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An assessment of the cage-culture turbidostat as an alternative algal bioassay
PII: S0043-1354(97)00302-3
Wat. Res. Vol. 32, No. 4, pp. 1162±1168, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/98 $19.00 + 0.00
AN ASSESSMENT OF THE CAGE-CULTURE TURBIDOSTAT AS AN ALTERNATIVE ALGAL BIOASSAY N. CLARKSON*{, J. W. LEFTLEY, D. T. MELDRUM and J. W. WATSON Centre for Coastal and Marine Sciences, Dunstanage Marine Laboratory, P.O. Box 3, Oban, Argyll PA34 4AD, U.K. (First received February 1997; accepted in revised form August 1997) AbstractÐA computer controlled cage-culture turbidostat (CCT) is described as an alternative to batch algal bioassays. The CCT is designed to monitor algal growth changes in response to the addition of toxicants including chronic toxic eects in comparison to batch culture toxicity tests which are most successful in assessing acute responses. The system is easily and quickly set up, with minimum operator time required once assembled. The alga Phaeodactylum tricornutum (CCAP 1052/6) was used as the test species. Stable growth of the alga could be maintained in the CCT for 14 days, sucient time to run bioassay experiments using both the herbicide Diuron2 (DCMU) and a complex industrial euent. Additions of various concentrations of both toxins to the chamber caused changes in the growth rate and the chl-a concentration of the culture. The Median Inhibitory Concentration of euent calculated to cause a 50% reduction in the growth rate (IC50) in the CCT was estimated to be 0.33 mM DCMU, and 15.33% of the euent. Comparable results were obtained when using the standard algal growth inhibition toxicity (AGIT) test, the 72 hIC50 values were 0.09 mM DCMU and 16.4% euent. This study indicates a potential use for the CCT for water quality monitoring, which may provide more environmentally relevant information than the commonly used AGIT test. # 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION
The most widely used algal bioassay is probably the Algal Growth Inhibition Toxicity (AGIT) test (Environment Agency, 1994), a procedure which measures the inhibition of growth in batch cultures due to toxicants. This method is widely used because of its simplicity, reasonable cost and acceptable reproducibility (Jensen, 1984). However, although generally satisfactory for lethal and acute toxicity, batch culture tests are of little value in assessing long-term chronic impacts (Rhee, 1989). In addition, when high sample throughput is required, such assays are labour intensive and time consuming and alternative protocols are constantly being sought (St-Laurent et al., 1992; Van der Heever and Grobbelaar, 1996). Continuous culture methods, in which fresh medium is added to the culture at a rate sucient to maintain it, are used as an alternative to batch culture methods. In the chemostat, fresh medium constantly ¯ows at a set rate through a vessel of ®xed volume which maintains the culture at a growth rate proportional to the medium ¯ow rate *Present address: The Northumbrian Water Ecology Centre, The University of Sunderland, Sunderland, Tyne and Wear SR1 3SD, U.K. {Author to whom all correspondence should be addressed. Tel.: 0191 5152526, Fax: 0191 5152531, Email: [email protected]
(Tempest, 1970). Turbidostats are a second continuous culture method, but with direct control, in which a photoelectric monitor detects deviations from a desired culture turbidity and induces an increase or decrease in dilution rate of the ¯ow of growth medium (Munson, 1970). The turbidostat has been further developed as the cage-culture turbidostat (CCT), ®rst described by Skipnes et al. (1980). It comprizes: (a) a vessel with porous membranes which contains the algae but allows free exchange of medium in and out of the vessel, and (b) a photosensor system which, via a feedback loop, maintains the culture density (number of cells per unit volume) at a pre-set level by diluting the culture with fresh medium. The frequency at which the dilution pump operates is used to calculate the growth rate. When using the CCT for toxicity testing, medium is continuously perfused through the growth vessel so that the algae are eectively exposed to a constant concentration of all the components of the medium, including pollutants. Constant exposure of the algae to low concentration of pollutants in this way may be a more realistic environmental situation than in the AGIT test in which the algae are eectively exposed to a single large dose of toxicant (Rhee, 1989). There have been relatively few reports of the use of the CCT for water quality monitoring, especially for pollutants (éstgaard et al., 1984; Wangersky
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and Maas, 1991). We therefore constructed a ``state-of-the-art'' apparatus and assessed its potential as an automated algal bioassay for pollutants, using both the herbicide Diuron2 as a model and samples of an industrial euent, and compared the results with those obtained form the standard AGIT test. MATERIALS AND METHODS
The cage-culture turbidostat system A brief description of the system is given here in full detail, including drawings of the culture vessel, inlet/outlet ®lter units, electronic circuit diagrams and computer program can be obtained from the senior author. A schematic diagram of the system is shown in Fig. 1. This CCT system based on the design of Skipnes et al. (1980). The borosilicate glass culture vessel (100 ml capacity) is in two parts which are sealed with a silicone rubber gasket and held together by a metal clamp; this allows easy dismantling for cleaning. Screw-threaded ports are ®tted to the vessel for the connection of the two inlet/outlet ®lter units and the sampling and over¯ow lines. The low dead volume, polycarbonate, in-line ®lter units were specially constructed; each unit held a 1 mm pore size, 47 mm diameter polycarbonate membrane (Cyclopore2, Whatman Labsales Ltd., Maidstone, U.K.). Medium was continuously pumped through the culture vessel at a rate of 18 ml hÿ1 with a multi-channel pump (Model 505U/RL, Watson-Marlow, Falmouth, U.K.) which simultaneously pumped fresh medium into the vessel via one channel and pumped out waste medium, at the same rate, via another channel. This arrangement kept back-pressure in the vessel to a minimum. Silicone rubber tubing (1.5 mm bore, 1 mm wall) connected the pump, ®lters and reservoirs (Altec Ltd., Alton, U.K.). To prevent build up of algae on the inlet/outlet ®lters the ¯ow of medium into and out of the chamber was alternated, changing direction every 10 min. This was achieved by opening and closing four solenoid pinch valves (normally open when not energized) which were activated by the ¯ow control unit (FCU). To ensure complete mixing in the CCT vessel the culture was continuously stirred magnetically by an orbiting magnetic follower (120 rpm) specially designed for ecient mixing in round-bottomed vessels (Whatman Labsales, Maidstone, U.K.). Illumination was provided by a standard 11 W bayonet-®tting white ¯uorescent lamp (100±
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120 mE mÿ2 sÿ1). The optical density of the culture was monitored by an LED/photodiode emitter/sensor circuit. To minimize the eect of extraneous visible light the emitter used a high intensity infra-red LED (peak emission 940 nm), modulated by a 1-kHz square wave, and the detector was an integrated infra-red light-to-voltage converter chip, ®tted with appropriate optical and electronic ®lters. The sensor voltage passed via an A/D converter to the computer capable of running DTVee2 software (Data Translation Ltd., Malboro, MA, U.S.A.). When the optical density (OD) of the culture exceeded the pre-set threshold the computer activated the dilution pump (¯ow rate 0.45 ml minÿ1; Watson±Marlow, model 101U/R MK 2) which simultaneously activated the solenoid pinch valve (normally closed when not energized) to allow excess culture to over¯ow into a sterile receiver. The pump and solenoid were deactivated when the OD returned to the pre-set level. The DTVee2 software recorded the OD of the culture every 15 min in tandem with the time the dilution pump operates during each hour. Growth rates were calculated according to Skipnes et al. (1980). The speci®c growth rate (m) was calculated as the proportion of the volume of the growth chamber (V) harvested, where H = volume harvested per hour (i.e. the time the dilution pump was activated multiplied by the ¯ow rate of medium). The number of cell divisions (d) per hour can then be calculated. The equations are: m H=V d m=ln2
