1-(5-ethyl-5-methylcyclohex-1-en-1-yl)pent-4-en-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:

24-08-2015 to 15-12-2015

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)

Qualifier:

according to guideline

Guideline:

other: Guidelines for the Testing of Chemicals, Effects on Biotic Systems, Version 2, 203 Fish Acute Toxicity Test, China Environmental Press, China (2013)

Deviations:

no

Principles of method if other than guideline:

The test was generally conducted in accordance with or equivalent to the following: China HJ/T153 - 2004 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: Nominal: 1.80, 2.32, 3.00, 3.87 and 5.00 mg/L (geometric series factor 1.291); with geometric mean measured equivalent concentrations of: 1.33, 1.82, 2.34, 3.10 and 3.97 mg/L, respectively.
- Sampling method: 50 mL extract from test solution.
- Sample storage conditions before analysis: 50mL test solution (after filtered with 0.22 µm filter membrane) was added in a separatory funnel, and 20 mL, 15 mL, 10 mL ethyl acetate were added respectively to extract three times (5min for each extraction). After that, each extraction was collected and transferred respectively into three 50mL volumetric flasks, and then diluted with ethyl acetate to 50mL. The actual concentrations of test substance in ethyl acetate extractions were immediately measured. The yet to be analyzed samples (the ethyl acetate extractions) were stored at 4 °C and had analyzed within one week.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 9.0mg of test item was obtained in a flask with bottle cap and dissolved in a small amount of test water with ultrasonication treatment for 15min. The mixture was transferred into the 5L -jar with bottle cap (ca. 1L headspace on the test solutions), then test water was added to 5000mL to obtain the nominal concentration of 1.80mg/L test suspension. Then the suspension was stirred with a magnetic stirrer for about 48h, after the test item fully dissolved, the nominal concentration of 1.80mg/L test item solution was obtained and used directly in the test. 11.6mg, 15.0mg, 19.4mg and 25.0mg of test item was prepared in the same way as the above method, the nominal concentrations of 2.32mg/L, 3.00mg/L, 3.87mg/L and 5.00mg/L, respectively.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: A negative/blank control without test item or reference item was also included. A previously conducted sensitivity test with potassium dichromate (positive control/reference item) was performed. Full information provided in the full study report.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): In the preliminary range finding test: solutions were observed to be clear colourless without any undissolved test item at up to 5.0 mg/L and/or the definitive test there was no evidence of undissolved test item. The test item in solubility testing was found to reach a steady stated after 24 hours magnetic stirring mixing period pre-exposure.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish (Brachydanio rerio)
- Strain: Not reported. Batch generation number FDr20150731-1
- 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 deviation of test fish total length and body weight were respectively 2.274 - 2.728 cm (meeting the requirement of 2.0±1.0 cm according to guideline) and 0.102 – 0.178 g. In the treated/control groups the loading was 0.263 g/L (i.e. < 1.0g fish/litre).
- Method of breeding: Not reported.
- Feeding during test: No.
- Food type: Feeding was conducted during acclimation (three times week) until 24-hours prior to test initiation.
- Amount: No feeding during exposure. Not applicable.
- Frequency: No feeding during exposure. Not applicable.

ACCLIMATION
- Acclimation period: 59 days (i.e. > 48 hours)
- 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). Oxygen concentration was in the range of the exposure duration (> 80% air saturation).
- Type and amount of food: Not reported.
- Feeding frequency: At three times a week until 24 hours before the start of the test.
- Health during acclimation (any mortality observed): None reported (0% during 7 days pre-test).

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

99.1 mg/L expressed as CaCO3; Test water (dechlorinated water)

Test temperature:

Temperature 21.9 - 24.2 °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 6.0 - 8.5 (actual: pH 7.90) as specified in China Guidelines for the testing of Chemicals (2nd Ed.) 203 test.
The pH was in the range of 7.61 to 8.03 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. Measured range: 81 to 105% ASV during the definitive test.

Nominal and measured concentrations:

- Preliminary range-finding test performed on semi-static test system with renewal every 24 hour intervals:
Semi-static exposure method was performed in the range-finding test with renewal of test solution every 24 hours. The range-finding test showed that there was no mortality observed at the nominal concentration of below 2 mg/L and 20% mortality at 3.16 mg/L and 100% mortality was observed at the nominal concentration of 5.0 mg/L within the test period of 96 hours.
- Definitive test performed on semi-static test system with renewal every 24 hour intervals:
Nominal: 0 (blank control), 1.80, 2.32, 3.00, 3.87 and 5.00 mg/L (geometric series factor 1.291)
with geometric mean measured equivalent concentrations of: 1.33, 1.82, 2.34, 3.10 and 3.97 mg/L, respectively.
During the test, the dissolved test material concentration analytically were determined in the freshly prepared solution/medium (t=0h, 24h, 48h, 72 h) and in the aged medium/solution before renewal (t=24h*, 48h*, 72h*, 96h*).

Details on test conditions:

TEST SYSTEM
- Test vessel: 5L wide-mouth jar with bottle cap
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass. 5 Litre fill volume (minimum headspace)
- Aeration: Not continuously aerated although DOC > 60% ASV during exposure.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): Renewal every 24 hours.
- No. of organisms per vessel: Ten (10) per vessel
- No. of vessels per concentration (replicates): None.
- No. of vessels per control (replicates): One (1)
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: < 1.0 g/L (Actual: 0.263 g/L)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water used in the test were purified by the reverse osmosis equipment. (Certified annually).
- Conductivity: Not reported. Typically, would be: Water: 500±100μs/cm) as specified in China Water quality standard for fisheries in GB-11607-89.
- Culture medium different from test medium: No.
- Intervals of water quality measurement: Every 24 hour intervals; before and after renewal

OTHER TEST CONDITIONS
- Adjustment of pH: None.
- Photoperiod: 14 hours photoperiod daily
- Light intensity: Not reported.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortalities and abnormalities; measured at 3h, 6h, 24h, 48h, 72h and 96h as applicable.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.291
- Justification for using less concentrations than requested by guideline: Not applicable. Five concentrations used.
- Range finding study: Semi-static exposure method was performed in the range-finding test with renewal of test solution every 24 hours. The range-finding test showed that there was no mortality observed at the nominal concentration of below 2 mg/L and 20% mortality at 3.16 mg/L and 100% mortality was observed at the nominal concentration of 5.0 mg/L within the test period of 96 hours.
- Test concentrations: Not reported (no mortality at < 2.0 mg/L was reported); 100% mortality at 5.0 mg/L
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

<= 1.33 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

2.09 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: C.I.1.75 - 2.43 mg/L

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

3.97 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

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 for control: 2.274 - 2.728 cm (meeting the requirement of 2.0±1.0 cm) with mean: 2.541 ± 0.165 cm and 0.132 ± 0.023 g.
- 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: 289 (C.I. 260 - 353) mg/L

Reported statistics and error estimates:

The LC50 values and the 95% confidence limits (LC5) at 24h, 72h and 96h after the start of the test were calculated by Probit Analysis software (US EPA: Probit Analysis Program Version 1.5). The LC50 value and the 95% confidence limits (L95) at 48h was calculated by Trimmed Spearman-Karber software (US EPA: Trimmed Spearman-Karber Program Version 1.5).

Sublethal observations / clinical signs:

Table 1. Median Lethal Concentration (LC50) and 95% confidence limits

Recommended observation times	LC50 (mg/L) based pn geometric mean measured concentrations	95% confidence limits (mg/L)
24 h	3.07	2.76 - 3.43
48 h	2.69	2.50 - 2.89
72 h	2.25	1.91 - 2.62
96 h	2.09	1.75 - 2.43

#1: LC50 based on geometric mean measured concentrations

 

Table 2. Cumulative mortality

Cumulative mortality
Group #1	Exposure duration (hours)
	3	6	24	48	72	96
Control	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%
1.33 mg/L	0.0%	0.0%	0.0%	0.0%	10.0%	10.0%
1.82 mg/L	0.0%	0.0%	0.0%	10.0%	30.0%	40.0%
2.34 mg/L	0.0%	0.0%	0.0%	0.0%	30.0%	50.0%
3.10 mg/L	0.0%	0.0%	50.0%	90.0%	90.0%	90.0%
3.97 mg/L	0.0%	0.0%	100.0%	100.0%	100.0%	100.0%

#1: geometric mean measured concentrations.

