Glycerol, propoxylated, esters with acrylic acid, reaction products with diethylamine

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-02-13 to

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

During the preliminary test, temperatures were outside the 21-25°C range controlled at +-2°C; test concentrations were not separated by a constant factor between 1.5 and 2.2. These deviations were not considered to have affected the outcome of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The test item analysis is known to be complex and not reliable due to its stability: when the test item is solubilised in aqueous or organic medium a rapid hydrolysis is observed leading to the predominant formation of 3-(diethylamino) propionic acid (DEAPA). Nevertheless, at the request of the Sponsor’s Representative, the Test Facility developed an analytical procedure with the objective to assess the test item concentrations in fresh solutions only (an additional analysis on old solutions at 24h was nevertheless proposed).

Vehicle:

no

Details on test solutions:

All testing was performed using a synthetic medium. Chemical reagents used for the preparation of this dilution water were of “analytical grade”; it is prepared according to the protocol described in standard NF EN ISO 6341:
The following stock solutions were prepared:
- CaCl2, 2H2O: 11.76 g per litre of water,
- MgSO4, 7H2O: 4.93 g per litre of water,
- NaHCO3: 2.59 g per litre of water,
- KCl: 0.23 g of per litre of water,
Dilution water was obtained by adding a concentration of 25 mL/L of each of the stock solutions and then aerating the solution until oxygen saturation.
The sum of Ca and Mg ions in dilution water was of 2.5 mmol/L and ratios of Ca/Mg et Na/K were 4 and 10 respectively.

Test solutions were prepared using the synthetic medium; chemical reagents used for the preparation of the dilution water were of “analytical grade”.

The control treatment was maintained under the same conditions, with no test item. No auxiliary substance was used.

PRELIMINARY TEST
Tests solutions were prepared from dilutions of a stock solution: a known volume of test item (490 µL) was poured into a volumetric flask, the volume was then made up to 500 mL with test medium. The solution was kept under high speed stirring at ambient temperature during approximately 1 hour with a magnetic stir bar. Although the stock solution was found to be slightly troubled but homogenous no filtration was necessary before dilution. The pH of the test solutions were not adjusted.

Fish were exposed under static conditions to a series of dilutions (1.0, 5.0, 10.0, 50.0 and 100.0 mg/L nominal concentration) of the stock solution in dilution water. One tank was prepared for each test concentration; each tank contained 7 fish. Test solutions were not renewed throughout the 96h test period (static test).

DEFINITIVE TEST
Based on the results of the range-finding test, the definitive test was conducted at the same series of dilutions with semi-static conditions. A new test item stock solution was thus prepared every day as follows: a known volume of test item (490 µL) was poured into a volumetric flask, the volume was then made up to 500 mL with test medium. The solution was kept under high speed stirring at ambient temperature during approximately 1 hour with a magnetic stir bar. Although the stock solution was found to be slightly trouble but homogenous no filtration was necessary before dilution. The pH of the test solutions were not adjusted.

One tank was prepared for each test concentration; each tank contained 7 fish. Test solutions were renewed every day throughout the 96h test period (semi-static test).

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The test organisms were Danio rerio (Teleostei, Cyprinidae), obtained on 28 July 2016 from a recognised supplier: La Grande Rivière France (69490 Saint-Forgeux, France). All fish were in good health and free from any apparent malformation. Mortality of the batch was less than 5% in the week preceding the start of the study. No disease treatments were administered throughout holding and testing.

Holding was maintained within the laboratory at a temperature of 21-25°C in glass tanks. A light cycle of 12 h light and 12 h dark was applied, illumination being provided by fluorescent tubes (intensity between 400 and 800 lux at the surface of the tanks). Tanks were aerated to ensure that the dissolved oxygen concentration is at least 60% of air saturation value in holding tanks and in test tanks. During holding, fish were fed twice per day with ground flake food TetraMin®.

The fish were not fed for a period of 24h prior to test commencement or throughout the duration of the test.

Fish were added to the test tanks within 30 min after the completion of the preparation of the test solutions. All test fish were weighed prior to the test and a representative number of test fish batch (10 at random) was measured after the test to assess compliance with guideline criteria.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

During the preliminary range-finding test, temperature was maintained in a range from 21°C to 25°C but did not remain constant within ± 2°C: mean measured temperature: 22.4°C, min.: 20.8°C, max.: 23.4°C.

During the definitive test the temperature was maintained in a range from 21°C to 25°C and remaining constant within ± 2°C during the test: mean measured temperature: 22.5°C, min.: 22.1°C, max.: 23.3°C.

pH:

PRELIMINARY TEST (STATIC)
0h: 7.8 - 8.5
24h: 7.6 - 7.7
48h: 7.5 - 7.7
72h: 7.6
96h: 7.5 - 7.6

DEFINITIVE TEST (SEMI-STATIC)
0h (Fresh solution): 8.0 - 8.1
24h (Old solution): 7.4 - 7.6
24h (Fresh solution): 8.0 - 8.1
48h (Old solution): 7.7 - 7.8
48h (Fresh solution): 8.0 - 8.2
72h (Old solution): 7.6 - 7.8
72h (Fresh solution): 8.0 - 8.2
96h (Old solution): 7.7

Dissolved oxygen:

All in % air saturation value.

PRELIMINARY TEST (STATIC)
0h: 100.4 - 101.1
24h: 81.9 - 93.2
48h: 80.8 - 85.2
72h: 83.2 - 88.5
96h: 80.1 - 85.0

DEFINITIVE TEST (SEMI-STATIC)
0h (Fresh solution): 98.6 - 99.5
24h (Old solution): 80.2 - 88.0
24h (Fresh solution): 100.1 - 100.9
48h (Old solution): 86.9 - 89.4
48h (Fresh solution): 97.5 - 98.5
72h (Old solution): 86.5 - 90.8
72h (Fresh solution): 98.5 - 99.7
96h (Old solution): 90.7 - 92.7

Nominal and measured concentrations:

All in mg/L
Nominal concentrations: 0 (Control), 1.0, 5.0, 10.0, 50.0, 100.0
Measured concentrations: highly inconsistent recovery rates due to analysis difficulties related to the test substance.

Details on test conditions:

Test tanks (filled with 2.5 L of test solution) containing fish were incubated with a photo period of 9 hours of darkness per 24 h, at a temperature ranging from 21°C to 25°C but remaining constant within ± 2°C during the test.

All test tanks were made of clear glass and covered by plates with ventilation holes to minimise evaporation and avoid the entry of dust into the solutions.

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

No mortality was observed and no visible abnormalities were reported in the preliminary range-finding test.
No mortality was observed and no visible abnormalities were reported in the definitive test, with the exception of a decrease of activity observed at the test solution renewal.

Sublethal observations / clinical signs:

Analytical results (definitive test)

Measured test item concentrations for the definitive test are summarised in the following table. Chemical analysis of test samples are clearly unreliable: measurements are not repeatable, leading to aberrant data. However, this confirmed the exposure of organisms at high test item concentrations.

Test item determination – Summary (Definitive test)

	Test item concentration (mg/L)
Test solution (mg/L)	T0/T24		T24/T48		T48/T72		T72/T96
	Fresh	Old	Fresh	Old	Fresh	Old	Fresh	Old
Control	< DL	< DL	< DL	ND	< DL	ND	< DL	ND
1.0	< DL	< QL	< DL	ND	< DL	ND	NA	ND
5.0	< QL	NA	< DL	ND	< QL	ND	NA	ND
10.0	< QL	9.8	< DL	ND	*	ND	23.7	ND
50.0	51.8	60.1	24.6	ND	104.2	ND	62.9	ND
100.0	79.2	130.0	53.5	ND	115.9	ND	696.5	ND

< DL (1.86 mg/L): concentration lower than the Detection Limit of the analytical method

< QL (6.2 mg/L): concentration lower than the Quantification Limit of the analytical method

NA: Not Applicable

ND: Not Determined at this sample time

*concentrations higher than the validated interval

 

Cumulative lethality (Definitive test)

Test solution (mg/L)	Cumulative dead fish					Mortality at the end of the period
	T0	T24h	T48h	T72h	T96h
Control	0	0	0	0	0	0.0%
1.0	0	0	0	0	0	0.0%
5.0	0	0	0	0	0	0.0%
10.0	0	0	0	0	0	0.0%
50.0	0	0	0	0	0	0.0%
100.0	0	0	0	0	0	0.0%

Reference Item Test

The sensitivity of the test system and the methodology are evaluated with every batch of fish by performing an acute toxicity test on potassium dichromate. The value of 24h-LC50 obtained with the batch used for the definitive test was as follows:

Batch 16/Br/01/2: 24h-LC50 = 173.6 mg/L (obtained on 11 August 2016)

 

This result is consistent with the laboratory control quality chart.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to Danio rerio exposed under semi-static conditions for 96h was assessed according to the OECD 203 Guideline. Fish were exposed to a series of dilutions of stock solutions in dilution water, which were renewed every day throughout the test period.

Although the test item analysis was known to be complex and unreliable, the Test Facility was asked to develop an analytical procedure to assess the test item concentrations in fresh solutions. As expected the test concentrations were aberrant but confirmed the exposure of organisms at high test item concentrations.

Due to raw data obtained (no mortality noticed in the test), no statistical analysis was necessary; the test item has no toxicity at the highest tested concentration under the test conditions.

Executive summary:

The acute toxicity of the test item to Danio rerio exposed under semi-static conditions for 96h was assessed according to the OECD 203 Guideline. Fish were exposed to a series of dilutions of stock solutions in dilution water, which were renewed every day throughout the test period.

 

Although the test item analysis was known to be complex and unreliable, the Test Facility was asked to develop an analytical procedure to assess the test item concentrations in fresh solutions. As expected, the test concentrations were aberrant but confirmed the exposure of organisms at high test item concentrations.

 

Due to raw data obtained (no mortality observed in the test), no statistical analysis was necessary; the test item has no toxicity at the highest tested concentration (100 mg/L) under the test conditions.

  

The definitive test met the validity criteria of the test guideline detailed as follows:

§ The mortality in the control did not exceed 10% at the end of the test;

§ The dissolved oxygen in the test tanks remained above 60% of the air saturation value throughout the test;

§ The pH did not vary by more than 1 unit.

Description of key information

An acute fish toxicity test according to the OECD Test Guideline 203 was conducted with Glycerol, propoxylated, esters with acrylic acid, reaction products with diethylamine (CAS 162492-10-6) under semi-static conditions.

As the test item analysis in water is complex and unreliable, recovery rates in the test solutions were highly inconsistent, although they confirmed sufficient exposure to the test item in fresh solutions. Up to the highest test item concentration tested (100 mg/L), no mortality was observed. Therefore, the LC50 is considered to be greater than this tested concentration.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

As a conservative approach for the chemical safety assessment, the LC50 is considered to be equal to the highest concentration tested (100mg/L), although no mortality was observed up to this concentration.