4-methylmorpholine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

read across from related substance

Justification for type of information:

Read-across from studies available on structural analogue (4-ethylmorpholine). Justification for the read-across approach is included in section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

61.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: P 95% = 31.6 to 118.3 mg/L

Key result

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

35.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: P 95% = 13.8 to 90.0 mg/L

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

291.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: P 95% = 113.5 to 747.4 mg/L

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

143 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: P 95% = 57.1 to 358.0 mg/L

Conclusions:

No reliable data is available with the target substance. Data generated with the related substance 4-ethylmorpholine was used for endpoint coverage. Short-term toxicity of 4-ethylmorpholine to algae was investigated by exposing Desmodesus subspicatus during 96 hours in a static test, according to the German Industrial Standard DIN 38412, part 9. This resulted in an EC50(96h) value of 291.2 mg/L and an EC10 (96h) of 61.2 mg/L, both based on growth rate. The same is assumed to be correct for the target substance.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

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:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: German Industrial Standard DIN 38412, part 9

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): 4-Ethylmorpholin
- Substance type: pure active substance
- Physical state: liquid
- Analytical purity: 99.5%

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: SAG 86.81

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

21 to 25 °C (constant within 2 °C)

Nominal and measured concentrations:

nominal: 0 (control), 10, 25, 50, 100, 250, and 500 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume:
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density:
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates):
- No. of vessels per vehicle control (replicates): 4

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH:
- Photoperiod:
- Light intensity and quality:
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: photometric determination (pulse-fluorimeter, chlorophyll fluorescence) after 0, 24, 48, 72 and 96 h
- Chlorophyll measurement: yes
- Other: pH

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

61.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: P 95% = 31.6 to 118.3 mg/L

Key result

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

35.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: P 95% = 13.8to 90.0 mg/L

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

291.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: P 95% = 113.5 to 747.4 mg/L

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

143 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: P 95% = 57.1 to 358.0 mg/L

Details on results:

The following 72h results were obtained, all based on nominal concentration and expressed as test material:
EC10 growth rate: 131.7 mg/L (P 95% = 4.3 to > 500 mg/L)
EC10 biomass: 162.2 mg/L (P 95% = 69.9 to 376.1 mg/L)
EC50 growth rate: 583.7 mg/L (P 95% = 2.2 to > 500 mg/L)
EC50 biomass: 270.7 mg/L (P 95% = 101.0 to 725.8 mg/L)

Reported statistics and error estimates:

The EC values were calculated according to Tallarida and Jacob (1979) from the concentration-response relationship
Tallarida RJ and Jacob LS (1979). The Dose-Response Relation in Pharmacology. Springer-Verlag Publishers, Berlin, Heidelberg, New York, 98-103.

Cell density values (cells * 10^3/ml):

Concentration (mg/l)	0 h	24 h	48 h	72 h	96 h
0	12	25	68	265	873
10	12	23	79	243	843
25	12	22	81	262	812
50	12	22	85	270	753
100	12	22	73	210	503
250	12	21	51	107	208
500	12	13	19	22	23

Inhibition of growth rate and biomass after 72 and 96 h:

Concentration (mg/l)	Inhibition (%)
	Growth rate		Biomass
	72 h	96 h	72 h	96 h
0	--	--	--	--
10	2.8	0.8	-3.4	4.9
25	0.4	1.7	-8.2	2.6
50	-0.6	3.4	-12.7	3.5
100	7.5	12.9	7.8	28.8
250	29.3	33.5	44.4	65.8
500	80.4	84.8	87.9	95.5

pH values at test start (w/o algae) and after 96 h (inoculated assays):

Concentration (mg/l)	pH
	0 h	96 h
0	7.60	8.13
10	7.66	7.83
25	7.73	7.86
50	7.92	7.87
100	8.06	7.90
250	8.25	7.94
500	8.43	8.13

The temperature was 21.4 °C in all test concentrations.

