7. EVALUATING THE APPLICATION OF THE STUDY IN WATER RESOURCE MANAGEMENT
7.2 A CRITICAL EVALUATION OF THE STUDY
7.2.2 Phase 2
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organisms that form homogenous suspensions in growth media, rather than those than organisms where cells clump together.
Eight single species cultures were successfully established (Figure 7.1) and identified according to morphology. The limitation was not to support this identification with molecular identification. However, for the purpose of the study, this was adequate since molecular identification can still be done at a later stage for confirmation. It is also important to note that the isolation technique plays a role in the outcome in terms of species that are able to out- compete others and grow successfully in the defined culture conditions. Some species may find it difficult to grow on agar plates. Due to the process of elimination used in this study, only three species out of the eight species that satisfied the selection criteria for toxicity testing (Scenedesmus bicaudatus, Chlorella sorokiniana and Chlorella vulgaris).
Even though there was a positive outcome in that three of the obtained species seemed to be suitable for toxicity testing, all of them were green algae (Chlorophyta). This was seen as a limitation of this study because the ideal scenario would have been to obtain species from different taxonomic groups (green, blue-green and diatoms). Different taxonomic groups would have contributed to improved representativity of the ecosystem (Hornstrom 1990).
Green algae are generally easy to culture and they are mostly used in toxicity bioassays (Hawxby et al. 1977, Gardner et al. 1997, Rioboo et al. 2002, Yan et al. 2002). Blue-green algae and diatoms have been known to be difficult to maintain in viable cultures and they typically grow relatively slowly (Lewis 1995) compared to green-algae. Species of Chlorella and Scenedesmus are the most common, well known and widespread green algae. They are truly cosmopolitan and can be found in freshwater bodies all around the world. They are well known for being particularly easy to isolate and maintain in culture, and they are available in most commercial culture collections (Lürling 2005, Silva et al. 2009).
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involved adapting the protocol to take into consideration the ideal growth conditions of the locally isolated species.
Algal toxicity testing was introduced in the 1980s in South Africa, and the only published protocol for the algal bioassay is described in the Direct Estimation of Ecological Effect Potential (DEEEP) Methods Manual (Slabbert 2004). This protocol is based on the standard US EPA Algal Growth Inhibition Test protocol (US EPA 1978), which was adjusted for use in routine toxicity testing in South Africa (Slabbert and Hinler 1990, Slabbert et al. 1998).
The DEEEP Methods Manual describes the key practical steps involved in the execution of this bioassay. Even though the method of this bioassay described in the DEEEP Manual is the only published method for use in South Africa, there are no quality requirements pertaining to the standard operating procedures for the practical execution of the protocol (Chapman et al. 2011a). The limitation in terms of quality assurance is that there is no recognised national test protocol for this bioassay endorsed by the South African Bureau of Standards (SABS) that can be used nationally by all laboratories (Chapman et al. 2011b). South African laboratories typically use different international test protocols of the bioassay (US EPA 1978, OECD 2006, ISO 1989).
The algal toxicity test protocol described in the DEEEP Methods Manual (Slabbert 2004) was adapted and refined for the use of locally isolated species (Scenedesmus bicaudatus, Chlorella sorokiniana and Chlorella vulgaris). Adaptations were based on the conditions for growth required by the selected locally isolated species. This refined method was then used to assess the suitability of these species for toxicity testing, by assessing their ability to withstand the experimental conditions prescribed in the refined protocol. The two most commonly used reference toxicants K2Cr2O7 and CdCl2 in South Africa and internationally for algal toxicity testing (Wang 1987, Slabbert et al. 1998, ISO 2004, Slabbert 2004) were selected as suitable chemicals for use in this phase of the study. There was relatively sufficient data available in literature on the toxicity of these two reference toxicants to Pseudokirchneriella subcapitata, to enable the comparison of the results from this study with existing data so as to validate the authenticity and feasibility of the refined method.
The tests with reference toxicants K2Cr2O7 and CdCl2 on the standard species P. subcapitata were used as the basis for comparison in this phase. Out of the three species, two (Chlorella sorokiniana and Chlorella vulgaris) adequately met the selection criteria. Scenedesmus
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bicaudatus was eliminated as a potential toxicity test species due high growth variability in controls. Constant and uniform growth was a prescribed requirement of toxicity test species in this study. There may have been physical and/or chemical experimental factors that contributed to the variability in the growth of S. bicaudatus. One of the weaknesses in this phase is that these factors were not quantified due to time constraints. It is unknown whether manipulation of one or two parameters in the growth environment would have improved the growth rate and variability of growth of controls in S. bicaudatus.
Another factor may be that the morphology of S. bicaudatus was different from that of the Chlorella species tested. The cells were either bi-cellular or colonies of up to 8 cells in pairs.
One observation was that the S. bicaudatus cells were polymorphic, being unicellular or colonial. These morphological changes depend on environmental conditions and may occur in Scenedesmus sp. without any effects on growth (Lürling 2005). This could mean that morphological changes may be a more relevant and sensitive endpoint for Scenedesmus than the traditional growth rate or biomass. The toxicity test method (algal growth inhibition assay), even though adapted and refined, was still originally based on supporting the growth of the standard species P. subcapitata bioassay (US EPA 1978, Slabbert 2004), and therefore could not necessarily be expected to adequately support the growth of some species.
Three Chlorella species (C. protothecoides, C. vulgaris and C. sorokiniana) that were also tested grew constantly and uniformly under the prescribed experimental conditions. It was therefore on the basis of comparing the growth of S. bicaudatus under those prescribed experimental conditions with that of the standard species P. subcapitata, the commercial species C. protothecoides and the two indigenous Chlorella species (C. sorokiniana and C.
vulgaris) that that this species was eliminated as a potential toxicity test species. It should be clarified that although S. bicaudatus was eliminated as a toxicity test species for the test protocol prescribed in this study, it may be a useful toxicity test species for other test methods with different experimental conditions and endpoints.
One aspect that can be pointed out as a weakness in this phase of the study was the lower light intensity in the experimental facility than the prescribed light intensity. However, the light intensity used in this study was adequate for the standard species P. subcapitata (and the other three species) to obtain balanced control growth and fulfil the established control growth variability requirements for test acceptability. The growth of the standard species
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provided a basis for comparison of results obtained for the other four species under the same test conditions. The test conditions used in this study were not outside the prescribed temperature and light culture conditions for the particular strain of P. subcapitata used in this study i.e. 20-25°C and 30-40 µE/m2/s (Appendix C). Moreover species such as Chlorella sorokiniana are also known to grow at light intensities of 30-60 µE/m2/s (de Bashan et al.
2008). If conditions are kept constant and within the bounds of survival, micro-algal cells will become acclimated to their environment and their growth will be balanced (MacIntyre and Cullen 2005). Standard light intensities between 10-30 µE/m2/s are appropriate for long- term culturing of most micro-algal taxa. Localised heating may be problematic and temperature needs to be carefully controlled, because as temperature increases, evaporation also increases (Lorenz et al. 2005), and this could affects the results of a toxicity test particularly where small volumes such as those of wells of a 24-well microplate are used.