Bis[2-[2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexan-1,2-diylbiscarbamate

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:

2012-03-16 to 2012-03-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(2006)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: "Alga growth inhibition test, Daphnia sp. acute immobilisation test, and fish acute toxicity test" Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No.110331009, March 31, 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Based on the results of the range-finding test, the maximum concentration for the definitive test was set to 100 mg/L.
- Sampling method: The test substance dissolved in acetone was collected to a vessel, then acetone was dried up under nitrogen flow, and the dilution water was added. It was stirred with a magnetic stirrer for 48 hours.
- Sample storage conditions before analysis: During the study, the test substance was stored in a desiccator in the laboratory (storage condition: room temperature, dark, nitrogen-sealed).
- Sampling method: The medium was sterilized by membrane filtration (pore diameter 0.22 μm) pH of the medium 8.0 ± 0.2.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Mixing method: the test substance 500 mg dissolved in aceton 20 mL was collected to a vessel. It was stirred with a magnetic stirrer for 48 hours before the start of exposure. Then acetone was dried up under nitrogen flow and the dilution water was added to prepare the different concentrations. An extra vessel for the pH measurement and color observations was prepared (1 vessel/test group, named extra vessel). In addition, vessels for analysis of the concentrations of the test substance were also prepared (1 vessel/test group, named vessels for collecting analysis sample at the beginning of exposure).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: ATCC22662
- Source: American Type Culture Collection
- Age of inoculum: receipt June 20, 1996
- Method of cultivation: Algae have been maintained by period subculture using Gorham medium. Algae are kept under sterile condition.
- Initial biomass: 5E3 cells/mL, algae of exponential growth phase (dried weight of cells at the exponential growth phase: 1.8E-8 mg/cell, n=14)

ACCLIMATION
- Acclimation period: From March 13 to 16, 2012
- Culturing media and conditions: Same as
- Any deformed or abnormal cells observed:No
- Temperature: 22 ± 2 °C
- Light: 60 to 65 μE/m2/s, continuously illuminated with white fluorescent lamp (on the surface of test culture)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 ± 2 °C

pH:

pHs of test solutions at the beginning of exposure were 7.8 to 8.7 and test cultures at 72 hours after the beginning of exposure were 8.4 to 9.8

Nominal and measured concentrations:

Nominal concentrations: 1.9, 7.2, 27, 100 mg/L
Measured concentrations (mean 0 h - 72 h): 1.66, 7.12, 24.3, 87.95 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask with stopper, Iwaki, Asahi Glass Co., Ltd.
- Type: closed system, static
- Material, size, headspace, fill volume: 100 mL/vessel
- No. of organisms per vessel: 5000 cells/mL (0 hour)
- No. of vessels per concentration: 3
- No. of vessels per control: 6
- Additional vessels: 1 extra vessel/test group, 1 vessel for collecting analysis sample at the beginning of exposure/test group

GROWTH MEDIUM
- Standard medium used: yes ,OECD medium recommended by the Test Guideline
The medium was sterilized by membrane filtration (pore diameter 0.22 μm).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium recommended by the Test Guideline was used. The medium was sterilized by membrane filtration (pore diameter 0.22 μm) pH 8.0 ± 0.2
- Total organic carbon: test concentration measured at the beginning and after the exposure
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: 60 to 65 μE/m2/s white fluorescent lamp (on the surface of test culture)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations:
Apparatous: Electronic particle counter and a hemacytometer on microscope
Frequency: 24 h, 48 h, 72 h
- Test solution measurement: Sum concentration of the test substance and the transformation products was quantified by total organic carbon (TOC) analyzer (0 hour and 72 hour)
- Other: Temperatures, light intensities and revolutions in the culturing apparatus were measured at least once a day

TEST CONCENTRATIONS
- Test concentrations: Nominal 100, 27, 7.2, 1.9 mg/L
- Results used to determine the conditions for the definitive study: Due to range-finding test the the test solution prepared by stirring for 48 hours without solvent was used in the definitive test.

Statistical method used for determining results:
No observed effect concentration (NOEC): Bartlett test for homogeneity of variances
50 % growth inhibition concentration (EC50): By the least squares linear regression analysis of points recognized as straight in the concentration-inhibition curves

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 16.0 – 21.6 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.026 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.194 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 0.125 − 0.233 mg/L)

Details on results:

Conditions of test solution and appearance
- pH (control group): Increased by more than 1.5 units during the test. In test cultures of closed system, the concentration of bicarbonate ion was decreased by carbon dioxide assimilation and therefore, the pH was increased.
- Appearance: Test solutions before inoculation were colorless, and showed no suspended solids, no floating solids, and no precipitates in all test groups.

Concentrations in Test Solutions and Test Cultures
The measured concentrations (sum of the test substance and the transformation products) in concentration group 1 to 8 at the beginning of exposure were 0.00700, 0.0259, 0.0980, 0.364, 1.33, 7.12, 24.8, and 90.6 mg/L, respectively. Since the measured concentrations in concentration group 1 to 4 were below 1 mg/L, these values were calculated based on both of the measured concentration of concentration group 5 and adding volume of stock solution.

