2-(3,3-dimethylcyclohex-1-enyl)-2,5,5-trimethyl-1,3-dioxane

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:

From November 23, 2015 to December 04, 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study was performed according to OECD Guideline 203 with GLP certificate. All validity criteria were fulfilled. However, this study is considered reliable with restrictions for the following reasons: - The treatment solutions were prepared with a solvent (acetone), which is not an appropriate method to conduct toxicity tests for substances with a solubility at the level of this substance (15.4 mg/L) but the concentration of solvent used was considered acceptable (0.125 mL/L). - A further drawback of this study was that it was performed under open conditions and loss of substance by volatilisation were observed.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

July 1992

Deviations:

yes

Remarks:

Range-finding test: the test was performed using 4 test item treatments instead of 5; test temperature during the definitive test was less than 21°C, with a difference over the period of 2°C.

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspection dates: 12 and 13 June 2014 / GLP certificate signed on 5 March 2015

Specific details on test material used for the study:

- Physical state: Colourless translucent liquid
- Storage condition: Room temperature protected from direct sun light

Analytical monitoring:

yes

Details on sampling:

The test item concentrations were measured before and after each renewal/adjustment

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The treatment solutions were prepared in acetone. The different treatment solutions are shown in Table 1 in "Any other information on materials and methods incl. tables", as they were prepared (nominal concentrations). The specimens were transferred daily into newly prepared test solutions. The volumes were further adjusted in the course of the day without changing the water volumes.
- Controls: water control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.125 mL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Oily material was observed at the surface of the aqueous volume, for the two highest test item treatments

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Strain: Brachidanio rerio
- Origin: The batch of fish was received on November 10, 2015 and previously validated.
- Length at study initiation (length definition, mean, range and SD): Mean lenth was measured out from 15% of the total population, taken at random: 2.6 +/- 0.2 cm
- Maintenance of the brood fish: Fish were maintained at 23 +/- 2 °C, with a 16h/8h light/dark cycle, in the same reconstituted water as used for testing.
- The fish were kept unfed during the exposure period.

Test type:

semi-static

Water media type:

other: ISO test water (according to ISO 6341) was freshly reconstituted by dilution of mineral stock solutions in pure water.

Limit test:

no

Total exposure duration:

96 h

Test temperature:

The temperature of the test media were within 20 and 22°C and did not deviate by more than 1.5°C in any one case throughout each successive period of when renewing the test medium except for the first day of testing (deviation of 2°C), because the temperature conditions were exceptionally approx. 0.5°C higher than what was observed later in the time course of the test.

pH:

The pH values remained within 7.3 and 7.9 at the beginning of each media renewal and decreased down to 7.2-7.4 at the end of each successive period.

Dissolved oxygen:

More than 80% of the saturation level for every test item treatment and for the controls in the new media and the old media.

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

Nominal test concentrations: 4.5, 6.3, 9.0, 12.6 and 18.0 mg/L
Geometric mean measured concentrations: 2.3, 3.0, 4.1, 5.4 and 5.3 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 8.0 L capacity glass containers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: filled with 4L of reconstituted water
- No. of organisms per vessel: 7 fish per aquarium
- No. of vessels per concentration (replicates): one replicate unit for each the five test treatments with 7 fish per aquarium
- No. of vessels per control (replicates): one replicate unit for the controls with 7 fish per aquarium
- No. of vessels per vehicle control (replicates): one replicate unit for the controls with 7 fish per aquarium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO test water(according to ISO 6341) was freshly reconstituted by dilution of mineral stock solutions in pure water.

OTHER TEST CONDITIONS
- Photoperiod: 16h light per day
- Light intensity: <500 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The test units were inspected for mortality 24h, 48h, 72h and 96h after initiation. Any abnormal behavior or appearance, if any, was reported.

TEST CONCENTRATIONS
- Range finding study : yes
- Test concentrations: 0.01 - 0.1 - 1.0 and 10.0 mg/L
- Results used to determine the conditions for the definitive study: The viability was not affected for the three lowest test item treatments, but at 10 mg/L one specimen died after 24 hours of exposure. The behaviour was not affected for the three lowest test item treatments, but at 10 mg/L the movements were considered as reduced.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 5.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

Highest tested concentration

Basis for effect:

mortality (fish)

Remarks on result:

not determinable

Duration:

96 h

Dose descriptor:

other: Highest tested concentration without observed effect

Effect conc.:

5.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

Highest tested concentration

Basis for effect:

mortality (fish)

Remarks on result:

not determinable

Details on results:

The 5.4 mg/L test item treatment was the highest test concentration based on geometric mean, even though it did not correspond to the highest nominal concentrations.
No regression curve was calculated because there was no signficant effect over the test range, and the 96h-LC50 was reported as > 5.4 mg/L which was the highest test substance concentration in the present sutdy (geometric mean of the measured concentrations).
No mortality was observed in the water control and the solvent control.

