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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:
11 October 2017 to 20 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Samples were taken from the control and each 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 at 0 hour and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Preliminary solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
- Based on this information the test material was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document, therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.
- A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 56, 32, 18 and 10 % v/v saturated solution. An aliquot (1 liter) of each of the stock solutions was separately inoculated with 7.3 mL of algal suspension to give the required test concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution.
- The stock solutions and each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.
- The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ± 1 °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 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.
- Culturing media and conditions: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ± 1 °C
pH:
7.1 - 7.7
Nominal and measured concentrations:
Nominal:10, 18, 32, 56 and 100 % v/v Saturated Solution
Measured 0 hours: Measured 72 hours: Geometric mean measured test concentrations: 0.0050, 0.012, 0.051 and 0.047 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: closed- the flasks were plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: 100 mL
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 6.87 x 10^5 cells per mL. Inoculation of 1 L of test medium with 7.3 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
- No. of vessels per concentration: 3
- No. of vessels per control: 6

TEST MEDIUM
- The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
- Culture medium: NaNO3 25.5 mg/L, MgCl2.6H2O 12.16 mg/L, CaCl2.2H2O 4.41 mg/L, MgSO4.7H2O 14.6 mg/L, K2HPO4 1.044 mg/L, NaHCO3 15.0 mg/L, H3BO3 0.186 mg/L, MnCl2.4H2O 0.415 mg/L, ZnCl2 0.00327 mg/L, FeCl3.6H2O 0.160 mg/L, CoCl2.6H2O 0.00143 mg/L, Na2MoO4.2H2O 0.00726 mg/L, CuCl2.2H2O 0.000012 mg/L and Na2EDTA.2H2O 0.30 mg/L.
- The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.
- Water Quality Criteria: The pH of the control and each test preparation 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.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm)
- Constantly shaken at approximately 150 rpm for 72 hours.

EFFECT PARAMETERS MEASURED:
- Test Organism Observations: Samples were taken at 23 and 48 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Due to technical constraints, at 72 hours cell densities were determined using a haemocytometer and light microscope. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
- To determine the potential effect of the test material 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.
- Test material concentrations were analytically verified.

TEST CONCENTRATIONS
- Range finding study: The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.025 mg/L could be obtained using a saturated solution method of preparation. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 1.0, 10 and 100 % v/v saturated solution for a period of 72 hours.
- A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10 and 1.0% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (5.7 mL) to give the required test concentrations of 1.0, 10 and 100% v/v saturated solution.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
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 used for each control and test concentration.
The control group was maintained under identical conditions but not exposed to the test item.
- Results used to determine the conditions for the definitive study: Yes, based on the results of the range-finding test an initial experiment was conducted at concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution. A concentration dose response was not observed at 56 and 100 % v/v saturated solution after 72 hours exposure and as such the test was repeated.

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:

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 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 material 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 material vessel was calculated using the following equation:

Ir = [(µc - µt) / µc] x 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 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 ] x 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 ECx 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). ECx values were then determined from the equation for the fitted line.
- It was not possible to calculate 95 % confidence limits for the EC50 values as the data generated did not fit the models available for the calculation of confidence limits.


GEOMETRIC MEAN MEASURED CONCENTRATIONS
The geometric mean measured test concentrations of the samples were calculated as follows:

GM = √(C0 x C1)

Where:
GM = geometric mean measured test concentration (mg/L)
C0 = measured concentration at the start of the test (mg/L)
C1 = measured concentration at the end of the test (mg/L)
Where a measured concentration of less than the LOQ was obtained, a value of ½ the LOQ was substituted into the equation.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.015 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.035 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.047 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: It was not possible to calculate an ErC50 value as no more that 23 % inhibition of growth rate occurred at the maximum attainable dissolved test material concentration.
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.005 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.012 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.01 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.011 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.014 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
RANGE-FINDING TEST
- The results showed no effect on growth at the test concentrations of 1.0 and 10 % v/v saturated solution. However, growth was observed to be reduced at 100 % v/v saturated solution. Based on this information test concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution were selected for the definitive test.
- Chemical analysis of the 10 and 100 % v/v saturated solution test preparations at 0 and 72 hours showed measured test concentrations of less than the LOQ of the analytical method employed were obtained which was determined to be 0.0099 mg/L. This does not infer that no test material was in solution, just that any dissolved test material present was at a concentration of less than the LOQ.

INITIAL EXPERIMENT
- Based on the results of the range-finding test an initial experiment was conducted at concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution. A concentration dose response was not observed at 56 and 100 % v/v saturated solution after 72 hours exposure and as such the test was repeated.

DEFINITIVE TEST
- Verification of Test Concentrations: Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the LOQ of the analytical method employed, determined to be 0.0099 mg/L, to 0.063 mg/L. A decline in measured test concentration was observed at 72 hours to between less than the LOQ and 0.036 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations only in order to give a “worst case” analysis of the data.
The geometric mean measured test concentrations were determined to be: 0.0050, 0.012, 0.051 and 0.047 mg/L.

