Reaction mass of 1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane and cineole

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 August 2016 to 29 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline No. 202. All validity criteria were fulfilled

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

5 march 2015

Analytical monitoring:

yes

Details on sampling:

- Chemical analyses: Single samples for analysis were taken from the control and all test concentrations at the start of the test (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for fresh media at t=0h and t=24h) were prepared under closed conditions and by slow-stirring to avoid production of a dispersion.
The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of each mixing vessel was approximately 5 L. A magnetic stirring bar was placed in each mixing vessel and 5.0 to 5.5 L of test water (depending on the brim capacity of the bottles) were added in order to use a maximum volume and to minimise headspace. The loading rates of the test item were weighed on glass slides that afterwards were placed under the surface of the test water contained in the mixing vessels through fishing wire. Then the mixing vessels were closed immediately. The mixing was carried out at a speed that was slow enough not to cause dispersion or emulsification of the undissolved fraction of the test item. Mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 ± 2 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for 1 hour before use. The first 100 mL were removed via the drain port. Then the WAFs were directly added into test vessels that were immediately sealed with screw caps after introduction of daphnids. No small bubble was observed in the test vessels. The test solution in test vessels was observed to be clear and colourless at all loading rates. The test was carried out without adjustment of the pH.

- Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna (Straus), clone 5
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Reason for selection: Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
- Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at study initiation: < 24 h
- Breeding Conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water was changed three times a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Feeding during test: No feeding

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

Total water hardness was approximately 250 mg/L (as CaCO3)

Test temperature:

between 20.6 and 20.8 °C throughout the test (average value: 20.8°C), and complied with the requirements (20°C ± 2°C, constant within 1°C).

pH:

7.60 - 8.18
pH: 6.0-9.0, not varying by more than 1.5 units

Dissolved oxygen:

7.42 - 8.41 mg O2/L
oxygen: ≥ 60% of the air-saturation value at the end of the test in controls and test vessels.

Salinity:

None

Nominal and measured concentrations:

Nominal : control, 5.1, 7.1, 10.0, 14.2, 20.0

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screw caps. Each test vessel will be uniquely identified with study code, replicate number, date of experimentation and concentration.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Renewal rate of test solution: A semi-static test was performed with renewal of test solutions after 24 h (because of the properties of the test item)
- No. of daphnids: 20 per treatment group (including controls), divided into 4 groups of 5 animals
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace.
- Introduction of Daphnids: Daphnids were introduced into the test vessels each completely filled with test solution and without headspace immediately after filling the test vessels with test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as prescribed by the OECD Guideline 202

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED:
- Immobility: Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Immobile animals were eliminated from the vessels as soon as they were discovered. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.
- pH and dissolved O2: At start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h) from all test concentrations and control.
- Temperature of Medium: Measured continuously in a temperature controlled vessel next to the test vessels, over the entire study period, beginning at the start of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.41
- Range finding study: Ten daphnids per concentration (5 per vessel, in duplicate) were exposed to the nominal loading rates 1.00, 5.00, 10.00 and 25.00 mg.L-1 and to a control.
- Results used to determine the conditions for the definitive study: The per cent immobility was 0, 0, 0, 0 and 100 (24 h) and 0, 0, 0, 30 and 100 (48 h) at 0, 1, 5, 10 and 25 mg/L, respectively. Based on the results of a range-finding test, the following nominal concentrations (spaced by a factor of approximately 1.41): 5.10, 7.10, 10.00, 14.20 and 20.00 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

11.61 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 10.95 - 12.32 mg/L

Details on results:

The evaluation was based on the nominal WAFs concentrations.
After 48 hours of exposure, immobilisation rates were 0% at 5.10 and 7.10 mg.L-1 (loading rates), 5% at 10.00 mg.L-1 (loading rate) and 100% at 14.20 and 20.00 mg.L-1 (loading rates).
The 48-hour EL50 including the 95% confidence interval using Probit-analysis was determined by the computer program ToxRat.

Results with reference substance (positive control):

On June 3, 2016 (most recent test), the 24h-EC50 was 1.04 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (1) (expected 24h-EC50: 0.6 mg.L-1 to 2.1 mg.L-1) at this time.

Reported statistics and error estimates:

The evaluation of the effects was based on the nominal WAFs concentrations (nominal loading values). The software ToxRat® Professional was used for the determination of the effective concentrations.

Analytical results

Chemical analyses were performed on the main constituents(1,4-cineol, 1,8-cineol) and one impurity (dl-limonene) to provide an indication of the WAFs stability.

The analytical results of this test showed that WAFs were overall stable throughout the test period. Indeed, analytical monitoring of1,4-cineole and 1,8-cineole revealed thatconcentrations of these major constituents were satisfactorily maintained within or close to ± 20-30% of the initial concentrationbetween the start and the end of each exposure period (exceptthe first day at the nominal test loading rate of 10mg.L^-1). However, these two main constituents of the test item (representing 76.7% of the mixture) were maintained to an overall level from 52 to 75% with regards to their expected nominal initial concentration. Moreover, dl-limonene levels were lower than their expected nominal concentration. It should be noted that aWAF is by definition a complex mixture for which the individual concentration of each constituent differs due to its properties (e.g. solubility, adsorption, volatilisation, bioaccumulation…). Due to these differences, interactions between certain constituents of the mixture may occur and affect the behaviour of a given constituent which consequently would not react in the same way that if it was alone in the mixture.

Therefore, results are provided as Effective Loading Rate 50, according to the OECD No. 23

 

Table 6.1.3/1: Acute immobilisation of daphnids after 24 and 48 h in the final test

 

Nominal concentration* (mg/L)	Replicate	Number of daphnids exposed	Response at 24 h		Response at 48 h
			Number	Total %	Number	Total %
Control	1	5	0	0	0	0
	2	5	0		0
	3	5	0		0
	4	5	0		0
5.1	1	5	0	0	0	0
	2	5	0		0
	3	5	0		0
	4	5	0		0
7.1	1	5	0	0	0	0
	2	5	0		0
	3	5	0		0
	4	5	0		0
10.0	1	5	0	0	0	5
	2	5	0		0
	3	5	0		0
	4	5	0		1
14.2	1	5	3	60	5	100
	2	5	3		5
	3	5	4		5
	4	5	2		5
20.0	1	5	5	100	5	100
	2	5	5		5
	3	5	5		5
	4	5	5		5

* WAF prepared at the given loading rate

 

Water quality parameter values throughout the test:

The results of measurement of pH and oxygen concentrations (mg.L^-1) are presented in Tables 2 and 3. These parameters remained within the limits prescribed (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen:≥ 60% of the air-saturation valueat the end of the test), suggesting that pH and oxygen concentrations were not influenced by the test item.

Furthermore, the temperature of the test medium was situated between 20.6 and 20.8°C throughout the test (average value: 20.8°C), and complied with the requirements (20°C ± 2°C, constant within 1°C).

 

Validity criteria of the study:

Controls: In the control, no daphnids became immobilized nor trapped at the surface of the water or showed signs of stress.

Dissolved [O2]: Dissolved oxygen concentration at the end of the test was ≥ 60 % of the air-saturation value in controls and test vessels.

Thus the validity criteria have been fulfilled in the present study.

Validity criteria fulfilled:

yes

Conclusions:

Based on nominal concentrations, the 48-hour EL50 value estimated was 11.61 mg test item.L-1 (loading rate).

In accordance with Regulation EC No. 1272/2008, the test substance has to be classified as chronic category 3. Hazard statement “H412: harmful to aquatic life with long lasting effects” is required.

Executive summary:

A study was performed to assess the acute toxicity of test item REACTION MASS OF 1,4-CINEOLE AND 1,8-CINEOLE to the freshwater invertebrate Daphnia magna. It was investigated in a 48h closed semi-static test using Water Accommodated Fractions according to OECD Guideline 202 and in compliance with GLP. 

 

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 5.1, 7.1, 10, 14.2 and 20 mg.L^-1 and to a control. The immobility of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. The concentrations of the test item, represented by analytical monitoring of the main constituents, were determined by chemical analyses at the start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

 

Analytical monitoring of 1,4-cineole and 1,8-cineole revealed that concentrations of these main constituents were satisfactorily maintained within or close to ± 20-30% of the initial concentration between the start and the end of each exposure period (t=0h-t24h_Oldand t=24h_Fresh-t=48h), suggesting that WAFs were overall stable throughout the test. After 48 hours of exposure, immobilisation rates were 0% at 5.10 and 7.10 mg.L^-1(loading), 5% at 10.00mg.L^-1(loading) and 100% at 14.20 and 20.00 mg.L^-1(loading). The 48 h EL50 at the end of the test was: 11.61 mg/L (95% CL 10.95 - 12.32 mg/L).

 

Therefore, based on nominal concentrations, the 48-hour EL50 value estimated was 11.61 mg test item.L-1 (loading rate).

Therefore, in accordance with Regulation EC No. 1272/2008, the test substance has to be classified as chronic category 3. Hazard statement “H412: harmful to aquatic life with long lasting effects” is required.

Description of key information

The registered substance exhibits a 48h-EL50 for freshwater invertebrates of 11.61 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

11.61 mg/L

Additional information

One reliable study is available for the registered substance. A study was performed to assess the acute toxicity of test item REACTION MASS OF 1,4-CINEOLE AND 1,8-CINEOLE to freshwater invertebrate Daphnia magna. It was investigated in a 48h closed semi-static test using Water Accommodated Fractions according to OECD Guideline 202 and in compliance with GLP. Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 5.1, 7.1, 10, 14.2 and 20 mg.L^-1and to a control.

The immobility of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. The concentrations of the test item, represented by analytical monitoring of the main constituents, were determined by chemical analyses at the start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

Analytical monitoring of 1,4-cineole and 1,8-cineole revealed that concentrations of these major constituents were satisfactorily maintained within or close to ± 20-30% of the initial concentration between the start and the end of each exposure period (t=0h-t=24h_Old and t=24h_Fresh-t=48h), suggesting that WAFs were overall stable throughout the test.

After 48 hours of exposure, immobilisation rates were 0% at 5.10 and 7.10 mg.L^-1(loading), 5% at 10.00 mg.L^-1(loading) and 100% at 14.20 and 20.00 mg.L^-1(loading).

The 48 h EL50 at the end of the test was: 11.61 mg/L (95% CL 10.95 - 12.32 mg/L).

Therefore, based on nominal concentrations, the 48-hour EL50 value estimated was 11.61 mg test item.L-1 (loading rate).

In accordance with Regulation EC No. 1272/2008, the test substance has to be classified as chronic category 3. Hazard statement “H412: harmful to aquatic life with long lasting effects” is required.