(1'R,2R)-2-Methyl-4-(2',2',3'-tri-nal and methylcyclopent-3'-en-1'-yl)-4-pent-enal

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:

16-08-2000 to 29-09-2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

No positive control conducted as part of the study.

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

yes

Remarks:

No positive control conducted as part of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: February 2000 ; signature: April 2000

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 0.600, 1.23, 2.19, 4.11, 7.70 mg/L analytically confirmed initial concentration
Time Weighted Mean Measured equivalent concentrations: 0 (control), of 0.255, 0.430, 0.747, 1.34 and 2.41 mg/L (measured ; based on measured concentrations in the definitive test taken at 24 hour intervals in old/fresh media respectively pre-/post- renewal)
- Sampling method: Water samples were taken from the control and all surviving test groups at 0 (fresh media); 24, 48, 72 (old and fresh media) and 96 (old media) hours for quantitative analysis. Where possible all samples were prepared for analysis on the day of sampling. Duplicate samples were taken and stored frozen (approximately -20°C) for further analysis if necessary.
- Sample storage conditions before analysis: See above.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct dissolution of test item in culture medium to prepare stock; serial dilution of stock to prepare test media. The test concentrations used in the definitive study were prepared by adding an amount of test item to the vortex created in a volume of water to give an initial dispersion at a concentration of 1000 mg/L. Stirring periods of 23 hours and 95 hours were employed, both with 1 hour standing periods. Vortex depths of 5% and 25% were used for both stirring periods and samples taken by mid-depth siphon for chemical analysis. This work showed that increasing the stirring period and/or the vortex depth did not significantly increase the measured test concentration of the saturated solution. Therefore, a stirring period of 23 hours with a 5% vortex depth was used for all further stirring work. To ensure that saturated solutions prepared by this method contained only dissolved test item, samples were removed by mid-depth siphon and analysed with and without filtration (0.2 micron
filters). A sample prepared as above was also subject to centrifugation at approximately 17000 G for periods of 10, 20 and 30 minutes prior to chemical analysis. Based on the results of the range-finding study the test item solutions for the definitive study were prepared by stirring an excess of test item (equivalent to 1000 mg/L) in dechlorinated tap water for a period of time and then removing the aqueous phase by siphon to produce a saturated solution with a measured concentration of 7. 7 mg/L. This saturated solution was then further diluted, as necessary, to produce the remaining measured test concentrations of 0.600, 1.23, 2.19 and 4.11 mg/L.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No precipitate reported.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout (Oncorhynchus mykiss)
- Strain: Not reported.
- Source: Recognised supplier (listed in full study report).
- Age at study initiation (mean and range, SD): Not reported.
- Length at study initiation (length definition, mean, range and SD): 4.3 cm (SD = 0.2)
- Weight at study initiation (mean and range, SD): 1.04 g (SD = 0.16) i.e. ca. 0.52 g bw/L loading rate
- Method of breeding: Not reported.
- Maintenance of the brood fish: Not reported.

ACCLIMATION
- Acclimation period: 12 days.
- Acclimation conditions (same as test or not): Yes.
- Type and amount of food during acclimation: certified pelleted diet
- Feeding frequency during acclimation: daily (until 24 hours prior to test).
- Health during acclimation (any mortality observed): < 1% mortality in 7 days pre-test

QUARANTINE (wild caught)
- Duration: Not applicable.
- Health/mortality: Not applicable.

FEEDING DURING TEST
- Food type: Not applicable.
- Amount: Not applicable.
- Frequency: Not applicable.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

According to OECD TG 203 guidelines.

Post exposure observation period:

none.

Hardness:

ca. 100 mg/L (expressed as CaCO3)

Test temperature:

14 ºC to 14.5 ºC

pH:

7.7 fresh media); 7.8-8.0 (expired media)

Dissolved oxygen:

8.8-8.9 mgO2/L (fresh media); 8.2-8.4 mgO2/L (expired media)

Salinity:

not applicable.

Nominal and measured concentrations:

Concentrations: 0 (control), 0.600, 1.23, 2.19, 4.11, 7.70 mg/L analytically confirmed initial concentration
Time Weighted Mean Measured equivalent concentrations: 0 (control), of 0.255, 0.430, 0.747, 1.34 and 2.41 mg/L (measured ; based on measured concentrations in the definitive test taken at 24 hour intervals in old/fresh media respectively pre-/post- renewal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 20 L for test and control vessels, glass fibre
- Type (delete if not applicable): open, covered to reduce evaporation
- Material, size, headspace, fill volume: 20L fill volume test media per test vessel
- Aeration: Yes.
- Renewal rate of test solution (frequency): 24 h
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: 0.52 g/L (mean)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Drinking water; chlorine removed by activated carbon filtration and partial softening (DI).
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: No.
- Intervals of water quality measurement: 24 hours.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16 h light / 8 h darkness
- Light intensity: Not reported.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and visible abnormality recorded at 3 h, 24 h, 48 h, 72 h and 96 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable. Concentrations justified from the results of the range finding study.
- Range finding study: yes (semi-static) ; 3 organisms per concentration.
- Test concentrations: blank control, 0.12, 1.2 and 12 mg/L nominal
- Results used to determine the conditions for the definitive study: Yes. No mortalities at 0 (control), 0.12 or 1.2 mg/L. Complete mortality at 24 hours in 12 mg/L.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95% CL: 1.4 - 1.9 mg/L) ; time weighted mean measured concentrations

Details on results:

