4-methylmorpholine

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

no

Specific details on test material used for the study:

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

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge, industrial

Details on inoculum:

- Source: activated sludge of an wwtp treating industrial sewage (BASF AG)
- Initial biomass concentration: dry matter: 1 g/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 min

Test temperature:

ambient temperature

Nominal and measured concentrations:

nominal: 15, 150, 2000 mg/L

Key result

Duration:

30 min

Dose descriptor:

other: EC20

Effect conc.:

> 2 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

- blank value (mean of two blanks): 15 mg 02/g dry matter

Concentration	Respiration rate	Change of respiration rate
(mg/L)	(mg O2/L*h)	(%)
15	14	-7
150	16	+7
2000	17	+13

No significant inhibition ot the respiration was observed with the test substance up to a concentration of 2000 mg/L. A stimulation rather than an inhibition of the respiration was observed.

Validity criteria fulfilled:

not specified

Conclusions:

In this study, the EC20 (30min) was concluded to be > 2000 mg/L.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Qualifier:

according to guideline

Guideline:

other: EC Directive 88/302/EEC Activated sludge: Testing of respiration inhibition

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): 4-methylmorpholine
- Substance type: pure active substance
- Physical state: liquid
- Analytical purity: >= 99%

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Source: activated sludge of a laboratory scale wwtp treating domestic and synthetic sewage
- Initial biomass concentration: dry matter: 1 g/L

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

30 min

Test temperature:

ambient temperature

Nominal and measured concentrations:

nominal: 1000 mg/L (related to TOC: 574 mg/L)

Details on test conditions:

- test volume: 250 mL

Reference substance (positive control):

yes

Remarks:

3,5-dichlorphenol (3,5-DCP); concentrations: 4 and 40 mg/L (1.86 and 18.6 mg/L related to TOC)

Key result

Duration:

30 min

Dose descriptor:

other: EC20

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

- blank value (mean): 29 mg 02/g dry matter

Concentration	Respiration rate	Change of respiration rate
(mg/L)	(mg O2/L*h)	(%)
1000	28	-3
Reference substance: 4	7	-76
Reference substance: 40	24	-17

No significant inhibition ot the respiration was observed with the test substance at a concentration of 1001 mg/l.

Validity criteria fulfilled:

not specified

Conclusions:

In this study, the EC20 (30min) was concluded to be > 1000 mg/L.

Reference 3

Endpoint:

activated sludge respiration inhibition testing

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:

other: see 'Remark'

Remarks:

Test performed according to a non-standard method (Pseudomonas putida O2 consumption test), which was demonstrated to yield similar results (for a series of test substances) as the Pseudomonas putida growth inhibition test (DIN 38 412, part 8). The test results can be considered reliable with restrictions, because not all details were reported in the publication.

Qualifier:

no guideline available

Principles of method if other than guideline:

Pseudomonas putida O2 consumption test (5-6 h incubation, O2 levels tested at start and end of exposure period).

GLP compliance:

no

Specific details on test material used for the study:

- Name: N-methylmorpholin
- No further details reported

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

see test conditions

Test organisms (species):

Pseudomonas putida

Details on inoculum:

- Laboratory culture: yes
- Method of cultivation: cultured on agar (incubated at 25°C), each 4-6 weeks transferred to fresh agar (containing D-glucose, NaNO3, K2HPO4, KH2PO4, MgSO4.6H2O, iron citrate, trace elements, vitamins)
- Preparation of inoculum for exposure: For inoculation of pre-cultures, 7-d old cultures were used. Pre-cultures (100 mL pre-culture medium in 250-mL closed Erlenmeyer flasks) were inoculated with 2 mL inoculum from the 7-d-old cultures and incubated for 19 h (continuous shaking). Pre-culture medium contained D-glucose, NaNO3, K2HPO4, KH2PO4, FeSO4.7H2O, MgSO4.6H2O, trace element solution, and water.
- Initial cell density: ca. 1E+06 cells/mL (cell density in pre-cultures measured photometrically at 436 nm)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

6 h

Remarks on exposure duration:

5-6 h, depending on O2 levels of blank controls (minimal O2 level in blank controls (without HgCl2) should be 3.0-3.5 mg O2/L - this was checked from 4.5 h on and as soon as these levels were attained the test was finished).

Hardness:

not applicable

Test temperature:

25°C

pH:

not reported

Dissolved oxygen:

not applicable

Salinity:

not applicable

Nominal and measured concentrations:

Control + a series of test substance concentrations <= 1000 mg/L. Test concentrations not reported.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100-mL sterilized glass bottles, fill-volume 100 mL, no headspace
- Type (delete if not applicable): closed
- Aeration: no
- Initial cell density: ca. 1E+06 cells/mL
- No. of vessels: 4 per test concentration (of which 2 with HgCl2), 4 blank controls with HgCl2, 5 blank controls without HgCl2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: bidistilled water
- Test medium contained D-glucose, NaNO3, K2HPO4, KH2PO4, FeSO4.7H2O, MgSO4.6H2O, trace element solution.
- Culture medium different from test medium: yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not reported
- Light intensity: not reported
- Continuous stirring by magnetic stirrer

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- O2 consumption was calculated based on measurements of O2 levels at start and end of testing.
- Test vessels were ended by addition of 1 mL HgCl2 solution (300 mg/L) to stop bacterial growth.
- O2 levels were measured using an O2 electrode.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not reported

Reference substance (positive control):

no

Key result

Duration:

6 h

Dose descriptor:

EC10

Effect conc.:

95 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: O2 consumption

Remarks on result:

other: mean of 3 tests

Details on results:

For a series of other substances, the outcome of the method was compared to that of the Pseudomonas putida growth inhibition test (DIN 38 412, part 8), resulting in good correlation (R2 = 0.982 and 0.983 for EC10 and EC50 values, respectively).

Reported statistics and error estimates:

EC10 and EC50 were determined using graphical methods or Probit analysis.

Validity criteria fulfilled:

yes

Conclusions:

In this study, the oxygen consumption in closed bottles inoculated with Pseudomonas putida was measured at the start and at the end of a 5 to 6 hour exposure period and compared to the oxygen consumption in blank controls. Three replicate tests yielded an average 6-h EC10 of 95 mg/L.

Description of key information

Three Klimisch 2 studies are available and taken forward in a weight-of-evidence approach. Given the presence of deviations in all three studies, the OECD 209-test result from BASF (1991-04-02) was chosen to derive PNEC-STP and not a test result which was not performed according to standardized test method. The respiration inhibition test from BASF was performed according to a guideline valid at that time including a test duration of 30 min. The 30 min. EC20 value of > 1000 mg/L is used for the CSA and derivation of the PNEC.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

A weight-of-evidence approach is applied to cover this endpoint.

The respiration inhibition study (OECD 209) by BASF AG (1981) reported a 30-min EC20 of >2000 mg/L. Three concentrations were tested (15, 150 and 2000 mg/L) using industrial sewage sludge from a BASF WWTP. There is no mentioning of any possible adaptation of the sludge. The EC10 value is not mentioned either, but the test results indicate that it lies between 150 en 2000 mg/L.

The respiration inhibition study (OECD 209) by BASF AG (1991) reported a 30-min EC20 of >1000 mg/L. The single concentration tested was 1000 mg/L using domestic/synthetic sewage sludge. The EC10 value is not mentioned but extrapolation from the data shows it is >1000 mg/L.

Both BASF studies tested for 30 minutes, while 3 hours is the standard according to OECD TG 209.

The Pseudomonas putida oxygen consumption test is not a standard test normally used for this endpoint either. However, the publication (Schöberl, 1995) reported a close similarity with results of the Pseudomonas putida growth inhibition test (DIN 38 412, part 8). The 6h-EC10 value was 95 mg/L.