1,5,10-trimethylcyclododeca-1,5,9-triene, epoxidised

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 10 April to 3 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 203 and EU Method C.1 with GLP compliance. All validity criteria were fulfilled but a minor deviation was observed. The dissolved oxygen concentration was at least 60% of the air saturation value throughout the test in all test vessels except at t=24h at the highest test concentration of 21.8 mg/L where dissolved [O2] was 54% of the air saturation value. It was assumed that this decrease was probably due to the quick mortality of fish, and therefore to a substantial bacterial growth depleting [O2] from the test medium due to fish decomposition. Besides, it should be noted that 100% mortality was also recorded at 11.0 mg/L within 24 hours and [O2] concentration was well higher than 60% of the air-saturation value, which supports the hypothesis stated above. Thus, this deviation was not considered to have affected the results and the integrity of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

minor deviation. See "Dissolved oxygen" section in the Endpoint Study Record.

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

yes

Remarks:

minor deviation. See "Dissolved oxygen" section in the Endpoint Study Record.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspection by COFRAC on July 4th, 5th and 6th 2016, signed on January 10th 2017

Specific details on test material used for the study:

No additional information

Analytical monitoring:

yes

Details on sampling:

- Chemical analyses : Single samples for analysis were taken from the control and all test concentrations at the start (t=0h), at t=48h (old and fresh solutions) and at the end of the test (t=96h).
- Nominal concentrations : 1.4, 2.8, 5.5, 11.0 and 21.8 mg test item/L
- Sampling method: The test samples were injected directly after a dilution by a factor of two with acetone (500µL sample + 500 µL of cyclohexanone 10 mg/L in acetone). If the sample concentration was too high and not included in the concentration range of the calibration, the samples were diluted appropriately with a test water/acetone (50/50 v/v) mixture.
- Sample storage conditions before analysis: The samples were injected directly after their preparation to avoid any degradation of the test item.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on the results of an acute toxicity test with D. magna (LPL Project D15-018) and an algal growth inhibition test (LPL Project A15-018), and regarding the measured concentration of the stock solution at t=0h (15.4 mg.L-1), test solutions used in the definitive test were prepared by direct addition of the required amounts of stock solution to test water to obtain the following nominal concentrations : 1.4, 2.8, 5.5, 11.0 and 21.8 mg test item/L [= the highest test concentration was expected to be close to the solubility of the test item in test water; after two weeks of slow-stirring, the concentration of the stock solution of the test item was 15.4 mg.L-1, and since concentrations values were calculated considering the test item major constituents included in the main chromatographic peak (representing 70.76% of the test item) => 15.4/70.76% = 21.8 mg.L-1].
- Controls: Test water without test substance but treated in the same way as the test substance solutions.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): The solvent used for the preparation of the spiking solutions was acetone.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Stock solutions of the test substance were prepared in acetone at concentrations of approximately 1 g/L.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Source: Elevage de la Grande Rivière - B. et C. Romano - La Fond Garel – 69490 SAINT FORGEUX
- Sex: Male and female
- Average length of the fish used during the final test: 2.87 cm
- Average weight of the fish used during the final test: 0.241 g

ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions (same as test or not): The fish were held in water of the same quality as used in the test (reconstituted water, as prescribed by the OECD Guideline 203) until the start of exposure. Test fish were held for at least 12 days prior to the test under similar temperature and light conditions as used in the test.
- Type and amount of food: Fish were fed daily with flaked food or live food (such as brine shrimp nauplii) throughout the holding period.
- Feeding frequency: once daily
- Health during acclimation (any mortality observed): In the batch of fish which was used for the test, no mortality during the seven days prior to the start of the test was observed.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

The water hardness was between 10 and 250 mg.L-1 (as CaCO3).

