4,4'-(4-methylpentane-2,2-diyl)bis((heptyloxy)benzene)

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:

11th April to 19th April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1-(heptyloxy)-4-{2-[4-(heptyloxy)phenyl]-4-methylpentan-2-yl}benzene

EC Number:

830-582-9

Cas Number:

1951440-04-2

Molecular formula:

C32H50O2

IUPAC Name:

1-(heptyloxy)-4-{2-[4-(heptyloxy)phenyl]-4-methylpentan-2-yl}benzene

Test material form:

liquid: viscous

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AS455433
- Expiration date of the lot/batch: 01 November 2020
- Purity: 98.5%
- CAS number: 1951440-04-2

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from the light
- Stability under test conditions: not stable under test conditions, with a drop in concentrations determined during the course of the experiment
- Solubility and stability of the test substance in the solvent/vehicle: the test substance was shown to be stable in the stock solutions with THF

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples taken from all test concentrations and controls
- Sampling method: 2.0 mL from the approximate centre of the test vessels.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- Other information: Extra samples of 2.0 mL for possible analysis were taken from the stock solutions in THF used in the preparation of test solutions. These were also analysed

Test solutions

Vehicle:

yes

Remarks:

tetrahydrofuran (THF)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: the solubility limit in test medium was determined to be below the LOD of <0.051 mg/L (Test Facility Study No. 20179825). As a result the stock solutions were prepared by dissolving the test item in tetrahydrofuran (THF) to obtain a stock solution with a nominal concentration of 1mg/ml. The solution was vigourously shaken which was adequate to completely dissolve the test item. Lower stock solutions were prepared by subsequent dilution. 100µl of the respective stocks were spiked to a 1L test medium. 50ml of the solutions were added to test vessels.
- Controls: 100µl THF with no test substance
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): tetrahydrofuran (THF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100µl/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All test solutions were clear and colourless at the end of the preparation procedure. No undissolved material was observed in the stocks or any test solution.
- Other relevant information: test solutions were magnetically stirred for 15 minutes post addition of dosing stock/THF (controls) to ensure homegenous test solutions.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: microalgae, unicellular
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In house laboratory culture
- Age of inoculum (at test initiation):cells from the algal stock culture were taken 3 days before the start of the test.

ACCLIMATION
- Acclimation period: not stated
- Culturing media and conditions (same as test or not): different, M1; according to the NPR 6505 (“Nederlandse Praktijk
Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (24 mg CaCO3/L) culture medium

Test temperature:

22 to 24°C

pH:

8.2 to 8.4

Dissolved oxygen:

not stated

Salinity:

not stated

Conductivity:

not stated

Nominal and measured concentrations:

Nominal concentrations: 1.0, 3.2, 10, 32, and 100 μg/L
Measured concentrations test solutions: A concentration of 85 μg/L was measured in the solution prepared at nominally 100 μg/L at the start of the test. The concentration decreased to 68% relative to the initial concentration in the first 24 hours and further below the LOD (i.e. <18 μg/L) by the end of the test. Based on the obtained results, the Time Weighted Average (TWA) concentration at nominally 100 μg/L was calculated to be 39 μg/L. The test substance was not measured in most of the other concentration samples. See Table for further information.
Measured concentrations stock solutions: 87 to 95% of nominal concentration in the 0.1 and 1mg/ml stock concentrations. Stocks were demonstrated stable for at least 8 days. See Table 2 for further information.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: headspace 50ml
- Aeration: no, during incubation the algal cells were kept in suspension by continuous shaking.
- Initial cells density: 1 mL of an algal suspension was added to each replicate providing a cell density of 10,000 cells/mL.
- Control end cells density: 1 mL of an algal suspension was added to each replicate providing a cell density of 10,000 cells/mL.
- No. of vessels per concentration (replicates): 3 replicates per test concentration
- No. of vessels per control (replicates): 6 replicates, test media only
- No. of vessels per vehicle control (replicates): 6 replicates, with solvent

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water
- Culture medium different from test medium: yes, M1, according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3 20 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

OTHER TEST CONDITIONS
- Sterile test conditions: not stated
- Adjustment of pH:
- Photoperiod: continuous lighting
- Light intensity and quality: 78 to 83 μE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : yield and growth inhibition.
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: a limit test at 100µg/l was carried out initially, which indicated inhibition of yield and growth at upto 21%.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

39 µg/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:

EC50

Effect conc.:

> 39 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

39 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 39 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the TWA concentration of 39 μg/L when compared to the blank control.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none noted
- Effect concentrations exceeding solubility of substance in test medium: The TWA concentration of 39 μg/L exceeded the water solubility of the test item (< 23 μg/L). Therefore, it can be stated that the test item was not toxic to algae at levels up to the maximum water solubility.

