3-morpholinopropylamine

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 17, 2017 - January 24, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016/266

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD No. 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss GLP Monitoring Authorities, Date of decission 2015 - 11- 16

Test material

Test material information

Constituent 1

Reference substance name:

3-morpholinopropylamine

EC Number:

204-590-2

EC Name:

3-morpholinopropylamine

Cas Number:

123-00-2

Molecular formula:

C7H16N2O

IUPAC Name:

3-morpholin-4-ylpropan-1-amine

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Test Item Name: N-morpholinopropylamine
Batch Number: 6H706
CAS-No.: 123-00-2
Purity: 99.7 %
Molecular Formula: C7H16N2O
Appearance: Colorless, clear liquid, substantially free of foreign matter
Relative Density: 0.99
Expiration Date: August 11, 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Storage Conditions: At room temperature, at 20 ± 5 °C. Keep container tightly closed in a dry and well-ventilated place.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampled concentrations: 6.25, 12.5, 25, 50 and 100 mg/L
- Sampling method: duplicate samples were taken from the test media of all test concentrations and the control at the start of the test (without algae) and at the end of the test (containing algae). For sampling at the end of the test, the test medium of the treatment replicates was pooled.
- Sample storage conditions before analysis: all samples were frozen (at -20 ± 5 °C)
- Analysed samples: The concentrations of the test item N-morpholinopropylamine were determined in one of the duplicate samples from the nominal test concentrations of 50 and 100 mg/L from both sampling times (0 and 72 hours). Th samples from the nominal test concentrations of 6.25 to 25 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test and thus not relevant for the interpretation of the biological results. From the control samples, one of the duplicate samples was analyzed per sampling time.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 101 µL of the test item completely in 1000 mL of test water. This volume is equivalent to a loading rate of 100 mg/L, considering the relative density of the test item of 0.99. The test item was dissolved by intense stirring for 15 minutes at room temperature. During this stirring period the pH was adjusted from pH 10 to 7.5 with 1 M hydrochloric acid solution.
- Eluate: The highest test concentration of 100 mg/L was further diluted with test water to prepare the test media with the lower test concentrations.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): After the dissolving procedure the highest test concentration of 100 mg/L was a clear solution.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (Raphidocelis subcapitata; formerly Selenastrum capricornutum)
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen/Germany)
- Age of inoculum (at test initiation): An inoculum culture was set up three days before the start of the exposure.
- Method of cultivation: The algae were cultivated at IES Laboratories under standardized conditions according to the test guidelines. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

ACCLIMATION
- Acclimation period: no acclimatization was necessary since the algae were cultivated under the same conditions as in the test.
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

other: AAP-medium according to the guideline

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

not performed

Test conditions

Hardness:

0.15 mmol/L (= 15 mg/L as CaCO3)

Test temperature:

23°C

pH:

7.5-8.6

Dissolved oxygen:

n.d.

Salinity:

n.d.

Conductivity:

n.d.

Nominal and measured concentrations:

nominal concentrations [mg/L]: 6.25, 12.5, 25, 50, 100
measured concentrations at 0 hour [mg/L]: n.d., n.d., n.d., 55.2, 105
measured concentrations after 72 hours [mg/L]: n.d., n.d., n.d., 55, 105

n.d.: not determined, since these concentrations were below the NOEC determined in this test and
thus not relevant for the interpretation of the biological results.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glas, 75 mL, open, 30 mL
- Aeration: No
- Initial cells density: 5000 cells/mL (corresponding 0.93E+04 relative fluorescence)
- Control end relative fluorescence: 182.2E+04
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP-Medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Macro-nutrients: Ingredients / Concentration
NaHCO3 / 15.0 mg/L
K2HPO4 / 1.044 mg/L
MgSO4 × 7 H2O / 14.6 mg/L
MgCl2 × 6 H2O / 12.16 mg/L
CaCl2 × 2 H2O / 4.41 mg/L
NaNO3 / 25.5 mg/L
Trace elements: Ingredients / Concentration
H3BO3 / 186.0 µg/L
MnCl2 × 4 H2O / 415.0 µg/L
ZnCl2 / 3.27 µg/L
CoCl2 × 6 H2O / 1.43 µg/L
CuCl2 × 2 H2O / 0.012 µg/L
Na2MoO4 × 2 H2O / 7.26 µg/L
FeCl3 × 6 H2O / 160.0 µg/L
Na2EDTA × 2 H2O: 300.0 µg/L

