Dihexyl fumarate

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 24 July 2018 and 13 Septermber 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Dihexyl fumarate (EC 242-833-4)
CAS Number: 19139-31-2
Molecular Formula: C16H28O4
Molecular Weight: 284.39
Batch: 800317920
Purity: 98.6%
Physical State/Appearance: Clear colorless liquid
Expiry Date: 25 January 2019
Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the solvent 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. A set of duplicate samples was taken at 0 and 72 hours and stored frozen for further analysis if necessary.
All sample bottles were fortified by the addition of 5 mL of acetonitrile in order to stabilize the test item.

Vehicle:

yes

Remarks:

dimethylformamide

Details on test solutions:

Preliminary Media Preparation Trials
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.
The EPIWIN predicted water solubility value for the test item was 0.089 mg/L.
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 media preparation trials were conducted in order to determine the solubility of the test item under test conditions.

Range-Finding Tests
The test concentrations to be used in the definitive test were determined by preliminary range-finding tests. The initial range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.0010,
0.010 and 0.10 mg/L for a period of 72 hours.
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, solvent control and test concentration. The test item was prepared using a preliminary solution in dimethylformamide.
A nominal amount of test item (20 mg) was dissolved in dimethylformamide and the volume adjusted to 20 mL to give a 1.0 mg/mL solvent stock solution from which a series of dilutions was made to give further solvent stock solutions of 0.10 and 0.010 mg/mL.
An aliquot (50 µL) of each of the solvent stock solutions was separately dispersed in 500 mL of culture medium with the aid of a magnetic stirring for approximately 5 minutes, to give the required test concentrations of 0.0010, 0.010 and 0.10 mg/L to which 1.9 mL of algal suspension was added.
The solvent stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 µL/L of dimethylformamide.
The results of the initial range-finding test showed poor growth in the solvent control cultures and so a second range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal test concentrations of 0.0010, 0.010 and 0.10 mg/L for a period of 72 hours.
A nominal amount of test item (20 mg) was dissolved in dimethylformamide and the volume adjusted to 20 mL to give a 1.0 mg/mL solvent stock solution from which a series of dilutions was made to give further solvent stock solutions of 0.10 and 0.010 mg/mL.
An aliquot (50 µL) of each of the solvent stock solutions was separately dispersed in 500 mL of culture medium with the aid of magnetic stirring for approximately 10 minutes, to give the required test concentrations of 0.0010, 0.010 and 0.10 mg/L to which 1.9 mL of algal suspension was added.
The solvent stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 µL/L of dimethylformamide.

Definitive Test
Based on the results of the second range-finding test the following test concentrations were assigned to the definitive test: 0.010, 0.018, 0.032, 0.056 and 0.10 mg/L.
A nominal amount of test item (50 mg) was dissolved in dimethylformamide and the volume adjusted to 50 mL to give a 1.0 mg/mL solvent stock solution from which a series of dilutions were made to give further solvent stock solutions of 0.56 mg/mL, 0.32 mg/mL, 0.18 mg/mL and 0.10 mg/mL. An aliquot (50 µl) of each of the solvent stock solutions was separately dispersed in 500 mL of culture medium with the aid of magnetic stirring for approximately 5 minutes prior to inoculation with 2.0 mL of algal suspension to give the required test concentrations of 0.010, 0.018, 0.032, 0.056 and 0.10 mg/L.
The solvent 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 (Section 3.3). The master cultures were maintained 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/

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The temperature was maintiained at 24 ±1 °C throughout the test.

pH:

The pH value of the control cultures was observed to range from pH 7.9 at 0 hours to pH 7.8 at 72 hours. A pH value of 7.9 was determined for the solvent control cultures at both 0 and 72 hours. The pH deviation in the control and solvent 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:

Range-Finding Tests:
Nominal concentration: 0.0010, 0.010 and 0.10 mg/L

Definitive Test:
Nominal concentration: 0.010, 0.018, 0.032, 0.056 and 0.10 mg/L

Details on test conditions:

Culture Medium
The culture medium used for the range-finding tests and definitive test was the same as that used to maintain the stock culture.
NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

Range-Finding Tests
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 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.
The results of the initial range-finding test showed poor growth in the solvent control cultures and so a second range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal test concentrations of 0.0010, 0.010 and 0.10 mg/L for a period of 72 hours.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 µL/L of dimethylformamide.
Exposure conditions in the second range-finding test were the same as those in the initial test.
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in fortified sample bottles in order to determine the stability of the test item under test conditions. Only concentrations within the range to be used for the definitive test were analyzed. A duplicate set of samples was taken at 0 and 72 hours from each test concentration. All samples were stored frozen prior to analysis.

Definitive Test
Based on the results of the second range-finding test the following test concentrations were assigned to the definitive test: 0.010, 0.018, 0.032, 0.056 and 0.10 mg/L.

Exposure Conditions
As in the range-finding tests 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and solvent control and three flasks each containing 100 mL were used for each treatment group.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 µL/L of dimethylformamide.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.22 x 10^6 cells per mL. Inoculation of 500 mL of test medium with 2.0 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
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 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

Range-finding Tests
The results showed no effect on growth at the test concentrations of 0.0010 and 0.010 mg/L. However, growth was observed to be reduced at 0.10 mg/L.
Based on this information test concentrations of 0.010, 0.018, 0.032, 0.056 and 0.10 mg/L were selected for the definitive test.
Chemical analysis of the 0.010 and 0.10 mg/L test preparations at 0 hours showed measured test concentrations of 0.0054 and 0.071 mg/L respectively were obtained. A decline in measured test concentrations was observed at 72 hours to less than the LOD in both the 0.010 and 0.10 mg/L test preparations indicating instability of the test item under the conditions of the test.

Growth Data
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 based on the geometric mean measured test concentrations:

Inhibition of Growth Rate
ErC10 (0 to 72 hour): 0.0061 mg/L
ErC20 (0 to 72 hour): >0.0064 mg/L
ErC50 (0 to 72 hour): >0.0064 mg/L
where ErCx is the test concentration that reduced growth rate by x%.
It was not possible to calculate an ErC20 or ErC50 value as no more than 14% inhibition of growth rate occurred at the maximum attainable dissolved test item concentration of 0.0064 mg/L.
Statistical analysis of the growth rate data was carried out for the solvent 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 (P≥0.05), between the solvent control, 0.0017, 0.0021, 0.0038 and 0.0049 mg/L test concentrations; however, the 0.0064 mg/L test concentration was significantly different (P<0.05) and, therefore the NOEC based on growth rate was 0.0049 mg/L. Correspondingly the LOEC based on growth rate was 0.0064 mg/L.

Inhibition of Yield
EyC10 (0 to 72 hour): 0.0044 mg/L
EyC20 (0 to 72 hour): 0.0050 mg/L
EyC50 (0 to 72 hour): 0.0063 mg/L
Where EyCx is the test concentration that reduced yield by x%.

There were no statistically significant differences (P≥0.05), between the solvent control, 0.0017, 0.0021, 0.0038 and 0.0049 mg/L test concentrations; however, the 0.0064 mg/L test concentration was significantly different (P<0.05) and, therefore the NOEC based on yield was 0.0049 mg/L. Correspondingly the LOEC based on yield was 0.0064 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 261 after 72 hours and the cell concentration of the solvent control cultures increased by a factor of 303 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours: 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 1.31 x 10^6 cells per mL
Nominal cell density of solvent control at 0 hours: 5.00 x 10^3 cells per mL
Mean cell density of solvent control at 72 hours: 1.51 x 10^6 cells per mL
The mean coefficients of variation for section by section specific growth rate for the control and solvent control cultures were 6% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficients of variation for average specific growth rate for the control and solvent control cultures over the test period (0 to 72 hour) were 3% and 1% respectively 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. There were no abnormalities detected in any of the control, solvent control or test cultures.

Water Quality Criteria
The pH values of the control, solvent control and each test preparation are given in Table 2. Temperature was maintained at 24 ±1 ºC throughout the test.
The pH value of the control cultures was observed to range from pH 7.9 at 0 hours to pH 7.8 at 72 hours. A pH value of 7.9 was determined for the solvent control cultures at both 0 and 72 hours. The pH deviation in the control and solvent 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, solvent control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, solvent control, 0.0017, 0.0021, 0.0038 and 0.0049 mg/L test cultures were observed to be green dispersions whilst the 0.0064 mg/L test cultures were observed to be slightly pale green dispersions.

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.

DefinitiveTest

Verification of Test Concentrations

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 82% to 89% with the exception of the 0.018 mg/L test preparation where a measured concentration of 0.0057 mg/L (32% of nominal) was obtained. This low value was considered to be due to a possible dosing error during preparation.

 

A marked decline in measured test concentrations was observed at 72 hours to less than the LOD, equivalent to 0.001 mg/L in all test preparations. This decline was in line with the results obtained from the study to determine the Abiotic Degradation: Hydrolysis as a Function of pH (Engivo Study Number XP76GX) whereby approximately 50% degradation occurred after 24 hours at pH 4 and 7. It was considered unnecessary to provide additional samples for analysis at 24 and 48 hours in order to better define the rate in decline given that analysis of the test preparations taken from the Acute Daphnia magna Test (Envigo Study Number LT47HW) showed a decline in measured test concentrations to less than the LOD occurred following 24 hours exposure.

Current regulatory advice is that in cases where a decline in measured concentrations isobserved, geometric mean measured concentrations should be used for calculating EC50values. 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 LOD of the analytical method following current regulatory advice a value of half the LOD (i.e. 0.00050 mg/L) was used to enable calculation of the geometric mean measured concentrations. The geometric mean measured test concentrations were determined to be:

 

Nominal Test Concentration (mg/L)	Geometric Mean Measured Test Concentration (mg/L)	Expressed as a Percentage ofthe Nominal Test Concentration (%)
0.010	0.0021	21
0.018	0.0017	9
0.032	0.0038	12
0.056	0.0049	9
0.10	0.0064	6

 

As the nominal 0.018 mg/L test group was below the level at which any significant biological effects were observed, the possible error during dosing was considered to have had no impact on the outcome of the test.

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

 

Nominal Concentration (mg/L)		Cell Densities*(cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	5.28E+03	2.04E+06	-	-
	R2	4.11E+03	1.95E+06
	Mean	4.69E+03	1.99E+06
Solvent Control	R1	4.40E+03	2.22E+06	-	-
	R2	5.05E+03	2.27E+06
	Mean	4.72E+03	2.24E+06
0.0010	R1	4.05E+03	2.29E+06	1	9
	R2	5.16E+03	1.80E+06
	Mean	4.61E+03	2.05E+06
0.010	R1	4.69E+03	2.04E+06	0	4
	R2	4.34E+03	2.27E+06
	Mean	4.52E+03	2.16E+06
0.10	R1	5.28E+03	1.24E+06	10	43
	R2	4.81E+03	1.32E+06
	Mean	5.05E+03	1.28E+06

 *  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 replicateflasks

R = Replicate

-  = Not applicable

Cell Densities and pH Values in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0 Hours	22 Hours	48 Hours	72 Hours	72 Hours
Control	R1	7.9	2.94E+04	1.97E+05	1.28E+06	7.8
	R2		2.89E+04	1.89E+05	1.09E+06
	R3		2.98E+04	1.91E+05	1.19E+06
	R4		2.86E+04	1.98E+05	1.57E+06
	R5		3.16E+04	2.03E+05	1.51E+06
	R6		2.74E+04	1.89E+05	1.20E+06
	Mean		2.93E+04	1.95E+05	1.31E+06
Solvent Control	R1	7.9	3.34E+04	2.18E+05	1.70E+06	7.9
	R2		3.14E+04	2.19E+05	1.43E+06
	R3		2.74E+04	1.98E+05	1.50E+06
	R4		2.77E+04	2.14E+05	1.42E+06
	R5		2.80E+04	2.16E+05	1.63E+06
	R6		3.17E+04	2.12E+05	1.40E+06
	Mean		2.99E+04	2.13E+05	1.51E+06
0.0021	R1	7.9	2.70E+04	2.19E+05	1.79E+06	8.0
	R2		2.51E+04	2.32E+05	1.78E+06
	R3		2.62E+04	2.02E+05	1.40E+06
	Mean		2.61E+04	2.18E+05	1.66E+06
0.0017	R1	7.8	2.84E+04	2.06E+05	1.42E+06	8.0
	R2		3.15E+04	2.02E+05	1.73E+06
	R3		2.52E+04	2.18E+05	1.37E+06
	Mean		2.84E+04	2.09E+05	1.51E+06
0.0038	R1	7.8	2.76E+04	1.97E+05	1.58E+06	8.1
	R2		2.61E+04	2.03E+05	1.37E+06
	R3		3.08E+04	2.10E+05	1.43E+06
	Mean		2.82E+04	2.04E+05	1.46E+06
0.0049	R1	7.8	2.49E+04	1.70E+05	9.94E+05	8.2
	R2		2.76E+04	2.06E+05	1.38E+06
	R3		2.45E+04	1.97E+05	1.29E+06
	Mean		2.57E+04	1.91E+05	1.22E+06
0.0064	R1	7.8	1.40E+04	1.19E+05	8.31E+05	8.2
	R2		1.45E+04	1.05E+05	6.66E+05
	R3		1.65E+04	1.18E+05	7.38E+05
	Mean		1.50E+04	1.14E+05	7.45E+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

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	0.081	0.073	0.078
	R2	0.080	0.072	0.073
	R3	0.081	0.071	0.076
	R4	0.079	0.074	0.086
	R5	0.084	0.071	0.084
	R6	0.077	0.074	0.077
	Mean	0.080	0.073	0.079
Solvent Control	R1	0.086	0.072	0.086
	R2	0.084	0.075	0.078
	R3	0.077	0.076	0.084
	R4	0.078	0.079	0.079
	R5	0.078	0.079	0.084
	R6	0.084	0.073	0.079
	Mean	0.081	0.076	0.082

