2-(1-(2-hydroxy-3,5-di-tert-pentyl-phenyl)ethyl)-4,6-di-tert-pentylphenyl acrylate

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:

23.02.2010 - 16.04.2010

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate included in the full study report

Test material

Test material information

Constituent 1

Reference substance name:

2-(1-(2-hydroxy-3,5-di-tert-pentyl-phenyl)ethyl)-4,6-di-tert-pentylphenyl acrylate

EC Number:

413-850-6

EC Name:

2-(1-(2-hydroxy-3,5-di-tert-pentyl-phenyl)ethyl)-4,6-di-tert-pentylphenyl acrylate

Cas Number:

123968-25-2

Molecular formula:

C37H56O3

IUPAC Name:

2-{1-[2-hydroxy-3,5-bis(2-methylbutan-2-yl)phenyl]ethyl}-4,6-bis(2-methylbutan-2-yl)phenyl prop-2-enoate

Constituent 2

Reference substance name:

ZIKANOX-549

IUPAC Name:

ZIKANOX-549

Details on test material:

Name: ZIKANOX-549
Manufacturer: ZIKO Ltd.
Supplier: ZIKO Ltd.
Manufacture date: 2009-09-10
Receipt date: 2009-10-27
Receipt quantity: 248.988 g (gross weight)
Appearence: off white powder
Lot Number: 10909001-003
CAS Number: 123968-25-2
Purity: 99.5 %
Storage condition: room temperature
Expiration date (after manufacture): 3 years

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The solution was leaved under same condition with the toxicity test and sampled 300 mL every 24 hours.

Test solutions

Vehicle:

no

Details on test solutions:

0.010, 0.030, 0.090, 0.271 and 0.814 g of test substance, respectively were put into 1000 mL of volumetric flask and then test medium was filled up to the 1000 mL marker to prepare stock solutions. And the stock solutions were sonicated for 30 minutes. The stock solution was filtered through 0.45 µm membrane filter. The stock solution was divided 100 mL each (test solution).

Test organisms

Test organisms (species):

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

Details on test organisms:

Freshwater algae (Pseudokirchnerella subcapitata, ATCC22662)
Supplier: American Type Culture Collection (ATCC)
Receipt date: November 17, 2006

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no data

Test conditions

Hardness:

no data

Test temperature:

The mean of temperature was 21.9 °C (21.6°C ~ 22.4 °C)

pH:

During the definitive test period, the mean of pH was (8.57 ± 0.24) (8.22 ~ 8.93)

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

- As the result of analysis at 0 hour, the concentrations of the test substance in test medium were under detection limit (0.002 mg/L) at 10.00 mg/L treatment group. At 30.00, 90.00, 270.00 and 810.00 mg/L treatment groups, the test substance in test medium were detected 0.003, 0.019, 0.060 and 0.323 mg/L and 0.0, 0.0, 0.0 and 0.0 % of nominal concentration, respectively.

- As the result of analysis at 24 hours, the concentrations of the test substance in test medium were under detection limit (0.002 mg/L) at 10.00 and 30.00 mg/L treatment groups. At 90.00, 270.00 and 810.00 mg/L treatment groups, the test substance in test medium were detected 0.011, 0.047 and 0.260 mg/L and 0.0, 0.0 and 0.0 % of nominal concentration, respectively.

- As the result of analysis at 48 hours, the concentrations of the test substance in test medium were under detection limit (0.002 mg/L) at 10.00 and 30.00 mg/L treatment groups. At 90.00, 270.00 and 810.00 mg/L treatment groups, the test substance in test medium were detected 0.010, 0.050 and 0.211 mg/L and 0.0, 0.0 and 0.0 % of nominal concentration, respectively.

- As the result of analysis at 72 hours, the concentrations of the test substance in test medium were under detection limit (0.002 mg/L) at 10.00 and 30.00 mg/L treatment groups. At 90.00, 270.00 and 810.00 mg/L treatment groups, the test substance in test medium were detected 0.003, 0.016 and 0.096 mg/L and 0.0, 0.0 and 0.0 % of nominal concentration, respectively.

- The geometric mean of concentrations were under detection limit (0.002 mg/L) at 10.00 mg/L treatment groups. At 30.00, 90.00, 270.00 and 810.00 mg/L treatment groups, the test substance in test solution were detected 0.003, 0.009 and 0.039 and 0.203 mg/L during the test periods.

