Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate

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:

15 Jan. 2018 to 14 Feb. 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guidelines for the Testing of Chemicals, Part 301 B, adopted 17. Jul. 1992 “CO2-Evolution-Test (Modified STURM Test)“

Deviations:

yes

Remarks:

Temperature range was 19.5 – 21.2 °C instead of 20.0 – 24.0 °C See "Any other information" for further details.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Council Regulation (EC) No. 440/2008, Method C.4-C, adopted 30. May 2008 “CO2-Evolution-Test”

Deviations:

yes

Remarks:

Temperature range was 19.5 – 21.2 °C instead of 20.0 – 24.0 °C. See "Any other information" for further details.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate

EC Number:

946-383-6

Molecular formula:

C17H24N2O3

IUPAC Name:

Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate

Test material form:

liquid: viscous

Details on test material:

Name: Trixene AS
Batch no.: OP544-153/1801239973
Appearance: clear, light yellow-green, viscous liquid
Composition: Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate
EINECS-No.: 946-383-6
Molecular formula: C17-H24-N2-O3
Purity: "multi-constituent substance made up of a mixture of isomers
Homogeneity: Homogeneous
Vapour pressure: not stated
Expiry date: 01. Sep. 2019
Storage Room: Temperature (20 ± 5°C)

Specific details on test material used for the study:

No further details specified in the study report.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Specification
Activated sludge from a biologic sewage treatment plant was used as inoculum. The chosen plant treats mostly domestic sewage.

Source and Pre-Treatment of inoculum
Source
The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf.
Date of collection: 12. Jan. 2018, batch no: 20180112.

Pre-Treatment
The sludge was filtrated, washed with test medium (2x) and re-suspended in test medium.
It was then aerated until use. The dry matter was determined to contain 4900 mg of suspended solids/L.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

>= 29.9 - <= 31.1 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preparations
The medium was prepared from the stock solutions. The stock solution of the positive control was prepared and its DOC was measured. The inoculum was taken from its source, washed, aerated and the dry matter was determined.
The test vessels were filled with medium and inoculum. Then, all flasks were aerated for 72 hours with purified, CO2-free, moistened air to purge the system of CO2.

Experimental Parameters
Flask volume: 1500 mL
Apparatus blanks: 2, containing mineral medium only
Blank Controls: 2, containing mineral medium and inoculum
Positive control flasks: 2, containing positive control, mineral medium and inoculum
Test flasks: 2, containing test item, mineral medium and inoculum
Abiotic control: 1, containing test item, mineral medium and HgCl2
Toxicity control: 1, containing test item, positive control, mineral medium and inoculum
Inoculum concentration: 25.0 mg/L
Temperature: 19.5 – 21.2 °C
Duration: 28 days
The test was performed with a nominal start concentration of 20 mg organic carbon/L.

Apparatus
The test vessels were aerated with purified (by activated charcoal), CO2-scrubbed, moistened air. The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1.5 M NaOH. To control the absence of CO2, the air was then led through a flask containing a solution of Ba(OH)2 before reaching the test vessels.
Magnetic stirrers were used to prevent deposition of inoculum.
The emitted CO2 was trapped in 0.25 M NaOH. Two scrubbers containing 100 mL each were connected in series to the test vessels. The initial IC value of the 0.25 M NaOH was separately determined in each flask.

Sampling
From each front scrubber flask, 10 samples were taken in order to determine the emitted CO2 (on day 0, 2, 4, 7, 9, 11, 14, 18, 23 and 29). The sample volume was 1 mL. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2.
On day 28, 5 mL HCl 2 M was added to each test flask in order to drive off dissolved CO2.
On day 29, samples from both scrubber flasks were taken.

