1-Octadecanamine, reaction products with 1,1'-methylenebis[4-isocyanatobenzene]

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 Starting Date: 24 July 2012 Experimental Completion Date: 22 August 2012

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

PU Thickener

IUPAC Name:

PU Thickener

Test material form:

other: Beige solid pieces

Details on test material:

The integrity of the supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor.

Identification: PU Thickener
Description: beige solid pieces
Batch: not provided
Purity: >98%
Expiry / retest date: 14 June 2015
Storage conditions: room temperature in the dark

Study design

Oxygen conditions:

aerobic

Remarks:

aqueous medium

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Test Species
A mixed population of activated sewage sludge micro-organisms was obtained on 23 July 2012 from the aeration stage of the Severn Trent Water Plc sewage
treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum
The activated sewage sludge sample was washed two times by settlement and resuspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of
approximately 21 ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then
continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.1 g/L prior to use.


Mineral Medium
The mineral medium used in this study (see attached Appendix 1) was that recommended in the OECD Guidelines.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

38.7 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Remarks:

Percentage biodegradation

Details on study design:

Test Species
A mixed population of activated sewage sludge micro-organisms was obtained on 23 July 2012 from the aeration stage of the Severn Trent Water Plc sewage
treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Pre-Study Solubility Work
Information provided by the Sponsor indicated that the test item was insoluble in water. Therefore pre-study solubility/dispersibility work was performed in
order to determine the most suitable method of preparation (see attached Appendix 2).


Experimental Preparation
For the purpose of the test, the test item was dispersed directly in mineral medium. In order to aid weighing the test item was crushed using a pestle and
mortar prior to weighing.

An amount of test item (38.7 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm,
15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of 12.9 mg/L, equivalent to
10 mg carbon/L.

A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

Data from the inoculum control vessels was shared with similar concurrent studies.


Reference Item
For the purposes of the test, a reference item, sodium benzoate (C6H5COONa), 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 10 minutes. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium and the volume adjusted to 3 liters to give a final test
concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure
homogeneity of the solution.

Data from the procedure control vessels was shared with similar concurrent studies.


Toxicity Control
For the purposes of the test, a toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.

An amount of test item (38.7 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm,
15 minutes) prior to dispersal in inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 liters to give a final concentration of 12.9 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.


Preparation of Test System
The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:

a) An inoculated control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control
(one vessel only).

Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a
temperature controlled room at approximately 21 °C, in darkness.

Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 42.9 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. The pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being
adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2 free air.

The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic
stirrer.

The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.

The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were
prepared using purified de-gassed water.

Reference substance

Reference substance:

other: sodium benzoate (C6H5COONa)

Results and discussion

Preliminary study:

Pre-Study Solubility Work
Information provided by the Sponsor indicated that the test item was insoluble in water. Therefore pre-study solubility/dispersibility work was
performed in order to determine the most suitable method of preparation (see the attached Appendix 2).

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Details on results:

RESULTS
Inorganic carbon values for the test item, procedure control, toxicity control and inoculum control vessels at each analysis occasion are given in Table 1.
Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 and the biodegradation curves are presented in the attached Figure 1. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3. The pH values of the test preparations on Days 0 and 28 are given in Table 4. Observations made on the contents of the test vessels are given in Table 5.

The total CO2 evolution in the inoculum control vessels on Day 28 was 28.73 mg/L and therefore satisfied the validation criterion given in the OECD Test
Guidelines.

The IC content of the test item suspension in the mineral medium at the start of the test (see Table 3) was below 5% of the TC content and hence satisfied
the validation criterion given in the OECD Test Guidelines.

The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion
given in the OECD Test Guidelines.

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test
Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any
additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the
exception of inoculum control replicates R1 and R2, procedure control replicate R1 and test item replicate R2.

Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second
absorber vessels occurred.

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

Statistical analysis of the Day 29 IC values for the control and test item vessels showed there were no statistically significant differences (P≥ 0.05) between the control and the test item. The test item was therefore considered not to have a toxic effect on the sewage sludge micro-organisms used in the study
and this was confirmed by the toxicity control results.

The toxicity control attained 33% degradation after 14 days and 33% degradation after 28 days thereby confirming that the test item was not toxic to the
sewage treatment micro-organisms used in the test.

Sodium benzoate attained 83% degradation after 14 days and 72% degradation after 28 days thereby confirming the suitability of the inoculum and test
conditions. The slight decrease in degradation between days 14 and 28 was considered to be due to sampling/analytical variation.

BOD5 / COD results

Results with reference substance:

Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 and the biodegradation curves are presented in the attached Figure 1

Any other information on results incl. tables

Tables

Table1     Inorganic Carbon Values on Each Analysis Occasion

Day	Inoculum Control (mg IC)				Sodium Benzoate Procedure Control (mg IC)				Test Item (mg IC)				Test Item plus Sodium Benzoate Toxicity Control (mg IC)
	R1		R2		R1		R2		R1		R2		R1
	Abs1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	1.05	1.05	0.93	1.05	1.05	1.40	0.93	1.05	1.05	1.05	1.05	1.05	1.05	1.52
2	7.66	-	7.42	-	13.22	-	24.25	-	6.61	-	4.52	-	20.42	-
6	17.65	-	12.69	-	34.25	-	35.06	-	12.80	-	9.80	-	35.06	-
8	11.24	-	11.47	-	32.91	-	35.09	-	12.61	-	8.94	-	32.11	-
10	11.97	-	12.31	-	32.94	-	34.65	-	11.63	-	12.65	-	32.94	-
14	16.09	-	15.87	-	40.01	-	41.48	-	15.07	-	16.77	-	35.81	-
21	21.63	-	19.04	-	43.60	-	42.70	-	19.27	-	17.01	-	41.57	-
28	24.30	-	22.74	-	44.91	-	45.02	-	21.84	-	21.17	-	43.12	-
29	23.16	2.67	22.71	4.18	44.53	2.32	45.98	2.78	22.27	2.44	20.93	3.83	43.34	2.78

 

R₁ – R₂          =   Replicates 1 and 2

Abs              =  CO₂ absorber vessels

Table 2     Percentage Biodegradation Values            

Day	% Degradation Sodium Benzoate Procedure Control	% Degradation Test Item	% Degradation Test Item plus Sodium Benzoate Toxicity Control
0	0	0	0
2	37	0	21
6	65	0	33
8	75	0	35
10	72	0	35
14	83	0	33
21	76	0	35
28	72	0	33
29*	74	0	34

*Day 29 values corrected to include any carry-over of CO₂ detected in Absorber 2

Table 3     Total and Inorganic Carbon Values in the Culture Vessels on Day 0       

Test vessel	Total Carbon*^+ (mg/L)	Inorganic Carbon^* (mg/L)	IC Content (% of TC)
Test Item 10 mg C/L R1	10.16	-0.01	0
Test Item 10 mg C/L R2	10.15	-0.01	0

 

R₁ – R₂     =  Replicates 1 and 2                                                                                                                                                    

*       Corrected for control values.

+      Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item

Table 4     pH Values of the Test Preparations on Days 0 and 28

Test Vessel	pH prior to adjustment on Day 0	pH after adjustment on Day 0	pH on Day 28
Inoculum ControlR1	7.9	7.5	7.4
Inoculum Control R2	7.8	7.5	7.4
Procedure Control 10 mg C/L R1	7.8	7.5	7.5
Procedure Control 10 mg C/L R2	7.8	7.5	7.6
Test Item 10 mg C/L R1	7.8	7.5	7.7
Test Item 10 mg C/L R2	7.8	7.5	7.7
Toxicity Control 20 mg C/L	7.8	7.5	7.8

 

R₁– R₂    =      Replicates 1 and 2

Table 5     Observations on the Test Preparations Throughout the Test Period

Test Vessel		Observations on Test Preparations
		Day 0	Day 6	Day 13	Day 20	Day 27
Inoculum Control	R1	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
	R2	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
Procedure Control	R1	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible
	R2	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible
Test Item	R1	Light brown dispersion, particles of test item visible on surface and dispersed throughout	Light brown dispersion, particles of test item visible on surface and dispersed throughout	Light brown dispersion, particles of test item visible on surface and dispersed throughout	Light brown dispersion, particles of test item visible on surface and dispersed throughout	Light brown dispersion, particles of test item visible on surface and dispersed throughout
	R2	Light brown dispersion, particles of test item visible on surface and dispersed throughout	Light brown dispersion, particles of test item visible on surface and dispersed throughout	Light brown dispersion, particles of test item visible on surface and dispersed throughout	Light brown dispersion, particles of test item visible on surface and dispersed throughout	Light brown dispersion, particles of test item visible on surface and dispersed throughout
Toxicity Control		Light brown dispersion, no undissolved reference item visible. Particles of test item visible on surface and dispersed throughout	Light brown dispersion, no undissolved reference item visible. Particles of test item visible on surface and dispersed throughout	Light brown dispersion, no undissolved reference item visible. Particles of test item visible on surface and dispersed throughout	Light brown dispersion, no undissolved reference item visible. Particles of test item visible on surface and dispersed throughout	Light brown dispersion, no undissolved reference item visible. Particles of test item visible on surface and dispersed throughout

 

R₁ – R₂ = Replicates 1 and 2

 

Applicant's summary and conclusion

Interpretation of results:

not inherently biodegradable

Conclusions:

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

Executive summary:

Introduction

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO₂ Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (m)).

 

 

Methods

The test item, at a concentration of 10 mg Carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at approximately 21 °C for 28 days.

 

The degradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

 

 

Results

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