Dipotassium hexacyanocobalt(II)-ferrate(II)

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:

18.12.2017-30.5.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed according to the recommended Guidelines (OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation (EC) No 761/2009) and GLP. The report fully and accurately reflects the procedures used and data generated. There were no circumstances considered to have affected the integrity of the study or the validity of the data.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(EC) No 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate is included in the study report.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control (0), 0.10, 0.32, 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
- Sampling method: Duplicate 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. The concentration of both cobalt and iron was determined from the samples.
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC, 1996 and OECD, 2000), is to expose organisms to a saturated solution of the test item in cases where the test item is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared.

At the request of the Sponsor, a four phase washing process was employed to replicate the end-point use of the test item.

A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of vigorous magnetic stirring for 15 minutes. After stirring the dispersion was filtered through a coarse filter paper and the filtrate discarded. The test item collected in the filter paper was re-suspended in culture medium and stirred for a further period of 15 minutes. This process was repeated a further three times. After the final stirring phase any undissolved test item present was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 1 liter discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. A series of dilutions was made from this saturated solution to give stock solutions of 32, 10, 3.2, 1.0, 0.32 and 0.10% v/v saturated solution. An aliquot (900 mL) of each of the stock solutions was separately inoculated with 4.7 mL of algal suspension to give the required test concentrations of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
The stock solutions and each prepared concentration 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 and 72 hours.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter prior to use to give a 100% v/v saturated solution.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Liquid cultures from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.

For the tests the algae were cultivated in flasks that 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. The culture medium used for the tests was the same as that used to maintain the stock culture.

ACCLIMATION
- Acclimation period: 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 (approx. 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 - 10^5 cells/mL.
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

All test and control cultures were inspected microscopically at 72 hours.

Test temperature:

Temperature was maintained at 24 ± 1 ºC throughout the test.

pH:

The pH value of the test solutions: 7.6-9.1. The pH value of the control cultures was observed to increase from pH 7.8 at 0 hours to pH 8.6 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.

Nominal and measured concentrations:

Analysis of the test preparations at 0 and 72 hours showed measured iron concentrations to be less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.010 mg/L.

Analysis of the 0.10, 0.32, 1.0 and 3.2% v/v saturated solution test preparations at 0 and 72 hours showed measured cobalt concentrations of less than the LOQ were obtained, determined to be 0.0010 mg/L. Measured cobalt concentrations in the range of 0.0022 to 0.023 mg/L; equivalent to dissolved test item concentrations of 0.013 to 0.14 mg/L were obtained from the 10, 32 and 100% v/v saturated solution test preparations at 0 hours. As measured concentrations at 72 hours were within 80-120% of the 0-Hour measured concentrations it was considered appropriate to calculate the results based on the equivalent test item concentrations determined at 0 hours only.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks plugged with polyurethane foam bungs
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 250 mL, fill volume 100 mL, headspace 150 mL
- Aeration: constant shaking at approximately 150 rpm
- Initial cells density: initial nominal cell density of 5 x 10^3 cells per mL
- Control end cells density: 1.27 x 10^6 cells per mL
- No. of vessels per concentration (replicates): Three flasks for each treatment group
- No. of vessels per control (replicates): Six flasks for the control
- No. of vessels per positive control (replicates): Three flasks for each positive control concentration (potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L)

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionized water (Elga Optima 15+ or Elga Purelab Option R-15 BP)
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No. The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure.
- Photoperiod: continuous illumination
- Light intensity and quality: intensity approximately 7000 lux, warm white lighting (380 – 730 nm)
- The appearance of the test media was recorded daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [electronic particle counter] Samples of the algal populations were taken at 0, 21, 49 and 72 hours and cell concentrations determined for each control and treatment group, 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.
- Other: 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.
Duplicate samples were also 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.

TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study: Information provided by results from earlier water solubility testing and aquatic toxicity testing indicated the water solubility of the test item to be less than or equal to 0.89 mg/L. Given this and the pure nature of the test item, a saturated solution method of preparation was considered most appropriate. An initial experiment conducted at concentrations of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100% v/v saturated solution was terminated after 24 hours exposure due to significant and variable levels of inhibition across the control and all test concentrations. As such testing was repeated.
- Test concentrations in the definitive study: 0.10, 0.32, 1.0, 3.2, 10, 32 and 100% v/v saturated solution.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Remarks:

It was not possible to calculate an ErC50 value as no more than 26% inhibition of growth rate occurred at the maximum dissolved test item concentration of 0.14 mg/L.