- Growth Data: The growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test material over the 72 Hour exposure period:
ErC10 (0 to 72 hour): 0.015 mg/L
ErC20 (0 to 72 hour): 0.035 mg/L
ErC50 (0 to 72 hour): >0.047 mg/L
It was not possible to calculate an ErC50 value as no more than 23 % inhibition of growth rate occurred at the maximum attainable dissolved test material concentration.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations 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, 10 and 18 % v/v saturated solution test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 18 % v/v saturated solution, equivalent to a geometric mean measured test concentration of 0.0050 mg/L. Correspondingly the Lowest Observed Effect Concentration (LOEC) based on growth rate 32 % v/v saturated solution, equivalent to a geometric mean measured test concentration of 0.012 mg/L.

- Inhibition of Yield:
EyC10 (0 to 72 hour): 0.0099 mg/L
EyC20 (0 to 72 hour): 0.011 mg/L
EyC50 (0 to 72 hour): 0.014 mg/L

There were no statistically significant differences between the control, 10 and 18 % v/v saturated solution test concentrations (P ≥ 0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 18 % v/v saturated solution, equivalent to a geometric mean measured test concentration of 0.0050 mg/L. Correspondingly the Lowest Observed Effect Concentration (LOEC) based on yield 32 % v/v saturated solution, equivalent to a geometric mean measured test concentration of 0.012 mg/L.


OBSERVATIONS ON CULTURES
- All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

WATER QUALITY CRITERIA
- The pH value of the control cultures was observed to range from pH 7.2 at 0 hours to pH 7.1 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

TEST MATERIAL SOLUBILITY
- At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, 10, 18 and 32 % v/v saturated solution test preparations were observed to be green dispersions. The 56 % v/v saturated solution test preparations were observed to be pale green dispersions whilst the 100 % v/v saturated solution test preparations were observed to be extremely pale green dispersions.
Results with reference substance (positive control):
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference material gave the following results:
ErC50 (0 to 72 hour): 1.6 mg/L; 95 % confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour): 0.77 mg/L; 95 % confidence limits 0.68 to 0.87 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference material.
Reported statistics and error estimates:
- 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 concentrations 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 following data show that the cell concentration of the control cultures increased by a factor of 298 after 72 hours.  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.

Nominal cell density of control at 0 hours: 5.00 x 10^3 cells per mL

Mean cell density of control at 72 hours: 1.49 x 10^6 cells per mL

- The mean coefficient of variation for section by section specific growth rate for the control cultures was 8 % 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 to 72 hours) was 3 % and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7 %.

Table 1: Cell Densities and pH Values in the Definitive Test

Nominal Concentration
(% v/v Saturated Solution)

pH

Cell Densities* (cells per mL)

pH

0 Hour

23 Hour

48 Hour

72 Hour

72 Hour

Control

R1

7.2

3.66E+04

2.55E+05

1.90E+06

7.1

R2

3.33E+04

2.31E+05

1.42E+06

R3

3.63E+04

2.19E+05

1.45E+06

R4

3.91E+04

2.38E+05

1.37E+06

R5

3.68E+04

2.29E+05

1.42E+06

R6

3.42E+04

2.48E+05

1.38E+06

Mean

3.61E+04

2.37E+05

1.49E+06

10

R1

7.2

3.48E+04

2.45E+05

1.62E+06

7.5

R2

3.84E+04

2.17E+05

1.28E+06

R3

3.64E+04

2.18E+05

1.28E+06

Mean

3.66E+04

2.27E+05

1.39E+06

18

R1

7.2

3.37E+04

2.36E+05

1.55E+06

7.5

R2

3.60E+04

2.40E+05

1.38E+06

R3

3.30E+04

1.88E+05

1.73E+06

Mean

3.42E+04

2.22E+05

1.55E+06

32

R1

7.2

3.11E+04

2.11E+05

1.07E+06

7.7

R2

3.16E+04

1.78E+05

1.05E+06

R3

2.83E+04

1.84E+05

9.34E+05

Mean

3.03E+04

1.91E+05

1.02E+06

56

R1

7.2

2.95E+04

1.61E+05

5.34E+05

7.7

R2

2.77E+04

1.49E+05

6.34E+05

R3

2.69E+04

9.78E+04

6.00E+05

Mean

2.81E+04

1.36E+05

5.89E+05

100

R1

7.3

2.31E+04

1.06E+05

5.00E+05

7.6

R2

2.08E+04

8.47E+04

3.17E+05

R3

2.05E+04

7.98E+04

3.84E+05

Mean

2.15E+04

9.00E+04

4.00E+05

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts or fields of view for each of the replicate flask

