4. THE RESPONSE OF FRESHWATER MICRO-ALGAE TO REFERENCE
4.1 INTRODUCTION
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(ANZECC and ARMCANZ 2000). Generally an estimate of the maximum concentration of a toxicant or chemical protective of most species (typically 95%) in the environment can be deduced from an SSD with laboratory toxicity data from a variety of species (Wheeler 2002).
If the toxicant is kept below such a concentration in nature, the assumption is that most species (95%) will not be harmed by the toxicant (Kefford et al. 2006). The use of SSDs in the context of this study will be as a tool to compare the sensitivity of a range of species to a particular toxicant or chemical.
Cadmium chloride (CdCl2)and potassium dichromate (K2Cr2O7) are the reference toxicants most commonly used internationally, and in South Africa, for toxicity testing (Wang 1987, Slabbert et al. 1998, ISO 2004, Slabbert 2004). A reference toxicant is a chemical that a particular organism is known to respond to. The reference toxicant is typically used as a positive control during the toxicity test to determine if the test organism is responding to the toxicant in the expected manner (US EPA 1996). These chemicals are used in the South African toxicity proficiency test scheme, which is an inter-laboratory evaluation of toxicity tests to ensure adequate standardization for routine use. Cadmium chloride is used for the daphnia proficiency test, while potassium dichromate is used for the algal growth inhibition proficiency test (Chapman et al. 2011a). Cadmium chloride was initially recommended as a reference toxicant for the algal growth inhibition test and used in inter-laboratory training exercises for this assay in South Africa (Slabbert et al. 1998, Slabbert 2004). The use of cadmium chloride as a reference toxicant was later substituted with potassium dichromate.
This was done to align South African algal toxicity testing with international trends and to enable comparison of our data with internationally generated data, since K2Cr2O7 is the reference toxicant most widely used internationally (US EPA 1996, NIWA 1998, ISO 2004, OECD 2006).
Cadmium is found in trace concentrations in freshwater, mostly as a result of industrial discharges from mining, metal smelters, agricultural fertilizers and manufacturing of metal plated containers (Terry and Stone 2002). It is a non-essential metal, potentially toxic to marine and freshwater aquatic life (Terry and Stone 2002, Tukaj et al. 2007). The toxicity of cadmium in water depends on the different parameters such as hardness, chemical speciation, pH, temperature and the presence of other metal ions in the water. According to the South African water quality guidelines for aquatic ecosystems (DWAF 1996), the target water quality range (TWQR) for cadmium is 0.15, 0.25 and 0.35 μg/L for soft, medium, and hard
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waters respectively. Measurements of cadmium for any particular site should be within the TWQR 90% of the time in order to comply with the guidelines. The chronic effect values that should not be exceeded at any site in order ensure protection of aquatic ecosystems in soft, medium, and hard waters are 0.3, 0.5 and 0.7 μg/L respectively (DWAF 1996).
Cadmium has been shown to have adverse effects on some vital processes of algal cells (Rebhun and Ben-Amotz 1986, Cepák 2002). Investigations on the toxicological response of micro-algae to cadmium have taken two approaches: (a) how cadmium as an environmental toxicant affects algae as primary producers in aquatic ecosystems and (b) biochemical- physiological studies determining the mode of action of toxicity to algae (Rebhun and Ben- Amotz 1986). Cadmium has been reported to impact on algal growth and reproductive processes by affecting the nuclei and chloroplasts of the cells. It may inhibit or inactivate some enzymes, thereby affecting growth, respiration or photosynthesis in algae (Tukaj et al.
2007). Loss of cell motility in flagellates and reduction in cell volume in some species, have also been reported (Cepák 2002). Although cadmium may be taken up and assimilated by green algae at low concentrations, it may be toxic to sensitive organisms and its effects are species specific and dependent on growth conditions (Terry and Stone 2002, Tukaj et al.
2007). The relevance of the use of CdCl2 in toxicity tests is supported by the fact that, Cd2+ is more readily taken up by aquatic plants than other cadmium ions (Rebhun and Ben-Amotz 1986).
Potassium dichromate is a well-known algal toxicant and an internationally recommended reference in algal growth inhibition tests (Wang 1987, Nyholm 1990, ISO 2004, European Chemicals Bureau 2005, OECD 2006, Labra et al. 2007). The toxicity of potassium dichromate to some microalgae may not only be dependent on concentration, but also on the duration of exposure (Aizdaicher and Markina 2011). At elevated levels, chromium is considered a mutagenic and carcinogenic agent for living organisms (Mortuza et al. 2009). It is genotoxic to micro-algae and genomic damage may alter gene transcription resulting in modification of cell physiological activity (Labra et al. 2007, Aizdaicher and Markina 2011).
In aqueous systems, chromium exists primarily in two biologically significant oxidation states, hexavalent (Cr (VI)) and trivalent chromium (Cr (III)), which differ in their toxicity (Labra et al. 2007, Mortuza et al. 2009, Vannini et al. 2009). Hexavalent chromium is extensively used in a variety of commercial processes, followed by unregulated discharge of chromium containing effluent into aquatic systems, in both developing and developed
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countries (Mortuza et al. 2009). In contrast to Cr (III), Cr (VI) may cause serious damage to living cells and induce growth inhibition to micro-algae because, not only is it more soluble but it can cross cellular membranes via non-specific anion carriers (Labra et al. 2007, Vignati et al. 2010). However, Vignati et al. (2010) argue that Cr (III) concentrations added to algal toxicity test media decrease over the duration of the test, due to the formation of hydrolysis products in the medium. This temporal decrease of Cr (III) may therefore underestimate its toxicity to algae during a test.
Potassium dichromate (K2Cr2O7) is one of the most soluble Cr (VI) salts. It is used extensively in the manufacture of paints and dyes, as well as in the leather industry as a tanning agent. The target water quality range and the chronic effect value for chromium (VI) according to the South African water quality guidelines are 7 and 14 μg/L, respectively (DWAF 1996). Notably, these values are much higher than those of cadmium (mentioned earlier), which means that chromium is perceived to be less of a threat to aquatic ecosystems than cadmium.
The ability of algal cells to accumulate cadmium and chromium as a defense mechanism has been reported (Tukaj et al. 2007, Mortuza et al. 2009). These algae produce cellular structures which form complexes with these metal ions. These complexes are then accumulated in vacuoles in the algal cells (Tukaj et al. 2007, Mortuza et al. 2009). Chromium also induces changes in cell morphology, chloroplast structure and enzyme activity in some micro-algal species (Aizdaicher and Markina 2011).
Unicellular algae are good test organisms for investigating aquatic environmental pollution because they are in immediate contact with the environment, and therefore respond readily to unfavorable conditions (Mortuza et al. 2009). The use of single phytoplankton test species under regulated laboratory conditions to assess ecosystem toxicity induced by different compounds has been criticized because the species-specific responses of test species to individual toxicants cannot mimic those of natural phytoplankton species living under highly variable environmental conditions (Chapman 1995a). However, the single-species phytoplankton responses observed in bioassays may provide potential answers to reasons for changes in phytoplankton community structures induced by pollutants in the natural environment (Oberholster et al. 2010).
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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.