4-(piperidin-4-yl)morpholine

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Nov - 01 Dec 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 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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, 100 mg/L
- Sampling method: Water samples were taken from the control and the 100 mg/L test vessel at 0 and 72 h from fresh media and at 24 and 96 h from old media for quantitative analysis. Duplicate samples at 24 (fresh media), 48 (old and fresh media) and 72 h (old media) were taken and stored frozen for further analysis if necessary.
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A nominal amount of test item (2000 mg) was dissolved in test water and the volume adjusted to 20 L to give the 100 mg/L test concentration. After pH adjustment, the prepared concentration was stirred with a flat bladed mixer to ensure adequate mixing and homogeneity.
- 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):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Source: Brow Well Fisheries Limited, Hebden, UK.
- Age at study initiation (mean and range, SD): juvenile
- Length at study initiation: 5 cm, S.D. = 0.2
- Weight at study initiation: 1.2 g, S.D. = 0.1
- Maintenance of the brood fish: Fish were maintained in-house since 06 Nov 2015 in a glass fiber tank with a "single pass" water renewal system.

ACCLIMATION
- Acclimation period: 15 - 23 Nov 2015
- Acclimation conditions: Same as test: 16 h ligh and 8 h darkness cycle with 20 minute dawn and dusk transition periods. The water temperature was 13 °C with a dissolved oxygen content of greater than or equal to 9.4 mg O2/L.
- Type and amount of food during acclimation: Commercial trout pellets.
- Health during acclimation: There was no mortality during the 7 d prior to the start of the test.

FEEDING DURING TEST
Feeding was discontinued approximately 24 h prior to the start of the definitive test. The fish received no food during exposure.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

140 mg/L CaCO3

Test temperature:

13 - 14 °C

pH:

control: 7.7 - 8.1
treatment: 7.7 - 8.0

Dissolved oxygen:

≥ 9.4 mg O2/L

Nominal and measured concentrations:

control, 100 mg/L (nominal)
< LOQ, 102 - 109 mg/L (measured in old and fresh media)

Details on test conditions:

TEST SYSTEM
- Test vessel: exposure vessels
- Type: covered
- Material, size, headspace, fill volume: Material: glass; Size: 25 - 30 L; Fill volume: 20 L, Headspace: 5 - 10 L
- Aeration: The test vessels were aerated via narrow bore glass tubes.
- Renewal rate of test solution: A daily renewal of the test preparations was employed to prevent the build up of nitrogenous waste products.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.42 g bodyweight/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Total organic carbon: 0.996 mg/L
- Chlorine: 0.248 mg/L
- Culture medium different from test medium: The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
- Intervals of water quality measurement: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 h, and after each test media renewal at 24, 48, and 72 h, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 h, represent those of the used or 24-h old test preparations.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the 100 mg/L test preparations were recorded at 9.6 to 9.7 at each media renewal period and then adjusted to match the pH of the control using hydrochloric acid.
- Photoperiod: 16 h light and 8 h darkness with 20 min dawn and dusk transition periods.

EFFECT PARAMETERS MEASURED:
- Mortality and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 h after the start of exposure.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: A limit test at 100 mg/L (nominal) was performed. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.
- Range finding study: yes
- Test concentrations: 1.0, 10 and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given no mortalities or sub-lethal effects of exposure were observed.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Other biological observations: There were no sub-lethal effects of exposure observed in the seven fish exposed to the limit concentration of 100 mg/L (nominal) for 96 h.
- Mortality of control: 0%
- Other adverse effects control: None of the control fish showed signs of stress during the test.

Reported statistics and error estimates:

An estimate of the LC50 values was given by inspection of the mortality data.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Analysis of the test preparations at 0, 24, 72 and 96 h showed measured test concentrations to be near nominal and so it was considered justifiable to estimate the LC50 values in terms of the nominal test concentration only. Chemical analysis of the fresh (at 0, 24, 48, and 72 h) and old (at 24, 48, 72, and 96 h) test preparations indicated that the test item was stable under test conditions (Table 1).

Table 1. Water Quality Measurements.

Nominal concentration (mg/L)	Time (h)
	0 h (fresh media)			24 h (old media)
	pH	mg O2/L	T (°C)	pH	mg O2/L	T (°C)
Control	7.7	10.5	13	7.9	9.7	14
100	7.7	10.4	13	7.9	9.6	14

 

Nominal concentration (mg/L)	Time (h)
	24 h (fresh media)			48 h (old media)
	pH	mg O2/L	T (°C)	pH	mg O2/L	T (°C)
Control	7.7	10.2	13	8.1	9.8	14
100	7.8	10.4	13	7.9	9.3	14

 

Nominal concentration (mg/L)	Time (h)
	48 h (fresh media)			72 h (old media)
	pH	mg O2/L	T (°C)	pH	mg O2/L	T (°C)
Control	7.7	10.3	13	7.9	9.8	14
100	7.7	10.3	13	8.0	9.9	14

 

Nominal concentration (mg/L)	Time (h)
	72 h (fresh media)			96 h (old media)
	pH	mg O2/L	T (°C)	pH	mg O2/L	T (°C)
Control	7.7	10.2	13	7.9	9.9	14
100	7.7	10.3	13	7.8	9.6	14

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes