methyl-bis(2-arylpropyl)dihydro-heteropolycycle

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:

2019-03-13 to 2019-08-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD No 23, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Remarks:

Gas chromatography with MS/MS detection

Details on sampling:

Quadruplicate 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, quadruplicate 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 pooling the contents of the test beakers of each treatment.
In addition, quadruplicate samples from the test media of all test concentrations and the control were taken after 24 and 48 hours test duration from the additional vessels prepared for analytical sampling.
All samples were adjusted to pH10 with 1 M sodium hydroxide solution before storage.

Test solutions

Vehicle:

no

Details on test solutions:

Test Concentrations:
An aqueous solution of the test item methyl-bis(2-arylpropyl)dihydro-heteropolycycle in test media with a concentration (nominal: 110 mg/L) as close to saturation as possible (referred to as a ´stock solution´) was obtained first (see ´Dosage of test item´ below). This stock solution and dilutions of this “stock solution” by 1:3, 1:9, 1:27 and 1:81 were prepared for testing. Additionally, a control was tested in parallel. These concentrations refer to nominal concentrations of 110, 36.7, 12.2, 4.07 and 1.36 mg test item/L.
Control: In the control, test water was used without addition of the test item.
Dosage of Test Item: Before test start, a ´stock solution´ (110 mg/L) of methyl-bis(2-arylpropyl)dihydro-heteropolycycle in test medium was prepared by mixing 111.7 mg test item into 1015.45 mL test water. The test medium was stirred with an excess of methyl-bis(2-arylpropyl)dihydro-heteropolycycle in a vessel for 24 hours in the dark. Afterwards the test medium was allowed to settle until phase separation for 1.5 hours. This ´stock solution´ was withdrawn from a tap at the center of the vessel, after first flushing the tap and tubing with a small aliquot, and then mixed with test medium to produce the desired dilutions of 1:3, 1:9, 1:27 and 1:81.
Appearance of the Test Item in Test Medium: There were no remarkable observations.

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
Origin: The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzen-wissenschaften, 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

Test conditions

Hardness:

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

Test temperature:

The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
Water temperature was: 21.6 to 21.9 °C

pH:

The pH was measured in all test item concentrations and the control at the start and the end of the test.
The pH in the control was 8.1 at test start and 9.1 at test end.
The pH at the different test concentrations was 8.0 to 8.1 at test start and 8.8 to 9.1 at test end.

Nominal and measured concentrations:

An aqueous solution of the test substance methyl-bis(2-arylpropyl)dihydro-heteropolycycle in test media with a concentration (nominal: 110 mg/L) as close to saturation as possible. This stock solution and dilutions of this “stock solution” by 1:3, 1:9, 1:27 and 1:81 were prepared for testing. Additionally, a control was tested in parallel. These concentrations refer to nominal concentrations of 110, 36.7, 12.2, 4.07 and 1.36 mg test item/L.

The mean measured values of the freshly prepared samples (0h) are:
2689 µg test item/L in the WAF stock solution of nominal 110 mg test item/L
783 µg test item/L in the 1:3 dilution of the stock solution
220 µg test item/L in the 1:9 dilution of the stock solution
80.8 µg test item/L in the 1:27 dilution of the stock solution
32.0 µg test item/L in the 1:81 dilution of the stock solution
The mean measured values of the aged samples (24h) are:
1769 µg test item/L in the WAF stock solution of nominal 110 mg test item/L
514 µg test item/L in the 1:3 dilution of the stock solution
159 µg test item/L in the 1:9 dilution of the stock solution
57.3 µg test item/L in the 1:27 dilution of the stock solution
22.7 µg test item/L in the 1:81 dilution of the stock solution
The mean measured values of the aged samples (48h) are:
1026 µg test item/L in the WAF stock solution of nominal 110 mg test item/L
324 µg test item/L in the 1:3 dilution of the stock solution
99.6 µg test item/L in the 1:9 dilution of the stock solution
36.8 µg test item/L in the 1:27 dilution of the stock solution
The mean measured values of the aged samples (72h) are:
653 µg test item/L in the WAF stock solution of nominal 110 mg test item/L
226 µg test item/L in the 1:3 dilution of the stock solution
74.6 µg test item/L in the 1:9 dilution of the stock solution

