4. THE RESPONSE OF FRESHWATER MICRO-ALGAE TO REFERENCE
4.2 MATERIALS AND METHODS
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This chapter is aimed at assessing the potential of the selected freshwater micro-algal species isolated from local rivers (Chapter 2) as toxicity test species, and having their sensitivity compared to that of the standard test species. This was done by exposing the locally isolated species, Scenedesmus bicaudatus, Chlorella sorokiniana, and Chlorella vulgaris to two toxicants, potassium dichromate and cadmium chloride using growth inhibition as toxicity test endpoint. Pseudokirchneriella subcapitata and Chlorella protothecoides obtained from commercial culture collections were also exposed to these toxicants for comparison with the local species, using the same toxicity test endpoint. Pseudokirchneriella subcapitata is the standard toxicity test species for the algal growth inhibition test, and C. protothecoides has been used in toxicity tests in a number of studies (Stauber 1995, Zeng et al. 2009, Neil et al.
2009). Chlorella protothecoides is a widely distributed species in rivers and lakes of Australia and has been recommended as a toxicity test species in that country (Stauber et al.
1994). All of the above-mentioned species are unicellular or coenobial green-algae. They have been shown to grow well under the defined laboratory conditions and form homogenous suspensions in defined test medium (Chapter 2), important characteristics of an algal toxicity test species.
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The toxicity of CdCl2 and K2Cr2O7 to five species of freshwater green micro-algae was tested using the growth inhibition assay (96 hrs). The general method used in these toxicity tests is described in Chapter 3 (section 3.4). A batch method was used for the bioassay using sterile 24-well micro-plates, covered with lids as test vessels (Slabbert 2004). Cultures in exponential growth phase were diluted with 10% BG-11 growth medium to an algal density of 3×105 cells per mL. Initial cell density was determined microscopically by counting cells in a sub-sample of the algal culture using a haemocytometer. Each well contained 200 μL mixture of medium and algal inoculum and 1.8 mL of test substance in appropriate concentration dilution. Micro-plates were incubated at 25±3 °C under continuous cool fluorescence light (35-40 µE/m2/s) for 96 hours. Cadmium chloride and potassium dichromate were applied at a series of eight concentrations respectively, for each species (0.007, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.5 and 1 mg/L). Each exposure of one species to a toxicant was repeated six times. The six exposures were treated as replicates, since they were performed simultaneously. Algal population growth was determined spectro- photometrically by measuring optical density at 450 nm for each plate on a micro-plate reader, at the beginning and the end of the toxicity test.
Test acceptability criteria were:
Coefficient of variation of control growth ≤10% for P. subcapitata and ≤20% for other species.
An average OD450nm reading of >0.10 for the controls at the end of the test
R2 of more than 0.8 in the linear regression for ECx calculations
Inhibition data between 10% and 90% growth inhibition used in the linear interpolation for ECx calculations
4.2.2 Analysis
4.2.2.1 Growth inhibition and effective concentration (ECx) determination
Data for all test replicates were assessed for validity using the above acceptability criteria, and those that did not meet all the criteria were not used. Percentage inhibition values were estimated for each experimental group (exposure of each species to each toxicant). EC50 and EC20 values were determined by linear regression and graphic interpolation on the percentage
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inhibition data (between 10% and 90%). Data belonging to the upper-most and lower-most part of the curve (< 10% and > 90%) may negatively influence the curve-fitting; exclusion of these data could minimize that influence (Mayer et al. 1997, Slabbert et al. 2004). The experimental data that did not fit the model well (R2<0.8) were not used in EC50 and EC20 calculations, as stated in the data acceptability criteria above (section 4.2.1). EC50 and EC20 values for all replicates of each toxicant on each species were pooled together and expressed as mean ECx (+standard deviation). The differences in sensitivity of species to each of the two toxicants were determined by statistically comparing the EC50 values. Data were tested for normality and homogenous variance, and a one-way-ANOVA (Analysis of Variance), with Unequal N HSD post-hoc test was used for normally distributed data (due to unequal replicates after quality control of the data). A non-parametric Kruskal-Wallis ANOVA by ranks was used for data that were not normally distributed.
4.2.2.2 Specific growth rate, no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC)
All the data were used (even those with higher than acceptable variability in control growth after 96 hrs) to determine specific growth rate for the control and each toxicant concentration treatment (see Chapter 3.4). This was done in order to include and present stimulation data, which was omitted in ECx calculations. The data were tested for normality and homogeneity, and a one-way ANOVA was used to determine which treatments differed significantly from the control, thus determining NOEC (no observed effect concentration) or LOEC (lowest observed effect concentration) values (using treatments with significantly reduced growth compared to the control). All statistical analyses were undertaken using STATISTICA (Version 8) software package with p≤0.05 as the level of significance.
4.2.2.3 Species sensitivity distributions
Species sensitivity distribution (SSD) curves were generated in order to compare the sensitivity of different micro-algae to the selected reference toxicants (CdCl2 and K2Cr2O7).
Median concentration effect (EC50 and LC50) data of each of the two toxicants to several algal species used for the SSDs were extracted from the US EPA ECOTOX database, as well as toxicity data generated in this study. The usefulness of an SSD is dependent on the quality of data used to generate the SSD curve. Including poor data could possibly result in
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misrepresentation and affect the interpretation of the SSD curve. The toxicity data extracted from the ECOTOX database for each of the two toxicants were pre-screened and filtered by only using laboratory generated toxicity data and excluding the following data:
seawater as the medium of exposure,
exposure duration longer than 96 hours and shorter than 24 hours, and
no reports on fields such as endpoint, effects measurement, exposure duration and/or concentration units.
Ecotoxicology databases often contain multiple entries for a single species and chemical, because data come from different sources. Therefore, after the pre-screening, in cases where there was more than one data point (or effect concentration value) for a species, the geometric mean of the different values was used to represent the species; where there was only one data point, that single value represented the species (Warne et. al. 2004). Species sensitivity distribution curves were drawn using SSD generator (US EPA 2005), which is an easy-to-use tool designed to create custom SSDs. This tool creates the curves by fitting a linearized log- normal distribution to effect concentration data (LC50 or EC50) for different species. The SSD curve enables the estimation of the proportion of species affected by the toxicant at different exposure concentrations. The SSD generator was used to construct SSD curves with single species toxicity data generated from this study, and pre-screened data extracted from the ECOTOX database.