFI values of the 24-hour test showed significantly higher sensitivity to 3,5-DCP

(EC50 of 1.38±0.57 mg l−1) and DCMU (EC50 of 0.01±0.002 mg l−1) compared to the

results after the 72-hour exposure (Table 1). The EC50 values of algae exposed to potas-

sium dichromate (0.35±0.06 mg l−1) and Cd2+(0.07±0.03 mg l−1) were similar after 24

and after 72 hours.

To compare the influence of different exposure conditions of the growth inhibition

test and the 24-hour test, we calculated EC50 values also from DFI of the samples, taken

from the growth inhibition tests after 24 hours. The EC50 value of potassium dichromate

was approximately the same as with the 24-hour test (0.35±0.03 mg l−1), whereas the

exposure to 3,5-DCP resulted in a slightly higher EC50 (2.02±0.20 mg l−1).

Exposure to potassium dichromate and DCMU for 30 minutes or one hour did not

influence the DFI. In contrast, the exposure to 3,5-DCP for 30 minutes caused 18.6±6%

inhibition of DFI already at the concentration of 3 mg l−1. After one hour, 3,5-DCP

effects were even more pronounced causing a 50% inhibition of DFI at 3.86 ± 0.42 mg

3,5-DCP l−1. Cadmium caused a 16.5±2% inhibition after one hour at the highest con-

centration (0.152 mg l−1).

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4 Discussion

In general, DFI was equally or more sensitive to the tested toxicants compared with the

cell concentration and absorbance, which are standard parameters in the algal growth

inhibition tests. Although all the three tested endpoints are a measure of the biomass,

DFI detects only living cells with active photosynthesis, whereas distinguishing live and

dead cells can be problematic under a microscope or with absorbance measurements.

Regarding this, the differences in sensitivity were not surprising.

The 24-hour test was even more sensitive, especially to DCMU, and to 3,5-DCP, which

influenced DFI within 30 minutes of the exposure. However, other tested toxicants did not

have any effects on DFI after 30 minutes. 3,5-DCP influenced the DFI of several other

unicellular algae almost instantly after having been added, for example in the marine

alga Dunaliella tertiolecta [23] and the freshwater alga Pseudokirchneriella subcapitata

(previously Selenastrum capricornutum) [21]. After one hour, cadmium also inhibited

the DFI of D. subspicatus.

A strong inhibition of DFI by potassium dichromate was expected due to the known

deleterious effects of Cr(VI) on photosynthesis [30]. DFI sensitivity to copper could be

the consequence of influence of Cu2+ ions on PSII system [33], although it is possible that

effects on dark reactions and plastoquinone pool prevailed [20]. The stimulating action

of 0.02 mg Cu l−1 is in agreement with other studies [7, 33, 36]. Cadmium concentrations

of more than 0.3 mg l−1clearly perturbed PSII function in the Barraza and Carballeira

study [37]. In our case, much lower concentrations resulted in the 50% DFI inhibition

within 24 hours.

The suitability of photosynthesis as an endpoint of algal toxicity tests has already been

shown on numerous occasions. The response of P. subcapitata to potassium dichromate

calculated from the fluorescence measurements was comparable to the response evaluated

from the measurements of the cell concentration, or even more sensitive for concentrations

higher than 1 mg l−1 [1]. The relative intensity of P. subcapitata DF after the 15-minute

exposure to Simazine (CAT) and 3,5-DCP was comparable to the results of a standard

algal inhibition test [21].

From the DF measurements, various parameters can be derived, such as DFI, DF

kinetics and DF excitation spectra. Their various responses can enable selectivity for

certain groups of toxicants that influence different sites in the photosynthetic electron

chain. In our study, herbicide DCMU, which directly blocks the electron transport in PSII

[31, 34], changed the DF relaxation kinetics in a dose dependent manner. Conversely,

Cr(VI) and copper did not influence DF relaxation kinetics of D. subspicatus, but inhibited

DFI more than the cell concentration and absorbance.

Our results support the findings of many authors who have already shown that short-

term algal toxicity tests are more sensitive to toxicants than standard growth inhibition

tests [2, 21, 27]. In comparison to many measurements of the photosynthesis response in

algal assays, DF measurements are much simpler and quicker, minimizing the handling of

samples and the associated errors. The additional advantage of the presented 24-hour test

178 M. Berden-Zrimec et al. / Central European Journal of Biology 2(2) 2007 169–181

is the use of small volumes of samples, incubated in a measuring cuvette, which enables

utilisation of numerous parallels under a more homogenous illumination compared to tests

using Erlenmayer flasks [6]. The illumination in algal toxicity tests is of major importance,

because uncontrollable variations in the light regime are probably the largest source of

experimental error [38]. Similar volumes have already been used in a miniscale algal

toxicity test suggested for standardization [39].

An important advantage of many short-term tests is also the utilization of low cell

concentrations. In the growth inhibition test ISO 8692, final cell concentrations after 72

hours are in the range of 106 cells ml−1. High algal densities are problematic, because

they change the physico-chemical properties of the medium and can directly affect the

bioavailability and toxicity of the tested substances [1]. DFI allows a precise determina-

tion of the living biomass for cell densities 103 cells ml−1 or less, but with the equipment

used in this study [20] concentrations of 104 cells ml−1 were necessary to follow also DF

relaxation kinetics. With further optimisation of the measuring device [20] for toxicity

testing, DF relaxation kinetics could be measured at lower cell concentrations.

Apart from the high cost equipment usually required for high sensitivity, short-term

tests are mostly more cost-effective as standard tests, which makes them very applicable

for extensive toxicity studies, rapid toxicity screening of large polluted areas or areas

considered for ecological agriculture, where the environment has to be non-toxic. Nu-

merous sampling sites are extremely important in assessing the extent of pollution in the

environment. Assessing toxicity of complex matrices with DF measurements excludes

problems with the fluorescent background [28], which can be problematic for example in

waste water effluents.

5 Conclusions

DF measurements are rapid, sensitive and well reproducible, which makes them a good

choice for toxicity tests.

Various endpoints can be derived from DF measurements, potentially enabling specific

detection of certain groups of toxicants.

DF measurements showed comparable or more sensitive results than the measured

standard growth inhibition tests’ endpoints for the tested toxicants.

Minimal sample handling is required for DF measurements, which reduces the sample

manipulation connected errors.

DF is sensitive enough to enable short-term miniscale tests with relatively low cell

concentrations and thus higher relevance of toxicity tests.

DF measurements in complex matrices exclude problems with fluorescent background.

Acknowledgment

This work was financed by the Slovenian Research Agency (grants L4-6222, V4-0106 and

P1-0237). We are grateful to Emil Meden and Tanja Urh for their assistance with the

measurements and Lidija Berden for revising the language.

M. Berden-Zrimec et al. / Central European Journal of Biology 2(2) 2007 169–181 179

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