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A simple in vitro fluorescence method for biomass measurements in algal growth inhibition tests
Pergamon Pii: S0043-1354(97)00084-5
War. Res. Vol. 31, No. 10, pp. 2525-2531, 1997 © 1997ElsevierScienceLtd. All rights reserved Printed in Great Britain 0043-1354/97$17.00 + 0.00
A SIMPLE IN VITRO FLUORESCENCE METHOD FOR BIOMASS MEASUREMENTS IN ALGAL GROWTH INHIBITION TESTS P H I L I P P M A Y E W * , RUSSELL C U H E L 2 and NIELS N Y H O L M @t ~Technical University of Denmark, Institute for Environmental Science and Technology, 2800 Lyngby, Denmark and 2Center for Great Lakes Studies, University of Wisconsin Milwaukee, Milwaukee, WI 53204, U.S.A. (Received September 1996; accepted in revised form March 1997)
Abstract--The estimation of biomass concentrations in algal growth inhibition tests from measurements of pigment fluorescence in extracts of 20% sample (final v/v) prepared by direct addition to dimethylsulfoxide/acetone solvent offers several advantages compared to currently used direct or indirect methods. The extraction stops the electron transfer and other processes which interact with chlorophyll fluorescence when measured in vivo. As a result the response is stabilized and the sensitivity improved. The injection method is very fast, has a high potential for automation, allows storage of samples and is suitable for small sample volumes (e.g. 0.2 ml). The typical initial cell density in standard toxicity tests of 104 cells ml-L of Selenastrum capricornutum was measured precisely with a standard fluorimeter set-up, and 103 cells ml -~ of S. capr&ornutum was measured reliably with a sensitive fluorimeter. At low levels of toxicity by the model test compound potassium dichromate, the proposed fluorescence method resulted in very similar inhibition figures as obtained with electronic particle counting. At high levels of toxicity, on the other hand, biomass determinations from pigment fluorescence readings were markedly affected by toxicant-induced changes of the algal physiology. The low effect part of a dose response curve is normally that one of major interest, and biomass estimation errors associated with fluorescence measurements on extracts are thus considered acceptable in most situations. When the entire dataset was applied for endpoint estimation by the Weibull model, EC-I estimates were markedly affected by the curve fitting to dala in the high inhibition range, while EC-10 and EC-20 were less and EC-50 almost unaffected. The method is expected to be less suitable for toxicity testing of herbicides specifically inhibiting photosynthesis. © 1997 Elsevier Science Ltd Key words---chlorophyll measurement method
extraction, in vitro fluorescence, algal toxicity testing, Weibull model,
INTRODUCTION The regulation of environmental pollutants is still based primarily on toxicity to aquatic animals, despite the importance of phytoplankton as the dominant primary producer in most aquatic habitats, and despite the tact that aquatic plants including algae are more sensitive than animals to a variety of potential toxicants (Lewis, 1993; Peterson and Nyholm, 1993). The use of algal toxicity tests is increasing, and today algal toxicity tests are frequently required by authorities for notifications of chemicals and are also increasingly being used to manage chemical discharges. The resistance for an extended use of algal toxicity tests might be partly of a practical nature, and the proposed method
*Author to whom all correspondence should be addressed at Environmental Chemistry Group, Research Institute of Toxicology, Utrecht University, P.O. Box 80058, 3508 TB Utrecht, The Netherlands [Fax: +31-30-2532837, E-maih p.mayer@ ritox.dgk.ruu.nl].
presented in this paper is believed to ease the performance of such tests. Additionally, the suggested method might permit toxicity testing at very low algal densities, particularly when applied in combination with a reduced test duration. Final biomass densities in standard growth inhibition tests with algae exceed those found in most natural waters and high algal densities give rise to physico-chemical medium changes during the test, which may affect the environmental relevance and reproducibility of laboratory-derived toxicity data. High algal densities can also directly affect the bio-availability and toxicity of a test substance. Therefore, it is generally desirable to use low algal densities in toxicity tests. (Petersen and Nyhoim, 1993; Nyholm and Petersen, 1997). Measurements of low algal densities, however, require highly sensitive methods, and if the biomass level of the current standard tests was to be reduced, many laboratories would have to adopt new techniques. For many years, electronic particle counting, preferably including determination of cell volume, has been the method of reference. The
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technique is precise and sensitive but also time-consuming, and the equipment costs are high. Further, the technique is difficult to automate and sample volumes of several milliliters are required for precise measurements at low algal densities. Optical densities by spectrophotometry or turbidity are inexpensive and frequently used indirect methods, but both methods offer a lower precision and sensitivity than that of electronic particle counting. Optical density is considered to have only borderline sensitivity and precision at the biomass levels of standard tests. Measurement of chlorophyll fluorescence, in vivo or in vitro, is a hitherto less often used but sensitive alternative. Eloranta (1978) reported a high correlation between the primary pigment chlorophyll a and cell counts as growth indicator in algal toxicity tests of different test substances with Ankistrodesmus falcatus. However, a more widely use of chlorophyll a fluorescence measurements in algal growth inhibition tests requires practical methods, which combine sensitivity, precision and reproducibility. In vivo fluorescence is re-radiation of light absorbed by algal pigments in intact cells. In living cells, active in photosynthesis only a very small portion (about 1%) of the absorbed light is re-emitted as fluorescence (Kirk, 1994), which limits the sensitivity of in vivo fluorescence measurements. Recent technical advances in fluorescence spectroscopy allow measurements on dilute samples. A drawback of measuring in vivo fluorescence, however, is that the signal varies with the state of the photosynthetical reaction center (PS 2), which among others depends on the previous light regime and also is influenced by many herbicides (Putt et al., 1987; B6ger and Sandemann, 1989). This makes appropriate and reproducible handling of samples for in vivo fluorescence measurements crucial. The addition of the herbicide DCMU can be used to standardize the redox state of PS 2 and can thereby stabilize and maximize the fluorescence signal. Another way to reduce the problems associated with in vivo fluorescence is the extraction of pigments with organic solvents, because chlorophyll a and other pigments in solvent extracts are uncoupled from the biochemical processes influencing the fluorescence signal of in vivo measurements. A major argument against the use of extracted fluorescence measurements in algal toxicity tests has been a purely practical one--measurements are traditionally preceded by a filtration or centrifugation step, which is both time-consuming and constitutes a source of errors. The whole water extract fluorescence method presented here is performed without any filtration or centrifugation step, and the technique is cost-effective, sensitive, fast and well suited for automation. The method is based on the comprehensive description of chlorophyll a extraction with acetone and DMSO by Shoaf and Lium (1976) with modifications by us. A comparable method for field
studies of phytoplankton was proposed by Phinney and Yentsch (1985) and by Greenberg and Watras (1989). Most published extracted fluorescence methods aim at quantifying chlorophyll a in field samples. Chlorophyll a concentrations are typically measured as the difference in fluorescence before and after acidification, to separate phaeophytin a and chlorophyll a, and to correct for background fluorescence (Holm Hansen et al., 1965). Extraction procedures are usually optimized to ensure high extraction efficiencies (near unity) for most algal species, as the total chlorophyll a concentration is the parameter of interest. The objective of a whole water extraction method for fluorescence measurements in algal growth inhibition tests is different. The detection limit and in particular the precision at low algal densities is here of great importance, because it determines the possible range of algal densities in the test. To obtain a high sensitivity, the fluorescence of the extract is maximized; and to obtain a high precision and reproducibility, consistency in the extraction efficiency is more important than a high absolute extraction efficiency. Acidification of extracts to distinguish chlorophyll a from other pigments is not necessary in toxicity tests, where the goal is to measure algal growth and not specifically to measure chlorophyll a. The aim of this paper is to present a simple in vitro fluorescence method for biomass measurements in algal growth inhibition tests. Documentation is provided with regard to choice of extractive solvent, sample/solvent ratio, extraction time, stability of extracts, precision and sensitivity. Finally, the potential of the method and the test results obtained are discussed, and a comparison of results obtained with the fluorescence method and with electronic particle counting is provided for a toxicity test with the model test compound potassium dichromate.
