2-Pentanone, 4-methyl-, reaction products with 2-(2-aminoethoxy)ethanol

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 17 August 2015 and 15 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):Not applicable

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary. Two further samples of each test concentration were incubated alongside the test to provide samples for analysis at 24 and 48 hours.

Vehicle:

no

Details on test solutions:

Range-Finding Test 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 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 (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.5 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.Definitive TestA 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 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 50, 25, 12.5 and 6.25% v/v saturated solution. An aliquot (500 mL) of each of the stock solutions was separately inoculated with 3.2 mL of algal suspension to give the required test concentrations of 6.25, 12.5, 25, 50 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.

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 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 1E+03 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 1E+04 - 1E+05 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

No data

Test temperature:

The temperature within the incubator was recorded daily. Temperature was maintained at 24 ± 1 ºC throughout the test.

pH:

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 pH value of the control cultures was observed to increase from pH 8.1 at 0 hours to pH 8.8 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.

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

Range-finding test: Nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solutionDefinitive: Nominal test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution.

Details on test conditions:

Culture MediumThe culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.Preliminary Media Preparation TrialPreliminary 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 degradation product concentration of approximately 86 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 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 (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.5 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 degradation product 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 TestBased on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100% v/v saturated solution.Experimental PreparationA 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 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 50, 25, 12.5 and 6.25% v/v saturated solution. An aliquot (500 mL) of each of the stock solutions was separately inoculated with 3.2 mL of algal suspension to give the required test concentrations of 6.25, 12.5, 25, 50 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 degradation product in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours.Exposure ConditionsAs 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 7.80E+05 cells per mL. Inoculation of 500 mL of test medium with 3.2 mL of this algal suspension gave an initial nominal cell density of 5E+03 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.EvaluationsTest Organism ObservationsSamples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.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.Data AnalysisComparison of Growth RatesThe average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:µ = (1n Nn – 1n N1) / (tn – t1)Where:µ = average specific growth rate from time t1 to tnN1 = cell concentration at t1Nn = cell concentration at tnt1 = time of first measurementtn = time of nth measurementThe average specific growth rate over the test duration was calculated for each replicate control and test item 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 item vessel was calculated using the following equation:Ir = ((µc - µt) / µc) x 100Where:Ir = percentage inhibition of average specific growth rateµc = mean average specific growth rate for the control culturesµt = average specific growth rate for the test cultureComparison of YieldYield is calculated as the increase in biomass over the exposure period using the following equation:Y = Nn – N0Where:Y = yieldN0 = cell concentration at the start of the testNn = cell concentration at the end of the testFor 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 100Where:Iy = percentage inhibition of yieldYc = mean value for yield in the control groupYt = mean value for yield for the treatment groupDetermination of ECx ValuesFor each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line.It was not possible to calculate 95% confidence limits for the EC50 values as the data generated did not fit the models available for the calculation of confidence limits.Time-Weighted Mean Measured Test ConcentrationsThe time-weighted mean measured test concentrations of the samples were calculated as follows:Area = ((C0 – C1) / (1n (C0) – 1n (C1))) x daysTWM = Total area / Total number of days of the testArea = area under the exponential curve for each renewal periodC0 = measured concentration at the start of each renewal period (mg/L)C1 = measured concentration at the end of each renewal period (mg/L)Days = number of days in the renewal periodTWM = time-weighted mean measured test concentration (mg/L)

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 24 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: It was not possible to calculate an ErC50 value as less than 50% inhibition of growth rate was observed to have occurred at the maximum attainable test item degradation product concentration of 24 mg/L.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

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.Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution were selected for the definitive test.Chemical analysis of the 10 and 100% v/v saturated solution test preparations at 0 hours showed measured concentrations of the test item degradation product to range from 7.3 to 82 mg/L. A decline in measured concentration was observed at 72 hours in the range of 2.2 to 3.5 mg/L indicating that the test item degradation product was unstable over the test duration.Definitive TestGrowth DataFrom the 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 degradation product over the 72-Hour exposure period.Accordingly the following results were determined from the data:Inhibition of Growth RateErC10 (0 - 72 h): 8.9 mg/LErC20 (0 - 72 h): 19 mg/LErC50 (0 - 72 h): >24 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, 1.2, 2.3 and 5.1 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 5.1 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 10 mg/L.Inhibition of YieldEyC10 (0 - 72 h): 3.4 mg/LEyC20 (0 - 72 h): 5.6 mg/LEyC50 (0 - 72 h): 13 mg/LWhere:EyCx is the test concentration that reduced yield by x%.There were no statistically significant differences between the control, 1.2, 2.3 and 5.1 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 5.1 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 10 mg/L.

Results with reference substance (positive control):

A positive control (Study Number 41501180) 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 – 72 h): 1.0 mg/L; 95% confidence limits 0.90 – 1.2 mg/LEyC50 (0 – 72 h): 0.49 mg/L; 95% confidence limits 0.42 – 0.58 mg/LNo Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/LNo Observed Effect Concentration (NOEC) based on yield: 0.25 mg/LLowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/LLowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/LThe 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 (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Verification of Test Concentrations

Analysis of the test preparations at 0 hours showed measured concentrations of the degradation product to range from 2.6 to 61 mg/L. A further decline in measured concentrations was observed in the range of 1.3 to 33 mg/L at 24 hours and 0.90 to 15 mg/L at 72 hours. Measured concentrations of less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 0.82 mg/L were observed in all test samples at 72 hours.

 

Current regulatory advice is that in cases where a decline in measured concentrations is observed, time-weighted mean measured concentrations should be used for calculating EC₅₀values. It was therefore considered justifiable to base the results on the time-weighted 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. 0.41 mg/L) was used to enable calculation of the time-weighted mean measured concentration. The time-weighted mean measured test concentrations were determined to be:

 

Nominal Test Concentration (% v/v Saturated Solution)	Time-Weighted Mean Measured Test Concentration (mg/L)	Expressed as a % of the 0-Hour Measured Test Concentration
6.25	1.2	46
12.5	2.3	45
25	5.1	43
50	10	42
100	24	39

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 113 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: 5.29 x 10³cells per mL

Mean cell density of control at 72 hours: 5.97 x 10⁵cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 27% 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 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

 

 

Observationson 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 1.2, 2.3 and 5.1 mg/L, however cell debris and enlarged cells were observed to be present at 10 mg/L and 24 mg/L. 

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, 1.2, 2.3 and 5.1 mg/L test cultures were observed to be green dispersions. The 10 mg/L test cultures were observed to be pale green dispersions whilst the 24 mg/L test cultures were observed to be extremely pale green dispersions.

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	5.09E+03	2.82E+05	-	-
	R2	4.91E+03	2.22E+05
	Mean	5.00E+03	2.52E+05
0.10	R1	5.33E+03	2.29E+05	[7]	[41]
	R2	5.52E+03	4.79E+05
	Mean	5.42E+03	3.54E+05
1.0	R1	5.34E+03	3.33E+05	[6]	[36]
	R2	5.74E+03	3.48E+05
	Mean	5.54E+03	3.40E+05
10	R1	5.41E+03	4.63E+05	[15]	[85]
	R2	5.18E+03	4.61E+05
	Mean	5.30E+03	4.62E+05
100	R1	5.14E+03	5.36E+04	35	76
	R2	5.12E+03	7.45E+04
	Mean	5.13E+03	6.40E+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

Cell Densities and pH Values in the Definitive Test

Time-Weighted Mean Measured Concentration (mg/L)		pH	Cell Densities* (cells permL)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	8.1	5.15E+03	2.06E+04	1.01E+05	6.07E+05	8.8
	R2		4.98E+03	1.97E+04	6.51E+04	6.13E+05
	R3		5.63E+03	1.78E+04	6.53E+04	6.68E+05
	R4		5.34E+03	1.70E+04	5.84E+04	6.02E+05
	R5		4.98E+03	2.12E+04	8.28E+04	6.41E+05
	R6		5.68E+03	1.79E+04	7.96E+04	4.52E+05
	Mean		5.29E+03	1.90E+04	7.54E+04	5.97E+05
1.2	R1	8.1	5.55E+03	2.00E+04	5.93E+04	6.96E+05	8.7
	R2		4.91E+03	1.84E+04	8.32E+04	5.93E+05
	R3		5.47E+03	2.01E+04	9.65E+04	7.05E+05
	Mean		5.31E+03	1.95E+04	7.97E+04	6.65E+05
2.3	R1	8.5	5.71E+03	1.83E+04	7.47E+04	6.90E+05	8.6
	R2		4.93E+03	1.41E+04	6.27E+04	4.29E+05
	R3		5.02E+03	1.49E+04	9.00E+04	6.82E+05
	Mean		5.22E+03	1.58E+04	7.58E+04	6.00E+05
5.1	R1	9.2	4.88E+03	1.58E+04	9.01E+04	5.46E+05	8.6
	R2		4.56E+03	1.29E+04	5.94E+04	4.65E+05
	R3		4.35E+03	1.14E+04	6.59E+04	5.01E+05
	Mean		4.60E+03	1.34E+04	7.18E+04	5.04E+05
10	R1	9.5	5.15E+03	1.35E+04	4.00E+04	2.81E+05	8.5
	R2		5.06E+03	1.18E+04	5.48E+04	3.24E+05
	R3		5.25E+03	1.20E+04	5.79E+04	3.10E+05
	Mean		5.15E+03	1.24E+04	5.09E+04	3.05E+05
24	R1	9.8	5.22E+03	9.82E+03	5.55E+04	2.00E+05	8.4
	R2		4.40E+03	1.12E+04	4.66E+04	2.33E+05
	R3		4.51E+03	1.16E+04	5.34E+04	1.90E+05
	Mean		4.71E+03	1.09E+04	5.18E+04	2.07E+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