Test temperature:

23°C ± 2°C, constant within 2°C

pH:

6.0-8.5, constant within 1 unit

Dissolved oxygen:

The dissolved oxygen concentration was at least 60% of the air saturation value throughout the test in all test vessels, except at t=24h at the highest test concentration of 21.8 mg.L-1 where dissolved [O2] was 54% of the air saturation value. It was assumed that this decrease was probably due to the quick mortality of fish, and therefore to a substantial bacterial growth depleting [O2] from the test medium due to fish decomposition. Besides, it should be noted that 100% mortality was also recorded at 11.0 mg.L-1 within 24 hours and [O2] concentration was well higher than 60% of the air-saturation value, which supports the hypothesis stated above. Thus, this deviation was not considered to have affected the results and the integrity of the study.

Salinity:

Not applicable

Conductivity:

Not mentionned

Nominal and measured concentrations:

Nominal concentrations : Test solutions used in the definitive test were prepared by direct addition of the required amounts of stock solution to test water to obtain the following nominal concentrations (spaced by a factor of approximately 1.98): 1.4, 2.8, 5.5, 11.0 and 21.8 mg test item.L-1 [= the highest test concentration was expected to be close to the solubility of the test item in test water; after two weeks of slow-stirring, the concentration of the stock solution of the test item was 15.4 mg.L-1, and since concentrations values were calculated considering the test item major constituents included in the main chromatographic peak (representing 70.76% of the test item): 15.4/70.76% = 21.8 mg.L-1].

Measured concentrations : The analytical results of this test showed that concentrations of test solutions were overall stable. Indeed, specific analyses of samples revealed that test item levels found were maintained around ± 30% of the initial concentration between the start and the end of the first part of the test (t=0h-t48hOld), and were satisfactorily preserved within ± 10% of the initial concentration during the second exposure period (t=48hFresh-t=96h). Moreover, geometric mean concentrations were closed to their corresponding nominal concentrations. Thus, and since the test item was a UVCB substance, the results were based on the nominal test concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass aquaria, of 3.5 L capacity filled with minimum headspace (approximately 1 cm) and closed by placing a glass plate on the aquarium and sealed with Vaseline. Each test vessel was uniquely identified with study code, date of experimentation and concentration.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: see above
- Aeration: No aeration of the test solutions occurred throughout the test.
- Type of flow-through (e.g. peristaltic or proportional diluter): A semi-static test was performed.
- Renewal rate of test solution (frequency/flow rate): one renewal of the test solutions and the control at t=48h.
- No. of organisms per vessel: 7 fish per vessel filled with minimum headspace
- 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: loading of fish not exceeding 1.0 g.L-1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water, as prescribed by the OECD Guideline 203. The pH of this solution was in the range of 6 to 8.5 and the water hardness was between 10 and 250 mg.L-1 (as CaCO3).

OTHER TEST CONDITIONS
- Adjustment of pH: The test was carried out without adjustment of the pH.
- Photoperiod: 16h light : 8 h dark
- Light intensity: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Fish were inspected at 24, 48, 72 and 96 hours following the start of exposure. Dead fish recorded were removed when observed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.98
- Range finding study: no
- Test concentrations: Based on the results of an acute toxicity test with D. magna (LPL Project D15-018) and an algal growth inhibition test (LPL Project A15-018), and regarding the measured concentration of the stock solution at t=0h (15.4 mg.L-1), test solutions used in the definitive test were prepared by direct addition of the required amounts of stock solution to test water to obtain the following nominal concentrations (spaced by a factor of approximately 1.98): 1.4, 2.8, 5.5, 11.0 and 21.8 mg.L-1 [= the highest test concentration was expected to be close to the solubility of the test item in test water; after two weeks of slow-stirring, the concentration of the stock solution of the test item was 15.4 mg.L-1, and since concentrations values were calculated considering the test item major constituents included in the main chromatographic peak (representing 70.76% of the test item) => 15.4/70.76% = 21.8 mg.L-1].
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

5.371 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL: not determined

Details on results:

See tables of results included in "Any other information on results incl. tables".