METHODS
Culturing
Cultures of the unicellular green alga Selenastrum capricornutum Printz were grown in ISO 8692 standard algal medium under continuously illumination of white fluorescence light at 110 + 4/Jmol PAR photons m--' s-~ (or/~E m-2s -' in the photosynthetically active range) at a temperature of 22 + 1°C, resulting in specific growth rates of about 1.9 day-t. The cultures were cultivated in 20 ml Scintillation vials with 4-5 ml liquid volume and shaken at 300 rpm. Direct injection method
Culture samples of 0.14).4 ml were extracted by direct injection into different organic solvents in a test-tube immediately followed by mixing. A 20% sample (final volume %) was extracted in a 1: 1 mixture of DMSO and acetone for at least 20 rain unless otherwise noted, and extracts were stored in the dark at room temperature. Fluorescence was measured at room temperature in
Algal fluorescence measurement arbitrary units on a Hitachi fluorescence spectrophotometer model F-2000 with an excitation wavelength of 430 nm and a measured emission wavelength of 671 + 10 nm. The signal changed less than 0.5% per °C. Reported fluorescence figures were corrected for background fluorescence measured on solvents mixed with algal medium. Background fluorescence of differnt commercially available DMSO and acetone products were tested. HPLC certified DMSO and acetone yielded lower or similar background fluorescence than more expensive fluorescence grade solvents. In the standtard set-up, measurements were made using a carousel with eight standard test-tubes with an outer diameter of 10 ram. With a high sensitivity set-up available from the manufacturer both excitation light and emitted light were amplified by reflectors, and fluorescence was measured in a 12 mm rectangular quartz cuvette. Reference algal population densities were measured in triplicates as total bio-volume (pm3/ml) and numbers (algal cells per milliter) by use of a~a electronic particle counter (Coulter Multisizer). Algae were counted in the size range 2.125-9 pm with an aperture of 70 pm, a counting volume of 0.5 ml and lstotone R as dilution medium. Monitoring of balanced growth Twelve replicate cultures with no test substance added were inoculated at 103 cells ml-' and in vitro fluorescence measured daily usingl the standard fluorimeter set-up. Toxicity test and data treatment A 72-h algal growth inhibition test was performed according to ISO 8692 (ISO, 1989) using potassium dichromate as reference toxicant. This test was fully randomized and the toxicant was dosed in a geometric dilution series of 16 concentrations in triplicates (data for one concentration were lost). During the test period, in vitro fluorescence of the control cultures were measured daily to verify exponential growth. After 72 h, in vitro fluorescence, cell number and total cell volume of all samples were measured. The specil~c growth rate (~) was defined as the rate constant in the following equation:
-~Nt=/J.N~N(t) = N0.e"' where N is the algal biomass measured as algal volume, algal numbers or in vitro fluorescence. The toxic response was expressed as growth inhibition (I) calculated as the relative reduction in growth rate caused by the test substance: I =
l - - ~inhibited ]./control
Concentration-respense curves were described by the Weibull equation (Christensen and Nyholm, 1984) which was fitted to data using weighted non-linear regression applying a computer program developed by Andersen (1994). The program uses the "Downhill Simplex Method" by Nelder and Mead (1965) to give a weighted least-squares fit to replicate means. Weighting factors are inversely proportional to the empirical variance as estimated from replicates. Confidence intervals around EC values are calculated by inverse estimation after a Taylor expansion.
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c
J l,
DMSO/Acetone
Acetone
95%Ethanol
+5%lsoprop.
Fig. 1. Fluorescence in different extracts of Selenastrum capricornutum after 20-rain and 48-h storage at room temperature in the dark. Average of triplicates with 95% confidence limits.
after 20 min extraction at room temperature and again 2 days later are shown in Fig. 1. Extraction with acetone was less than with D M S O / a c e t o n e after 20 minutes (t-test, p < 0.001), but reached the same fluorescence after 2 days. Fluorescence of ethanol extracts was about 30% less than obtained in D M S O / a c e t o n e extracts (t-test, p < < 0.0001), even when increasing the extraction temperature up to the boiling point. Fluorescence of ethanol extracts decreased within 2 days (t-test, p < 0.05), even when adding isopropanol to inhibit enzymatic degradation of pigments (t-test, p < 0.07). Extraction of S. capricornutum with D M S O / acetone was complete within a few minutes and fluorescence was stable for several days when stored in the dark at room temperature (Fig. 2). O f the maximal fluorescence, 99% was established after 3 min and 89% was still remaining after 6 weeks. Exposure to even low light (5 #mol P A R photons m -2 s - ' ) for 1 day caused up to 30% degradation o f photopigments in extracts. Fluorescence increased less than proportional with sample volumes of 5-30%, when the ratio between sample and solvent volume was varied for D M S O / acetone and D M S O / e t h a n o l as shown on Fig. 3. The fluorescence signal of the presently proposed method
Ir
100%
,,
II
RESULTS A m o n g the solvents investigated D M S O / a c e t o n e (1:1) provided both the highest and most stable fluorescence. Triplicates of 0 . 4 m l culture at 3.105 algal cells/ml were extracted in 1.6 ml of D M S O / acetone, acetone, ethanol and ethanol + 5 % isopropanol. Fluorescence maxima for all extracts were found to be within a narrow wavelength interval of 671 + 2 nm. Fluo:rescence measured at 671 _ 10 nm
Ethanol
"o,
II II .I.ll...I.1.1 .
1 rn 3m 6111 l O m 2 0 m 1 d 2 d
. . . . 7 d 14d 42d
.
. I 4"C 21"C21"C dalk dw'k I~t,I
Solvent extraction time
Fig. 2. Fluorescence of one acetone/DMSO extract of Selenastrum capricornutum as function of extraction time. The extract was stored in the dark at room temperature. Three bars to the right show fluorescence of extract after 1 day storage under different conditions (m, minutes; d, days).
2528
P. Mayer et al. 140-
120.
~
80-
~
60-
= DMSO/Acetona ] • DMSO/Ethanol [ - - Poly. (DMSO/Ethano0 I
. ~
~
LT. 40O ._1
2001
0
I
I
I
5
10
15
/
I
20
25
30
Sample fraction as % of final extract (vol/vol)
-1
I 48
24
Fig. 3. Average fluorescenceof triplicate extracts as function of sample fraction (v/v) in final extract. Curves are least-square fitted second-order polynomia.
(20% H20 v/v in extract) was 3.3 times higher than obtained with the composition of the original method (5% H20 v/v in extract) by Shoaf and Lium (1976). The proposed injection method accurately recovered chlorophyll from samples containing as little as 500 cells Selenastrum per milliliter, and fluorescence increased linearly with cell density up to at least 5' 105 cells ml -~ as illustrated in Fig. 4. Standard curves based on extraction of diluted samples with DMSO/acetone were made for the standard set-up as well as for the high sensitivity set-up. This test of sensitivity and linearity was based on dilutions of a single algal culture of 5.105 algal cells per milliliter. Precise triplicate measurements (standard deviation among replicates < 5%) using the standard setting were obtained beginning at population densities of 5000cells ml-L This corresponds to about 1/~g chlorophyll a per liter in original samples and 0.2/~g liter-' in extracts assuming a chlorophyll a content of 0.2 pg per cell, as reported by Mayer and Jensen (1995) for exponentially growing S. capricornutum under conditions very similar to those of the present study. A higher sensitivity with a detection limit of
2
•
Standard
j / ~ - -
,,=o,_//.,_._ 2
3
4
5
6
L~j(~lls/mL) Fig. 4. Dilutions of one algal culture were extracted with DMSO/acetone. Extracts of 20% sample volume and 80% DMSO/acetone were measured for the range of 500-5'105 cells ml-~. Averages of three replicate extracts of each cell density were corrected for background fluorescenceof 0.56 in the standard set-up and 0.70 in the high sensitivitymode. Error bars are smaller than symbols.
72
Time (h)
Fig. 5. Growth in 12 replicate cultures is illustrated as the average fluorescence and standard deviations among replicates. The initial algal density of 101cells ml-~ (nominal density) and the initial fluorescence (nominal fluorescence) were both calculated from the inoculum dilution factor.
500 cells ml -~ was obtained when excitation and emission light was amplified by reflectors. The method is suitable for precise monitoring of algal biomass in cultures with balanced growth and can be used for small volume cultures with low inoculum densities as shown in Fig. 5. Twelve control cultures of each 5 ml at an initial nominal density of 10~ cells ml-' were incubated for 3 days with daily sampling of 0.2 ml for fluorescence measurements. A straight line in the time-log(fluorescence) plot indicate exponential growth with constant chlorophyll content; and low deviations among replicate cultures indicate that both growth rate and algal chlorophyll content are reproducible among replicates. To obtain this curve it was necessary to maintain constant and homogenous test conditions with regard to temperature and light, as both growth rate and chlorophyll content vary considerably with light and temperature. The response of S. capricornutum to the reference toxicant potassium dichromate calculated from fluorescence measurements was very similar to the response evaluated from measurements of cell number, except for very high inhibitions (Fig. 6a). The toxic response calculated from measurements of total cell volume was similar at low, slightly less at moderate and markedly less at high toxicity levels, when compared to responses based on fluorescence and cell number. Differences are due to a toxicant induced increase in cell size and a toxicant induced reduction of the internal chlorophyll concentration (chlorophyll/biovolume ratio), while the chlorophyll content per cell seems to be less affected by potassium dichromate. Weibull model effect concentration estimates (EC-x) were similar for all three measurement methods, when estimations were based on low end responses with up to 50% as shown on Fig. 6b. This is normally the interesting part of the concentration-response curve. Figure 6c shows effect concentration estimates for curve fitting to the entire dataset; and a comparison of EC-I estimates in
Algal fluorescence measurement 120% '
~
100%'
- . - ~ - f l u o r u ~ of - - w - nXtr~lurn~r L
~ ,
80%,
t'-
._o so*/.. .m
..c 4 0 % .
t-
20%, 0%
00 -20%
Potassium dichromate in pg/I Fig. 6a. Growth inhibition of Selenastrum capricornutum by potassium dichromate at 15 concentrations. Inhibition averages of 2-3 replicates as estimated from measurements of fluorescence (1), cell number (2) or cell volume (3). 100OO
._.q 1000
E 2t-
._O "O 100
10 EC-1
EC-10
EC-20
EC-50
Fig. 6b. Growth inhibition of Selenastrum capricornutum by potassium dichromate. Estimated effect concentration and their 95% confidence limits as based on curve fitting to inhibitions of up to 50%. 10000. t~
.-.q 1000-
E 2e-
.o "o .6
100.
ft.. 10,
EC-1
EC..IO
EC-20
EC-50
EC-80
EC-90
Fig. 6c. Growth inhibition of Selenastrum capricornutum by potassium dichromate. Estimated effect concentration and their 95% confidence limits as based on curve fitting to the end:ire inhibition range.
Fig. 6c with Fig. 6b indicates that "high effect" data points affect the curve fit to "low effect" data points. DISCUSSION
The proposed method is a modification of a fluorimetric field method for chlorophyll analysis made suitable for routine alga! toxicity testing. The
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method is fast, with about 30 s processing time per sample. Extraction is completed after a few minutes and extracts are ready for measurement. Costs are relatively low, and even more when glassware is re-used. With a standard fluorimeter the needed sub-sample volume was 0.2ml with 10-12-mm cuvettes, which is markedly less than required for electronic particle counting of low algal densities. Thus, testing is feasible in mini-scale size. Extracts can be stored stable in the dark for days and fluorescence measurements can be automated. The feasibility of the method is demonstrated with Selenastrum, but the method is believed to be rather generally applicable to micro algae. Chlorophyll a is an universal photo-pigment in phytoplankton and DMSO/acetone has been found an effective extractive solvent (Shoaf and Lium, 1976; Speziale et al., 1984; Phinney and Yentsch, 1985; Webb et al., 1992). If generally applicable the method may also be used to facilitate toxicity tests with algal species that are difficult to measure with electronic particle counters. Should the extraction efficiency of DMSO/acetone be insufficient with certain species, the stronger extractant N, N-dimethylformamide (DMF) may be applied (Speziale et al., 1984). The precision and sensitivity of the in vitro fluorescence method is high, even in comparison with electronic particle counting. The proposed method thus makes it possible to conduct algal toxicity tests with low biomass densities, thereby eliminating many of the practical problems associated with algal tests (Nyholm and K/illqvist, 1989). Algal densities in growth inhibition test may easily be reduced by 1-2 orders of magnigude compared to current standards. As an example, tests of 2 days duration were successfully carried out with Selenastrum using 5 ml test volume and calculated nominal initial algal densities of 103 cells ml-L Final measured algal densities were 1-6 × 10 4 cells ml-L Initial densities in 3 days standard tests are typically 104 cells ml -~ and final densities in controls are typically 1-6 x 106 cells ml-L Another possible field of application may be the testing of complex materials, such as wastewater. In order not to "loose toxicity", samples may be tested unfiltered, which is recommended practice by the ISO (1996) for wastewater. Particulates in unfiltered samples give rise to interferences with algal biomass determinations--in some situations making measurements, e.g. with electronic particle counting, virtually impossible. Measurements of extracted pigment fluorescence is obviously an attractive alternative when testing algal toxicity of particle-rich test materials. With direct injection into a solvent as proposed here, a blank with no algae should be measured for each concentration of the test material, and quenching of the fluorescence signal by the test material should be investigated. The method might even be applied for toxicity testing of aqueous soil or sediment suspensions. The soil and sediment
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concentration in algal cultures must then be kept under a critical level, to avoid inhibition of growth and alterations of the pigmentation due to shading. However, the effect of shading can be minimized, by a reduced light pass (e.g. to 5 ram) and by operating at high light intensities. For satisfactory application of the presented method, a number of details should be observed. A stable fluorescence/biomass ratio is only obtained with balanced growth, therefore homogenous and constant conditions with regard to temperature, light and nutrients are important. It must be pointed out that, with very dense cultures and long light path, self-shading may result in an increased chlorophyll/ biomass ratio due to a reduced effective light intensity. One should also be aware of the fact that light exposure of extracts results in a decreased fluorescence signal due to photo-degradation of pigments. At low algal densities correct measurements of blank fluorescence are of course crucial, and blanks should be measured on the actual solvent batch and the actual water content. Finally, the observation of strictly exponential growth in controls (straight line in a semilog plot as illustrated in Fig. 5) can be taken as a preliminary validity criteria on the measurements. One must be aware of the fact that pigment fluorescence is only an indirect measurement which is strictly proportional to the algal biomass only under conditions of balanced growth. Toxic substances induce changes in algal physiology, and growth in affected cultures will never be strictly balanced. Chlorophyll a is the major contributor to the measured fluorescence in extracts and any changes in the chlorophyll-a/biomass ratio will of course affect the biomass estimation. At low toxicity levels, however, photosynthesis is near its uninhibited level and changes in the content of chlorophyll a and hence the fluorescence/biomass ratio in extracts can likewise be expected to be low, unless the toxicant specifically gives rise to significant alterations of the photosynthetic energy trapping system. Thus, for most chemicals at low toxicity levels, pigment fluorescence of extracts is probably a good measure of biomass. For herbicides specifically inhibiting photosynthesis, on the other hand, the algal chlorophyll content may be altered significantly even at low toxicity levels. At moderate and high toxicity levels the overall algal metabolism is highly affected by the toxicant, and also the production and content of photopigments may therefore be significantly altered. Examples of toxicant-induced changes of the algal chlorophyll a content in S. capricornutum were reported by Mayer and Jensen (1995). Exposure to the herbicide atrazine resulted in significant increase in the chlorophyll content in S. capricornutum cells at moderate and high levels of growth inhibition, while three other toxicants without known specific mode of action caused a marked decrease of chlorophyll a at moderate and high toxicity levels. They concluded
that the selection of the biomass parameter monitored is of significance at moderate and high levels of growth inhibition, whereas the detection and quantification of small inhibitions are mainly dependent on the sensitivity and precision of the methed of measurement. Therefore, it can be expected that with the proposed fluorescence method, growth inhibitions in the low range will be practically identical to inhibitions generated from measurements by means of electronic particle counting. At high levels of inhibition, on the other hand, significant differences can be expected. Such differences are normally rela.fively unimportant, as for most purposes only the lower part of the concentration-response curve is of practical interest. Unfortunately, incorrect data belonging to the upper part of the curve may influence the curve fitting of the lower curve part, if care is not taken to minimize this influence, e.g. by exclusion of data representing the high level of inhibition. This is exemplified by results of the toxicity test with potassium dichromate, where inhibitions based on biomass determinations by fluorescence, cell number and cell volume were compared. Inhibitions of less than 50% were very similar for all three measures as shown in Fig. 6a. EC-1, EC-10 and EC-20 estimates for all three measures were also almost the same, when the Weibull curve was fitted to data points beionging in the range 0-50% inhibition, as illt~strated on Fig. 6b. The EC-estimates on Fig. 6c are derived from a Weibull curve fit to the whole data range. It is seen that the EC-I estimation is markedly affected by data from the high range as a consequence of the data fitting procedure using a model with only two parameters. SUMMARY
The proposed in vitro fluorescence method has a high potential as a surrogate biomass estimation method in algal toxicity tests. At low and in most situations also at moderate levels of toxicity (including EC-50), inhibitions calculated from such measurements are generally expected to be very similar to inhibitions calculated from measurements by particle counting. Algal growth inhibitions at high toxicity levels and for herbicides specifically affecting photosynthesis should be evaluated with caution, when calculated from measurements of extracted fluorescence. SAFETY AND HEALTH HAZARDS
DMSO penetrates skin quickly (Rammler and Zaffaroni, 1967) and is suspected to be carcinogenic (KBS, 1994). Additionally, DMSO can facilitate penetration of other organics dissolved in the extract (KBS, 1994) and, because many substances tested in algal toxicity tests are hazardous to human health,
Algal fluorescence measurement any contact with the extract should be avoided. This was achieved with the fluorimeter used in the standard set-up by closing test-tubes immediately after injection, which took place in a ventilated hood. Volatized D M S O can easily be detected due its strong and unpleasant odor. For some applications extraction with pure acetone or ethanol without D M S O might be considered for safety reasons. Excluding D M S O , however, reduces extraction speed and possibly also extr~Lction yield and the stability of extracts, and is therefore not recommended unless work-place exposure is unavoidable. Acknowledgements--(Fhanks are given to Susanne Kruse for technical assistance throughout the project, to Anders Baun for assistance in d~.ta treatment and to Bent Hailing S~rensen and Dick Sijm for many helpful comments on the manuscript. This work was funded in part by the Danish Environmental Research Program (Danish Center for Ecotoxicological Research, project 7.1), and in part by the Center for Great Lakes Studies of the Universtiy of Wisconsin-Milwaukee (contribution number 404). REFERENCES
Andersen H. (1994) Statisiske metoder til vurdering af spildevand toxicitet. Master's thesis, Institute for Mathematical Modelling, Technical University of Denmark, Lyngby. B6ger P. and Sandemann G. (1989) Target Sites of Herbicide Action. CRC Press, Boca Raton, FL. Christensen E. R. and Nyholm N. (1984) Ecotoxicological assays with algae: Weibull dose-response curves. Environ. Sci. Technol. 18, 713-718. Eloranta V. (1978) O11biomass monitoring methods in toxic assays of algae. NORDFORSK, University of Jyvaskyla, Finland. pp. 147-156. Greenberg E. and Watras C. J. (1989) Field evaluation of a micro-extraction technique for measuring chlorophyll in lakewater without filtration. Hydorbiologia 173, 193-197. Holm-Hansen O., l_,orenzen C. J., Holmes R. W. and Strickland J. D. H. (1965) Fluorometric determination of chlorophyll. J. Cons. Perm. Int. Explor. Mer. 30, 3-15. ISO 8692 (1989) Water quality--Fresh Water Algal Growth Inhibition Test wJ~th Scenedesmus subspicatus or Selenastrum capricornutum. International Organization for Standardization, Geneva. ISO (1996) ISO TC/147/SC6/WGI2NI, Wateranalysis-General Guidance for the Biotesting of water and Waste
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Water--Sampling, Pretreatment, Performance and Evaluation. International Organization for Standardization, Geneva. KBS (1994) Arbejdspladsbrugsanvisning for Dimethylsulfoxid (Guideline for handling Dimethylsulfoxide). Kemikalie-Brugsanvisnings-Sekretariatet, Technical University of Denmark, Lyngby. Kirk J. T. O. (1994) Light and Photosynthesis in Aquatic Ecosystems. Cambridge University Press, Cambridge, U.K. Lewis M. A. (1993) Freshwater primary producers. In Handbook of Ecotoxicology Vol. 1 (Edited by Calow P.), pp. 28-38. Blackwell Scientific, Oxford. Mayer P. and Jensen J. F. (1995) Factors affecting results of algal toxicity tests. MS thesis, Technical University of Denmark, Institute for Environmental Science and Technology, Lyngby. Nelder J. A. and Mead R. (1965) A simplex method for function minimisation. Computer J. 7, 308-313. Nyholm N. and K/illqvist T. (1989) Methods for growth inhibition toxicity tests with freshwater algae. Environ. Toxicol. Chem. 8, 689-703. Nyholm N. and Petersen H. G. (1997) Laboratory bioassays with microalgae. Chapter 9. Wang W., Gorsuch J. W. and Hughes J. S. (eds), Plants for Environmental Studies. Lewis Publishers, Boca Raton, FL. Petersen H. G. and Nyholm N. (1993) Algal bioassays for metal toxicity identification. Bat. Pollut. Res. J. Canada 28, 129-153. Phinney D. A. and Yentsch C. S. (1985) A novel phytoplankton chlorophyll technique: toward automated analysis. J. Plankton Res. 7, 633-642. Putt M., Harris G. P. and Cuhel R. L. (1987) Photoinhibition of DCMU-enhanced fluorescence in Lake Ontario phytoplankton. Can. J. Fish. Aquat. Sci. 44, 2144-2154. Rammler D. H. and Zaffaroni A. (1967) Biological implications of DMSO based on a review of its chemical properties. Ann. N.Y. Acad. Sci. 141, 13-23. ShoafW. T. and Lium B. W. (1976) Improved extraction of chlorophyll a and b from algae using dimethyl sulfoxide. Limnol. Oceanogr. 21, 926-928. Speziale B. J., Schreiner S. P., Giammatteo P. A. and Schindler J. E. (1984) Comparison of N, N-dimethylformamide, dimethyl sulfoxide, and acetone for extraction of phytoplankton chlorophyll. Can. J. Fish. Aquat. Sci. 41, 1519 1522. Webb D. J., Burnison B. K., Trimbee A. M. and Prepas E.E. (1992) Comparison of chlorophyll a extractions with ethanol and dimethyl sulfoxide/acetone, and a concern about spectrophotometric phaeopigment correction. Can. J. Fish. Aquat. Sci. 49, 2331-2336.
