Reaction mass of 6-butyl-3,6-dihydro-2,4-dimethyl-2H-pyran and 2-butyl-3,6-dihydro-4,6-dimethyl-2H-pyran and 2-butyltetrahydro-6-methyl-4-methylene-2H-pyran

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 June - 03 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-butyl-3,6-dihydro-4,6-dimethyl-2H-pyran

EC Number:

246-099-6

EC Name:

2-butyl-3,6-dihydro-4,6-dimethyl-2H-pyran

Cas Number:

24237-01-2

Molecular formula:

C11H20O

IUPAC Name:

2-butyl-4,6-dimethyl-3,6-dihydro-2H-pyran

Constituent 2

Reference substance name:

6-butyl-3,6-dihydro-2,4-dimethyl-2H-pyran

EC Number:

246-098-0

EC Name:

6-butyl-3,6-dihydro-2,4-dimethyl-2H-pyran

Cas Number:

24237-00-1

Molecular formula:

C11H20O

IUPAC Name:

6-butyl-2,4-dimethyl-3,6-dihydro-2H-pyran

Constituent 3

Reference substance name:

2-butyltetrahydro-6-methyl-4-methylene-2H-pyran

EC Number:

246-101-5

EC Name:

2-butyltetrahydro-6-methyl-4-methylene-2H-pyran

Cas Number:

24237-02-3

Molecular formula:

C11H20O

IUPAC Name:

2-butyl-6-methyl-4-methylenetetrahydro-2H-pyran

Specific details on test material used for the study:

Material Identification (as stated in the report) : Gyrane
Appearance: Colourless to pale yellow liquid
Batch: SC00016010
Expiry date: 27 August 2017

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

The batch of GYRANE tested was a colorless to pale yellow liquid mixture and not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium. Thereafter, the resulting mixture was allowed to stabilize for one hour. Subsequently, the Saturated Solution (SS) was siphoned off and used as the highest test concentration. Lower concentrations were prepared by subsequent dilutions of the highest concentration in test medium. Due to the light sensitivity of the test item, preparation of test solutions was performed under dimmed light and used glassware was wrapped in aluminum foil. All final test solutions were clear and colorless.
After preparation, volumes of 120 mL were added to each replicate of the respective test concentration. Subsequently, 2.4 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pseudokirchneriella subcapitata, strain: NIVA CHL 1 (In-house laboratory culture). This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Test conditions

Nominal and measured concentrations:

At the start of the test, the actual concentrations were 0.32, 1.1, 2.9, 11 and 36 mg/L in 1.0, 3.2, 10, 32 and 100% of the SS. The measured concentrations were at the level of 27-49% and at 7.4-15% of initial after 24 and 72 hours of exposure, respectively. The Time Weighted Average (TWA) concentrations were calculated to be 0.11, 0.43, 1.1, 3.4 and 13 mg/L, respectively, and used to determine the effect parameters

Details on test conditions:

A final test was performed based on the results of a preceding range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to 1.0, 3.2, 10, 32 and 100% of the SS. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. The test item was suspected to be volatile and hence testing was performed in closed airtight vessels with minimum headspace and with adjusted medium.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.5 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Effects based on yield were also reported (see executive summary below and attached full study report). However, the preferred observational endpoint in the algal inhibition study is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. Thus only the effects based on growth rate are presented in the above "effects concentration" table.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata,
The EC10 for growth rate inhibition (72h-ERC10) was 4.3 mg/L with a 95% confidence interval ranging from 3.6 to 4.9 mg/L.
The EC50 for growth rate inhibition (72h-ERC50) was 8.5 mg/L with a 95% confidence interval ranging from 7.9 to 9.1 mg/L.

Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with GYRANE.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of theCommissionRegulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009 and the OECD series on testing and assessment number 23, 2000.

The batch of GYRANE tested was a colorless to pale yellow liquid mixture and not completely soluble in test medium at the loading rate initially prepared.

A Saturated Solution (SS) was prepared at 100 mg/L and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.Due to the light sensitivity of the test item, preparation of test solutions was performed under dimmed light and used glassware was wrapped in aluminum foil. All final test solutions were clear and colorless.

A final test was performed based on the results of a preceding range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to 1.0, 3.2, 10, 32 and 100% of the SS. The initial algal cell density was 10⁴cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. The test item was suspected to be volatile and hence testing was performed in closed airtight vessels withminimum headspace and with adjusted medium.

Samples taken from all concentrations were analyzed. At the start of the test, the actual concentrations were 0.32, 1.1, 2.9, 11 and 36 mg/L in 1.0, 3.2, 10, 32 and 100% of the SS. The measured concentrations were at the level of 27-49% and at 7.4-15% of initial after 24 and 72 hours of exposure, respectively. The Time Weighted Average (TWA) concentrations were calculated to be 0.11, 0.43, 1.1, 3.4 and 13 mg/L, respectively, and used to determine the effect parameters. The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized

The EC10 for growth rate inhibition (72h-ERC10) was 4.3 mg/L with a 95% confidence interval ranging from 3.6 to 4.9 mg/L. The EC50 for growth rate inhibition (72h-ERC50) was 8.5 mg/L with a 95% confidence interval ranging from 7.9 to 9.1 mg/L. The EC10 for yield inhibition (72h-EYC10) was 2.8 mg/L with a 95% confidence interval ranging from 1.6 to 3.1 mg/L. The EC50 for yield inhibition (72h-EYC50) was 4.9 mg/L with a 95% confidence interval ranging from 4.1 to 11 mg/L. The 72h-NOEC for growth rate inhibition was 3.4 mg/L, while the NOEC for yield inhibition was 1.1 mg/L.

The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus the 72-h EC50 and EC10 based on growth rate are used for classification purposes, which were determined in this study to be 8.5 mg/L and 4.7 mg/L respectively.