Details on test conditions:

Water Temperature:
The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
Water temperature was: 21.6 to 21.9 °C) pH-Values: The pH was measured in all test item concentrations and the control at the start and the end of the test.
The pH in the control was 8.1 at test start and 9.1 at test end.
The pH at the different test concentrations was 8.0 to 8.1 at test start and 8.8 to 9.1 at test end. Light Regime: Continuous illumination
Light Intensity: 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: 4975 lux (range: 4780 to 5150 lux)

Reference substance (positive control):

yes

Remarks:

reference item potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

80.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

36.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

36.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The 72-hour EyC50 was calculated to be > 110 mg test item/L and the ErC50 > 110 mg test item/L. The 72-hour EyC10 was calculated to be 80.7 mg test item/L and the ErC10 > 110 mg test item/L. The 72-hour NOEyC was determined to be 36.7 mg test item/L and the associated 72-hour LOEyC of 110 mg test item/L. The 72-hour NOErC was determined to be 36.7 mg test item/L and the associated 72-hour LOErC is 110 mg test item/L.
Microscopic Observations:
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 110 mg test item/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):

Results with reference substance Potassium dichromate:
Cell Density
Increase in Control Cultures: 206.1-fold increase within 72 hours and thus, the validity criterion was met.
Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: 6.9 % and thus, the validity criterion was met. Coefficient of Variation of Average Growth between Control Replicates: 1.0 % and thus, the validity criterion was met.
Growth Inhibition:
The 72-hour EyC50 was calculated to be 0.445 mg test item/L, the ErC50 0.941 mg test item/L and the EbC50 0.494 mg test item/L. The 72-hour EyC10 was calculated to be 0.192 mg test item/L, the ErC10 0.479 mg test item/L and the EbC10 0.207 mg test item/L.
The 72-hour NOEyC was determined to be < 0.094 mg test item/L and the associated 72-hour LOEyC of ≤ 0.094 mg test item/L. The 72-hour NOErC was determined to be 0.094 mg test item/L and the associated 72-hour LOErC is 0.21 mg test item/L. The 72-hour NOEbC was determined to be < 0.094 mg test item/Land the associated 72-hour LOEbC is ≤ 0.094 mg test item/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 three parametric normal concentration distribution function (CDF).
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 3.2.1, ToxRat Solutions GmbH.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Cell Density Increase: 144.2-fold increase within 72 hours; Coefficient of Variation of Sectional (Daily) Growth Rates: 11.3 %; Coefficient of Variation of Average Growth: 1.0 %

Conclusions:

The influence of the test substance methyl-bis(2-arylpropyl)dihydro-heteropolycycle on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be > 110 mg test item/L and the 72-hour ErC50 value was calculated to be > 110 mg test item/L. The 72-hour NOEyC was determined to be 36.7 mg test item/L and the associated 72-hour LOEyC was 110 mg test item/L. The 72-hour NOErC was determined to be 36.7 mg test item/L and the associated 72-hour LOErC was 110 mg test item/L. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to nominal values (loading rate) since a supersatured stock solution via a water accommodated fraction was prepared.

Executive summary:

The influence of the test substance methyl-bis(2-arylpropyl)dihydro-heteropolycycle on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be > 110 mg test item/L and the 72-hour ErC50 value was calculated to be > 110 mg test item/L. The 72-hour NOEyC was determined to be 36.7 mg test item/L and the associated 72-hour LOEyC was 110 mg test item/L. The 72-hour NOErC was determined to be 36.7 mg test item/L and the associated 72-hour LOErC was 110 mg test item/L. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to nominal values (loading rate) since a supersatured stock solution via a water accommodated fraction was prepared.

This study is classified acceptable and satisfies the guideline requirements for growth inhibition algae studies.