6. THE SENSITIVITY OF FRESHWATER MICRO-ALGAE TO TWO EFFLUENTS AND A HERBICIDE FORMULATION
6.3 RESULTS
6.3.3 Herbicide formulation (Roundup®)
There was no growth of all the species in the Roundup® exposures at the highest concentrations (6.25 mg/L to 50 mg/L). The EC50 values for Roundup® on P. subcapitata could not be calculated because no toxicity test replicates satisfied all the validity criteria.
The variability in the control growth was above the acceptable 10% for all six test replicates (with a minimum coefficient of variation of 18% and a maximum of 42%).
The pesticide highly inhibited the growth of P. subcapitata with mean ±standard deviation percentage growth inhibition values of 73±3%, 50±17%, 87±6 and 94±3 by the following concentrations 0.39, 0.78, 1.56, 3.13 mg/L, respectively. Figure 6.3d illustrates the effect of Roundup® on the specific growth rate of P. subcapitata, and indicates a significant decrease in the specific growth rate of this species at the lowest concentration (0.39 mg/L). The specific growth rate of P. subcapitata was highly variable for the six test replicates at the 0.78 mg/L concentration of the pesticide, with specific growth rate not significantly different from the control. The two highest concentrations (1.56 mg/L and 3.13 mg/L) of Roundup®
significantly decreased the specific growth rate of P. subcapitata in comparison to the control (Figure 6.3d).
Only three out of the six toxicity test replicates with C. protothecoides were considered valid according to the prescribed criteria, and therefore only EC50 values for those tests could be determined. The mean EC50 of Roundup® on C. protothecoides was 1.2±0.1 mg/L. There was high variability in the response of C. protothecoides to the lowest concentration (0.39 mg/L) of the pesticide for the three valid test replicates. In one of the test replicates the growth inhibition was 5.7%, while the other two tests showed growth stimulation of 107%
and 146% respectively.
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(a)
(b)
(c)
(d)
Figure 6.3 Mean specific growth rate (OD450nm per hour) (+ standard deviation (n=6)) of (a) Chlorella vulgaris, (b) Chlorella sorokiniana, (c) Chlorella protothecoides and (d) Pseudokirchneriella subcapitata after 96 hours of exposure to a pesticide (Roundup® ).
*=significantly lower than the control, **=significantly higher than the control.
0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016
C. vulgaris
**
Growth rate (OD450nm / hour)
0 0.002 0.004 0.006 0.008 0.01 0.012 0.014
C. sorokiniana
Growth rate (OD450nm / hour)
* * *
*
0 0.005 0.01 0.015 0.02 0.025
C. protothecoides
* Growth rate (OD450nm / hour)
0 0.002 0.004 0.006 0.008 0.01 0.012 0.014
Control 0.39 0.78 1.56 3.13
P. subcapitata
*
* *
Roundup® concentration (mg/L) Growth rate (OD450nm / hour)
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The stimulation data was omitted for the EC50 calculations. The specific growth rate of C.
protothecoides was slightly increased at the lowest concentration (0.39 mg/L) of Roundup®
compared to the control, although this increase was not statistically significant. At the three highest concentrations (0.78 mg/L, 1.56 mg/L and 3.13 mg/L) of Roundup® , the specific growth rate of C. protothecoides decreased, in comparison to the control, although only growth at the highest concentration (3.13 mg/L) was significantly different from the control (Figure 6.3c).
The mean percentage growth inhibition of C. sorokiniana by Roundup® was high (74%, 86%, 85% and 93%) at all four concentrations (0.39, 0.78, 1.56and 3.13 mg/L) respectively.
The validity criteria state that the inhibition data to be used in the linear interpolation for EC50
calculations must be between 10% and 90%, and the growth inhibition of C. sorokiniana by the herbicide formulation was between 74% and 93%, which could. This high growth inhibition, coupled with the low R2 (less than 0.8) for the regression, led to none of the test replicates satisfying the validity criteria, and therefore EC50 values could not be determined.
However, the specific growth rate of C. sorokiniana was significantly decreased at all concentrations (Figure 6.3b), compared to the control.