Effect conc.:

> 0.14 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Remarks:

Cobalt

Basis for effect:

growth rate

Remarks on result:

not determinable

Remarks:

It was not possible to calculate an ErC50 value as no more than 26% inhibition of growth rate occurred at the maximum attainable dissolved test item concentration of 0.14 mg/L.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.073 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Remarks:

Cobalt

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Remarks:

Cobalt

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

The NOEC based on growth rate was 10% v/v saturated solution (equivalent to 0.013 mg/L as test item).

Effect conc.:

0.013 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Remarks:

Cobalt

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

The LOEC based on growth rate was 32% v/v saturated solution (equivalent to 0.042 mg/L as test item).

Effect conc.:

0.042 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Remarks:

Cobalt

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.081 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Remarks:

Cobalt

Basis for effect:

cell number

Remarks:

Yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.029 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Remarks:

Cobalt

Basis for effect:

cell number

Remarks:

Yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.042 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Remarks:

Cobalt

Basis for effect:

cell number

Remarks:

YIeld

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

The NOEC based on yield was 10% v/v saturated solution (equivalent to 0.013 mg/L as test item).

Effect conc.:

0.013 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Remarks:

Cobalt

Basis for effect:

cell number

Remarks:

Yield

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

The Lowest Observed Effect Concentration (LOEC) based on yield was 32% v/v saturated solution (equivalent to 0.042 mg/L as test item).

Effect conc.:

0.042 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Remarks:

Cobalt

Basis for effect:

cell number

Remarks:

Yield

Details on results:

- Exponential growth in the control (for algal test): yes. The cell concentration of the control cultures increased by a factor of 255 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.
- Observation of abnormalities (for algal test): After 72 hours there were no abnormalities detected in the control or test cultures at 0.32, 1.0, 10 and 100% v/v saturated solution.
- Unusual cell shape: Some enlarged cells were observed to be present in the 0.10, 3.2 and 32% v/v saturated solution test cultures after 72 hours of exposure.
- Colour differences: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.10, 0.32, 1.0, 3.2, 10 and 32% v/v saturated solution test cultures were observed to be green dispersions whilst the 100% v/v saturated solution test cultures were observed to be light green dispersions.
- Validation criteria: the validation criteria in terms of cell concentration increase, the mean of the coefficients of variation of the section by section daily growth rates in the control cultures and the coefficient of variation of the average specific growth rate in replicate control cultures were satisfied. 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 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.
- Any stimulation of growth found in any treatment: Slight increase in growth rate as compared to controls observed in singular or all replicates of concentrations 0.1, 0.32, 1.0, 3.2and 10 % v/v Saturated Solution (see Table 3)
- Other: 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. It was not possible to calculate an ErC50 value as no more than 26% inhibition of growth rate occurred at the maximum dissolved test item concentration of 0.14 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Growth rate ErC50 (0 to 72 hour) : 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
- Yield EyC50 (0 to 72 hour) : 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L
- Other: The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control 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.

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 h		21 h	49 h	72 h	72 h
Control	R1	7.8		2.83E+04	2.37E+05	1.24E+06	8.6
	R2			2.82E+04	2.21E+05	1.14E+06
	R3			3.01E+04	2.21E+05	1.17E+06
	R4			2.80E+04	2.45E+05	1.24E+06
	R5			2.95E+04	2.43E+05	1.45E+06
	R6			2.92E+04	2.67E+05	1.41E+06
	Mean			2.89E+04	2.39E+05	1.27E+06
0.10	R1	7.9		2.88E+04	2.15E+05	1.30E+06	8.5
	R2			2.34E+04	2.01E+05	1.23E+06
	R3			2.63E+04	2.29E+05	1.61E+06
	Mean			2.62E+04	2.15E+05	1.38E+06
0.32	R1	7.9		2.74E+04	2.41E+05	1.37E+06	8.5
	R2			2.75E+04	2.22E+05	1.50E+06
	R3			3.08E+04	2.38E+05	1.63E+06
	Mean			2.86E+04	2.34E+05	1.50E+06
1.0	R1	7.8		3.11E+04	2.12E+05	1.22E+06	9.1
	R2			2.91E+04	2.19E+05	1.36E+06
	R3			2.96E+04	2.05E+05	1.29E+06
	Mean			2.99E+04	2.12E+05	1.29E+06
3.2	R1	7.7		2.75E+04	2.40E+05	1.42E+06	9.0
	R2			3.07E+04	2.47E+05	1.50E+06
	R3			2.95E+04	2.09E+05	1.21E+06
	Mean			2.92E+04	2.32E+05	1.38E+06
10	R1	7.7		3.05E+04	2.42E+05	1.37E+06	9.0
	R2			3.14E+04	2.51E+05	1.38E+06
	R3			3.09E+04	1.96E+05	1.21E+06
	Mean			3.09E+04	2.30E+05	1.32E+06
32	R1	7.7		2.82E+04	1.86E+05	9.26E+05	8.6
	R2			2.87E+04	2.13E+05	1.04E+06
	R3			2.96E+04	1.91E+05	1.05E+06
	Mean			2.88E+04	1.97E+05	1.00E+06
100	R1	7.6		1.64E+04	5.60E+04	3.16E+05	8.2
	R2			2.18E+04	6.63E+04	3.25E+05
	R3			1.92E+04	4.82E+04	2.94E+05
	Mean			1.91E+04	5.68E+04	3.12E+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 2. Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.083	0.076	0.072
	R2	0.082	0.074	0.071
	R3	0.085	0.071	0.072
	R4	0.082	0.077	0.071
	R5	0.084	0.075	0.078
	R6	0.084	0.079	0.072
	Mean	0.083	0.075	0.073

R1 - R6 = Replicates 1 to 6

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 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.077	-	1.23E+06	-
	R2	0.075		1.14E+06
	R3	0.076		1.17E+06
	R4	0.077		1.23E+06
	R5	0.079		1.44E+06
	R6	0.078		1.41E+06
	Mean	0.077		1.27E+06
	SD	0.001		1.25E+05
0.1	R1	0.077	0	1.30E+06
	R2	0.076	1	1.22E+06
	R3	0.080	[4]	1.60E+06
	Mean	0.078	[1]	1.38E+06	[8]
	SD	0.002		2.01E+05
0.32	R1	0.078	[1]	1.36E+06
	R2	0.079	[3]	1.50E+06
	R3	0.080	[4]	1.63E+06
	Mean	0.079	[3]	1.50E+06	[18]
	SD	0.001		1.32E+05
1.0	R1	0.076	1	1.22E+06
	R2	0.078	[1]	1.35E+06
	R3	0.077	0	1.29E+06
	Mean	0.077	0	1.28E+06	[1]
	SD	0.001		6.60E+04
3.2	R1	0.078	[1]	1.41E+06
	R2	0.079	[3]	1.49E+06
	R3	0.076	1	1.21E+06
	Mean	0.078	[1]	1.37E+06	[8]
	SD	0.002		1.46E+05
10	R1	0.078	[1]	1.37E+06
	R2	0.078	[1]	1.38E+06
	R3	0.076	1	1.20E+06
	Mean	0.077	[0]	1.32E+06	[4]
	SD	0.001		9.98E+04
32	R1	0.073	5	9.21E+05
	R2	0.074	4	1.03E+06
	R3	0.074	4	1.04E+06
	Mean	0.074	4	9.98E+05	21
	SD	0.001		6.70E+04
100	R1	0.058	25	3.11E+05
	R2	0.058	25	3.20E+05
	R3	0.057	26	2.89E+05
	Mean	0.058	25	3.07E+05	76
	SD	0.001		1.63E+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

[Increase in growth as compared to controls]

Validity criteria fulfilled:

yes

Remarks:

Validation criteria fulfilled: cell concentration increase, mean coefficients of variation of daily growth rates and the coefficient of variation of average specific growth rate in the control cultures

Conclusions:

The effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata has been investigated over a 72-hour period.

Exposure of Pseudokirchneriella subcapitata to the saturated solution of the test item gave results based on the 0-Hour measured test concentrations for growth rate.

The No Observed Effect Concentration for growth rate was 0.013 mg/L. The Lowest Observed Effect Concentration for growth rate was 0.042 mg/L. It was not possible to calculate an ErC50 value as no more than 26% inhibition of growth rate occurred at the maximum attainable dissolved test item concentration of 0.14 mg/L (EC50 > 100 % v/v saturated solution).

The EC50 values for yield were 0.081 mg/L, with a No Observed Effect Concentration of 0.013 mg/L and a Lowest Observed Effect Concentration of 0.042 mg/L for yield.

Significant effects on growth rate were only seen at 100% v/v saturated solution. As the EC50 vallue for the test item based on this test would be EC50 > 100 % v/v saturated solution, the results warrant a hazard classification of Aquatic Chronic 4, H413 for the substance, as some concern still remains.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in 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) No 761/2009.

Methods

Information based on earlier aquatic toxicity and water solubility testing indicated the water solubility of the test item to be less than or equal to 0.89 mg/L. Given this and the pure nature of the test item, a saturated solution method of preparation was considered most appropriate. At the request of the Sponsor, a four phase washing process was employed to replicate the final step of the manufacturing process.

Following an initial experiment, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. The test item solutions were prepared following a four phase washing process. A saturated solution of the test item was prepared by stirring an excess (100 mg/L) of test item in culture medium via vigorous magnetic stirring for 15 minutes. After stirring the dispersion was filtered through a coarse filter paper and the filtrate discarded. The test item collected in the filter paper was re-suspended in culture medium and stirred for a further period of 15 minutes. This process was repeated a further three times. After the final stirring phase any undissolved test item present was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 1 liter discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. 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.

Results

Analysis of the test preparations was performed for the concentration of iron and cobalt present. The equivalent test item concentrations were then determined based on the cobalt content of test item.

Analysis of the test preparations at 0 and 72 hours showed measured iron concentrations to be less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.010 mg/L.

Analysis of the 0.10, 0.32, 1.0 and 3.2% v/v saturated solution test preparations at 0 and 72 hours showed measured cobalt concentrations of less than the LOQ were obtained, determined to be 0.0010 mg/L.

Measured cobalt concentrations in the range of 0.0022 to 0.023 mg/L; equivalent to dissolved test item concentrations of 0.013 to 0.14 mg/L were obtained from the 10, 32 and 100% v/v saturated solution test preparations at 0 hours. As measured concentrations at 72 hours were within 80-120% of the 0-Hour measured concentrations it was considered appropriate to calculate the results based on the equivalent test item concentrations determined at 0 hours only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the 0-Hour measured test concentrations:

Growth Rate - EC50 >0.14 mg/L (It was not possible to calculate an ErC50 value as no more than 26% inhibition of growth rate occurred at the maximum attainable dissolved test item concentration of 0.14 mg/L), NOEC = 0.013 mg/L, LOEC = 0.042 mg/L

Yield - EC50 = 0.081 mg/L, NOEC = 0.013 mg/L, LOEC = 0.042 mg/L

Description of key information

Static 72-hour test for freshwater algae Pseudokirchneriella subcapitata:  ErC50 > 100 % v/v saturated solution, NOEC = 0.013 mg/L, LOEC = 0.042 mg/L(OECD 201, EU Method C.3, GLP)

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.013 mg/L

Additional information

The effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata has been investigated over a 72-hour period. Exposure of Pseudokirchneriella subcapitata to the saturated solution of the test item gave NOEC values based on the 0-Hour measured test concentrations of 0.013 mg/L for growth rate. The Lowest Observed Effect Concentration for growth rate was 0.042 mg/L. It was not possible to calculate an ErC50 value as no more than 26% inhibition of growth rate occurred at the maximum attainable dissolved test item concentration of 0.14 mg/L (EC50 > 100 % v/v saturated solution).

The EC50 values for yield were 0.081 mg/L, with a No Observed Effect Concentration of 0.013 mg/L and a Lowest Observed Effect Concentration of 0.042 mg/L for yield.

Significant effects on growth rate were only seen at 100% v/v saturated solution. As the EC50 value for the test item based on this test would be EC50 > 100 % v/v saturated solution, the results warrant a hazard classification of Aquatic Chronic 4, H413 for the substance, as some concern still remains.

Based on the water solubility test the substance is highly insoluble in water, and can be regarded as a poorly soluble substance. Nevertheless, long-term aquatic studies are not considered necessary, as aquatic toxicity is unlikely to occur: the substance is manufactured and used only in closed, controlled environments, where water that might contain the substance in detectable amounts is pretreated before release into further treatment or the sewage system. As such, there is no emission to water. The substance is also unreactive. Therefore, there are no data gaps in aquatic toxicity.