Ethyl 4-dimethylaminobenzoate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March 2017 to 22 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentrations
- Sampling method: Samples were taken from the control and each test group from the freshly prepared bulk test preparation at 0 hours and from the old or expired pooled replicates at 48 hours for quantitative analysis.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preliminary solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. Based on this information the test material was categorised as being a ‘difficult substance’ as defined by OECD. Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.
Following the trial, the maximum dissolved test material concentration was obtaining following a 24-hour preparation period. There were no significant differences between the pre-treatment types (centrifugation under different conditions and filtration). As near nominal concentrations were obtained, the initial loading rate of the saturated solution was increased to 100 mg/L to ensure the maximum dissolved test material concentration was obtained.
Based on this information the test material was prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test material by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded) to give a nominal test concentration of approximately 46 mg/L.
In the definitive test, a nominal amount of test material (1100 mg) was dispersed in 11 litres of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres 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 the required test concentrations of 1.0, 2.5, 6.4 16 and 40 % v/v saturated solution. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: in house
- Age: First instar; gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- Feeding during test: no

ACCLIMATION
- Acclimation conditions: Adult daphnids were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a cycle of 16 hours of light and 8 hours of darkness with 20 minute dawn and dusk transition periods. Culture conditions ensured that reproduction was by parthenogenesis.
- Type and amount of food: Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.
- Feeding frequency: Daily

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

21 to 22 °C

pH:

7.6 to 7.7

Dissolved oxygen:

8.3 to 8.9 mg O2/L

Nominal and measured concentrations:

- Nominal: 1.0, 2.5, 6.4, 16 and 40 % (v/v) saturated solution
- Measured: 0.34, 1.1, 3.1, 8.1 and 21 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass beakers
- Type: The test vessels were covered to reduce evaporation
- Material, size, headspace, fill volume: 100 mL of test preparation
- Renewal of test solution: The test preparations were not renewed during the exposure period
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water,Elendt M7, contained the following substances in mg/L: H3BO3 0.715, MnCl2.4H2O 0.090, LiCl 0.077, RbCl 0.018, SrCl2.6H2O 0.038, NaBr 0.004, Na2MoO4.2H2O 0.016, CuCl2.2H2O 0.004, ZnCl2 0.013, CoCl2.6H2O 0.010, KI 0.0033, Na2SeO3 0.0022, NH4VO3 0.00058, Na2EDTA.2H2O 0.625, FeSO4.7H2O 0.249, CaCl2.2H2O 293.8, NaHCO3 64.8, MgSO4.7H2O 123.3, Na2SiO3.9H2O 10, KCl 5.8, NaNO3 0.274, K2HPO4 0.184, KH2PO4 0.143, Thiamine hydrochloride 0.075, Cyanocobalamine (vitamin B12) 0.0010 and D(+) biotin (vitamin H) 0.00075.
-Water quality assessments: The water temperature was recorded daily throughout the test; pH and dissolved oxygen concentrations were recorded at the start and termination of the test. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of darkness with 20 minute dawn and dusk transition periods
- Light intensity: 515 to 553 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilised if they were unable to swim within 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 % v/v saturated solution.
- Results used to determine the conditions for the definitive study: Yes. No immobilisation was observed at the test concentrations of 0.10 and 1.0 % v/v saturated solution, however, immobilisation was observed at 10 and 100 % v/v saturated solution. Based on this information, the test concentrations were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

8.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CL 6.2 to 11 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CL 4.0 to 5.2 mg/L

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

DEFINITIVE TEST
Cumulative immobilisation data and other observations from the exposure of Daphnia magna to the test material during the definitive test are given in Tables 1 and 2. Analysis of the immobilisation data at 24 hours by Probit analysis using Linear Maximum-Likelihood regression and by Trimmed Spearman-Karber method at 48 hours using the 0-hour measured test concentrations gave the following results: The EC50 values at 24 and 48 hours of exposure were 8.1 and 4.5 mg/L, respectively. The No Observed Effect Concentrations after 24 and 48 hours of exposure were 3.1 and 1.1 mg/L respectively. The Lowest Observed Effect Concentrations after 24 and 48 hours exposure were 8.1 and 3.1 mg/L, respectively.
Sub-lethal effects of exposure were observed in the control and the 1.1, 3.1, 8.1 and 21 mg/L test concentrations. These responses were trapping at the surface and reduced mobility (see Table 1).

The test was considered to be valid given that no more than 10 % of the control daphnids showed immobilisation or other signs of disease or stress at 48 hours and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.
At the start and throughout the test all control and test solutions were observed to be clear colourless solutions.

Results with reference substance (positive control):

- Results with reference substance valid: Yes
The 48 h-EC50 for the positive control was 1.2 mg/L with 95 % CL of 1.1 to 1.3 mg/L; the NOEC was 0.56 mg/L. The results from the positive control with potassium dichromate were within the normal range for this reference material.

