4-(piperidin-4-yl)morpholine

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Oct - 18 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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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, 10, 18, 32, 56, and 100 mg/L (nominal)
- 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 48 h for quantitative analysis. Duplicate samples were taken at 0 and 48 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 order to aid handling. A nominal amount of test item (200 mg) was dissolved in test waster and the volume adjusted to 2 L to give the 100 mg/L test concentration from which a series of dilutions was made to give the further test concentrations. Prior to diluting, the pH of the 100 mg/L stock solution was recorded at 9.8 and adjusted to 7.7 using hydrochloric acid.
- Differential loading: no, a stock solution of 100 mg/L was prepared and diluted to the further test concentrations.
- 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 and throughout the test all control and test solutions were observed to be clear colorless solutions.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: in-house laboratory cultures
- Feeding during test: The daphnids received no food during exposure.
- Food type: A mixture of algal suspension (Desmodesmus subspicatus) and Tetramin flake food suspension.
- Frequency: daily
- Culture conditions: Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 19 - 20 °C. The lighting cycle was controlled to a 16 h light and 8 h darkness cycle with 20 min dawn and dusk transition periods. The culture conditions ensured that reproduction was by parthenogenesis.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 h old. These young were removed from the cultures and used for testing.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

250 mg/L CaCO3 (reconstituted water - ISO medium)

Test temperature:

21 - 22 °C

pH:

control: 8.1 (0 h), 7.9 (48 h)
treatments: 7.6 - 8.0 (0 h), 8.0 (48 h)

Dissolved oxygen:

control: 9.1 mg O2/L (0 h), 8.9 mg O2/L (48 h)
treatments: 8.9 - 9.1 (0 h), 8.0 mg O2/L (48 h)

Nominal and measured concentrations:

control, 10, 18, 32, 56, and 100 mg/L (nominal)
< LOQ, 10.3/9.28, 17.1/17.4, 30.4/29.8, 53.3/51.2, and 93.6/94.7 mg/L (measured in: fresh media at 0 h/in old media at 48 h)

Details on test conditions:

TEST SYSTEM
- Test vessel: jars
- Type: covered
- Material, size, headspace, fill volume: Material: glass; Size: 150 mL; Fill volume: 100 mL; Headpace: 50 mL
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Daphnia were maintained in reconstituted water - Elendt M7 medium. Reconstituted water - ISO medium was used for both the range-finding and definitive tests.
- Intervals of water quality measurement: Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: Prior to diluting, the pH of the 100 mg/L stock solution of the test substance was adjusted to 7.7.
- Photoperiod: 16 h light and 8 h dark with 20 min dawn and dusk transition periods.
- Light intensity: Between 200 and 1200 lux.

EFFECT PARAMETERS MEASURED:
- Immobilization: recorded after 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: control, 0.10, 1.0, 10, and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: No immobilization was observed at the test concentrations of 0.10, 1.0 and 10 mg/L. However, immobilization was observed at 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 71 - 79 mg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Other biological observations: Sub-lethal effects of exposure were observed in the 10, 56 and 100 mg/L concentrations. These responses were trapping at the surface and reduced mobility.
- Mortality of control: One single daphnid was observed to be immobilized in the control (mortality = 5%). However, it was considered to be due to natural causes as less than 10% immobilization was observed.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (48 h): 0.47 mg/L, 95% confidence limits: 0.47 - 0.63 mg/L (potassium dichromate, 07 - 23 Sep 2015)

Reported statistics and error estimates:

The EC50 values and associated confidence limits at 48 h was calculated by linear interpolation. The Lowest Observed Effect Concentration and the No Observed Effect Concentration at 24 and 48 h were calculated using the Fisher´s Exact Binomial Test with Bonferroni correction. All results were calculated using the ToxCalc Professional computer software package (TOXCalc).

Any other information on results incl. tables

Analysis of the test preparations at 0 and 48 h showed measured test concentrations to be near nominal. Thus, the results are based on nominal test concentrations (Table 1).

Table 1. Results for Test Samples.

Time point [h]	Nominal concentration of test item [mg/L]	Sample preparation factor F	Determined Concentration of Test Item in Test Sample C [mg/L]	% of Nominal Concentration [%]
0 (fresh)	Control	1	< LOQ	-
	10	20	10.3	103
	18	33.3	17.1	95
	32	50	30.4	95
	56	100	53.3	95
	100	200	93.6	94
48 (old)	Control	1	< LOQ	-
	10	20	9.28	93
	18	33.3	17.4	97
	32	50	29.8	93
	56	100	51.2	91
	100	200	94.7	95

LOQ = Limit of Quantification

-   = not applicable

Table 2. Cumulative Immobilization Data and Observations in the Definitive Test.

Nominal concentration (mg/L)	48 h
	Cumulative Immobilized Daphnia (Initial population: 5 per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	1*	0	0	1	5	5N	4N	5N	5N
10	0	0	0	0	0	0	5N	4N, IT	5N	5N
18	0	0	0	0	0	0	5N	5N	5N	5N
32	0	0	0	0	0	0	5N	5N	5N	5N
56	1	0	0	1	2	10	4N	4N, IT	5N	4N
100	5	5	5	5	20	100	A/I	A/I	A/I	A/I

* A single daphnia observed to be immobilized, considered to be due to natural causes as less than 10% immobilization is observed.

R1-R4 = Replicates 1-4

N = No sub-lethal effects observed

T = Trapped at the surface

A/I = All daphnia immobilized

R = Reduced Mobility

Applicant's summary and conclusion

Validity criteria fulfilled:

yes