2-ethylhexyl 4-(dimethylamino)benzoate

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:

06 January 2017 to 07 February 2017

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)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Solvent control and limit concentration
- Sampling method: Samples were taken from the solvent control and the 0.077 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples were taken.
- Sample storage conditions before analysis: Samples were stored frozen prior to analysis

Vehicle:

yes

Remarks:

Dimethylformamide

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preliminary solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation such as ultrasonication and high shear mixing. A test concentration of 0.40 mg/L (by visual inspection) was obtained using a preliminary solution in dimethylformamide (DMF).
An amount of test material (40.6 mg) was dissolved in a final volume of 10 mL of DMFto give a 40 mg/10 mL solvent stock solution. An aliquot (100 µL) of the 40 mg/10 mL solvent stock solution was dispersed in 2 litres of culture medium with the aid of magnetic stirring for approximately 30 minutes prior to the removal of any undissolved test material by centrifugation at 40 000 g for 30 minutes. An aliquot of algal suspension (12.8 mL) was added to 1 L of the centrifuged test preparation to give the required nominal concentration of 0.077 mg/L. The stock solutions and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
- Controls: Control and solvent control groups were maintained under identical conditions but not exposed to the test material.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide (DMF)
- Concentration of vehicle in test medium: The solvent control group was exposed to 100 µL/L of DMF.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No; any undissolved test material was removed by centrifugation.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Method of cultivation: 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 illumination at 21 ± 2 °C.

ACCLIMATION
- Culturing media and conditions: 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 (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 – 10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

7.6 to 9.1

Nominal and measured concentrations:

- Nominal concentration: 0.077 mg/L
- Geometric mean measured concentration: 0.015 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks
- Type: Plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: Filled with 100 mL of test preparation
- Aeration: Yes; constantly shaken at approximately 150 rpm
- Initial cell density: 5.00 x 10^3 cells/mL
- Control end cell density: 1.28 x 10^6 cells/ mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per solvent control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP) containing the following: 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 and Na2EDTA.2H2O 0.30mg/L. The pH was adjusted to 7.5 with 0.1 N NaOH or HCl.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH values of the control and each test preparation were determined at 0 hours and 72 hours. the temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the medium was adjusted to 7.5 with 0.1 N NaOH or HCl.
- Photoperiod: Continuous
- Light intensity and quality: Approximately 7000 lux intensity provided by warm white lighting (380-730 nm).

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Samples were taken at 21, 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 10^3 cells/mL) was taken as the starting cell density.
- Other: To determine the potential effect of the test material 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable (limit test)
- Range finding study; Yes
- Test concentration: 0.077 mg/L (3 replicates)
- Results used to determine the conditions for the definitive study: Yes. The results showed no effect on growth.

DATA EVALUATION
- Comparison of Growth Rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:
µ = (ln Nn – ln N1) / (tn – t1)
Where:
µ = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement

The average specific growth rate over the test duration was calculated for each replicate control and test material 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 100
Where:
Ir = percentage inhibition of average specific growth rate
µc = mean average specific growth rate for the solvent control cultures
µt = average specific growth rate for the test culture

- Comparison of Yield
Yield is calculated as the increase in biomass over the exposure period using the following equation:
Y = Nn – N0
Where:
Y = yield
N0 = cell concentration at the start of the test
Nn = cell concentration at the end of the test

For 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 100
Where:
Iy = percentage inhibition of yield
Yc = mean value for yield in the solvent control group
Yt = mean value for yield for the treatment group

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.015 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.:

>= 0.015 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.015 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.015 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

GROWTH DATA
Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 1.
The growth rate (r) and yield (y) of Pseudokirchneriella subcapitata were not affected by the presence of the test material at a geometric mean measured test concentration of 0.015 mg/L over the 72-hour exposure period.
The test concentration of 0.015 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guideline.
Accordingly the following results were determined from the data:
-Inhibition of growth rate
ErC10 (0 - 72 h) : >0.015 mg/L
ErC20 (0 - 72 h) : >0.015 mg/L
ErC50 (0 - 72 h) : >0.015 mg/L
Where ErCx is the test concentration that reduced growth rate by x %.
There were no statistically significant differences (P=0.05), between the solvent control and 0.015 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.015 mg/L.

-Inhibition of Yield
EyC10 (0 - 72 h) : >0.015 mg/L
EyC20 (0 - 72 h) : >0.015 mg/L
EyC50 (0 - 72 h) : >0.015 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 and 0.015 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.015 mg/L.

VALIDATION CRITERIA
The results of the test are considered valid if the following performance criteria are met:
1) The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
2) The mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-hour tests) must not exceed 35 %.
3) The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7 %.
The cell concentration of the control cultures increased by a factor of 255 after 72 hours and the cell concentration of the solvent control cultures increased by a factor of 248 after 72 hours. The mean coefficient of variation for section by section specific growth rate for the control and solvent control cultures was 13 %. The coefficients of variation for average specific growth rate for the control and solvent control cultures over the test period (0 – 72 h) were 3 and 1 %, respectively.

