4-[3-(1-naphthylamino)propyl]morpholine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 24, 1998 to May 28, 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: While the study was conducted according to OECD TG 201 and in accordance with the Principles of Good Laboratory practice (GLP), the stability of the test substance under test conditions was not verified analytically.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Minor deviations which did not have an adverse effect on the outcome of the study were noted

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

yes

Details on test solutions:

For the definitive test, a 100000 mg/l primary stock solution was prepared by combining 2.5 g 4-(3-(1-naphthylamino)propyl) morpholine and dimethylformamide in a 25 ml Class A volumetric flask and adjusting the final solvent volume to 25 ml. A secondary stock solution was prepared at the highest tested concentration (5.0 mg/l) by bringing 0.05 ml of the primary stock solution to a total volume of 1000 ml with dilution water in 1000 ml Class A glass volumetric flask. Test solutions were prepared by combining appropriate amounts of the secondary stock solution and dilution water. The solvent control contained the same solvent concentration as the highest tested concentration (47 mg/l dimethlformamide).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: freshwater alga
- Strain: Selenastrum capricornutum, UTEX 1648
- Source (laboratory, culture collection): in house cultured from an original culture procured from the Culture Collection Alage at the University of Texas at Austin. The culture was transferred to sterile enriched media identical to media used for the test and maintained at test conditions for at least of 14 days before the definitive test. The subsample of algae used to inoculate media at the start of the definitive test came from a 5-day old culture.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

not applicable

Hardness:

not applicable

Test temperature:

23.5 - 24.4 °C

pH:

7.5 - 10.2

Dissolved oxygen:

not applicable

Salinity:

not applicable

Nominal and measured concentrations:

Range finding test - dilution water control, solvent control, 0.10, 1.0, 5.0, 10.0 and 20.0 mg/l (nominal concentration)
Definitive test - dilution water control, solvent control (87 mg/l dimethylformamide), 0.31, 0.63, 1.3, 2.5 and 5.0 mg/l (nominal concentration), this test was invalidated because solvent control contained less solvent than the highest tested concentration
Final definitive test - dilution water control, solvent control (47 mg/l dimethylformamide), 0.31, 0.63, 1.3, 2.5 and 5.0 mg/l (nominal concentration)

Details on test conditions:

A range finding test with a dilution water control, a solvent control, and 5 concentrations of 4-(3-(1-naphthylamino)propyl) morpholine (0.10, 1.0, 5.0, 10.0 and 20.0 mg/l) was conducted. At the end of the 72-hour test, the cell growth in test solutions was equal to the following percents of control growth: solvent growth - 112%, 0.10 mg/l - 101%, 1.0 mg/l - 89%, 5.0 mg/l - 1% and 10 and 20 mg/l - < 1%.

A definitive test was conducted with a dilution water control, a solvent control (87 mg/l dimethylformamide), and 5 concentrations of 4-(3-(1-naphthylamino)propyl) morpholine (0.31, 0.63,1.3, 2.5 and 5.0 mg/l). Because the solvent control contained less solvent than the highest tested concentration (87 mg/l versus 95 mg/l), the test was determined to be invalid and was repeated.

The final definitive test was conducted with a dilution water control, a solvent control (47 mg/l dimethylformamide), and 5 concentrations of 4-(3-(1-naphthylamino)propyl) morpholine (0.31, 0.63,1.3, 2.5 and 5.0 mg/l). The test was conducted under static conditions at 24 ± 1 °C. Algae was distributed among 6 replicates of the control and 3 replicates of each solvent control and treatment at the rate of approximately 10000 cells/ml. The test vessels were 250 ml Erlenmeyer flasks that contained 50 ml of test solution (water depth was approximately 1 cm) and were capped with inverted glass beakers. Test vessels were randomly arranged on a rotary shaker that was adjusted to 100 rpm and was located in an incubator during the test. Test vessels were repositioned daily to minimize spatial differences. A 24 hour light period was maintained.
The number of algal cells/ml in each test vessel and the occurence of relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers or aggregation of cells was determined visually by means of direct microscopic examination with a haemocytometer.
Cell counts were made and recorded daily. Temperature of the incubator was measured and recorded daily, pH was determined in each test vessel at the begining and end of the test.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

No insoluble material was noted during the test. The algal population grew well, resulting in an average of 3347000 cells/ml in the control after 72 hours. No effects (size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers or aggregation of cells) were noted during the test. The 72-hour no observed effect concentration was 0.63 mg/l 4-(3-(1-naphthylamino)propyl) morpholine. The 72-hour EC50 was 1.0 mg/l when calculated using the number of cells/ml and 2.6 mg/l when calculated using the average specific growth rate.
A determination on whether toxic effects were algistatic or algicidal was performed at the conclusion of the toxicity test and results indicated that 4-(3-(1-naphthylamino)propyl) morpholine at the highest tested concentration was algistatic.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

The average specific growth rate was calculated as the natural log of the number of cells/ml at 24, 48, and 72 hours of exposure, minus the natural log of the number of cells/ml at 0 hour of exposure, divided by the hour of exposure. Results of the toxicity test were interpreted using a weighted least squares non-linear regression. The no observed effect cocncentration (NOEC) is the highest concentration of test substance that was not statistically different than the control (alpha = 0.05). Data normality was determied using a Chi-square test and the homogeneity of variance was determed using a Bartlett's test. The NOEC was calculated using Bonferoni's test, which includes an analysis of variance (ANOVA). Because no growth occurred at the highest tested concentration, data from that treatment were exluded from the NOEC calculation.

None

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 72-hour no observed effect concentration was 0.63 mg/l 4-(3-(1-naphthylamino)propyl) morpholine. The 72-hour EC50 was 1.0 mg/l when calculated using the number of cells/ml and 2.6 mg/l when calculated using the average specific growth rate.

Executive summary:

The acute toxicity of 4-(3-(1-naphthylamino)propyl) morpholine to freshwater algae, Selenastrum capricornutum, was performed as per OECD TG 201 and in accordance with the Principles of Good Laboratory Practice (GLP). The test was performed at 24 ± 1 °C under static conditions with a control (0 mg/l), a solvent control (47 mg/l dimethylformamide) and 5 nominal concentrations of 4-(3-(1-naphthylamino)propyl) morpholine (0.31, 0.63, 1.3, 2.5 and 5.0 mg/l). The dilution water was sterile enriched media adjusted to a pH of 7.5 ± 0.1. No insoluble material was noted during the test. Under the conditions of the study, the 72-hour no observed effect concentration was 0.63 mg/l 4-(3-(1-naphthylamino)propyl) morpholine. The 72-hour EC50 was 1.0 mg/l when calculated using the number of cells/ml and 2.6 mg/l when calculated using the average specific growth rate.

Description of key information

In a growth inhibition test with Selenastrum capricornutum,the 72-hour E_rC50 (based on growth rate) for 4-(3-(1-naphthylamino)propyl) morpholine was 2.6 mg/L. The 72-hour NOEC was determined to be 0.63 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

2.6 mg/L

EC10 or NOEC for freshwater algae:

0.63 mg/L

Additional information

This study measured both growth rate (r) and biomass as indicators of growth inhibition over 72 hours of exposure to 4-(3-(1-naphthylamino)propyl) morpholine.  The study was performed as per OECD TG 201 and in accordance with the Principles of Good Laboratory Practice (GLP). The test was performed at 24 ± 1 °C under static conditions with a control (0 mg/l), a solvent control (47 mg/l dimethylformamide) and 5 nominal concentrations of 4-(3-(1-naphthylamino)propyl) morpholine (0.31, 0.63, 1.3, 2.5 and 5.0 mg/l). The dilution water was sterile enriched media adjusted to a pH of 7.5 ± 0.1. No insoluble material was noted during the test. Under the conditions of the study, the 72-hour no observed effect concentration was 0.63 mg/l 4-(3-(1-naphthylamino)propyl) morpholine. The 72-hour EC50 was 1.0 mg/l when calculated using the number of cells/ml and 2.6 mg/l when calculated using the average specific growth rate.