Reaction mass of N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethyldocosan-1-aminium chloride and N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethylicosan-1-aminium chloride

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 27 July 2016. Experimental completion date: 24 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

yes

Remarks:

please see any other info on materials and methods section

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

yes

Remarks:

please see any other info on materials and methods section

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethyldocosan-1-aminium chloride and N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethylicosan-1-aminium chloride

EC Number:

947-079-6

IUPAC Name:

Reaction mass of N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethyldocosan-1-aminium chloride and N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethylicosan-1-aminium chloride

Test material form:

other: paste

Specific details on test material used for the study:

Identification: Methoxycinnamidopropyl Behendimonium Chloride
Physical state/Appearance: off-white paste
Batch: INV-1603010
Purity: not provided
Expiry Date: 01 February 2017
Storage Conditions: room temperature in the dark

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

Test System and Supporting Information
A mixed population of sewage treatment micro-organisms was obtained on 25 July 2016 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum
The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at temperatures of between 20 and 22 ºC prior to use.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

29.37 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Test Item Preparation
The test item contained a solids content of 29.37%, therefore the test concentrations employed were corrected for this. The test item was dispersed directly in mineral medium.
A nominal amount of test item (50 mg) was dispersed in mineral medium (350 mL) and subjected to high shear mixing (approximately 7500 rpm, 15 minutes) prior to the addition of inoculum (5 mL) and mineral medium to a final volume of 500 mL to give the test concentration of 29.37 mg ai/L.
A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guidelines. However on correction for the solids content of 29.37%, the test concentration employed was 29.37 mg ai/L with a Theoretical Oxygen Demand value (ThOD) of 25 mg O2/L. This was a deviation to the study plan but was considered to have no effect on the study given that the test item demonstrated an inhibitory effect at the test concentration employed.

Reference Item Preparation
A reference item, aniline (C6H5NH2), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium with the aid of ultrasonication for approximately 5 minutes. An aliquot (50 mL) of this stock solution was diluted with mineral medium (445 mL) and the inoculum (5 mL), to give the test concentration of 100 mg/L. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity.

Toxicity Control
A toxicity control, containing the test item and aniline, was prepared in order to assess any toxic effect of the test item on the sewage treatment micro-organisms used in the test.
A nominal amount of test item (50 mg) was dispersed in mineral medium (350 mL) and subjected to high shear mixing (approximately 7500 rpm, 15 minutes). After high shear mixing an aliquot (50 mL) of the 1000 mg/L aniline stock solution (see Section 3.5.2) along with inoculum (5 mL) to a final volume of 500 mL with mineral medium. This gave a test concentration of 29.37 mg ai test item/L and 100 mg aniline/L.

Preparation of Test System
The following test preparations were prepared and inoculated in 500 mL bottles:
a) Three replicate bottles containing inoculated mineral medium to act as the inoculum control.
b) Two replicate bottles containing inoculated mineral medium and the reference item, aniline, at a concentration of 100 mg/L.
c) Three replicate bottles containing inoculated mineral medium and the test item at a concentration of 29.37 mg ai/L.
d) Two replicate bottles containing inoculated mineral medium the reference item, aniline, at a concentration of 100 mg/L and the test item at a concentration of 29.37 mg ai/L to act as toxicity control vessels.

Data from the inoculum control and procedure control vessels was shared with similar concurrent studies up to Day 27.
All vessels were inoculated with the prepared inoculum at a rate of 1% v/v.
On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter. The pH values were adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the addition of the inoculum and the volume in all the vessels being adjusted to 500 mL by the addition of mineral medium.
In order to confirm that the aniline item stock solution was prepared correctly, a diluted,
100 mg/L stock solution (in reverse osmosis water) was also sampled for Dissolved Organic Carbon (DOC) analysis.
All remaining inoculum control, test item, procedure control and toxicity control vessels were placed in a CES Multi-Channel Aerobic Respirometer.
The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the test with a magnetically coupled stirrer.
As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into the ethanolamine solution (50% v/v) causing a net reduction in gas pressure within the sample flask. The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data generated from the respirometer’s own battery backed memory was collected on the hard disk drive of a non-dedicated computer.
The test was conducted in diffuse light at a temperatures of between 21 and 22 ºC.
On Day 28, two inoculum control, one procedure control, two test item and one toxicity control vessel were sampled for pH analysis.
The remaining vessels which were not sampled were discarded and are not reported. Additional replicate vessels were prepared and incubated in order that in the event of a leak in the test system a replicate vessel could be discarded without jeopardizing the integrity of the test.

Reference substance

Reference substance:

aniline

Results and discussion

Test performance:

See details on result for validation criteria.

% Degradation

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

Validation Criteria
The mean BOD of the inoculated mineral medium (control) was 22.02 mg O2/L after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The pH of the inoculated test item vessels on Day 28 ranged from 7.5 to 7.6 and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between extremes of replicate BOD values at the end of the test was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.

Biodegradation
The test item attained 0% biodegradation after 28 days, calculated from the oxygen consumption values, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.
There were statistically significant differences (P≥0.05) between the control and test item Day 28 BOD values. The test item was therefore considered to have had an adverse effect on the sewage treatment micro-organisms used in the test.
The toxicity control attained 1% biodegradation after 14 and 28 days thereby confirming that the test item was inhibitory to the sewage treatment micro-organisms used in the test.

BOD5 / COD results

Results with reference substance:

Aniline (procedure control) attained 70% biodegradation after 14 days and 75% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

Table       Percentage Biodegradation Values

Day	Procedure Control	Test Item			Toxicity Control
		R1	R2	Mean
0	0	0	0	0	0
1	0	6	6	6	1
2	0	1	2	2	0
3	1	0	0	0	0
4	21	0	0	0	0
5	45	0	0	0	0
6	58	0	0	0	0
7	61	0	0	0	1
8	63	0	0	0	1
9	65	0	0	0	1
10	66	0	0	0	1
11	67	0	0	0	1
12	68	0	0	0	1
13	69	0	0	0	1
14	70	0	0	0	1
15	71	0	0	0	1
16	71	0	0	0	1
17	72	0	0	0	2
18	72	0	0	0	2
19	73	0	0	0	1
20	73	0	0	0	1
21	74	0	0	0	1
22	74	0	0	0	1
23	74	0	0	0	1
24	74	0	0	0	1
25	75	0	0	0	1
26	75	0	0	0	1
27	75	0	0	0	1
28	75	0	0	0	1

 

R₁– R₂= Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.
The toxicity control attained 1% biodegradation after 14 and 28 days thereby confirming that the test item was inhibitory to the sewage treatment micro-organisms used in the test.
Care should be taken in the interpretation of these results due to the inhibitory nature of the test item to the sewage treatment micro-organisms at a test concentration of 29.37 mg/L.

Executive summary:

Introduction:

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301F, “Ready Biodegradability; Manometric Respirometry Test” referenced as method C.4-D of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (q)).

Methods:

The test item at a concentration of 29.37 mg active ingredient (ai)/L was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at temperatures of between 21 and 22 ºC for 28 days. 

The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values on Days 0 to 28. Control solutions with inoculum and the reference item, aniline, together with a toxicity control were used for validation purposes.

Results:

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

The toxicity control attained 1% biodegradation after 14 and 28 days thereby confirming that the test item was inhibitory to the sewage treatment micro-organisms used in the test.

Care should be taken in the interpretation of these results due to the inhibitory nature of the test item to the sewage treatment micro-organisms at a test concentration of 29.37 mg ai/L.