2-tert-butylcyclohexyl ethyl carbonate

Hydrolysis

Administrative data

Endpoint:

hydrolysis

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 22-29, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 201 (Alga, Growth Inhibition Test)

Deviations:

no

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:

September 24, 2013

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Density: 0.974 g/mL

Analytical monitoring:

yes

Details on sampling:

Sampling: Duplicate samples (20 mL) 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 (20 mL) 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 beakers of each treatment.
The samples were immediately extracted with cyclohexane.
Additional samples of the control blank 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 both sampling times (0 and 72 hours).
Storage of Samples: All samples (the cyclohexane extracts) were stored in a fridge (4 ± 4°C), protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 20 mg test item/L was prepared by mixing 20.5 µL test item into 1000 mL test water by stirring for 48 hours.
Afterwards the test medium was allowed to settle for 24 hours and the lower fraction of the stock solution was used. Adequate volumes of the lower fraction of the stock solution were mixed with test water to prepare the test media of the desired test concentrations. The test media were prepared just before introduction of the algae (= start of the test).
A saturated stock solution and dilutions of 1:3, 1:9, 1:27 and 1:81 of this were tested together with a control.
- Controls: In the control, test water was used without addition of the test item.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name:
- Strain: 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: Algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3.

Test temperature:

Water temperature: 22-23 °C

pH:

pH values at test start 8.0, at the end of the test 8.7 to 10.1

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

Initial measured concentrations: 0.145, 0.419, 1.35, 3.35 and 10.8 mg/L

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: The test was started (0 hours) by inoculation of a biomass of 5000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Volumes of 50 mL of algal suspension per replicate were continuously stirred with magnetic stirrers in 50 mL Erlenmeyer flasks. The flasks were covered with glass stoppers and incubated in a water bath. The flasks were placed in a random order and were repositioned each day to minimize differences in test conditions.
- 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) according to OECD 201

OTHER TEST CONDITIONS
- Adjustment of pH: pH was adjusted with 1 M HCl to pH 8.1
- Photoperiod: Continuous illumination
- Light intensity and quality: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media. Mean light intensity: 5058 lux (range: 4630 to 5380 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
- Pre-experiments (Non-GLP) were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
- Test concentrations: A saturated stock solution and dilutions of 1:3, 1:9, 1:27 and 1:81 of this were tested together with a control.
Corresponding to following initial measured concentrations of the test item: 10.8, 3.35, 1.35, 0.419 and 0.145 mg/L, and a control.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (Project No.: 84840210)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.01 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % CI: 1.55-2.34 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.57 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % CI: 2.17-2.87 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.13 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % CI: 3.76-4.67 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.35 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.35 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.76 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 3.10-4.34 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

5.38 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 4.73-5.94 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10.7 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 10.02 - 11.47 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.35 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.35 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Growth Inhibition: The 72 -hour EyC50 was calculated to be 4.13 mg/L and the ErC50 10.7 mg/L. The 72-hour EyC10 was calculated to be 2.01 mg/L and the ErC10 3.76 mg/L. The 72-hour NOEyC was determined to be 1.35 mg/L and the associated 72 -hour LOEyC of 3.35 mg/L. The 72-hour NOErC was determined to be 1.35 mg/L and the associated 72-hour LOErC is 3.35 mg/L.

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 nominal test concentration of 9.96 mg/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, 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 (see Definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by non-linear 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 (yield and growth rate). The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Validity Criteria of the Study

Cell density increase in control cultures: 300.6-fold increase within 72 hours and thus, the validity criterion was met.

Coefficient of variation of sectional (daily) growth rates in control cultures: 16.6 % and thus, the validity criterion was met.

Coefficient of variation of average growth between control replicates: 0.9 % and thus, the validity criterion was met.

Analytical Results

Limit of Detection: 0.2 mg test item/L

Limit of Quantification: 0.1 mg test item/L (corresponding to 1 mg test item/L after an enrichment factor of 10);106% (n = 10, RSD 11%)

Mean recovery in the test samples: Freshly prepared:    56% (n = 10, RSD 7%); Aged test media: 47% (n = 10, RSD 10%)

 

Table 6.1.5/1: Summary of Analytical Results

 

Sample description (mg/L)	Fresh			Aged			Initial measured concentration
	% of nominal^1	RSD %	n	% of nominal^1	RSD %	n
Control	n.a.	n.a.	2	n.a.	n.a.	2	n.a.
1:81	59	3	2	54	10	2	0.145
1:27	57	2	2	43	7	2	0.419
1:9	61	1	2	49	4	2	1.35
1:3	50	7	2	44	0	2	3.35
Filtrate, 20 mg/L	54	2	2	46	5	2	10.8

 

¹mean value of all measured samples per treatment group
RSD: relative standard deviation per treatment group
n: number of analysed samples
n.a.: not applicable

 

At the start of the test 56% of the nominal test concentrations were found (average of all test concentrations). The saturated stock solution of 20 mg/L nominal (also corresponding to the highest test concentration) was found to contain 10.8 mg/L corresponding closely to the water solubility value of 10.9 mg/L determined in an OECD 105 study.

After 72 hours test duration, 47% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 52% of nominal. The 1:27 concentration degraded slightly more than 20% of the initial measured concentrations. All other concentrations remained within 20% of their initial concentrations. As the effects observed in the 1:27 concentration were lower than the NOEC/ EC10 concentrations, it was considered acceptable to report all results as initial concentrations.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, 72-hour EyC50 was calculated to be 4.13 mg/L, and the 72-hour ErC50 value was calculated to be 10.7 mg/L. The 72-hour NOEyC was determined to be 1.35 mg/L and the associated 72-hour LOEyC was 3.35 mg/L. The 72-hour NOErC was determined to be 1.35 mg/L and the associated 72-hour LOErC was 3.35 mg/L. As the test item was found to be stable over the study period, all results are based on initial concentrations.

Executive summary:

In an algal growth inhibition study performed according to OECD Guideline 201/ EU method C.3 and in compliance with GLP, the freshwater green algae species Pseudokirchneriella subcapitata was exposed to the test item at the initial measured concentrations of 0.145, 0.419, 1.35, 3.35 and 10.8 mg/L for 72 hours under static conditions.

This study encompassed 6 treatment groups (5 concentrations 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.

Test Conditions: Water temperature: 22-23 °C; pH values at test start 8.0, at the end of the test 8.7 to 10.1; continuous illumination; mean light intensity: 5058 lux (4630 to 5380 lux).

The quantification of the test item was performed using gas chromatography with flame ionization detection. At the start of the test 56% of the nominal test concentrations were found (average of all test concentrations). The saturated stock solution of 20 mg/L (also corresponding to the highest test concentration) was found to contain 10.8 mg/L.

After 72 hours test duration, 47% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 52% of nominal. The 1:27 concentration degraded slightly more than 20% of the initial measured concentrations. All other concentrations remained within 20% of their initial concentrations. All reported results therefore refer to initial concentrations.

Under the test conditions, 72-hour EyC50 was calculated to be 4.13 mg/L, and the 72-hour ErC50 value was calculated to be 10.7 mg/L. The 72-hour NOEyC was determined to be 1.35 mg/L and the associated 72-hour LOEyC was 3.35 mg/L. The 72-hour NOErC was determined to be 1.35 mg/L and the associated 72-hour LOErC was 3.35 mg/L. As the test item was found to be stable over the study period, all results are based on initial concentrations.