War. Res. Vol. 31, No. 10, pp. 2525-2531, 1997 © 1997ElsevierScienceLtd. All rights reserved Printed in Great Britain 0043-1354/97$17.00 + 0.00
A SIMPLE IN VITRO FLUORESCENCE METHOD FOR BIOMASS MEASUREMENTS IN ALGAL GROWTH INHIBITION TESTS P H I L I P P M A Y E W * , RUSSELL C U H E L 2 and NIELS N Y H O L M @t ~Technical University of Denmark, Institute for Environmental Science and Technology, 2800 Lyngby, Denmark and 2Center for Great Lakes Studies, University of Wisconsin Milwaukee, Milwaukee, WI 53204, U.S.A. (Received September 1996; accepted in revised form March 1997)
Abstract--The estimation of biomass concentrations in algal growth inhibition tests from measurements of pigment fluorescence in extracts of 20% sample (final v/v) prepared by direct addition to dimethylsulfoxide/acetone solvent offers several advantages compared to currently used direct or indirect methods. The extraction stops the electron transfer and other processes which interact with chlorophyll fluorescence when measured in vivo. As a result the response is stabilized and the sensitivity improved. The injection method is very fast, has a high potential for automation, allows storage of samples and is suitable for small sample volumes (e.g. 0.2 ml). The typical initial cell density in standard toxicity tests of 104 cells ml-L of Selenastrum capricornutum was measured precisely with a standard fluorimeter set-up, and 103 cells ml -~ of S. capr&ornutum was measured reliably with a sensitive fluorimeter. At low levels of toxicity by the model test compound potassium dichromate, the proposed fluorescence method resulted in very similar inhibition figures as obtained with electronic particle counting. At high levels of toxicity, on the other hand, biomass determinations from pigment fluorescence readings were markedly affected by toxicant-induced changes of the algal physiology. The low effect part of a dose response curve is normally that one of major interest, and biomass estimation errors associated with fluorescence measurements on extracts are thus considered acceptable in most situations. When the entire dataset was applied for endpoint estimation by the Weibull model, EC-I estimates were markedly affected by the curve fitting to dala in the high inhibition range, while EC-10 and EC-20 were less and EC-50 almost unaffected. The method is expected to be less suitable for toxicity testing of herbicides specifically inhibiting photosynthesis. © 1997 Elsevier Science Ltd Key words---chlorophyll measurement method
extraction, in vitro fluorescence, algal toxicity testing, Weibull model,
INTRODUCTION The regulation of environmental pollutants is still based primarily on toxicity to aquatic animals, despite the importance of phytoplankton as the dominant primary producer in most aquatic habitats, and despite the tact that aquatic plants including algae are more sensitive than animals to a variety of potential toxicants (Lewis, 1993; Peterson and Nyholm, 1993). The use of algal toxicity tests is increasing, and today algal toxicity tests are frequently required by authorities for notifications of chemicals and are also increasingly being used to manage chemical discharges. The resistance for an extended use of algal toxicity tests might be partly of a practical nature, and the proposed method
*Author to whom all correspondence should be addressed at Environmental Chemistry Group, Research Institute of Toxicology, Utrecht University, P.O. Box 80058, 3508 TB Utrecht, The Netherlands [Fax: +31-30-2532837, E-maih p.mayer@ ritox.dgk.ruu.nl].
presented in this paper is believed to ease the performance of such tests. Additionally, the suggested method might permit toxicity testing at very low algal densities, particularly when applied in combination with a reduced test duration. Final biomass densities in standard growth inhibition tests with algae exceed those found in most natural waters and high algal densities give rise to physico-chemical medium changes during the test, which may affect the environmental relevance and reproducibility of laboratory-derived toxicity data. High algal densities can also directly affect the bio-availability and toxicity of a test substance. Therefore, it is generally desirable to use low algal densities in toxicity tests. (Petersen and Nyhoim, 1993; Nyholm and Petersen, 1997). Measurements of low algal densities, however, require highly sensitive methods, and if the biomass level of the current standard tests was to be reduced, many laboratories would have to adopt new techniques. For many years, electronic particle counting, preferably including determination of cell volume, has been the method of reference. The
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technique is precise and sensitive but also time-consuming, and the equipment costs are high. Further, the technique is difficult to automate and sample volumes of several milliliters are required for precise measurements at low algal densities. Optical densities by spectrophotometry or turbidity are inexpensive and frequently used indirect methods, but both methods offer a lower precision and sensitivity than that of electronic particle counting. Optical density is considered to have only borderline sensitivity and precision at the biomass levels of standard tests. Measurement of chlorophyll fluorescence, in vivo or in vitro, is a hitherto less often used but sensitive alternative. Eloranta (1978) reported a high correlation between the primary pigment chlorophyll a and cell counts as growth indicator in algal toxicity tests of different test substances with Ankistrodesmus falcatus. However, a more widely use of chlorophyll a fluorescence measurements in algal growth inhibition tests requires practical methods, which combine sensitivity, precision and reproducibility. In vivo fluorescence is re-radiation of light absorbed by algal pigments in intact cells. In living cells, active in photosynthesis only a very small portion (about 1%) of the absorbed light is re-emitted as fluorescence (Kirk, 1994), which limits the sensitivity of in vivo fluorescence measurements. Recent technical advances in fluorescence spectroscopy allow measurements on dilute samples. A drawback of measuring in vivo fluorescence, however, is that the signal varies with the state of the photosynthetical reaction center (PS 2), which among others depends on the previous light regime and also is influenced by many herbicides (Putt et al., 1987; B6ger and Sandemann, 1989). This makes appropriate and reproducible handling of samples for in vivo fluorescence measurements crucial. The addition of the herbicide DCMU can be used to standardize the redox state of PS 2 and can thereby stabilize and maximize the fluorescence signal. Another way to reduce the problems associated with in vivo fluorescence is the extraction of pigments with organic solvents, because chlorophyll a and other pigments in solvent extracts are uncoupled from the biochemical processes influencing the fluorescence signal of in vivo measurements. A major argument against the use of extracted fluorescence measurements in algal toxicity tests has been a purely practical one--measurements are traditionally preceded by a filtration or centrifugation step, which is both time-consuming and constitutes a source of errors. The whole water extract fluorescence method presented here is performed without any filtration or centrifugation step, and the technique is cost-effective, sensitive, fast and well suited for automation. The method is based on the comprehensive description of chlorophyll a extraction with acetone and DMSO by Shoaf and Lium (1976) with modifications by us. A comparable method for field
studies of phytoplankton was proposed by Phinney and Yentsch (1985) and by Greenberg and Watras (1989). Most published extracted fluorescence methods aim at quantifying chlorophyll a in field samples. Chlorophyll a concentrations are typically measured as the difference in fluorescence before and after acidification, to separate phaeophytin a and chlorophyll a, and to correct for background fluorescence (Holm Hansen et al., 1965). Extraction procedures are usually optimized to ensure high extraction efficiencies (near unity) for most algal species, as the total chlorophyll a concentration is the parameter of interest. The objective of a whole water extraction method for fluorescence measurements in algal growth inhibition tests is different. The detection limit and in particular the precision at low algal densities is here of great importance, because it determines the possible range of algal densities in the test. To obtain a high sensitivity, the fluorescence of the extract is maximized; and to obtain a high precision and reproducibility, consistency in the extraction efficiency is more important than a high absolute extraction efficiency. Acidification of extracts to distinguish chlorophyll a from other pigments is not necessary in toxicity tests, where the goal is to measure algal growth and not specifically to measure chlorophyll a. The aim of this paper is to present a simple in vitro fluorescence method for biomass measurements in algal growth inhibition tests. Documentation is provided with regard to choice of extractive solvent, sample/solvent ratio, extraction time, stability of extracts, precision and sensitivity. Finally, the potential of the method and the test results obtained are discussed, and a comparison of results obtained with the fluorescence method and with electronic particle counting is provided for a toxicity test with the model test compound potassium dichromate.
