Reaction mass of (3E)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-1-en-1-yl)butan-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)butan-2-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:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP; minor deviation not considered to impacted the reliability of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

The temperature range during exposure was > 2°C; actual 2.5°C. This by expert judgement is not considered to have significantly affected the validity of the study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see above

Qualifier:

according to guideline

Guideline:

other: China National Technical Committee for Standardization of Dangerous Chemicals Management: GB/T 27861-2011, Chemicals Fish Acute Toxicity Test. (2012)

Deviations:

yes

Remarks:

see above

Qualifier:

according to guideline

Guideline:

other: State Environmental Protection Administration of China: HJ/T 153-2004, The guidelines for the testing of chemicals. China (2004)

Version / remarks:

The test was conducted in accordance with 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).

Deviations:

yes

Remarks:

see above

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23 (2000)

Deviations:

not applicable

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 6.80 mg/L, 10.6 mg/L, 16.4 mg/L, 25.6 mg/L and 40.0 mg/L prepared as WAFs
- Sampling method: 50 ml extract from test solution.
- Sample storage conditions before analysis: Actual concentrations of test substance in hexyl hydride were immediately measured. Otherwise the samples (the hexyl hydride extractions) would be stored at 4°C until being analysed within one week. Upon which constituent 1 was measured as tracer.

Vehicle:

no

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 25.10 mm±0.87 mm and 0.126 g±0.008 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: 48 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:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

118 mg/L expressed as CaCO3

Test temperature:

Temperature 21.3 - 23.8 °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 7.55 to 8.06 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 Jess than 60 % of the air saturation value.

Dissolved oxygen (control): 8.7 mg/L. Dissolved oxygen (start; all test item concentrations): 8.3 - 8.7 mg/L. Dissolved oxygen (end 96-hours; all test item concentrations): 9.3 - 9.6 mg/L.

Nominal and measured concentrations:

Preliminary range-finding test performed on static test system:
Nominal loading rates of 0 (blank control), 25.0 mg/L, 50.0 mg/L, and 100.0 mg/L prepared as WAFs
Definitive test performed on semi-static test system with renwal at 48 hours intervals:
Nominal loading rates of 0 (blank control), 6.80 mg/L, 10.6 mg/L, 16.4 mg/L, 25.6 mg/L and 40.0 mg/L prepared as WAFs

Otherwise indicated as: 2.08 mg/L, 3.24 mg/L, 5.03 mg/L, 7.85 mg/L and 12.2 mg/L calculated based on constituent 1 tracer; which in turn was measured (after and before the renewal) by Gas Chromatography-Mass Spectrometer (GC-MS) whereas the blank control was measured at 0h (new solution) and 48h (old solution). Measured concentrations are provided in 'Any other information on materials and methods incl. tables' field.

Details on test conditions:

TEST SYSTEM
- Test vessel: 5L vessel. Closed with plug.
- Material, size, headspace, fill volume: 4L test medium for each test concentration
- Aeration: None.
- Renewal rate of test solution (frequency/flow rate): Semi-Static; renewal 48 hour intervals.
- No. of organisms per vessel: 10 / test group (60 total).
- No. of vessels per concentration (replicates): no replicates; 1 exposure per test concentration

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Local tap water (dechlorinated tap water)
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH: None.
- Photoperiod: 14 hours light / 10 hours dark.
- Light intensity: Start: 4 lx, 76 lx and 92 lx (84 lx on average) / At the end of the test, the intensities of illumination of test environment were 92 lx, 84 lx and
88 lx (88 lx on average).

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

TEST CONCENTRATIONS
- Test concentrations: test concentrations were based on the results of a range-finding tests.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

96 h

Dose descriptor:

LL0

Effect conc.:

16.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAF

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

30.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAF

Basis for effect:

mortality (fish)

Remarks on result:

other: 28.1-33.3 mg/L

Duration:

96 h

Dose descriptor:

LL100

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAF

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.
- 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.

Sublethal observations / clinical signs:

Table 1. Median Lethal Loading Rate (LL50) and 95% confidence limits

Exposure Duration	LL50 (mg/L) #1	C.I. 95% (mg/L) #1
24 h	32.0	Not available
48 h	32.0	Not available
72 h	30.6	28.1 – 33.3
96h	30.6	28.1 – 33.3

Note #1: means the median lethal loading rate was calculated based on the nominal loading rate

 

Table 2. Cumulative mortality

Nominal loading rate (mg/L)	Nominal loading rate constituent 1 (mg/L)	Number of organisms	Cumulative mortality (%)
			3h	24h	48h	72h	96h
Blank control	0	10	0	0	0	0	0
6.89	2.08	10	0	0	0	0	0
10.60	3.24	10	0	0	0	0	0
16.40	5.03	10	0	0	0	0	0
25.60	7.85	10	0	0	0	10	10
40.0	12.2	10	60	100	100	100	100

 

Table 3. Observed abnormal responses

Nominal loading rate (mg/L)	Result of observations Number affected / Total surviving ; Abnormal response (number)
		3h		24h		48h		72h		96h
Blank Control	0/10	N	0/10	N	0/10	N	0/10	N	0/10	N
6.80	3/10	RA (3)	3/10	RA (3)	4/10	RA (4)	4/10	RA (4)	3/10	RA (3)
10.60	6/10	RA (6)	7/10	RA (5) IMM(2)	10/10	RA (9) AB (1)	10/10	RA (7) IMM (3)	6/10	RA (6) IMM (2)
16.40	8/10	AB (8)	10/10	RA (3) (IMM7)	10/10	RA (4) AB (6)	10/10	RA (4) LB (6)	8/10	RA (3) IMM (7)
25.60	10/10	LB (3)	10/10	LB (8) OLE(2)	10/10	LB (10)	9/9	LB (9)	9/9	LB (9)
40.00	4/4	LB (4)	4/4	-	-	-	-	-	-	-

 

Table 4. Abnormal responses descriptions of abbreviations

Abbreviation	Description
N	Normal
RA	Reduction in activity
IMM	Immobilised at the bottom
LB	Lying on the bottom
AB	Swimming at the bottom
OLE	Overturn and loss of equilibrium

 

Table 5. Measured concentration of constituent 1 (tracer) in test solutions

Nominal loading rate (mg/L)	Nominal loading rate constituent 1 (mg/L)	Measured concentration of the 1^stconstituent of test substance (mg/L) (Percent of measured concentrations versus the initial concentrations %)				Measured concentration in geometric mean
		0h	48h		96h
		New	Old	New	Old	-
Blank control	0	ND	ND	-	-	-
6.89	2.08	1.40	1.32 (94)	1.68	1.48 (88)	1.46
10.60	3.24	2.02	2.11 (104)	2.34	2.52 (108)	2.24
16.40	5.03	2.79	2.94 (105)	3.44	4.04 (117)	3.27
25.60	7.85	4.22	4.94 (117)	5.24	5.32 (102)	4.91
40.0	12.2	5.68	5.84 (103)	-	-	5.76

Where:

ND = lower than the LOD (LOD = 0.00411 mg/L)

 -= test solution not measured.

Validity criteria fulfilled:

yes

Conclusions:

The 96 hour LL50 for the test substance to Danio rerio was determined to be 30.6 mg/L with a 95% confidence interval from 28.1 - 33.3 mg/L based on nominal loading rate as WAF.

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 CRC MEP guidelines, effects on Biotic Systems, 203 Fish Acute toxicity test (2013) under GLP. Based on the results of range finding tests, the nominal loading rates of 6.80, 10.6, 16.4, 25.6 and 40.0 mg/L WAFs with a geometric series factor of 1.56 were selected for exposure to 10 fish groups (60 total). One control containing no test substance was also performed. The test item was renewed at 48 hour intervals within a 96hour semi-static test system in response to ensuring adequate stability of exposure to test item. The actual concentrations were verified by measurement of constituent 1 used as a tracer in the nominal loading rates of 6.80, 10.6mg/, 16.4mg/, 25.6 and 40.0 mg/L WAFs at 0h, 96h, and 48h of new and old solutions (after and before the renewal) were measured by Gas Chromatography-Mass Spectrometer (GC-MS) the blank control was measured at 0h (new solution) and 48h (old solution) respectively. These measured concentrations ranged from 1.32 to 5.84mg/L (or 88% to 117% of the initial concentration constituent 1). During the renewal period of 48h, the measured concentrations of the tracer constituent in the old solutions could be maintained ±20% of the initial concentrations. Behavioural abnormalities and biological effects were reported during the course of the study. The 96h highest loading rate causing no mortality was 16.4 mg/L and the 96h median LL50 was 30.6 (C.I. 28.1 – 33.3) mg/L based on the nominal loading rate WAF.

Description of key information

LL50 (fish) = 30.6 (C.I. 28.1-33.3) mg/L based on WAF loading rates, 96hour, freshwater, OECD TG 203, 2015

LL0 (fish)= 16.4 mg/L based on loading WAF loading rates, 96hour, freshwater, OECD TG 203, 2015

Additional information

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 CRC MEP guidelines, effects on Biotic Systems, 203 Fish Acute toxicity test (2013) under GLP. Based on the results of range finding tests, the nominal loading rates of 6.80, 10.6, 16.4, 25.6 and 40.0 mg/L WAFs with a geometric series factor of 1.56 were selected for exposure to 10 fish groups (60 total). One control containing no test substance was also performed. The test item was renewed at 48 hour intervals within a 96hour semi-static test system in response to ensuring adequate stability of exposure to test item. The actual concentrations were verified by measurement of constituent 1 used as a tracer. These measured concentrations ranged from 1.32 to 5.84mg/L (or 88% to 117% of the initial concentration constituent 1). During the renewal period of 48h, the measured concentrations of the tracer constituent in the old solutions could be maintained ±20% of the initial concentrations. Behavioural abnormalities and biological effects were reported during the course of the study. The 96h highest loading rate causing no mortality was 16.4 mg/L and the 96h median LL50 was 30.6 (C.I. 28.1 – 33.3) mg/L based on the nominal loading rate WAF.