3. REFINING THE TOXICITY TEST METHODS FOR USE OF SOUTH AFRICAN TAXA IN TOXICITY TESTS: The Algal Growth Inhibition Assay
3.2 THE ALGAL GROWTH INHIBITION ASSAY
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3. REFINING THE TOXICITY TEST METHODS FOR USE OF SOUTH AFRICAN
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respond by stimulation, inhibition, or both, to a variety of contaminants (Sbrilli et al. 2005).
The Algal Growth Inhibition Assay is the most widely used algal assay, because of its simplicity, reasonable cost and accepted reproducibility (US EPA 1978, OECD 1984, ISO 1989, Slabbert et al 1998, Slabbert 2004). This assay was developed in the mid-1960s as the Algal Assay Procedure Bottle Test (U.S. EPA 1969) and has since been modified and accepted by various regulatory agencies as a standard toxicity test assay with Pseudokirchneriella subcapitata as the standard species (US EPA 1978, OECD 1984, Slabbert et al 1998, Slabbert 2004). One of the most important improvements of the efficiency of the test is the miniaturization of test vessels by substituting the traditional 250 mL flasks with 24-well or 96-well micro-plates, with no deleterious effect on the precision, reliability and reproducibility of the test (Thellen et al. 1989). The use of micro- plates simplifies the test because smaller sample volumes are used, incubation space is reduced and more samples can be tested at a time (Slabbert 2004, Paixão et al. 2008).
Protocols for the growth inhibition test with green algae have been published and are routinely used in water resource management internationally and nationally (US EPA 1978, OECD 1984, ISO 1989, Slabbert 2004).
3.2.1 Summary of the standard algal growth inhibition test methodology and experimental design (ISO 1989, US EPA 1996, OECD 2006)
In summary, a culture of known algal cell density of the standard species e.g.
Pseudokirchneriella subcapitata, in its exponential growth phase is exposed to a range of test concentrations in defined test vessels under specified static conditions in a temperature and photo-period controlled environment. An untreated control is maintained to measure the normal growth of the algal cells. The effect of the test material is evaluated by comparing the growth of the test culture to the control. Algae are exposed to a minimum of five concentrations of the test substance in a geometric series of the ratio between 1.5 and 2.0 to generate a concentration-response (US EPA 1996).
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The assay is performed in Erlenmeyer flasks of any volume between 125 and 500 mL (as long as flasks of the same size are used for each test, the volume of test substance does not exceed half of the total flask volume. Test vessels should contain an initial cell inoculum of 1×104 cells per mL of P. subcapitata. The constituents of the recommended test medium for this test are listed on Table 3.1. The experimental design should include sufficient replication
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to allow statistical evaluation of results (Parrish1985). A minimum of three replicates per concentration is required for this assay (US EPA 1996).
Table 3.1: Chemical composition of the EPA assay medium (EPA 1978) and the 10% BG-11 medium (Rippka et al. 1979, Slabbert 2004).
MACRONUTRIENTS
Compound EPA Algal Assay Medium 10% BG-11 Medium Concentration (mg/L) Concentration (mg/L)
CaCl2.2H2O 4.41 3 600
NaNO3 25.5 15 000
K2HPO4 1.044 4 000
MgSO4.7H2O 14.7 7 500
NaHCO3 15 2 000
MgCl2.6H2O 12.164
MICRONUTRIENTS
Concentration (mg/L) Concentration (mg/L)
H3BO3 0.185 286
MnCl2.4H2O 0.415 181
Na2MoO4.2H2O 0.00726 39
Na2EDTA.2H2O 0.3 100
CuSO4.5H2O 7.9
CuCl2.2H2O 0.00012
ZnSO4.7H2O 22.2
ZnCl2 0.00327
Co(NO3)2.6H2O 4.94
CoCl2.2H2O 0.00143
FeCl3.6H2O 0.16
Fe(NH3)-citrate 600
Citric acid 600
The conventional response endpoints applied in the algal growth assay include final yield (biomass or cell density), growth rate, chlorophyll a content or total biovolume (Lin et al.
2005). Algal growth may be recorded using a spectrophotometer to measure optical density, a
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particle counter to count the algal cells, a fluorometer to measure chlorophyll fluorescence or by counting cells under the microscope. Measuring chlorophyll may be combined with particle counting to enable correlation between chlorophyll concentration and cell volume.
This could be a valuable parameter since cell volume may be influenced by toxicity without chlorophyll concentration being changed. Counting cells under the microscope also allows further information on cell deformities or structural modifications to be obtained (Hornstrom 1990). The qualitative and descriptive microscopic observations are not used in endpoint calculations, but they can be useful in determining additional effects of the tested chemical (US EPA 1996).
Toxicity of a test solution is determined as reduced growth rate or reduced final biomass in relation to the control. The most common parameter to measure is the EC50, that is, the effective concentration of the test substance that inhibits growth by 50%. However, the EC50
value alone may be unsatisfactory since the dose-response curve may vary. A substance may inhibit growth at a lower concentration but have no effect on growth at higher concentrations, or stimulate growth at lower concentrations and have no effect or inhibit growth at higher concentrations. A no observed effect concentration (NOEC) or lowest observed effect concentration (LOEC) may also be used to exhibit stimulation responses to substances, which are usually omitted while determining ECx values (Hornstrom 1990, Slabbert 2004).
The NOEC is derived by hypothesis testing, where treatment responses are compared with control responses to test the null hypothesis that the responses are the same. Determination of NOEC is widely used as an endpoint for chronic toxicity data. However it is criticized for several reasons, the most important is that NOEC depends on the choice of test concentrations and the number of replicates (Sbrilli et al. 2005). Precision of the NOEC increases with the number of concentrations tested, while confidence in the NOEC value increases with the number of replicates (Kooijman et al. 1996). In order to overcome the difficulty in statistically deriving NOEC using hypothesis testing, a regression-based estimation process (ECx) should also be used (Sbrilli et al. 2005, Chen et al. 2009). Most NOEC values have been found to be close to EC10 – EC30 values. It is therefore recommended that the regression-based approach using ECx is a better tool than the hypothesis testing (NOEC) for estimating toxic effects lower than the EC50 (Chen et al.
2009).
41 3.2.2 Important physico-chemical parameters
It is important to control physico-chemical parameters for the duration of the toxicity test. A controlled environment room that can maintain the specified air temperature, light intensity, and photoperiod is required (US EPA 1996). Physical and chemical parameters that are not controlled adequately may be the primary cause of variability (and thus reduced confidence in either EC50 or NOEC/LOEC values) in algal growth during a toxicity test. Thus, homogenous and constant conditions with regard to temperature, light and nutrients are important in the growth inhibition assay to ensure test precision and test reproducibility (Altenburger et al. 2008).
3.2.2.1 Temperature
The growth rate and cell morphology of the standard species Pseudokirchneriella subcapitata ATCC® 22662 are temperature dependent. This species can grow at a temperature range of 6 to 33 °C, with an optimum temperature of 28 °C. The standard growth inhibition test is performed at temperatures between 23 °C and 25 °C with an allowable temperature variability of 2 °C during the test period (ISO 1989, US EPA 1996, Slabbert 2004). Most processes of algal metabolism are dependent on temperature, and effects of toxicity therefore could be temperature dependent. More homogenous temperature requirements could reduce variability in growth rate, resulting in more reproducible and precise results (Mayer et al.
1998).
3.2.2.2 Light
Light intensity is another important parameter for this assay because algal growth is correlated to the amount of light available for photosynthesis. Variability in light intensity may be less crucial at light saturation conditions of the standard growth inhibition test, than under environmental light- limiting conditions. Prescribed light intensity for the standard algal assay is within the range of 60-120 µEm-2s-1 with cool white fluorescent light as the source (Mayer et al. 1998). One of the requirements of the standard algal growth inhibition assay is continuous illumination during the test period. However, a daily rhythm of light-dark cycles may be more suitable, particularly with regard to simulating natural conditions (Hornstrom 1990). Unicellular green algae allocate their resources towards biomass
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accumulation via photosynthesis during the light period, and achieve their biomass gain by cell-division in the dark period. Given this growth pattern, unicellular algae may not follow a simple exponential growth function during a toxicity test with continuous illumination. This may result in problems such as growth stimulation or variability of growth in controls during a test (Altenburger et al. 2008). The observation of exponential growth with relatively low variability in the control is an important validity criterion in assessing the validity of the results of the growth inhibition assay (Mayer et al. 1997).
3.2.2.3 Nutrients and pH
The bioassay is sensitive to nitrogen, phosphorus, potassium and magnesium, and growth is affected when one of these nutrients is omitted from the medium (Payne 1975). Generally, toxic effects of contaminants may be more severe under limited nutrient conditions.
However, the toxic effects under nutrient limitation are species specific and depend on the degree of nutrient limitation (Interlandi 2002). Another parameter of the growth inhibition test to be considered is pH. Pseudokirchneriella subcapitata can tolerate a pH range of 6 to 9 (Mayer et al. 1998). The initial pH in the standard test medium should be buffered to values of 7 to 8.3 (Mayer et al. 1998, Slabbert 2004). The US EPA standard method recommends and initial pH of 7.5 (± 0.1) of the test medium for the test with P. subcapitata (US EPA 1996). Sample pre-treatment, surface to volume ratio, as well as amount and age of the inoculums (Payne 1975) should also be taken into account during the test as these may affect the physic-chemistry of the test medium.