R  = Replicate

Inhibition of Growth Rate and Yield in the DefinitiveTest

Geometric Mean Measured Test Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.077	-	1.27E+06	-
	R2	0.075		1.09E+06
	R3	0.076		1.19E+06
	R4	0.080		1.56E+06
	R5	0.079		1.50E+06
	R6	0.076		1.20E+06
	Mean	0.077		1.30E+06
	SD	0.002		1.89E+05
Solvent Control	R1	0.081	-	1.70E+06	-
	R2	0.079		1.43E+06
	R3	0.079		1.49E+06
	R4	0.078		1.41E+06
	R5	0.080		1.63E+06
	R6	0.078		1.40E+06
	Mean	0.079		1.51E+06
	SD	0.001		1.25E+05
0.0021	R1	0.082	[4]	1.79E+06
	R2	0.082	[4]	1.77E+06
	R3	0.078	1	1.39E+06
	Mean	0.081	[2]	1.65E+06	[9]
	SD	0.002		2.25E+05
0.0017	R1	0.078	1	1.42E+06
	R2	0.081	[3]	1.73E+06
	R3	0.078	1	1.36E+06
	Mean	0.079	[0]	1.50E+06	1
	SD	0.002		1.96E+05
0.0038	R1	0.080	[1]	1.58E+06
	R2	0.078	1	1.37E+06
	R3	0.079	0	1.43E+06
	Mean	0.079	0	1.46E+06	3
	SD	0.001		1.08E+05
0.0049	R1	0.074	6	9.89E+05
	R2	0.078	1	1.37E+06
	R3	0.077	3	1.29E+06
	Mean	0.076	3	1.22E+06	19
	SD	0.002		2.02E+05
0.0064	R1	0.071	10	8.26E+05
	R2	0.068	14	6.61E+05
	R3	0.069	13	7.33E+05
	Mean	0.069	12	7.40E+05	51
	SD	0.002		8.30E+04

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

R  = Replicate

SD = Standard Deviation

[ ] = Increase in growth compared to solvent controls

-    = Notapplicable

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 following results:
Response Variable EC50(mg/L) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate >0.0064* 0.0049 0.0064
Yield 0.0063 0.0049 0.0064

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 was designed to be compatible with the 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.

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 EPIWIN predicted water solubility value for the test item was 0.089 mg/L.

The results obtained from the preliminary media preparation trials conducted indicated that a dissolved test item concentration of approximately 0.092 mg/L could be obtained using a solvent spike method of preparation in culture medium.

Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 0.010, 0.018, 0.032, 0.056 and 0.10 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

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 82% to 89% with the exception of the 0.018 mg/L test preparation where a measured concentration of 0.0057 mg/L (32% of nominal) was obtained. This low value was considered to be due to a possible dosing error during preparation. A marked decline in measured test concentrations was observed at 72 hours to less than the limit of detection (LOD), equivalent to 0.001 mg/L in all test preparations. This decline was in line with the results obtained from the study to determine the Abiotic Degradation: Hydrolysis as a Function of pH (Engivo Study Number XP76GX) whereby approximately 50% degradation occurred after 24 hours at pH 4 and 7.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The geometric mean measured test concentrations were determined to be 0.0021, 0.0017, 0.0038, 0.0049 and 0.0064 mg/L. As the nominal 0.018 mg/L test group was below the level at which any significant biological effects were observed, the possible error during dosing was considered to have had no impact on the outcome of the test.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric 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	>0.0064*	0.0049	0.0064
Yield	0.0063	0.0049	0.0064

Description of key information

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 was designed to be compatible with the 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.

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 EPIWIN predicted water solubility value for the test item was 0.089 mg/L.

The results obtained from the preliminary media preparation trials conducted indicated that a dissolved test item concentration of approximately 0.092 mg/L could be obtained using a solvent spike method of preparation in culture medium.

Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 0.010, 0.018, 0.032, 0.056 and 0.10 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

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 82% to 89% with the exception of the 0.018 mg/L test preparation where a measured concentration of 0.0057 mg/L (32% of nominal) was obtained. This low value was considered to be due to a possible dosing error during preparation. A marked decline in measured test concentrations was observed at 72 hours to less than the limit of detection (LOD), equivalent to 0.001 mg/L in all test preparations. This decline was in line with the results obtained from the study to determine the Abiotic Degradation: Hydrolysis as a Function of pH (Engivo Study Number XP76GX) whereby approximately 50% degradation occurred after 24 hours at pH 4 and 7.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The geometric mean measured test concentrations were determined to be 0.0021, 0.0017, 0.0038, 0.0049 and 0.0064 mg/L. As the nominal 0.018 mg/L test group was below the level at which any significant biological effects were observed, the possible error during dosing was considered to have had no impact on the outcome of the test.

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

Key value for chemical safety assessment

EC50 for freshwater algae:

0.006 mg/L

EC10 or NOEC for freshwater algae:

0.005 mg/L