Analytical results:
The analytical results of this test showed that concentrations of test solutions prepared following the outlined procedures were overall stable. Indeed, specific analyses of samples revealed that test item levels found were maintained around ± 30% of the initial concentration between the start and the end of the first part of the test (t=0h-t48hOld), and were satisfactorily preserved within ± 10% of the initial concentration during the second exposure period (t=48hFresh-t=96h). Moreover, geometric mean concentrations were closed to their corresponding nominal concentrations. Thus, and since the test item was a UVCB substance, the results were based on the nominal test concentrations.

Biological results :
After 24 and 48 hours of exposure, mortality was 0% at 1.4 and 2.8 mg/L, 14.3% at 5.5 mg/L, and 100% at 11.0 and 21.8 mg/L. After 72 and 96 hours of exposure, mortality was 0% at 1.4 and 2.8 mg/L, 57.1% at 5.5 mg/L, and 100% at 11.0 and 21.8 mg/L.
Therefore, the highest concentration without effect was 2.8 mg/L and the lowest concentration with 100% mortality was 11.0 mg/L.
The software ToxRat® Professional (8) was used for the determination of the effective concentrations.

Results with reference substance (positive control):

On April 18, 2017 (most recent test), the 24h-LC50 was 205.042 mg/L. Hence, the sensitivity of this batch of Danio rerio was in agreement with internal historical data.

Sublethal observations / clinical signs:

Table 6.1.1/1: Dissolved oxygen concentrations (mg/L) and corresponding air saturation values (%) during the final test

		Nominal concentration(mg test item.L^-1)
		Control	1.4	2.8	5.5	11.0	21.8
Start t=0h		8.53	8.41	8.39	8.29	8.26	7.96 [92.7%]
t=24h	Old	7.10 [88.0%]	6.72 [84%]	6.67 [84.9%]	6.28 [78%]	6.80 [85%]	4.50 [53.9%]
t=48h	Old	7.12 [80%]	7.80 [84.5%]	7.78 [89%]	7.35 [78.5%]	7.87* [88.4%]	6.46* [73.2%]
	Fresh	8.50 [98.8%]	8.41 [95.2%]	8.50 [97.1%]	8.43 [96.4%]
t=72h	Old	7.90 [91.2%]	6.96 [80.4%]	6.80 [78.4%]	6.67 [75.4%]
End t=96h		7.96 [93%]	7.30 [82.9%]	6.98 [78.7%]	6.84 [77.7%]

Grey boxes: no further analysis was performed for concentrations in which all fish were dead.

* All fish were dead at t=24h and removed from these test vessels.

Table 6.1.1/2: Incidence of mortality and total mortality during the final test

	Concentration (mg test item.L^-1)
Nominal Cumulative Mortality	Control M     S	1.4 M     S	2.8 M     S	5.5 M     S	11.0 M     S	21.8 M     S
t=24h	0      0	0      0	0      0	1      6^b	7^a      0	7^a     0
24h Total Mortality (%)	0	0	0	14.3	100	100
t=48h	0      0	0      0	0      0	1      6^b	7      0	7    0
48h Total Mortality (%)	0	0	0	14.3	100	100
t=72h	0      0	0      0	0      0	4      3^b	7      0	7    0
72h Total Mortality (%)	0	0	0	57.1	100	100
t=96h	0      0	0      0	0      0	4      3^b	7      0	7    0
96h Total Mortality (%)	0	0	0	57.1	100	100

M: Number of dead fish; S: Number of surviving fish but with sublethal effects

Max. Sol.: Maximum Solubility

^aAt t=0h, sublethal effects were observed directly after the fish were introduced into test vessels.

^bFish with pronounced sublethal effects (distributed on the side and at the bottom of the aquarium, with small opercula movements).

The initial number of fish in each test vessel was 7.

Validity criteria fulfilled:

yes

Remarks:

Except for the dissolved oxygen concentration (see "Dissolved oxygen" section in the Endpoint Study Record). This deviation was not considered to have affected the integrity of the study.

Conclusions:

The toxic effect of test item 1,5,10-trimethylcyclododeca-1,5,9-triene, epoxidised to the zebrafish (Danio rerio) was investigated in a closed semi-static test. Under the experimental conditions and based on the nominal test concentrations, the 96-hour LC50 value estimated was 5.371 mg/L.