METHODS
Culturing
Cultures of the unicellular green alga Selenastrum capricornutum Printz were grown in ISO 8692 standard algal medium under continuously illumination of white fluorescence light at 110 + 4/Jmol PAR photons m--' s-~ (or/~E m-2s -' in the photosynthetically active range) at a temperature of 22 + 1°C, resulting in specific growth rates of about 1.9 day-t. The cultures were cultivated in 20 ml Scintillation vials with 4-5 ml liquid volume and shaken at 300 rpm. Direct injection method
Culture samples of 0.14).4 ml were extracted by direct injection into different organic solvents in a test-tube immediately followed by mixing. A 20% sample (final volume %) was extracted in a 1: 1 mixture of DMSO and acetone for at least 20 rain unless otherwise noted, and extracts were stored in the dark at room temperature. Fluorescence was measured at room temperature in
Algal fluorescence measurement arbitrary units on a Hitachi fluorescence spectrophotometer model F-2000 with an excitation wavelength of 430 nm and a measured emission wavelength of 671 + 10 nm. The signal changed less than 0.5% per °C. Reported fluorescence figures were corrected for background fluorescence measured on solvents mixed with algal medium. Background fluorescence of differnt commercially available DMSO and acetone products were tested. HPLC certified DMSO and acetone yielded lower or similar background fluorescence than more expensive fluorescence grade solvents. In the standtard set-up, measurements were made using a carousel with eight standard test-tubes with an outer diameter of 10 ram. With a high sensitivity set-up available from the manufacturer both excitation light and emitted light were amplified by reflectors, and fluorescence was measured in a 12 mm rectangular quartz cuvette. Reference algal population densities were measured in triplicates as total bio-volume (pm3/ml) and numbers (algal cells per milliter) by use of a~a electronic particle counter (Coulter Multisizer). Algae were counted in the size range 2.125-9 pm with an aperture of 70 pm, a counting volume of 0.5 ml and lstotone R as dilution medium. Monitoring of balanced growth Twelve replicate cultures with no test substance added were inoculated at 103 cells ml-' and in vitro fluorescence measured daily usingl the standard fluorimeter set-up. Toxicity test and data treatment A 72-h algal growth inhibition test was performed according to ISO 8692 (ISO, 1989) using potassium dichromate as reference toxicant. This test was fully randomized and the toxicant was dosed in a geometric dilution series of 16 concentrations in triplicates (data for one concentration were lost). During the test period, in vitro fluorescence of the control cultures were measured daily to verify exponential growth. After 72 h, in vitro fluorescence, cell number and total cell volume of all samples were measured. The specil~c growth rate (~) was defined as the rate constant in the following equation:
-~Nt=/J.N~N(t) = N0.e"' where N is the algal biomass measured as algal volume, algal numbers or in vitro fluorescence. The toxic response was expressed as growth inhibition (I) calculated as the relative reduction in growth rate caused by the test substance: I =
l - - ~inhibited ]./control
Concentration-respense curves were described by the Weibull equation (Christensen and Nyholm, 1984) which was fitted to data using weighted non-linear regression applying a computer program developed by Andersen (1994). The program uses the "Downhill Simplex Method" by Nelder and Mead (1965) to give a weighted least-squares fit to replicate means. Weighting factors are inversely proportional to the empirical variance as estimated from replicates. Confidence intervals around EC values are calculated by inverse estimation after a Taylor expansion.
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c
J l,
DMSO/Acetone
Acetone
95%Ethanol
+5%lsoprop.
Fig. 1. Fluorescence in different extracts of Selenastrum capricornutum after 20-rain and 48-h storage at room temperature in the dark. Average of triplicates with 95% confidence limits.
after 20 min extraction at room temperature and again 2 days later are shown in Fig. 1. Extraction with acetone was less than with D M S O / a c e t o n e after 20 minutes (t-test, p < 0.001), but reached the same fluorescence after 2 days. Fluorescence of ethanol extracts was about 30% less than obtained in D M S O / a c e t o n e extracts (t-test, p < < 0.0001), even when increasing the extraction temperature up to the boiling point. Fluorescence of ethanol extracts decreased within 2 days (t-test, p < 0.05), even when adding isopropanol to inhibit enzymatic degradation of pigments (t-test, p < 0.07). Extraction of S. capricornutum with D M S O / acetone was complete within a few minutes and fluorescence was stable for several days when stored in the dark at room temperature (Fig. 2). O f the maximal fluorescence, 99% was established after 3 min and 89% was still remaining after 6 weeks. Exposure to even low light (5 #mol P A R photons m -2 s - ' ) for 1 day caused up to 30% degradation o f photopigments in extracts. Fluorescence increased less than proportional with sample volumes of 5-30%, when the ratio between sample and solvent volume was varied for D M S O / acetone and D M S O / e t h a n o l as shown on Fig. 3. The fluorescence signal of the presently proposed method
Ir
100%
,,
II
RESULTS A m o n g the solvents investigated D M S O / a c e t o n e (1:1) provided both the highest and most stable fluorescence. Triplicates of 0 . 4 m l culture at 3.105 algal cells/ml were extracted in 1.6 ml of D M S O / acetone, acetone, ethanol and ethanol + 5 % isopropanol. Fluorescence maxima for all extracts were found to be within a narrow wavelength interval of 671 + 2 nm. Fluo:rescence measured at 671 _ 10 nm
Ethanol
"o,
II II .I.ll...I.1.1 .
1 rn 3m 6111 l O m 2 0 m 1 d 2 d
. . . . 7 d 14d 42d
.
. I 4"C 21"C21"C dalk dw'k I~t,I
Solvent extraction time
Fig. 2. Fluorescence of one acetone/DMSO extract of Selenastrum capricornutum as function of extraction time. The extract was stored in the dark at room temperature. Three bars to the right show fluorescence of extract after 1 day storage under different conditions (m, minutes; d, days).
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P. Mayer et al. 140-
120.
~
80-
~
60-
= DMSO/Acetona ] • DMSO/Ethanol [ - - Poly. (DMSO/Ethano0 I
. ~
~
LT. 40O ._1
2001
0
I
I
I
5
10
15
/
I
20
25
30
Sample fraction as % of final extract (vol/vol)
-1
I 48
24
Fig. 3. Average fluorescenceof triplicate extracts as function of sample fraction (v/v) in final extract. Curves are least-square fitted second-order polynomia.
(20% H20 v/v in extract) was 3.3 times higher than obtained with the composition of the original method (5% H20 v/v in extract) by Shoaf and Lium (1976). The proposed injection method accurately recovered chlorophyll from samples containing as little as 500 cells Selenastrum per milliliter, and fluorescence increased linearly with cell density up to at least 5' 105 cells ml -~ as illustrated in Fig. 4. Standard curves based on extraction of diluted samples with DMSO/acetone were made for the standard set-up as well as for the high sensitivity set-up. This test of sensitivity and linearity was based on dilutions of a single algal culture of 5.105 algal cells per milliliter. Precise triplicate measurements (standard deviation among replicates < 5%) using the standard setting were obtained beginning at population densities of 5000cells ml-L This corresponds to about 1/~g chlorophyll a per liter in original samples and 0.2/~g liter-' in extracts assuming a chlorophyll a content of 0.2 pg per cell, as reported by Mayer and Jensen (1995) for exponentially growing S. capricornutum under conditions very similar to those of the present study. A higher sensitivity with a detection limit of
2
•
Standard
j / ~ - -
,,=o,_//.,_._ 2
3
4
5
6
L~j(~lls/mL) Fig. 4. Dilutions of one algal culture were extracted with DMSO/acetone. Extracts of 20% sample volume and 80% DMSO/acetone were measured for the range of 500-5'105 cells ml-~. Averages of three replicate extracts of each cell density were corrected for background fluorescenceof 0.56 in the standard set-up and 0.70 in the high sensitivitymode. Error bars are smaller than symbols.