Algal culture The diatom Phaeodactylum tricornutum Bohlin (Strain 1052/6, Culture Collection of Algae and Protozoa, Oban, U.K.) was used as it is fast growing, easily cultured and well suited to electronic particle counting. It is one of the recommended species for marine algal growth tests (Environment Agency, 1994). Stock cultures were maintained in f/2 enriched seawater medium (Guillard, 1975), at 208C under continuous illumination (warm white ¯uorescent light, ca. 100 mE mÿ2 sÿ1, at the surface of the culture). f/2 medium was prepared with seawater ®ltered through Whatman glass ®bre ®lters (GF/F 0.7 mm with a GF/B 1.0 mm pre-®lter; Whatman International Ltd., Maidstone, U.K.). Before autoclaving the salinity was standardized to 30 22%0 by dilution with distilled water and the pH adjusted to 8.020.1 with 1 M HCl. All medium was autoclaved (1158C, 10 min, or 1 h for large volumes). Stock P. tricornutum cultures were periodically checked by microscope for bacterial contamination. All inocula were from 3-day-old, exponentially growing cultures. DCMU solutions The herbicide Diuron2, DCMU (3-(3,4 dichlorophenyl)1,1-dimethyl urea) (Sigma, Poole, UK) is a potent inhibitor of photosystem II, inhibiting ®xation of CO2 (Renger, 1986). A stock solution of DCMU was prepared in acetone and added aseptically to sterile modi®ed f/2 medium to give the range of concentrations used in the CCT and algal growth inhibition test. The controls were f/ 2 enriched reference seawater (RSW) with and without acetone. The volumes of acetone added were found to have no eect on the cells and all results shown are f/2 enriched seawater with acetone. Euent samples
Fig. 1. Schematic diagram of the CCT system. FCU = ¯ow control unit; M = magnetic stirrer; F = magnetic follower; L = ¯uorescent lamp; E = photodiode emitter; D = photodiode sensor; F = ®lters; V1-5 = solenoid pinch valves.
A sample of industrial euent, discharged into the Firth of Clyde, was collected as a 25 l snap sample by personnel of the Scottish Environmental Protection Agency, Western Region (SEPAWR) as part of a routine monitoring pro-
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gramme. Euent samples were prepared for toxicity tests within 48 h of collection according to recommended guidelines (Environment Agency, 1994). Suspended solids were removed by ®ltration through glass ®bre membranes as described above. The pH of the euent (initially 1.30) was adjusted to 8.0, the pH of the reference seawater, and its salinity (initially 10-), measured with a hydrometer, was adjusted to 30- by addition of solid NaCl. The euent was sterilized by pressure ®ltration through 90 mm, 0.2 mm CN membranes (Whatman Labsales Ltd.) and stored in the dark at 88C. Euent concentrations were prepared by dilution with seawater and sterile f/2 nutrient enrichments were added aseptically, the control medium was f/2 enriched RSW. For the AGIT tests euents were used within 48 h of receipt. Because of the time necessary for the CCT to stabilize, euents could not be assayed within 48 h, but were used as soon as possible after preparation. Operation of the CCT The culture vessel, ®lters and associated tubing were autoclaved and assembled aseptically. The apparatus was then transferred to a constant temperature incubator (208C) and connected to the pumps and electronic system (see Fig. 1). The vessel was ®lled aseptically with the control medium, the voltage baseline set and inoculated with algae to give an initial density of ca. 5 104 cells mlÿ1. The algae were then allowed to grow until they exceeded the pre-set threshold, about 2 V (ca. 2 105 cells mlÿ1) to trigger the dilution pump. After allowing the system to stabilize at a constant growth rate for 48 h toxicant (DCMU or euent) was added aseptically to the reference medium in the reservoir to give a known concentration. With a ¯ow rate of 18 ml hÿ1 the total volume of the vessel was replaced in approximately 5 h with the new concentration. This would mean that the initial response of the cells was to a sub-optimal concentration. However, any possible eect of this was reduced by using a total of 24 hourly results as the response time. Each of the series of concentrations (DCMU and euent) was carried out once. A 2 ml sample was taken aseptically from the vessel each day for the determination of cell number and chlorophyll-a (chl-a) per unit volume. Cell numbers were determined with a Coulter Counter Model ZM (Coulter Electronics Ltd, Luton) ®tted with a 50 mm ori®ce, counts were a mean of ®ve replicates. Chl-a was extracted in 90% acetone and measured ¯uorometrically (Turner Instruments Model III Fluorometer, Turner Designs Inc. USA) according to the method of Tett (1987), values were a mean of three measurements. The algal growth inhibition toxicity test The algal growth inhibition (AGIT) test was carried out as a comparative bioassay for both DMCU and the euent. The protocol used was that for marine algae as described by Environment Agency (1994) using cultures of P. tricornutum. DMCU was prepared as described above and added to f/2 medium. For the euent, a dilution series was prepared by addition of a known volume of euent to sterile 1 l ¯asks. Sterile nutrients were added to f/2 concentrations and the solution made up to volume with sterile RSW. The concentration range chosen was 0, 0.1, 0.5, 1, 5, 10, 22, 46 and 100% (v/v) euent, the control was f/2 enriched RSW, the salinity of the samples was 30-. The tests were considered valid if the control cell density increased by more than 16 times in 72 h and the control pH did not vary by more than 1 unit during the test (Environment Agency, 1994). The assessment of inhibition of growth in the test is based on a comparison of test and control growth rates and the data used to determine the 72 h inhibitory concentration (IC50) value, the concen-
tration of test substance estimated to cause 50% inhibition of growth after 72 h. Speci®c growth rates were calculated for each individual ¯ask by linear regression of logarithmic biomass data from the exponential growth phase (Nyholm, 1985). The data were then normalized by dividing by the mean value of the control test. Dose-response curves were obtained by non-linear least squares ®t to the logistic equation: y1ÿ
1 1
eÿ abz
where z = log (concentration) and Y = the normalized response (Nyholm, 1990). Calculations were performed using the SAS procedure NLIN (SAS, 1990) assuming a constant variance of Y and therefore equal weighting of data points. The 72 h-IC50 value corresponds to a relative speci®c growth rate of 0.5. RESULTS AND DISCUSSION
Algal growth in the CCT The growth of the P. tricornutum culture was monitored over a 14 day period incubated in f/2 RSW to assess the long-term functioning of the CCT and its ability to maintain a constant growth rates. Figure 2 shows the daily growth rates and chl-a concentrations of the cells. Growth rate was maintained between 0.72 and 1.16, with generally small ¯uctuations between days. After the initial 2 days the chl-a values settled down and were relatively constant for the remaining days. A comparison of the dierences in growth rate between f/2 nutrient enriched RSW and concentrations of toxicant (DCMU or euent) can be achieved using relative growth values. A mean of the ®rst two daily growth rates in f/2 RSW gave an estimate of the control algal growth rate for that CCT run. The growth rate at each toxicant concentration tested was then expressed as a percentage value relative to this initial calculated mean RSW growth rate. It was then possible to estimate, by simple graphical interpolation of the % inhibition at each concentration, the concentration of toxicant that would cause a 50% inhibition in growth rate.
