Cyclooctane

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-01-11 to 2022-01-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified via GC-MS at the start (0 hours) and at the end of the exposure (72 hours) with algae. Separate replicates for the test item analysis after 0 and 72 hours were prepared in the original test vessels with algae at the start of the exposure and incubated under test conditions until analysis.

Vehicle:

yes

Remarks:

Dilution water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal loading rate of 100 mg test item/L was prepared at room temperature 24 ± 1 hour prior to the start of the exposure and the renewal of the test solutions. An appropriate amount of the test item was pipetted onto the surface of the dilution water. For this purpose, the density was taken into account. A slow stirring procedure was applied for 24 ± 1 hour at room temperature. After a separation phase of 1 hour at room temperature, the saturated solution was removed by siphoning from the bottom of the glass bottle and was used in the test. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. Out of the saturated solution 5 concentrations were tested in a test item solutions geometric series with a dilution factor of 2.8: 0.349, 0.976, 2.73, 7.65, 21.4, 60 % of the saturated solution.
- Controls:
Control: Dilution water without test item incubated under the same conditions as the test groups.
Reference Test: Potassium dichromate was tested as a reference item in a separate study. The toxicity to the unicellular freshwater green alga Pseudokirchneriella subcapitata wasdetermined over a period of 72 hours (with and without headspace).
- Test concentration separation factor: 2.8
- Evidence of undissolved material: No.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata, green algae
- Strain: HINDÁK CCAP 278/4 (axenic)
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Age of inoculum (at test initiation): A four days old preculture, prepared in dilution water and incubated under test conditions, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lux for 24 hours per day.

ACCLIMATION
Acclimatization was not necessary, because the preculture was prepared in dilution water and incubated under test conditions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

40 mg CaCO3/L

Test temperature:

21.0 - 22.0 °C

pH:

8.09 - 9.67

Dissolved oxygen:

Not specified

Salinity:

Not applicable

Conductivity:

136 µS/cm

Nominal and measured concentrations:

5.76, 20.0, 63.1, 184, 636, 1880 μg/L (geometric mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks, with aluminium tops with PTFE seals
- Type: closed
- Material, size, headspace, fill volume: size and volume: 59 mL, no headspace
- Aeration: Not specified
- Renewal rate of test solution: Not applicable
- Initial cells density: Nominal: approximately 5 x 10^3 – 10^4 cells/mL, actual: 6864 cells/mL
- Control end cells density:
- No. of vessels per concentration (replicates): 3, Separate replicates for each measuring time were prepared.
- No. of vessels per control (replicates): 6, Separate replicates for each measuring time were prepared.

GROWTH MEDIUM
- Standard medium used: yes, composition of Dilution water was according to OECD TG 201 medium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Ultrapure water was used to prepare the dilution water (conductivity max. 0.1 μS/cm).
- Culture medium different from test medium: No.
- Intervals of water quality measurement: Not specified
- Total oranic cabon: < 2.0 mg C/L
- Acidity: 0.2 mmol/L
- Alkalinity: 0.6 mmol/L

OTHER TEST CONDITIONS
- Sterile test conditions: A sterile flask was used.
- Adjustment of pH: No.
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux (measured: 4483 to 5750 lux), corresponding to 60 to 120 μE*m-2*s^-1; light homogeneity: within ± 15 % over incubation area
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm. The flasks were positioned randomly and repositioned daily.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: fluorimeter
- Chlorophyll measurement: 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 observed in the preliminary range finding test at the saturated solution (100 mg/L).
- Other: 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.8
- Range finding study: A preliminary range finding test under static conditions over a period of 72 hours in a closed system without headspace was conducted at the test facility with a saturated solution with a nominal loading rate of 100 mg/L and two dilution levels. Two replicates per concentration and four for the control were tested. All concentration levels were visually clear throughout the exposure period.
- Test concentrations of range finding: 0.800, 8.00 and 80.0 % of the saturated solution (100 mg/L); equal to mean measured: 34.9, 372, 3077 µg/L
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate was tested as a reference item in a separate study.

