Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-07-12 to 2017-02-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 17. Jul. 1992

Deviations:

yes

Remarks:

Temperature range was 18.9 – 23.4 °C instead of 20.0 – 24.0 °C.

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:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from a biologic sewage treatment plant was used. The chosen plant is treating mostly domestic sewage. The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf.
- Storage conditions: 18.9 – 23.4 °C
- Storage length: 28 days
- Pretreatment: The sludge was filtrated, washed with tap water (2x), then washed with and re-suspended in test medium. It was then aerated until use.
- Concentration of sludge: The dry matter was determined with 3600 mg suspended solids/L. Inoculum concentration: 25.0 mg/L.
- Test item concentration: nominally 20 mg organic carbon/L (corresponding to 25.7 mg VOELOFA monomer/L)
- Date of collection: 05. Aug. 2016
- batch no: 20160805

Duration of test (contact time):

28 d

Initial conc.:

25.7 mg/L

Based on:

test mat.

Remarks:

Test item Loading Rate (nominal)

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The medium was freshly prepared.
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with 1000 mL demin. water; The pH-value was 7.4.
b) 27.5 g CaCl2 filled up with 1000 mL demin. water
c) 22.5 g MgSO4 x 7 H2O filled up with 1000 mL demin. water
d) 0.25 g FeCl3 x 6 H2O, 0.4 g Na2EDTA x 2 H2O filled up with 1000 demin. water
10 mL of solution a, 1 mL of solution b, 1 mL of solution c, 1 mL of solution d were combined and filled up with 1000 mL demin. water
(see also Tabe 1 in "Any other information on materials and methods incl. tables".

- Additional substrate:
- Solubilising agent (type and concentration if used):
- Test temperature: 18.9 – 23.4 °C
- pH: 7.3 (measured at the end of the test before addition of HCl)
- pH adjusted: no
- CEC (meq/100 g):
- Aeration of dilution water: no
- Suspended solids concentration: The dry matter was determined with 3600 mg suspended solids/L; concentration in the test 25.0 mg dry matter/L
- Continuous darkness: yes
- Other:

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration:
- Method used to create aerobic conditions: all flasks were aerated for 72 hours with purified, CO2-free, moistened air to purge the system of CO2.
- Method used to create anaerobic conditions:
- Measuring equipment: Data logger for temperature, ebro; Analytical scales Mettler Toledo XS 205 DU; Precision scales Mettler Toledo XS 6001S; Adjustable pipettes with one-way tips Rainin®; Carbon analyser TOC multi N/C 2100S, Analytik Jena; Magnetic stirrers; pH-meter 3310 wtw; Heating chamber Memmert; Ultrasonic bath SONOREX RK 510H Bandelin
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used:
- Other: 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
- Sampling frequency: on day 0, 2, 4, 7, 9, 11, 14, 18, 23 and 29
- Sampling method: From each front scrubber flask, 10 samples were taken in order to determine the emitted CO2. 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.
- Sterility check if applicable:
- Sample storage before analysis:
- Other:
- Analyses: Analyses of the emitted CO2 were made by IC measurement using the carbon analyser TOC multi N/C 2100S, Analytik Jena.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2, containing mineral medium and inoculum
- Abiotic sterile control: 1, containing test item, mineral medium and HgCl2
- Toxicity control: 1, containing test item, positive control, mineral medium and inoculum
- Other:
- Positive control flasks: 2, containing positive control, mineral medium and inoculum
- Apparatus blanks: 2, containing mineral medium only

STATISTICAL METHODS:

Test Parameters:
- CO2 Determination: Analyses of the emitted CO2 were made by IC measurement using the carbon analyser TOC multi N/C 2100S, Analytik Jena.
- pH-Value: measured with the pH-meter 3310 wtw

Preparation of positive control:
- Aniline was chosen as positive control.
- Activated sludge was used as inoculum (concentration in the test 25.0 mg dry matter/L).
- A stock solution containing 2108.8 mg/L in deionised water was prepared and its organic carbon content was measured with 1604.6 mg/L, corresponding to an organic carbon content of the positive control of 76.1 %.

