4-(2-methylpropyl)benzyl propyl ether

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 22 March 2018 and 17 April 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study conducted according to OECD TG 201 in compliance with GLP, without deviations that influence the quality of the results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 11-0539
Physical state/Appearance: clear colorless liquid
Storage Conditions: room temperature in the dark

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. An additional 200 mL of each test concentration containing no algal cells was also incubated to provide samples for uninoculated analysis at 72 hours.

Vehicle:

no

Details on test solutions:

Preliminary Media Preparation Trial
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.

Range-Finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 2.0 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 (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 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 (900 mL) of each of the stock solutions was separately inoculated with algal suspension (5.3 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.

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.
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 24 hours. After 24 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 stock solutions of 32, 10, 3.2 and 1.0% v/v saturated solution. An aliquot (1600 mL) of each of the stock solutions was separately inoculated with 4.0 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 and 72 hours.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 kept under constant agitation by orbital shaker (approximately 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 (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 ± 1 °C throughout the test.

pH:

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was maintained within 7.8 - 8.0.

Nominal and measured concentrations:

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.

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. The media used in both the range-finding and definitive tests was prepared with the addition of 250 mg/L of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with testing in an enclosed system (Herman et al 1990).

Range-Finding Test:
The test was conducted in 250 mL glass conical flasks each completely filled with test preparation and sealed with ground glass stoppers 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 sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ±1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 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 immediate chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions.

Definitive Test:
Exposure Conditions:
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each completely filled with solution were used for the control and three flasks each completely filled 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 1.99 x 106 cells per mL. Inoculation of 1600 mL of test medium with 4.0 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ±1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.095 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.26 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.25 mg/L

Nominal / measured:

meas. (initial)

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 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 hours showed measured test concentrations to range from between less than the LOQ, determined to be 0.045 mg/L and 2.2 mg/L. A concentration dependent decline in measured test concentrations was observed at 72 hours in the range of less than the LOQ to 2.3 mg/L (53% to 105% of the 0-Hour measured test concentrations). This was considered to be due to possible adsorption of the test item to the algal cells present given that the decline in measured concentrations followed a concentration dependent decline, there being more algal cells present in the lower test concentrations to which adsorption could occur.

Definitive Test
Verification of Test Concentrations
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 0.045 mg/L to 2.8 mg/L. An overall decline in measured test concentration was observed at 72 hours to between less than the LOQ and 2.1 mg/L (49% to 127% of the 0-Hour measured test concentrations). Analysis of test preparations at 72 hours which contained no algal cells showed measured test concentrations to range from less than the LOQ to 2.3 mg/L (81% to 95% of the 0-Hour measured test concentrations) indicating that the decline in measured concentrations observed in the samples which contained algae was due to adsorption of the test item to the algal cells present rather than instability. As such it was considered appropriate to calculate the results based on the 0-Hour measured test concentrations only.

Growth Data
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.
Accordingly the following results were determined from the data:
Inhibition of Growth Rate
ErC10 (0 to 72 hour): 0.11 mg/L
ErC20 (0 to 72 hour): 0.16 mg/L
ErC50 (0 to 72 hour): 0.30 mg/L; 95% confidence limits 0.26 to 0.35 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 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, 1.0 and 3.2% 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 3.2% v/v saturated solution (0.095 mg/L based on the 0-Hour measured test concentrations). Correspondingly the LOEC based on growth rate was 10% v/v saturated solution (0.30 mg/L based on the 0-Hour measured test concentrations).

Inhibition of Yield
EyC10 (0 to 72 hour): 0.23 mg/L
EyC20 (0 to 72 hour): 0.24 mg/L
EyC50 (0 to 72 hour): 0.26 mg/L*
Where EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences between the control, 1.0 and 3.2% 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 3.2% v/v saturated solution (0.095 mg/L based on the 0-Hour measured test concentrations). Correspondingly the LOEC based on yield was 10% v/v saturated solution (0.30 mg/L based on the 0-Hour measured test concentrations).

Results with reference substance (positive control):

A positive control (Envigo study number RD71YQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 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 item.

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 93 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 10³ cells per mL

Mean cell density of control at 72 hours:              4.63 x 10⁵ cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 25% 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 5% 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 1.0 and 3.2% v/v. Some misshapen cells were observed in the 10% v/v test cultures whilst no algal cells and some cell debris was observed to be present in the 32 and 100% v/v test cultures.

Water Quality Criteria

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

The pH value of the control cultures was observed to increase from pH 7.8 at 0 hours to pH 9.7 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the Test Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion 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, 1.0 and 3.2% v/v saturated solution test cultures were observed to be green dispersions. The 10% v/v saturated solution test cultures were observed to be very pale green dispersions whilst the 32 and 100% v/v saturated solution test cultures were observed to remain as clear colorless solutions.

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	6.10E+03	8.56E+05	-	-
	R2	5.43E+03	8.61E+05
	Mean	5.76E+03	8.58E+05
0.10	R1	6.07E+03	9.89E+05	[1]	[14]
	R2	6.28E+03	9.68E+05
	Mean	6.17E+03	9.78E+05
1.0	R1	5.51E+03	9.68E+05	[3]	[1]
	R2	5.07E+03	7.57E+05
	Mean	5.29E+03	8.63E+05
10	R1	4.87E+03	1.73E+05	35	86
	R2	5.07E+03	7.30E+04
	Mean	4.97E+03	1.23E+05
100	R1	5.43E+03	1.12E+04	86	99
	R2	5.49E+03	1.12E+04
	Mean	5.46E+03	1.12E+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

