4-(piperidin-4-yl)morpholine

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:

12 Oct - 30 Nov 2015

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 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, London, UK

Test material

Test material information

Constituent 1

Reference substance name:

4-(piperidin-4-yl)morpholine

EC Number:

627-580-3

Cas Number:

53617-35-9

Molecular formula:

C9H18N2O

IUPAC Name:

4-(piperidin-4-yl)morpholine

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 1.0, 3.2, 10, 32, and 100 mg/L
- Sampling method: Samples were taken from the control and each test group from the bulk test preparation at 0 h and from the pooled replicates at 72 h for quantitative analysis. Duplicate samples were taken at 0 and 72 h and stored frozen for further analysis if necessary.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Prior to use, the test item was heated to 70 °C in an oven in order to ensure homogeneity and aid handling. Nominal amounts of test item (100 and 32 mg) were each separately dissolved in culture medium and the volumes adjusted to 1 L to give 100 and 32 mg/L stock solutions respectively. A series of dilutions was made from these stock solutions to give further stock solutions of 10, 3.2 and 1.0 mg/L. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (9.0 mL) to give the required test concentration of 1.0, 3.2, 10, 32 and 100 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. Inoculation of 900 mL of test medium with 9.0 mL of this algal suspension had no significant dilution effect on the final test concentration.
- Differential loading: no, Stock solutions (100 and 32 mg/L, nominal) were prepared from which a series of dilutions was made.
- Controls: yes, The control group was maintained under identical conditions but not exposed to the test item.
- Evidence of undissolved material: no, at the start of the test all control and test cultures were observed to be clear colorless solutions.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: freshwater unicellular green alga
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd. Scottish Marine Institute, Oban, Scotland
- Age of inoculum (at test initiation): Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 1E-03 cells/mL. The flasks were kept under controlled conditions until the algal cell density was approximately 1E-04 cells/mL.
- Method of cultivation: Master cultures (liquid) were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 1 °C

pH:

control: 8.0 (0 h), 8.7 (72 h)
treatment: 8.1 - 10.1 (0 h), 8.4 - 8.7 (72 h)

Nominal and measured concentrations:

control, 1.0, 3.2, 10, 32, and 100 mg/L (nominal)
control, 1.2, 3.3, 11, 33, and 156 mg/L (0 h measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks
- Type: plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: Material: glass; Size: 250 mL; Fill volume: 100 mL; Headspace: 150 mL
- Initial cells density: 5E+03 cells/mL
- Control end cells density: 9.81E+05 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified water (Elga Optima 15+ or Elga Purelab Option R-15 BP)
- Culture medium different from test medium: No, the culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at study initiation (0 h) and termination (72 h) of the test. The temperature in the incubator was recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the culture medium was adjusted to 7.5 with 0.1 N NaOH or HCl.
- Photoperiod: constant illumination
- Light intensity and quality: Intensity of approximately 7000 lux, provided by warm white lighting (380 - 730 nm).
- The test flasks were placed in an incubator (INFORS Multitron, Version 2) and constantly shaken at approx. 150 rpm.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cells densities were measured by Coulter Multisizer Particle Counter at 0, 24, 48, and 72 h.
- Other: The shape and size of the algal cells from test and control cultures was inspected microscopically at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: A preliminary range-finding test and an initial experiment were performed.
- Test concentrations: Range-finding test: control, 0.1, 1.0, 10, and 100 mg/L (nominal); Initial experiment: control, 6.25, 12,5, 25, 50, and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: The test concentrations for the initial experiment were based on the results of the range-finding test. In the initial experiment significant inhibition was observed to occur at the lowest test concentration employed of 6.25 mg/L and therefore, the test concentration range for the definitive test were selected accordingly.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

56 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

120 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

33 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

156 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

30 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

51 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

33 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

156 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): After 72 h there were no abnormalities detected in the control or test cultures at 1.2, 3.3, 11, and 33 mg/L (0 h measured concentrations). However, no intact cells were observed to be present in the test cultures at 156 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- ErC50 (72 h): 1.0 mg/L; 95% confidence limits 0.90 - 1.2 mg/L (potassium dichromate, 12 - 15 May 2015)

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one-way analysis of variance incorporating Bartlett´s test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett´s multiple comparison procedure for comparing several teratments with a control (Dunnett, 1955). There were no statistically significant differences (p ≥ 0.05), between the control, 1.2, 3.3, 11 and 33 mg/L test concentrations. However, the 156 mg/L test concentration was significantly different (P < 0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate ws 33 mg/L. Correspondingly, the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 156 mg/L.

Any other information on results incl. tables

Analysis of the test preparations at 0 and 72 h showed measured test concentrations to be near nominal with the exception of the 100 mg/L test preparations where measured concentrations of 156% and 161% of nominal were observed at 0 and 72 h, respectively. Examination of the data could find no cause for these slightly higher than expected results as the correct quantity of test item was weighed out and all subsequent dilutions made were near nominal.

Given this, and that no significant decline in measured concentrations was observed at 72 h, it was considered justifiable to base the results on the 0-hour measured test concentrations.

Table 1. Results for Test Samples.

Time point [h]	Nominal concentration of Test Item in Test Sample Cnom [mg/L]	Sample Preparation Factor F	Determined Concentration of Test item in Test Sample C [mg/L]	% of Nominal Concentration       [%]
0	Control	1	<LOQ	-
	1.0	2	1.18	118
	3.2	5	3.33	104
	10	20	10.9	109
	32	50	32.5	102
	100	200	156*	156
72	Control	1	<LOQ	-
	1.0	2	1.13	113
	3.2	5	3.19	100
	10	20	10.9	109
	32	50	32.8	103
	100	200	161*	161

 LOQ     = Limit of Quantification

-           = not applicable

*            = outside expected 80 - 120% range. Value accepted and discussed above.

Table 2. Inhibition of Growth Rate and Yield in the Definitive Test.

0 –hour Measured Concentration (mg/L)		Growth Rate (cells/mL/h)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.073		9.25E+05
	R2	0.071		8.43E+05
	R3	0.075		1.10E+06
	R4	0.075	-	1.11E+06	-
	R5	0.073		9.35E+05
	R6	0.073		9.34E+05
	Mean	0.073		9.75E+05
	SD	0.002		1.08E+05
1.2	R1	0.075	[3]	1.13E+06
	R2	0.074	[1]	1.04E+06
	R3	0.074	[1]	1.04E+06
	Mean	0.074	[2]	1.07E+06	[9]
	SD	0.001		4.99E+04
3.3	R1	0.071	3	8.37E+05
	R2	0.073	0	9.51E+05
	R3	0.073	0	9.39E+05
	Mean	0.072	1	9.09E+05	7
	SD	0.001		6.26E+04
11	R1	0.071	3	8.03E+05
	R2	0.076	[4]	1.21E+06
	R3	0.076	[4]	1.17E+06
	Mean	0.074	[2]	1.06E+06	[9]
	SD	0.003		2.24E+05
33	R1	0.071	3	8.19E+05
	R2	0.072	1	8.70E+05
	R3	0.071	3	8.34E+05
	Mean	0.071	2	8.41E+05	14
	SD	0.001		2.62E+04
156	R1	0.025	66	2.40E+04
	R2	0.015	79	8.37E+03
	R3	0.025	66	2.42E+04
	Mean	0.022	70	1.89E+04	98
	SD	0.006		9.09E+03

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R1 –R6=Replicates1to 6

SD=Standard Deviation

[Increase in growth as compared to controls]

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes