4-vinylcyclohexene

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:

2017-04-18 to 2017-04-21

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted March 23, 2006, corrected July 28, 2011

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

dated August 24, 2009

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

4-vinylcyclohexene

EC Number:

202-848-9

EC Name:

4-vinylcyclohexene

Cas Number:

100-40-3

Molecular formula:

C8H12

IUPAC Name:

4-ethenylcyclohex-1-ene

Test material form:

liquid

Details on test material:

4-vinylcyclohexene produced by Evonik Degussa GmbH, Batch 117, purity >98.0 % w/w

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The concentrations of test item were determined at the start of the exposure (0 hours) and at the end of the exposure (72 hours) in all tested concentrations and the control via GC-MS.

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution: 30.0 mg test item/L, freshly prepared with dilution water
Dispersion treatment: The stock solution was stirred for 2 hours at room temperature (1100 rpm).
Test concentrations: Five dilution levels out of the stock solution were tested in a (nominal) geometrical series with a dilution factor of √10: 0.150 - 0.474 - 1.50 - 4.74 - 15.0 mg/L, corresponding to the geometric mean measured test item concentrations 0.138 – 0.390 – 1.02 – 3.22 – 10.2 mg/L. The dilution levels were based on the results of a preliminary range finding test (non-GLP, closed bottles without headspace).

Test organisms

Test organisms (species):

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

Details on test organisms:

Test System
Species: Pseudokirchneriella subcapitata HINDÁK, SAG 61.81
Synonyms: Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2016)
Origin: The algae were supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
Cultivation at test facility: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux for 24 hours per day.
Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

Water Temperature:
Temperature (target): Nominal range: 21 - 24°C, controlled at ± 2°C

pH:

The pH-values at the start of exposure were measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-values were measured from pooled samples of each test item concentration and the control.

Nominal and measured concentrations:

Five dilution levels out of the stock solution were tested in a (nominal) geometrical series with a dilution factor of √10: 0.150 - 0.474 - 1.50 - 4.74 - 15.0 mg/L,
corresponding to the geometric mean measured test item concentrations 0.138 – 0.390 – 1.02 – 3.22 – 10.2 mg/L.
The measured concentrations of test item were between 61 to 91% of the nominal value at the start of the exposure (0 hours) and between 67 – 92% of the nominal value at the end of the exposure (72 hours). Therefore the geometric mean value was calculated.
see table "overall remarks"

Details on test conditions:

Non-GLP Preliminary Range Finding Test
A non-GLP preliminary range finding test without headspace (closed system) under static conditions over a period of 72 hours was conducted at the test facility. A stock solution of 50 mg/L was prepared in dilution water (see below), stirred for 2 hours at room temperature (1100 rpm) and used as the highest concentration in the test. The other test concentrations were prepared by diluting the stock solution with dilution water.
All test item concentrations were clear and colourless throughout the exposure period.
In the range finding test, two replicates per concentration and four for the control were tested.
Nominal test item concentration [mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
50.0 100 100
5.00 87 99
0.500 2 9

Experimental Procedure
Stock solution: 30.0 mg test item/L, freshly prepared with dilution water.
Dispersion treatment: The stock solution was stirred for 2 hours at room temperature (1100 rpm).
Test concentrations: Five dilution levels out of the stock solution were tested in a (nominal) geometrical series with a dilution factor of √10: 0.150 - 0.474 - 1.50 - 4.74 - 15.0 mg/L, corresponding to the geometric mean measured test item concentrations 0.138 – 0.390 – 1.02 – 3.22 – 10.2 mg/L. The dilution levels were based on the results of a preliminary range finding test (non-GLP, closed bottles without headspace).
Control: Six replicates (without test item) were incubated under the same conditions as the test concentrations.
Reference Item: Potassium dichromate was tested as a reference item
Duration of the test: 72 hours
Performance of the test: A static test was carried out. With regard to the volatility of the test item glass flasks without headspace were used to reduce losses of test item.
Replicates: Three replicates per concentration and six for the control. Separate replicates for each measuring time were prepared.
Test container: Sterile headspace flasks, volume: 59 mL, with aluminium tops with PTFE seals
Test volume: 59 mL
Preculture: A three days old preculture, prepared in dilution water, was used as inoculum.
Initial cell density: Nominal: approximately 5 x 103 - 104 cells/mL
Current: 6839 cells/mL
Application: Application was carried out by adding appropriate volumes of the test solution to the replicate test vessels.
Incubation: The flasks were positioned randomly and repositioned daily.
Temperature (target): Nominal range: 21 - 24°C, controlled at ± 2°C
Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
Light intensity (target): Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1
Light regime: 24 hours/day light
Light homogeneity (target): Within ± 15% over incubation area

Biological Parameters
Chlorophyll a-fluorescence: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test at the concentration 50.0 mg/L.

Microscopic evaluation: Microscopic evaluation of the cells at the start and the end of the incubation period was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

Endpoints: Yield and growth rate of the algae

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.138 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.138 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

Growth Rate Inhibition
Geometric mean measured test item concentration [mg/L]
NOEC 0.138
LOEC 0.390
ErC10 1.89 (1.71 – 2.06)
ErC20 3.15 (3.03 – 3.28)
ErC50 6.15 (5.74 – 6.62)

Yield Inhibition
Geometric mean measured test item concentration [mg/L]
NOEC 0.138
LOEC 0.390
EyC10 0.592 (0.479 – 0.725)
EyC20 1.04 (0.896 – 1.19)
EyC50 2.49 (2.31 – 2.70)

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours. The reference item toxicity is in the valid range following test facility SOPS.

Set-up and
Experimental Date with Headspace without Headspace Valid Range
(average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.559 0.811 0.773 ± 0.540
95% confidence interval 0.538 - 0.581 0.763 - 0.883

Yield Inhibition
EyC50 0.320 0.387 0.406 ± 0.287
95% confidence interval 0.242 - 0.341 0.347 - 0.428

Any other information on results incl. tables

Physico-chemical Data

The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits. The test media were clear throughout the exposure phase.

Geometric mean measured test item concentration		pH-values
[mg/L]		Start; 0 hours	End; 72 hours
10.2		8.14	8.37
3.22		8.10	8.77
1.02		8.15	9.31
0.390		8.22	9.38
0.138		8.20	9.41
Control		8.26	9.42
Room temperature [°C]:	min.: 22.0	max.: 23.0		mean value: 22.5
Light intensity [lux]	n = 31	mean value: 5787 range of the measured values: 4966 to 6412 equalling -14.2 to 10.8 %		CV [%]: 7.27

CV = Coefficient of variation                   n = number of measuring points

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean measured test item concentration):
The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 6.15 (5.74 – 6.62) mg/L and 2.49 (2.31 – 2.70) mg/L, respectively.
The NOEC-values for both inhibition of growth rate and yield after 72 hours were 0.138 mg/L, respectively.

Executive summary:

The toxicity of test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2017-04-18 to2017-04-21 at the test facility.The aim of the study was the determination of NOEC, LOEC, EC10, EC20- and EC50-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions in closed test vessels with an initial cell density of 6839 cells/mL. A stock solution with a nominal loading of 30 mg test item/L was prepared directly prior to the start of the exposure. Five dilution levels of the stock solution were tested in a geometrical series with a dilution factor of √10:0.150 - 0.474 - 1.50 - 4.74 - 15.0 mg/L, corresponding to the geometric mean measured test item concentrations 0.138 – 0.390 – 1.02 – 3.22 – 10.2 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

 

The concentrations of test item were determined at the start of the exposure (0 hours) and at the end of the exposure (72 hours) in all tested concentrations and the control via GC-MS.

The measured concentrations of test item were in the range of 61 – 91% at test start and between 67 and 92% at test end.

 

All effect values given are based on the geometric mean measured test item concentrations.

The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 6.15 (5.74 – 6.62) mg/L and 2.49 (2.31 – 2.70) mg/L, respectively.

The NOEC-values for both inhibition of growth rate and yield after 72 hours were 0.138 mg/L, respectively.