Reference substance:

aniline

Remarks:

Phenylamine, C6H5NH2, CAS-No. 62-53-3

Parameter:

% degradation (CO2 evolution)

Value:

10

Sampling time:

4 d

Parameter:

% degradation (CO2 evolution)

Value:

44

Sampling time:

14 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

52

Sampling time:

28 d

Details on results:

Degradation behaviour of positive control and toxicity control was normal. Abiotic degradation reached 3 %. Both replicates of the test item showed very good correspondence. If degradation in the toxicity flask is below 25% after 14 days, the test item can be considered as toxic towards the inoculum. As degradation in the toxicity flask was 61% after 14 days, the test item can be stated as “not toxic towards the inoculum in a concentration of26.1 mg/L”.
For pure substances ready degradability is defined in the guidelines as degradation surpassing 60% within 10 days after reaching a level of 10%. Because the test item is a mixture, the 10-day window has not to be considered. Still, the test item is considered as “not readily biodegradable” as the pass level of 6O % was missed at the end of the test.
The IC content of test item in medium was more than 5 % of TC as demanded in the guideline. This is because the test item is very poorly soluble in water. Therefore, the validity criterion is not applicable for the test item.
No observations were made which might cause doubts concerning the validity of the study
outcome.

The result of the test can be considered valid.

Results with reference substance:

Degradation of the positive control was 69 % after 9 days.

In the following tables, the IC values which were measured are stated.

Table 2: 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	2.71	2.70	2.98	2.88
2	4.26	4.29	15.67	8.00
4	4.82	4.79	24.32	13.53
7	7.11	6.72	27.82	24.99
9	8.79	8.06	32.72	33.76
11	16.01	10.54	38.44	40.90
14	12.16	10.41	36.58	41.50
18	14.13	12.45	49.65	55.90
23	19.34	16.03	57.15	63.08
29	24.93	22.32	68.28	76.59

Table 3: 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	2.87	2.78	2.75	2.73	2.60	3.03
2	9.63	6.10	20.99	15.11	11.58	15.35
4	24.70	34.90	70.82	47.61	14.62	162.35
7	211.53	197.07	123.39	51.18	16.74	315.57
9	251.74	248.55	148.76	136.29	19.21	397.46
11	293.18	282.36	170.58	183.44	20.52	444.05
14	314.59	303.38	188.72	174.89	23.16	428.82
18	330.60	311.53	207.76	207.66	24.09	485.43
23	342.68	320.75	219.16	220.36	28.07	502.26
29	374.83	355.24	245.24	245.49	33.23	543.85

Table 4 IC values in mg/L of blank controls, apparatus blanks, back scrubber.

Day	Apparatus blank 1	Apparatus blank 2	Blank Control 1	Blank Control 2
0	2.80	3.02	2.70	2.88
29	3.41	3.14	3.45	3.80

Table 5 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	2.69	2.53	3.03	2.79	2.93	2.82
29	3.63	3.26	3.70	3.90	3.98	2.81

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

In order to evaluate aerobic elimination and degradation potential of VOELOFA Monomer in a test for ready biodegradability, a test item concentration of nominally 20 mg organic carbon/L (corresponding to 25.7 mg VOELOFA Monomer/L) was used. The mean degradation rate of the test item VOELOFA Monomer was 52% after 28 days. Therefore, regardless of the 10-day-window, VOELOFA Monomer is not readily biodegradable following OECD 301B/EU C.4-C.

Executive summary:

The aerobic ready biodegradability of VOELOFA Monomer in the CO2 Evolution Test following OECD 301B resp. EU C.4 -C was studied in a mineral medium (pH 7.3 in test flasks) inoculated and incubated under aerobic conditions in the dark with activated sludge from a domestic sewage treatment plant (stock suspension of 3600 mg/L on dry matter base and a final sludge concentration in test flasks of 25.0 mg sludge/L). VOELOFA Monomer was applied with 25.7 mg/L (corresponding to 20 mg organic carbon/L). The experiment was conducted in accordance with the OECD test guideline 301 B ”CO2-Evolution-Test (Modified STURM Test)”, and in compliance with the OECD-GLP standards. The test system was aerated with purified (by activated charcoal), CO2 -scrubbed, moistened air. Magnetic stirrers were used to prevent deposition of inoculum. The emitted CO2 was trapped in two scrubbers containing 100 mL 0.25 M NaOH each. The initial IC value of the 0.25 M NaOH was separately determined in each flask. Analyses of the emitted CO2 were made by IC measurement using the carbon analyser TOC multi N/C 2100S, Analytik Jena.

 

The degree of biodegradation reached 52 % after 28 days. The 10-day-window began on day 4, at its end, 44 % degradation were reached, missing the pass level of 60 % given in the OECD guideline. Because the test item is a mixture, the 10-day window is not considered. Degradation missed 60% within 28 days. Therefore, the test item is considered as “not readily biodegradable within 28 days”. The abiotic degradation reached 3 %.

Description of key information

The mean degradation rate of the test item VOELOFA Monomer was 52 % after 28 days. Therefore, VOELOFA Monomer is not readily biodegradable following OECD 301B/EU C.4-C. The 10-day-window began on day 4, at its end, 44 % degradation were reached, fulfilling the criterion for the 10-d window. The 10-day window is not considered relevant as the test item is a UVCB.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater