Ethyl 2-methyl-4-oxo-6-pentylcyclohex-2-ene-1-carboxylate

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:

16 June to 30 Novembre 2016

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

modified by use of sealed vessels for assessment of volatile substances

Principles of method if other than guideline:

The test medium was supplemented with sodium bicarbonate to increase the CO2 concentration in the test medium to achieve maximum algal growth rates without pH drift (Mayer et al. (2000), ISO 14442 (2006)). A closed-bottle test system was used to reduce losses of volatile components of the test item. Deviating from the study plan the pH in the test medium exceeded the required upper limit (pH = 9) by 0.6 pH units. This deviation has no influence on the integrity of the study results, since all cell growth related validity criteria were fulfilled and the pH drift throughout all well growing treatment levels were always below 1.3 pH units. Otherwise, the study was performed according to the guideline, the study plan and its amendment(s).

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

rel-(1R,6S)-ethyl 2-methyl-4-oxo-6-pentylcyclohex-2-enecarboxylate

Molecular formula:

C15H24O3

IUPAC Name:

rel-(1R,6S)-ethyl 2-methyl-4-oxo-6-pentylcyclohex-2-enecarboxylate

Constituent 2

Reference substance name:

rel-(1R,6R)-ethyl 2-methyl-4-oxo-6-pentylcyclohex-2-enecarboxylate

Molecular formula:

C15H24O3

IUPAC Name:

rel-(1R,6R)-ethyl 2-methyl-4-oxo-6-pentylcyclohex-2-enecarboxylate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material (as stated in the report): Calyxol
- Batch No. VE00412958
- Expiration date of the lot/batch: 15.11.2017

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples of stock solution and test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations. Control and test solutions were sampled in duplicate. The duplicate samples were kept separately as a reserve. The volume of each sample was recorded. After sampling and before shipment, all samples were stored in glass bottles in the dark at a temperature of g -18°C if not advised otherwise. Samples of untreated test medium were provided to the analytical laboratory in order to determine the recovery of the analytical method with the specific test medium. Samples were transferred to the test site for chemical analysis under the required storage conditions.

Test solutions

Vehicle:

no

Details on test solutions:

One day before the test start a stock solution (Si) was prepared by suspending 0.0800 g of the test item in 2000 mL of growth medium, resulting in a nominal loading rate of 40 mg test item/L. This stock solution was shaken three times overhead and thereafter intensively stirred for 4 hours on a magnetic stirrer at room temperature in the dark. After 0.5 hour without stirring, the stock solution was visually observed for undissolved test item particles. In the stock solution, no test item particles were visible on the surface of the stock solution. Therefore, this stock solution was used to prepare the test solution used for the conditioning of the test vessels and the exposure. To reduce potential adsorption process of the test item to the glass of the preparation flasks during preparation of test solutions and to the test vessels used for later exposure both materials were pre-conditioned with the corresponding test solutions.

Test organisms

Test organisms (species):

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

Details on test organisms:

The test system used in this study was the freshwater algae Pseudokirchneriella subcapitata (SAG 86.81).
The organisms were originally supplied by Sammlung von Algenkulturen, Albrecht-von- Haller-Institut, Universität Göttingen, Germany. To adapt the algae to the test conditions a pre-culture was inoculated by a liquid algal culture and incubated under test conditions.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.0 - 22.8, mean 22.3 °C

pH:

8.3 - 9.6

Nominal and measured concentrations:

Test concentrations (nominal): 0.40, 1.26, 4.00, 12.6, and 40.0 mg test item/L and a control
Measured concentrations (test period: 0 day) : 0.28, 1.10, 3.52, 11.1 and 35.4 mg Calyxol/L
Measured concentrations (test period: 3 days) : 0.25, 0.968, 3.49, 11.2 and 33.4 mg Calyxol/L

Details on test conditions:

The test item was applied once at the beginning of the exposure period. Three replicate vessels were used per test item concentration, and six replicate vessels for the control.
Two additional test vessels without algae were prepared for chemical analysis of the intermediate test item concentration (C3, 4.00 mg test item/L) at the end of the test, in order to assess test item stability during exposure without the presence of algae (stability samples). Two additional test vessels without algae were prepared for chemical analysis of the intermediate test item concentration (C3, 4.00 mg test item/L) at the end of the test. The test vessels were exposed in darkness, in order to assess test item stability during exposure without the presence of algae and light (light/dark stability samples).
The additional vessels were maintained under the same test conditions as the vessels with algae, with the exceptions described above. The test period (exposure of test organisms to the test solutions in a static system) was 72 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr207)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

8.66 ng/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 7.44-9.63

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

18.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 17.3-21.3

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 6.62-8.40

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 15.5-18.4

Details on results:

Effects based on yield were also reported (see 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. Furthermore, 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, only the EC10 is presented in the above "effects concentration" table. The NOEC values are available in the full study report.

Results with reference substance (positive control):

The ErC50 value for the toxic reference item, potassium dichromate, was determined as 0.913 mg/L (standard system) and 0.803 mg/L (closed system). These values are within the historical range of the general reference test results (standard open test system) of our laboratory (mean ErC50 value = 1.088 ± 0.364 mg/L) and within the narrow upper (1.463 mg/L) and lower (0.722 mg/L) warning limit.
Therefore, the results of these reference tests are acceptable and the test conditions are reliable.

Any other information on results incl. tables

Samples from the test solutions were analysed to determine actual levels of the test item at start of the exposure period (time 0) and following 72 hours of exposure in comparison to nominal concentrations. The measured concentrations ranged between 70-89% at start (0 hours), and 63-89% at end of the exposure period (72 hours), based on nominal concentrations. Therefore, the measured concentrations of the test item were not within 80- 120% of the nominal concentrations throughout the test period. Also a slight decrease of test item concentration throughout the exposure period was determined. Nevertheless, the test item concentration based on initial measured concentration remained stable within ±20% throughout the exposure period. Since all concentration levels were measured, the biological results were additionally calculated and reported based on geometric mean measured concentrations at each concentration level.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were fulfilled as required by the study plan, the study is therefore considered to be valid.

Conclusions:

The ErC50 Calyxol of Pseudokirchneriella subcapitata was determined to be 16.7 mg/L (15.5-18.4) based on geometric-mean measured concentrations.
The ErC10 Calyxol of Pseudokirchneriella subcapitata was determined to be 7.59 mg/L (6.62-8.40) based on geometric-mean measured concentrations.

Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with Calyxol was determined according to OECD guideline No. 201. The batch of Calyxol tested was a colorless to pale yellow liquid. One day before the test start a stock solution (Si) was prepared by suspending 0.0800 g of the test item in 2000 mL of growth medium, resulting in a nominal loading rate of 40 mg test item/L. This stock solution was shaken three times overhead and thereafter intensively stirred for 4 hours on a magnetic stirrer at room temperature in the dark. After 0.5 hour without stirring, the stock solution was visually observed for undissolved test item particles. In the stock solution, no test item particles were visible on the surface of the stock solution. Therefore, this stock solution was used to prepare the test solution used for the conditioning of the test vessels and the exposure.  To reduce potential adsorption process of the test item to the glass of the preparation flasks during preparation of test solutions and to the test vessels used for later exposure both materials were pre-conditioned with the corresponding test solutions.

Six growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to the nominal test concentraions 0.40, 1.26, 4.00, 12.6, and 40.0 mg test item/L.

Two additional test vessels without algae were prepared for chemical analysis of the intermediate test item concentration (C3, 4.00 mg test item/L) at the end of the test, in order to assess test item stability during exposure without the presence of algae (stability samples). Two additional test vessels without algae were prepared for chemical analysis of the intermediate test item concentration (C3, 4.00 mg test item/L) at the end of the test. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test ( Day 0 and Day 3).

Samples from the test solutions were analysed to determine actual levels of the test item at start of the exposure period (time 0) and following 72 hours of exposure in comparison to nominal concentrations. The measured concentrations ranged between 70-89% at start (0 hours), and 63-89% at end of the exposure period (72 hours), based on nominal concentrations. Therefore, the measured concentrations of the test item were not within 80- 120% of the nominal concentrations throughout the test period. Also a slight decrease of test item concentration throughout the exposure period was determined. Nevertheless, the test item concentration based on initial measured concentration remained stable within ±20% throughout the exposure period. Since all concentration levels were measured, the biological results were additionally calculated and reported based on geometric mean measured concentrations at each concentration level.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The ErC50 Calyxol of Pseudokirchneriella subcapitata was determined to be 16.7 mg/L (15.5 -18.4) based on geometric-mean measured concentrations.

The ErC10 Calyxol of Pseudokirchneriella subcapitata was determined to be 7.59 mg/L (6.62 -8.40) based on geometric-mean measured concentrations.