Results with reference substance (positive control):

The study met the acceptability criteria prescribed by the study plan and was considered valid.
Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 1.11 mg/L with a 95% confidence interval ranging from 1.10 to 1.13 mg/L.
The 72h-ERC50 was within the expected range of 0.92 and 1.46 mg/L as specified in ISO International Standard 8692, February 2012, and the historical range of 0.86 and 2.3 mg/L, which is based on reference tests performed at the Test Facility during the last ten years.

Reported statistics and error estimates:

GROWTH rate statistics:
Shapiro-Wilk´s Test on Normal Distribution carried out to test for differences between the blanks and the solvent control. Data showed treatment data do not significantly deviate from normal distribution.
NOEC statistics: pre-tests, test for normality of distribution, treatment data do not significantly deviate from normal distribution; test for homogeneity of variance, showed normal distribution and variance-homogeneity requirements are fulfilled. Dunnetts multiple t-test carried out to determine NOEC, showed NOEC appears higher or equal to the nominally 100µg/l.

YIELD STATISTICS
Shapiro-Wilk´s Test on Normal Distribution carried out to test for differences between the blanks and the solvent control. Data showed treatment data do not significantly deviate from normal distribution.
NOEC statistics: pre-tests, test for normality of distribution, treatment data do not significantly deviate from normal distribution; test for homogeneity of variance, showed normal distribution and variance-homogeneity requirements are fulfilled. Dunnetts multiple t-test carried out to determine NOEC, showed NOEC appears higher or equal to the nominally 100µg/l.

Any other information on results incl. tables

Table 3. Growth Rate and Percentage Inhibition for the Total Test Period

Nominal conc. (µg/L)   Pooled control 1.798 0.0189 12   1.0 1.810 0.0157 3 -0.63 3.2 1.784 0.0155 3 0.81 10 1.799 0.0240 3 -0.059 32 1.787 0.0059 3 0.65 100 [39] 1.821 0.0125 3 -1.3

[ ] – TWA concentration (µg/L)

 

Table 4. Growth Rate and Percentage Inhibition at Different Time Intervals

 

Nominal conc. (µg/L) Mean %Inhibition Mean %Inhibition Mean %Inhibition Pooledcontrol         12 1.953   1.795   1.647   1.0                   3 1.996 -2.2 1.757 2.1 1.677 -1.8 3.2                   3 1.943 0.48 1.751 2.4 1.657 -0.59 10                    3 1.998 -2.3 1.770 1.4 1.631 1.0 32                    3 1.874 4.0 1.790 0.31 1.697 -3.0 100 [39]               3 1.930 1.2 1.915 -6.7 1.620 1.7

Table 5. Yield and Percentage Inhibition for the Total Test Period

Nominal conc. (µg/L)   Pooled control 219.7 12.70 12   1.0 227.2 10.72 3 -3.4 3.2 210.1 9.89 3 4.4 10 220.4 15.70 3 -0.34 32 211.7 3.77 3 3.6 100 [39] 235.3 8.91 3 -7.1

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of 4,4’-(4-methylpentane-2,2-diyl)bis((heptyloxy)benzene) to Raphidocelis subcapitata algae was measured with time weighted average NOEC's and EC50's of 39 and >39 µg/l calculated for both growth and yield, which was above the water solubility limit.

Executive summary:

The toxicity of 4,4’-(4-methylpentane-2,2-diyl)bis((heptyloxy)benzene) to Raphidocelis subcapitata algae was investigated according to the OECD201 guidelines and to GLP. The test substance was shown to have a very low solubility in a preliminary study and therefore the substance was dissolved in tetrahydofuran (THF) initially and dosed into the test vessels. Controls and solvent controls were also included. Nominal test concentrations of 1, 3.2, 10, 32 and 100 µg/l were included. Analytical measurement of the test solutions was only able to determine the concentration at the nominal 100 µg/l test concentraion, with a time weighted average (TWA) concentration determined of 39 µg/l over the 72 hour period. No significant effects on yield or growth were found compared to the controls at any concentration. NOEC's and EC50's of 39 and >39 µg/l were determined for both growth and yield, which was above the water solubility limit.