Observation of Algae
In control group and concentration group 1 to 6, color of test cultures observed with naked eye showed a tendency to get more greenish during the exposure period. Although the colour of test cultures slightly changed in concentration group 7, there was no tendency to get greenish. The color change was not observed in the concentration group 8. By microscopic observation at the end of exposure, in all concentration groups neither unusual cell shape of algae (contraction, expansion, damaged cell etc.) nor agglutination was observed, and the algae looked normal compared with that in the control group.

Results with reference substance (positive control):

Growth inhibition test of a reference substance (potassium dichromate, guaranteed reagent) latest value of ErC50 = 0.878 mg/L (95 % confidence limit: 0.828 - 0.931 mg/L).

Reported statistics and error estimates:

The EC50 value is determined by the least squares linear regression analysis of points recognized as straight in the concentration-inhibition curves. The 95 % confidence limits were also calculated. The NOECr value was determined by an analysis of variance (ANOVA), Williams test, subsequent to Bartlett test for homogeneity of variances. All tests of significance level were set at α = 0.05, except Bartlett test, which was at α = 0.01.

Validity criteria fulfilled:

yes

Conclusions:

The algae growth inhibition test was conducted according to the OECD guideline no. 201. The test item Incozol 4 inhibits the growth of 50 % of the alga at a concentration of ErC50 (0 - 72 h): 18.6 mg/L (95 % confidence limits: 16.0 – 21.6 mg/L). The no observed effect concentration, NOECr (0 - 72 h) was estimated to be 0.0259 mg/L. The EC10 was calculated to be 0.194 mg/L.

Executive summary:

The study was conducted in accordance with the test method relating to new chemical substances “Alga growth inhibition test, Daphnia sp. acute immobilisation test, and fish acute toxicity test” (Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No.110331009, March 31, 2011) and the OECD Guidelines for the Testing of Chemicals No.201. The test substance is poorly water-soluble. In range-finding tests, a relationship between stirring time after adding the test substance to the dilution water and effect on algae was examined. As a result, a greater growth inhibition was observed in the test solution prepared by stirring for 48 hours than 5 minutes and 24 hours stirring. Therefore, the test solution prepared by stirring for 48 hours was used in the definitive test. The test organism Pseudokirchneriella subcapitata, unicellular green algae was exposed by a static, closed system for a period of 72 hours with the test item. The following substance concentrations (nominal) were used: 100, 27, 7.2, 1.9 mg/L. Based on the growth rate (μ) the percent inhibition (Iμ) for each concnetration was calculated. The 50 % growth inhibition concentration ErC50 (0 - 72 h) was measured to be 18.6 mg/L and the associated 95 % confidence limits 16.0 – 21.6 mg/L. These values were determined by least squares linear regression analysis of the logarithm of test concentration against the growth inhibition (%) relative to the control. The NOECr (0 - 72 h) value was determined to be 0.0259 mg/L. The EC10 was calculated to be 0.194 mg/L. Due to the big diffenrence between EC50 and NOEC value the classification and labelling is based on the more reliable EC10 value.

Description of key information

The algae growth inhibition test was conducted according to the OECD guideline no. 201. The test item Incozol 4 inhibits the growth of 50 % at a concentration of ErC50 (0 - 72 h): 18.6 mg/L (95 % confidence limits: 16.0 – 21.6 mg/L). The no observed effect concentration, NOECr (0 - 72 h) was estimated to be 0.0259 mg/L. The EC10 was calculated to be 0.194 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

18.6 mg/L

EC10 or NOEC for freshwater algae:

0.194 mg/L

Additional information

The study was conducted in accordance with the test method relating to new chemical substances “Alga growth inhibition test, Daphnia sp. acute immobilisation test, and fish acute toxicity test” (Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No.110331009, March 31, 2011) and the OECD Guidelines for the Testing of Chemicals No.201. The test substance is poorly water-soluble. In range-finding tests, a relationship between stirring time after adding the test substance to the dilution water and effect on algae was examined. As a result, a greater growth inhibition was observed in the test solution prepared by stirring for 48 hours than 5 minutes and 24 hours stirring. Therefore, the test solution prepared by stirring for 48 hours was used in the definitive test. The test organism Pseudokirchneriella subcapitata, unicellular green algae was exposed by a static, closed system for a period of 72 hours with the test item. The following substance concentrations (nominal) were used: 100, 27, 7.2, 1.9 mg/L. Based on the growth rate (μ) the percent inhibition (Iμ) for each concnetration was calculated. The 50 % growth inhibition concentration ErC50 (0 - 72 h) was measured to be 18.6 mg/L and the associated 95 % confidence limits 16.0 – 21.6 mg/L. These values were determined by least squares linear regression analysis of the logarithm of test concentration against the growth inhibition (%) relative to the control. The NOECr (0 - 72 h) value was determined to be 0.0259 mg/L. The EC10 was calculated to be 0.194 mg/L. Due to the big diffenrence between EC50 and NOEC value the classification and labelling is based on the more reliable EC10 value.