2-[3-(4-propyl-heptyl)-morpholin-4-yl]-ethanol hydrochloride

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:

September 06, 2016 - October 17, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment No 23 - Guidance Document On Aquatic Toxicity Testing Of Difficult Substances And Mixtures ENV/JM/MONO(2000)6.

Deviations:

no

Principles of method if other than guideline:

Was investigated the influence of the Delmopinol HCl on the growth of the green algal species Pseudokirchneriella subcapitata in a 72-hour test. The algae were exposed to aqueous test media containing test item at five different nominal concentrations.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-[3-(4-propylheptyl)morpholin-4-yl]ethan-1-ol hydrochloride

EC Number:

688-947-1

Cas Number:

98092-92-3

Molecular formula:

C16H34NClO2

IUPAC Name:

2-[3-(4-propylheptyl)morpholin-4-yl]ethan-1-ol hydrochloride

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Test item name: Delmopinolo HCl
Batch number: 2510468
Internal number: 2929564-001
Chemical name 2-[3-(4-propylheptyl)-4-morpholinyl) ethanol hydrochloride (1:1)
Purity : 100.3% w/w

Stability under storage conditions: protect from moisture

Storage condition: The test item will be stored at room temperature without particular precaution to avoid the light exposure following Test Substance Data Sheet (TSDS) supplied by the Sponsor.

Sampling and analysis

Analytical monitoring:

not specified

Details on sampling:

not specified

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc.):

Test organisms

Test organisms (species):

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

Details on test organisms:

test system
The study was performed with the unicellular, fresh-water green algae Pseudokirchneriella subcapitata strain (formerly known as Selenastrum capricornutum),
cultured in the laboratories of the Test Facility and originally purchased from the Institute of Plant Physiology of the University of Göttingen, Germany.
The algae were cultured in a climatic chamber at 24 °C ± 2 °C under continuous uniform illumination in the range 4440-8880 Lux in the spectral range 400-700 nm.
The culture medium was the same of the test medium; the stock cultures were weekly transferred to fresh medium and maintained in continuous shaking to ensure the necessary amount of CO2 and to keep algae in suspension. Cultures containing deformed or abnormal cells were discarded. Only exponentially growing algal cultures have been used to start the test.

Study design

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Not specified

Test temperature:

Test temperature: 23.8 – 24.1 °C

pH:

pH of test medium:
7.81 – 8.12 at the test start
7.72 – 9.44 at the test end

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Test concentrations:
Negative control (algal medium without test item) and 1.0, 1.7,3.1, 5.6 and 10.0 mg test item/L.

Details on test conditions:

Dosage and Concentrations
The test concentrations were chosen in accordance with the results of a pre-test (range finding test), whose relevant Raw Data were archived under the code of the present study.
The main test included five test item concentrations in a geometric series, namely 1.0, 1.7, 3.1, 5.6 and 10.0 mg/L.

The test medium
The test medium was prepared as follows: prior to the test start, 250 mL of a 100.0 mg/L test item stock solution were prepared by direct weighing into algal growth medium (0.0250 g of test item into 250 mL of algal growth medium). The obtained solution appeared clear and transparent with the test item completely solubilized; pH was measured and then, from this stock solution, the test item solutions were prepared by dilution with algal growth medium.
After pH check the test was start.
A negative control with algal growth medium only was also tested.

Experimental Conditions
The treatment vessels were identified by experiment number, group and replicate number. All the described procedures were conducted under a laminar flow bench to ensure the sterility of the algal cultures. For the test concentrations and for the negative control, 100 mL capacity conical glass flasks capped with air permeable stoppers were used. Three replicates for each test concentrations, six for negative control were prepared. At test start, 50 mL of test solutions were poured into the test flasks and inoculated with algae to obtain a starting cell density of about 104 cells/mL. The algal inoculum was taken from a pre-culture, started 3 days before the beginning of the test in order to ensure that the assay was performed with exponentially growing algal population. The cell density of the inoculum was around 106 cells/mL. The test flasks were incubated in a climatic chamber, under continuous illumination and constant shaking. The flasks were randomly distributed under the light and were replaced every day during the test.