There was high variability in the response of C. vulgaris to Roundup®. At the following concentrations, 0.39, 1.56 and 3.13 mg/L, some test replicates exhibited growth inhibition, while others showed growth stimulation. Roundup® stimulated growth in Chlorella vulgaris at the 0.78 mg/L concentration, for all six test replicates as shown in Figure 6.3a. There was a slight decrease in the specific growth rate of C. vulgaris at the lowest concentration of 0.39 mg/L, compared to the control, even though this decrease was not statistically significant.
The Roundup® concentration of 0.78 mg/L significantly increased the specific growth rate of C. vulgaris, compared to the control, and the specific growth rate at the other concentrations (0.39, 1.56 and 3.13 mg/L) was not significantly different from the control.
Of the four micro-algal species exposed to Roundup®, the growth of two species P.
subcapitata and C. sorokiniana was inhibited by the pesticide. The growth of the other two species, C. protothecoides and C. vulgaris, was not greatly affected by the pesticide, except for a slight stimulation of C. vulgaris at a low concentration (0.78 mg/L) of the pesticide and high inhibition of C. protothecoides at the highest concentration (3.13 mg/L).
115 6.4 DISCUSSION
Algal species are useful indicators of pollution in aquatic systems because they are able to respond by stimulation, inhibition, stimulation at low concentrations and inhibition at higher concentrations, or inhibition at low concentrations and stimulation at high concentrations.
Algal growth stimulation may have implications for alteration of the species composition in aquatic communities (Ma 2005), which could potentially result in eutrophication in natural water bodies (Sbrilli et al. 2005).The variability in response of algae to the complex effluents could be attributed to the bioactivity and the related interaction of effluent components (Sbrilli et al. 2005), coupled with the differences in the morphology and metabolic activities of the different algal species.
In South Africa, coal mining is the second largest mining sector after gold. Most of the active coal mining in South Africa produces coal for power generation which supports the country’s power generation for export and domestic consumption (Hobbs et al. 2008). The two effluents from coal-based operations stimulated the growth of the algal species, especially at low concentrations. This algal growth stimulation could have serious implications for environmental water quality and in-stream ecological responses. Industries are permitted to discharge effluent into the receiving waters, and for the most part these discharges are allowed during periods of high rainfall, high run-off and increased water levels (Hobbs et al.
2008, Naddy et al. 2011). These effluent releases, although permitted and controlled by legislation, could lead to low levels of effluent present in the receiving water resources, and stimulating algal growth in those aquatic systems.
Phosphate concentrations in the effluent may be higher than in the receiving surface water (Naddy et al. 2011), and may thus result in the stimulation of algal growth in the receiving environment. Although phosphate and nitrate levels in the effluents were not measured, high nutrient levels in the effluents may be a possible explanation for the observed growth stimulation by the effluent at low concentration levels compared to the controls. The problem of excess phosphate is typically manifested through stimulation of algal growth, especially when it is accompanied by high nitrogen concentrations (Interlandi 2002, Naddy et al. 2011).
The two locally isolated species C. vulgaris and C. sorokiniana were stimulated by the petro- chemical industrial effluent at all concentrations (except the highest concentration for C.
sorokiniana).
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The standard species, P. subcapitata, was stimulated by the power plant effluent at all concentrations, whereas the petro-chemical industrial effluent stimulated the algae at the lowest concentrations, with no effect at the medium concentrations and stimulation at the highest concentrations. A similar response of this species was also observed by Sbrilli et al.
(2005), in their evaluation of the effects of surface and ground water samples on the growth of P. subcapitata. The alteration of the chemistry of the algal growth medium by the chemical components of the effluent during the toxicity test must also be taken into account (Interlandi 2002), as this could also explain the variability in response of the algal species to the different effluent concentrations. However, no chemical analysis was undertaken to corroborate this statement.
Chlorella protothecoides was stimulated by the power plant effluent at all concentrations, but responded slightly differently to the petro-chemical effluent, with growth inhibition at the lowest concentrations, no significant response at the medium concentrations and stimulation at the highest concentrations. There was concern of the petro-chemical effluent being slightly volatile, as observed during the toxicity test, because this may have led to the instability of concentrations over the exposure period and potentially resulted in the pronounced growth inhibition of C. protothecoides at the lowest concentration. Evaporated substances may evoke effects on adjacent wells in a micro-plate test and the evaporation loss of volatile substances may influence observation in algal growth inhibition tests (Riedl and Altenburger 2007).