Fig. 2. The daily growth rate and the cellular chl-a concentration per cell of a continuous culture of P. tricornutum cells in the CCT in control f/2 medium over a 14 day growth period.
The cage-culture turbidostat algal bioassay
This calculation of the Median Inhibitory Concentration of euent (IC50) allows the results from the CCT to be compared with the AGIT test data. Addition of DCMU The daily growth rate and the chl-a concentration of algae grown in a range of concentrations of DCMU, is shown in Fig. 3(a), and the relative growth rate in Fig. 3(b). DCMU was added to the chamber for separate 24 h periods at concentrations of 0.1 mM and 1.0 mM with a 48 h period of RSW medium between the additions to allow for cell recovery. The growth rate decreased on addition of 0.1 mM DCMU by 35.3% subsequently recovering on removal of the DCMU to pre-exposure levels within 48 h. Addition of 1 mM DCMU caused a decrease in the relative growth rate by 92.9% from which the cells did not recover for the remainder of the experiment. The IC50 value was estimated as described above to be 0.33 mM DCMU. The chl-a concentration also decreased, but did not recover after the ®rst addition, and continued to decrease for the duration of the run.
Fig. 3. (a) The daily growth rate and cellular chl-a concentration per cell of a continuously growing culture of P. tricornutum grown in reference seawater (RSW) and dierent concentrations of DCMU. Times; 1±3 days in RSW; day 3 in 0.1 mM DCMU; 4±5 days in RSW; day 6 in 1 mM DCMU; 7±10 days in RSW. (b) The daily relative growth rates compared to f/2 control values of P. tricornutum grown in 0.1 and 1.0 mM DCMU with an intervening recovery period of 48 h in f/2 RSW.
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Addition of euent Figure 4(a) shows the growth rate and chl-a concentration of algae grown in RSW and various concentrations of euent, and Fig. 4(b) the corresponding relative growth rate of the algae. Additions of the euent caused marked eects on algal growth. A concentration of 1% (v/v) caused an immediate stimulation in growth rate by 27% compared to the control. Additions of 5 and 10% (v/v) euent both caused an initial increase in growth within 24 h followed by a decrease after continued exposure to the euent over 48 h. Addition of 22% euent caused an immediate decrease in growth rate, after 48 h m was only 30% of that measured initially in f/2. For concentrations of 1, 5, and 10% the growth rate returned to initial levels within 48 h in RSW, this was not the case with the addition of 22%, although after 48 h in RSW the growth rate increased slightly indicating possible cell recovery. An inhibition of growth by 50% was estimated to be 15.33% euent, calcu-
Fig. 4. (a) The daily growth rate and the chl-a concentration per cell of a continuous culture of P. tricornutum cells grown in reference seawater (RSW) and dierent dilutions (v/v) of euent. Times: 1±2 days in RSW; 3± 4 days in 1% euent; 5±7 days in RSW; 8±9 days in 5% euent; 10±11 days in RSW; 12±13 days in 10% euent; 14±15 days in RSW; 16±17 days in 22% euent; 18± 19 days in RSW. (b) Daily relative growth rates compared to the mean of the initial two RSW samples of P. tricornutum culture grown in euent.
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lated as described above. Addition of low concentrations of the euent caused an increase in the growth rate which may be related to the presence of stimulatory compounds in the euent such as phosphorous and nitrogen. Statistical analysis of the relatedness of the variables measured over the time period of the experiment was carried out using Spearman Rank Correlation. For the DCMU data, a signi®cant correlation was found between the cell count and the chl-a values (rs=0.82; n = 10; P < 0.01) and the growth rate and the chl-a concentration per cell values (rs=0.80; n = 10; P < 0.01). For the euent a similar result was found between the cell count and the chl-a values (rs=0.70; n = 19; P < 0.01) and between the growth rate and the chl-a concentration per cell (rs=0.40; n = 19; P < 0.05). Of the parameters measured, growth rate was the most sensitive for detection of changes in medium, an eect also noted by éstgaard et al. (1984). The results of the AGIT test for DCMU and the euent are shown in Figs 5 and 6 respectively. Figure 5 shows the dose response curve of the relative growth of P. tricornutum cells grown in DCMU for 3 days. The curve clearly indicates the inhibition of growth rate caused by the addition of DCMU and gave a 72 h-IC50 value of 0.09 mM. The euent dose response curve gave a 72h-IC50 value of 16.4% euent. These results can be compared to the IC50 values derived from the CCT data, 0.33 mM for DCMU and 15.33% for the euent. Due to dierences in the tests such as time of exposure to the toxicant and determination of the eect of the toxin, the results cannot be qualitatively compared. This inability for comparing results from dierent toxicity tests means that other studies in this area are infrequent. When testing the euent, the two methods gave relatively close values, with good agreement between the outcome of the two tests. In
Fig. 5. Algal growth inhibition test growth response curve of the relative growth rate after 3 days of P. tricornutum exposure to various concentrations of DCMU.
Fig. 6. Algal growth inhibition toxicity test dose response curve of the relative growth rate after 3 days of P. tricornutum exposure to various dilutions (v/v) of euent.
comparative studies of dierent bioassay techniques, Miller et al. (1985) and Nyholm (1992) stated that algal growth tests are sensitive when assessing euent toxicity. However, when comparing the two results obtained when testing DCMU, the CCT value was approximately 3.5 times higher than the AGIT test. Further studies assessing comparisons between dierent toxicity tests would be helpful to enable more thorough evaluation of data collected from such tests. The AGIT test is a bioassay procedure based on the inhibition of growth in batch cultured algae compared to a control. It relies on the use of standard laboratory equipment, it is inexpensive and relatively rapid to perform, being completed within 4 days. In each test a range of concentrations of the toxicant can be tested simultaneously, and dose-response curves constructed. The data can be statistically analyzed using standard techniques, and response indicator values such as the 72 h-IC50 can be determined. In addition, due to the simplicity of the test, it is relatively easy to standardize and to assess the precision (the variability between repeated tests) using reference toxicants such as zinc. Thus the test has been accepted as a standard operating procedure for ecotoxicological studies (OECD, 1984; ISO, 1988; EEC, 1990), and is widely used to provide data for regulatory purposes. However, using batch cultures for AGIT testing has a number of inherent problems. The test solutions are added to the cultures once, as a single dose, and the concentration can be modi®ed by a range of factors, including adsorption onto the algae and the vessel and sequestration by chelating agents in the medium. Surviving algae are subject to less stress than their parents and the concentration of the toxicant in the medium can be variable (Maestrini et al., 1984). In addition, in batch culture as the nutrients are consumed the nutri-
The cage-culture turbidostat algal bioassay
tional state of the organisms will alter, resulting in possible changes in sensitivity to toxicants (Wangersky and Maas, 1991). Batch cultures commonly predict the eect of relatively concentrated pulses of toxicants, and give little indication of chronic responses (Maestrini et al., 1984). The CCT described here was designed as an automated alternative to the AGIT test. Once the apparatus is assembled (2±3 h), the system is largely autonomous, with minimal operator time involved for daily sampling. The original CCT was run for 10±14 days with a stabilization period of 2±3 h on set-up or changing the medium (Skipnes et al., 1980). A 14 day running period has also been used by other researchers (éstgaard et al., 1982, 1984; Wangersky and Maas, 1991). In this design, it was found that the system could be run for up to 20 days, although after about 14 days algal attachment to the chamber walls aected the sensor readings. Maestrini et al. (1984) suggested that low biomass concentration in the CCT vessel would help reduce cell attachment and thus increase the operational time of each culture, and therefore, a CCT should be run on as low a biomass as the sensitivity of the monitoring device can allow. Our system was sensitive enough to record accurately changes of approximately 20,000 cells mlÿ1 enabling the cultures to be maintained at relatively low densities. Other CCTs had cell densities in the ranges of 1±5 105 cells mlÿ1 (éstgaard et al., 1984) to 3 108 cells mlÿ1 (Lombardi and Wangersky, 1991). When adding toxicants to the chamber, Skipnes et al. (1980) suggested that the CCT would respond to changes in medium within 2±3 h. In our CCT, electronic measurements were taken hourly and using reference medium a constant overall growth rate was attained, although there appeared to be hourly ¯uctuations. To achieve reproducible results it was necessary to use the integrated daily growth rate instead of hourly data. As a result, the duration of each exposure was relatively long because each toxicant concentration was maintained for at least 24 h followed by a return to the reference medium for at least 48 h to allow the possibility of cell recovery to approximately initial values, allowing only 3±4 concentrations to be tested per experiment. In comparison to the limits on batch cultures, using continuous cultures in algal testing has signi®cant advantages in terms of the relevance of the results to natural algal populations. The experiments in this study tested brief 24 or 48 h exposure of the cells to toxicants. If the cells in the CCT had been exposed to the toxicant for a longer period, dierences in results may be expected. The automated nature of the CCT allows hourly monitoring of the eect of additions of toxicant to the algal population. This feature could be potentially useful in modelling the eects of the constant release of chemicals to natural algal populations. Such studies
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and an assessment of the decay kinetics of the intracellular concentration of toxicant in the CCT cultures would be potentially interesting areas for future studies. CONCLUSIONS
The potential of the cage culture turbidostat as an algal bioassay was assessed. Exposure to the herbicide DCMU and an industrial euent produced a signi®cant decrease in both growth rate and chlorophyll content of the algae compared to the control medium. IC50 values for DCMU and euent determined with the CCT were similar to those determined by the standard Algal Growth Inhibition Toxicity Test. A limitation of the CCT in its present form is the number of concentrations of toxicant that can be tested per experimental run, thus there is insucient data for rigorous statistical analysis. However, the CCT remains a promising automated algal bioassay system which overcomes some of the inherent problems associated with batch culture toxicity testing. In addition, it allows evaluation of chronic levels of toxicants which may be of environmental relevance.
AcknowledgementsÐFunded under contract EPG1/9/2 to the U.K. Department of the Environment as a contribution to its co-ordinated programme of marine research for the North East Atlantic. The authors would like to thank Dr John Redshaw and sta of the Scottish Environment Protection Agency, Western Region for providing the euent sample. Thanks also to The Culture Collection of Algae and Protozoa for donation of algal cultures. REFERENCES
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Rhee G.-Y. (1989) Continuous culture algal bioassays for organic pollutants in aquatic ecosystems. Hydrobiologia 188/189, 247±257. S.A.S. Institute Inc. (1990) SAS/STAT Users Guide, Version 6, Fourth Edition, Volume 2. S.A.S. Cary, U.S.A. St-Laurent C., Blaise C., MacQuarrie P., Scroggins R. and Trottier B. (1992) Comparative assessment of herbicide phytotoxicity to Selenastrum capricornutum using microplate and ¯ask bioassay procedures. Environ. Toxicol. Water Qual. 7, 35±48. Skipnes O., Eide I. and Jensen A. (1980) Cage culture turbidostat: a device for rapid determination of algal growth. Appl. Environ. Microbiol. 40, 318±325. Tempest D. W. (1970) The continuous cultivation of microorganisms. In Methods in Microbiology. Volume 2. (Edited by Norris J. R. and Ribbons D. W.), pp. 259± 276. Academic Press, London. Tett P. B. (1987) Plankton. In Biological Surveys of Estuaries and Coasts. (Edited by Baker J. M. and Wolfe W. J.), pp. 280±341. Cambridge University Press, Cambridge. Van der Heever J. A. and Grobbelaar J. U. (1996) Evaluation of a short-incubation-time small-volume radiocarbon-uptake algal toxicity test. J. Appl. Phycol. 8, 65±71. Wangersky P. J. and Maas R. L. (1991) Bioavailability: the organism as sensor. Mar. Chem. 36, 199±213.