72
Time (h)
Fig. 5. Growth in 12 replicate cultures is illustrated as the average fluorescence and standard deviations among replicates. The initial algal density of 101cells ml-~ (nominal density) and the initial fluorescence (nominal fluorescence) were both calculated from the inoculum dilution factor.
500 cells ml -~ was obtained when excitation and emission light was amplified by reflectors. The method is suitable for precise monitoring of algal biomass in cultures with balanced growth and can be used for small volume cultures with low inoculum densities as shown in Fig. 5. Twelve control cultures of each 5 ml at an initial nominal density of 10~ cells ml-' were incubated for 3 days with daily sampling of 0.2 ml for fluorescence measurements. A straight line in the time-log(fluorescence) plot indicate exponential growth with constant chlorophyll content; and low deviations among replicate cultures indicate that both growth rate and algal chlorophyll content are reproducible among replicates. To obtain this curve it was necessary to maintain constant and homogenous test conditions with regard to temperature and light, as both growth rate and chlorophyll content vary considerably with light and temperature. The response of S. capricornutum to the reference toxicant potassium dichromate calculated from fluorescence measurements was very similar to the response evaluated from measurements of cell number, except for very high inhibitions (Fig. 6a). The toxic response calculated from measurements of total cell volume was similar at low, slightly less at moderate and markedly less at high toxicity levels, when compared to responses based on fluorescence and cell number. Differences are due to a toxicant induced increase in cell size and a toxicant induced reduction of the internal chlorophyll concentration (chlorophyll/biovolume ratio), while the chlorophyll content per cell seems to be less affected by potassium dichromate. Weibull model effect concentration estimates (EC-x) were similar for all three measurement methods, when estimations were based on low end responses with up to 50% as shown on Fig. 6b. This is normally the interesting part of the concentration-response curve. Figure 6c shows effect concentration estimates for curve fitting to the entire dataset; and a comparison of EC-I estimates in
Algal fluorescence measurement 120% '
~
100%'
- . - ~ - f l u o r u ~ of - - w - nXtr~lurn~r L
~ ,
80%,
t'-
._o so*/.. .m
..c 4 0 % .
t-
20%, 0%
00 -20%
Potassium dichromate in pg/I Fig. 6a. Growth inhibition of Selenastrum capricornutum by potassium dichromate at 15 concentrations. Inhibition averages of 2-3 replicates as estimated from measurements of fluorescence (1), cell number (2) or cell volume (3). 100OO
._.q 1000
E 2t-
._O "O 100
10 EC-1
EC-10
EC-20
EC-50
Fig. 6b. Growth inhibition of Selenastrum capricornutum by potassium dichromate. Estimated effect concentration and their 95% confidence limits as based on curve fitting to inhibitions of up to 50%. 10000. t~
.-.q 1000-
E 2e-
.o "o .6
100.
ft.. 10,
EC-1
EC..IO
EC-20
EC-50
EC-80
EC-90
Fig. 6c. Growth inhibition of Selenastrum capricornutum by potassium dichromate. Estimated effect concentration and their 95% confidence limits as based on curve fitting to the end:ire inhibition range.
Fig. 6c with Fig. 6b indicates that "high effect" data points affect the curve fit to "low effect" data points. DISCUSSION
The proposed method is a modification of a fluorimetric field method for chlorophyll analysis made suitable for routine alga! toxicity testing. The
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method is fast, with about 30 s processing time per sample. Extraction is completed after a few minutes and extracts are ready for measurement. Costs are relatively low, and even more when glassware is re-used. With a standard fluorimeter the needed sub-sample volume was 0.2ml with 10-12-mm cuvettes, which is markedly less than required for electronic particle counting of low algal densities. Thus, testing is feasible in mini-scale size. Extracts can be stored stable in the dark for days and fluorescence measurements can be automated. The feasibility of the method is demonstrated with Selenastrum, but the method is believed to be rather generally applicable to micro algae. Chlorophyll a is an universal photo-pigment in phytoplankton and DMSO/acetone has been found an effective extractive solvent (Shoaf and Lium, 1976; Speziale et al., 1984; Phinney and Yentsch, 1985; Webb et al., 1992). If generally applicable the method may also be used to facilitate toxicity tests with algal species that are difficult to measure with electronic particle counters. Should the extraction efficiency of DMSO/acetone be insufficient with certain species, the stronger extractant N, N-dimethylformamide (DMF) may be applied (Speziale et al., 1984). The precision and sensitivity of the in vitro fluorescence method is high, even in comparison with electronic particle counting. The proposed method thus makes it possible to conduct algal toxicity tests with low biomass densities, thereby eliminating many of the practical problems associated with algal tests (Nyholm and K/illqvist, 1989). Algal densities in growth inhibition test may easily be reduced by 1-2 orders of magnigude compared to current standards. As an example, tests of 2 days duration were successfully carried out with Selenastrum using 5 ml test volume and calculated nominal initial algal densities of 103 cells ml-L Final measured algal densities were 1-6 × 10 4 cells ml-L Initial densities in 3 days standard tests are typically 104 cells ml -~ and final densities in controls are typically 1-6 x 106 cells ml-L Another possible field of application may be the testing of complex materials, such as wastewater. In order not to "loose toxicity", samples may be tested unfiltered, which is recommended practice by the ISO (1996) for wastewater. Particulates in unfiltered samples give rise to interferences with algal biomass determinations--in some situations making measurements, e.g. with electronic particle counting, virtually impossible. Measurements of extracted pigment fluorescence is obviously an attractive alternative when testing algal toxicity of particle-rich test materials. With direct injection into a solvent as proposed here, a blank with no algae should be measured for each concentration of the test material, and quenching of the fluorescence signal by the test material should be investigated. The method might even be applied for toxicity testing of aqueous soil or sediment suspensions. The soil and sediment
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concentration in algal cultures must then be kept under a critical level, to avoid inhibition of growth and alterations of the pigmentation due to shading. However, the effect of shading can be minimized, by a reduced light pass (e.g. to 5 ram) and by operating at high light intensities. For satisfactory application of the presented method, a number of details should be observed. A stable fluorescence/biomass ratio is only obtained with balanced growth, therefore homogenous and constant conditions with regard to temperature, light and nutrients are important. It must be pointed out that, with very dense cultures and long light path, self-shading may result in an increased chlorophyll/ biomass ratio due to a reduced effective light intensity. One should also be aware of the fact that light exposure of extracts results in a decreased fluorescence signal due to photo-degradation of pigments. At low algal densities correct measurements of blank fluorescence are of course crucial, and blanks should be measured on the actual solvent batch and the actual water content. Finally, the observation of strictly exponential growth in controls (straight line in a semilog plot as illustrated in Fig. 5) can be taken as a preliminary validity criteria on the measurements. One must be aware of the fact that pigment fluorescence is only an indirect measurement which is strictly proportional to the algal biomass only under conditions of balanced growth. Toxic substances induce changes in algal physiology, and growth in affected cultures will never be strictly balanced. Chlorophyll a is the major contributor to the measured fluorescence in extracts and any changes in the chlorophyll-a/biomass ratio will of course affect the biomass estimation. At low toxicity levels, however, photosynthesis is near its uninhibited level and changes in the content of chlorophyll a and hence the fluorescence/biomass ratio in extracts can likewise be expected to be low, unless the toxicant specifically gives rise to significant alterations of the photosynthetic energy trapping system. Thus, for most chemicals at low toxicity levels, pigment fluorescence of extracts is probably a good measure of biomass. For herbicides specifically inhibiting photosynthesis, on the other hand, the algal chlorophyll content may be altered significantly even at low toxicity levels. At moderate and high toxicity levels the overall algal metabolism is highly affected by the toxicant, and also the production and content of photopigments may therefore be significantly altered. Examples of toxicant-induced changes of the algal chlorophyll a content in S. capricornutum were reported by Mayer and Jensen (1995). Exposure to the herbicide atrazine resulted in significant increase in the chlorophyll content in S. capricornutum cells at moderate and high levels of growth inhibition, while three other toxicants without known specific mode of action caused a marked decrease of chlorophyll a at moderate and high toxicity levels. They concluded
