Bis(6-methylalkyl-5-en-2-yl)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:

18.05.2020 - 15.10.2020

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)

Deviations:

no

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:

The samples were taken from the biological phase of the study. Collecting, storage and handing over of the samples were the Study Director’s responsibility. The information concerning the samples was provided by the Study Director.
Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at test start.
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, samples of the test vessels of all treatment groups were taken in duplicate at the end of the test (after the 72 hours test period) by pooling the content of the test beakers of all replicates for each treatment.
Additionally, samples were taken after 24 and 48 hours from the additional vessels.
The pH value was adjusted to pH 10 directly after sampling.

Test solutions

Vehicle:

no

Details on test solutions:

An aqueous solution of the test item in test water with a concentration (nominal: 110 mg/L) as close to saturation as possible (referred to as a ´stock solution´) was obtained first. This stock solution represents the highest test concentration. From this solution, dilutions of 1:3.16, 1:9.99, 1:31.6 and 1:99.7 were prepared and tested. These test concentrations refer to nominal concentrations of 110, 34.8, 11.0, 3.49 and 1.10 mg test item/L. Additionally, a control was tested.

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG
Origin: The algae were supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der 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. The culture medium was prepared 9 days before test start to allow pH to stabilise.

Test temperature:

22 to 23°C

pH:

7.5 to 8.0 at teststart and 9.5 to 9.9 at test end

Nominal and measured concentrations:

An aqueous solution of the test item in test water with a concentration (nominal: 110 mg/L) as close to saturation as possible (referred to as a ´stock solution´) was obtained first. This stock solution represents the highest test concentration. From this solution, dilutions of 1:3.16, 1:9.99, 1:31.6 and 1:99.7 were prepared and tested. These test concentrations refer to nominal concentrations of 110, 34.8, 11.0, 3.49 and 1.10 mg test item/L. Additionally, a control was tested.

Details on test conditions:

Water temperature: 21.6 to 22.9 °C;
pH value in the control at test start: 8.0,
pH value in the control at test end: 9.8;
pH values in the test item treatments at test start: 7.5 to 7.7,
pH values in the test item treatments at test end: 9.5 to 9.9;
continuous illumination; mean light intensity: 5702 lux (5460 to 5950 lux).

Reference substance (positive control):

yes

Remarks:

Reference Item potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 110 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:

NOEC

Effect conc.:

> 110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

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 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The influence of the test item 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 ≥ 110 mg test item/L and the associated 72-hour LOEyC was > 110 mg test item/L. The 72-hour NOErC was determined to be ≥ 110 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 examined in the analytical part. At the end of test no test item concentrations above the limit of detection/quantification of the analytical method were found. All reported results refer to the nominal loading rate.

Results with reference substance (positive control):

The results with the reference substance were valid
72-hour EC50
0.470 Yield [mg test item/L]
0.858 Growth rate [mg test item/L]
0.495 Biomass [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 a 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 Dunnett’s t-test.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Cell Density Increase in Control Cultures: At least by a factor of 16 within 72 hours Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: Must not exceed 35 % Coefficient of Variation of Average Growth Must not exceed 7 %

Conclusions:

The influence of the test item 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 ≥ 110 mg test item/L and the associated 72-hour LOEyC was > 110 mg test item/L. The 72-hour NOErC was determined to be ≥ 110 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 examined in the analytical part. At the end of test no test item concentrations above the limit of detection/quantification of the analytical method were found. All reported results refer to the nominal loading rate.

Executive summary:

Purpose:	The purpose of this test was to determine the inhibitory effect of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. The test method of application and the test system are recommended by the test guidelines and Pseudokirchneriella subcapitata is one of the recommended test species. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.
Guidelines/Recommendations:	-      OECD Guidelines for the Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted March 23, 2006, corrected July 28, 2011 -      OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals", 2^nd Ed., February 08, 2019 -      SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414
Materials and Methods
Test Item:	according to substance identity and purity report.
Test Species:	Pseudokirchneriella subcapitata, Strain No. 61.81 SAG formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV). Cultivated in the laboratories of ibacon; original source: "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissen-schaften, Universität Göttingen", 37073 Göttingen, Germany.
Test Design:	This study encompassed 6 treatment groups (5 dose rates 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 media were inoculated with nominal 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 spectrophotometric measurement.
Endpoints:	Yield and growth rate of the algae
Test Concentrations:	An aqueous solution of test item in test water with a concentration (nominal: 110 mg/L) as close to saturation as possible (referred to as a ´stock solution´) was obtained first. This stock solution represents the highest test concentration. From this solution, dilutions of 1:3.16, 1:9.99, 1:31.6 and 1:99.7 were prepared and tested. These test concentrations refer to nominal concentrations of 110, 34.8, 11.0, 3.49 and 1.10 mg test item/L. Additionally, a control was tested.
Test Conditions:	Water temperature: 21.6 to 22.9 °C; pH value in the control at test start: 8.0, pH value in the control at test end: 9.8; pH values in the test item treatments at test start: 7.5 to 7.7, pH values in the test item treatments at test end: 9.5 to 9.9; continuous illumination; mean light intensity: 5702 lux (5460 to 5950 lux).
Results
Biological Results:
Validity Criteria:	272.3-fold increase of cell density within 72 hours; CV of sectional (daily) growth rate of control: 9.7 %; CV of average growth of control replicates: 2.3 %; and thus, the validity criteria were met.
Analytical Results:	The quantification of the test item in the test samples was performed using liquid/ liquid extraction followed by analysis via gas chromatography with MS/MS detection. At the start the mean measured value ranged between below of limit of detection and 114 µg test item/L (average of all test concentrations). After 72 hours test duration, the mean measured value ranged between below of limit of detection and below of limit of quantification.
Conclusion:	The influence of the test item 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 ≥ 110 mg test item/L and the associated 72-hour LOEyC was > 110 mg test item/L. The 72-hour NOErC was determined to be ≥ 110 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 examined in the analytical part. At the end of test no test item concentrations above the limit of detection/quantification of the analytical method were found. All reported results refer to the nominal loading rate.