O-(ethoxycarbonyl)-N-(1-methyl-2-oxo-2-phenylethylidene)hydroxylamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 November 2017 to 14 December 2017

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)

Version / remarks:

2006

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 immediate quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
- Two additional samples of each test preparation were incubated alongside to provide samples for immediate analysis at 24 and 48 hours.
- All sample bottles were fortified with 0.1 mL of formic acid prior to addition of the test sample.

Vehicle:

no

Details on test solutions:

- 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 on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore media preparation trials were conducted in order to determine the solubility of the test material under test conditions
- Based on the results from the Preliminary Media Preparation Trials, in order to attain the maximum dissolved concentration of the parent test material, a saturated solution of the test material was prepared at an initial loading rate of 100 mg/L, stirred for a period of 4 hours prior to the removal of any undissolved test material by filtration through a 0.45 μm Gelman Acrocap filter (first ~100 mL discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution with a nominal concentration of approximately 60 mg/L.

Definitive Test Experimental Preparation
nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 4 hours. After 4 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.45 μm filter (first approximate 100 mL 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 L) of each of the stock solutions was separately inoculated with 3.2 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 were 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, 24, 48 and 72 hours

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 ºC

pH:

pH 5.0 to 7.5 at 0 hours to pH 4.8 to 7.6 at 72 hours.

Nominal and measured concentrations:

Nominal: 10, 18, 32, 56 and 100 % v/v saturated solution.
Geometric mean measured test concentrations: 0.074, 0.097, 0.13, 0.17 and 1.5 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 1.58 x 10^6 cells per mL. Inoculation of 1 L of test medium with 3.2 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 0.1 N 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 21, 46 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 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 trials conducted indicated that a dissolved test material concentration of approximately 60 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 0.10, 1.0, 10 and 100 % v/v saturated solution for a period of 72 hours.
- A nominal amount of test material (1100 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm for 4 hours. After 4 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.45 μm Gelman Acrocap filter (first approximate 100 mL 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, 1.0 and 0.10 % v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (5.6 mL) to give the required test concentrations of 0.10, 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. 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 material.
- Results used to determine the conditions for the definitive study: Yes, based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 % v/v saturated solution.

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.
- Where appropriate 95 % confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).


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)
- The geometric mean for each 24-Hour period, 0-24, 24-48 and 48-72 hours was determined and the arithmetic mean of the three values calculated.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits 0.14 to 0.15 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.074 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.:

0.097 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.094 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence limits 0.088 to 0.10 mg/L

Details on results:

RANGE-FINDING TEST
- The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10 % v/v saturated solution. However, growth was observed to be reduced at 100 % v/v saturated solution.
- Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the LOQ, determined to be 0.032 mg/L, to 25 mg/L. A decline in measured test concentrations was observed at 72 hours in the range of less than the LOQ to 0.11 mg/L confirming that the test material was unstable under test conditions.
- The measured concentration obtained from the 100 % v/v saturated solution at 0 hours was significantly less than that obtained from the media preparation trial (60 mg/L). This was considered to be due to a reduction in solubility due to the salts present in the test media and had no adverse effect on the outcome of the test given that a significant response to exposure was observed at 100 % v/v saturated solution.


DEFINITIVE TEST
Verification of Test Concentrations
- Analysis of the test preparations at 0 hours showed measured test concentrations to range from 2.3 to 29 mg/L. An overall decline in measured test concentrations was observed after each 24-Hour period in the range of less than the limit of quantification (LOQ), determined to be 0.032 mg/L, to 0.62 mg/L at 24 hours, less than the LOQ to 0.13 mg/L at 48 hours and from less than the LOQ to 0.17 mg/L at 72 hours.
- Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.016 mg/L) was used to enable calculation of the geometric mean measured concentration. The geometric mean measured test concentrations were determined to be: 0.074, 0.097, 0.13, 0.17 and 1.5 mg/L at 10, 18, 32, 56 and 100 % v/v saturated solutions, respectively.

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. Accordingly the following results were determined from the data based on the geometric mean measured test concentrations:
- Inhibition of Growth Rate:
ErC10 (0 to 72 hour): 0.12 mg/L
ErC20 (0 to 72 hour): 0.13 mg/L
ErC50 (0 to 72 hour): 0.14 mg/L; 95 % confidence limits 0.14 to 0.15 mg/L
Where ErCx is the test concentration that reduced growth rate by x %.
Statistical analysis of the growth rate data was carried out for the control, 0.074, 0.097, 0.13 and 0.17 mg/L test groups 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 0.074 mg/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 0.074 mg/L. Correspondingly the Lowest Observed Effect Concentration (LOEC) based on growth rate was 0.097 mg/L.

- Inhibition of Yield:
EyC10 (0 to 72 hour): 0.078 mg/L
EyC20 (0 to 72 hour): 0.081 mg/L
EyC50 (0 to 72 hour): 0.094 mg/L; 95 % confidence limits 0.088 to 0.10 mg/L
Where:
EyCx is the test concentration that reduced yield by x %.
Statistical analysis of the yield data was carried out as above. There were no statistically significant differences between the control and 0.074 mg/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 0.074 mg/L. Correspondingly the LOEC based on yield was 0.097 mg/L.

Validation Criteria
- The following data show that the cell concentration of the control cultures increased by a factor of 128 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: 6.41 x 10^5 cells per mL
- The mean coefficient of variation for section by section specific growth rate for the control cultures was 11% 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 %.

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 test cultures at 0.074 mg/L. Some misshapen cells were observed to be present in the 0.13 mg/L test cultures whilst cell debris was observed to be present in the 0.17 and 1.5 mg/L test cultures. Due to a technical oversight, no observations were made on the 0.097 mg/L test cultures, this was considered to have had no adverse effect on the outcome of the test.

Water Quality Criteria
- Temperature was maintained at 24 ± 1 °C throughout the test.
- The pH value of the control cultures was observed to range from pH 7.5 at 0 hours to pH 7.3 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.

Observations on 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 and 0.074 mg/L test cultures were observed to be green dispersions. The 0.097 and 0.13 mg/L test cultures were observed to be extremely pale green dispersions whilst the 0.17 and 1.5 mg/L test cultures were observed to remain clear colourless solutions.

Results with reference substance (positive control):

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, 0.074, 0.097, 0.13 and 0.17 mg/L 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). The 1.5 mg/L test concentration was not included in the analysis as visual inspection of the data showed a significant effect on growth.

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

Geometric Mean Measured Test Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0 Hours	21 Hour	46 Hour	72 Hour	72 Hours
Control	R1	7.5	1.99E+04	1.13E+05	6.78E+05	7.3
	R2		1.76E+04	8.80E+04	7.23E+05
	R3		1.63E+04	9.89E+04	6.46E+05
	R4		1.87E+04	1.12E+05	6.27E+05
	R5		1.58E+04	1.03E+05	5.04E+05
	R6		1.67E+04	1.02E+05	6.69E+05
	Mean		1.75E+04	1.03E+05	6.41E+05
0.074	R1	7.2	2.19E+04	1.30E+05	7.57E+05	7.6
	R2		2.21E+04	1.20E+05	6.76E+05
	R3		1.60E+04	1.08E+05	6.93E+05
	Mean		2.00E+04	1.19E+05	7.09E+05
0.097	R1	6.6	1.43E+04	1.04E+05	2.74E+05	7.5
	R2		1.70E+04	8.33E+04	1.73E+05
	R3		1.67E+04	1.08E+05	4.18E+05
	Mean		1.60E+04	9.86E+04	2.89E+05
0.13	R1	6.4	1.58E+04	7.52E+04	1.94E+05	7.5
	R2		1.83E+04	5.38E+04	2.24E+05
	R3		1.90E+04	7.39E+04	2.05E+05
	Mean		1.77E+04	6.77E+04	2.08E+05
0.17	R1	5.6	4.90E+03	4.66E+03	1.01E+04	6.4
	R2		4.90E+03	2.67E+03	4.87E+03
	R3		4.40E+03	2.46E+03	2.26E+03
	Mean		4.73E+03	3.27E+03	5.75E+03
1.5	R1	5.0	2.20E+03	2.46E+03	1.09E+03	4.8
	R2		2.29E+03	3.05E+03	1.58E+03
	R3		2.58E+03	3.78E+03	1.06E+03
	Mean		2.36E+03	3.10E+03	1.24E+03