 

Conclusions:

Short-term toxicity of 4-ethylmorpholine to algae was investigated by exposing Desmodesmus subspicatus during 96 hours in a static test, according to the German Industrial Standard DIN 38412, part 9. This resulted in an EC50(96h) value of 291.2 mg/L and an EC10(96h) of 61.2 mgL, both based on growth rate.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2003

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

According to the SIAR presented at SIAM 21 (Oct 2005), at the concentration of 53 mg/L aggregation of algal cells occurred partially, and at the highest concentration of 80 mg/L it was observed heavily. Therefore the growth rate based on cell counts measured using a particle counter should be incorrect. The formation of cell aggregates was not observed at the BASF study even at much higher test item concentrations. Therefore, we grade the Japanese study with reliability 4. It cannot be excluded that effects observed in the Japanese study were (partly) caused by inexplicable physical effects rather than by intrinsic properties of the chemical.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

See "Remarks on result"

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): 4-ethylmorpholine
- Molecular formula (if other than submission substance): C6H13NO
- Molecular weight (if other than submission substance): 115.18
- Analytical purity: 99.9%

Details on properties of test surrogate or analogue material:
PHYSICO-CHEMICAL PROPERTIES
- Melting point: -63°C
- Boiling point: 140 °C
- Water solubility (under test conditions): high

Analytical monitoring:

yes

Details on sampling:

- Concentrations:control, 10.0, 15.0, 23.0, 35.0, 53.0 and 80.0 mg/L
- Sampling method:
- Sample storage conditions before analysis:

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Test type:

static

Water media type:

not specified

Test temperature:

23°C ± 1°C

pH:

8.0-8.9

Details on test conditions:

TEST SYSTEM
- Test vessel:100 mL medium in a 300mL Erlenmeyer flask with a ventilative silicon cap.
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume:
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density:
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates):
- No. of vessels per vehicle control (replicates):

GROWTH MEDIUM
- Standard medium used: yes/no
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH:
- Photoperiod:
- Light intensity and quality:
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 53 mg/L

Nominal / measured:

nominal

Conc. based on:

not specified

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

According to the SIAR presented at SIAM 21 (Oct 2005), at the concentration of 53 mg/L aggregation of algal cells occurred partially, and at the highest concentration of 80 mg/L it was observed heavily. Therefore the growth rate based on cell counts measured using a particle counter should be incorrect. The formation of cell aggregates was not observed at the BASF study even at much higher test item concentrations. Therefore, we grade the Japanese study with reliability 4. It cannot be excluded that effects observed in the Japanese study were (partly) caused by inexplicable physical effects rather than by intrinsic properties of the chemical.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

23 mg/L

Nominal / measured:

nominal

Conc. based on:

not specified

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

According to the SIAR presented at SIAM 21 (Oct 2005), at the concentration of 53 mg/L aggregation of algal cells occurred partially, and at the highest concentration of 80 mg/L it was observed heavily. Therefore the growth rate based on cell counts measured using a particle counter should be incorrect. The formation of cell aggregates was not observed at the BASF study even at much higher test item concentrations. Therefore, we grade the Japanese study with reliability 4. It cannot be excluded that effects observed in the Japanese study were (partly) caused by inexplicable physical effects rather than by intrinsic properties of the chemical.

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50: 0.43 mg/L
- Other:

Reported statistics and error estimates:

a) Data Analysis: Inhibition rates were based on growth rate inhibition. Probit method for EC50 if applicable. 1-way ANOVA and Dunnett's method for NOEC.
b) Method of Calculating Mean Measured Concentrations (i.e. arithmetic mean, geometric mean, etc.): Not used

Conclusions:

The study is considered as Klimisch 4 (not assignable). According to the SIAR presented at SIAM 21 (Oct 2005), at the concentration of 53 mg/L aggregation of algal cells occurred partially, and at the highest concentration of 80 mg/L it was observed heavily. Therefore the growth rate based on cell counts measured using a particle counter should be incorrect. The formation of cell aggregates was not observed at the BASF study even at much higher test item concentrations. Therefore, we grade the Japanese study with reliability 4. It cannot be excluded that effects observed in the Japanese study were (partly) caused by inexplicable physical effects rather than by intrinsic properties of the chemical.

Description of key information

Following the read-across strategy, it is considered appropriate to cover this endpoint by a key study performed with the structural analogue 4-ethylmorpholine, yielding an EC50(96h) value of 291.2 mg/L and an EC10(96h) of 61.2 mg/L based on growth rate. The same is assumed to be correct for the target substance.

Key value for chemical safety assessment

EC50 for freshwater algae:

291.2 mg/L

EC10 or NOEC for freshwater algae:

61.2 mg/L

Additional information

Short-term toxicity of 4-ethylmorpholine to algae was investigated by exposing Desmodesmus subspicatus during 96 hours in a static test, according to the German Industrial Standard DIN 38412, part 9. This resulted in an EC50(96h) value of 291.2 mg/L and an EC10(96h) of 61.2 mg/L, both based on growth rate.

The justification for read-across within this endpoint can be found in section 13.