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	0.059	0.066	0.075
	R2	0.057	0.050	0.093
	R3	0.053	0.054	0.097
	R4	0.051	0.051	0.097
	R5	0.060	0.057	0.085
	R6	0.053	0.062	0.072
	Mean	0.056	0.057	0.087

R₁- R₆= Replicates 1 to 6

Inhibition of Growth Rate and Yield in the Definitive Test

Time-Weighted Mean Measured Concentration (mg/L))		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.067		6.02E+05
	R2	0.067		6.08E+05
	R3	0.068		6.62E+05
	R4	0.067	-	5.96E+05	-
	R5	0.067		6.36E+05
	R6	0.063		4.47E+05
	Mean	0.067		5.92E+05
	SD	0.002		7.52E+04
1.2	R1	0.069	[3]	6.90E+05
	R2	0.066	1	5.88E+05
	R3	0.069	[3]	7.00E+05
	Mean	0.068	[2]	6.59E+05	[11]
	SD	0.002		6.20E+04
2.3	R1	0.068	[1]	6.84E+05
	R2	0.062	7	4.24E+05
	R3	0.068	[1]	6.77E+05
	Mean	0.066	2	5.95E+05	[1]
	SD	0.003		1.48E+05
5.1	R1	0.065	3	5.41E+05
	R2	0.063	6	4.61E+05
	R3	0.064	4	4.97E+05
	Mean	0.064	4	5.00E+05	16
	SD	0.001		4.00E+04
10	R1	0.056	16	2.76E+05
	R2	0.058	13	3.19E+05
	R3	0.057	15	3.05E+05
	Mean	0.057	15	3.00E+05	49
	SD	0.001		2.18E+04
24	R1	0.051	24	1.95E+05
	R2	0.053	21	2.28E+05
	R3	0.051	24	1.85E+05
	Mean	0.052	23	2.03E+05	66
	SD	0.001		2.26E+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 the controls]

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item degradation product on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the time-weighted mean measured test concentrations gave the following results:The EC50 (growth rate) was > 24* mg/L with a NOEC of 5.1 mg/L and LOEC of 10 mg/L.* It was not possible to calculate an ErC50 value as less than 50% inhibition of growth rate was observed to have occurred at the maximum attainable test item degradation product concentration of 24 mg/L.The EC50 (yield) was > 13 mg/L.

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…….

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.

 

The test item was known to rapidly degrade in water. Given this, and in accordance with current regulatory guidance, the concentration of the degradation product was determined. A preliminary media preparation trial indicated that a dissolved degradation product concentration of approximately 86 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of the degradation product under test conditions.

 

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item degradation product at nominal concentrations of 6.25, 12.5, 25, 50 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 24 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 degradation product. 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 concentrations of the degradation product to range from 2.6 to 61 mg/L. A further decline in measured concentrations was observed in the range of 1.3 to 33 mg/L at 24 hours and 0.90 to 15 mg/L at 72 hours. Measured concentrations of less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 0.82 mg/L were observed in all test samples at 72 hours.

 

Given this decline in measured concentrations it was considered appropriate to calculate the results based on the time-weighted mean measured test concentrations only in order to give a “worst case” analysis of the data.

 

Exposure of Pseudokirchneriella subcapitata to the test item degradation product gave the following results based on the time-weighted mean measured test concentrations:

 

Response Variable	EC50(mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	>24*	5.1	10
Yield	13	5.1	10

*It was not possible to calculate an E_rC₅₀value as less than 50% inhibition of growth rate was observed to have occurred at the maximum attainable test item degradation product concentration of 24 mg/L.

Description of key information

A study was performed to assess the toxicity of the test item to the a representative algal species. The method followed was designed to be compatible with the OECD 201 Guideline for Testing of Chemicals.

Based on mean measured concentrations the following endpoints were determined

72 -hour ErC50s > 24 mg/L

NOEC of 5.1 mg/L

LOEC of 10 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

24 mg/L

EC10 or NOEC for freshwater algae:

5.1 mg/L

Additional information

The test substance is not toxic to Algea.