4,8-Cyclododecadien-1-one

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:

28-11-2014 to 05-02-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, effects on Biotic Systems, 203 Fish Acute toxicity test [M]. (2013)

Deviations:

no

Principles of method if other than guideline:

The test was conducted in accordance with China GB/T 27861-2011 Chemical Test Guideline stipulations and Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, effects on Biotic Systems, 203 Fish Acute toxicity test [M]. Second Edition. Beijing: China
Environmental Press. 2013: 30-36. The test was also completed under OECD TG 203: Fish Acute Toxicity Test (1992).

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 6.79, 9.84, 14.3, 20.7 and 30 mg/L (nominal); with analytically confirmed 0 h concentrations of: 6.53, 9.84, 14.3, 20.7 and 30.7 mg/L, respectively.
At 96 hours, the measured concentrations ranged from 5.84 to 30.1 mg/L (84 to 102% nominal, respectively at lowest and highest concentrations).
- Sampling method: 50 ml test solution was added in the separator funnel and 20 ml, 15 ml, 10 ml n-hexane were added respectively to extract for three times and extracted for 5 min each time. After that the extractions were collected and transferred into the 50 ml measuring flask, and then diluted with n-hexane to 50 ml. The actual concentrations of test substance in n-hexane were measured.
- Sample storage conditions before analysis: The samples were analysed immediately. Otherwise the samples (the n-hexane extractions) were stored at 4°C until analysis within one week.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test stock solution: 0.535g test substance was obtained in a small amount of dechlorinated tap water with ultra-sonication treatment for 10 min and then transferred into 5 L jar. Test water was added to 5350 mL and the bottle cap was covered (there was no headspace on the test suspension). Then the nominal concentration of 100 mg/L suspension was obtained. The suspension was stirred with a magnetic stirrer for about 24h in dark conditions. The nominal concentration of 100 mg/L stock solution (98.8 mg/L calculated based on the purity) was obtained. Preparation of test solution: 340 mL, 492 mL, 715 mL, 1035 mL and 1500 mL volumes of test stock solutions were taken and allocated into the five 5 L jars respectively. Then 4660 mL, 4508 mL, 4285 mL, 3965 mL and 3500 mL volumes of test water were added. After stirring well the nominal concentrations of 6.79 mg/L, 9.84 mg/L, 14.3 mg/L, 20.7 mg/L and 30.0 mg/L test solutions (6.71 mg/L, 9.72 mg/L, 14.3 mg/L, 20.5 mg/L and 29.6 mg/L calculated based on the purity, respectively) were obtained. During the test period all test vessels were covered with bottle cap.
- Controls: Dechlorinated tap water with no test substance.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Strain: Not reported. Batch number FDr20141023-1
- Source: Recognised supplier (recorded in full study report) located in China.
- Age at study initiation (mean and range, SD): Not reported.
- Length at study initiation (length definition, mean, range and SD): Not reported. The body weight and total length of test fish in blank control were measured when the test was finished. The average and standard deviation of test fish body weight and total length were respectively 24.82 mm±0.86 mm and 0.134g±0.011 g.
- Method of breeding: Not reported.
- Feeding during test
- Food type: No feeding during exposure. Not applicable.
- Amount: No feeding during exposure. Not applicable.
- Frequency: No feeding during exposure. Not applicable.

ACCLIMATION
- Acclimation period: 40 days
- Acclimation conditions (same as test or not): Yes. Same water quality, temperature 23 ± 2°C and photoperiod as used during the test (14 hours light; 10 hours dark).
- Type and amount of food: Not reported.
- Feeding frequency: At least three times a week until 24 hours before the start of the test.
- Health during acclimation (any mortality observed): None reported.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

118 mg/L expressed as CaCO3

Test temperature:

Temperature 22 - 23.9 °C during test period, the test was performed in a temperature controlled environment with continuous monitoring and was maintained at 23 ± 2°C.

pH:

Test water (dechlorinated water): pH 7.95.
The pH was in the range of 7.44 to 7.98 during the study.

Dissolved oxygen:

The test solutions were not aerated during the exposure. The dissolved oxygen concentration in the test solutions during the test was kept at not less than 60 % of the air saturation value. (Range 0h: 95 to 96% ; 96h: 70 – 84% and 86% in the control).

Nominal and measured concentrations:

Nominal: 6.79, 9.84, 14.3, 20.7 and 30 mg/L (nominal); with analytically confirmed 0 h concentrations of: 6.53, 9.84, 14.3, 20.7 and 30.7 mg/L, respectively.
Measured: 6.79, 9.84, 14.3, 20.7 and 30.0 mg/L test groups at 0 h ranged from 6.53 mg/L to 30.6 mg/L (96% to 103% of the nominal concentration of the main constituent of test substance). At 96 h, the measured concentrations of old solutions ranged from 5.84 mg/L to 30.1 mg/L (84% to 102% of the nominal concentration).

Details on test conditions:

TEST SYSTEM
- Test vessel: 5 L flask; Closed with plug.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Glass, 3 Litre fill volume per vessel.
- Aeration: None
- No. of organisms per vessel: 10 / test group
- No. of vessels per concentration (replicates): no replicates; 1 exposure per test concentration.
- No. of vessels per control (replicates): no replicates; 1 exposure
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: 0.268 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water
- Culture medium different from test medium: Not reported
- Intervals of water quality measurement: Temperature, pH and dissolved oxygen were measured at 0 h, 24 h, 48 h, 72 h and 96 h after the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: None.
- Photoperiod: 14 hours light/ 10 hours dark
- Light intensity: 86 - 93 lx (mean = 86 lx)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortalities and abnormalities; measured at 3h, 24h, 48h, 72h and 96h as applicable.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.45
- Justification for using less concentrations than requested by guideline: Note applicable.
- Range finding study
- Test concentrations: 3.05, 9.77, 31.2 and 100 mg/L and blank control.
- Results used to determine the conditions for the definitive study: Yes. 0% mortality in the 3.05 and 9.77 mg/L concentrations. 100% mortality in the 31.2 and 100 mg/L concentrations.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

23.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Concentrations based on analytically confirmed nominal concentration of the test substance. 95% confidence limits 21.1 mg/l to 25.4 mg/l

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

14.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Concentrations based on analytically confirmed nominal concentration of the test substance.

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Concentrations based on analytically confirmed nominal concentration of the test substance.

Details on results:

- Behavioural abnormalities: There were no abnormal responses of test fish in the blank control. Observed abnormal responses are reported in table 3.
- Observations on body length and weight: Body weight and length were recorded at the end of the observation period.
- Mortality of control: None.
- Other adverse effects control: Yes. None reported.
- Abnormal responses: Observed abnormal responses are reported in table 3.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported.
- Effect concentrations exceeding solubility of substance in test medium: Not applicable.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 24h LC50: 252 mg/L (C.I. 223 -284 mg/L)
- Other: The results were kept at the value ranging from 200 mg/L to 400 mg/L, which met the criterion for the validity of the test.

Reported statistics and error estimates:

The LC50 values and the 95% confidence limits (L95) at 24 h, 48 h, 72 h and 96 h after the start of the test were calculated by Trimmed Spearman-Karber software (US EPA: Trimmed Spearman-Karber Program Version 1.5).

Sublethal observations / clinical signs:

Table 1: Nominal and Measured Concentrations

Nominal concentration (mg/L)	Nominal concentration of main constituent (mg/L)	Measured concentrations of teat substance (mg/L) (Percent of measured concentrations versus the analytically confirmed nominal concentration of main constituent %)
		0h	96h
Black control	0	ND	ND
6.79	6.71	6.53 (97)	5.84 (87)
9.84	9.72	9.58 (99)	8.20 (84)
14.3	14.1	13.6 (96)	11.8 (84)
20.7	20.5	20.4 (100)	19.4 (95)
30.0	29.6	30.6 (103)	30.1* (102)

Note:

1) ND means lower than the LOD (LOD=0.113 mg/L).

2) * was the measured value at 3h, due to complete mortality in the test group at 30.0 mg/L at 3h, so the test samples were collected and measured immediately.

Table 2: Cumulative mortality

Nominal concentration (mg/l)	Number of test fish	Cumulative mortality (%)
		3h	24h	48h	72h	96h
Blank control	10	0	0	0	0	0
6.79	10	0	0	0	0	0
9.84	10	0	0	0	0	0
14.3	10	0	0	0	0	0
20.7	10	20	20	20	20	20
30.0	10	100	100	100	100	100

Table 3; Observed abnormal responses

Nominal concentration (mg/L)	Result of observation (Left: Number of affected fish / Right: Total survival number and Symptom detail)
	3h		24h		48h		72h		96h
Blank control	0/10	N	0/10	N	0/10	N	0/10	N	0/10	N
6.79	6/10	RA(6)	6/10	IMM(6)	5/10	RA(5)	6/10	RA(6)	4/10	RA(4)
9.84	8/10	IMM(4) RA(4)	10/10	IMM(9) RA(1)	10/10	IMM(10)	10/10	IMM(8) RA(2)	10/10	IMM(6) RA(4)
14.3	10/10	IMM(3) RA(7)	10/10	IMM(4) LB(6)	10/10	LB(10)	10/10	LB(10)	10/10	LB(8) IMM(2)
20.7	10/10	LB(7) OLE(3)	8/8	LB(8)	8/8	LB(8)	8/8	LB(8)	8/8	LB(8)
30.0	-	-	-	-	-	-	-	-	-	-

Where:

N : Normal,

- : Complete mortality

RA : Reduction of activity

IMM : Immobilized at the bottom

OLE : Overturn and loss of equilibrium

LB : Lying on the bottom

Validity criteria fulfilled:

yes

Conclusions:

The 96 hour LC50 for the test item to Danio rerio was determined to be 23.1 mg/L with a 95% confidence interval from 21.1 – 25.4 mg/L based on nominal concentrations that were analytically confirmed.

Executive summary:

The acute toxicity of the test item to Zebra fish (Danio rerio) was determined in a 96 hour static test according to OECD TG 203 and the China Guidelines for the Testing of Chemicals, Effects on Biotic Systems, Version 2, 203 Fish Acute Toxicity Test (2013) under GLP. Based on the results of range finding tests the nominal concentrations of 0 (control), 6.79, 9.84, 14.3, 20.7 and 30.0 mg/L (geometric series with a factor of 1.45) were selected for exposure to 10 fish per group. One control containing no test item was also performed. Observations were made at 3, 6, 24, 48, 72 and 96 hours after the start of the exposure. Analytical measurements of test item concentration were made at 0 and 96 hours by GC-FID analysis. The measured concentrations were determined to remain within the (±20%) limit range of 96 – 103% during the test period which were within the guideline limit range. Therefore effect concentrations were based on nominal concentrations that are analytically confirmed. Statistical analysis was used for LC50 value estimation and 95% confidence intervals for the observation times of 24h, 48h, 72h and 96h after the start of the exposure. During the definitive test the water temperature was in the 22.0 – 23.9 °C, pH was 7.44 – 7.98 and air saturation was 70 – 84% indicating that environmental conditions were adequately maintained. Behavioural abnormalities and biological effects were reported during the course of the study. No abnormalities were observed in the control group. The 96h highest nominal concentration causing no mortality was 14.3 mg/L. The 96h median LC50 was 23.1 (C.I. 21.1 – 25.4) mg/L based on the nominal concentrations that were analytically confirmed.

Description of key information

LC50 (fish) =  23.1 (C.I. 21.1 - 25.4) mg/L based on analytically confirmed nominal concentrations, 96-hour, freshwater, OECD TG 203, 2015

LC0 (fish) = 14.3 mg/L based on analytically confirmed nominal concentrations, 96-hour, freshwater, OECD TG 203, 2015

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

23.1 mg/L

Additional information

The acute toxicity of the test item to Zebra fish (Danio rerio) was determined in a 96 hour static test according to OECD TG 203 and the China Guidelines for the Testing of Chemicals, Effects on Biotic Systems, Version 2, 203 Fish Acute Toxicity Test (2013) under GLP. Based on the results of range finding tests the nominal concentrations of 0 (control), 6.79, 9.84, 14.3, 20.7 and 30.0 mg/L (geometric series with a factor of 1.45) were selected for exposure to 10 fish per group. One control containing no test item was also performed. Observations were made at 3, 6, 24, 48, 72 and 96 hours after the start of the exposure. Analytical measurements of test item concentration were made at 0 and 96 hours by GC-FID analysis. The measured concentrations were determined to remain within the (±20%) limit range of 96 – 103% during the test period which were within the guideline limit range. Therefore effect concentrations were based on nominal concentrations that are analytically confirmed. Statistical analysis was used for LC50 value estimation and 95% confidence intervals for the observation times of 24h, 48h, 72h and 96h after the start of the exposure. During the definitive test the water temperature was in the 22.0 – 23.9 °C, pH was 7.44 – 7.98 and air saturation was 70 – 84% indicating that environmental conditions were adequately maintained. Behavioural abnormalities and biological effects were reported during the course of the study. No abnormalities were observed in the control group. The 96h highest nominal concentration causing no mortality was 14.3 mg/L. The 96h median LC50 was 23.1 (C.I. 21.1 – 25.4) mg/L based on the nominal concentrations that were analytically confirmed.