Wat. Res. Vol. 32, No. 4, pp. 1162±1168, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/98 $19.00 + 0.00
AN ASSESSMENT OF THE CAGE-CULTURE TURBIDOSTAT AS AN ALTERNATIVE ALGAL BIOASSAY N. CLARKSON*{, J. W. LEFTLEY, D. T. MELDRUM and J. W. WATSON Centre for Coastal and Marine Sciences, Dunstanage Marine Laboratory, P.O. Box 3, Oban, Argyll PA34 4AD, U.K. (First received February 1997; accepted in revised form August 1997) AbstractÐA computer controlled cage-culture turbidostat (CCT) is described as an alternative to batch algal bioassays. The CCT is designed to monitor algal growth changes in response to the addition of toxicants including chronic toxic eects in comparison to batch culture toxicity tests which are most successful in assessing acute responses. The system is easily and quickly set up, with minimum operator time required once assembled. The alga Phaeodactylum tricornutum (CCAP 1052/6) was used as the test species. Stable growth of the alga could be maintained in the CCT for 14 days, sucient time to run bioassay experiments using both the herbicide Diuron2 (DCMU) and a complex industrial euent. Additions of various concentrations of both toxins to the chamber caused changes in the growth rate and the chl-a concentration of the culture. The Median Inhibitory Concentration of euent calculated to cause a 50% reduction in the growth rate (IC50) in the CCT was estimated to be 0.33 mM DCMU, and 15.33% of the euent. Comparable results were obtained when using the standard algal growth inhibition toxicity (AGIT) test, the 72 hIC50 values were 0.09 mM DCMU and 16.4% euent. This study indicates a potential use for the CCT for water quality monitoring, which may provide more environmentally relevant information than the commonly used AGIT test. # 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION
The most widely used algal bioassay is probably the Algal Growth Inhibition Toxicity (AGIT) test (Environment Agency, 1994), a procedure which measures the inhibition of growth in batch cultures due to toxicants. This method is widely used because of its simplicity, reasonable cost and acceptable reproducibility (Jensen, 1984). However, although generally satisfactory for lethal and acute toxicity, batch culture tests are of little value in assessing long-term chronic impacts (Rhee, 1989). In addition, when high sample throughput is required, such assays are labour intensive and time consuming and alternative protocols are constantly being sought (St-Laurent et al., 1992; Van der Heever and Grobbelaar, 1996). Continuous culture methods, in which fresh medium is added to the culture at a rate sucient to maintain it, are used as an alternative to batch culture methods. In the chemostat, fresh medium constantly ¯ows at a set rate through a vessel of ®xed volume which maintains the culture at a growth rate proportional to the medium ¯ow rate *Present address: The Northumbrian Water Ecology Centre, The University of Sunderland, Sunderland, Tyne and Wear SR1 3SD, U.K. {Author to whom all correspondence should be addressed. Tel.: 0191 5152526, Fax: 0191 5152531, Email: [email protected]
(Tempest, 1970). Turbidostats are a second continuous culture method, but with direct control, in which a photoelectric monitor detects deviations from a desired culture turbidity and induces an increase or decrease in dilution rate of the ¯ow of growth medium (Munson, 1970). The turbidostat has been further developed as the cage-culture turbidostat (CCT), ®rst described by Skipnes et al. (1980). It comprizes: (a) a vessel with porous membranes which contains the algae but allows free exchange of medium in and out of the vessel, and (b) a photosensor system which, via a feedback loop, maintains the culture density (number of cells per unit volume) at a pre-set level by diluting the culture with fresh medium. The frequency at which the dilution pump operates is used to calculate the growth rate. When using the CCT for toxicity testing, medium is continuously perfused through the growth vessel so that the algae are eectively exposed to a constant concentration of all the components of the medium, including pollutants. Constant exposure of the algae to low concentration of pollutants in this way may be a more realistic environmental situation than in the AGIT test in which the algae are eectively exposed to a single large dose of toxicant (Rhee, 1989). There have been relatively few reports of the use of the CCT for water quality monitoring, especially for pollutants (éstgaard et al., 1984; Wangersky
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and Maas, 1991). We therefore constructed a ``state-of-the-art'' apparatus and assessed its potential as an automated algal bioassay for pollutants, using both the herbicide Diuron2 as a model and samples of an industrial euent, and compared the results with those obtained form the standard AGIT test. MATERIALS AND METHODS
The cage-culture turbidostat system A brief description of the system is given here in full detail, including drawings of the culture vessel, inlet/outlet ®lter units, electronic circuit diagrams and computer program can be obtained from the senior author. A schematic diagram of the system is shown in Fig. 1. This CCT system based on the design of Skipnes et al. (1980). The borosilicate glass culture vessel (100 ml capacity) is in two parts which are sealed with a silicone rubber gasket and held together by a metal clamp; this allows easy dismantling for cleaning. Screw-threaded ports are ®tted to the vessel for the connection of the two inlet/outlet ®lter units and the sampling and over¯ow lines. The low dead volume, polycarbonate, in-line ®lter units were specially constructed; each unit held a 1 mm pore size, 47 mm diameter polycarbonate membrane (Cyclopore2, Whatman Labsales Ltd., Maidstone, U.K.). Medium was continuously pumped through the culture vessel at a rate of 18 ml hÿ1 with a multi-channel pump (Model 505U/RL, Watson-Marlow, Falmouth, U.K.) which simultaneously pumped fresh medium into the vessel via one channel and pumped out waste medium, at the same rate, via another channel. This arrangement kept back-pressure in the vessel to a minimum. Silicone rubber tubing (1.5 mm bore, 1 mm wall) connected the pump, ®lters and reservoirs (Altec Ltd., Alton, U.K.). To prevent build up of algae on the inlet/outlet ®lters the ¯ow of medium into and out of the chamber was alternated, changing direction every 10 min. This was achieved by opening and closing four solenoid pinch valves (normally open when not energized) which were activated by the ¯ow control unit (FCU). To ensure complete mixing in the CCT vessel the culture was continuously stirred magnetically by an orbiting magnetic follower (120 rpm) specially designed for ecient mixing in round-bottomed vessels (Whatman Labsales, Maidstone, U.K.). Illumination was provided by a standard 11 W bayonet-®tting white ¯uorescent lamp (100±
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120 mE mÿ2 sÿ1). The optical density of the culture was monitored by an LED/photodiode emitter/sensor circuit. To minimize the eect of extraneous visible light the emitter used a high intensity infra-red LED (peak emission 940 nm), modulated by a 1-kHz square wave, and the detector was an integrated infra-red light-to-voltage converter chip, ®tted with appropriate optical and electronic ®lters. The sensor voltage passed via an A/D converter to the computer capable of running DTVee2 software (Data Translation Ltd., Malboro, MA, U.S.A.). When the optical density (OD) of the culture exceeded the pre-set threshold the computer activated the dilution pump (¯ow rate 0.45 ml minÿ1; Watson±Marlow, model 101U/R MK 2) which simultaneously activated the solenoid pinch valve (normally closed when not energized) to allow excess culture to over¯ow into a sterile receiver. The pump and solenoid were deactivated when the OD returned to the pre-set level. The DTVee2 software recorded the OD of the culture every 15 min in tandem with the time the dilution pump operates during each hour. Growth rates were calculated according to Skipnes et al. (1980). The speci®c growth rate (m) was calculated as the proportion of the volume of the growth chamber (V) harvested, where H = volume harvested per hour (i.e. the time the dilution pump was activated multiplied by the ¯ow rate of medium). The number of cell divisions (d) per hour can then be calculated. The equations are: m H=V d m=ln2
