3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

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:

The study was conducted between 16 March 2016 and 13 April 2016.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 201 in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: ES421 Pinyl Nitrile

SOURCE OF TEST MATERIAL
- Appearance/physical state: white solid
- Source and lot/batch No.of test material: SM15077102
- Expiration date of the lot/batch: 22 July 2016
- Purity: 98.08%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approximately 4 °C, in the dark

Analytical monitoring:

yes

Details on sampling:

Verification of Test Concentrations
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 and 72 hours and stored frozen for further analysis if necessary.
Two additional samples of each test concentration were prepared at 0 hours and incubated alongside the test to provide samples for analysis at 24 and 48 hours.

Vehicle:

no

Details on test solutions:

Preliminary Media Preparation Trial
Information provided by the Sponsor indicated the water solubility of the test item to be 13 mg/L. Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000).

Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions:

Saturated Solution Preparation
A nominal amount of test item (1100 mg) was dispersed, in duplicate, in 11 liters of deionized reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. Prior to use the test item was heated to 45 ºC in order to aid handling. After stirring samples were taken for chemical analysis after the following pre-treatments:
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 1 liter discarded in order to pre-condition the filter)
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 2 liters discarded in order to pre-condition the filter)

Results
Stirring Period and Treatment/Concentration Found:
24 Hours Centrifuged 10000 g 3.08 mg/L
24 Hours Centrifuged 40000 g 2.29 mg/L
24 Hours Filtered ~ 1 liter discarded 4.11 mg/L
24 Hours Filtered ~ 2 liters discarded 4.48 mg/L
48 Hours Centrifuged 10000 g 4.61 mg/L
48 Hours Centrifuged 40000 g 3.37 mg/L
48 Hours Filtered ~ 1 liter discarded 5.35 mg/L
48 Hours Filtered ~ 2 liters discarded 5.80 mg/L

Discussion
The results obtained indicated that there was a slight increase in the dissolved test item concentrations obtained when the preparation period was extended to 48 hours. The maximum dissolved test item concentration was obtained following filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded). Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L, stirred for a period of 48 hours prior to the removal of any undissolved test item by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded) to give a nominal test concentration of approximately 5.8 mg/L.

References
Environment Directorate, Organisation for Economic Co-Operation and Development (OECD) (2000) Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.

Range-Finding Test

The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 5.8 mg/L could be obtained using a saturated solution method of preparation. The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to 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 item (1100 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, 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 (4.5 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% (v/v) saturated solution. 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. 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. A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100% (v/v) saturated solution.

Experimental Preparation
A 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 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 32, 10, 3.2 and 1.0% (v/v) saturated solution. An aliquot (900 mL) of each of the stock solutions was separately inoculated with 8.0 mL of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 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 (see details on analytical method).

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 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 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100–150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104-105 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Details on test conditions:

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture. For details refer to 'Any other information on matererials and methods incl. tables'.

Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control and three flasks each containing 100 mL were used for each treatment group. The control group was maintained under identical conditions but not exposed to the test item. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 5.66 x 105 cells per mL. Inoculation of 900 mL of test medium with 8.0 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 25, 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 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.

Verification of Test Concentrations
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 and 72 hours and stored frozen for further analysis if necessary.
Two additional samples of each test concentration were prepared at 0 hours and incubated alongside the test to provide samples for analysis at 24 and 48 hours.
The method of analysis, recovery and test solution analyses are described under details on analytical methods.

Reference substance (positive control):

yes

Remarks:

A positive control 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

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given under 'Any other information on results icl. tables'. The results showed no effect on growth at the test concentrations of 0.10 and 1.0% (v/v) saturated solution. However, growth was observed to be reduced at 10 and 100% (v/v) saturated solution. Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100% (v/v) saturated solution were selected for the definitive test. Chemical analysis of the test preparations at 0 and 72 hours (see details on analytical methods) showed a decline in measured concentrations occurred indicating that the test item was unstable under test conditions.

Definitive Test

Verification of Test Concentrations
Analysis of the test preparations at 0 hours (see details on analytical methods) showed measured test concentrations to range from 0.066 to 6.8 mg/L. A decline in measured concentrations was observed after each 24-Hour interval in the range of 0.056 to 5.6 mg/L at 24 hours, 0.045 to 4.5 mg/L at 48 hours and less than the limit of quantification (LOQ), determined to be 0.0034, to 0.098 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. 0.0017 mg/L) was used to enable calculation of the geometric mean measured concentration. The geometric mean measured test concentrations are shown under 'Any other information on results icl. tables'.

Growth Data
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given under 'Any other information on results icl. tables'. Daily specific growth rates for the control cultures are given under 'Any other information on results icl. tables'. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given under 'Any other information on results icl. tables'.
From the data presented, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

Inhibition of Growth Rate
ErC10 (0 - 72 h): 1.8 mg/L
ErC20 (0 - 72 h): 2.4 mg/L
ErC50 (0 - 72 h): 4.1 mg/L*

* It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

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 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, 0.040, 0.15, 0.37 and 1.4 mg/L test concentrations (P>=0.05), however the 4.0 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 1.4 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 4.0 mg/L.

