Feb 18, 1986 - 9 !991 SprJnger-Veflag New Yerk Inc. Evaluation of a ... pollution assessment and control are discussed. Tests with ... Danish environmental authorities for a toxicity test of inhibi- ... Stock solutions of test materials were pH adjusted to that of the ..... American Society for Testing and Materials, Philadelphia,.
Ar'ohive,m of
E novritr~, omni rr n~ ec ni o~n. a i c
Arch. Environ. Contam. Toxicol. 20, 375-379 (1991)
9 !991 SprJnger-Veflag New Yerk Inc.
Evaluation of a Phytoplankton Toxicity Test for Water Pollution Assessment and Control Kresten Ole Kusk and Niels Nyholm 1 Water Quality Institute, l 1 Agern Alle, DK-2970 HCrsholm, Denmark Abstract. A suggested standard test protocol for the short term 14C assimilation algal toxicity test method (photosynthesis inhibition test) has been evaluated with natural phytoplankton and cultures of the marine diatom S k e l e t o n e m a c o s t a t u m . A number of test technical factors as well as the variability in the sensitivity of natural phytoplankton have been investigated, using potassium dichromate as a reference toxicant in all tests. Some supplementary experiments were carried out with pentachlorophenol and with an industrial effluent. The sensitivity of the test increased with increasing incubation time, but for practical reasons 6 hours of incubation time is recommended. The method showed good reproducibility. The sensitivity of natural phytoplankton, however, varied considerably, both between sampling locations and with time. For testing of effluents it was found necessary to measure the COz alkalinity and correct for changes in the specific 14C activity caused by the deviating alkalinity of the effluent. Advantages and disadvantages of using natural phytoplankton or cultured algal species in toxicity tests for water pollution assessment and control are discussed. Tests with natural phytoplankton are not suitable for use in effluent control schemes or for similar regulatory purposes because their sensitivity is too variable. However, such tests are considered realistic indicators of the actual acute toxic effects on the phytoplanktonic community of a receiving water body. Laboratory tests with cultures of S k e l e t o n e m a costature are reproducible and can be performed all year round and are therefore well suited for regulatory uses.
In recent years several requests have been made by the Danish environmental authorities for a toxicity test of inhibition of photosynthesis in microalgae as part of eftluent control schemes or as part of testing schemes prescribed for drilling muds or chemicals to be used in off shore operations. A preliminary standard method for this test has been proposed (Nyholm et al. 1981). The photosynthesis inhibi-
1 Present Address: Region of StorstrCm, Environmental Office, 37 Parkvej, DK-4800 NykCbing E, Denmark.
tion test is based on the techniques used in measurements of primary production and measures 14C assimilation at different concentrations of test compounds. The test has been used to assess toxicity of both pure chemicals and complex samples. It is conducted in closed experimental bottles placed in an incubator constructed for primary productivity measurements (Steeman NMsen and Hansen 1961; Steeman Nielsen and Willemo6s 1971; Gargas et al. 1976). The use of closed bottles and the relatively short test period make the method well suitable for testing complex mixtures containing volatiles and easily biodegradable compounds. When natural phytoplankton is used as test organisms, maintenance of stock cultures is not necessary, and the test is therefore cost effective for occasional use and can easily be carried out by laborator/es that perform primary production measurements, but which may not necessarily specialize in bioassays. In addition, tests with natural phytoplankton of a particular receiving water body are considered to possess a high degree of ecological realism. Although test methodologies have been described by others (Giddings 1981; Kusk 1981; Lumsden and Florence 1983; Giddings et al. 1983; Eloranta and Haittunen-Keyril~iinen 1984; Madsen 1984; Kuivasniemi et al. 1985; Florence and Stauber 1986; Lewis and Harem 1986; Davis e t a / . 1988), no one standard method has yet been accepted. This paper describes a proposed standard method as well as experimental results obtained with the method and results from investigations of the influence of various technical test factors. It is based upon a technical report (Kusk and Nyholm 1988) prepared for the Danish Agency of Environmental Protection. The report includes a test protocol which is proposed as a Danish Standard. The utility of the method as used with either natural phytoplankton or with cultured single species of microalgae is discussed, and it is evaluated for which purpose either the natural phytoplankton test or the test with cultured algae may be preferable for use in water pollution assessment and control. Materials and M e t h o d s
Tests were conducted with laboratory cultures of the marine diatom Skeletonema costatum and with natural marine phytoplankton populations collected from different locations and at different times of
376
K. Ole Kusk and N. Nyholm Test org(~nlsm : Natural pnytopIankton from S KattegQt Test type : Photesynthesis inhibition test
the year. Potassium dichromate was used as a reference toxicant in all tests. Supplementary tests were conducted with pentachlorophenol and with a sample of industrial effluent. The influences of the incubation time, nutrient supplements, and changes in CO2 alkalinity caused by the addition of a complex sample were evaluated.
" 0 96
CorreLGtion
39
~9
Laboratory Cultures The marine diatom Skeletonema costatum (Grey.), Cleve. (clone from the Norwegian Institute for Water Research) was used as the test organism. The growth medium was filtered natural seawater adjusted to 20%~ salinity and supplemented with the following nutrients: NaNO 3 240 mg/L, K/HPO 4 32 nag/L, FeCI 3 9 6H20 80 Fg/L, NaEDTA 9 2H20 100 ~zg/L, H3BO~ 185 Ixg/L, MnCI2 ' 4H20 415 ~g/L, ZnCI2 3 Ixg/L, CaCI2 96H20 1.5 Ixg/L, CuCI2 92H20 0.01 Ixg/L, NazMoO 4 92H20 7 tzg/L, Na2SiO 3 99H20 10 mg/L. The medium was briefly heated to 73~ to avoid bacterial contamination problems. The diatom was cultured at 15 _+ I~ in Kluyver flasks exposed to continuous white fluorescent light (Philips 18 W/33) of an intensity of 7 x 10~5 quanta • c m - 2 X s e c - 1 . Toxicity tests were conducted with exponentially growing cells diluted to a density of approximately 104 cells • m1-1 in the test flasks.
Natural Phytoplankton Field samples of natural phytoplankton were collected at a depth of 0.5-2 m less than 24 h before the start of each test. Immediately after collection the samples were filtered through a 100 t~m filter to remove zooplankton, and were then as soon as possible transported to the laboratory, where they were held at or near the temperature of the sampling locality. The samples were gently aerated until use. Prior to use, the sampling container was shaken vigorously to resuspend the sedimented phytoplankton.
~
J
5 0 - -
3o
.................ii............. ..t~.............
20
..---'~.....
~ 1.9
I8.2 2
5 Potcssium
10 D i c h r o m o t e - 4 hours
2O
5O
IO0
(rag/t)
Fig. 1. Linearized dose-response curve (log probability transformation) for the inhibition of the photosynthesis of natural phytoplankton exposed for potassium dichromate for 4 h. Data points represent mean of three replicates
When the long pre-incubation period of 18 h was used, the algae were kept in darkness for the first 16 h in order to restrict growth. At the end of the test, the contents of the flasks were filtered on membrane filters (0.2 txm Sartorius Cellulose Nitrate Filters). The wet filters with the algae retained on the filter were stacked in drying cassettes, which were placed for 5 min in a dessicator with fuming hydrochloric acid to remove any residual 14C left on the filters, and then dried. The dried filters were dissolved in scintillation liquid, and the activity of the fixed ~4C on each filter was counted in a liquid scintillation counter. Inhibition of ~4C-assimilation was expressed as a linear function of the test concentration in a log-normal probability plot (probit transformation) from which the EC 10 and EC 50 values were calculated by weighted linear regression (Christensen and Nyholm 1984).
Toxicity Tests Stock solutions of test materials were pH adjusted to that of the dilution water (pH 89 - 0.1). A dilution series of the test material were prepared, and phytoplankton or cultured algal suspensions were added to a fixed cell density. Subsamples of the dilution series were pre-incubated in 25 ml-flasks which were placed on a rotating wheel submerged in a thermostatically-controlled water bath illuminated by fluorescent light tubes. The standard test set-up included 6 - 8 dilutions each tested in triplicate and 3-5 controls. The test temperature was identical to the field/culture temperature. The light intensity was above saturation for photosynthesis (33 x 1015 quanta x cm -z x sec-~). For description of the incubator see Gargas et al. (1976) or Steeman Nielsen and Willemoes (1971). The dilution water used in the toxicity tests with cultured algae was identical to the growth medium except that FeCI3 9 6H/O and NazEDTA were omitted to avoid chelation of toxicants, while in tests with natural phytoplankton the dilution water was prepared by filtering with a 1 ixm membrane filter a portion of the field sample. In natural phytoplankton tests with nutrient supplements, N, P and Si were added to the following concentrations: N H 3 - N 800 Fg/L, o-P 73 Ixg/L and Si 500 p,g/L as NH4C1, K2HPO4 and NazSiO 3 9 9H20 , respectively. The test performance was investigated with pre-incubation periods of 2, 4, or 18 h. At the end of each pre-incubation period, the flasks were opened and 200 ~1 NaH~4CO3 solution (4 ixCi) was added, After addition of I4C, incubation was continued for 2 h.
