2,2-bis[(allyloxy)methyl]butyl (3-{[({2,2-bis[(allyloxy)methyl]butoxy}carbonyl)amino]methyl}-3,5,5-trimethylcyclohexyl)carbamate

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:

29 August 2009 to 13 October 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Version / remarks:

1989

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 201 Alga Growth inhibition Test. China Environmental Science Press.

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentration: 100 mg/L
- Sampling method: the actual concentration of the 100mg/L test material was analysed at the start and the end of the test.
- Sample storage conditions before analysis: -20°C

Vehicle:

yes

Remarks:

Carbinol

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
2 g of test material was dissolved in 2 g Carbinol. The total volume of the solution was 4.1 mL, measured with a 10 mL graduated cylinder. A 1:1 (mass ratio) test stock solution of test material and carbinol was obtained for the limit test.

Test organisms (species):

Selenastrum sp.

Details on test organisms:

TEST ORGANISM
- Strain: Selenastrum capricornutum
- Source (laboratory, culture collection): bought from FACHB-Collection and cultured in the laboratory
- Age of inoculum: Logarithmic growth phase alga cells

ACCLIMATION
- the pre-culture was incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about 3 days. When the alga cultures contained deformed or abnormal cells, they were be discarded.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

20 ± 2 °C

pH:

7.74 to 9.23

Nominal and measured concentrations:

Nominal: 100 mg/L
Measured: 1.95 (0h) and 1.84 (72 h) µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: The test vessels were cleaned, sterilized, and marked before the test..
- Initial cells density: 10^4 cells/mL (± 25 %)
- No. of vessels per concentration (replicates): 7
- No. of vessels per blank control (replicates): 7
- No. of vessels per solvent control (replicates): 7
- No. of vessels per reference control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Test water- Sterile deionised water
- The deionised water was used to prepare the following solutions:
NH4Cl: 15 mg/L
MgCl2.6H2O: 12 mg/L
CaCl2.2H2O: 18 mg/L
MgSO4.7H2O: 15 mg/L
KH2PO4: 1.6 mg/L
FeCl3.6H2O: 80 µg/L
Na2EDTA.2H2O: 100 µg/L
H3BO3: 185 µg/L
MnCl2.4H2O: 415 µg/L
ZnCl2: 3 µg/L
CoCl2.6H2O: 1.5 µg/L
CuCl2.2H2O: 0.01 µg/L
Na2MoO4.2H20: 7 µg/L
NaHCO3: 50 mg/L.

- The stock solutions were sterilised by membrane filtration (mean pore diameter 0.45 µm) or autoclaving (120°C, 15 min) and were stored in the dark at approximately 4°C until use.

OTHER TEST CONDITIONS
- Light intensity and quality: 8120 to 8230 Lux

METHOD
- Alga culture: alga was pre-cultured, and the alga biomass was measured and diluted to 2 X 10^4 cells/mL by culture medium.
-Test solutions:
Test substance group : 50 mL algal culture and 20 mL stock solution were added to a vessel . The solution was diluted with culture medium to 100 mL, with final algal concentration of 10^4 cells/mL.
Solvent control group: 50 mL algae and 10 mg carbinol were added to a vessel and the solution was diluted with culture medium to 100 mL.
Blank control was composed of 50 mL algae solution and 50 mL culture medium.

-The test vessels were shaken and placed in the culturing apparatus to start the test.
-The algae biomass in each flask was determined at 0, 24, 48 and 72 hours. Algae biomass was measured by spectrophotography
- Measurements: pH, temperature and light intensity were determined at 0, 24, 48 and 72 hours.
- Analysis of test concentrations: the actual concentration of the 100 mg/L test solution was analysed at the start and the end of the test.

TEST CONCENTRATIONS
- Test concentration: 100 mg/L

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- The growth inhibition of nominal concentration of 100 mg/L of the test material and solvent control group on the test algae (Selenastrum capricornutum) were both below 50%.
- The 72h-EC50 of the test material based on the nominal concentration was > 100 mg/L.

QUALITY ASSURANCE
- The concentration of solvent had not exceeded 100 mg/L.
- The biomass in the control cultures had increased by a factor of more than 16 within the 72-hour test period.
- The 72h-EC50 of the reference substance on the selected alga was 0.88 mg/L, within the range of 0.84 mg/L ± 0.12 mg/L that meets the requirement of the tests.

Results with reference substance (positive control):

The 72h-EC50 of the reference substance on the selected alga was 0.88 mg/L, within the range of 0.84 mg/L ± 0.12 mg/L that meets the requirement of the tests.

Table 1: Percentage inhibition of algal growth during the test

Group	Density of algae cells (mL)				72 h inhibition percentage of algae growth (%)
	0 h	24 h	48 h	72 h
Blank control	0.94 x 10^4	5.42 x 10^4	1.80 x 10^5	5.24 x 10^5	-
Solvent control	1.00 x 10^4	5.39 x 10^4	1.97 x 10^5	5.19 x 10^5	-
Test material	0.97 x 10^4	5.64 x 10^4	1.89 x 10^5	5.65 x 10^5	-

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 72 h EC50 of the test material based on the nominal concentration was > 100 mg/L.

Executive summary:

The acute toxicity of the test material to algae was determined in accordance with the standardised guidelines "State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 201 Alga Growth inhibition Test" and OECD 201 under GLP conditions. The limit test was conducted with a normal concentration of 100 mg/L, a blank control and a solvent control group with seven replicates for each group. The effects of the test material on the growth of algae was measured by spectrophotography and were recorded at 24, 48 and 72 hours after starting the test. Analysis of the samples taken from the test solution showed that measured concentration of the test material was 1.95 µg/L and 1.84 µg/L at 0 and 72 hours, respectively. All of the quality assurance criteria of the study were met making it valid. Under the conditions of the study the 72 h EC50 of the test material, based on the nominal concentration, was > 100 mg/L.

Description of key information

Under the conditions of the study the 72 h EC50 of the test material, based on the nominal concentration, was > 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

Additional information

The acute toxicity of the test material to algae was determined in accordance with the standardised guidelines "State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 201 Alga Growth inhibition Test" and OECD 201 under GLP conditions. The limit test was conducted with a normal concentration of 100 mg/L, a blank control and a solvent control group with seven replicates for each group. The effects of the test material on the growth of algae was measured by spectrophotography and were recorded at 24, 48 and 72 hours after starting the test. Analysis of the samples taken from the test solution showed that measured concentration of the test material was 1.95 µg/L and 1.84 µg/L at 0 and 72 hours, respectively. All of the quality assurance criteria of the study were met making it valid. Under the conditions of the study the 72 h EC50 of the test material, based on the nominal concentration, was > 100 mg/L.