N-(butoxymethyl)acrylamide

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:

28 November 2001 to 01 March 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Correction factor: Test article is handled by correcting the purity. Procured sample was handled as 100 % pure organic substance.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions of test material: Refrigerated storage
- Stability under test conditions: The infrared absorption spectrum of the sample was measured before and after; both spectra match and it is confirmed that the substance is stable under storage conditions.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Sludge picked up from the following 10 places throughout Japan in September 2001:
Fushikogawa Treatment Plant (Hokkaido Sapporo City)
Nakahama Treatment Plant (Osaka Prefecture Osaka City)
Kitakamikawa (Miyazaki Prefecture Ishinomaki City)
Yoshinokawa (Tokushima Prefecture, Tokushima City)
Hiroshima bay (Hiroshima prefecture Hiroshima city)
Fukashiba Treatment Plant (Ibaraki Prefecture Kagoshima District)
Ochiai Treatment Plant (Tokyo Metropolitan Shinjuku Ward)
Shinano River (Niigata Prefecture Niigata City)
Lake Biwa (Shiga Prefecture, Otsu City)
Dokai Bay (Fukuoka Prefecture Kitakyushu City)
- Picking sludge: Sewage treatment plant: sludge was return sludge; River, lake and sea: top soil of water edge that is in contact with surface water and air.

- Laboratory culture: In order to retain the uniformity of the sludge, 5 L filtrate of sludge mixture picked from each location above and activated sludge filtrate 5 L that is cultured for around 3 months is mixed to make 10 L, it is adjusted to pH 7.0 ± 1.0 and aerated by fermentor (the outside air is passed through pre-filter and used in aeration).
The 10 L filtrate of sludge mixture that is picked from each location mentioned above is the activated sludge that is cultured as mentioned below.

- Method of cultivation: After around 30 minutes of stopping the aeration of fermentor, 1/3rd of the total quantity of clear supernatant liquid is removed. De-chlorinated water is added to it and the total quantity made as 10 L, it is again aerated (30 minutes or more) in order that the synthetic sewage concentration in de-chlorinated water becomes 0.1 wt % and 50 g/L synthetic sewage was added. This operation was repeated once every day and activated sludge cultured. Culture temperature was 25 ± 2 °C.
Synthetic sewage was as follows: Glucose, peptone and potassium di-hydrogen phosphate are dissolved in de-chlorinated water such that it becomes 50 g/L respectively and pH is adjusted to 7.0 ± 1.0 by sodium hydroxide.
In order to maintain normal status of activated sludge during culture, along with observing the creation state of activated sludge and external appearance of clear supernatant liquid, submergibility is excellent, pH, temperature and dissolved oxygen level are measured and it is confirmed that it is within the range of management standards. The biota of activated sludge is observed using appropriate optical microscope and after confirming that there is no abnormality, it is offered for the test.

- Concentration of sludge: The concentration of suspended matter was measured in order to decide the added quantity of activated sludge. Carried out according to ‘Industrial sewage test method, suspended matter’ (14.1 of JIS K 0102-1998). The concentration of suspended matter of activated sludge was 3500 mg/L.

- Adjusting basic culture medium: By adding pure water to liquid A, liquid B, liquid C and liquid D which is 3 mL each and which is defined in ‘Industrial sewage test method, biochemical oxygen demand’ (21. of JIS K 0102-1998) and mixing, its pH is adjusted to 7.0.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Test temperature: 25 ± 1 °C
- pH: 6.2 to 8.1
- pH adjusted: the pH was adjusted when preparing the activated sludge
- Aeration of dilution water: aeration took place when preparing the activated sludge
- Suspended solids concentration: 30 mg/L
- Test container: 300 mL culture flask
- Carbon case absorbent: Soda lime, No. 1.
- Other: End-over-end shaking by magnetic stirrer. The condition of the test solutions was visually observed every day during the culture period. The appropriateness of the operation condition of the device was also checked.

TEST SYSTEM
- Culturing apparatus: Closed system oxygen consumption measurement device. Thermostatic bath and measurement unit with data processing equipment.
- Measuring equipment: The change in BOD of test solution was continuously and automatically recorded by data processing equipment during the culture period and measured. The temperature in the flask was measured and recorded every day.
Six test containers are provided and test solution is adjusted by the following method.
Adding test articles and aniline: The impurities in the procured sample might contribute to BOD; hence, the procured sample is added such that the organic substance concentration becomes 100 mg/L.

(Water + test article) system (1 unit, test container 1)
In the test container, 300 mL purified water is added, procured sample is divided by micro syringe to 30.5 µL [Organic substance addition quantity 29.9 mg = 30.5 µL x 0.9790 g/cm3 (density)] such that the organic concentration of the substance becomes 100 mg/L and after that the pH is measured.

(Sludge + Test article) system (3 units, test containers 2, 3 and 4)
In the test container, basic culture medium [activated sludge added liquid (2.57 mL) is subtracted from 300 mL] is added, procured sample is divided by micro syringe to 30.5 µL [Organic substance addition quantity 29.9 mg = 30.5 µL x 0.9790 g/cm3 (density)] such that the organic concentration of the substance becomes 100 mg/L and after that the pH is measured.

(Sludge + aniline) system (1 unit, test container 6):
In the test container, basic culture medium [activated sludge added liquid (2.57 mL) is subtracted from 300 mL] is added, aniline is divided by micro syringe to 29.5 µL [Added quantity 30 mg = 29.5 µL x 1.022 g/cm3 (density)] such that it becomes 100 mg/L and then added.

Sludge blank system (1 unit, test container 5):
In the test container, basic culture medium [activated sludge added liquid (2.57 mL) is subtracted from 300 mL] is added.

Reference substance:

aniline

Key result

Parameter:

% degradation (O2 consumption)

Value:

76

Sampling time:

28 d

Parameter:

% degradation (TOC removal)

Value:

95

Sampling time:

28 d

Details on results:

In this test, the average degradation by BOD, TOC and HPLC is 76, 95 and 100 %, respectively. In procured test sample there is around 7 % impurity, in HPLC analysis of (sludge + test article) system, test article did not remain however insoluble matter on HPLC chromatogram nearly remained. Identification was carried out with high-performance liquid chromatography mass analysis meter for insoluble matter in procured sample, however, structural estimation could not be done. From this result, it appears that there is insoluble matter in the procured sample but the degradability of test article is good.

Results with reference substance:

This test is considered to be valid when degradation of aniline derived by BOD after 7 days and after 14 days exceeds 40 and 65 %, respectively.
As the degradation of aniline after 7 and 14 days derived by BOD 69 and 76 %, respectively, it is confirmed that the conditions of the test are valid.

Table 1: Degradation after 28 days

	Degradation (%)
	[2]	[3]	[4]	Average
Result by BOD	75	75	78	76
Result by TOC	95	95	94	95
Result by HPLC	100	100	100	100

 

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The average degradation by BOD was 76 % and the substance is therefore considered readily biodegradable.

Description of key information

The biodegradation of the test material was assessed over 28 days according to OECD Test Guideline 301C using a modified MITI test. The average degradation by BOD was 76 % and is therefore considered readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

The biodegradation of the test material was assessed over 28 days according to OECD Test Guideline 301C using a modified MITI test. Non-adapted activated sludge was used as the inoculum and the study was performed under aerobic conditions. In the procured test sample there was around 7 % impurity and this insoluble matter remained in HPLC analysis, however, structural estimation could not be done. The test article did not remain. The degradation after 7 and 14 days of the reference substance (aniline) derived by BOD is 69 and 76 %, respectively, thereby confirming that the conditions of the test are valid.

The average degradation by BOD of the test material was 76 % and it is therefore considered to be readily biodegradable.

 

The biodegradation of the read-across material, acrylamide, was assessed over 28 days according to OECD test guideline 301D using a closed bottle test. Non-adapted activated sludge was used as the inoculum and the study was performed under aerobic conditions.

Stock solution was prepared by adding 2 g of sample to 1 L of distilled water. Degradation was followed by oxygen analyses with the YSI Dissolved Oxygen Analyzer 54A over a 28-day period. Degradability was based on a comparison of readings of actual oxygen demand to the theoretically expected oxygen demand. A parallel control with inoculum, but without test material, was run as a blank correction factor. The procedure was validated by means of a reference substance (aniline, 2 mg/L) of known biodegradability.

The reference material degraded approximately 98 % in concurrent testing and was considered readily biodegradable because it achieved degradation of greater than 70 %.

The test material was found to degrade approximately 100 % and is also considered readily biodegradable.