Results with reference substance (positive control):

The positive control used potassium dichromate 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 to the reference material gave the following results:
ErC50 (0 – 72 h): 1.4 mg/L; 95 % confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h): 0.60 mg/L; 95 % confidence limits 0.52 – 0.69 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference material.

Reported statistics and error estimates:

ECx values were determined by inspection of the growth rate and yield data after 72 hours.
A Student’s t-test incorporating Bartlett's test for homogeneity of variance was carried out on the growth rate and yield data after 72 hours for the solvent control and the 0.077 mg/L test concentration 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).

Table 1: 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	0.077	-	1.30 E+06
	R2	0.077		1.26 E+06
	R3	0.077		1.32 E+06
	R4	0.079		1.45 E+06
	R5	0.073		9.55 E+05
	R6	0.078		1.35 E+06
	Mean	0.077		1.27 E+06
	SD	0.002		1.67 E+05
Solvent Control	R1	0.076	-	1.22 E+06
	R2	0.078		1.34 E+06
	R3	0.077		1.26 E+06
	R4	0.076		1.15 E+06
	R5	0.076		1.20 E+06
	R6	0.077		1.25 E+06
	Mean	0.077		1.24 E+06
	SD	0.001		6.21 E+04
0.015	R1	0.076	1	1.15 E+06
	R2	0.075	3	1.09 E+06
	R3	0.076	1	1.19 E+06
	R4	0.076	1	1.19 E+06
	R5	0.077	0	1.31 E+06
	R6	0.078	[1]	1.37 E+06
	Mean	0.076	1	1.22 E+06	1
	SD	0.001		1.02 E+05

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

R1 -R6 = Replicates 1 to 6

SD = Standard Deviation

[ ] = Increase in growth as compared to solvent controls

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 72 hour EC50 value for growth was greater than 0.015 mg/L. The No Observed Effect Concentration (NOEC) was 0.015 mg/L.

Executive summary:

Algal growth inhibition of the test material was tested was determined in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions using Pseudokirchneriella subcapitata.

A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 0.077 mg/L was obtained from a solvent spike method of preparation, indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test material at a nominal concentration of 0.077 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

An initial solvent stock solution was prepared by dissolving 40 mg of test material in a final volume of 10 mL of dimethylformamide to give a 40 mg/10 mL solvent stock solution. An aliquot (200 µL) of this solvent stock solution was dispersed in 2 litres of culture medium with the aid of magnetic stirring for approximately 30 minutes prior to the removal of any undissolved test material by centrifugation at 40 000 g for 30 minutes to give the required test concentration of 0.077 mg/L. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the 0.077 mg/L test preparation at 0 hours showed a measured test concentration of 0.030 mg/L was obtained. A decline in measured concentration was observed at 72 hours to less than the limit of quantification (LOQ) of the analytical method employed which was determined to be 0.015 mg/L. Given this decline in measured test concentration it was considered justifiable to base the results on the geometric mean measured test concentration.

The growth rate and yield were not affected by exposure to the test material. All validity criteria for the test were fulfilled.

Under the conditions of this study, the 72 hour EC50 values for both growth and yield were greater than 0.015 mg/L. The No Observed Effect Concentration (NOEC) for growth and yield was 0.015 mg/L. The test concentration of 0.015 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material. Under the conditions of this study there were no toxic effects of the test material at the limit of solubility.

Description of key information

Algal growth inhibition of the test material was tested was determined in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions using Pseudokirchneriella subcapitata.

An initial solvent stock solution was prepared by dissolving 40 mg of test material in a final volume of 10 mL of dimethylformamide to give a 40 mg/10 mL solvent stock solution. An aliquot (200 µL) of this solvent stock solution was dispersed in 2 litres of culture medium with the aid of magnetic stirring for approximately 30 minutes prior to the removal of any undissolved test material by centrifugation at 40 000 g for 30 minutes to give the required test concentration of 0.077 mg/L. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the 0.077 mg/L test preparation at 0 hours showed a measured test concentration of 0.030 mg/L was obtained. A decline in measured concentration was observed at 72 hours to less than the limit of quantification (LOQ) of the analytical method employed which was determined to be 0.015 mg/L. Given this decline in measured test concentration it was considered justifiable to base the results on the geometric mean measured test concentration.

The growth rate and yield were not affected by exposure to the test material. All validity criteria for the test were fulfilled.

Under the conditions of this study, the 72 hour EC50 values for both growth and yield were greater than 0.015 mg/L. The No Observed Effect Concentration (NOEC) for growth and yield was 0.015 mg/L. The test concentration of 0.015 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material. Under the conditions of this study there were no toxic effects of the test material at the limit of solubility.