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

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis is based on “different compounds which have similar properties”.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- The source substance is identified as Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, also known as Sodium 3-[(2-carboxyethyl)(2-ethylhexyl)amino]propanoate (CAS no. 94441-92-6 | EC no. 305-318-6). It is a UVCB substance whose major constituent is sodium 2-ethylhexylimino-di-propionate. Minor constituents are sodium 2-ethylhexylimino-mono-propionate, unreacted acrylic acid and unreacted 2-ethylhexylamine.
- The target substance is identified as Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide (methanol free) (CAS no. 68610-44-6| EC no. 271-865-1). It is a UVCB substance whose major constituents are sodium 2-ethylhexylimino-mono-propionate and sodium 2-ethylhexylimino-di-propionate. Minor constituents are unreacted acrylic acid and unreacted 2-ethylhexylamine.

3. ANALOGUE APPROACH JUSTIFICATION
The target and source substances are essentially the same: both are UVCB substances composed of the exact same constituents with the exact same functional groups (i.e. carboxylic acid groups and secondary/tertiary amine groups). They are expected to have the same ADME profile and to share common mode of action and breakdown products. The target and source substances only differ in the overlapping ranges of their constituents, the content of sodium 2-ethylhexylimino-mono-propionate being especially higher in the target substance. This difference is expected to have no or very limited impact on the potency of effects exerted on exposed living organisms.

4. DATA MATRIX
Cf. read-across justification document attached in §13. Assessment reports.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Test organisms (species):

activated sludge

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 246.5 g/L

Nominal / measured:

nominal

Conc. based on:

other: structural analogue

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Conclusions:

By analogy with Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate (source substance), 2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide (methanol free) is not expected to exert significant toxic effects on aquatic microorganisms/activated sludge (3h-EC50 > 246.5 mg/L).

Executive summary:

2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide (methanol free) has not been tested for its toxicity to aquatic microorganisms. The datagap is filled for this endpoint using a read-across from a supporting substance (analogy with Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS 94441-92-6, EC 305-318-6).

The toxicity of Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate (source substance) to activated sludge was investigated in a GLP-compliant study performed in accordance with OECD Guideline 209. A 3-hour 50% effective concentrations EC50 of > 246.5 mg/L (expressed as active ingredients) was obtained based on nominal concentrations; no effects were observed at the highest test concentration. By analogy, 2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide (methanol free) is not expected to exert significant toxic effects on aquatic microorganisms/activated sludge (3h-EC50 > 246.5 mg/L).

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-04-2012 - 25-04-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test performed under GLP according guidelines, all quality criteria were met

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Sodium capryliminodipropionate and 3,5-dichlorophenol was added to the incubation vessels using a stock solution of approximately 1.0
g/L. The stock solution of sodium capryliminodipropionate was prepared by dissolving 0.92 g sodium capryliminodipropionate in 920 mL deionized water.

pH stock solution sodium capryliminodipropionate = 9.5

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Unadapted secondary activated sludge was obtained (24-04-2012) from the WWTP Nieuwgraaf in Duiven. The WWTP Duiven is an activated sludge plant treating predominantly domestic wastewater. Prior to use the activated sludge was homogenized with a syringe. The dry weight of the homogenized activated sludge was determined and subsequently concentrated by settlement to the required dry weight concentration. The dry weight of the activated sludge in the incubation vessels was 1.5 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

Temperature varied from 19.8 to 20.5°C.

pH:

The pH of the reaction mixtures after the incubation period ranged from 7.2 to 7.3.

Dissolved oxygen:

aerobic during test

Details on test conditions:

-The test was performed in 300 ml Erlenmeyers.
-The homogenized activated sludge was incubated in a shaking water bath (100 rpm, 20°C) for 3 hours with various concentrations of the test
compound and synthetic sewage.
-Synthetic sewage was added to the activated sludge (1.6 ml on a total volume of 50 ml).
Synthetic sewage contained per liter: 16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.3 g CaCl2
0.2 g MgSO4.7H2O
2.8 g K2HPO4
-Different concentrations (spaced by a factor two) of test compound and reference compound where added using stock solutions both of
1.0 g/l
-Tap-water was used to fill up the total test volume to 50 ml.
- The dry weight concentration of the activated sludge in the test was 1.5 g/l
-The tested concentrations of the reference compound 3,5-dichlorophenol where: 2.5 mg/l, 5.0 mg/l,10.0 mg/l, 20.0 mg/l and 40.0 mg/l
-The tested concentrations of hydrogenated (tallow alkyl) trimethyl ammonium chloride where : 10 mg/l, 20 mg/l, 40 mg/l, 80 mg/l and 160 mg/l.
-Controls without test substance (or reference compound) are measured at the beginning and at the end of the test
-The inhibitory effect of the test substance/reference compound is expressed as a percentage of the mean of the two control respiration rates.
-An abiotic control was not performed because neither reduction of oxygen by the test substance nor formation of oxygen due to chemical
breakdown of the test substance is expected.
-No specific analyses were performed

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 640 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Test conditions
The pH of the reaction mixtures after the incubation period ranged from 7.2 to 7.3. Temperature varied from 19.8 to 20.5°C. These conditions allow
respiration of the activated sludge used.

