2-ethoxy-2,6,6-trimethyl-9-methylenebicyclo[3.3.1]nonane

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

rel-(1R,2R,5S)-2-ethoxy-2,6,6-trimethyl-9-methylenebicyclo[3.3.1]nonane

Cas Number:

125673-86-1

Molecular formula:

C15H26O

IUPAC Name:

rel-(1R,2R,5S)-2-ethoxy-2,6,6-trimethyl-9-methylenebicyclo[3.3.1]nonane

Constituent 2

Reference substance name:

rel-(1R,2S,5S)-2-ethoxy-2,6,6-trimethyl-9-methylenebicyclo[3.3.1]nonane

Molecular formula:

C15H26O

IUPAC Name:

rel-(1R,2S,5S)-2-ethoxy-2,6,6-trimethyl-9-methylenebicyclo[3.3.1]nonane

impurity 1

Reference substance name:

4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

EC Number:

250-657-4

EC Name:

4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

Cas Number:

31499-72-6

Molecular formula:

C13H22O

IUPAC Name:

4-(2,6,6-trimethylcyclohex-2-en-1-yl)butan-2-one

Test material form:

liquid

Specific details on test material used for the study:

Test item name: Boisiris
Batch No.: VE00461936
Physical Appearance: Liquid
Storage Conditions: In a dry, well ventilated dark location at ambient temperature / 10-30 °C
Expiry date: December 25, 2018

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. Samples were taken from the test solutions at the start of the exposure period (0 hours) and at the end of the exposure (72 hours).

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 ≤ -18°C if not advised otherwise. A record was kept for each sample.

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. The dates of transfer of the samples from ECT to the laboratory for chemical analysis were recorded in the raw data.

Analysis was performed according to an analytical method developed and validated by the laboratory for chemical analysis. Further details describing the analytical procedures are contained in the analytical phase report. The analytical work was reported by the principal investigator, and is included in the final report of this study.

Test solutions

Vehicle:

no

Details on test solutions:

One day before the test start a stock solution (Si) was prepared by adding 0.3009 g of the test item in 3000 mL of growth medium, resulting in a nominal loading rate of 100 mg test item/L. This stock solution was slowly stirred at 300 rpm for 22 hours on a magnetic stirrer at room temperature in the dark. After one hour without stirring, the stock solution was visually observed for undissolved test item particles. In the stock solution (Si), droplets (oil film) of the test item (oil film) was visible on the surface of the stock solution. The lower water phase was drained of in a preconditioned Erlenmeyer flask, no oil film was visible in the drained solution (S2). Therefore, this stock solution (S2) was used to prepare the test solution used for the conditioning of the test vessels and the exposure.

The stock solution (Si and S2 ) was prepared once to prepare the test solutions.

The volume of the stock solution (S2) was large enough to prepare the test solutions and all analytical samples and solutions used for conditioning.
The vessels containing the test solutions were stirred for ca. 5 minutes on a magnetic stirrer at room temperature and placed into a temperature controlled room for temperature adaptation.

To reduce potential adsorption loss 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. The Erlenmeyer flask used for collecting of S2 and the measuring flasks used to prepare the test solution were rinsed with approx. 100 mL of S2. Thereafter, the preparation flasks and test vessels were conditioned with the corresponding test solutions. The preparation flasks were conditioned for approx. 15 minutes and the test vessels were conditioned for approx. 2.5 hours. The following table describes the procedure for preparing the solutions used for the conditioning of the test vessels.

After temperature adaptation of the test solutions, 0.246 or 0.308 mL of the algal pre-culture (Pseudokirchneriella subcapitata) were added to each of the volumetric flasks containing 400 or 500 mL of the test solutions, respectively to achieve an algal cell concentration of approximately 0.3x104 cells/mL. These manipulations were performed under a laminar flow box. The test solutions were homogenised by manual shaking 20 times overhead, before adding 60±5 mL of the test media to each test vessel to ensure a homogeneous distribution of the algal cells.

The test vessels were placed onto a shaker under light and temperature controlled conditions.

The medium was sterilised by sterile filtration (pore size 0.2 pm) before use. All glassware and materials used for testing purposes were sterilised for at least 3 hours at *150 °C using a heating furnace.

Test organisms

Test organisms (species):

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

Details on test organisms:

The green alga Pseudokirchneriella subcapitata was chosen as a representative of freshwater algae. The selection of the test system is based on the test guideline.
The organisms were originally supplied by Sammlung von Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Germany.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21–24 °C, controlled at ±2 °C

pH:

pH of test medium: 8.3
pH in test solutions: 8.3–9.3

Nominal and measured concentrations:

Test concentrations (nominal): 4.27, 9.39, 20.7, 45.5, and 100% of a saturated solution at a loading rate of 100 mg/L and a control
Geometric mean measured concentration over 72h: 0.133, 0.331, 0.678, 1.59 and 3.56 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 3.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 30% inhibition seen at the highest tested concentration level (100% of a saturated solution at a loading rate of 100 mg/L, equivalent to 3.56 mg Boisiris/L).

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.331 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.46 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 1.37-1.54

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 v 2.0, OECD 201 Guidelines). The guideline includes the additional response variable of yield, to satisfy current regulatory requirements in some countries. 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, the EC10 has been selected as the key value for long-term (chronic) aquatic hazard classification.

The %-inhibition seen for the parameter growth rate was <50%. Therefore, the EC50, was stated to be higher than the highest tested concentration level (100% of a saturated solution at a loading rate of 100 mg/L, equivalent to 3.56 mg Boisiris/L).

Samples from the test solutions were analysed to determine actual levels of the test item at start of exposure period (time 0 hours) and at the end of the exposure at 72 hours. The latter were 87-96% of the initial measured concentrations. Thus, 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 are calculated and reported based on geometric mean measured concentrations at each concentration level.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study with Pseudokirchneriella subcapitata, Boisiris gave the following results based on measured concentrations:
The EC10 for growth rate inhibition (72h-ErC10) was 1.46 mg/L with a 95% confidence interval ranging from 1.37 to 1.54 mg/L.
The EC50 for growth rate inhibition (72h-ErC50) was > 3.56 mg/L (30% inhibition seen at highest tested concentration level, 100% of a saturated solution at a loading rate of 100 mg/L, equivalent to 3.56 mg Boisiris/L)
The 72h-NOEC for growth rate inhibition was 0.331 mg/L.

The preferred observational end point in the algal growth inhibition test 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. 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 the 72-h EC50 and EC10 based on growth rate are used for classification purposes, which were determined in this study to be >3.56 mg/L and 1.46 mg/L respectively.