3-ethoxy-1,1,5-trimethylcyclohexane

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:

14-19 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline No. 201. All validity criteria were fulfilled

Cross-reference

Reason / purpose for cross-reference:

other: no cross-reference

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on 19 March, 24-26 June, 22 July 2013 / signed on 24 September 2013

Test material

Test material information

Constituent 1

Reference substance name:

3-ethoxy-1,1,5-trimethylcyclohexane

EC Number:

266-722-5

EC Name:

3-ethoxy-1,1,5-trimethylcyclohexane

Cas Number:

67583-77-1

Molecular formula:

C11H22O

IUPAC Name:

3-ethoxy-1,1,5-trimethylcyclohexane

Test material form:

liquid

Details on test material:

- Physical state/Appearance: Clear colorless liquid
- Storage condition of test material: Stored at room temperature in the dark
- Expiration date of the lot/batch: 09 July 2016

Specific details on test material used for the study:

- Density: 0.841 g/mL

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling: Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples at 24 and 48 hours of all treatment groups were taken from an additional replicate set up. Duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test media of each treatment.
All samples (20 mL) were diluted with 2.5 mL acetonitrile, and extracted on the same day via a liquid-liquid extraction with cyclohexane.
Additional samples of the control blank and the dilution solvent were taken at test start and test end without any sample treatment.
- Analyses: The concentrations of the test item were analysed in the duplicate test media samples from all test concentrations, and in the duplicate control samples, from all sampling times (0, 24, 48, and 72 hours).
- Storage of Samples: All samples were extracted stand-by with cyclohexane directly after sampling at test start and test end. The extracts were stored in a fridge (4 ± 4 °C), protected from light until analysis was performed.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item is not well soluble in test medium. Therefore, supersaturated stock suspensions of the test item were prepared by suspending test item in test water at nominal concentrations of 100, 32, 10, 3.2 and 1.0 mg test item/L. 119, 38, 12, 3.8 and 1.2 µL were dissolved in 1000 ml, respectively. These suspensions were shaken in an over-head shaker for approximately 24 hours to dissolve as much test item as possible. Afterwards the suspensions settled for 2 hours to separate insoluble test item. The test solutions were extracted from below the water surface after the first 50 ml were discarded. Each concentration was prepared separately.
The test media were prepared just before introduction of the algae (= start of the test).
Appearance of the Test Item in Test Medium: The test item caused foaming in all test concentrations
- Controls: In the control, test water was used without addition of the test item.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: KORSHIKOV, Strain No. 61.81 SAG
- Source: Algae were supplied by the Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test conditions

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

21-22 °C

pH:

pH values at test start 8.0 to 8.1, at the end of the test 8.6 to 9.8

Nominal and measured concentrations:

Due to the low limit of water solubility of the test item Water accommodated Fractions (WAF) with nominal loading rates of 100, 32, 10, 3.2 and 1.0 mg/L and a control were tested;
corresponding to following geometric mean measured concentrations of the test item: 9.90, 7.90, 3.07, 3.11 and 0.476 mg/L, and a control.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 50 mL volume with 50 mL of test medium.
- Type: closed
- Initial cells density: Test was started (0 hours) by inoculation of a biomass of 5000 algal cells per mL test medium
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- One additional replicate for each concentration group and the control was incubated for the analytical measurements. These further replicates were treated in the same way as the regular replicates.
- Additionally, one replicate of each test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometric measurements.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (OECD Medium)

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Light intensity and quality: Mmean light intensity: 6448 lux (6060 to 6780 lux).

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae).
Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.

TEST CONCENTRATIONS
- Range finding study: Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.41 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % CI: 2.72 - 4.24 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 7.65 - > 9.90 mg/L

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.97 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 1.65 - 2.35 mg/L

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

4.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 3.88 – 4.99 mg/L

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.47 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % CI: 1.24 - 1.76 mg/L

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 2.67-3.42 mg/L

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.476 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.476 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 9

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

<= 0.476 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 10

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Growth Inhibition: The 72-hour EyC50 was calculated to be 3.41 mg/L and the ErC50 9.04 mg/L. The 72-hour EyC10 was calculated to be 1.47 mg/L and the ErC10 3.02 mg/L. The 72-hour NOEyC was determined to be < 0.476 mg/L and the associated 72 hour LOEyC of ≤ 0.476 mg/L. The 72-hour NOErC was determined to be 0.476 mg/L and the associated 72 hour LOErC is 3.11 mg/L. All concentrations refer to geometric mean measured concentrations.
- Microscopic Examination: The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a geometric mean measured test concentration of 9.90 mg/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to the highest concentration tested.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Project No.: 84840210 (August 2015).
72 h EC 50 (yield): 0.545 mg/L, (95 % confidence limits: 0.320 – 0.775 mg/L)
72 h EC 50 (growth rate): 1.65 mg/L, (95 % confidence limits: 1.56 – 1.75 mg/L)
72 h EC 50 (biomass): 0.562 mg/L, (95 % confidence limits: 0.391 – 0.819 mg/L)
72 h NOEC (yield): 0.45 mg/L
72 h NOEC (growth rate): 0.45 mg/L
72 h NOEC (biomass): < 0.076 mg/L

Reported statistics and error estimates:

Based on the calculated cell densities, the 72-hour ErC50 and the 72-hour EyC50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by nonlinear regression.
For the determination of the 72-hour LOEC and the 72-hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.1.0, ToxRat Solutions GmbH.

Any other information on results incl. tables

Validity Criteria of the Study

Cell density increase in control cultures: 215.0-fold increase within 72 hours and thus, validity criterion was met.

Coefficient of variation of sectional (daily) growth rates in control cultures: 23.4 % and thus, validity criterion was met.

Coefficient of variation of average growth between control replicates: 1.1 % and thus, the validity criterion was met.

 

Analytical Results

Limit of Detection: 0.04 mg test item/L

Limit of Quantification: 0.2 mg test item/L after concentration by factor 10 (corresponding to fortification level of nominal 0.02 mg test item/L); 86% (n = 5, RSD 6%)

Mean recovery in the test samples: Freshly prepared: 15.9 mg/L; Aged test media:6.16 mg/L  

The quantification of the test item was performed using gas chromatography with flame ionization detector.

The concentrations of the test item were determined in the duplicate test media samples from all test concentrations (test item stock solution and its dilutions) and the duplicate control samples from all sampling times. All reported results refer to geometric mean concentrations, since the test item concentrations were not within ± 20 % of the measured initial concentrations during the test.

At the start of the test 15.9 mg test item/L was detected in the highest test concentration (the saturated stock solution). After 72 hours test duration, 6.16 mg test item/L was detected in the highest test concentration.

In the lowest test concentration at test start 0.769 mg test item/L was detected. After 72 hours test duration 0.222 mg test item/L was detected. The test item therefore never decreased to below the Limit of Detection or the Limit of Quantification over the course of the test in any test concentration.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the 72-hour EyC50 was calculated to be 3.41 mg/L and the 72-hour ErC50 value was calculated to be 9.04 mg/L. The 72-hour NOEyC was determined to be < 0.476 mg/L and the associated 72-hour LOEyC was ≤ 0.476 mg/L. The 72-hour NOErC was determined to be 0.476 mg/L and the associated 72-hour LOErC was 3.11 mg/L. All concentrations refer to geometric mean measured concentrations

Executive summary:

In a algal growth inhibition study performed according to OECD Guideline 201/ EU method C.3 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to test item at 100, 32, 10, 3.2 and 1.0 mg/L corresponding to following geometric mean measured concentrations of the test item: 9.90, 7.90, 3.07, 3.11 and 0.476 mg/L, and a control for 72 hours under static conditions.

 

This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement. The flasks were covered with glass stoppers (closed vessel). One additional replicate of each treatment group was set up for analytical measurements treated in the same way as the regular replicates. The quantification of the test item was performed using gas chromatography with flame ionization detector.

 

Test Conditions: Water temperature: 21-22°C; pH values at test start 8.0 to 8.1, at the end of the test 8.6 to 9.8; continuous illumination; mean light intensity: 6448 lux (6060 to 6780 lux).

 

The concentrations of the test item were determined in the duplicate test media samples from all test concentrations (test item stock solution and its dilutions) and the duplicate control samples from all sampling times. All reported results refer to geometric mean concentrations, since the test item concentrations were not within ± 20 % of the measured initial concentrations during the test. At the start of the test 15.9 mg test item/L was detected in the highest test concentration (the saturated stock solution). After 72 hours test duration, 6.16 mg test item/L was detected in the highest test concentration. In the lowest test concentration at test start 0.769 mg test item/L was detected. After 72 hours test duration 0.222 mg test item/L was detected thus a little over two thirds of the test substance was lost over the course of the three day study. This was thought to be due to hydrolysis as similar losses were observed in the daphnid study. However, the test item therefore never decreased to below the Limit of Detection or the Limit of Quantification over the course of the test in any test concentration.

Under the test conditions, the 72-hour EyC50 was calculated to be 3.41 mg/L and the 72-hour ErC50 value was calculated to be 9.04 mg/L. The 72-hour NOEyC was determined to be < 0.476 mg/L and the associated 72-hour LOEyC was ≤ 0.476 mg/L. The 72-hour NOErC was determined to be 0.476 mg/L and the associated 72-hour LOErC was 3.11 mg/L. All concentrations refer to geometric mean measured concentrations