R   = Replicate

-    = Not applicable

[ ] = Increase in growth compared to controls

Cell Densities and pH Values in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)		pH	Cell Densities* (cells per mL)			pH
		0 Hours	22 Hour	49 Hour	72 Hour	72 Hours
Control	R1	7.8	1.73E+04	8.31E+04	4.62E+05	9.7
	R2		1.67E+04	8.11E+04	5.50E+05
	R3		1.83E+04	6.37E+04	4.24E+05
	R4		2.01E+04	8.02E+04	5.53E+05
	R5		1.84E+04	6.09E+04	4.91E+05
	R6		1.68E+04	6.20E+04	3.00E+05
	Mean		1.79E+04	7.18E+04	4.63E+05
1.0	R1	7.9	1.92E+04	9.27E+04	5.46E+05	9.9
	R2		2.07E+04	7.25E+04	5.15E+05
	R3		1.61E+04	5.18E+04	2.98E+05
	Mean		1.87E+04	7.23E+04	4.53E+05
3.2	R1	7.9	1.82E+04	6.75E+04	5.09E+05	9.8
	R2		1.77E+04	7.25E+04	5.50E+05
	R3		1.36E+04	7.35E+04	4.60E+05
	Mean		1.65E+04	7.12E+04	5.06E+05
10	R1	7.9	1.19E+04	2.17E+04	5.34E+04	8.9
	R2		1.36E+04	1.40E+04	2.75E+04
	R3		1.16E+04	1.43E+04	3.92E+04
	Mean		1.24E+04	1.67E+04	4.00E+04
32	R1	7.9	7.54E+03	9.42E+03	1.00E+04	8.0
	R2		8.15E+03	6.04E+03	1.23E+04
	R3		6.86E+03	6.78E+03	6.98E+03
	Mean		7.52E+03	7.41E+03	9.78E+03
100	R1	8.0	9.50E+03	8.27E+03	8.89E+03	8.0
	R2		7.80E+03	8.51E+03	9.33E+03
	R3		8.57E+03	8.54E+03	1.80E+04
	Mean		8.62E+03	8.44E+03	1.21E+04

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from3 counts for each of the replicate flasks

R = Replicate

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	0.057	0.058	0.075
	R2	0.055	0.059	0.083
	R3	0.059	0.046	0.082
	R4	0.063	0.051	0.084
	R5	0.059	0.044	0.091
	R6	0.055	0.048	0.069
	Mean	0.058	0.051	0.081

R = Replicate

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	0.063	-	4.57E+05	-
	R2	0.065		5.45E+05
	R3	0.062		4.19E+05
	R4	0.065		5.48E+05
	R5	0.064		4.86E+05
	R6	0.057		2.95E+05
	Mean	0.063		4.58E+05
	SD	0.003		9.42E+04
1.0	R1	0.065	[3]	5.41E+05
	R2	0.064	[2]	5.10E+05
	R3	0.057	10	2.93E+05
	Mean	0.062	2	4.48E+05	2
	SD	0.004		1.35E+05
3.2	R1	0.064	[2]	5.04E+05
	R2	0.065	[3]	5.45E+05
	R3	0.063	0	4.55E+05
	Mean	0.064	[2]	5.01E+05	[9]
	SD	0.001		4.51E+04
10	R1	0.033	48	4.84E+04
	R2	0.024	62	2.25E+04
	R3	0.029	54	3.42E+04
	Mean	0.029	55	3.50E+04	92
	SD	0.005		1.30E+04
32	R1	0.010	84	5.00E+03
	R2	0.013	79	7.35E+03
	R3	0.005	92	1.98E+03
	Mean	0.009	85	4.78E+03	99
	SD	0.004		2.69E+03
100	R1	0.008	87	3.89E+03
	R2	0.009	86	4.33E+03
	R3	0.018	71	1.30E+04
	Mean	0.012	81	7.09E+03	98
	SD	0.006		5.16E+03

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

R   = Replicate

-    = Not applicable

SD = Standard Deviation

[ ]  = Increase in growth compared to controls

Validity criteria fulfilled:

yes

Conclusions:

The ErC50, ErC10 and NOEC were 0.3, 0.11 and 0.095 mg/l

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was 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 (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period 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 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. 

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 0.045 mg/L to 2.8 mg/L. An overall decline in measured test concentration was observed at 72 hours to between less than the LOQ and 2.1 mg/L (49% to 127% of the 0-Hour measured test concentrations). Analysis of test preparations at 72 hours which contained no algal cells showed measured test concentrations to range from less than the LOQ to 2.3 mg/L (81% to 95% of the 0-Hour measured test concentrations) indicating that the decline in measured concentrations observed in the samples which contained algae was due to adsorption of the test item to the algal cells present rather than instability. As such it was considered appropriate to calculate the results based on the 0-Hour measured test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the 0-Hour 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	0.30	0.26	-	0.35	0.095	0.30
Yield	0.26		*		0.095	0.30

*It was not possible to calculate 95% confidence limits for the EyC50 value 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 material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was 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 (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period 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 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. 

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 0.045 mg/L to 2.8 mg/L. An overall decline in measured test concentration was observed at 72 hours to between less than the LOQ and 2.1 mg/L (49% to 127% of the 0-Hour measured test concentrations). Analysis of test preparations at 72 hours which contained no algal cells showed measured test concentrations to range from less than the LOQ to 2.3 mg/L (81% to 95% of the 0-Hour measured test concentrations) indicating that the decline in measured concentrations observed in the samples which contained algae was due to adsorption of the test item to the algal cells present rather than instability. As such it was considered appropriate to calculate the results based on the 0-Hour measured test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the 0-Hour 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	0.30	0.26	-	0.35	0.095	0.30
Yield	0.26		*		0.095	0.30

*It was not possible to calculate 95% confidence limits for the E_yC₅₀ value 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:

0.3 mg/L

EC10 or NOEC for freshwater algae:

0.11 mg/L