- CaCO3: 15 mg/L
- Water hardness: 0.15 mmol/L (= 15 mg/L as CaCO3)
- Culture medium different from test medium: No
- Intervals of water quality measurement: it is a reconstituted test water

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: The pH of the stock solution 100 mg/L was adjusted from pH 10 to pH 7.5 with 1M HCl
- Photoperiod: continuously illuminated
- Light intensity and quality: 66-70 µEs-1m-2, LED

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of biomass: fluorimeter (0, 24, 48 and 72 hours). (For the inoculum culture: electronic particle counter for determination of cell concentration)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0

RANGE-FINDING STUDY with pH adjustment: The pH was adjusted in the 100 mg/L stock solution from pH10 to pH 7.5
- Test concentrations: 0.1 / 1.0 / 10 / 100 mg/L
- Results used to determine the conditions for the definitive study:
Concentration / Inhibition of Average Growth after 72 hours / Inhibition of Yield after 72 hours
0.1 mg/L / 0.0% / -0.1%
1.0 mg/L / -1.2% / -6.7%
10 mg/L / -1.0% / -5.35%
100 mg/L / 5.3% / 25%

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, tested as reference study under GLP twice a year.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: > the highest tested concentration

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: > the highest tested concentration

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Inhibition of growth rate was 5.4 % at the highest tested concentration of 100 mg/L (nominal).
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no difference between the algae growing at the highest concentration of 100 mg/L and the algae cells in the control
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 72-hour EC50 for growth rate: 1.1 mg potassium dichromate/L.

Reported statistics and error estimates:

Growth rate and yield were calculated for each test flask. The mean values for growth rate and yield were calculated for each treatment.

The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95 % confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression.

For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test and Welch t-test with Bonferroni-Holm-adjustment, where appropriate.

Any other information on results incl. tables

Table: Analytical results

 

Nominal Test Item Concentration	Measured Concentration of the Test Item [mg/L]	Measured Concentration of the Test Item [mg/L]
[mg/L]	0 hours	72 hours
50	55.2	55.0
100	105	105

LOQ:     Limit of quantification (LOQ = 19.8 mg test item/L).

Table: Biological results

Nominal Test Item Concentration Repl. No. Biomass of Algae* Biomass of Algae* Biomass of Algae* [mg/L] 24 Hours 48 Hours 72 Hours Control 1 5.5 35.9 197.6   2 4.4 35.2 186.5   3 5.3 34.1 188.8   4 5.2 35.1 184.4   5 5.3 35.6 168.8   6 5.1 35.2 167.0   Mean 5.1 35.2 182.2   SD 0.4 0.6 12.0 6.25 1 4.3 34.8 203.3   2 4.7 38.8 192.6   3 4.9 36.8 195.8   Mean 4.6 36.8 197.2   SD 0.3 2.0 5.5 12.5 1 5.3 38.4 174.5   2 4.5 38.3 194.1   3 3.8 36.6 173.1   Mean 4.5 37.8 180.6   SD 0.7 1.0 11.8 25 1 5.2 33.3 170.5   2 4.2 33.6 170.5   3 4.0 32.0 160.8   Mean 4.4 32.9 167.3   SD 0.6 0.8 5.6 50 1 5.9 34.5 177.7   2 3.9 37.4 179.4   3 4.0 23.7 155.8   Mean 4.6 31.9 170.9   SD 1.2 7.2 13.2 100 1 4.0 23.9 129.7   2 3.9 26.8 143.0   3 3.9 22.0 138.1   Mean 3.9 24.2 136.9   SD 0.1 2.4 6.7

 SD:  Standard deviation

*:     The biomass was determined by fluorescence measurement (mean of duplicate measurements per replicate) and is given as relative fluorescence units (x 10000). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 0.93 x 10000 relative fluorescence units. 