Algal culture The diatom Phaeodactylum tricornutum Bohlin (Strain 1052/6, Culture Collection of Algae and Protozoa, Oban, U.K.) was used as it is fast growing, easily cultured and well suited to electronic particle counting. It is one of the recommended species for marine algal growth tests (Environment Agency, 1994). Stock cultures were maintained in f/2 enriched seawater medium (Guillard, 1975), at 208C under continuous illumination (warm white ¯uorescent light, ca. 100 mE mÿ2 sÿ1, at the surface of the culture). f/2 medium was prepared with seawater ®ltered through Whatman glass ®bre ®lters (GF/F 0.7 mm with a GF/B 1.0 mm pre-®lter; Whatman International Ltd., Maidstone, U.K.). Before autoclaving the salinity was standardized to 30 22%0 by dilution with distilled water and the pH adjusted to 8.020.1 with 1 M HCl. All medium was autoclaved (1158C, 10 min, or 1 h for large volumes). Stock P. tricornutum cultures were periodically checked by microscope for bacterial contamination. All inocula were from 3-day-old, exponentially growing cultures. DCMU solutions The herbicide Diuron2, DCMU (3-(3,4 dichlorophenyl)1,1-dimethyl urea) (Sigma, Poole, UK) is a potent inhibitor of photosystem II, inhibiting ®xation of CO2 (Renger, 1986). A stock solution of DCMU was prepared in acetone and added aseptically to sterile modi®ed f/2 medium to give the range of concentrations used in the CCT and algal growth inhibition test. The controls were f/ 2 enriched reference seawater (RSW) with and without acetone. The volumes of acetone added were found to have no eect on the cells and all results shown are f/2 enriched seawater with acetone. Euent samples
Fig. 1. Schematic diagram of the CCT system. FCU = ¯ow control unit; M = magnetic stirrer; F = magnetic follower; L = ¯uorescent lamp; E = photodiode emitter; D = photodiode sensor; F = ®lters; V1-5 = solenoid pinch valves.
A sample of industrial euent, discharged into the Firth of Clyde, was collected as a 25 l snap sample by personnel of the Scottish Environmental Protection Agency, Western Region (SEPAWR) as part of a routine monitoring pro-
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gramme. Euent samples were prepared for toxicity tests within 48 h of collection according to recommended guidelines (Environment Agency, 1994). Suspended solids were removed by ®ltration through glass ®bre membranes as described above. The pH of the euent (initially 1.30) was adjusted to 8.0, the pH of the reference seawater, and its salinity (initially 10-), measured with a hydrometer, was adjusted to 30- by addition of solid NaCl. The euent was sterilized by pressure ®ltration through 90 mm, 0.2 mm CN membranes (Whatman Labsales Ltd.) and stored in the dark at 88C. Euent concentrations were prepared by dilution with seawater and sterile f/2 nutrient enrichments were added aseptically, the control medium was f/2 enriched RSW. For the AGIT tests euents were used within 48 h of receipt. Because of the time necessary for the CCT to stabilize, euents could not be assayed within 48 h, but were used as soon as possible after preparation. Operation of the CCT The culture vessel, ®lters and associated tubing were autoclaved and assembled aseptically. The apparatus was then transferred to a constant temperature incubator (208C) and connected to the pumps and electronic system (see Fig. 1). The vessel was ®lled aseptically with the control medium, the voltage baseline set and inoculated with algae to give an initial density of ca. 5 104 cells mlÿ1. The algae were then allowed to grow until they exceeded the pre-set threshold, about 2 V (ca. 2 105 cells mlÿ1) to trigger the dilution pump. After allowing the system to stabilize at a constant growth rate for 48 h toxicant (DCMU or euent) was added aseptically to the reference medium in the reservoir to give a known concentration. With a ¯ow rate of 18 ml hÿ1 the total volume of the vessel was replaced in approximately 5 h with the new concentration. This would mean that the initial response of the cells was to a sub-optimal concentration. However, any possible eect of this was reduced by using a total of 24 hourly results as the response time. Each of the series of concentrations (DCMU and euent) was carried out once. A 2 ml sample was taken aseptically from the vessel each day for the determination of cell number and chlorophyll-a (chl-a) per unit volume. Cell numbers were determined with a Coulter Counter Model ZM (Coulter Electronics Ltd, Luton) ®tted with a 50 mm ori®ce, counts were a mean of ®ve replicates. Chl-a was extracted in 90% acetone and measured ¯uorometrically (Turner Instruments Model III Fluorometer, Turner Designs Inc. USA) according to the method of Tett (1987), values were a mean of three measurements. The algal growth inhibition toxicity test The algal growth inhibition (AGIT) test was carried out as a comparative bioassay for both DMCU and the euent. The protocol used was that for marine algae as described by Environment Agency (1994) using cultures of P. tricornutum. DMCU was prepared as described above and added to f/2 medium. For the euent, a dilution series was prepared by addition of a known volume of euent to sterile 1 l ¯asks. Sterile nutrients were added to f/2 concentrations and the solution made up to volume with sterile RSW. The concentration range chosen was 0, 0.1, 0.5, 1, 5, 10, 22, 46 and 100% (v/v) euent, the control was f/2 enriched RSW, the salinity of the samples was 30-. The tests were considered valid if the control cell density increased by more than 16 times in 72 h and the control pH did not vary by more than 1 unit during the test (Environment Agency, 1994). The assessment of inhibition of growth in the test is based on a comparison of test and control growth rates and the data used to determine the 72 h inhibitory concentration (IC50) value, the concen-
tration of test substance estimated to cause 50% inhibition of growth after 72 h. Speci®c growth rates were calculated for each individual ¯ask by linear regression of logarithmic biomass data from the exponential growth phase (Nyholm, 1985). The data were then normalized by dividing by the mean value of the control test. Dose-response curves were obtained by non-linear least squares ®t to the logistic equation: y1ÿ
1 1
eÿ abz
where z = log (concentration) and Y = the normalized response (Nyholm, 1990). Calculations were performed using the SAS procedure NLIN (SAS, 1990) assuming a constant variance of Y and therefore equal weighting of data points. The 72 h-IC50 value corresponds to a relative speci®c growth rate of 0.5. RESULTS AND DISCUSSION
Algal growth in the CCT The growth of the P. tricornutum culture was monitored over a 14 day period incubated in f/2 RSW to assess the long-term functioning of the CCT and its ability to maintain a constant growth rates. Figure 2 shows the daily growth rates and chl-a concentrations of the cells. Growth rate was maintained between 0.72 and 1.16, with generally small ¯uctuations between days. After the initial 2 days the chl-a values settled down and were relatively constant for the remaining days. A comparison of the dierences in growth rate between f/2 nutrient enriched RSW and concentrations of toxicant (DCMU or euent) can be achieved using relative growth values. A mean of the ®rst two daily growth rates in f/2 RSW gave an estimate of the control algal growth rate for that CCT run. The growth rate at each toxicant concentration tested was then expressed as a percentage value relative to this initial calculated mean RSW growth rate. It was then possible to estimate, by simple graphical interpolation of the % inhibition at each concentration, the concentration of toxicant that would cause a 50% inhibition in growth rate.
Fig. 2. The daily growth rate and the cellular chl-a concentration per cell of a continuous culture of P. tricornutum cells in the CCT in control f/2 medium over a 14 day growth period.
The cage-culture turbidostat algal bioassay
This calculation of the Median Inhibitory Concentration of euent (IC50) allows the results from the CCT to be compared with the AGIT test data. Addition of DCMU The daily growth rate and the chl-a concentration of algae grown in a range of concentrations of DCMU, is shown in Fig. 3(a), and the relative growth rate in Fig. 3(b). DCMU was added to the chamber for separate 24 h periods at concentrations of 0.1 mM and 1.0 mM with a 48 h period of RSW medium between the additions to allow for cell recovery. The growth rate decreased on addition of 0.1 mM DCMU by 35.3% subsequently recovering on removal of the DCMU to pre-exposure levels within 48 h. Addition of 1 mM DCMU caused a decrease in the relative growth rate by 92.9% from which the cells did not recover for the remainder of the experiment. The IC50 value was estimated as described above to be 0.33 mM DCMU. The chl-a concentration also decreased, but did not recover after the ®rst addition, and continued to decrease for the duration of the run.
Fig. 3. (a) The daily growth rate and cellular chl-a concentration per cell of a continuously growing culture of P. tricornutum grown in reference seawater (RSW) and dierent concentrations of DCMU. Times; 1±3 days in RSW; day 3 in 0.1 mM DCMU; 4±5 days in RSW; day 6 in 1 mM DCMU; 7±10 days in RSW. (b) The daily relative growth rates compared to f/2 control values of P. tricornutum grown in 0.1 and 1.0 mM DCMU with an intervening recovery period of 48 h in f/2 RSW.
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Addition of euent Figure 4(a) shows the growth rate and chl-a concentration of algae grown in RSW and various concentrations of euent, and Fig. 4(b) the corresponding relative growth rate of the algae. Additions of the euent caused marked eects on algal growth. A concentration of 1% (v/v) caused an immediate stimulation in growth rate by 27% compared to the control. Additions of 5 and 10% (v/v) euent both caused an initial increase in growth within 24 h followed by a decrease after continued exposure to the euent over 48 h. Addition of 22% euent caused an immediate decrease in growth rate, after 48 h m was only 30% of that measured initially in f/2. For concentrations of 1, 5, and 10% the growth rate returned to initial levels within 48 h in RSW, this was not the case with the addition of 22%, although after 48 h in RSW the growth rate increased slightly indicating possible cell recovery. An inhibition of growth by 50% was estimated to be 15.33% euent, calcu-
Fig. 4. (a) The daily growth rate and the chl-a concentration per cell of a continuous culture of P. tricornutum cells grown in reference seawater (RSW) and dierent dilutions (v/v) of euent. Times: 1±2 days in RSW; 3± 4 days in 1% euent; 5±7 days in RSW; 8±9 days in 5% euent; 10±11 days in RSW; 12±13 days in 10% euent; 14±15 days in RSW; 16±17 days in 22% euent; 18± 19 days in RSW. (b) Daily relative growth rates compared to the mean of the initial two RSW samples of P. tricornutum culture grown in euent.
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lated as described above. Addition of low concentrations of the euent caused an increase in the growth rate which may be related to the presence of stimulatory compounds in the euent such as phosphorous and nitrogen. Statistical analysis of the relatedness of the variables measured over the time period of the experiment was carried out using Spearman Rank Correlation. For the DCMU data, a signi®cant correlation was found between the cell count and the chl-a values (rs=0.82; n = 10; P < 0.01) and the growth rate and the chl-a concentration per cell values (rs=0.80; n = 10; P < 0.01). For the euent a similar result was found between the cell count and the chl-a values (rs=0.70; n = 19; P < 0.01) and between the growth rate and the chl-a concentration per cell (rs=0.40; n = 19; P < 0.05). Of the parameters measured, growth rate was the most sensitive for detection of changes in medium, an eect also noted by éstgaard et al. (1984). The results of the AGIT test for DCMU and the euent are shown in Figs 5 and 6 respectively. Figure 5 shows the dose response curve of the relative growth of P. tricornutum cells grown in DCMU for 3 days. The curve clearly indicates the inhibition of growth rate caused by the addition of DCMU and gave a 72 h-IC50 value of 0.09 mM. The euent dose response curve gave a 72h-IC50 value of 16.4% euent. These results can be compared to the IC50 values derived from the CCT data, 0.33 mM for DCMU and 15.33% for the euent. Due to dierences in the tests such as time of exposure to the toxicant and determination of the eect of the toxin, the results cannot be qualitatively compared. This inability for comparing results from dierent toxicity tests means that other studies in this area are infrequent. When testing the euent, the two methods gave relatively close values, with good agreement between the outcome of the two tests. In
Fig. 5. Algal growth inhibition test growth response curve of the relative growth rate after 3 days of P. tricornutum exposure to various concentrations of DCMU.
