(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

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:

2017-09-24 to 2017-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

- Sampling method: 10 mL of the test item concentration and the control were filled into a vial.
- Sample storage conditions before analysis: All samples were stored at 6 +- 2 °C until sample preparation and at room temperature until the start of the analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal loading of 5.00 mg/L test item which corresponds to 5.97 µL/L of the test item was prepared with dilution water one day prior to the start of exposure in two glass flasks with screw caps (each 5000 mL). These dispersions were stirred for 24 hours with 1100 rpm at room temperature. After a separation phase of 1 hour, the saturated solution was removed by siphoning (from the approximate bottom of the flasks) and used in the test. The solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative.
- Controls: Blank control with dilution water
- Evidence of undissolved material: No (Tyndall effect was negative, see above)

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Gained at Noack Laboratorien GmbH from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin)
- Length at study initiation: 2.59 cm
- Weight at study initiation: 0.11 g

ACCLIMATION
- Acclimation period: 12 days
- Type and amount of food during acclimation: Sera Vipan, Sera GmbH, 52518 Heinsberg, Germany; 4% of the fish body weight per feeding day
- Feeding frequency during acclimation: 3 times per week; not fed 24 h before test start
- Health during acclimation: No mortality observed

FEEDING DURING TEST: No

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

64 mg/L as CaCO3 (day 0, limit concentration and control)

Test temperature:

22.3 - 23.1 °C (mean: 22.7 °C)

pH:

7.19 - 7.50

Dissolved oxygen:

96 - 100 % saturation (new medium), 83 - 90 % saturation (old medium)

Conductivity:

138 µS/cm

Nominal and measured concentrations:

Nominal loading rate: 5 mg/L
Measured (geometric mean): 590 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquaria
- Material, size, headspace, fill volume: Glass, 13 L nominal volume, filled with approximately 8 L of dilution water (dimensions 25.5/18/28 cm, depth of water: 20 cm), covered with a floating lid
- Aeration: No aeration was provided
- Renewal rate of test solution: Daily
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Biomass loading rate: 0.0963 g fish per liter test solution
- Introduction of fish: Randomly

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water; filtered on activated charcoal and aerated for at least 24 hours to remove chlorine. Nominal water parameters:
Total hardness: 64 mg CaCO3/L
pH-value: 6.0 - 8.5
Acidity: 0.1 mmol/L
Alcalinity: 0.6 mmol/L
Conductivity: 138 µS/cm

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light, 8 hours darkness
- Light intensity: 7 - 750 Lux

EFFECT PARAMETERS MEASURED : Mortality (animals were considered dead if there was no visible movement and if touching of the caudal peduncle produced no reaction); sublethal effects, observations were made after 3, 24, 48, 72 and 96 hours

TEST CONCENTRATIONS
- Range finding study: Not performed.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 0.59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

0.59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Mortality of control: No
- Other adverse effects control: No
- Abnormal responses: No
- Any observations that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

VALIDITY OF THE TEST
- O2 saturation => 83%
- No mortality in control
- Deviation of pH value in control group over test duration: 0.31 units
- Measured concentrations of the test item in the range of 67 - 81% of the initially measured concetrations at the end of the test

The test is considered to be valid.

Results with reference substance (positive control):

Positive control was not performed

Reported statistics and error estimates:

LC50 values do not have to be calculated in a limit test.

Sublethal observations / clinical signs:

Table 1: Measured concentrations and percent of the initial concentrations of the test item

Sampling date	0 hours (Fresh medium)	24 hours (Old medium)		24 (hours (Fresh medium)	48 hours (Old medium)		48 hours (Fresh medium)
Nominal loading of the test item [mg/L]	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]
5	718	478	67	591	477	81	689
Control	< LOQ	< LOQ		< LOQ	< LOQ		< LOQ

Sampling date	72 hours (Old medium)		72 (hours (Fresh medium)	96 hours (Old medium)		Geometric mean measured concentration [µg/L]
Nominal loading of the test item [mg/L]	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%
5	484	70	779	584	75	590
Control	< LOQ		< LOQ	< LOQ		< LOQ

Table 2: Observations of sub lethal effects and normal behavior in the test vessels

Geometric mean measured concentration of the test item [µg/L]	Effect	Number of fish effected at abservation time [hours]
		3	24	48	72	96
590	(1)	0	0	0	0	0
Control	(1)	0	0	0	0	0

(1) = Normal behaviour

Table 3: Cumulative mortality [%] in the test vessels

Geometric mean measured concentration of the test item [µg/L]	Cumulative mortality [%] after different exposure periods [hours]
	3	24	48	72	96
590	0	0	0	0	0
Control	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

The 96h LC50 of the test item was determined to be > 0.59 mg/L (based on geometric mean measured concentration).

Executive summary:

To assess the toxicity to fish, a GLP-compliant acute toxicity test according to OECD Guideline 203 and EU Method C.1 was conducted under semi-static conditions with daily renewal of the test media. Fish (Danio rerio) were exposed to the limit loading of 5.00 mg/L (corresponding to a geometric mean measured concentration of 590 µg test item/L) over a test duration of 96 hours. Seven test organisms were exposed to the threshold loading and the control along with a negative control. The concentrations of the test item were analytically verified via GC-MS at the start (0, 24, 48, 72 h) and at the end (24, 48, 72, 96 h) of the exosure intervals in the tested concentration level and the control. The measured concentrations of the test item at the end of each exposure interval were in the range of 67 to 81% of the initially measured concentrations. All effect values given are based on the geometric mean measured test item concentration of the test item. In result, the 96 h LC50 of the test item was determined to be > 0.59 mg/L. All validity criteria of the test guidelines were met.

Description of key information

The 96h LC50 of the substance was determined to be > 0.59 mg/L (based on geometric mean measured concentrations).

Additional information

To assess the toxicity to fish, a GLP-compliant acute toxicity test according to OECD Guideline 203 and EU Method C.1 was conducted under semi-static conditions with daily renewal of the test media. Fish (Danio rerio) were exposed to the limit loading of 5.00 mg/L (corresponding to a geometric mean measured concentration of 590 µg test item/L) over a test duration of 96 hours. Seven test organisms were exposed to the threshold loading and the negative control (without the test item). The concentrations of the test item were analytically verified via GC-MS at the start (0, 24, 48, 72 h) and at the end (24, 48, 72, 96 h) of the exosure intervals in the tested concentration level and the control. The measured concentrations of the test item at the end of each exposure interval were in the range of 67 to 81% of the initially measured concentrations. All effect values given are based on the geometric mean measured test item concentration of the test item. In result, the 96-h LC50 of the test item was determined to be > 0.59 mg/L. All validity criteria of the test guidelines were met.