References
Dunnett, C.W. (1955) A Multiple Comparison Procedure for Comparing Several Treatments With a Control. J Am Stat Assoc 50, 1096-1121.
Sokal, R.R. and Rohlf, F.J. (1981) Biometry. New York: W H Freeman and Company.

Inhibition of Yield
EyC10 (0 - 72 h): 1.0 mg/L
EyC20 (0 - 72 h): 1.3 mg/L
EyC50 (0 - 72 h): 1.9 mg/L; 95% confidence limits 1.7–2.2 mg/L
Where:
EyCx is the test concentration that reduced yield by X%. Statistical analysis of the yield data was carried out as described above. There were no statistically significant differences between the control, 0.040, 0.15, 0.37 and 1.4 mg/L test concentrations (P>=0.05), however the 4.0 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 1.4 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 4.0 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 137 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.
Mean cell density of control at 0 hours: 5.00 x 103 cells per mL
Mean cell density of control at 72 hours: 6.84 x 105 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 (0–72 h) was 1% 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 0.040, 0.15, 0.37 and 1.4 mg/L, however cell debris was observed to be present in the test cultures at 4.0 mg/L.

Water Quality Criteria
The pH values of the control and each test preparation are shown under 'Any other information on results icl. tables'. Temperature was maintained at 24 ± 1 ºC throughout the test.
The pH value of the control cultures (see 'Any other information on results icl. tables') was observed to increase from pH 7.4 at 0 hours to pH 8.2 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 Item Solubility
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, 0.040, 0.15 and 0.37 mg/L test cultures were observed to be green dispersions. The 1.4 mg/L test cultures were observed to be pale green dispersions whilst the 4.0 mg/L test cultures were observed to be clear colorless solutions.

Results with reference substance (positive control):

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): 1.5 mg/L; 95% confidence limits 1.3–1.7 mg/L
EyC50 (0 – 72 h): 0.79 mg/L; 95% confidence limits 0.70–0.89 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 1.0 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:

Please refer to details on results.

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

Nominal Concentration (% v/v Saturated Solution)		Cell Densities* (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	5.10E+03	9.84E+05	-	-
	R2	4.21E+03	1.03E+06
	Mean	4.66E+03	1.01E+06
0.10	R1	3.66E+03	1.03E+06	[4]	0
	R2	3.62E+03	9.76E+05
	Mean	3.64E+03	1.00E+06
1.0	R1	7.26E+03	1.33E+06	0	[27]
	R2	4.61E+03	1.24E+06
	Mean	5.94E+03	1.28E+06
10	R1	5.17E+03	7.49E+05	4	9
	R2	4.96E+03	1.08E+06
	Mean	5.06E+03	9.16E+05
100	R1	5.10E+03	6.02E+04	53	94
	R2	4.58E+03	6.03E+04
	Mean	4.84E+03	6.02E+04

*    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.

R₁and R₂= Replicates 1 and 2; [Increase in growth compared to controls]

The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration (% v/v Saturated Solution)	Geometric Mean Measured Test Concentration (mg/L)	Expressed as a % of the 0-Hour Measured Test Concentration
1.0	0.040	61
3.2	0.15	57
10	0.37	56
32	1.4	59
100	4.0	59

Cell Densities and pH Values in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0 h	25 h	48 h	72 h	72 h
Control	R1	7.4	3.05E+04	1.50E+05	6.06E+05	8.2
	R2		3.17E+04	1.45E+05	6.88E+05
	R3		3.88E+04	1.83E+05	6.89E+05
	R4		3.15E+04	1.33E+05	6.10E+05
	R5		3.12E+04	1.70E+05	7.85E+05
	R6		3.07E+04	1.58E+05	7.28E+05
	Mean		3.24E+04	1.57E+05	6.84E+05
0.040	R1	7.5	2.56E+04	1.50E+05	5.27E+05	8.2
	R2		2.80E+04	1.55E+05	7.06E+05
	R3		3.42E+04	1.73E+05	8.86E+05
	Mean		2.93E+04	1.59E+05	7.07E+05
0.15	R1	7.6	2.99E+04	1.69E+05	8.16E+05	8.2
	R2		2.80E+04	1.49E+05	7.32E+05
	R3		3.08E+04	1.64E+05	6.03E+05
	Mean		2.95E+04	1.61E+05	7.17E+05
0.37	R1	7.4	3.41E+04	1.86E+05	7.10E+05	8.3
	R2		3.04E+04	1.82E+05	8.19E+05
	R3		3.37E+04	1.87E+05	5.94E+05
	Mean		3.28E+04	1.85E+05	7.07E+05
1.4	R1	7.5	2.16E+04	1.22E+05	5.80E+05	8.2
	R2		2.14E+04	1.15E+05	5.24E+05
	R3		1.92E+04	9.16E+04	4.12E+05
	Mean		2.07E+04	1.10E+05	5.05E+05
4.0	R1	7.4	2.00E+03	9.41E+03	5.64E+04	7.8
	R2		1.22E+03	6.66E+03	5.09E+04
	R3		2.14E+03	1.35E+04	7.60E+04
	Mean		1.79E+03	9.87E+03	6.11E+04