 

Table 3. Observed abnormal responses

	Observations
Concentration (geometric mean measured)	3h		24h		48h		72h		96h
Control	0/10	N	0/10	N	0/10	N	0/10	N	0/10	N
1.33 mg/L	0/10	N	10/10	RA(2) OLE(2) AB(6)	10/10	IMM(3) AB(7)	9/9	AB(4) IMM(5)	9/9	AB(7) IMM(1) OLE(1)
1.82 mg/L	0/10	N	10/10	AS(2) AB(3 IMM(2) OLE(3)	9/9	IMM(3) OLE(3) AB(3)	7/7	AS(3) IMM(2) OLE(2)	6/6	AB(1) OLE(2) AS(3)
2.34 mg/L	0/10	N	10/10	AB(3) OLE(5) IMM(2)	10/10	OLE(10)	7/7	OLE(3) IMM(4)	5/5	OLE(5)
3.10 mg/L	0/10	N	5/5	AS(1) SS(4)	1/1	AB(1)	1/1	AB(1)	1/1	AB(1)
3.97 mg/L	10/10	RA (10)	1/1	OLE(1)	-	-	-	-	-	-

Note: "N": Normal. "-": All test fish died. "RA": Reduction of activity, "IMM": Immobilized at the bottom. 'LB'': Lying on the bottom;

"AB": swimming at the bottom; "AS": swimming at the surface; "SS": spasmodic swimming; "OLE": overturn and loss of equilibrium.

Validity criteria fulfilled:

yes

Conclusions:

The 96 hour LC50 for the test item to Danio rerio was determined to be 2.09 mg/L with a 95% confidence interval from 1.75 - 2.43 mg/L based on geometric mean measured concentrations.

Executive summary:

The acute toxicity of the test item to Zebra fish (Danio rerio) was determined in a 96 hour semi-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), 1.80, 2.32, 3.00, 3.87 and 5.00 mg/L (geometric series with a factor of 1.291) were selected for exposure to fish groups of 10 individuals. The test item was renewed at 24 hour intervals within a 96 hour semi-static test system in response to ensuring adequate stability of exposure to test item. 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 every 24 hours, pre- and post- medium renewal and the end of the exposure by GC-FID analysis. The measured concentrations ranged from 65% to 83% of analytically confirmed nominal concentrations before renewal. On this basis effect levels were indicated based on geometric mean measured concentrations. The corresponding geometric mean measured concentrations were: 0 (control), 1.33, 1.82, 2.34, 3.10 and 3.97 mg/L, respectively. During the definitive test the water temperature was in the 21.9 - 24.2°C, pH was 7.61 to 8.03 and air saturation was 81 to 105% ASV 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. All validity criteria were considered to be met. The 96h median LC50 was 2.09 (C.I. 1.75 – 2.43) mg/L based on geometric mean measured concentrations.

Description of key information

LC50 (fish) =  2.09 (C.I. 1.75 - 2.43) mg/L based on geometric mean measured concentrations, 96-hour, freshwater, OECD TG 203, 2015

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2.09 mg/L

Additional information

Key Study : OECD TG 203, 2015 : The acute toxicity of the test item to Zebra fish (Danio rerio) was determined in a 96 hour semi-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), 1.80, 2.32, 3.00, 3.87 and 5.00 mg/L (geometric series with a factor of 1.291) were selected for exposure to fish groups of 10 individuals. The test item was renewed at 24 hour intervals within a 96 hour semi-static test system in response to ensuring adequate stability of exposure to test item. 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 every 24 hours, pre- and post- medium renewal and the end of the exposure by GC-FID analysis. The measured concentrations ranged from 65% to 83% of analytically confirmed nominal concentrations before renewal. On this basis effect levels were indicated based on geometric mean measured concentrations. The corresponding geometric mean measured concentrations were: 0 (control), 1.33, 1.82, 2.34, 3.10 and 3.97 mg/L, respectively. During the definitive test the water temperature was in the 21.9 - 24.2°C, pH was 7.61 to 8.03 and air saturation was 81 to 105% ASV 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. All validity criteria were considered to be met. The 96h median LC50 was 2.09 (C.I. 1.75 – 2.43) mg/L based on geometric mean measured concentrations.