- Behavioural abnormalities: See table 1 and 2
- Observations on body length and weight: Not reported
- Other biological observations: Not reported
- Mortality of control: 0%
- Other adverse effects control: No abnormalities observed in the control.
- Abnormal responses: increased pigmentation, loss of equilibrium, loss of equilibrium with increased pigmentation, swimming at the bottom, Moribund
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The reason for the decline in test item concentrations exposure period is speculated to be due to adsorption and/or biological factors.
- Effect concentrations exceeding solubility of substance in test medium: Not applicable

Sublethal observations / clinical signs:

Table 1.0 - Cumulative mortality

Time Weighted Mean Measured Concentration (mg/L)	Cumulative mortality (n=10)						Total mortality %
	3h	6h	24h	48h	72h	96h
0.00	0	0	0	0	0	0	0
0.255	0	0	0	0	0	0	0
0.430	0	0	0	0	0	0	0
0.747	0	0	0	1	1	1	10
1.34	0	0	7*	10	10	10	100
2.41	10	10	10	10	10	10	100

 * the remaining 3 fish were observed as moribund and humanely terminated. Included in 24h LC50 determination as mortality.

Table 2.0 – Sub-lethal effects/responses

Time Weighted Mean Measured Concentration (mg/L)	Sub-lethal effects	Time
		3h	6h	24h	48h	72h	96h
0.00	No abnormalities
0.255	No abnormalities
0.430	No abnormalities
0.747	Increased pigmentation Loss of equilibrium Loss of equilibrium with increased pigmentation Swimming at bottom with increased pigmentation Moribund	4/10 5/10 1/10	8/10 8/10 1/10	9/10 1/10	3/9 6/9	4/9 4/9	4/9 4/9 1/9
1.34	Loss of equilibrium Loss of equilibrium with increased pigmentation Moribund	4/10 6/10	1/10 9/10	3/3**	CM
2.41	loss of equilibrium and gasping, moribund	2/4 2/4**	CM

Where:

** = humanely euthanized and indicated as mortalities

CM = complete mortality

Validity criteria fulfilled:

yes

Conclusions:

The 96 hour LC50 for the test item to Rainbow Trout (Oncorhynchus mykiss) was determined to be 1.6 (C.I. 1.4 - 1.9) mg/L based on time weighted mean measured concentrations.

Executive summary:

The acute toxicity of the test item to Rainbow Trout (Oncorhynchus mykiss) was determined in a 96 hour semi-static test according to OECD TG 203 and EU Method C.1 guidelines under GLP. Following a preliminary range-finding test, fish were exposed, in groups often, to an aqueous solution of the test item at time-weighted mean measured test concentrations of 0.255, 0.430, 0.747, 1.34 and 2.41 mg/L for a period of 96 hours under semi-static test conditions. Based on the results of the range-finding study the test item solutions for the definitive study were prepared by stirring an excess of test item (equivalent to 1000 mg/L) in dechlorinated tap water for a period of time and then removing the aqueous phase by siphon to produce a saturated solution with a measured concentration of 7. 7 mg/L. This saturated solution was then further diluted, as necessary, to produce the remaining measured test concentrations of 0.600, 1.23, 2.19 and 4.11 mg/L.Chemical analysis of the freshly prepared test media throughout the study showed measured concentrations ranging from 0.646 to 8.24 mg/L for the uncentrifuged samples and from 0.522 to 7.70 mg/L for the centrifuged samples. A marked decline was shown in the measured concentrations of the old or expired test media with values ranging from less than the limit of quantitation (LOQ) to 4.53 mg/L for uncentrifuged samples and less than the LOQ to 4.04 mg/L for the centrifuged samples. The reason for the decline in test item concentrations exposure period is speculated to be due to adsorption and/or biological factors. On this basis the test item was not maintained at ±20% of the initial concentrations therefore the effect levels were estimated based on time weighted mean measured concentrations. Under the conditions of this study, the 96h LC50 was 1.6 (C.I. 1.4 – 1.9) mg/L based on time weighted mean measured concentrations.

Description of key information

LC50 (fish) = 1.6 (C.I. 1.4 – 1.9) mg/L based on time weighted mean measured concentrations, 96hour, freshwater, OECD TG 203, 2000

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.6 mg/L

Additional information

Key study : OECD TG 201, 2000 : The acute toxicity of the test item to Rainbow Trout (Oncorhynchus mykiss) was determined in a 96 hour semi-static test according to OECD TG 203 and EU Method C.1 guidelines under GLP. Following a preliminary range-finding test, fish were exposed, in groups often, to an aqueous solution of the test item at time-weighted mean measured test concentrations of 0.255, 0.430, 0.747, 1.34 and 2.41 mg/L for a period of 96 hours under semi-static test conditions. Based on the results of the range-finding study the test item solutions for the definitive study were prepared by stirring an excess of test item (equivalent to 1000 mg/L) in dechlorinated tap water for a period of time and then removing the aqueous phase by siphon to produce a saturated solution with a measured concentration of 7. 7 mg/L. This saturated solution was then further diluted, as necessary, to produce the remaining measured test concentrations of 0.600, 1.23, 2.19 and 4.11 mg/L.Chemical analysis of the freshly prepared test media throughout the study showed measured concentrations ranging from 0.646 to 8.24 mg/L for the uncentrifuged samples and from 0.522 to 7.70 mg/L for the centrifuged samples. A marked decline was shown in the measured concentrations of the old or expired test media with values ranging from less than the limit of quantitation (LOQ) to 4.53 mg/L for uncentrifuged samples and less than the LOQ to 4.04 mg/L for the centrifuged samples. The reason for the decline in test item concentrations exposure period is speculated to be due to adsorption and/or biological factors. On this basis the test item was not maintained at ±20% of the initial concentrations therefore the effect levels were estimated based on time weighted mean measured concentrations. Under the conditions of this study, the 96h LC50 was 1.6 (C.I. 1.4 – 1.9) mg/L based on time weighted mean measured concentrations.