Details on test conditions:

- Monitoring of culturing conditions: The temperature in the incubator was recorded automatically every 1 hour using TR-71U thermo-recorder (TND Co., Japan). The light intensities were measured at the 5 positions in the incubator using ANA-11 digital lux meter twice a week (Tokyo Koden Co., Japan)
- Test concentration: According to the results of range-finding test; 0.10, 1.00, 10.00 and 100.00 mg/L treatment, the percent inhibition in average specific growth rate were -5.6, -3.9, -1.5 and 4.2 %, respectively. The percent inhibition of yield were -9.1, -2.5, -5.7 and 24.0 %, respectively. Therefore, the test was performed in 10.00, 30.00, 90.00, 270.00 and 810.00 mg/L (geometric series 3.0, nominal concentration).
Control:
- Negative control: M4 medium was used for negative control.
- Positive control: The test was performed in control, 0.10, 0.18, 0.32, 0.58 and 1.05 mg/L (geometric series 1.8, nominal concentration) as active ingredient of Potassium dichromate (Sigma-aldrich, Lot No. 09514PA) as positive control (January 26, 2010 ~ January 29, 2010).
- Exposure condition: The pre-culture was incubated under the conditions of the test and used when exponentially growing, after an incubation period of 5 days. The pre-cultured algae was inoculated in test medium with concentration of 10 000 cells/mL. The vessels were shaken and placed in the culturing apparatus (J-SCI, Jisico Co., Korea). The test mediums were maintained at a temperature in the range of (21 ~ 24) °C. The surface of cultures were received continuous, uniform fluorescent illumination in the range of (4440 ~ 8880) Lux. The test vessels were 250 mL of glass flasks.
- Growth medium: The growth medium was prepared by Annex 3, OECD test guideline 201.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

426.88 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (231.204 ~ 1118.894) mg/L of 95 % confidence limits

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

26 424.547 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (5352.576 ~ 1849584.250) mg/L of 95 % confidence limits

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

90 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

270 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

64.271 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: (36.522 ~ 96.152) mg/L of 95 % confidence limits

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1 689.233 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: (958.200 ~ 4169.265) mg/L of 95 % confidence limits

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

90 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Cell concentration and growth inhibition rate: The mean of the initial cell concentration in the test medium was (1.13 ± 0.11) X 10 000 cells/mL.
The definitive test was conducted in nominal concentrations of 10.00, 30.00, 90.00, 270.00 and 810.00 mg/L. After 72 hours exposure, the percent inhibition in average specific growth rate were -1.3, 1.3, 4.9, 11.6 and 10.4 %, respectively.
- The percent inhibiton of yield were -5.1, 4.9, 16.9, 29.4 and 32.4 %, respectively.
- The growth curves of cell concentration in each test medium were described (please refer to the full study report).

Results with reference substance (positive control):

no data

Reported statistics and error estimates:

Refer to the attached full report.

Any other information on results incl. tables

- No abnormal appearance was observed in the control. The lysis was observed in 810.00 mg/L treatment group during the test periods.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

- The cell concentration in the control cultures were increased exponentially by a factor of 39 within the 72-hours test periods. - The change of pH in the control medium was 0.42. - The coefficient of variation of average specific growth rates during t

Conclusions:

- The 72 hours-ErC10 value of ZIKANOX-549 to Pseudokirchneriella subcapitata were investigated as 426.880 mg/L with (231.204 ~ 1118.894) mg/L of 95 % confidence limits, the 72 hours-ErC10 value were investigated as 26424.547 mg/L with (5352.576 ~ 1849584.250) mg/L of 95 % confidence limits. The NOEC of test substance to Pseudokirchneriella subcapitata was 90.00 mg/L and LOEC was 270.00 mg/L at 72 hours.
- The 72 hours-EyC10 value of test substance to Pseudokirchneriella subcapitata were investigated as 64.271 mg/L with (36.522 ~ 96.152) mg/L of 95 % confidence limits, the 72 hours-EyC50 value were investigated as 1689.233 mg/L with (958.200 ~ 4169.265) mg/L of 95 % confidence limits. The NOEC of test substance to Pseudokirchneriella subcapitata was 30.00mg/L and LOEC was 90.00 mg/L at 72 hours. Determinations of EC10, EC50, NOEC and LOEC were based on nominal concentration. During the test periods, there was no disturbance which had influenced results.

Executive summary:

- The freshwater alga, growth inhibition test of ZIKANOX-549 was studied using Pseudokirchneriella subcapitata for 72 hours under the static system. Observations were made on the concentration of algae in the test medium, abnormal response of Pseudokirchneriella subcapitata.

- This study was conducted in accordance with the OECD guideline for the testing of chemicals 201, "Freshwater Alga and Cyanobacteria, growth inhibition test" (Adopted: 23 March, 2006)

- The toxicity test was performed in control, 10.00, 30.00, 90.00, 270.00 and 810.00 mg/L (geometric series 3.0, nominal concentration) as active ingredient of ZIKANOX-549 as definitive test. The analyses of test substance in the test medium were performed at the test start (0 hour), 24 hours, 48 hours and the test end (72 hours).

- As the analysis of the ZIKANOX-549 in test solution, the mean concentrations of ZIKANOX-549 were under detection limit (0.002 mg/L) at 10.00 mg/L treatment groups. At 30.00, 90.00, 270.00 and 810.00 mg/L treatment groups, the test substance in test solution were detected 0.003, 0,009, 0,039 and 0,203 mg/L.

- The lysis was observed in 810.00 mg/L treatment group during the test periods

- The mean of pH was (8.57 ± 0.24) (8.22 ~ 8.93)

- The mean of light intensity was 6687 Lux (5740 Lux ~ 7910 Lux)

- The mean of temperature was 21.9 °C (21.6 °C ~ 22.4 °C)