*    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

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	0.072	0.069	0.058
	R2	0.074	0.066	0.065
	R3	0.082	0.068	0.055
	R4	0.074	0.063	0.063
	R5	0.073	0.074	0.064
	R6	0.073	0.071	0.064
	Mean	0.075	0.069	0.062

R₁- R₆= Replicates 1 to 6

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 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.067		6.01E+05
	R2	0.068		6.83E+05
	R3	0.068		6.84E+05
	R4	0.067	-	6.05E+05	-
	R5	0.070		7.80E+05
	R6	0.069		7.23E+05
	Mean	0.068		6.79E+05
	SD	0.001		6.88E+04
0.040	R1	0.065	4	5.22E+05
	R2	0.069	[1]	7.01E+05
	R3	0.072	[6]	8.81E+05
	Mean	0.069	[1]	7.02E+05	[3]
	SD	0.004		1.80E+05
0.15	R1	0.071	[4]	8.11E+05
	R2	0.069	[1]	7.27E+05
	R3	0.067	1	5.98E+05
	Mean	0.069	[1]	7.12E+05	[5]
	SD	0.002		1.07E+05
0.37	R1	0.069	[1]	7.05E+05
	R2	0.071	[4]	8.14E+05
	R3	0.066	3	5.89E+05
	Mean	0.069	[1]	7.02E+05	[3]
	SD	0.003		1.13E+05
1.4	R1	0.066	3	5.75E+05
	R2	0.065	4	5.19E+05
	R3	0.061	10	4.07E+05
	Mean	0.064	6	5.00E+05	26
	SD	0.003		8.54E+04
4.0	R1	0.034	50	5.14E+04
	R2	0.032	53	4.59E+04
	R3	0.038	44	7.10E+04
	Mean	0.035	49	5.61E+04	92
	SD	0.003		1.32E+04

*    In accordance with the OECD test guideline only thean 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

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the results shown under 'Any other information on results icl. tables'.

Executive summary:

Introduction

A study was performed to assess the effect of the test item, ES421 Pinyl Nitrile, on the growth of the green algaPseudokirchneriella 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 provided by the Sponsor indicated the water solubility of the test item to be 13 mg/L. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 5.8 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to solutions of the test item at nominal concentrations of 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 by stirring an excess (100 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters 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 at 0 hours showed measured test concentrations to range from 0.066 to 6.8 mg/L. A decline in measured concentrations was observed after each 24-Hour interval in the range of 0.056 to 5.6 mg/L at 24 hours, 0.045 to 4.5 mg/L at 48 hours and less than the limit of quantification (LOQ), determined to be 0.0034, to 0.098 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 concentrations in order to give a “worst case” analysis of the data.

 

Exposure of Pseudokirchneriella subcapitatato the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable	EC50(mg/L)	95% Confidence Limits (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	4.1	*	1.4	4.0
Yield	1.9	1.7-2.2	1.4	4.0

*It was not possible to calculate 95% confidence limits for the E_rC₅₀value as the data generated did not fit the models available for the calculation of confidence limits.

Description of key information

A study was performed to assess the effect of the test item, ES421 Pinyl Nitrile, on the growth of the green algaPseudokirchneriella 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 provided by the Sponsor indicated the water solubility of the test item to be 13 mg/L. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 5.8 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to solutions of the test item at nominal concentrations of 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 by stirring an excess (100 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters 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 at 0 hours showed measured test concentrations to range from 0.066 to 6.8 mg/L. A decline in measured concentrations was observed after each 24-Hour interval in the range of 0.056 to 5.6 mg/L at 24 hours, 0.045 to 4.5 mg/L at 48 hours and less than the limit of quantification (LOQ), determined to be 0.0034, to 0.098 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 concentrations in order to give a “worst case” analysis of the data.

 

Exposure ofPseudokirchneriella subcapitatato the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable	EC50(mg/L)	95% Confidence Limits (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	4.1	*	1.4	4.0
Yield	1.9	1.7-2.2	1.4	4.0

*It was not possible to calculate 95% confidence limits for the E_rC₅₀value as the data generated did not fit the models available for the calculation of confidence limits.

Key value for chemical safety assessment

EC50 for freshwater algae:

4.1 mg/L

EC10 or NOEC for freshwater algae:

1.4 mg/L