Results with reference substance (positive control):

Sensitivity to Potassium dichromate was assessed (CAS No. 7778-50-9, AnalaR, Prolabo Ref. 26784.231, batch No 07J050002). The measured values for 96-LC50 were within the expected interval of 50-250 mg/L.

Sublethal observations / clinical signs:

Table 6.1.1/2: Definitive test - % recovery of the test item treatments throughout the test period

	Nominal treatment concentrations, mg a.i./L
	4.5	6.3	9.0	12.6	18.0
Initial (T0)	3.81 mg/L 84.6%	5.63 mg/L 89.3%	7.53 mg/L 83.7%	8.43 mg/L 66.9%	11.46 mg/L 63.7%
T0+5h – Old medium	2.51 mg/L 55.7%	3.02 mg/L 48.0%	3.48 mg/L 38.7%	3.36 mg/L 26.6%	3.61 mg/L 20.1%
T0+5h – New medium	3.39 mg/L 75.4%	4.52 mg/L 71.7%	6.49 mg/L 72.1%	8.48 mg/L 67.3%	13.27 mg/L 73.7%
T0+20h – Old medium	1.46 mg/L 32.5%	1.89 mg/L 30.0%	2.71 mg/L 30.1%	2.80 mg/L 22.2%	2.37 mg/L 13.2%
T0+20h – New medium	3.61 mg/L 80.1%	5.18 mg/L 82.2%	6.86 mg/L 76.2%	11.28 mg/L 89.5%	12.86 mg/L 71.4%
T0+28h – Old medium	1.89 mg/L 42.1%	2.47 mg/L 39.1%	2.67 mg/L 29.7%	4.80 mg/L 38.1%	3.01 mg/L 16.7%
T0+28h – New medium	3.02 mg/L 67.1%	3.87 mg/L 61.5%	5.78 mg/L 64.2%	12.05 mg/L 95.6%	9.42 mg/L 52.4%
T0+44h – Old medium	1.51 mg/L 33.6%	1.85 mg/L 29.3%	2.31 mg/L 25.7%	3.26 mg/L 25.9%	2.35 mg/L 13.0%
T0+44h – New medium	3.75 mg/L 83.3%	4.89 mg/L 77.7%	6.49 mg/L 72.1%	8.70 mg/L 69.1%	13.87 mg/L 77.1%
T0+52h – Old medium	2.28 mg/L 50.7%	2.57 mg/L 40.8%	3.41 mg/L 37.9%	4.27 mg/L 33.9%	4.10 mg/L 22.8%
T0+52h – New medium	3.09 mg/L 68.6%	4.41 mg/L 70.0%	6.44 mg/L 71.5%	7.28 mg/L 57.8%	10.59 mg/L 58.8%
T0+68h – Old medium	1.58 mg/L 35.0%	1.94 mg/L 30.9%	2.84 mg/L 31.5%	3.21 mg/L 25.4%	2.77 mg/L 15.4%
T0+68h – New medium	3.56 mg/L 79.1%	5.67 mg/L 89.9%	8.16 mg/L 90.6%	10.92 mg/L 86.7%	13.01 mg/L 72.3%
T0+76h – Old medium	2.25 mg/L 50.0%	2.69 mg/L 42.7%	3.02 mg/L 33.6%	3.65 mg/L 29.0%	3.26 mg/L 18.1%
T0+76h – New medium	3.46 mg/L 76.8%	4.76 mg/L 75.5%	6.75 mg/L 75.0%	10.21 mg/L 81.1%	12.02 mg/L 66.8%
End of test	1.34 mg/L 29.7%	1.71 mg/L 27.2%	2.38 mg/L 26.5%	2.83 mg/L 22.5%	2.28 mg/L 12.7%
Geometric mean	2.3 mg/L	3.0 mg/L	4.1 mg/L	5.4 mg/L	5.3 mg/L

 Table 6.1.1/3: Definitive test - Observed mortality

	Mortality %
	24 h	48 h	72 h	96 h
Water control	0.0	0.0	0.0	0.0
Solvent control	0.0	0.0	0.0	0.0
2.3 mg a.i./L	0.0	0.0	0.0	0.0
3.0 mg a.i./L	0.0	0.0	0.0	0.0
4.1 mg a.i./L	0.0	0.0	0.0	0.0
5.4 mg a.i./L	0.0	0.0	0.0	0.0
5.3 mg a.i./L	0.0	0.0	0.0	0.0

Validity criteria fulfilled:

yes

Remarks:

Mortality in the controls did not exceed one fish at the end of the test; Constant environmental conditions were maintained throughout the test; The dissolved oxygen concentration was at least 60 per cent of saturation value throughout the test

Conclusions:

No mortality or abnormal behaviour was observed in any one case. Thefore, the 96 hours LC50 was defined as referred to the highest geometric mean of the measured concentrations, even though it did not correspond to the highest nominal concentration.
- 96h-LC50 > 5.4 mg a.i/L;
- The highest concentration without observed effect was 5.4 mg a.i./L.

Executive summary:

This study was performed to assess the acute toxicity of the test material to Brachidanio rerio (now Danio rerio). The method followed was the OECD Guideline No. 203.

Following a preliminary range-finding study, fish were exposed, in groups of 7 fish per aquarium, to an aqueous dispersion of the test material (with solvent) over a range of nominal concentrations of 4.5, 6.3, 9.0, 12.6 and 18.0 mg/L for a period of 96 hours, under open conditions. Due to high volatility of the test substance, the test solutions were alternatively renewed/adjusted twice per day (semi-static test conditions). Despite this, the test concentrations were not maintained within 80-120% of the nominal values and resulted in 2.3, 3.0, 4.1, 5.4 and 5.3 mg/L, respectively as the geometric means of the measured concentrations in the old and the new media.

No mortality or abnormal behaviour was observed in any one case. Thefore, the 96 hours LC50 was defined as referred to the highest geometric mean of the measured concentrations, even though it did not correspond to the highest nominal concentration.

- 96h-LC50 > 5.4 mg a.i/L;

- The highest concentration without observed effect was 5.4 mg a.i./L.

All validity criteria were fulfilled. However, this study is considered reliable with restrictions for the following reasons:

- The treatment solutions were prepared with a solvent (acetone), which is not an appropriate method to conduct toxicity tests for substances with a solubility at the level of this substance (15.4 mg/L)but the concentration of solvent used was considered acceptable (0.125 mL/L).

- A further drawback of this study was that it was performed under open conditions and loss of substance by volatilisation were observed.

Description of key information

OECD Guideline 203, GLP, key study, validity 2:

96h-LC50 (Danio rerio) > 5.4 mg/L, based on geometric mean measured concentrations.

Additional information

One experimental study is available to assess the short-term toxicity of the registered substance to fish.

This study (Phytosafe, 2016), performed according to OECD Guideline 203 with GLP statement, assess the acute toxicity of the registered substance to Danio rerio. Following a preliminary range-finding study, fish were exposed, in groups of 7 fish per aquarium, to an aqueous dispersion of the test material (with solvent) over a range of nominal concentrations of 4.5, 6.3, 9.0, 12.6 and 18.0 mg/L for a period of 96 hours, under open conditions. Due to high volatility of the test substance, the test solutions were alternately renewed/adjusted twice per day (semi-static test conditions). Despite this, the test concentrations were not maintained within 80 -120% of the nominal values and resulted in 2.3, 3.0, 4.1, 5.4 and 5.3 mg/L, respectively as the geometric means of the measured concentrations in the old and the new media.

No mortality or abnormal behaviour was observed in any case. Thefore, the 96 hour LC50 was defined to be greater than 5.4 mg/L, the highest geometric mean of the measured concentrations, even though it did not correspond to the highest nominal concentration.

All validity criteria were fulfilled. However, this study is considered reliable with restrictions for the following reasons:

- The treatment solutions were prepared with a solvent (acetone), which is not an appropriate method to conduct toxicity tests for substances with a solubility at the level of this substance (15.4 mg/L) but the concentration of solvent used was considered acceptable (0.125 mL/L).

- A further drawback of this study was that it was performed under open conditions and loss of substance by volatilisation were observed.