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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 January 2017 to 23 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- A sample of each loading rate WAF containing no algal cells was taken for Total Organic Carbon (TOC) analysis at 0 and 72 hours.
- Duplicate samples were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
CULTURE MEDIUM
- The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
- The culture medium is defined in Annex 3 (attached).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST SYSTEM
- The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 2 °C.
- Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E03 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10E04 – 10E05 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Hardness:
Not reported
Test temperature:
24 ± 1 °C
pH:
7.3 to 8.9 in the definitive test (see Table 2, attached)
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal test item concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L
Details on test conditions:
EXPERIMENTAL DESIGN AND STUDY CONDUCT
- Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.

VALIDATION OF MIXING PERIOD
- Preliminary work (see Annex 4, attached) was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances, in the WAF.

RANGE FINDING TEST
- The loading rates to be used in the definitive test were determined by a preliminary range-finding test.
- The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 1.0, 10 and 100 mg/L for a period of 72 hours.
- The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.
- Nominal amounts of test item (5, 50 and 500 mg) were each separately added to a glass slide and suspended in the water column of 5 L of culture medium to give the 1.0, 10 and 100 mg/L loading rates respectively.
- After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
- An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (6.4 mL) to give the required test concentrations of 1.0, 10 and 100 mg/L loading rate WAF.
- The control group was maintained under identical conditions but not exposed to the test item.
- At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
- After 72 hours the cell density of each flask was determined using a Coulter Multisizer Particle Counter.

DEFINITIVE TEST
- Based on the results of the range-finding test, loading rates of 6.25, 12.5, 25, 50 and 100 mg/L were assigned to the definitive test.

EXPERIMENTAL PREPARATION
- A nominal amount of test item (50 mg) was weighed onto a glass slide and suspended within 5 liters of culture medium to give the 10 mg/L loading rate.
- After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface.
- The stirring was stopped after 95 hours and the mixture allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal, and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. A series of dilutions was made from the 10 mg/L loading rate WAF to give the 0.625, 1.25, 2.5 and 5.0 mg/L loading rate WAFs.
- An aliquot (500 mL) of each of the loading rates was separately inoculated with algal suspension (5.3 mL) to give the required test concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L loading rate WAF.
- Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 and 72 hours (see Annex 5).

EXPOSURE CONDITIONS
- As in the range-finding test, 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.
- The control group was maintained under identical conditions, but not exposed to the test item.
- Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 4.70 x 10E05 cells per mL. Inoculation of 500 mL of test medium with 5.3 mL of this algal suspension gave an initial nominal cell density of 5 x 10E03 cells per mL, and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

TEST ORGANISM OBSERVATIONS
- Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10E03 cells/mL) was taken as the starting cell density.
- To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

WATER QUALITY CRITERIA
- The pH of the control and each test concentration was determined at initiation of the test and after 72 hours exposure.
- The pH was measured using a Hach HQ30d Flexi handheld meter.
- The temperature within the incubator was recorded daily.
- The appearance of the test media was recorded daily.

VORTEX DEPTH MEASUREMENTS
- The vortex depth was recorded at the start and end of the mixing period.

TOTAL ORGANIC CARBON ANALYSIS
- Analysis of the WAF was carried out by Total Organic Carbon (TOC) analysis.
- Samples were taken from the uninoculated control and 100 mg/L loading rate WAF test group at 0 and 72 hours for this analysis (see Annex 5, attached).

COMPARISON OF GROWTH RATES
- The average specific growth rate for a specified period was calculated as the logarithmic increase in biomass using the equation µ = ln Nn – ln N1 / tn – t1 where µ = average specific growth rate from time t1 to tn; N1 = cell concentration at t1; Nn = cell concentration at t0; t1 = time of first measurement; tn = time of nth measurement.
- The average specific growth rate over the test duration was calculated for each replicate control and test item vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.
- In addition, the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.
- Percentage inhibition of growth rate for each replicate test item vessel was calculated using the equation Ir = (µc - µt / µc) * 100 where Ir = percentage inhibition of average specific growth rate; µC = mean average specific growth rate for the control cultures; µt = average specific growth rate for the test culture.

COMPARISON OF YIELD
- Yield was calculated as the increase in biomass over the exposure period using the equation Y = Nn – N0 where Y = yield; N0 = cell concentration at the start of the test; Nn = cell concentration at the end of the test.
- For each test concentration and control the mean value for yield along with the standard deviation was calculated.
- The percentage inhibition of yield was calculated using the equation Iy = [(Yc – Yt) / Yc] * 100 where Iy = percentage inhibition of yield; Yc = mean value for yield in the control group; Yt = mean value for yield for the treatment group.

DETERMINATION OF ELx VALUES
- For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). ELx values were then determined from the equation for the fitted line.
- Where appropriate 95% confidence limits for the EL50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

VALIDATION CRITERIA
- The results of the test are considered valid if the following performance criteria are met:
(i) Cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
(ii) The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
(iii) The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
1.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidence limits 1.6 to 2.0 mg/L loading rate WAF
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
1.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.625 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
1.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
other: yield
Remarks on result:
other: 95 % confidence limits 1.2 to 1.2 mg/ loading rate WAF
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
1.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.625 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
other: yield
Details on results:
VALIDATION OF MIXING PERIOD
- Preliminary investigational work (see Annex 4, attached) indicated that there was a significant increase in the amount of total organic carbon when the preparation period was extended from 24 to 96 hours.
- Therefore, for the purpose of testing the WAF was prepared using a stirring period of 95 hours followed by a 1-Hour settlement period.

RANGE FINDING TEST
- The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1 (attached).
- The results showed no effect on growth at 1.0 mg/L loading rate WAF. However, growth was observed to be reduced at 10 and 100 mg/L loading rate WAF.
- Based on this information loading rates of 0.625, 1.25, 2.5, 5.0 and 10 mg/L were selected for the definitive test.
- Total Organic Carbon (TOC) analysis of the WAFs conducted at 0 hours (see Annex 5) and showed measured concentrations to range from 1.08 to 12.8 mg C/L. Measured concentrations in the range of less than the limit of quantification (LOQ), determined to be 1.0 mg C/L, and 11.9 mg/L were obtained at 72 hours.

DEFINITIVE TEST TOTAL ORGANIC CARBON ANALYSIS
- Total Organic Carbon (TOC) analysis of the WAFs containing no algal cells was conducted at 0 hours (see Annex 5, attached) and showed measured concentrations to range from less than the LOQ to 3.28 mg C/L. There was not a significant change in the measured concentrations at 72 hours.
- The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

DEFINITVE TEST GROWTH DATA
- Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.
The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage inhibition values are plotted against loading rate in Figure 2 and Figure 3.
- From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

INHIBITION OF GROWTH RATE
- The following results were determined: ErL10 (0-72h) 0.81 mg/L loading rate WAF; ErL20 (0-72h) 1.1 mg/L loading rate WAF; ErL50 (0-72h) 1.8 mg/L loading rate WAF (95 % confidence limits 1.6 to 2.0 mg L loading rate WAF) where ErLx is the loading rate that reduced growth by x%.
- Statistical analysis of the growth rate data was carried out for the control and all loading rates using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control and 0.625 mg/L loading rate WAF (P ≥ 0.05), however all other loading rates were significantly different (P < 0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 0.625 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 1.25 mg/L loading rate WAF.

INHIBITION OF YIELD
- The following results were determined: EyL10 (0-72h) 1.1 mg/L loading rate WAF; EyL20 (0-72h) 1.1 mg/L loading rate WAF; EyL50 (0-72h) 1.2 mg/L loading rate WAF (95 % confidence limits 1.2 to 1.2 mg L loading rate WAF) where EyLx is the loading rate that reduced yield by x%.
- Statistical analysis of the yield data was carried out as for growth rate. There were no statistically significant differences between the control and 0.625 mg/L loading rate WAF (P ≥ 0.05), however all other loading rates were significantly different (P < 0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 0.625 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 1.25 mg/L loading rate WAF.

VALIDATION CRITERIA
- The cell concentration of the control cultures increased by a factor of 154 after 72 hours (mean cell density of control at 0 hours was 5.00 x 10E03 cells/mL and mean cell density of control at 72 hours was 7.70 x 10E05 cells/mL). This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
- The mean coefficient of variation for section by section specific growth rate for the control cultures was 12% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35 %.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3 % and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7 %.

OBSERVATIONS ON CULTURES
- All test and control cultures were inspected microscopically at 72 hours.
- After 72 hours, there were no abnormalities detected in the control or 0.625 mg/L loading rate WAF test cultures.
- Some mutated cells were observed to be present in the test cultures at 1.25 mg/L loading rate WAF, and many mutated cells were observed to be present at 2.5 and 5.0 mg/L loading rate WAF test cultures.
- No intact cells were observed in the 10 mg/L loading rate WAF test cultures.

WATER QUALITY CRITERIA
- The pH values of the control and each test concentration are given in Table 2 (attached).
- Temperature was maintained at 24 ± 1 °C throughout the test.
- The pH value of the control cultures (see Table 2, attached) was observed to increase from pH 7.3 at 0 hours to pH 8.9 at 72 hours. The pH deviation in the control cultures was 1.6 units after 72 hours, and therefore, exceeded the increase of 1.5 units permitted in the Test Guidelines. The control cultures met all other validity criteria, and the test groups increased in pH at similar amounts, so the pH deviation is not considered to have affected the outcome of the study.

VORTEX DEPTH MEASUREMENTS
- The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

OBSERVATIONS ON TEST ITEM SOLUBILITY
- Observations on the test media were carried out during the mixing and testing of the WAFs.
- At the start of mixing, the 10 mg/L loading rate WAF was observed to have formed a clear, colourless media column with test item attached to a glass slide suspended in the media. At the end of stirring, and following a 1-Hour standing period, the WAF was observed to have formed a clear, colourless media column with test item attached to a glass slide suspended in the media and test item floating at the media surface. Microscopic examination of the WAF showed there to be no micro-dispersions of test item present.
- At the start of the test all control and test cultures were observed to be clear, colourless solutions. After the 72-Hour test period, the control and 0.625 mg/L loading rate WAF test cultures were observed to be green dispersions, and the 1.25 mg/L loading rate WAF test cultures were very pale green dispersions. The 2.5, 5.0 and 10 mg/L loading rate WAF test cultures were clear, colourless solutions.
Results with reference substance (positive control):
- The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
STATISTICAL ANALYSIS
- One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates to determine any statistically significant differences between the test and control groups.
- All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).
Validity criteria fulfilled:
yes
Conclusions:
Based on growth rate, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 (72h) value of 1.8 mg/L loading rate WAF, the No Observed Effect Loading Rate (NOELR) was reported as 0.625 mg/L and the Lowest Observed Effect Loading Rate (LOELR) was determined to be 1.25 mg/L. Based on yield, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 (72h) value of 1.2 mg/L loading rate WAF, the No Observed Effect Loading Rate (NOELR) was reported as0.625 mg/L and the Lowest Observed Effect Loading Rate (LOELR) was determined to be 1.25 mg/L.
Executive summary:

GUIDELINE

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

METHODS

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.625, 1.25, 2.5, 5.0 and 10 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

 

RESULTS

Total Organic Carbon (TOC) analysis of the 5.0 and 10 mg/L loading rate WAF test preparations performed at 0 and 72 hours showed measured concentrations to range from 1.59 to 3.29 mg C/L. The control, 0.625, 1.25 and 2.5 mg/L loading rate WAF test preparations showed measured concentrations of less than the limit of quantification (LOQ) determined to be 1.0 mg C/L. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

 

CONCLUSION

Based on growth rate, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 (72h) value of 1.8 mg/L loading rate WAF, the No Observed Effect Loading Rate (NOELR) was reported as 0.625 mg/L and the Lowest Observed Effect Loading Rate (LOELR) was determined to be 1.25 mg/L. Based on yield, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 (72h) value of 1.2 mg/L loading rate WAF, the No Observed Effect Loading Rate (NOELR) was reported as0.625 mg/L and the Lowest Observed Effect Loading Rate (LOELR) was determined to be 1.25 mg/L.

Description of key information

Based on growth rate, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 (72h) value of 1.8 mg/L loading rate WAF, the No Observed Effect Loading Rate (NOELR) was reported as 0.625 mg/L and the Lowest Observed Effect Loading Rate (LOELR) was determined to be 1.25 mg/L. Based on yield, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 (72h) value of 1.2 mg/L loading rate WAF, the No Observed Effect Loading Rate (NOELR) was reported as 0.625 mg/L and the Lowest Observed Effect Loading Rate (LOELR) was determined to be 1.25 mg/L (OECD 201 and EU Method C.3).

Key value for chemical safety assessment

EC50 for freshwater algae:
1.8 mg/L
EC10 or NOEC for freshwater algae:
0.625 mg/L

Additional information

GUIDELINE

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

METHODS

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.625, 1.25, 2.5, 5.0 and 10 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

 

RESULTS

Total Organic Carbon (TOC) analysis of the 5.0 and 10 mg/L loading rate WAF test preparations performed at 0 and 72 hours showed measured concentrations to range from 1.59 to 3.29 mg C/L. The control, 0.625, 1.25 and 2.5 mg/L loading rate WAF test preparations showed measured concentrations of less than the limit of quantification (LOQ) determined to be 1.0 mg C/L. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

 

CONCLUSION

Based on growth rate, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 (72h) value of 1.8 mg/L loading rate WAF, the No Observed Effect Loading Rate (NOELR) was reported as 0.625 mg/L and the Lowest Observed Effect Loading Rate (LOELR) was determined to be 1.25 mg/L. Based on yield, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 (72h) value of 1.2 mg/L loading rate WAF, the No Observed Effect Loading Rate (NOELR) was reported as0.625 mg/L and the Lowest Observed Effect Loading Rate (LOELR) was determined to be 1.25 mg/L.