2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 30, 2018 - July 16, 2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Composition of test material: Sodium-2-ethylhexyliminomonopropionate and Sodium-2-ethylhexyliminodipropionate
- Analytical purity: >=99%
- Purity test date: 13 December 2017
- Lot/batch No.: WI6K21X06-FD1
- Expiration date of the lot/batch: 05 December 2018
- Appearance: White paste
- Storage: At room temperature

Analytical monitoring:

yes

Details on sampling:

- Sampling method: At t = 0h and t = 48h from all test concentrations and the control
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was completely soluble in test medium at the concentrations tested. Preparation of test solutions started with the highest concentration of 100 mg/L applying a 18 minute period of magnetic stirring to accelerate dissolution of the test item in medium. The pH of the stock solution was adjusted from 9.6 to 8.0 with 1 mol HCl/L. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820) at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions
- Source: Daphnids originated from a healthy stock (in-house laboratory culture), 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals
- Age: For the test, selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

19-20°C

pH:

8.0-8.2

Dissolved oxygen:

8.8-9.1 mg O2/L

Nominal and measured concentrations:

Nominal concentrations: control, 0.10, 1.0, 10 and 100 mg/L (combined limit/range-finding test)

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass vessels
- Fill volume: 50 mL of test solution
- Aeration: No
- Renewal of test solution: No (static test)
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 (100 mg/L) or 2 (0.1, 1.0 and 10 mg/L)
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Test medium: Adjusted ISO medium
- Dilution water: Tap water purified by reverse osmosis (RO-water)
- Culture medium different from test medium: Yes (M7 medium = ajusted ISO medium supplemented with trace elements, macronutrients and vitamins)

OTHER TEST CONDITIONS
- Adjustment of pH: Yes
- Photoperiod: 16 hours light / 8 hours dark (room light)
- Feeding during test: No

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobility (including mortality): At 24 hours and at 48 hours

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: based on analytically confirmed nominal concentrations

Details on results:

MEASURED CONCENTRATIONS
Samples taken from the highest test concentration and the control were analysed. The final test item exposure concentrations were calculated based on the responses of the two constituents separately measured. The measured concentrations for the test item were at the level of nominal (89-113%) throughout the test, based on the responses of both constituents. Based on these results, the effect parameters were based on analytically confirmed nominal concentrations.

INHIBITION OF MOBILITY
No immobility was observed at any of the test concentrations during the test period.

ACCEPTABILITY OF THE TEST
1. In the control, no daphnids became or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.
2. The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.
The study met the acceptability criteria prescribed by the study plan and was considered valid.

Results with reference substance (positive control):

The EC50 for mobility inhibition in the reference test with potassium dichromate (48h-EC50) was 0.33 mg/L with a 95% confidence interval ranging from 0.29 to 0.38 mg/L. The historical ranges for mobility inhibition lie between 0.3 and 1.0 mg/L. Hence, the 48h-EC50 for the dapnid batch tested corresponds with this range.

Number of introduced daphnids and incidence of immobility

Time (h)	Replicate	Test item Nominal conc. (mg/L)
		Control	0.10	1.0	10	100
0	A	5	5	5	5	5
	B	5	5	5	5	5
	C	5				5
	D	5				5
	Total introduced	20	10	10	10	20
24	A	0	0	0	0	0
	B	0	0	0	0	0
	C	0				0
	D	0				0
	Total immobilised	0	0	0	0	0
	Effect %	0	0	0	0	0
48	A	0	0	0	0	0
	B	0	0	0	0	0
	C	0				0
	D	0				0
	Total immobilised	0	0	0	0	0
	Effect %	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

The 48h-EC50 for Daphnia magna exposed to the test item was beyond the range tested, i.e. exceeded the analytically confirmed nominal concentration of 100 mg/L.

Executive summary:

The acute toxicity of the test item to water fleas (Daphnia magna) was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 202, using a combined limit/range-finding test. No immobility was observed at any of the test concentrations during the test period. As a result, a 48-hour 50% effective concentration EC50 exceeding 100 mg/L was obtained based on analytically confirmed nominal concentrations.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

7/9/1995 - 9/9/1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP, quality criteria met. lack of details on substance identity, exposure concentrations not analytically verified.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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 relevant

Analytical monitoring:

no

Details on sampling:

Not relevant

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution was prepared by dissolving a known quantity of the sample in a specified volume of dilution water. The test solutions were prepared by adding the stock solution to the dilution water in appropriate quantities to obtain the desired concentrations for the test.
- Controls: Yes,

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: Shell research laboratories
- Age at study initiation (mean and range, SD): less than 24 hours
- Method of breeding: culturing under semi-static conditions at 20 +/- 2ºC, oxygen at >75% of air saturation value and 16/8 light/dark photoperiod. Daphnids were fed daily with a Chlorella vulgaris suspension at a rate of 1 mg organic carbon / litre.
- Feeding during test: no feeding during the test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not relevant

Hardness:

280 mg/l CaCO3

Test temperature:

20.0 - 20.8ºC

pH:

7.7 - 8.1

Dissolved oxygen:

95 - 97% of air saturation value

Salinity:

Not relevant

Nominal and measured concentrations:

A preliminary study had indicated that the 48 hour EC50 was greater than 1000 mg/l. Since the OECD guidelines state that the highest test concentration should not exceed 1000 mg/l, definitive nominal test concentrations were prepared as 100, 180, 320, 560 and 1000 mg/l. Corrected for active ingredient nominal test concentrations were: 40, 72, 128, 224 and 400 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: crystallising dishes
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 50 ml containing 25 ml of test medium
- Aeration: None
- 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: dechlorinated mains tap water
- Culture medium different from test medium: no
- Intervals of water quality measurement: at the begin and end of the test (0 and 48 hours)

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16/8 hours light/dark period

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): immobilisation (24 hour intervals)

TEST CONCENTRATIONS

- Test concentrations: 0, 100, 180, 320, 560, 1000 mg/l

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 400 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 400 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: 0%

Results with reference substance (positive control):

Not relevant

Reported statistics and error estimates:

Where possible, the EC50 values were estimated graphically, and 95% confidence limits calculated according to the method of Grammo and Larsstuvold (1976).

For detailed results, see attached file "Results.docx".

Validity criteria fulfilled:

yes

Remarks:

Control mortality 0%, abiotic conditions within acceptable limits. no analytical details on test substance.

Conclusions:

The acute toxicity(48h-EC50) of Lakeland AMA LF40 towards Daphnia magna is > 400 mg a.i./l.

Executive summary:

The acute aquatic toxicity of the substance towards Daphnia magna was investigated according to OECD guideline 202 under GLP. Daphnids were expsoed to nominal concentrations (based on active ingredient) of 40, 72, 128, 224 and 400 mg/L and observed for 48 hours. The 24h- and 48 -EC50 were found to be > 400 mg a.i./L.

Description of key information

The acute toxicity of 2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide (methanol free) to water fleas(Daphnia magna)was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 202, using a combined limit/range-finding test. No immobility was observed at any of the test concentrations during the test period. As a result, a 48-hour 50% effective concentration EC50 exceeding 100 mg/L was obtained based on analytically confirmed nominal concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

The toxicity of 2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide (methanol free) to freshwater fleaDaphnia magnawas investigated in one GLP-compliant study performed in accordance with standard methods, without deviations. The study is considered as reliable (Klimisch 1) and is selected as a key study for the endpoint.

The endpoint study summary for 2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide (methanol free) is linked to study record(s) on the substance itself but also to study record(s) on an analogue substance, a.k.a. Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate (CAS 94441-92-6 / EC 305-318-6). The latter is used as source substance to fill datagaps on 2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide (methanol free) for particular endpoints pertaining to environmental fate, aquatic toxicity and human health toxicity. Several aquatic toxicity data are thus reported for the source substance (e.g.toxicity to aquatic invertebrates, toxicity to aquatic plants – identified as “supporting studies”) to support the read-across approach (cf. read-across justification document attached in "13 Assessment reports").