Tetrahydro-2-isobutyl-4-methyl-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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study according to GLP criteria.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (2S-cis)-tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran and Tetrahydro-4-methyl-2-(2-methylpropen-1-yl)pyran (2R,4R) and Tetrahydro-4-methyl-2-(2-methylpropen-1-yl)pyran (2S,4S) and (2R-cis)-tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran

EC Number:

939-429-1

Cas Number:

16409-43-1

Molecular formula:

C10H18O

IUPAC Name:

Reaction mass of (2S-cis)-tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran and Tetrahydro-4-methyl-2-(2-methylpropen-1-yl)pyran (2R,4R) and Tetrahydro-4-methyl-2-(2-methylpropen-1-yl)pyran (2S,4S) and (2R-cis)-tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran

Details on test material:

- Name of test material (as cited in study report): Rose oxide 90
- Physical state: liquid / colorless, clear
- Analytical purity: 99.0 - 99.1 area-%
- Lot/batch No.: 40730216K0
- Expiration date of the lot/batch: 23 Sep 2013
- Water solubility: 0.9 g/L at 20°C
- Storage condition of test material: at ambient temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.0, 3.2, 10, 32, 100 mg/L as nominal concentrations based on test substance mass without correction for purity.
- Sampling method: Sampling was performed at the start of the exposure (0 h) from the inoculated replicate 7 of
each concentration and the control. At the end of the exposure (72 h) samples were taken from the combined inoculated (+
algae) replicates of the test concentrations and of the combined inoculated control group
replicates. Additionally, samples were collected from the uninoculated (-algae) replicate 0 of
each test concentration and the control at the end of the exposure (72 h)
- Sample storage conditions before analysis: Samples were transported to the Analytical Laboratory on the day of sampling

Test solutions

Vehicle:

no

Details on test solutions:

The test solutions were prepared separately for each test concentration. The stock solutions were prepared by directly
adding test substance to 2L of test medium and stirring for approximately 10 min at 20 ± 2 °C until visibly dissolved. The
stock test solution was prepared at 111% of the nominal concentration to compensate for the dilution by the later
addition of algal inoculum. All test solutions were visibly clear following preparation.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Pseudokirchneriella subcapitata KORSHIKOV (SAG 61.81)
- Source (laboratory, culture collection): Collection of algal cultures in University of Göttingen /Germany
- Age of inoculum (at test initiation): A stock algal culture is maintained continuously at the test facility. Before the exposure an inoculum culture is prepared from the stock culture and incubated for 4 days at 21 – 24 °C (max. temperature difference 2 °C). After this time, the inoculum
culture is in exponential growth phase and can be used to initiate the test (study day 0).

ACCLIMATION
- Any deformed or abnormal cells observed: The inoculum culture was observed microscopically at the start of the test to verify normal and healthy cells. At test termination, a pooled sample from each test concentration was examined and any abnormal appearance of the algae noted. Result: No abnormal cells observed.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.8 – 23.4 °C

pH:

7.6 - 9.7

Nominal and measured concentrations:

0 (control), 1.0, 3.2, 10, 32, 100 mg/L as nominal concentrations based on test substance mass without correction for purity.
0 (control), 0.68, 2.13, 7.26, 25.7, 80.9 mg/L analytically measured concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel:Erlenmeyer flasks (nominal volume 250 mL)
- Type (delete if not applicable): closed with glass plugs
- Material, size, headspace, fill volume: approx. 300 mL (Erlenmeyer flasks were completely filled)
- Initial cells density: 0.3 x 104 cells/mL
- Illumination: Artificial light, type universal white (OSRAM L 25), permanent illumination. To minimize the potential effect of slight variations
in illumination, the test vessels were rearranged daily
- Test temperature: 22.8 – 23.4 °C
- Shaking rate: Continuous (approx. 100 rpm)
- No. of vessels per concentration (replicates): 3 replicates for each test substance concentration
- No. of vessels per control (replicates): 6 replicates for the Control
1 additional replicate per test group for initial concentration control analysis only
1 additional uninoculated (without algae) replicate per test group for background fluorescence correction

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECD Guideline for Testing of Chemicals, No. 201 Algal growth inhibition test

OTHER TEST CONDITIONS

- Adjustment of pH: yes
- Light intensity and quality: Average 6074 lux (within ± 15% variability) at a wave length of
400 - 700 nm
-Test parameter: Algal growth measured as in vivo chlorophyll-a fluorescence (pulsed excitation with light flashes having a wavelength of 430
nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Inhibition of growth rate
- Determination of cell concentrations: in vivo chlorophyll-a fluorescence
-Fluorescencemeasurement: The fluorescence of aliquots from each test vessel was measured using a Tecan Infinite 200Pro fluorometer in a 96-well
flat bottom black plate with the following parameters: fluorescence top reading; excitation / emission wavelength = 430 / 670 nm; excitation / emission bandwidth = 20 / 25 nm; flashes = 5; integration time = 20 μs; shaking duration = 15s; shaking amplitude = 6 mm.
-Temperature measurement: Continuous measurement during the whole test period in the climate chamber in a separate deionized water filled flask.
-Determination of correlation between fluorescence and cell density: After the end of the exposure the control replicates were mixed
and serially diluted by factor 2. The fluorescence of aliquots from the undiluted mixture and the dilutions were measured and in parallel cell density was determined by a direct microscopic count (two counts in a Neubauer haemocytometer). These data were used to derive a linear correlation between fluorescence and cell density.
- Light measurement: Light homogeneity was evaluated by measuring light intensity at 5 locations within the incubation area at the start and end of
the test. The light intensity did not vary by more than ± 15% over the incubation area.

TEST CONCENTRATIONS
- The test concentrations were selected on the basis of a range finding test

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

19.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 18.3-20.9 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

24.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 23.0-25.3 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 34.1-37.9 mg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

38.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 35.1-41.4 mg/L

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

49.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 46.5-51.9 mgIL

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

79.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 78.3-81.1 mg/L

Details on results:

Since the measured concentrations deviated markedly from the nominal concentrations, the effect concentration, which is based on the mean measured concentrations should be preferably used for the evaluation of the test substance.

Results with reference substance (positive control):

The ErC50 (72 h) of the control substance potassium dichromate was 0.94 mg/L
(Date of the last control experiment: 23 Mar 2012 , project number: 60E0063/043031)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes