4-methylmorpholine

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed according to OECD guideline 302B (modified Zahn-Wellens test). The test was well performed and the results can be considered reliable (Klimisch 2, because no information on GLP and not all information on materials and methods is reported in a detailed way).

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): N-methylmorpholine
- Analytical purity: analytical grade
- Impurities (identity and concentrations): not reported
- Source: obtained from Fluka (Switzerland)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly industrial, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Lenzing AG's WWTP, at that time receiving pulp and paper mill waste waters and a small portion of domestic waste water
- The sludge age in the plant was only 2-3 days
- No data on sampling, cultivation, and preparation for inoculation
- Concentration of sludge: no data
- Initial cell/biomass concentration: no data

Duration of test (contact time):

28 d

Initial conc.:

ca. 1 000 mg/L

Based on:

COD

Parameter followed for biodegradation estimation:

other: COD

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: nutrient cocktail as in guideline OECD 302B
- Additional substrate: none
- Test temperature: room temperature
- pH: no data
- Aeration only if oxygen concentration < 2 mg/L
- Suspended solids concentration: not reported
- Continuous darkness: yes
- Continuous stirring

BIODEGRADATION MEASUREMENTS
- Biodegradation was monitored in the liquid phase via COD (in some cases also DOC) analysis. The samples were filtered to separate the sludge from the liquid.

CONTROL AND BLANK SYSTEM
- No data
- No toxicity was observed during the tests

Key result

Parameter:

other: COD

Value:

ca. 90

Sampling time:

28 d

Details on results:

Test using unadapted sludge from a WWTP with high loading and low sludge age (2-3 days, no slowly reproducing organisms present).
Biodegradation started after long lag period of about 20 days, indicating the necessity of an adaptation phase.

Validity criteria fulfilled:

not specified

Interpretation of results:

inherently biodegradable, not fulfilling specific criteria

Conclusions:

This study was performed according to the Zahn-Wellens method (OECD guideline 302B) using unadapted sludge from a WWTP with high loading and low sludge age (2-3 days, no slowly reproducing organisms present). After a long lag phase of about 20 days, biodegradation started, indicating the necessity of an adaptation phase. After 28 days ca. 90% of the substance was degraded. This serves as evidence for inherent, ultimate biodegradation (> 70%).

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): 4-Methylmorpholin

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, industrial, non-adapted

Duration of test (contact time):

30 d

Initial conc.:

400 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

TOC removal

Details on study design:

TEST CONDITIONS
- Test temperature: room temperature (20-25 °C)
- pH: 6.6 to 7.8
- pH adjusted: yes
- incubation: at room temperature (20-25 °C) on a magnetic stirrer, aerated with air (sparging)
- test substance concentration: calculated initial test item concentration: 400 mg/L DOC (=reference point) measured initial test item concentration: 420 mg/L DOC

TEST SYSTEM
- Culturing apparatus: all glass bottles (volume: 5 L; liquid volume: 3000.0 mL)
- Number of culture flasks/concentration: 1
- Measuring equipment: no data available
- Test performed in open system: yes


SAMPLING
- Sampling: 3 h, 1 d, 3 d, 5 d, 7 d, 12 d, 14 d, 19 d, 22 d, 26 d, 29 d and 30 d
- measurements: pH, TOC (total organic carbon)


CONTROL AND BLANK SYSTEM
- Inoculum blank: no (TOC (DOC) not determined, but a statistically obtained mean value of 17 mg/l TOC (inoculum: activated sludge from the industrial wwtp of BASF) was used to calculate the TOC-elimination)
- Abiotic sterile control: no
- Toxicity control: no

Key result

Parameter:

% degradation (DOC removal)

Value:

90

Sampling time:

29 d

Key result

Parameter:

% degradation (DOC removal)

Value:

-1

Sampling time:

3 h

Key result

Parameter:

% degradation (DOC removal)

Value:

2

Sampling time:

5 d

Key result

Parameter:

% degradation (DOC removal)

Value:

13

Sampling time:

12 d

Key result

Parameter:

% degradation (DOC removal)

Value:

54

Sampling time:

22 d

Validity criteria fulfilled:

not specified

Interpretation of results:

other: easily eliminated from water

Conclusions:

The test substance is considered easily eliminated from water.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2011-08-19 - 2011-09-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study performed according to OECD 301F guideline. According to ECHA guidance on IR&CSA chapter R.7b (2008, p.173-174), an enhanced test was performed by increasing the concentration of biomass. No direct conclusion can be drawn on ready biodegradability of the test substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

yes

Remarks:

according to ECHA Guidance R.7b the inoculum concentration was increased from 30 to 50 mg suspended solids/L

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): NMM (Morpholine, 4-methyl-)
- Substance type: clear pale yellow liquid
- Physical state: liquid
- Analytical purity: 99.54%
- Impurities (identity and concentrations): 0.03% water
- Purity test date: 1.06.2007
- Lot/batch No.: LL0707604
- Expiration date of the lot/batch: 1 Dec 2012
- Stability under test conditions: responsibility of the sponsor
- Storage condition of test material: room temperature in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: a mixed population of activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treated predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of DOC that may have been present. The washed sample was then maintained on continuous aeration at ca. 21°C and used on the day of collection.
- Concentration of sludge: the suspended solids concentration in the sludge sample was 2.8 g/L prior to use.
- Initial cell/biomass concentration: 50 mg suspended solids/L

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
- Composition of medium: as recommended by the OECD guideline
- Test temperature: 22.3-22.6°C
- pH: 6.8-7.6
- pH adjusted: no
- Aeration of dilution water: no
- Suspended solids concentration: 50 mg/L
- Continuous darkness: no, diffuse light was used

TEST SYSTEM
- Culturing apparatus: CES Multichannel Aerobic Respirometer. The sample flask is sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath.
- Number of culture flasks/concentration: 4
- Method used to create aerobic conditions: stirring for the duration of the test
- Measuring equipment: As biodegradation progresses, oxygen is converted to CO2 which is absorbed into the ethanolamine solution (50% v/v) in the sensor head, causing a net reduction in gas pressure within the sample flask. The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolysing current and the duration of the current is proportional to the amount of oxygen supplied to the microorganisms.

SAMPLING
- Sampling frequency: DOC on day 0 and 28. BOD daily.
- Sampling method: 30 ml samples were taken and filtered through 0.45 µm Gelman AcroCap filters. Analysis for DOC using a Shimadzu TOC-Vcph TOC analyser. Total Carbon and Inorganic Carbon was measured in triplicate.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 4 replicate bottless.
- Toxicity control: 100 mg/L test item and 100 mg/L aniline, plus 50 mg suspended solids/L: 3 replicate bottles

STATISTICAL METHODS: not applicable

Reference substance:

aniline

Remarks:

100 mg/L

Preliminary study:

Test item did not adsorb to filter matrices or to activated sewage sludge. Therefore the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test item.

Test performance:

The difference between extremes of replicate BOD values at the end of the test was less than 20% and therefore satisfied the validation criterion given in the OECD 301 guideline.
The toxicity control attained 34% degradation after 14 days and 86% degradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment microorganisms used in the test.

Key result

Parameter:

% degradation (O2 consumption)

Value:

75

St. dev.:

4

Sampling time:

28 d

Remarks on result:

other: 10-day window satisfied

Key result

Parameter:

% degradation (DOC removal)

Value:

91

Sampling time:

28 d

Details on results:

The test item attained 75% degradation after 28 days and satisfied the 10-day window, whereby 60% degradation must be attained within 10 days of the degradation rate exceeding 10%. Analysis of the test media from the test item vessels on day 0 and 28 for DOC showed that the test item attained a mean value of 91% degradation after 28 days and the toxicity control attained 88% degradation.
The test item starts to degrade only after 19 days.

Results with reference substance:

Aniline attained 67% degradation after 14 days and 93% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Aniline attained 100% degradation after 28 days, calculated from the results of the DOC analyses performed on days 0 and 28. The degradation rate calculated from the results of the DOC analyses for aniline was higher than that calculated from oxygen consumption values. This is considered to be due to incorporation of the aniline into the microbial biomass prior to degradation, and hence oxygen consumption, occurring.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

As the current OECD 301F test was enhanced by increasing the amount of biomass from 30 to 50 mg suspended solids/L, one cannot conclude on the ready biodegradability of the test substance. The results of the study show that the test substance rapidly degrades within 28 days (71-79%). In addition, the 10-day window criterion normally used in OECD 301 tests was fulfilled.

Description of key information

A weight-of-evidence approach is used to cover the endpoint.

Available data include inherent biodegradation tests (Zahn-Wellens test, OECD302B) and an enhanced ready biodegradability test according to the manometric respirometry method (OECD301F). All information together justifies the conclusion that the substance is ultimately and inherently biodegradable, not fulfilling specific criteria.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Additional information

Clarke (2012) investigated the ready biodegradability of the test substance by performing an enhanced manometric respirometry test (OECD301F). The test was enhanced by increasing the amount of biomass from 30 to 50 mg suspended solids/L. The results of the study show that the test substance rapidly degrades within 28 days (71-79%). In addition, the 10-day window criterion normally used in OECD 301 tests was fulfilled. However, as the study was enhanced, one cannot conclude on the ready biodegradability of the test substance (according to ECHA guidance R.7b). This study was considered reliable (Klimisch 2).

BASF (1981) performed a Zahn-Wellens test (OECD302B) to investigate the inherent biodegradation potential of the test substance. After 29 days, the test substance was degraded for 90% (as DOC removal), indicating that the test substance is inherently biodegradable. However, the pass level of 70% was not reached within 7 days. So it can be concluded that the test substance is inherently biodegradable, but is not fulfilling the specific criteria. This study was also considered reliable (Klimisch 2).

A third reliable study (Klimisch 2) is the study of Meister and Wechsler (1998), in which the results of several experiments with NMMO, the test substance NMM and morpholine were described. A Zahn-Wellens test (OECD302B) with unadapted sludge (only containing short-living organisms) also indicated that the substance is inherently biodegradable (not fulfilling specific criteria): after a lag phase of about 20 days, almost complete degradation was reached by day 28. This test indicates the need for adapation of the sludge to ensure fast breakdown. Several lab-scale and pilot-plant studies performed by the same authors further investigated the degradation of NMMO, NMM and morpholine. These experiments indicated that the main requirement for a successful adaptation is a high sludge age, as the slowly reproducing organisms in the sludge are the ones that most efficiently degrade the test substance. The slow step is the demethylation of the test substance to morpholine. Once morpholine has been formed, ring cleavage and further degradation occurs much faster. Overall, it can be concluded that the substance can be considered ultimately and inherently biodegradable.