4-methylcyclohexanone

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-12-10 to 2016-05-18

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 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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 96 hours; test item concentrations were analysed in one of the duplicate test medium samples taken from all test concentrations at the sampling times of 0 and 96 hours
- 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 2000 mg/L was freshly prepared by mixing 4 g of the test item into 2 litres of test water by vigorously stirring for 15 minutes with a magnetic stirrer. Adequate volumes of this stock solution were added to the test water in the test vessels and were mixed for 1 minute with a flat-bladed stirrer to prepare the test media with the test concentrations. The test media were freshly prepared just before introduction of the fish (i.e. start of the exposure).
- Controls: Blank control
- Evidence of undissolved material (e.g. precipitate, surface film, etc): All test media were clear solutions throughout the entire test duration

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Age at study initiation (mean and range, SD): Juvenile
- Length at study termination (length definition, mean, range and SD): Mean standard length of 5.9 cm (sd = 0.2)
- Weight at study termination (mean and range, SD): Mean weight of 1.81 g (sd = 0.21)
- Feeding during test: Fish were not fed during the test

ACCLIMATION
- Acclimation period: 1 week
- Acclimation conditions (same as test or not): During holding and acclimatization, the water temperature was controlled at 13 to 14 °C with a dissolved oxygen content of greater than or equal to 9.9 mg O2/L.
- Type and amount of food: Commercial trout pellets, amount not reported
- Feeding frequency: Feeding frequency not reported; feeding was discontinued approximately 24 hours prior to the start of the definitive test
- Health during acclimation (any mortality observed): During holding and acclimation, the mortality rate in the test fish batch was lower than 5 % and all fish were healthy

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Approximately 140 mg/L as CaCO3

Test temperature:

13 - 14 °C

pH:

Test start: 7.7 - 7.9
Test end: 7.6 - 8.0

Dissolved oxygen:

Test start: 10.8 - 11.0 mg/L
Test end: 7.3 - 9.4 mg/L

Nominal and measured concentrations:

6.3, 12.5, 25, 50 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass fish bowl (approximate diameter of 33 cm)
- Type: Closed
- Material, size, headspace, fill volume: Glass fish bowl with 20 litres of test medium
- Aeration: Measurements made at 6 hours indicated that dissolved oxygen concentrations were declining, therefore aeration was provided from 6 hours through to the end of the test
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.63 g fish wet weight per litre test medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated and softened laboratory tap water
- Total organic carbon: 1.060 mg/L (mean)
- Pesticides: 0.012 μg/L total pesticides (mean)
- Chlorine: 0.333 mg/L total chlorine (mean), 0.245 mg/L free chlorine (mean)
- Conductivity: 392.342 μS/cm at 20 °C (mean)
- Culture medium different from test medium: The test water used for the definitive test was the same as that used to maintain the stock fish
- Intervals of water quality measurement: The water temperature, pH, dissolved oxygen concentrations and appearance of the test media were recorded at the start of the test and once every day during the test at each test concentration with surviving fish and in the control. Additional dissolved oxygen concentrations were measured 6 hours after the start of the test. The light intensity during the light period was also measured.

OTHER TEST CONDITIONS
- Adjustment of pH: None reported
- Photoperiod: 16-hour light to 8-hour dark photoperiod, with a 20-minute transition period was used
- Light intensity: Approximately 293 to 309 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The test fish were observed after approximately 1, 3, 6, 24, 48, 72 and 96 hours test duration for mortality and visible abnormalities

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes
- Test concentrations: 100 mg/L.
- Results used to determine the conditions for the definitive study: 43 % mortality after 48 hours

Reference substance (positive control):

not required

Remarks:

In accordance with the UK Animal Protection Law, no positive control is tested for evaluation of the fish quality and of the experimental conditions

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

78 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % confidence interval: 65 - 94 mg/L

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: At the test concentration of 50 mg/L at observations 3 and 6 hours after exposure two fish were swimming at the surface, at observations 24 and 48 hours after exposure one fish was swimming at the surface. At the test concentration of 100 mg/L at observations 3 and 6 hours after exposure all fish had increased pigmentation, at observations after 24 hours one fish was found dead and six fish had increased pigmentation, at observations after 48 hours three fish were found dead and three fish had increased pigmentation and at observations after 72 hours two fish were found dead and one fish had increased pigmentation. In the 100 mg/L test concentration at the 96 hour observation there was one fish alive and recorded without visible abnormalities. At previous observations in the 100 mg/L test concentration surviving fish were recorded with increased pigmentation; however, this sub-lethal effect was no longer evident in the surviving fish at 96 hours.
- Mortality of control: No mortality in the control was observed
- Abnormal responses: In the control and at the test concentrations up to and including 25 mg/L, all fish survived until the end of the test and no visible abnormalities were observed in the test fish
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: All test media were clear solutions throughout the entire test duration. The measured concentrations were between 87 and 98 % of the nominal values at the start of the test and were between 81 to 98 % of nominal at the end of the test

Reported statistics and error estimates:

The LC50 values at the observation points at 1, 3, 6 and 24 hours were estimated by inspection of the data. The LC50 values at the observation points at 48, 72 and 96 hours were calculated by the Trimmed Spearman-Karber method. The NOEC and LOEC were determined by inspection of the data. Statistical analyses were performed using ToxRat Professional (Version 3.2.1).

Sublethal observations / clinical signs:

Table 1. Mortality and visible abnormalities observed in the test fish

Nominal test item concentrations (mg/L)	Number of fish showing abnormalities / Cumulative dead fish (n = 7) (Type of visible abnormalities)
	Observation time
	1 hour	3 hours	6 hours	24 hours	48 hours	72 hours	96 hours
Control	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
6.3	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
12.5	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
25	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
50	0 / 0	2 / 0(S)	2 / 0(S)	1 / 0(S)	1 / 0(S)	0 / 0	0 / 0
100	0 / 0	7 / 0 (I)	7 / 0 (I)	6 / 1(I)	3 / 4(I)	1 / 6(I)	0 / 6
LC50	> 100	> 100	> 100	> 100	95	78	78
95 % confidence interval	n.d.	n.d.	n.d.	n.d.	73 - 123	65 - 94	65 - 94

n.d. not determined

S: Swimming at surface

I: Increased pigmentation

Validity criteria fulfilled:

yes

Remarks:

The test was considered to be valid, since no mortality in the control was observed and the validity criterion of at least 60 % oxygen saturation was fulfilled

Conclusions:

The 96-hour LC50 was calculated to be 78 mg/L. The 96-hour NOEC and LOEC values were determined to be 50 and 100 mg/L, respectively.

Executive summary:

The toxicity of the test item to rainbow trout (Oncorhynchus mykiss) was assessed according to EU Method C.1 and OECD Guideline 201. The study is reliable without restrictions, as it is GLP-compliant and was conducted according to standard guidelines. Following a preliminary range-finding test, juvenile rainbow trout were exposed to nominal test item concentrations of 6.3, 12.5, 25, 50 and 100 mg/L, alongside a blank control, under static conditions for 96 hours. The 96-hour LC50 was calculated to be 78 mg/L. The 96-hour NOEC and LOEC values were determined to be 50 and 100 mg/L, respectively.

Description of key information

The toxicity of the test item to rainbow trout (Oncorhynchus mykiss) was assessed according to EU Method C.1 and OECD Guideline 201. The study is reliable without restrictions, as it is GLP-compliant and was conducted according to standard guidelines. Following a preliminary range-finding test, juvenile rainbow trout were exposed to nominal test item concentrations of 6.3, 12.5, 25, 50 and 100 mg/L, alongside a blank control, under static conditions for 96 hours. The 96-hour LC50 was calculated to be 78 mg/L. The 96-hour NOEC and LOEC values were determined to be 50 and 100 mg/L, respectively.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

78 mg/L