R=  Replicate

Table 2: Daily Specific Growth Rates for the Control Cultures in the Definitive Test

Treatment

Daily Specific Growth Rate (cells/mL/hour)

Day 0 to 1

Day 1 to 2

Day 2 to 3

Control

R1

0.087

0.078

0.084

R2

0.082

0.078

0.076

R3

0.086

0.072

0.079

R4

0.089

0.072

0.073

R5

0.087

0.073

0.076

R6

0.084

0.079

0.072

Mean

0.086

0.075

0.077

R= Replicate

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

Nominal Concentration
(% v/v Saturated Solution)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 to 72 Hour

% Inhibition

0 to 72 Hour

% Inhibition*

Control

R1

0.083

-

1.90E+06

-

R2

0.078

1.42E+06

R3

0.079

1.45E+06

R4

0.078

1.37E+06

R5

0.078

1.42E+06

R6

0.078

1.38E+06

Mean

0.079

1.49E+06

SD

0.002

2.03E+05

10

R1

0.080

[1]

1.62E+06

 

R2

0.077

3

1.28E+06

 

R3

0.077

3

1.28E+06

 

Mean

0.078

2

1.39E+06

7

SD

0.002

 

1.96E+05

 

18

R1

0.080

[1]

1.55E+06

 

R2

0.078

1

1.38E+06

 

R3

0.081

[3]

1.73E+06

 

Mean

0.080

[1]

1.55E+06

[4]

SD

0.002

 

1.75E+05

 

32

R1

0.075

5

1.07E+06

 

R2

0.074

6

1.05E+06

 

R3

0.073

8

9.29E+05

 

Mean

0.074

6

1.01E+06

32

SD

0.001

 

7.34E+04

 

56

R1

0.065

18

5.29E+05

 

R2

0.067

15

6.29E+05

 

R3

0.066

16

5.95E+05

 

Mean

0.066

16

5.84E+05

61

SD

0.001

 

5.08E+04

 

100

R1

0.064

19

4.95E+05

 

R2

0.058

27

3.12E+05

 

R3

0.060

24

3.79E+05

 

Mean

0.061

23

3.95E+05

73

SD

0.003

 

9.26E+04

 

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

R = Replicate

SD = Standard Deviation

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, exposure of Pseudokirchneriella subcapitata to the test material gave the following results based on the geometric mean measured test concentrations: Growth rate EC50 >0.047 mg/L and yield EC50 0.014 mg/L. The NOEC for growth rate and yield was 0.0050 mg/L and the LOEC for growth rate and yield was 0.012 mg/L.
Executive summary:

The effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 0.025 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test and initial experiment, Pseudokirchneriella subcapitata was exposed to solutions of the test material at nominal concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution (3 replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. The test material solutions were prepared by stirring an excess (50 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a 100 % v/v saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

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

Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0099 mg/L, to 0.063 mg/L. A decline in measured test concentration was observed at 72 hours to between less than the LOQ and 0.036 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations only in order to give a “worst case” analysis of the data. The geometric mean measured test concentration for the 18, 32, 56 and 100 % v/v saturated solution test preparations were determined to be 0.0050, 0.012, 0.015 and 0.047 mg/L.

Under the conditions of this study, exposure of Pseudokirchneriella subcapitata to the test material gave the following results based on the geometric mean measured test concentrations: Growth rate EC50 >0.047 mg/L, yield EC50 0.014 mg/L. The NOEC for growth rate and yield was 0.0050 mg/L and the LOEC for growth rate and yield was 0.012 mg/L.

Description of key information

Under the conditions of this study, exposure of Pseudokirchneriella subcapitata to the test material gave the following results based on the geometric mean measured test concentrations: Growth rate EC50 >0.047 mg/L, yield EC50 0.014 mg/L. The NOEC for growth rate and yield was 0.0050 mg/L and the LOEC for growth rate and yield was 0.012 mg/L.

Key value for chemical safety assessment

Additional information

The effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 0.025 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test and initial experiment, Pseudokirchneriella subcapitata was exposed to solutions of the test material at nominal concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution (3 replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. The test material solutions were prepared by stirring an excess (50 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

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

Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0099 mg/L, to 0.063 mg/L. A decline in measured test concentration was observed at 72 hours to between less than the LOQ and 0.036 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations only in order to give a “worst case” analysis of the data. The geometric mean measured test concentration for the 18, 32, 56 and 100 % v/v saturated solution test preparations were determined to be 0.0050, 0.012, 0.015 and 0.047 mg/L.

Under the conditions of this study, exposure of Pseudokirchneriella subcapitata to the test material gave the following results based on the geometric mean measured test concentrations: Growth rate EC50 >0.047 mg/L, yield EC50 0.014 mg/L. The NOEC for growth rate and yield was 0.0050 mg/L and the LOEC for growth rate and yield was 0.012 mg/L.