Reaction mass of 4-tert-butyl-2,5-xylenol and 6-tert-butyl-2,4-xylenol

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:

25 January to 24 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Meets the requirements of GLP. There are no deviations from the recommended guideline.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide 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): The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf.
Date of collection: 22. Jan 2009, batch no: 20100122.
- Storage conditions: 22 ± 2 °C
- Pretreatment: The sludge was filtrated, washed with tap water twice, then washed with and resuspended in test medium. It was then aerated for > 12 hours.
- Concentration of sludge: 3780 mg suspended solids/L

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

other: organic carbon

Initial conc.:

24.9 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
Solution a:
Potassium dihydrogenephosphate (KH2PO4) 8.5 g
Di-potassium hydrogenephosphate (K2HPO4) 21.75 g
Di-sodiumhydrogenephosphate dihydrate (Na2HP04*2H20) 33.4 g
Ammonia chloride (NH4CI) 0.5 g
H20 demin. ad 1000 mL
The pH was adjusted to 7.4 ±0.1.
Solution b:
Calcium chloride dihydrate (CaCI2*2H20) 36.4 g
H20 demin. ad 1000 mL
Solution c:
Magnesium sulfate heptahydrate (MgS04*7H20) 22.5 g
H20 demin. ad 1000 mL
Solution d:
Iron(lll) chloride hexahydrate (FeCI3*6H20) 0.25 g
Di-sodium-ethylendiamintetraacetate dihydrate (Na2EDTA*2H20) 0.4 g
H20 demin. ad 1000 mL

Test Medium: The medium was freshly prepared:
Solution a 10 mL
Solution b 1 mL
Solution c 1 mL
Solution d 1 mL
H20 demin. ad 1000 mL

- Test temperature: 22 ± 2 °C
- pH: determined at the end of the test, from 6.8 to 7.3
- pH adjusted: no
- Suspended solids concentration: The dry matter of the activated sludge was determined with 3780 mg suspended solids/litre.

TEST SYSTEM
- Culturing apparatus: 2000 mL-SCHOTT-flasks were used as test vessels, 100 mL scrubber flasks as absorbent vessels
- Number of culture flasks/concentration: 2 test flasks, containing test item, mineral medium and inoculum
- Method used to create aerobic conditions:
The medium was prepared from the stock solutions. 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 24 hours with purified, C02-free, moistened air to purge the system of C02.
The test vessels were aerated with purified (by activated charcoal), C02-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 C02, the air was then led through a flask containing a solution of Ba(OH)2 before reaching the test vessels.
- Measuring equipment: data logger for temperature, analytical scales Mettler Toledo 184 SA, precision scales Sartorius 1403, adjustable pipettes with one-way tips Rainin® (for volumes 1 mL, 1-10 mL); Carbon analyser TOC multi N/C 2100S, Analytik Jena, pH-meter 340i wtw.
- Details of trap for CO2 and volatile organics if used: The emitted C02 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 days 0, 2, 4, 7, 9, 12, 14, 18, 22 and 29
- Sampling method: from each front scrubber flask, ten samples were taken in order to determine the emitted C02. The sample volume was 1 mL. The resulting change in the volume of the front flask was considered in the calculation of emitted C02. On day 28, 5 mL HCI 2-m were added to each test flask in order to drive off dissolved C02. On day 29, samples from both scrubber flasks were taken.

CONTROL AND BLANK SYSTEM
- Abiotic sterile control: 1, containing test item, mineral medium and HgCI2
- Toxicity control: 1, containing test item, positive control, mineral medium and inoculum
- Other:
Controls: 2, containing mineral medium and inoculum
Positive control flasks: 2, containing positive control, mineral medium and inoculum

Reference substance:

aniline

Key result

Parameter:

% degradation (CO2 evolution)

Value:

2.6

Sampling time:

2 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

1.8

Sampling time:

4 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

2.5

Sampling time:

7 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0.2

Sampling time:

9 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

-3

Sampling time:

29 d

Details on results:

- The test item is considered as "not readily biodegradable".
- No biodegradation was observed after 28 days.
- A 10-day-window was not observed. The pass level of 60 % given in the OECD guideline was missed.
- No abiotic degradation was observed

Results with reference substance:

Degradation of the positive control was 68 % after nine days.

All validity parameters and values are presented in the following table:

Validity:

Parameter	Criterion	Found	Assessment
IC content of test item solution in medium	<5%ofTC	< 1 %	valid
C02emitted by the controls	< 70 mg/L	8.2 mg/L	valid
Difference within replicates	< 20%	4%	valid
Degradation of positive control > 60%	< 14 days	9 days	valid
Degradation in the toxicity flask on day 14	> 25%	36%	valid

 

All validity criteria were met.

Degradation behaviour of positive control and toxicity control was normal. Abiotic degrada­tion was not observed. Both replicates of the test item showed very good correspondence, as well as the two replicates of the positive control.

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 36% after 14 days, the test item can be stated as "not toxic towards the inoculum in a concentration of 25.0 mg/L".

The result of the test can be considered valid.

 

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

When applying the 10-day-window, the test substance is not readily biodegradable following OECD 301B/EU C.4-C.

Executive summary:

The test item was tested using a concentration of nominally 20 mg organic carbon/L (corresponding to 24.9 mg test substance/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 mg dry matter/L). The test was left running for 28 days. All validity criteria were met. Degradation of the positive control was 68 % after nine days. The following data were determined for the test item:

10-day-window: none
degradation at the end of 10-day-window: not detected

degradation at the end of the test: no degradation

pass level: 60% at the end of 10-day-window

Therefore, when applying the 10-day-window, the test substance is not readily biodegradable following OECD 301B/EU C.4-C.

Description of key information

Key study: Experimental result: Test according to OECD guideline 301B and EU method C.4.C. GLP study.

When applying the 10-day-window, the test substance is not readily biodegradable following OECD 301B/EU C.4-C.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

Key study: Experimental result: Test according to OECD guideline 301B and EU method C.4.C.

The test item was tested for aerobic ready biodegradability in the CO2 evolution test. At the end of 10 -day-window degradation was not detected, nor at the end of the test. Therefore, it can be concluded that when applying the 10-day-window, the test substance is not readily biodegradable.