Fig. 6. Algal growth inhibition toxicity test dose response curve of the relative growth rate after 3 days of P. tricornutum exposure to various dilutions (v/v) of euent.
comparative studies of dierent bioassay techniques, Miller et al. (1985) and Nyholm (1992) stated that algal growth tests are sensitive when assessing euent toxicity. However, when comparing the two results obtained when testing DCMU, the CCT value was approximately 3.5 times higher than the AGIT test. Further studies assessing comparisons between dierent toxicity tests would be helpful to enable more thorough evaluation of data collected from such tests. The AGIT test is a bioassay procedure based on the inhibition of growth in batch cultured algae compared to a control. It relies on the use of standard laboratory equipment, it is inexpensive and relatively rapid to perform, being completed within 4 days. In each test a range of concentrations of the toxicant can be tested simultaneously, and dose-response curves constructed. The data can be statistically analyzed using standard techniques, and response indicator values such as the 72 h-IC50 can be determined. In addition, due to the simplicity of the test, it is relatively easy to standardize and to assess the precision (the variability between repeated tests) using reference toxicants such as zinc. Thus the test has been accepted as a standard operating procedure for ecotoxicological studies (OECD, 1984; ISO, 1988; EEC, 1990), and is widely used to provide data for regulatory purposes. However, using batch cultures for AGIT testing has a number of inherent problems. The test solutions are added to the cultures once, as a single dose, and the concentration can be modi®ed by a range of factors, including adsorption onto the algae and the vessel and sequestration by chelating agents in the medium. Surviving algae are subject to less stress than their parents and the concentration of the toxicant in the medium can be variable (Maestrini et al., 1984). In addition, in batch culture as the nutrients are consumed the nutri-
The cage-culture turbidostat algal bioassay
tional state of the organisms will alter, resulting in possible changes in sensitivity to toxicants (Wangersky and Maas, 1991). Batch cultures commonly predict the eect of relatively concentrated pulses of toxicants, and give little indication of chronic responses (Maestrini et al., 1984). The CCT described here was designed as an automated alternative to the AGIT test. Once the apparatus is assembled (2±3 h), the system is largely autonomous, with minimal operator time involved for daily sampling. The original CCT was run for 10±14 days with a stabilization period of 2±3 h on set-up or changing the medium (Skipnes et al., 1980). A 14 day running period has also been used by other researchers (éstgaard et al., 1982, 1984; Wangersky and Maas, 1991). In this design, it was found that the system could be run for up to 20 days, although after about 14 days algal attachment to the chamber walls aected the sensor readings. Maestrini et al. (1984) suggested that low biomass concentration in the CCT vessel would help reduce cell attachment and thus increase the operational time of each culture, and therefore, a CCT should be run on as low a biomass as the sensitivity of the monitoring device can allow. Our system was sensitive enough to record accurately changes of approximately 20,000 cells mlÿ1 enabling the cultures to be maintained at relatively low densities. Other CCTs had cell densities in the ranges of 1±5 105 cells mlÿ1 (éstgaard et al., 1984) to 3 108 cells mlÿ1 (Lombardi and Wangersky, 1991). When adding toxicants to the chamber, Skipnes et al. (1980) suggested that the CCT would respond to changes in medium within 2±3 h. In our CCT, electronic measurements were taken hourly and using reference medium a constant overall growth rate was attained, although there appeared to be hourly ¯uctuations. To achieve reproducible results it was necessary to use the integrated daily growth rate instead of hourly data. As a result, the duration of each exposure was relatively long because each toxicant concentration was maintained for at least 24 h followed by a return to the reference medium for at least 48 h to allow the possibility of cell recovery to approximately initial values, allowing only 3±4 concentrations to be tested per experiment. In comparison to the limits on batch cultures, using continuous cultures in algal testing has signi®cant advantages in terms of the relevance of the results to natural algal populations. The experiments in this study tested brief 24 or 48 h exposure of the cells to toxicants. If the cells in the CCT had been exposed to the toxicant for a longer period, dierences in results may be expected. The automated nature of the CCT allows hourly monitoring of the eect of additions of toxicant to the algal population. This feature could be potentially useful in modelling the eects of the constant release of chemicals to natural algal populations. Such studies
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and an assessment of the decay kinetics of the intracellular concentration of toxicant in the CCT cultures would be potentially interesting areas for future studies. CONCLUSIONS
The potential of the cage culture turbidostat as an algal bioassay was assessed. Exposure to the herbicide DCMU and an industrial euent produced a signi®cant decrease in both growth rate and chlorophyll content of the algae compared to the control medium. IC50 values for DCMU and euent determined with the CCT were similar to those determined by the standard Algal Growth Inhibition Toxicity Test. A limitation of the CCT in its present form is the number of concentrations of toxicant that can be tested per experimental run, thus there is insucient data for rigorous statistical analysis. However, the CCT remains a promising automated algal bioassay system which overcomes some of the inherent problems associated with batch culture toxicity testing. In addition, it allows evaluation of chronic levels of toxicants which may be of environmental relevance.
AcknowledgementsÐFunded under contract EPG1/9/2 to the U.K. Department of the Environment as a contribution to its co-ordinated programme of marine research for the North East Atlantic. The authors would like to thank Dr John Redshaw and sta of the Scottish Environment Protection Agency, Western Region for providing the euent sample. Thanks also to The Culture Collection of Algae and Protozoa for donation of algal cultures. REFERENCES
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Rhee G.-Y. (1989) Continuous culture algal bioassays for organic pollutants in aquatic ecosystems. Hydrobiologia 188/189, 247±257. S.A.S. Institute Inc. (1990) SAS/STAT Users Guide, Version 6, Fourth Edition, Volume 2. S.A.S. Cary, U.S.A. St-Laurent C., Blaise C., MacQuarrie P., Scroggins R. and Trottier B. (1992) Comparative assessment of herbicide phytotoxicity to Selenastrum capricornutum using microplate and ¯ask bioassay procedures. Environ. Toxicol. Water Qual. 7, 35±48. Skipnes O., Eide I. and Jensen A. (1980) Cage culture turbidostat: a device for rapid determination of algal growth. Appl. Environ. Microbiol. 40, 318±325. Tempest D. W. (1970) The continuous cultivation of microorganisms. In Methods in Microbiology. Volume 2. (Edited by Norris J. R. and Ribbons D. W.), pp. 259± 276. Academic Press, London. Tett P. B. (1987) Plankton. In Biological Surveys of Estuaries and Coasts. (Edited by Baker J. M. and Wolfe W. J.), pp. 280±341. Cambridge University Press, Cambridge. Van der Heever J. A. and Grobbelaar J. U. (1996) Evaluation of a short-incubation-time small-volume radiocarbon-uptake algal toxicity test. J. Appl. Phycol. 8, 65±71. Wangersky P. J. and Maas R. L. (1991) Bioavailability: the organism as sensor. Mar. Chem. 36, 199±213.