that the selection of the biomass parameter monitored is of significance at moderate and high levels of growth inhibition, whereas the detection and quantification of small inhibitions are mainly dependent on the sensitivity and precision of the methed of measurement. Therefore, it can be expected that with the proposed fluorescence method, growth inhibitions in the low range will be practically identical to inhibitions generated from measurements by means of electronic particle counting. At high levels of inhibition, on the other hand, significant differences can be expected. Such differences are normally rela.fively unimportant, as for most purposes only the lower part of the concentration-response curve is of practical interest. Unfortunately, incorrect data belonging to the upper part of the curve may influence the curve fitting of the lower curve part, if care is not taken to minimize this influence, e.g. by exclusion of data representing the high level of inhibition. This is exemplified by results of the toxicity test with potassium dichromate, where inhibitions based on biomass determinations by fluorescence, cell number and cell volume were compared. Inhibitions of less than 50% were very similar for all three measures as shown in Fig. 6a. EC-1, EC-10 and EC-20 estimates for all three measures were also almost the same, when the Weibull curve was fitted to data points beionging in the range 0-50% inhibition, as illt~strated on Fig. 6b. The EC-estimates on Fig. 6c are derived from a Weibull curve fit to the whole data range. It is seen that the EC-I estimation is markedly affected by data from the high range as a consequence of the data fitting procedure using a model with only two parameters. SUMMARY
The proposed in vitro fluorescence method has a high potential as a surrogate biomass estimation method in algal toxicity tests. At low and in most situations also at moderate levels of toxicity (including EC-50), inhibitions calculated from such measurements are generally expected to be very similar to inhibitions calculated from measurements by particle counting. Algal growth inhibitions at high toxicity levels and for herbicides specifically affecting photosynthesis should be evaluated with caution, when calculated from measurements of extracted fluorescence. SAFETY AND HEALTH HAZARDS
DMSO penetrates skin quickly (Rammler and Zaffaroni, 1967) and is suspected to be carcinogenic (KBS, 1994). Additionally, DMSO can facilitate penetration of other organics dissolved in the extract (KBS, 1994) and, because many substances tested in algal toxicity tests are hazardous to human health,
Algal fluorescence measurement any contact with the extract should be avoided. This was achieved with the fluorimeter used in the standard set-up by closing test-tubes immediately after injection, which took place in a ventilated hood. Volatized D M S O can easily be detected due its strong and unpleasant odor. For some applications extraction with pure acetone or ethanol without D M S O might be considered for safety reasons. Excluding D M S O , however, reduces extraction speed and possibly also extr~Lction yield and the stability of extracts, and is therefore not recommended unless work-place exposure is unavoidable. Acknowledgements--(Fhanks are given to Susanne Kruse for technical assistance throughout the project, to Anders Baun for assistance in d~.ta treatment and to Bent Hailing S~rensen and Dick Sijm for many helpful comments on the manuscript. This work was funded in part by the Danish Environmental Research Program (Danish Center for Ecotoxicological Research, project 7.1), and in part by the Center for Great Lakes Studies of the Universtiy of Wisconsin-Milwaukee (contribution number 404). REFERENCES
Andersen H. (1994) Statisiske metoder til vurdering af spildevand toxicitet. Master's thesis, Institute for Mathematical Modelling, Technical University of Denmark, Lyngby. B6ger P. and Sandemann G. (1989) Target Sites of Herbicide Action. CRC Press, Boca Raton, FL. Christensen E. R. and Nyholm N. (1984) Ecotoxicological assays with algae: Weibull dose-response curves. Environ. Sci. Technol. 18, 713-718. Eloranta V. (1978) O11biomass monitoring methods in toxic assays of algae. NORDFORSK, University of Jyvaskyla, Finland. pp. 147-156. Greenberg E. and Watras C. J. (1989) Field evaluation of a micro-extraction technique for measuring chlorophyll in lakewater without filtration. Hydorbiologia 173, 193-197. Holm-Hansen O., l_,orenzen C. J., Holmes R. W. and Strickland J. D. H. (1965) Fluorometric determination of chlorophyll. J. Cons. Perm. Int. Explor. Mer. 30, 3-15. ISO 8692 (1989) Water quality--Fresh Water Algal Growth Inhibition Test wJ~th Scenedesmus subspicatus or Selenastrum capricornutum. International Organization for Standardization, Geneva. ISO (1996) ISO TC/147/SC6/WGI2NI, Wateranalysis-General Guidance for the Biotesting of water and Waste
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Water--Sampling, Pretreatment, Performance and Evaluation. International Organization for Standardization, Geneva. KBS (1994) Arbejdspladsbrugsanvisning for Dimethylsulfoxid (Guideline for handling Dimethylsulfoxide). Kemikalie-Brugsanvisnings-Sekretariatet, Technical University of Denmark, Lyngby. Kirk J. T. O. (1994) Light and Photosynthesis in Aquatic Ecosystems. Cambridge University Press, Cambridge, U.K. Lewis M. A. (1993) Freshwater primary producers. In Handbook of Ecotoxicology Vol. 1 (Edited by Calow P.), pp. 28-38. Blackwell Scientific, Oxford. Mayer P. and Jensen J. F. (1995) Factors affecting results of algal toxicity tests. MS thesis, Technical University of Denmark, Institute for Environmental Science and Technology, Lyngby. Nelder J. A. and Mead R. (1965) A simplex method for function minimisation. Computer J. 7, 308-313. Nyholm N. and K/illqvist T. (1989) Methods for growth inhibition toxicity tests with freshwater algae. Environ. Toxicol. Chem. 8, 689-703. Nyholm N. and Petersen H. G. (1997) Laboratory bioassays with microalgae. Chapter 9. Wang W., Gorsuch J. W. and Hughes J. S. (eds), Plants for Environmental Studies. Lewis Publishers, Boca Raton, FL. Petersen H. G. and Nyholm N. (1993) Algal bioassays for metal toxicity identification. Bat. Pollut. Res. J. Canada 28, 129-153. Phinney D. A. and Yentsch C. S. (1985) A novel phytoplankton chlorophyll technique: toward automated analysis. J. Plankton Res. 7, 633-642. Putt M., Harris G. P. and Cuhel R. L. (1987) Photoinhibition of DCMU-enhanced fluorescence in Lake Ontario phytoplankton. Can. J. Fish. Aquat. Sci. 44, 2144-2154. Rammler D. H. and Zaffaroni A. (1967) Biological implications of DMSO based on a review of its chemical properties. Ann. N.Y. Acad. Sci. 141, 13-23. ShoafW. T. and Lium B. W. (1976) Improved extraction of chlorophyll a and b from algae using dimethyl sulfoxide. Limnol. Oceanogr. 21, 926-928. Speziale B. J., Schreiner S. P., Giammatteo P. A. and Schindler J. E. (1984) Comparison of N, N-dimethylformamide, dimethyl sulfoxide, and acetone for extraction of phytoplankton chlorophyll. Can. J. Fish. Aquat. Sci. 41, 1519 1522. Webb D. J., Burnison B. K., Trimbee A. M. and Prepas E.E. (1992) Comparison of chlorophyll a extractions with ethanol and dimethyl sulfoxide/acetone, and a concern about spectrophotometric phaeopigment correction. Can. J. Fish. Aquat. Sci. 49, 2331-2336.