Validity of the test
The validity of the test is demonstrated by three criteria. First, the coefficient of variation of the replicates of the control oxygen uptake rates was 10%, which
fulfils the maximum prescribed variation of <30%. Secondly, the prescribed average oxygen uptake rate of > 20 mg O2/g dry weight/hour for the control
measurements was achieved (30 mg O2/g dry weight/hour). The third criterion was met as shown by the EC50 of the reference compound of 15 mg/L, which
is within the prescribed range of 2 to 25 mg/L.

Activated sludge respiration inhibition test
The inhibitory effect of sodium capryliminodipropionate at a particular concentration is expressed as a percentage of the two controls. From the
results EC values were calculated. The EC50 of sodium capryliminodipropionate for activated sludge after 3 hours contact time is >640 mg/L.
Inhibition of the activated sludge respiration by sodium capryliminodipropionate does not follow a classical dose-response curve and
varied from 11% to 19% over the measured concentration range.

Results with reference substance (positive control):

The EC50 of the reference compound was 15 mg/L at a contact time of 3 hours, respectively. These EC50 values is within the prescribed range of
2 to 25 mg/L.

Validity criteria fulfilled:

yes

Remarks:

see "details on results"

Conclusions:

The test is valid as shown by the EC50 of the reference compound 3,5- dichlorophenol (15 mg/L), the coefficient of variation of the replicates of the
control oxygen uptake rates (10%) and the average oxygen uptake rate for the control replicates (30 mg O2/g dry weight/hour).
The toxicity of sodium capryliminodipropionate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test
substance. The inhibitory effect of sodium capryliminodipropionate at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of sodium capryliminodipropionate for activated sludge after 3 hours contact time is >640 mg/L. Inhibition of the
activated sludge respiration by sodium capryliminodipropionate does not follow a classical dose-response curve and varied from 11% to 19% over the
measured concentration range (Figure 2). Sodium capryliminodipropionate is therefore considered not harmful to activated sludge.

Executive summary:

In order to predict effects of chemicals in the environment and in biological waste water treatment plants, an activated sludge respiration inhibition test was performed. The toxicity to activated sludge was determined in accordance with OECD Test Guideline 209, and in compliance with the OECD principles of Good Laboratory Practice.

The test is valid as shown by the EC50 of the reference compound 3,5 -dichlorophenol (15 mg/L), the coefficient of variation of the replicates of the control oxygen uptake rates (10%) and the average oxygen uptake rate for the control replicates (29 mg O2/g dry weight/hour). The toxicity of sodium capryliminodipropionate to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of sodium capryliminodipropionate at a particular

concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of sodium capryliminodipropionate for activated sludge after 3 hours contact time is >640 mg/L. Inhibition of the activated sludge respiration by sodium capryliminodipropionate does not follow a classical dose-response curve and varied from 11% to 19% over the measured concentration range (Figure 2). Sodium capryliminodipropionate is therefore considered not harmful to activated sludge.

Description of key information

2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide (methanol free) has not been tested for its toxicity to aquatic microorganisms. The datagap is filled for this endpoint using a read-across from a supporting substance (analogy with Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS 94441-92-6, EC 305-318-6).

The toxicity to activated sludge of Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate (source substance) was investigated in a GLP-compliant study performed in accordance with OECD Guideline 209. A 3-hour 50% effective concentrations EC50 of > 246.5 mg/L (expressed as active ingredients) was obtained based on nominal concentrations; no effects were observed at the highest test concentration. By analogy, 2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide (methanol free) is not expected to exert significant toxic effects on aquatic microorganisms/activated sludge (3h-EC50 > 246.5 mg/L).

Key value for chemical safety assessment

EC50 for microorganisms:

246.5 mg/L

Additional information

2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide (methanol free) has not been tested for its toxicity to aquatic microorganisms. The datagap is filled for this endpoint using a read-across from a supporting substance (analogy with Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS 94441-92-6, EC 305-318-6).

The toxicity to activated sludge of Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate (source substance) was investigated in a 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.