* =   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

R =  Replicate

Table 2: Inhibition of Growth Rate and Yield in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.068	-	6.73E+05	-
	R2	0.069		7.18E+05
	R3	0.068		6.41E+05
	R4	0.067		6.22E+05
	R5	0.064		4.99E+05
	R6	0.068		6.64E+05
	Mean	0.067		6.36E+05
	SD	0.002		7.46E+04
0.074	R1	0.070	[4]	7.52E+05
	R2	0.068	[1]	6.71E+05
	R3	0.068	[1]	6.88E+05
	Mean	0.069	[2]	7.04E+05	[11]
	SD	0.001		4.26E+04
0.097	R1	0.056	16	2.69E+05
	R2	0.049	27	1.68E+05
	R3	0.061	9	4.13E+05
	Mean	0.055	17	2.84E+05	55
	SD	0.006		1.23E+05
0.13	R1	0.051	24	1.89E+05
	R2	0.053	21	2.19E+05
	R3	0.052	22	2.00E+05
	Mean	0.052	22	2.03E+05	68
	SD	0.001		1.53E+04
0.17	R1	0.010	85	5.12E+03
	R2	0.000	100	-1.31E+02
	R3	-0.011	116	-2.74E+03
	Mean	0.000	100	7.49E+02	100
	SD	0.011		4.00E+03
1.5	R1	-0.021	131	-3.91E+03
	R2	-0.016	124	-3.42E+03
	R3	-0.022	133	-3.94E+03
	Mean	-0.020	129	-3.76E+03	101
	SD	0.003		2.97E+02

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

R = Replicate

SD = Standard Deviation

[ ] = Increase in growth compared to controls

- = Not applicable

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 0.14 mg/L (95 % Confidence Limits 0.14-0.15) and yield EC50 0.094 mg/L (95 % Confidence Limits: 0.088 - 0.10). The NOEC for growth rate and yield was 0.074 mg/L and the LOEC for growth rate and yield was 0.097 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 guideline OECD 201 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.

Preliminary media preparation trials indicated that a dissolved test material concentration of approximately 60 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, 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 (100 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm for 4 hours. After the stirring period any undissolved test material was removed by filtration (0.45 μm Gelman Acrocap filter, first approximate 100 mL 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.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 2.3 to 29 mg/L. An overall decline in measured test concentrations was observed after each 24-Hour period in the range of less than the limit of quantification (LOQ), determined to be 0.032 mg/L, to 0.62 mg/L at 24 hours, less than the LOQ to 0.13 mg/L at 48 hours and from less than the LOQ to 0.17 mg/L at 72 hours.

Given this decline in measured concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.074, 0.097, 0.13, 0.17 and 1.5 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 0.14 mg/L (95 % Confidence Limits 0.14-0.15) and yield EC50 0.094 mg/L (95 % Confidence Limits: 0.088 - 0.10). The NOEC for growth rate and yield was 0.074 mg/L and the LOEC for growth rate and yield was 0.097 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 0.14 mg/L (95 % Confidence Limits 0.14-0.15) and yield EC50 0.094 mg/L (95 % Confidence Limits: 0.088 - 0.10). The NOEC for growth rate and yield was 0.074 mg/L and the LOEC for growth rate and yield was 0.097 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.14 mg/L

EC10 or NOEC for freshwater algae:

0.074 mg/L

Additional information

The effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata was investigated in accordance with the standardised guideline OECD 201 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.

Preliminary media preparation trials indicated that a dissolved test material concentration of approximately 60 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, 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 (100 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm for 4 hours. After the stirring period any undissolved test material was removed by filtration (0.45 μm Gelman Acrocap filter, first approximate 100 mL 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.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 2.3 to 29 mg/L. An overall decline in measured test concentrations was observed after each 24-Hour period in the range of less than the limit of quantification (LOQ), determined to be 0.032 mg/L, to 0.62 mg/L at 24 hours, less than the LOQ to 0.13 mg/L at 48 hours and from less than the LOQ to 0.17 mg/L at 72 hours.

Given this decline in measured concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.074, 0.097, 0.13, 0.17 and 1.5 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 0.14 mg/L (95 % Confidence Limits 0.14-0.15) and yield EC50 0.094 mg/L (95 % Confidence Limits: 0.088 - 0.10). The NOEC for growth rate and yield was 0.074 mg/L and the LOEC for growth rate and yield was 0.097 mg/L.