Reported statistics and error estimates:

The EC50 values and associated confidence limits at 24 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The EC50 values and associated confidence limits at 48 hours were calculated by Trimmed Spearman-Karber method. The Lowest Observed Effect Concentration and the No Observed Effect Concentration at 24 and 48 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Table 1: Cumulative Immobilisation Data and Observations in the Definitive Test over 24 hours

0-Hour Measured Concentration (mg/L)	24 Hours
	Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5N	5N	1T 4N	2T 3N
0.34	0	0	0	0	0	0	5N	5N	5N	5N
1.1	0	0	0	0	0	0	5N	5N	5N	2T 3N
3.1	1	0	0	0	1	5	1R 3N	1T 4N	1T 4N	1R 4N
8.1	4	3	0	3	10	50	1R	2T	1R 2T 2N	2R
21	5	5	4	5	19	95	A/I	A/I	1R	A/I

N = No sub-lethal effects observed

T = Trapping at the surface

R = Reduced mobility

A/I = All daphnia immobilised

Table 3: Cumulative Immobilisation Data and Observations in the Definitive Test over 48 hours

0-Hour Measured Concentration (mg/L)	48 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5N	5N	5N	1T 4N
0.34	0	0	0	0	0	0	5N	5N	5N	5N
1.1	0	0	0	0	0	0	5N	5N	1T 4N	5N
3.1	2	0	0	0	2	10	3N	1R 4N	5N	5N
8.1	5	5	5	5	20	100	A/I	A/I	A/I	A/I
21	5	5	5	5	20	100	A/I	A/I	A/I	A/I

N = No sub-lethal effects observed

T = Trapping at the surface

R = Reduced mobility

A/I = All daphnia immobilised

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 48-hour EC50 value was 4.5 mg/L with 95 % CL of 4.0 to 5.2 mg/L. The No Observed Effect Concentration was 1.1 mg/L.

Executive summary:

The potential toxicity of the test material to Daphnia magna was determined in accordance with the standardised guidelines OECD 202 and EU Method C.2 under GLP conditions, using an acute immobilisation test.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 46 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at nominal concentrations of 1.0, 2.5, 6.4, 16 and 40 % v/v saturated solution for 48 hours at a temperature of 21 to 22 °C under static test conditions. The test material solutions were prepared by stirring an excess of test material in test water using a propeller stirrer for 24 hours; any undissolved test material was removed by filtration to produce a 100 % v/v saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the required test concentrations. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the freshly prepared test preparations at 0 hours showed measured test concentrations to be 0.34, 1.1, 3.1, 8.1 and 21 mg/L. There was no significant change in the measured concentrations at 48 hours and so the results are based on 0-hour measured test concentrations only.

Complete immobilisation was observed after 48 hours in the two highest test concentrations; 10 % immobilisation was observed in the 3.1 mg/L test concentration. Sub-lethal effects of exposure were observed in the control and the 1.1, 3.1, 8.1 and 21 mg/L test concentrations. These responses were trapping at the surface and reduced mobility.

The validity and water quality criteria of the test were both met.

Under the conditions of the study, the 48-hour EC50 value was 4.5 mg/L with 95 % CL of 4.0 to 5.2 mg/L. The No Observed Effect Concentration was 1.1 mg/L.

Description of key information

In the key study, the potential toxicity of the test material to Daphnia magna was determined in accordance with the standardised guidelines OECD 202 and EU Method C.2 under GLP conditions, using an acute immobilisation test. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 46 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test material at nominal concentrations of 1.0, 2.5, 6.4, 16 and 40 % v/v saturated solution for 48 hours at a temperature of 21 to 22 °C under static test conditions. The test material solutions were prepared by stirring an excess of test material in test water using a propeller stirrer for 24 hours; any undissolved test material was removed by filtration to produce a 100 % v/v saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the required test concentrations. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the freshly prepared test preparations at 0 hours showed measured test concentrations to be 0.34, 1.1, 3.1, 8.1 and 21 mg/L. There was no significant change in the measured concentrations at 48 hours and so the results are based on 0-hour measured test concentrations only.

Complete immobilisation was observed after 48 hours in the two highest test concentrations; 10 % immobilisation was observed in the 3.1 mg/L test concentration. Sub-lethal effects of exposure were observed in the control and the 1.1, 3.1, 8.1 and 21 mg/L test concentrations. These responses were trapping at the surface and reduced mobility.

The validity and water quality criteria of the test were both met.

Under the conditions of the study, the 48-hour EC50 value was 4.5 mg/L with 95 % CL of 4.0 to 5.2 mg/L. The No Observed Effect Concentration was 1.1 mg/L.

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

4.5 mg/L

Additional information

In the supporting study, the potential of the test material to cause acute toxicity to Daphnia magna was determined broadly in accordance with the standardised guideline OECD 202 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Daphnia magna were exposed to the test material at nominal concentrations of 1.0, 10.0 and 100.0 mg/L alongside a vehicle (0.1 mL/L acetone) and negative control under static conditions. The Daphnia were in duplicate sealed conical flasks (5 per flask) for a period of 48 hours. Daphnids were observed after 24 and 48 hours and were considered to be immobile if they did not swim during a 15 second period of observation.

The lowest concentration causing 100 % immobilisation was 100 mg/L and the highest concentration causing no immobilisation was 10 mg/L.

Under the conditions of this study, the 48 h EC50 was 31.8 mg/L.