CO2 Determination
Analyses of the emitted CO2 were made by IC measurement using the carbon analyser TOC multi N/C 2100S, Analytik Jena. Each sample was measured in duplicate or triplicate, respectively (depending on the variation between the measured values). The carbon analyser was calibrated with freshly prepared reference solutions containing potassium hydrogen phthalate (TC), sodium hydrogen carbonate and sodium carbonate (IC) every month.
After every start, quality control samples were measured.

Reference substance

Reference substance:

aniline

Results and discussion

Test performance:

The degree of biodegradation reached 12 % after 28 days.
The 10-day-window was not detected.
The abiotic degradation reached 3 %.

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

12

Sampling time:

28 d

Details on results:

The test item Trixene AS is considered as “not readily biodegradable“.
The degree of biodegradation reached 12 % after 28 days.
The 10-day-window was not detected.
Because the test item is a mixture, the 10-day window has not to be taken into account.
Therefore, regardless of the 10-day-window the test item is considered as “not readily biodegradable within 28 days”.
The abiotic degradation reached 3 %.

BOD5 / COD results

Results with reference substance:

Degradation of the positive control was 80 % after 9 days.

Any other information on results incl. tables

IC values in mg/L of apparatus blanks, blank controls, front scrubber

Day	Apparatus blank 1	Apparatus blank 2	Blank Control 1	Blank Control 2
0	2.44	2.42	2.31	2.61
2	3.55	4.63	4.33	18.77
4	5.99	7.76	8.42	32.68
7	7.67	10.03	13.32	44.63
9	11.19	13.07	21.05	54.89
11	12.55	12.94	24.17	59.55
14	13.36	15.55	30.22	64.19
18	16.50	17.81	28.27	64.15
23	21.23	20.49	41.06	75.80
29	26.97	25.50	47.58	84.69

 

IC values in mg/L of positive control, test flasks, front scrubber

Day	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
0	2.43	2.60	3.12	2.79	2.41	2.24
2	10.39	11.70	30.90	18.90	14.44	11.59
4	30.28	88.79	43.51	29.14	17.45	80.69
7	205.49	207.72	63.12	41.92	18.52	176.19
9	289.60	288.39	74.64	57.09	21.52	261.25
11	317.93	334.57	73.26	60.94	22.04	297.92
14	351.11	354.88	80.68	62.83	22.65	306.58
18	354.10	360.27	83.92	68.70	24.42	315.41
23	393.17	387.86	94.82	80.75	28.32	346.26
29	398.56	399.70	111.44	103.71	34.33	363.30

 

IC values in mg/L of apparatus blanks, blank controls, back scrubber

Day	Apparatus blank 1	Apparatus blank 2	Blank Control 1	Blank Control 2
0	1.78	2.29	2.31	2.26
29	2.86	2.87	3.01	3.45

 

IC values in mg/L of positive control, test flasks, back scrubber

Day	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
0	2.21	1.96	2.33	2.30	2.29	3.84
29	3.18	3.62	3.58	5.40	3.95	2.77

 

 

For each flask, the net IC was calculated by subtracting the mean IC value of the apparatus blanks of the corresponding sampling date from the remaining IC values. Exception: Values of day 0 do not need to be corrected.

 

Net IC-values in mg/L of front scrubber flasks

Day	Blank Control 1	Blank Control 2	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
0	2.3	2.6	2.4	2.6	3.1	2.8	2.4	2.2
2	2.7	17.1	8.7	10.3	29.2	17.2	12.8	9.9
4	4.0	28.2	25.8	84.3	39.1	24.7	13.0	76.2
7	6.9	38.2	199.1	201.3	56.7	35.5	12.1	169.8
9	11.4	45.2	279.9	278.7	64.9	47.4	11.8	251.6
11	13.9	49.2	307.6	324.3	62.9	50.6	11.7	287.6
14	18.2	52.2	339.1	342.9	68.7	50.8	10.6	294.6
18	13.5	49.4	339.4	345.5	69.2	54.0	9.7	300.7
23	22.6	57.4	374.7	369.4	76.4	62.3	9.9	327.8
29	23.8	60.9	374.8	375.9	87.6	79.9	10.5	339.5

