(+)-14-diethoxycarbonyl-1α-ethyl-1,2,3,4,5,6,12,12b-octahydro-indolo-[2,3-a]-tetrahydro-pyranyl-[2,3-c]-quinolizine

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:

72 h

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

(+)-14-diethoxycarbonyl-1α-ethyl-1,2,3,4,5,6,12,12b-octahydro-indolo-[2,3-a]-tetrahydro-pyranyl-[2,3-c]-quinolizine

EC Number:

692-750-6

Cas Number:

300661-83-0

Molecular formula:

C26H34N2O5

IUPAC Name:

(+)-14-diethoxycarbonyl-1α-ethyl-1,2,3,4,5,6,12,12b-octahydro-indolo-[2,3-a]-tetrahydro-pyranyl-[2,3-c]-quinolizine

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

For the stock solution 100.10 and 100.00 mg of test item was taken into 2 x 1 000 ml OECD medium. The dissolution of the test item was promoted by shaking on orbital shaker for 24 hours in thermostat at 21 ± 1 ºC. After stirring the suspensions were filtered through a 0.2 μm WhatmanTM ME24 (Mixed cellulose ester) filter. The first 50 ml filtrate was disposed. The two clear filtrates were pooled. The stock solution was prepared by mixing filtrates. The measured concentration of stock solution was 10.38 mg/L

Test organisms

Test organisms (species):

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

Details on test organisms:

Test species: Pseudokirchneriella subcapitata (Korshikov) F.Hindák (formerly known as Selenastrum capricornutum and Raphidocelis subcapitata)
Source: Culture Collection of Algae and Protozoa, Scottish Marine Institute (www.ccap.ac.uk)
Batch: CCAP 278/4
Date of arriving: 27 October 2017

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

The temperature inside the incubator at the test vessels was measured and registered continuously by Extech SD200 thermometer and datalogger. Throughout the test the temperature was nearly constant.
The vessels were closed with silicone stoppers with a filter for ensuring the proper air exchange. The vessels were kept in incubator at 21 ± 2 °C with continuous illumination. The treatments of control and test vessels were identical.

pH:

At the beginning of the test the pH values were measured in the remaining test solution of the concurrent test vessels at each dilution level. At the end of the test the pH values were measured in each test vessel. The pH of test solutions was not adjusted at the beginning of the test.

Nominal and measured concentrations:

The toxic property of test item was investigated at six concentrations which were prepared by different dilutions of stock solution of test item. OECD medium was used for dilution of stock solution. In the test the following calculated concentrations were applied: 1.04; 2.08; 3.11; 5.19; 7.27 and 10.38 mg/L.
The concentration of test item was measured at each concentration level at start and at the end of the test by a validated HPLC method. The geometric means of measured concentrations were determined. At the end of the test the concentrations of the test item in the dilution levels No 5 and 6 were below the detection limit, so these values were omitted from the calculation of mean concentrations. The following concentrations were measured: 1.93; 3.84; 5.51 and 8.10 mg/L. The test item concentrations of the abiotic controls were 8.19 (in light) and 9.12 (in darkness) mg/L. The following concentrations were applied for the statistical evaluation in the test: 1.93; 3.84; 5.51 and 8.10 mg/L.

Details on test conditions:

Preparation and breeding of inoculum culture: The inoculum culture was prepared four days before starting the test and it was bred in incubator at 21 ± 2 °C. At the end of incubation the cell density of the inoculum culture was determined with a Bürker chamber. The cell concentration was 958 333 cell/ml
Preparation of stock solution: The preparation of stock and test solutions was based on the preliminary non GLP range finding test [9]. As the Range Finding Test was not performed in compliance with the GLP-Regulations it is excluded from the Statement of Compliance in the final report, but the raw data of this test are archived under the study code of present study. For the stock solution 100.10 and 100.00 mg of test item was taken into 2 x 1 000 ml OECD medium. The dissolution of the test item was promoted by shaking on orbital shaker for 24 hours in thermostat at 21 ± 1 ºC. After stirring the suspensions were filtered through a 0.2 μm WhatmanTM ME24 (Mixed cellulose ester) filter. The first 50 ml filtrate was disposed. The two clear filtrates were pooled. The stock solution was prepared by mixing filtrates. The measured concentration of stock solution was 10.38 mg/L
Preparation of test solutions: Six test solutions of different concentration were prepared by mixing appropriate volume of OECD medium and stock solution of the test item. The maximum separation factor between of test vessels was 2.0. Two additional replicates were prepared for controlling the abiotic degradation of the test substance. These test vessels were not inoculated with alga inoculum culture. The following table shows the preparation of test solutions. The volume of the prepared dilution levels was 300 ml except the control solution which was 600 ml. The alga inoculum culture was diluted 193.55 times so the initial alga cell concentration in every dilution and the control levels were 4 951 cells/ml.
Preparation of test vessels: Three replicates were prepared at every test, two at the abiotic control and six at the control level. The vessels contained 75 ml test media.
Test procedure: The test vessels were closed with silicone stopper and kept in the Binder KBW 400 incubator. For the proper mass transfer of carbon dioxide glass tubes with filter were placed through silicone stoppers. The test was maintained under static conditions for a period of 72 hours. The temperature during the test remained in the 21 ± 2 °C range. The temperature data were harvested by Extech SD200 datalogger. The vessels were shaken and illuminated continuously in the incubator. The vessels were placed randomly into the incubator and were repositioned daily after sampling. One of the vessels of abiotic controls was taken into the incubator with a complete aluminium foil cover to check the stability of test item in the absence of light. This vessel was signed as 7/B (vessel No: 20). The abiotic control on light were signed as 7/A (vessel No: 19).

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

3.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

According to the Council Regulation (EC) 440/2008 Alpha-Ethyl pseudo adduct base is hazardous to the aquatic environment and classified into the second acute toxic hazardous category [GHS Acute aquatic hazard Category Acute II. – 72 h EC50 (for algae or other aquatic plants) > 1 mg/L but ≤ 10 mg/L]