Table: Average Growth Rates(µ) Nominal Test Item Conc. Average Growth Rate µ (day^-1) Inhibition of µ (Ir) Average Growth Rate µ (day^-1) Inhibition of µ (Ir) Average Growth Rate µ (day^-1) Inhibition of µ (Ir) 0-24 h 0-24 h 0-48 h 0-48 h 0-72 h 0-72 h [mg/L] µ Ir[%] µ Ir[%] µ Ir[%] Control 1.711 0.0 1.819 0.0 1.760 0.0 6.25 1.607 6.1 1.841 -1.2 1.787 -1.5 12.5 1.580 7.7 1.854 -1.9 1.757 0.2 25 1.563 8.6 1.786 1.8 1.732 1.6 50 1.584 7.4 1.760 3.2 1.739 1.2 100 1.440* 15.8 1.631 10.3 1.665* 5.4   *:           Mean value statistically significantly lower than in the control (according to a Williams t-test one-sided smaller,a= 0.05).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Biomass increased by a factor of 197 over 72h. Mean coeff. of variation of the daily growth rates (section-by-section) was 8.8 % (72h). Coeff. of variation of the average specific growth rates in the replicates was 1.3 % (72h)

Conclusions:

In a valid, reliable and conclusive study according to OECD 201, the impact of the test item N-morpholinopropylamine on the growth of the freshwater green algal species Pseudokirchneriella subcapitata is summarized below. The results are based on nominal concentrations of the test item:

Growth rate:
EC10 (0 -72 h) [mg/L]: >100 (95 % confidence interval not determined)
EC20 (0 -72 h) [mg/L]: >100 (95 % confidence interval not determined)
EC50 (0 -72 h) [mg/L]: >100 (95 % confidence interval not determined)
NOEC [mg/L]: 50
LOEC [mg/L]: 100

Yield:
EC10 (0 -72 h) [mg/L]: 46 (95 % confidence interval 26 - 60)
EC20 (0 -72 h) [mg/L]: 85 (95 % confidence interval 68 -115)
EC50 (0 -72 h) [mg/L]: >100 (95 % confidence interval not determined)
NOEC [mg/L]: 50
LOEC [mg/L]: 100

Executive summary:

The impact of the test item N-morpholinopropylamine on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No. 2016/266, C3.

The nominal test item concentrations tested were 6.25, 12.5, 25, 50 and 100 mg/L. A control (test water without test item) was tested in parallel.

The measured concentrations of N-morpholinopropylamine in the test media of the test concentrations of 50 and 100 mg/L were 110 and 105 % of the nominal values at the start and the end of the test, respectively.

Thus, the correct dosing of the test item N-morpholinopropylamine was confirmed. The test item was stable in the test media over the test period of 72 hours. The biological results were related to the nominal concentrations of the test item. The samples from the nominal test concentrations of 6.25 to 25 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test and thus not relevant for the interpretation of the biological results.

Growth rate:

EC10 (0 -72 h) [mg/L]: >100 (95 % confidence interval not determined)

EC20 (0 -72 h) [mg/L]: >100 (95 % confidence interval not determined)

EC50 (0 -72 h) [mg/L]: >100 (95 % confidence interval not determined)

NOEC [mg/L]: 50

LOEC [mg/L]: 100

Yield:

EC10 (0 -72 h) [mg/L]: 46 (95 % confidence interval 26 - 60)

EC20 (0 -72 h) [mg/L]: 85 (95 % confidence interval 68 -115)

EC50 (0 -72 h) [mg/L]: >100 (95 % confidence interval not determined)

NOEC [mg/L]: 50

LOEC [mg/L]: 100