7. EVALUATING THE APPLICATION OF THE STUDY IN WATER RESOURCE MANAGEMENT
7.2 A CRITICAL EVALUATION OF THE STUDY
7.2.1 Phase 1
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country as it plays a vital role in power generation as well as fuel and chemical production.
Coal-based power generation and its effects on the environment, as well as acid mine drainage from coal mines have recently become points of concern and discussion in South Africa. The use of these effluents contributed to the applicability, site-specificity and environmental realism of the study. The response and sensitivity of these local species to the effluents was compared to that of the standard toxicity test species P. subcapitata and C.
protothecoides from a culture collection in order to consider the potential of using of the selected local micro-algae in addition to the standard species in routine toxicity testing to determine industrial effluent impacts on local aquatic resources. Furthermore, the sensitivity of the locally isolated micro-algae to a commonly used herbicide Roundup was assessed. This herbicide was selected for the important role it plays in controlling weeds and alien vegetation. This herbicide is used by commercial farmers in the agricultural sector as well as in formal national programmes that are designed to control invasive alien species. The use of the herbicide in this study further contributed to the environmental realism and applicability of the study to the country’s water resource management as the use of this herbicide may negatively impact on non-target organisms such as micro-algae in the aquatic ecosystem.
125 PHASE 1:
To develop capacity to use SA micro- algae in toxicity tests.
PHASE 2:
To refine toxicity test methods for using SA algal taxa.
PHASE 3:
To assess the application and value of using local micro-algal species in toxicity
tests for use in water resource management in SA OBJECTIVES
Developing and refine protocol to isolate and culture micro-algae for use in toxicity testing.
Isolate and culture micro-algae from the natural environment.
Select suitable species for toxicity testing.
Refining existing algal toxicity test methods for the use of South African species.
Exposing selected species to reference toxicants to assess their ability to withstand toxicity test conditions.
METHOD
Exposing the selected species to toxicants relevant to SA water resource management.
Assessing the sensitivity of the SA species to the toxicants in comparison to the standard toxicity test species, and a species obtained from a commercial culture collection.
Using SSDs to compare the sensitivity of the SA micro-algal species to other micro-algal species and other taxonomic groups such as micro- invertebrates.
Chlorella vulgaris
Chlorella sorokiniana
Scenedesmus bicaudatus
Scenedesmus arcautus
Stichococcus minutissimus
Monoraphidium minutum
Oocystis lacustris
Nitzschia sp.
OUTCOMES
Scenedesmus bicadatus
Chlorella vulgaris
Chlorella sorokiniana
Pseudokirchneriella subcapitata
Chlorella protothecoides
Sensitivity ranking:
1. Pseudokirchneriella subcapitata 2. Chlorella sorokiniana
3. Chlorella vulgaris 4. Chlorella protothecoides Figure 7.1: Schematic overview of the study, summarising objectives, methods and results of
each Phase.
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In South Africa, micro-algal cultures have typically been used to advance research towards understanding the physiological and genetic characteristics, as well as taxonomy of the organisms. Although isolating micro-algal cultures for use in toxicity tests has been used for some time in developed countries such as Australia, Canada and the United States of America, it is a fairly new phenomenon in South Africa. There are currently no local species isolated and cultured for use in toxicity test in South Africa. This is largely due to the lack of capacity and facilities to establish the cultures and maintain them.
As expected the isolation and establishing of cultures from environmental samples was a labour intensive and complicated task. Various isolation techniques were attempted but ultimately isolation on agar plates was selected as the method of choice for his study due to it being a relatively easy technique that requires minimum technical skill. It is a commonly used technique by small-scale laboratories that isolate cultures for academic and research purposes. It seemed to be the appropriate isolation and culturing method for the purpose and objective of growing the cultures.
When isolating micro-algae to use in toxicity tests, consideration must be given to the morphology, eco-physiology and growth properties of the organism. This is to ensure compatibility of the test organisms with the endpoint of the toxicity test protocol. In addition to the isolating and culturing, there is the maintenance of the cultures axenically once they are established. One of the common problems with this isolation and general maintenance of cultures with isolation on agar plates is contamination of axenic cultures by bacteria and fungi. This was encountered during this study, and periodically cleaning up the cultures was one of the setbacks of the process. However, improved laboratory facilities, such as obtaining a laminar flow system and a growth chamber remarkably reduced the rate of contamination and improved the success of the isolation and culturing process.
Given that biomass and growth rate are measures of the growth endpoint of the micro-algal toxicity test protocol, useful organisms toxicity testing are those that grow relatively well under defined laboratory conditions. Suitable organisms must also form homogenous suspensions in growth media, for easy quantitative determination of growth. The two techniques selected for quantifying micro-algal growth in this study were counting cells on a haemocytometer under the microscope and determining optical density, both of which require
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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).