Executive summary:

This study was performed to assess the acute toxicity of the test item 1,5,10-trimethylcyclododeca-1,5,9-triene, epoxidisedto the zebrafish Danio rerio. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 203, "Fish Acute Toxicity Test" (1), referenced as Method C.1 of Commission Regulation No. 440/2008 (amended by Commission Regulation (EU) 2016/266)(2) and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23). The criterion measured was the LC₅₀ (Median Lethal Concentration), a statistically derived concentration which is expected to cause death in 50% of test animals within a period of 96 hours.

Groups of seven fish were exposed to an aqueous solution of the test itemover a range ofnominal test concentrations of 1.4, 2.8, 5.5, 11.0 and 21.8 mg/L (a concentration close to the solubility of the test item in test water) in aquaria closed over the study period with a minimum of headspace. The total test period was 96 hours and test solutions were renewed at t=48h. The mortality of the fish was determined by visual observation after 24, 48, 72 and 96 hours. The concentrations of the test item were determined by chemical analysesat the start (t=0h), at t=48h (old and fresh solutions) and at the end of the test (t=96h).

The analytical results showed that concentrations of test solutions were overall stable. Indeed, specific analyses of samples revealed that test item levels found were maintained around ± 30% of the initial concentration between the start and the end of the first part of the test (t=0h-t48h_Old), and were satisfactorily preserved within ± 10% of the initial concentration during the second exposure period (t=48h_Fresh-t=96h). Besides, geometric mean concentrations were closed to their corresponding nominal concentrations. Since the test item was a UVCB substance, the results were based on the nominal test concentrations. After 24 and 48 hours of exposure, mortality was 0% at 1.4 and 2.8 mg/L, 14.3% at 5.5 mg/L, and 100% at 11.0 and 21.8 mg/L. After 72 and 96 hours of exposure, mortality was 0% at 1.4 and 2.8 mg/L, 57.1% at 5.5 mg/L, and 100% at 11.0 and 21.8 mg/L.

Under the experimental conditions and based on the nominal test concentrations, the 96-hour LC50 value estimated was 5.371 mg/L.

Description of key information

OECD 203, EU Method C.1, GLP, key study, validity 1:

96h-LC50 (Danio rerio) = 5.371 mg/L, based on nominal concentrations (as UVCB substance)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

5.371 mg/L

Additional information

One key study is available to assess the acute toxicity of the registered substance to zebra fish (Danio rerio).

This study was performed, according to the OECD Guideline 203 and EU Method C.1 with GLP compliance, under semi-staitc conditions for 96 hours. Groups of seven fish were exposed to an aqueous solution of the test substance over a range of nominal test concentrations of 1.4, 2.8, 5.5, 11.0 and 21.8 mg/L (a concentration close to the solubility of the test item in test water) in aquaria closed over the study period with a minimum of headspace. The mortality of the fish was determined by visual observation after 24, 48, 72 and 96 hours. The concentrations of the test item were determined by chemical analysesat the start (t=0h), at t=48h (old and fresh solutions) and at the end of the test (t=96h).

The analytical results showed that concentrations of test solutions were overall stable. Indeed, specific analyses of samples revealed that test substance levels found were maintained around ± 30% of the initial concentration between the start and the end of the first part of the test (t=0h-t48h_Old), and were satisfactorily preserved within ± 10% of the initial concentration during the second exposure period (t=48h_Fresh-t=96h). Since the test item was a UVCB substance, the results were based on the nominal test concentrations. After 24 and 48 hours of exposure, mortality was 0% at 1.4 and 2.8mg/L, 14.3% at 5.5 mg/L, and 100% at 11.0 and 21.8 mg/L. After 72 and 96 hours of exposure, mortality was 0% at 1.4 and 2.8 mg/L, 57.1% at 5.5 mg/L,and 100% at 11.0 and 21.8 mg/L. Therefore, under the experimental conditions and based on the nominal test concentrations, the 96-hour LC50 value estimated was 5.371 mg/L.