The two locally isolated algae (C. vulgaris and C. sorokiniana) responded relatively similarly to both effluents, and their response was slightly different to that of the other two species P.
subcapitata and C. protothecoides. The local isolates were stimulated at low concentrations and inhibited at high concentrations of the power plant effluent, whereas the other two species were stimulated at all concentrations. The two locally isolated species were stimulated by the petro-chemical industrial effluent at all concentrations, a slightly different response to that of C. protothecoides, which was inhibited at low concentrations and stimulated at high concentrations, and P. subcapitata which was stimulated a low concentrations, not affected by the medium concentrations and stimulated again by high concentrations. The algal growth stimulation could be the result of high nutrient load in the effluent coupled with the synergistic or additive interactions of the effluent components.
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Because invasive exotic plants have become increasingly widely distributed, herbicides to control the exotic vegetation are also being increasingly used. Herbicide formulations with glyphosate as an active ingredient, such as Roundup® used in this study, are used extensively. Glyphosate reduces plant growth by inhibiting aromatic amino acid biosynthesis (Gardner 1997).
The pesticide Roundup® (a formulation of glyphosate) showed relatively high toxicity to the locally isolated C. sorokiniana, even at the relatively low concentrations used in the test. The sensitivity of C. sorokiniana to glyphosate was also reported by Christy et al. (1981), where an EC50 value of 0.017 mg/L was determined. The other local isolate C. vulgaris was not adversely affected by the pesticide at the tested concentrations, except to show slight stimulation at low concentration. The standard species P. subcapitata was also inhibited by the pesticide, while the growth of C. protothecoides was not significantly affected, compared to the control, except being significantly inhibited at the highest concentration. The EC50
value of Roundup® on C. protothecoides was 1.2±0.1 mg/L. Given that the concentrations tested were much lower than the application concentration of two – four percent (equivalent to 7200 mg/L -14400 mg/L active ingredient) recommended by the manufacturer, it can be deduced from this study that Roundup® is relatively toxic to the tested micro-algae, except C. vulgaris. Another Chlorella species (C. pyrenoidosa) was reported to be relatively insensitive to glyphosate (Anton et al. 1993).
The concern about herbicides is that they may impact non-target organisms. Micro-algae are at risk of being affected by pesticide spray-drift and runoff since they have physiological similarities with the intended target organisms (invasive plants) (Dorigo et al. 2004).
Herbicide input into surface water generally occurs in pulses, often highly concentrated, with low levels persisting during the intervals between the pulses. As shown in this study, these low levels may have deleterious effects on non-target organisms such as micro-algae when they enter aquatic environments. The response of micro-algae to herbicides may be a useful tool to monitor herbicide impacts on aquatic resources, and using more than one species could be more appropriate since algae respond variably depending on tested concentrations, and the species tested (Dorigo et al. 2004). The toxicity of herbicides to micro-algae may also depend on the ability of the toxic components to permeate the algal cells. Water quality conditions may influence the stability and solubility of the pesticide, subsequently affecting toxicity (Gardner et al. 1997).
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Until recently there have been no water quality guidelines determined to control and monitor the entry of these herbicides into water resources (Mensah et al. 2013), despite their extensive use in South Africa. Water quality guidelines are the determined minimum acceptable levels of a chemical to protect aquatic biota. These guidelines are generally based on toxicity data compiled in various forms to determine a nominated environmental level of the chemical required to protect the aquatic ecosystem (Chapman 1995b). Toxicity data from this study were used in SSDs that developed water quality guidelines for Roundup® in South Africa (Mensah et al. 2013). The contribution of a taxonomic group is one of the most important parameters for the SSD (Schmitt-Jansen et al. 2007), and this study was the main source of micro-algal data to the Roundup® SSDs.
Although it is understood that responses to toxicants, of single species of algae under regulated laboratory conditions, cannot adequately mimic those of natural phytoplankton communities, bioassays provide useful information on the effect of toxicants at biochemical, physiological, morphological and perhaps population levels (Oberholsteret al. 2010).