3. REFINING THE TOXICITY TEST METHODS FOR USE OF SOUTH AFRICAN TAXA IN TOXICITY TESTS: The Algal Growth Inhibition Assay
3.3 THE ALGAL GROWTH INHIBITION TEST PROTOCOL IN SOUTH AFRICA
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accumulation via photosynthesis during the light period, and achieve their biomass gain by cell-division in the dark period. Given this growth pattern, unicellular algae may not follow a simple exponential growth function during a toxicity test with continuous illumination. This may result in problems such as growth stimulation or variability of growth in controls during a test (Altenburger et al. 2008). The observation of exponential growth with relatively low variability in the control is an important validity criterion in assessing the validity of the results of the growth inhibition assay (Mayer et al. 1997).
3.2.2.3 Nutrients and pH
The bioassay is sensitive to nitrogen, phosphorus, potassium and magnesium, and growth is affected when one of these nutrients is omitted from the medium (Payne 1975). Generally, toxic effects of contaminants may be more severe under limited nutrient conditions.
However, the toxic effects under nutrient limitation are species specific and depend on the degree of nutrient limitation (Interlandi 2002). Another parameter of the growth inhibition test to be considered is pH. Pseudokirchneriella subcapitata can tolerate a pH range of 6 to 9 (Mayer et al. 1998). The initial pH in the standard test medium should be buffered to values of 7 to 8.3 (Mayer et al. 1998, Slabbert 2004). The US EPA standard method recommends and initial pH of 7.5 (± 0.1) of the test medium for the test with P. subcapitata (US EPA 1996). Sample pre-treatment, surface to volume ratio, as well as amount and age of the inoculums (Payne 1975) should also be taken into account during the test as these may affect the physic-chemistry of the test medium.
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The recommended test medium is modified 10% BG-11 (Rippka et al. 1979) since it has been shown to result in more improved growth than the EPA assay medium (EPA 1978, Slabbert et al. 1998, Slabbert 2004). The improved growth is no surprise, given that the BG-11 medium is generally used for growth and maintenance of green and blue-green algae (Olvera- Ramirez et al. 2000, Wang et al. 2004, Slabbert 2004, Tripathi et al. 2006). The chemical composition of both test media is shown in Table 3.1.
Table 3.2 Summary of test conditions for the algal growth inhibition: comparing the DEEEP protocol (Slabbert 2004) to the protocol modified for the selected locally isolated organisms in this study
Parameters DEEEP Protocol Protocol for indigenous organisms
Test species P. subcapitata P. subcapitata, S. bicaudatus, C. vulgaris and C. sorokiniana Test vessel 24 well micro-plates 24 well micro-plates
Temperature 24±2 25±3
Light quality Cool white fluorescent Cool white fluorescent
Photoperiod Continuous Continuous
Light intensity 80-95 µE/m2/s 35-40 µE/m2/s
pH 7.1-7.2 adjusted 7.0-7.6 not adjusted
Test medium modified BG-11 modified BG-11
Volume of test sample 2 mL 2 mL
Age of algal culture 3-4 days 4-6 days
Inoculum size 200 000 cells/mL 300 000 cells/mL
No. of replicate wells 6 controls + 3 test samples 6 controls + 3 test samples Control or dilution
water Autoclaved Milli-Q® Autoclaved Milli-Q®
Test duration 72 hours 96 hours
measured endpoint EC20 and EC50 EC20, EC50, NOEC
44 3.3.1 Summary of test protocol
The unicellular alga P. subcapitata is maintained axenically in batch culture using 250 mL Erlenmeyer flasks according to Slabbert (2004), as described in Chapter 2. The culture is maintained at 24±2 °C under cool fluorescence light of 16:8 hour light/dark cycle, without agitation. Algae are kept in exponential growth phase by sub-culturing weekly in order to keep a constant supply cells for a toxicity test. A summary of test conditions for this assay is given in Table 3.2. The cultures are used for toxicity tests three to four days after sub- culturing. The medium used for culture maintenance and toxicity testing is a modified version of 10% BG-11 (Rippka et al. 1979, Slabbert 2004). The initial pH of the test medium should be 7.1 ±0.1 and must be adjusted to with NaOH or HCl.
The algal suspension is prepared by removing the supernatant medium from the undisturbed culture vessel and re-suspending cells in fresh medium. The fresh 10% BG-11 medium is added to the algal suspension to obtain a concentration of approximately 2×105 cells per mL of cell inoculum. The prepared algal inoculum is added at a ratio of 1:1 to 20-times concentrated BG-11 medium for improved nutrient saturation, thereby improving growth. A volume of 200 µl of algal suspension is added to 180 µl of test substance in each well of the 24-well micro-plate. Sterile Milli-Q® water is used for control wells and as a diluent for test treatments. A mixture of appropriate concentrations of test substance and medium is used for blank wells. Plates are covered with lids and a layer of dark insulation tape is taped around the sides of each plate (to avoid over-illumination of outer wells). The tape is cut to enable opening of the lid and to allow air circulation during incubation. Plates are incubated for 72 hours at 24±2 °C under continuous illumination of approximately 80-95 µE/m2/s. Optical density measurements are read at 450 nm in the beginning (0 hours) and at the end (72 hours) of the test. Results are expressed as percentage growth inhibition (or stimulation). Linear regression is applied to the inhibition data within the 16-84% growth inhibition range, to determine EC20 and EC50 values. R2 values are determined to establish how the regression line fits the measured data. Stimulation data are omitted in the ECx calculation. A toxicity test with a reference toxicant (CdCl2 or K2Cr2O7) must be performed parallel to every toxicity test.
45 3.3.2 Test validity criteria
The algal toxicity test must meet certain criteria to be considered valid. A summary of validity criteria as described on the DEEEP Methods Manual, and validity criteria for the modified method used in this study for indigenous species is shown in Table 3.3. The validity criteria for the toxicity test with indigenous species (Table 3.3) were determined in accordance with the growth characteristics of these identified species.
Table 3.3 A summary of test validity criteria for the reliability of data: the DEEEP methodology (Slabbert 2004) and the method adapted for the selected locally isolated species.
Criteria DEEEP methodology Methodology with locally isolated species
(this study) 1. Coefficient of
variation of controls
Less than 10% Less than 10% for P.
subcapitata, less than 20%
for commercial culture collection species and indigenous species.
2. OD450nm readings of