4-methylcyclohexanone

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-12-10 to 2016-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP-compliant study, performed according to guideline, fully adequate for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and all test concentrations
- Sampling method: Duplicate samples were taken from each treatment before the start of the test and after 48 hours; test item concentrations were analysed in one of the duplicate test medium samples
- Sample storage conditions before analysis: All samples were stored frozen immediately after sampling

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: At the start of the test, a concentrated stock solution of nominal 200 mg/L was freshly prepared by dissolving approximately 200 mg of the test item into 1 litre of test water. The required test concentrations were prepared as dilutions from this stock solution. The following test concentrations were assigned to the definitive test; 6.25, 12.5, 25, 50 and 100 mg/L. The stock solution and each test concentration were inverted several times to ensure homogeneity and mixing of the solutions. The test media were prepared just before introduction of the daphnids (i.e. start of the exposure).
For each treatment, 20 daphnids were used divided into four replicates of five daphnids each. The test was performed in 100 mL conical flasks completely filled with test medium and sealed with ground glass stoppers. The daphnids were randomly distributed to the test vessels at the initiation of the test.
- Controls: Blank control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None reported

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Straus, clone 5
- Age at study initiation (mean and range, SD): Less than 24 hours old, not first brood progeny
- Feeding during test: No

ACCLIMATION - Not reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L as CaCO3 (corresponding to 2.5 mmol/L)

Test temperature:

21 to 22 °C

pH:

Start of test: 7.8 - 8.0
End of test: 7.7 - 7.8

Dissolved oxygen:

Start of test: 8.9 to 9.3 mg/L
End of test: 8.6 to 8.8 mg/L

Nominal and measured concentrations:

6.25, 12.5, 25, 50 and 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks sealed with ground glass stoppers
- Type: closed
- Material, size, headspace, fill volume: 100 mL flasks completely filled with test medium
- Aeration: The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO reconstituted test water
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4:1 (based on molarity)
- Culture medium different from test medium: Culture medium (with respect to pH, main ions, and total hardness) was identical to the test medium
- 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. The appearance of the test media was visually inspected at the start of the test and after 24 and 48 hours.

OTHER TEST CONDITIONS
- Adjustment of pH: Not reported
- Photoperiod: 16-hour light to 8-hour dark cycle with a 20 minute transition period
- Light intensity: 548 to 621 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
The immobility of the daphnids was determined by visual inspection after 24 and 48 hours of exposure

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Results from the ranger finder showed no immobilization at 1.0 mg/L, 10 % immobilization was recorded at 0.10 and 10 mg/L and 60 % immobilization was observed at 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Other biological observations: No signs of disease or stress (e.g. discoloration or unusual behaviour such as trapping at the water surface) were observed in the control
- Mortality of control: No immobilized Daphnia were determined in the control at the end of the test
- Abnormal responses: At 100 mg/L test concentration, 5 test animals were observed to be trapped at the surface at 48 hours
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The control and all test concentrations were observed to be clear, colourless solutions. The measured concentrations were between 97 and 102 % of the nominal values at the start of the test and were between 89 and 97 % of the nominal test concentrations after 48 hours.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50/LC50: 0.54 mg/L (48-h)

Reported statistics and error estimates:

The 24 and 48-hour EC50 values were estimated by inspection of the data. The NOEC was determined using Fisher’s Exact Test using the ToxRat Professional computer software package (TOXRAT).

Table 1. Immobilisation and observations of Daphnia magna

Nominal test item concentration (mg/L)	No. of daphnids tested	No. of immobilised daphnids after 24 hours	% of immobilised daphnids after 24 hours	No. of immobilised daphnids after 48 hours	% of immobilised daphnids after 24 hours
Control	20	0	0	0	0
6.25	20	0	0	0	0
12.5	20	0	0	0	0
25	20	0	0	0	0
50	20	0	0	0	0
100	20	0	0	2 (5a)	10

Values in parenthesis: number of test animals with adverse effects

a: Trapped at the surface

Validity criteria fulfilled:

yes

Remarks:

The test is considered to be valid, as no daphnids showed immobilization or other signs of disease or stress in the control, and the dissolved oxygen concentration at the end of the test was at least 3 mg/L in the control and test vessels.

Conclusions:

The 24- and 48-h EC50 were determined to be > 100 mg/L, the highest concentration tested. The 48-h NOEC was 100 mg/L.

Executive summary:

The toxicity of the test item to Daphnia magna was assessed according to EU Method C.2 and OECD Guideline 202. The study is reliable without restrictions, as it is GLP-compliant and was conducted according to standard guidelines. Following a preliminary range-finding test, daphnids (less than 24 hours old) were exposed to an aqueous solution of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L, alongside a control, under static conditions for 48 hours. The 24- and 48-h EC50 were determined to be > 100 mg/L, the highest concentration tested. The 48-h NOEC was 100 mg/L.

Description of key information

The toxicity of the test item to Daphnia magnawas assessed according to EU Method C.2 and OECD Guideline 202. The study is reliable without restrictions, as it is GLP-compliant and was conducted according to standard guidelines. Following a preliminary range-finding test, daphnids (less than 24 hours old) were exposed to an aqueous solution of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L, alongside a control, under static conditions for 48 hours. The 24- and 48-h EC50 were determined to be > 100 mg/L, the highest concentration tested. The 48-h NOEC was 100 mg/L.