4. R 2 for the regression line
3.4 THE ALGAL GROWTH INHIBITION ASSAY ADAPTED FOR THE LOCALLY ISOLATED ORGANISMS IDENTIFIED IN THIS STUDY
The methodology used in this study is based on the DEEEP Methods Manual (Slabbert 2004), summarized above (Section 3.3.1, Tables 3.1 and 3.2), with adaptations to accommodate species specific requirements of the selected locally isolated organisms.
3.4.1. Culture maintenance
Batch cultures of the test species Pseudokirchneriella subcapitata CCAP 278/4 (Appendix B) were maintained axenically under controlled conditions at 24±2 °C under cool fluorescence light of 16:8 hour light/dark cycle, without agitation. Cultures were maintained in the exponential growth phase by sub-culturing once a week in 10% BG-11 medium, and used in toxicity tests four to six days after sub-culturing. Culture medium was sterilized in an autoclave at 121 °C for 15 minutes, and toxicity test medium was filter-sterilized through a sterile 0.22 μm syringe filter membrane. The initial pH of the toxicity test and culture medium ranged between 7.0 and 7.6 without adjustment.
3.4.2. Inoculum preparation and exposure to test solutions
Test solutions were inoculated with an initial density of approximately 3×105 cells per mL.
The initial cell density was determined by counting cells in the counting chamber of the Neubauer haemocytometer under an Olympus BX51 research light microscope at 400 times magnification in phase contrast illumination. Cell density was expressed as number of cells counted × 101(10 fold dilution) × 104 (factor for haemocytometer) (Slabbert 2004). The algal inoculum volume and the volume of BG-11 medium required for the number of treatment and control wells of six replicate 24-well microplates was determined. The determined volumes of the aforementioned respective substances were combined to form an inoculum suspension (Appendix D).
Six replicate microplates were prepared for each test substance and marked according to the plate configuration on Table 3.4. A range of eight concentrations of test substance in 1:2 serial dilutions were prepared. There were two microplates for each replicate of each contaminant to accommodate eight concentrations (see Table 3.4), and six replicates of each
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Sterile (autoclaved at 121 °C for 15 minutes) Milli-Q® water was used for controls and substance dilutions. A volume of 1.8 mL of control and treatment solutions was was dispensed into each of the appropriated wells of each of the six replicate microplates (Table 3.4, Appendix D). A volume of 0.2 mL of the inoculum suspension was dispensed into each of the controls and treatment wells of the six replicate microplates (see Table 3.4 for plate configuration).
Table 3.4 Configuration for a toxicity test micro-plate with four concentration treatments
Microplate 1
Blank Blank Blank Blank Blank Blank
Control Conc. 1 Conc. 2 Conc. 3 Conc. 4 Control
Control Conc. 1 Conc. 2 Conc. 3 Conc. 4 Control
Control Conc. 1 Conc. 2 Conc. 3 Conc. 4 Control
Microplate 2
Blank Blank Blank Blank Blank Blank
Control Conc. 5 Conc.6 Conc. 7 Conc. 8 Control
Control Conc. 5 Conc. 6 Conc. 7 Conc. 8 Control
Control Conc. 5 Conc. 6 Conc. 7 Conc. 8 Control
Two micro-plates, with four concentrations on each, were used for the eight concentrations of each toxicant on each species. Blanks of treatment samples without inoculum were prepared for each concentration treatment and control (Table 3.4). A volume of 2 mL of cell inoculums with test substance was added in each well of the 24-well micro-plates. There were three replicate wells for each concentration treatment and six replicate wells for the control on each 24-well micro-plate (Table 3.4).
Micro-plates were incubated for 96 hours at 25±3 °C under continuous illumination of approximately 35-40 µE/m2/s. This low light intensity was due to increased distance between the test vessels and the light source. Although cool white fluorescent light bulbs were used, when the distance between the test vessels and the light source was decreased, the temperature on the test surface increased, so there had to be a trade-off between light intensity and temperature.
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Light intensity was measured at the test surface with a Lutron LX101 lux meter, using the illumination unit, lux, which is gives an indication of illumination intensity to the human eye.
Since algae and plants do not see light the ways human do, illumination intensity was converted to photosynthetic active radiation (PAR) which is expressed in units of µE/m2/s (Aldos et al. 1995, Pearcy, 2000). The exposure time was extended from 72 to 96 hours to accommodate the indigenous species that did not show sufficient growth, as specified in the DEEEP method (Table 3.3) after 72 hours.
3.4.3. Growth measurement
Growth was determined spectrophotometrically by measuring optical density on a Biotek (ELx800 UV) micro-plate reader at 450 nm at the beginning and the end (96 hours) of the test. Results were expressed as percentage growth inhibition (or stimulation) compared to the control, using the following formula:
[(ODC-OD0) - (ODT-OD0-ODB>0.005)]/(ODC-OD0) × 100 (Slabbert 2004), where
ODC = mean optical density of the control wells at the end of the experiment
OD0 = mean optical density of the test wells for each concentration treatment at the beginning of the experiment.
ODT = mean optical density of the test wells for each concentration treatment at the end of the experiment (96hrs)
ODB>0.005 = the blank optical density measurements of the test well >0.005 (to compensate for precipitation and/colour interference).
The inhibitory concentration to reduce growth by 20% and 50% (EC20 and EC50 values) was determined using linear regression and graphic interpolation (using inhibition data between 10% and 90% as stated on the test validity criteria). The growth inhibition was read off the linear scale (Y axis) and the toxicant concentration on the logarithmic scale (X axis). R2 values were determined to establish how the regression line fitted the measured data, and data that did not fit the model well (with R2 values less than 0.8) were not used. Stimulation data were omitted in the ECx calculation. Specific growth rate at each concentration treatment and control was also calculated for all species, using the initial (0 hours) and final (96 hours) optical density readings with the formula:
49 μ = ln(x2/x1)/t2-t1 where:
μ=specific growth rate, x2= OD450nm at 96 hours, x1=OD450nm at 0 hours, t2=96 hours and t1= 0 hrs
3.4.4. Data Analysis
The 96 hour EC50 values for each toxicant for each species were averaged to obtain a single EC50 value. Some of the replicate tests did not meet all of the test validity criteria described on Table 3.4, and therefore not all of the six replicates for each toxicity test could be used to calculate EC50 values. This resulted in uneven replication of exposures (unequal number of averaged EC50 values) of each toxicant to each species. These uneven numbers of replicates were used in statistical analyses to establish if there were any significant differences in the EC50 values of each toxicant between species, and also to determine significant differences in the sensitivity (EC50 values) of each species to the different toxicants. Data were checked for homogeneity of variance using Levene’s Test. If there were no significant differences in the variance of the EC50 values, normality of data was determined using the Kolmogorov- Smirnov test. If data were normal, one-way-ANOVA was used, with the Unequal N HSD post-hoc test; the Kruskal-Wallis ANOVA by ranks was used for data that were not normal.
The specific growth rate response of each species to each toxicant at each concentration and control was determined, for all six toxicity test replicates. The six replicates were averaged to give one specific growth rate value for each toxicant concentration and control for each species. Statistically significant differences in specific growth rate between the control and toxicant concentrations were determined in order to determine the NOEC (No Observed Effect Concentration) of each toxicant for each species. The LOEC (Lowest Observed Effect Concentration) was determined in instances where the NOEC could not be determined.
Toxicant concentrations resulting in significantly lower specific growth rate than the control (indicating inhibition) were used to determine the NOEC or the LOEC, not concentrations with specific growth rates significantly higher than the control (indicating stimulation).
Specific growth rate data were checked for homogeneity of variance with Levene’s Test, and normality using the Kolmogorov-Smirnov test. If there were significant differences in variance, or the data were not normal, a non-parametric Mann-Whitney U test was used to
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compare control specific growth rate for each species to specific growth rate at each toxicant concentration. If there were no significant differences in homogeneity of variance, and data were normally distributed, the Dunnett’s test was used to determine treatments with specific growth rates that were significantly different from the control. P≤0.05 was used as the level of significance for all analyses.
3.4.5 General Discussion
Deviations from the currently defined protocols were to accommodate the growth of the locally isolated species so that they could be compared to the standard species Pseudokirchneriella subcapitata. Preliminary experiments with the locally isolated species were used to define the protocol for using these species and the deviations from the standard protocols such as the EPA and OECD methods.
For example the major deviation from the protocol on the document: Environment Canada, March 2007 - EPS 1/RM/25 Second Edition, mentioned are as follows
The use of 96 well microplate vessels instead the 24 well microplates used in this study, which means smaller volume of the test medium.
Higher light intensity of 50-62 µE/m2/s as opposed to 35-40 µE/m2/s used in this study.
This was a practical issue with the test facility that needs to be addressed. It should be borne in mind that the light intensity used in the study is still within the acceptable growth range of the species used.
Starting inoculum of 10 000 cells/mL as opposed to 300 000 cells/mL used in this study.
This was based on the preliminary experiments which showed 300 000 cells/mL to be the optimal starting inoculum given the conditions of light intensity and temperature.
The toxicity tests in the study were extended to 96 hours instead of the conventional 72 hrs, to ensure sufficient growth of controls at the end of the experiment, particularly for the local isolates.
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