Results and Discussion A linearized d o s e - r e s p o n s e c u r v e (log p r o b a b i l i t y transform a t i o n ) f r o m a 4-h e x p e r i m e n t with natural p h y t o p l a n k t o n a n d p o t a s s i u m d i c h r o m a t e is s h o w n in F i g u r e 1. Table 1 s h o w s the results of e x p e r i m e n t s w i t h S. costatum and pot a s s i u m d i c h r o m a t e using test d u r a t i o n s of 4, 6 a n d 20 h. T h e l o n g e r the e x p o s u r e d u r a t i o n , the l o w e r w e r e t h e E C values a n d the m o r e sensitive was the test s y s t e m to p o t a s s i u m d i c h r o m a t e 9 T h e sensitivity o f n a t u r a l p h y t o p l a n k t o n (Table 2) t o w a r d s p o t a s s i u m d i c h r o m a t e also i n c r e a s e d with longer i n c u b a t i o n time. P r a c t i c a l c o n s i d e r a t i o n s , h o w e v e r , led to a r e c o m m e n d a t i o n of a total t e s t p e r i o d of 6 h, w h i c h m a d e it p o s s i b l e to c o n d u c t the test in the c o u r s e of o n e w o r k i n g day a n d to o b t a i n sufficient sensitivity 9 T h e r e p r o d u c i b i l i t y of the test is illustrated b y the results g i v e n in Table 1 with c u l t u r e d S. costatum. Only small diff e r e n c e s are s e e n b e t w e e n the EC values e s t i m a t e d for e a c h i n c u b a t i o n period. T h e coefficient of v a r i a t i o n in s a m p l e s i n c u b a t e d for 6 h was 16% for E C 50 e s t i m a t e s a n d 14% for E C 10 estimates 9 T h u s , t h e test s h o w s good reproducibility. T h e r e was a large v a r i a t i o n in sensitivity a m o n g samples of n a t u r a l p h y t o p l a n k t o n collected at different localities a n d
Phytoplankton Toxicity Test for Water Pollution
377
Table 1. Sensitivity of the laboratory-grown diatom Skeletonema costaturn to potassium dichromate at 15~ and different durations of exposure. Figures in brackets are 95% confidence limits
Date
Test duration (h)
2 Feb. t986 4 25 Mar. 1986 4 18 Feb. 1986 6 7 May 1986 6 10 June 1986 6 7 Jan. 1987 6 9 Dec. 1987 6 15 Dec. 1987 6 28 Jan. 1988 6 18 Feb. 1986 20
EC 10 (rag/L)
EC 50 (rag/L)
2.9 (2.6-3.2) 2.7 (2.2-3.1) 1.6 (1,0-2. l) 1.4 (0.9-1,8) 1.4 (1.0-1.7) 1.1 (0.8-1.3) 1.3 (1.0-1.5) 1.7 (1.3-1.9) 1.4 (1.2-1.7) 0.13 (.08-.17)
5.8 (5.6-6,1) 7.7 (7.2-8,4) 4.4 (3.7-5.3) 5.0 (4.1-6,1) 3.6 (3.2-3,9) 3.5 (3.2-3.9) 3.2 (2.9-3.6) 3.6 (3.3 -4.0) 3.8 (3.5-4.2) 0.50 (.42-.58)
Table 2. Sensitivity of natural phytoplankton to potassium dichro-
mate at different durations of exposure. The sample was taken from KCge Bay on 19 September 1985 and was tested at 15~ The salinity was 9%~. Figures in brackets are 95% confidence limits Test duration (h)
4
EC 10 (mg/L) (1.0-1.8) 2.3 (1.3-3.5) 1.2
6 6 20 20
Date/ Test temperature 9 Sept. 85 15 ~ 8 Apr. 86 9~ 23 Apr. 86 8~ 24 Apr. 86 8~ 6 June 86 12 ~ 12 June 86 12 ~ 15 Aug. 86 15 ~ 18 Aug. 86 15 ~ !7 Sep. 86 15 ~ 26 Oct. 86 10 ~
Sampling location
Salinity %o 9
KCge Bay 9 KCge Bay 9 KCge Bay 17 S. Kattegat 10 Kctge Bay 24 Little Belt 12 Great Belt 9 Kcige Bay 10 KCge Bay 9 KCge Bay
EC 10 (rag/L)
EC 50 (rag/L)
1.4 (1.0-1.8) 3.2 (2.4-3.9) 0.81 (.49-1.2) t.9 (l.2-2.6) 1.9 (1.1-2.2) 4.3 (1.9-6.9) 2.2 (1.5-3.0) 25 (15-31) 16 (13- t9) 0.9 (0.2-1.9)
22 (20-25) 39 (35-44) 10 (8.7-12) 18 (t6-21) >60 (87a) 41 (34-53) 25 (22-28) >60 (93a) 54 (50-60) 13 (9.1 - 17)
a The calculated value was higher than the highest examined concentration
Table 4. Variations in the sensitivity of natural phytoplankton to-
1.4
4
Table 3. Variation in the sensitivity of natural phytoplankton towards potassium dichromate Phytoplankton samples were collected at different times of the year at different locations and were incubated for four hours. Figures in brackets are 95% confidence limits
(.9-1.5) 0.93 (.21-2.0) 0.13 (.08-.20) 0.10 (.06-. 16)
EC 50 (rag/L) 22 (20-25) 25 (21-29) 15 (13-17) 12 (8,0-18) 4.2 (3.6-5.0) 4.5 (3.8-5.3)
times of the year. For potassium dichromate (Table 3), the range between the lowest and the highest EC 50 values was greater than a factor of 6 and for the EC 10 values about a factor of 30. With pentachlorophenol (Table 4) the range of the EC 50 and the EC 10 values was a factor of 6; however, fewer results were obtained and compared than for potassium dichromate. The observed variation can result from changes in sensitivity within a short time as indicated by the results obtained with samples collected in KCge Bay in April 1986 (Table 3). Variation may also be found between different localities as demonstrated by the results obtained with samples from Great Belt and KCge Bay collected in the middle of August
wards pentachlorophenol. Phytoplankton samples were collected at different times of the year at different locations and were incubated for four hours. Figures in brackets are 95% confidence limits Date/ Test temperature 12 Nov. 85 10 ~ 23 Apr. 86 8~ 24 Apr. 86 8~ 6 June 86 12 ~ 12 June 86 12 ~
Sampling location
Salinity %o
EC 10 (p,g/L)
EC 50 (Ixg/L)
20
13 (8-19) 3.8 (2.6-5.1) 22 (b_ 47) 13 (1,1 - 16) 9 (2-16)
195 (156-261) 34 (29-42) >100
Kattegat 9 KCge Bay 17 S. Kattegat i0 KCge Bay 24 Little Belt
>t00 (131a) 56 (37-93)
a The calculated value was higher than the highest examined concentration b The value could not be estimated
1986 and samples from KCge Bay and Southern Kattegat collected in April 1986 (Table 3). The main reason for the observed variability is believed to be a variation in the species c o m p o s i t i o n of the phytoplankton. Lewis and Harem (1986), working with surfactants, found that the photosynthetic response of lake phytoplankton to the same surfactant varied as much as by a factor of 80 and attributed this partly to changes in water temperature and partly to sea-
378
K. Ole Kusk and N. Nyhoim
Table 5. Influence of nutrient addition on EC values found in tests with potassium dichromate and natural phytoplankton from KCge Bay (salinity: 9-10%o). Figures in brackets are 95% confidence limits Test duration (h)
Date/ Test temperature
Nutrient supply
19 Sept. 85 15 ~ 19 Sept. 85 15 ~ 8 Apr. 86 9~ 8 Apr. 86 9~ 18 Aug. 86 15 ~ 18 Aug. 86 15 ~ 17 Sept. 86 15 ~ 17 Sept. 86 15 ~ 19 Sept. 85 15 ~ 19 Sept. 85 15 ~ 19 Sept. 85 15 ~ 19 Sept. 85 15 ~ 8 Apr. 86 9~ 8 Apr. 86 9~ 18 Aug. 86 15 ~ 18 Aug. 86 15 ~
-
4
+
4
-
4
+
4
-
4
+
4
-
4
+
4
-
6
+
6
-
20
+
20
-
20
+
20
-
20
+
20
sonal differences in p h y t o p l a n k t o n composition. Kusk (1981) found that fresh water phytoplankton collected in the spring and dominated by centric diatoms was more sensitive to aromatic hydrocarbons than phytoplankton collected in the autumn and dominated by green algae and attributed this to the different species composition. One important observation in this study was that the slope of the concentration-response curve was generally smaller for natural phytoplankton than for S. c o s t a t u m , but this is not surprising. In an algal culture, the cells originate from the same clone and have identical genetic properties, while natural phytoplankton populations consist of many species with different sensitivities towards toxicants. The addition of combined nutrients (N, P, Si) to natural phytoplankton samples (Table 5) did not to have any significant effect on the resulting EC values, but in some instances the addition resulted in a decrease in ~4C-assimilation. When testing complex samples that may contain nutrients, it is recommended to add nutrients to mask a possible interference from such nutrients. This obviously applies only to natural phytoplankton samples, since tests with cultured algae are always conducted with nutrient-supplemented medium. A high alkalinity sample (industrial effluent) was tested with natural phytoplankton (15%o salinity). The alkalinities
EC 10 (mg/L)
EC 50 (mg/L)
1.4 (1.0-1.8) 2.3 (1.3-3.5) 3.2 (2.4-3.9) 3.6 (2.5-4.7) 25 (15-31) 13 (7.8-19) 16 (13-19) 10 (0-19) 1.2 (.9 - 1.5) 0.93 (.21-2.0) 0.13 (.08-.20) 0.10 (.06-. 16) 0.54 (.32-.76) 1.3 (1.0-1.7) 0.41 (.20-.65) 0.62 (.29-1.0)
22 (20-25) 25 (21-29) 39 (35-44) 46 (39-55) >60
Control-DPM/ 100 p.1 14C-solution 6000 6000 8500 8500 6800
>60 5800 54 (50-61) 63 (41-400) 15 (13 - 17) 12 (8.0-18) 4.2 (3.6-5.0) 4.5 (3.8-5.3) 5.0 (3.9-6.8) 4.7 (4.2-5.3) 7.4 (5.4-11) 7.8 (5.6-12)
9600 7800 7000 7000 6000 6000 7000 7000 7300 6900
in the field sample and the effluent were 1. l mmol/L and 5.0 mmol/L, respectively. Reduced 14C-assimilations were found in the dilution series of the effluent sample (Figure 2), which circumstance results from a decreased specific 14Cactivity at higher concentrations of the sample (higher proportion of unlabelled carbon). When the 14C-activities measured on the filters were corrected for changes in the molar concentrations of CO2-H2CO3 as calculated from CO/alkalinity, the variations were less than 10% of the control 14Cassimilation except for the highest concentration where a slight stimulation was observed (Figure 2). Changes in COz alkalinity due to the addition of test material and the resulting changes in specific 14C activity between test solutions pose a general problem with the ~4C-technique for testing complex samples. As demonstrated, this can be corrected. It is recommended to adjust the pH of the stock solution of the test material to the pH of the dilution water and then measure the CO/alkalinity after a sufficient period of time, which would allow equilibrium for the COz-system. Proportional correction of the ~4C-assimilation data can then be made. The measurement of alkalinity should preferably be done by CO2 measurements, e.g., by infra-red spectrometry, because this procedure is not influenced by the presence of
Phytoplankton Toxicity Test for Water Pollution
379 Acknowledgments. This work was supported by the Danish Agency of Environmental Protection and the Danish Council for Technical and Scientific Research.
50-
40-
30-
J
J
References
20-
o~
lo 0
-10 I
-20 10
ML SAMPLE / L
0
CoFrecteddata
Fig. 2. Dose-response curves for the photosynthesis inhibition test with natural phytoplankton and a high alkalinity sample uncorrected and corrected for deviations in specific ~4C-activity.Data points represent mean of three replicates
weak acid or bases, which may affect the more traditional measurements of alkalinity by titration. The ~4C-assimilation test with natural phytoplankton is believed to assess the acute effect on algae in a water body with great ecological relevance, and is thus useful for environmental impact assessments. Repeated testing may be necessary, however, to account for the variation in sensitivity. The test may also be used as a simple and inexpensive first screen with the object of detecting toxicity. Conversely, the variable sensitivity makes the test unsuitable for describing changes in the toxicity of an effluent or for hazard evaluation of chemicals (unless tested with a number of different phytoplankton samples). For such purposes, the use of cultured algae such as S. costatum is preferred because the test results are reproducible.