Duration:

72 min

Dose descriptor:

LOEC

Effect conc.:

<= 5.76 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 5.76 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

227 µg/L

95% CI:

191 - 271

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

73.2 µg/L

95% CI:

52.5 - 98.7

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

25.2 µg/L

95% CI:

>= 5.76 - 47.5

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

63.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

20 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

264 µg/L

95% CI:

234 - 294

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

717 µg/L

95% CI:

674 - 767

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

128 µg/L

95% CI:

104 - 156

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes, increase in cell growth was 44 fold (growth rate 1.26/day).
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start and at the end revealed no morphological abnormalities.
- Any stimulation of growth found in any treatment: Yes. In one replicate treated with 5.76 µg test item/L a negative inhibition growth rate of -1, which equals a growth stimulation, was detected. No other replicates at the same concentration nor any other concentration showed this effect.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No.

Results with reference substance (positive control):

The toxicity for the reference item potassium dichromate (SIGMA-ALDRICH, batch
number BCCC1619, purity 100 %, CAS RN 7778-50-9) was determined over a period of 72 hours in a separate test without headspace. The EC50 was 1.28 mg/L (95 % confidence interval 1.04 - 1.36 mg/L) for growth rate inhibition and 0.661 mg/L (95 % confidence interval 0.573 - 0.783 mg/L) for yield inhibition.

Validity criteria fulfilled:

yes

Conclusions:

The EC50-value for inhibition of growth rate (ErC50) after 72 hours was 717 (674 – 767) μg/L and the EC10-value for inhibition of growth rate (ErC10) after 72 hours was 128 (104 – 156) μg/L.

Executive summary:

The toxicity of the 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. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions in a closed system without headspace with an initial cell density of 6864 cells/mL. A saturated solution with a nominal concentration of 100 mg test item/L was freshly prepared with dilution water. Out of the saturated solution six concentration levels were tested in a geometrical series with a dilution factor of 2.8: 0.349, 0.976, 2.73, 7.65, 21.4, 60.0 % of the saturated solution, corresponding to the geometric mean measured test item concentrations of 5.76, 20.0, 63.1, 184, 636, 1880 μg/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 test item concentrations and the control of the test item were determined in fresh media (0 hours) and at the end of the exposure interval (72 hours) via GC-MS/MS. At the start of the exposure the measured concentrations of the test item were in the range of 6.43 μg/L to 2220 μg/L. At the end of exposure after 72 hours, the measured concentrations were in the range of 72 % to 88 % of the initially measured concentrations. The geometric mean measured concentrations were calculated. All effect values given are based on the geometric mean measured test item concentrations. The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 717 (674 – 767) μg/L and 227 (191 – 271) μg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 128 (104 – 156) μg/L and 25.2 (< 5.76 – 47.5) μg/L, respectively. The NOEC for growth rate was 20.0 μg/L. The NOEC for yield was < 5.76 μg/L.

Description of key information

The EC50-value for inhibition of growth rate (ErC50) after 72 hours was 717 (674 – 767) μg/L and the EC10-value for inhibition of growth rate (ErC10) after 72 hours was 128 (104 – 156) μg/L (reference 6.1.5-1).

Key value for chemical safety assessment

EC50 for freshwater algae:

717 µg/L

EC10 or NOEC for freshwater algae:

128 µg/L

Additional information

The toxicity of the 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. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions in a closed system without headspace with an initial cell density of 6864 cells/mL. A saturated solution with a nominal concentration of 100 mg test item/L was freshly prepared with dilution water. Out of the saturated solution six concentration levels were tested in a geometrical series with a dilution factor of 2.8: 0.349, 0.976, 2.73, 7.65, 21.4, 60.0 % of the saturated solution, corresponding to the geometric mean measured test item concentrations of 5.76, 20.0, 63.1, 184, 636, 1880 μg/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 test item concentrations and the control of the test item were determined in fresh media (0 hours) and at the end of the exposure interval (72 hours) via GC-MS/MS. At the start of the exposure the measured concentrations of the test item were in the range of 6.43 μg/L to 2220 μg/L. At the end of exposure after 72 hours, the measured concentrations were in the range of 72 % to 88 % of the initially measured concentrations. The geometric mean measured concentrations were calculated. All effect values given are based on the geometric mean measured test item concentrations. The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 717 (674 – 767) μg/L and 227 (191 – 271) μg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 128 (104 – 156) μg/L and 25.2 (< 5.76 – 47.5) μg/L, respectively. The NOEC for growth rate was 20.0 μg/L. The NOEC for yield was < 5.76 μg/L.