Temperature
The incubation temperature was continuously monitored during the course of the study by a PT100 probe installed in the environmental test chamber. It was in the range 23.8 – 24.1 °C with a mean value of 24.0 °C and a standard deviation of 0.03°C. The temperature range provided by OECD guideline is 24 ± 2 C. The temperature values during the test were within the provided range by guideline.

Light conditions
Continuous illumination; light intensity was ranged between 5217 and 5784 Lux, within the OECD recommended range (4440 - 8880 Lux).

Test duration
72 hours

Test medium
Reconstituted water according to OECD Guideline No. 201, 2011. Test medium pH was measured with a portable pH meter at the beginning of the test and in each test flask at the end of the test. For fresh solutions the pH values were measured sampling an aliquot immediately after test solutions preparation, before pouring it in the test flasks. For spent solutions (after 72 hours), pH were measured in each test flask.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 1.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 1.2 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.:

ca. 1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 1.7 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:

EC10

Effect conc.:

ca. 1.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 1.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 9

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 10

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Observations and determination of EyCx , ErCx, NOEC and LOEC
Algal cell density was measured every 24 hours taking aliquots from each test concentration replicate from negative control, diluting them in a NaCl 9 g/L solution and reading with an electronic particle counter.
The measured values were used to calculate the percentage inhibition of cell growth as yield and as growth rate in comparison to the negative control.

The CETIS v.1.8.7.7 software was used to carry out the statistical analysis: it automatically chooses the most appropriate analysis for the pool of data.

The EyCx and ErCx values were determined by Linear Interpolation (ICPIN) analysis for growth rate and yield endpoint. The NOEC and LOEC values for growth rate and yield endpoint were determined by comparison with the control by Bonferroni Adj t Test.
As input data, nominal test item concentrations were used.

Results with reference substance (positive control):

Not relevant

Any other information on results incl. tables

Results

Growth rate

In test item solutions, the algal growth rate (r) after 72 hours of exposure was inhibited by a minimum value of 52.3 % for the concentration 1.7 mg test item/L to a maximum value of 94.9 % for the highest tested concentration, 10.0 mg test item/L, compared to the negative control.

For the lowest test item concentration (1.0 mg/L) no significant biological effect was shown in comparison to the negative control for end-point growth rate.

Yield

After 72 hours of exposure, the yield (Y), as difference of algal density between the end and the beginning of the test, was inhibited by a minimum value of 96.3% for the concentration of 1.7 mg test item/L to a maximum value of 99.9% for the highest test item concentration of 10.0 mg/L, compared to the negative control. For the lowest test item concentration (1.0 mg/L) no significant biological effect was shown in comparison to the negative control for end-point yield.

Endpoint	0 -72 h EC10 (mg/L)	0 -72 h EC20 (mg/L)	0 -72 h EC50 (mg/L)	0 -72 h NOEC   (mg/L)	0 -72 h LOEC   (mg/L)
Growth rate	1.1	1.2	1.7	1.0	1.7
Yield	1.1	1.1	1.3	1.0	1.7

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of test item Delmopinolo HCl was tested on Pseudokirchneriella subcapitata.

The EC50 value, calculated in terms of nominal test item concentrations, after 72 hours of exposure, were as follows:
- EC50 (72h): 1.7 mg/L (Growth rate)
- EC50 (72h): 1.3 mg/L (Yield)

According to CLP Regulation criteria, the EC50 (72h) is included in the range > 1 up to ≤ 10 mg/l; therefore the substance is classified as Aquatic Chronic 2; H411.

Executive summary:

Delmopinolo HCl is classified as Aquatic Chronic 2; H411 according to acute toxicity to daphnia magna in a 48-hour immobilization test under static exposure.