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Ecotoxicological information

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:
2017-03-9 - 2017-07-10
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)
Version / remarks:
OECD Guidelines for Testing of Chemicals (2006)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Analytical monitoring:
yes
Details on sampling:
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 103 cells/mL) was taken as the starting cell density.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low aqueous solubility and complex nature of the test item for the purposes of testing the test item was prepared as a Water Accommodated Fraction (WAF). A study to determine the General Physico-Chemical Properties of the test item indicated that the water solubility of the test item was less than 1.0 mg C/L. Given this it was considered appropriate to test up to a maximum loading rate of 50 mg/L to prevent overloading the aqueous phase with undissolved test item. A 23-Hour stirring period, followed by a 1-Hour standing period was deemed sufficient to ensure equilibration between the test item and aqueous phase.
A nominal amount of test item (125 mg) was added to the surface of 2.5 liters of culture medium to give the 50 mg/L loading rate. A nominal amount of mineral oil (66.3 mg) was added to the surface of 2.5 liters of culture medium to give the mineral oil control at a nominal concentration of 26.5 mg/L. After the addition of the test item and mineral oil, 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 23 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 mineral oil control and the 50 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
- Controls: Mineral oil control and a control group that was maintained under identical conditions but not exposed to the test item
Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. After siphoning, the control, mineral oil control and the 50 mg/L loading rate test concentration were observed to be clear, colorless solutions, whilst at 72 hours were observed to be green dispersions.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none - Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. After siphoning, the control, mineral oil control and the 50 mg/L loading rate test concentration were observed to be clear, colorless solutions, whilst at 72 hours were observed to be green dispersions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Liquid cultures of Pseudokirchneriella subcapitata were obtained from the 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.

TEST ORGANISM
- Common name: Green alga
- Strain: CCAP 278/4.
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation):
- Method of cultivation: 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.

ACCLIMATION
- Culturing media and conditions (same as test or not): The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
none
Hardness:
No details available.
Test temperature:
24 ± 1 ºC
pH:
7.5 - 8.3
Dissolved oxygen:
No details available.
Salinity:
Not applicable.
Conductivity:
Not applicable.
Nominal and measured concentrations:
Range finding test: 5 and 50 mg/L loading rate WAF
Definitve test: 50 mg/L loading rate WAF
Details on test conditions:
Range-Finding Test
The loading rate to be used in the definitive test was determined by a preliminary range-finding test.
The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 5.0 and 50 mg/L for a period of 72 hours. The test item was a complex mixture containing 53% mineral oil. At the Sponsors request additional vessels were prepared containing mineral oil at the same concentration as in the test item preparation; 26.5 mg/L.
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 (10 and 100 mg) were each separately added to the surface of 2 liters of culture medium to give the 5.0 and 50 mg/L loading rates respectively. A nominal amount of mineral oil control (53 mg) was added to the surface of 2 liters to give a nominal loading rate of 26.5 mg/L. 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 23 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 mineral oil control, 5.0 and 50 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 the mineral oil control and the 5.0 and 50 mg/L loading rate WAFs were separately inoculated with algal suspension (2.8 mL) to give the required test concentrations of mineral oil control, 5.0 and 50 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 ºC 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 result of the range-finding test a "limit test" was conducted at a single loading rate of 50 mg/L to confirm that no effect on algal growth was observed.

Experimental Preparation
A nominal amount of test item (125 mg) was added to the surface of 2.5 liters of culture medium to give the 50 mg/L loading rate. A nominal amount of mineral oil (66.3 mg) was added to the surface of 2.5 liters of culture medium to give the mineral oil control at a nominal concentration of 26.5 mg/L. After the addition of the test item and mineral oil, 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 23 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 mineral oil control and the 50 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
An aliquot (1 liter) of the mineral oil control and the 50 mg/L loading rate WAF was separately inoculated with algal suspension (5.2 mL).
The concentration of boron in the test preparations was verified by chemical analysis at 0 and 72 hours.
Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 and 72 hours.

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, mineral oil control and 50 mg/L loading rate WAF 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 9.56 x 105 cells per mL. Inoculation of 1 liter of test medium with 5.2 mL of this algal suspension gave an initial nominal cell density of 5 x 103 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 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.

Assessments
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 103 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, mineral oil control and the 50 mg/L loading rate WAF 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.
Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period.
Chemical Analysis of Test Loading Rates
Samples were taken from the control, mineral oil control and the 50 mg/L loading rate WAF test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken each occasion and stored frozen for further analysis if necessary.
Total Organic Carbon Analysis
Analysis of the WAFs was also carried out by Total Organic Carbon (TOC) analysis. Samples were taken from the uninoculated control, mineral oil control and 50 mg/L loading rate WAF test group at 0 and 72 hours for this analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Data Evaluation
Comparison of Growth Rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:
u = (ln Nn - ln N1) / (tn - t1)
Where: u = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
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 following equation:
Ir = ((uc - ut) / (uc)) * 100
Where: Ir = percentage inhibition of average specific growth rate
uc = mean average specific growth rate for the control cultures
ut = average specific growth rate for the test culture
Comparison of Yield
Yield is calculated as the increase in biomass over the exposure period using the following 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. Percentage inhibition of yield was calculated using the following 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
ELx values were determined by inspection of the growth rate and yield data after 72 hours.

Statistical Analysis
A Student’s t-test was carried out on the growth rate and yield data after 72 hours for the mineral oil control and the 50 mg/L loading rate 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).

Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
• The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
• 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%.
• 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:
EL10
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
biomass
Details on results:
Range-finding Test
The results showed no effect on growth at 5.0 and 50 mg/L loading rate WAF.
Based on this information a single loading rate of six replicates of 50 mg/L was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.

Definitive Test
Chemical Analysis of Test Loading Rates
Analysis of the test preparation at 0 hours showed that a measured concentration of 0.38 mg/L as boron was obtained (equivalent to 10.1 mg/L as test item), and at 72 hours a measured concentration of 0.40 mg/L as boron was obtained (equivalent to 10.7 mg/L as test item).

Total Organic Carbon Analysis
Total Organic Carbon (TOC) analysis of the test preparations performed at 0 and 72 hours showed measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method employed, determined to be 1.0 mg C/L, were obtained.
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.
Results with reference substance (positive control):
A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 – 72 h) : 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h) : 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L
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 of the growth rate data was carried out for the mineral oil control and the 50 mg/L loading rate WAF test group using a Student’s t-test. There were no statistically significant differences (P≥0.05), between the mineral oil control and the 50 mg/L loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 50 mg/L loading rate WAF.
Statistical analysis of the yield data was carried out. There were no statistically significant differences between the mineral oil control and the 50 mg/L loading rate WAF (P≥0.05), therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 50 mg/L loading rate WAF.

Table 1. Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate (mg/L) Cell Densities* (cells per mL) Inhibition Values(%)
0Hours 72 Hours Growth Rate Yield
Control R1 5.52E+03 8.64E+05 - -
R2 6.06E+03 8.68E+05
Mean 5.79E+03 8.66E+05
Mineral Oil Control R1 5.38E+03 8.08E+05 - -
R2 4.75E+03 9.06E+05
Mean 5.06E+03 8.57E+05
5.0 R1 3.44E+03 6.77E+05 [4] 5
R2 4.58E+03 9.49E+05
Mean 4.01E+03 8.13E+05
50 R1 4.29E+03 7.63E+05 [3] [11]
R2 5.39E+03 1.14E+06
Mean 4.84E+03 9.53E+05

* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1 and R2 = Replicates 1 and 2

[Increase in growth compared to controls ]

Table 2 Cell Densities and pH Values in the Definitive Test

Nominal Loading Rate (mg/L) pH Cell Densities* (cells per mL) pH
0h 24h 48h 72 h 72 h
Control R1 7.4 1.83E+04 1.03E+05 5.88E+05 8.3
R2 1.74E+04 1.13E+05 6.33E+05
R3 2.09E+04 1,11E+07 6.50E+05
R4 1.90E+04 1.25E+05 5.66E+05
R5 1.56E+04 9.83E+04 5.48E+05
R6 1.54E+04 1.15E+05 6.46E+05
Mean 1.78E+04 1.11E+05 6.05E+05
Mineral Oil Control R1 7.4 1.89E+04 1.27E+05 6.85E+05 8.5
R2 1.93E+04 1.07E+05 6.51E+05
R3 1.97E+04 9.64E+04 5.23E+05
R4 1.67E+04 9.48E+04 5.25E+05
R5 1.84E+04 1.18E+05 6.51E+05
R6 2.01E+04 1.32E+05 5.98E+05
Mean 1.89E+04 1.12E+05 6.05E+05
50 R1 7.5 1.50E+04 1.09E+05 5.84E+05 8.6
R2 1.87E+04 1.03E+05 5.30E+05
R3 1.94E+04 1.14E+05 5.04E+05
R4 1.35E+04 1.10E+05 5.71E+05
R5 2.00E+04 1.08E+05 5.26E+05
R6 1.53E+04 8.17E+04 4.30E+05
Mean 1.70E+04 1.04E+05 5.24E+05

* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1 - R6 = Replicates 1 to 6

Table 3 Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate Growth Rate (cells/mL/hour) Yield (cells/mL)
(mg/L)   0 - 72h %Inhibition 0 - 72h %Inhibition*
Control R1 0.066   5.83E+05  
R2 0.067   6.28E+05  
R3 0.068   6.45E+05  
R4 0.066 - 5.61E+05 -
R5 0.065   5.43E+05  
R6 0.068   6.41E+05  
Mean 0.067   6.00E+05  
SD 0.001   4.38E+04  
Mineral Oil Control R1 0.068   6.80E+05  
R2 0.068   6.46E+05  
R3 0.065   5.18E+05  
R4 0.065 - 5.20E+05 -
R5 0.068   6.46E+05  
R6 0.066   5.93E+05  
Mean 0.067   6.00E+05  
SD 0.002   6.91E+04  
50 R1 0.066 1 5.79E+05  
R2 0.065 3 5.25E+05  
R3 0.064 4 4.99E+05  
R4 0.066 1 5.66E+05  
R5 0.065 3 5.21E+05  
R6 0.062 7 4.25E+05  
Mean 0.065** 2** 5.38E+05** 10**
SD 0.0008**   3.33E+04**  

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R1 -R6 = Replicates 1 to 6

SD = Standard Deviation

* *Results based on Replicates 1-5 only as replicate 6 determined to be an outlier.

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 50 mg/L loading rate WAF. The No Observed Effect Loading Rate was 50 mg/L loading rate WAF.
Executive summary:

A GLP 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.

Due to the low aqueous solubility and complex nature of the test item for the purposes of testing the test item was prepared as a Water Accommodated Fraction (WAF). A study to determine the General Physico-Chemical Properties of the test item indicated that the water solubility of the test item was less than 1.0 mg C/L. Given this it was considered appropriate to test up to a maximum loading rate of 50 mg/L to prevent overloading the aqueous phase with undissolved test item.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 50 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

The test item was a complex mixture containing 53% mineral oil. At the Sponsors request additional vessels were prepared containing mineral oil at the same concentration as in the test item preparation; 26.5 mg/L.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the test preparation at 0 hours showed that a measured concentration of 0.38 mg/L as boron was obtained (equivalent to 10.1 mg/L as test item), and at 72 hours showed that a measured concentration of 0.40 mg/L as boron was obtained (equivalent to 10.7 mg/L as test item). Concentrations of less than the limit of quantification of the analytical method, determined to be 0.050 mg/L as boron (equivalent to 1.33 mg/L as test item), were obtained in the control and mineral oil control at 0 and 72 hours.

Total Organic Carbon (TOC) analysis of the test preparations performed at 0 and 72 hours showed measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method employed, determined to be 1.0 mg C/L, were obtained.

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. Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 values of greater than 50 mg/L loading rate WAF. The No Observed Effect Loading Rate was 50 mg/L loading rate WAF.

Description of key information

Algal Growth Inhibition Test, OECD Guideline 201, static, freshwater, 72h: ErL50 > 50 mg/L loading rate WAF, NOELr 50 mg/L loading rate WAF

Key value for chemical safety assessment

EC50 for freshwater algae:
50 mg/L
EC10 or NOEC for freshwater algae:
50 mg/L

Additional information

A GLP study (Ablitt, 2017a) 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.

Due to the low aqueous solubility and complex nature of the test item for the purposes of testing the test item was prepared as a Water Accommodated Fraction (WAF). A study to determine the General Physico-Chemical Properties of the test item indicated that the water solubility of the test item was less than 1.0 mg C/L. Given this it was considered appropriate to test up to a maximum loading rate of 50 mg/L to prevent overloading the aqueous phase with undissolved test item.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 50 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

The test item was a complex mixture containing 53% mineral oil. At the Sponsors request additional vessels were prepared containing mineral oil at the same concentration as in the test item preparation; 26.5 mg/L.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the test preparation at 0 hours showed that a measured concentration of 0.38 mg/L as boron was obtained (equivalent to 10.1 mg/L as test item), and at 72 hours showed that a measured concentration of 0.40 mg/L as boron was obtained (equivalent to 10.7 mg/L as test item). Concentrations of less than the limit of quantification of the analytical method, determined to be 0.050 mg/L as boron (equivalent to 1.33 mg/L as test item), were obtained in the control and mineral oil control at 0 and 72 hours.

Total Organic Carbon (TOC) analysis of the test preparations performed at 0 and 72 hours showed measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method employed, determined to be 1.0 mg C/L, were obtained.

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. Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 values of greater than 50 mg/L loading rate WAF. The No Observed Effect Loading Rate was 50 mg/L loading rate WAF.