Conclusions The ~4C-assimilation algal toxicity test with either natural phytoplankton or cultured unialgal species has been found sensitive to toxicants and well-suited for testing of both chemicals and complex test materials such as effluent samples. Practical considerations in combination with findings in the present study led to a suggestion of a test period of 6 hours. Tests with natural phytoplankton are considered well suited for assessment of the environmental impact on a receiving water body and the test also constitutes an inexpensive tool for occasional toxicity screening, maintenance of stock cultures not being necessary. Due to variations in the sensitivity of natural phytoplankton, however, the test may not be suitable for detecting changes in effluent toxicity or for hazard evaluation of chemicals. If the objective is to monitor variations in effluent toxicity, to control set limits of toxicity, or to initially evaluate the toxicity of a chemical, it is preferable to use a sensitive cultured microalga as the test organism. Such tests have been demonstrated to show good reproducibility.
Christensen ER, Nyholm N (1984) Ecotoxicological assays with algae: Weibull dose-response curves. Environ Sci Technol 18:713-718 Davis TM, Vance BD, Rodgers JH Jr (1988) Productivity response of periphyton and phytoplankton to bleach-kraft miil effluent. Aquat Toxicol 12:83-106 Eloranta VA, Halttunen-Keyrilfiinen L (1984) A comparison of the Selenastrum bottle test and the natural phytoplankton assay in algal toxicity tests. Arch Hydrobiol Suppl 67:447-459 Florence TM, Stauber JL (1986) Toxicity of copper complexes to the marine diatom Nitzschia ctosterium. Aquat Toxicol 8:11-26 Gargas E, Nielsen CS, Lenholdt J (1976) An incubator method for estimating the actual daily plankton algae primary production. Water Res 10:853-860 Giddings JM (1981) Four-hour bioassays for assessing the toxicity of coN-derived materials. In: Levins PL, Harris JC, Dvewitz KD (eds) Proceedings: Second Symposium on Process Measurements for Environmental Assessment, Georgia, February 24-27, 1980. EPA 600/9-81-018. US Environmental Protection Agency, Washington, DC Giddings JM, Stewart AJ, O'Neitl RV, Gardner RH (1983) An efficient algal bioassay on short-term photosynthetic response. In: Bishop WE, Cardwell RD, Heidolph BB (eds) Aquatic Toxicology and Hazard Assessment: Sixth Symposium. ASTM STP 802. American Society for Testing and Materials, Philadelphia, pp 445-459 Kuivasniemi K, Eloranta V, Knuwtinen J (1985) Acute toxicity of some chlorinated phenolic compounds to Selenastrum capricornutum and phytoplankton. Arch Environ Contam Toxicol 14:43-49 Kusk KO (1981) Comparison of the effects of aromatic hydrocarbons on a laboratory alga and natura! phytoplankton. Bot Mar 24:611-613 Kusk KO, Nyholm N (1988) Algal toxicity test. Development and documentation of a method based on measurement of 14C-assimilation, Environmental Research Project No. 91. Agency of Environmental Protection, Denmark (in Danish) pp t-112 Lewis MA, Hamm BG (1986) Environmental modification of. the photosynthetic response of lake plankton to surfactants and significance to a laboratory-field comparison. Water Res 20:15751582 Lumsden BR, Florence TM (1983) A new algal assay procedure for the determination of the toxicity of copper species in seawater. Environ Tech Lett 4:271-276 Madsen L (1984) Problems connected with the use of algal tests in ecotoxicology. Ecol Bull 36: 165-170 Nyholm N, Kusk KO, Damgaard BM (1981) Water quality--Toxicity test based on measurements of the carbon assimilation of phytoplankton. Water Quality Institute, 1t Agern A!le, DK 2970-Hc~rsholm (In Danish) pp 1-29 Steeman Nielsen E, Hansen VK (1961) Influence of surface illumination on plankton photosynthesis in Danish waters (56~ throughout the year. Phys Plant 14:595-613 Steeman Nielsen E, Willemoes M (1971) How to measure the illumination rate when investigating the rate of photosynthesis of unicellular algae under various light conditions. Int Revue ges Hydrobiol 56:541-556 Manuscript received December 10, 1989 and in revised form September 17, 1990.