 

Net IC-values in mg/L of back scrubber flasks

Day	Blank Control 1	Blank Control 2	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
0	2.3	2.3	2.2	2.0	2.3	2.3	2.3	3.8
29	2.2	2.6	2.4	2.8	2.8	4.6	3.1	1.9

 

pH in Test flasks on Day 28

Day	Blank Control 1	Blank Control 2	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
28	7.5	7.5	7.3	7.3	7.5	7.5	7.0	7.6

 

Emitted carbon in mg/L

Day	Blank Control 1	Blank Control 2	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
2	0.02	0.96	0.42	0.49	1.72	0.95	0.68	0.51
4	0.11	1.67	1.53	5.34	2.35	1.43	0.69	4.83
7	0.30	2.30	12.72	12.85	3.46	2.12	0.63	10.83
9	0.58	2.73	17.76	17.67	3.96	2.85	0.60	15.96
11	0.73	2.95	19.33	20.37	3.79	3.03	0.59	18.07
14	1.00	3.11	21.10	21.32	4.11	3.01	0.51	18.32
18	0.70	2.90	20.89	21.26	4.10	3.17	0.45	18.50
23	1.25	3.36	22.84	22.50	4.49	3.65	0.46	19.97
29	1.29	3.56	22.60	22.70	5.15	4.83	0.55	20.33

 

Degradation values in %

Day	Positive Control 1	Positive Control 2	Positive Control Mean	Test 1	Test 2	Test Mean	Abiotic Control	Toxicity Control
2	-0.4	0.0	-0.2	5.8	2.3	4.1	3.3	0.0
4	3.2	22.2	12.7	6.9	2.7	4.8	3.3	9.7
9	57.1	57.7	57.4	10.2	4.0	7.1	3.0	23.5
11	80.5	80.1	80.3	10.9	5.9	8.4	2.9	35.3
14	95.2	96.3	95.8	9.7	4.7	7.2	2.5	40.1
18	95.4	97.3	96.4	10.9	6.8	8.8	2.2	41.2
23	102.6	101.0	101.8	10.4	6.6	8.5	2.2	43.5
29	100.8	101.3	101.1	12.9	11.8	12.4	2.6	44.1

 

Because the values of day 29 are the sum of the IC values in both scrubber flasks, an increase (IC values in flasks B of the test higher than in those of the control) or a decrease (IC values in flasks B of the test lower than in those of the control) of degradation can be observed.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

All validity criteria were met.
Because the test item Trixene AS is a mixture, the 10-day window has not to be taken into account. Therefore, the test item is considered as “not readily biodegradable within 28 days”.
No observations were made which might cause doubts concerning the validity of the study outcome.
The result of the test can be considered valid.

Executive summary:

Title of Study: Determination of the aerobic ready biodegradability of Trixene AS in the CO2 Evolution Test following OECD 301B resp. EU C.4-C

 

Findings and Results:

The test item Trixene AS was tested using a concentration of nominally 20 mg organic carbon/L (corresponding to 29.5 mg Trixene AS/L) in test medium following OECD 301B and EU-Method C.4-C.

 

Aniline was chosen as positive control.

Activated sludge was used as inoculum (concentration in the test 25.0 mg dry matter/L).

The test was left running for 28 days.

All validity criteria were met. Degradation of the positive control was 80 % after 9 days.

 

The following data were determined for the test item Trixene AS:

10-day-window: not detected degradation at the end of the test 12 % pass level following guideline: 60 % at the end of 10-day-window for pure substances respective 60 % at the end of the test for mixtures

 

Therefore, when applying the 10-day-window, Trixene AS is not readily biodegradable following OECD 301B and EU C.4-C respectively.

 

Because the test item is a mixture, the 10-day-window does not have to be taken into account.

As degradation missed 60% in the course of the test, Trixene AS is considered as not readily biodegradable.