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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-12-05 to 2002-03-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
A certain amount of the solution was collected, at the starting of exposure, from an extra container prepared separately from three containers for each concentration group, and a certain amount of the solution was collected from three containers for each concentration and mixed at the end of exposure (72h), and the concentration of the test solution for each concentration group was analyzed by gas chromatography.

Vehicle:
no
Details on test solutions:
500 mg of 3-methoxy-3-methyl-1-butanol was placed into a 50 mL measuring flask and diluted with test medium to prepare 1000 mg/L of test stock solution. After filtering and sterilization of the test stock solution, 10 mL of the solution was placed into a 300 ml conical flask containing 90 mL sterilized test medium to prepare the test solution
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: ATCC22662
- Source (laboratory, culture collection): American Type Culture Collection
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Aseptic subculture using C medium

ACCLIMATION
- Acclimation period: 2002-01-25 to 2002-01-29
- Culturing media and conditions (same as test or not): same as test, medium indicated in the OECD Guideline for testing of chemicals was used.
- Any deformed or abnormal cells observed: During preculture period, the alga showed logarithmic growth
Test type:
other: static, shake culture (100rpm)
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
23 to 23.1°C
pH:
pH of the test solution ranged from 8.8 to 8.9 at the starting of exposure and 9.9 to 10.0 at the end of the test.

pH of the test solution in an extra container prepared separately from three containers for each concentration group was measured at the starting of exposure, and pH of the test solution of three containers measured at the end of exposure (72 hours).
Nominal and measured concentrations:
The measured concentration of the test substance was within ±20% of the nominal value
Details on test conditions:
TEST SYSTEM
- Test vessel: 300mL glass conical flask
- Material, size, headspace, fill volume: 300 mL
- Initial cells density: Pre-cultured alga, 1 X 10^4 cells/mL
- No. of organisms per vessel: 300 * 10^4
- No. of vessels per concentration (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Special grade chemical, for HPLC, Wako Pure Chemical Industries, Ltd.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: Continuous lighting
- Light intensity and quality: 4000 to 5000 lx (near the liquid surface in the flask)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Measurement of the cell density was done 24, 48 and 72 hours after starting. The cell density was measured, using a hemocytometer, after collection of about 1mL of the test solution from each test container.

TEST CONCENTRATIONS
- Test concentrations: 1000 mg/L
- Results used to determine the conditions for the definitive study: Based on the results of the preliminary test, the 100 mg/L concentration group and control group were set.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (0.82 mg/L); not under test conditons, data from the quality control of the organisms (n=8)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Basis for effect:
biomass
Remarks on result:
other: Inhibition concentrations by comparison of area under the growth curve; The concentraion obtained by the area method
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Basis for effect:
biomass
Remarks on result:
other: Inhibition concentrations by comparison of area under the growth curve; The concentraion obtained by the area method
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Basis for effect:
growth rate
Remarks on result:
other: Inhibition concentrations by comparison of growth rate for EC50 (24-48); The concentration obtained by the velocity method
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Basis for effect:
growth rate
Remarks on result:
other: Inhibition concentrations by comparison of growth rate for EC50 (24-48); The concentration obtained by the velocity method
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Basis for effect:
growth rate
Remarks on result:
other: Inhibition concentrations by comparison of growth rate for EC50 (24-72); The concentration obtained by the velocity method
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Basis for effect:
growth rate
Remarks on result:
other: Inhibition concentrations by comparison of growth rate for EC50 (24-72); The concentration obtained by the velocity method
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Environmental factors considered to affect reliability of the test results not rcognized
Results with reference substance (positive control):
Labour literature data: EbC50 = 0.50 - 1.02 mg/L (n=8) since December 1997
Reported statistics and error estimates:
For statistics, one-way anlysis of variance was used
Calculation of restults according to OECD 201

- Measured Concentrations: The tested concentrations were
measured at the start and the 72nd hour.  For all of them,
the deviations from the nominal concentration were less than
+/-20%.
-----------------------------------------------------------
Nominal   Measured Conc., mg/L     Percent of nominal
conc.     
-----------------------------------------------------------
mg/L      0 Hour   72 Hour        0 Hour   72 Hour
-----------------------------------------------------------
Control    <2      <2              ---       ---
1,000      1,050   1,040           105      104
-----------------------------------------------------------



-Effect Data: biomass 
Area Method
    EbC50(0-72hr) >1,000 mg/L (nc)
    NOEbC (0-72hr) = 1,000 mg/L (nc)

Rate Method
    ErC50(24-48hr) > 1,000 mg/L (mc)
    NOErC (24-48hr) = 1,000 mg/L (mc)
    ErC50(0-72hr) > 1,000 mg/L (mc)
    NOErC (0-72hr) = 1,000 mg/L (mc)
    nc: nominal concentration


- Percent Growth Inhibition of Selenastrum capricornutum
-----------------------------------------------------------
Nominal      Area under the growth curves (Average)
Conc.            Area           Inhibition (%)*
mg/L             A (0-72hr)      IA (0-72hr)
-----------------------------------------------------------
Control          1,596.0            ---
1,000            1,519.0            4.82
-----------------------------------------------------------

-----------------------------------------------------------
       Growth rates and percent inhibition (Average)
Nominal  --------------------------------------------------
Conc.           Rate         Inhibition(%)
mg/L            u(0-72hr)    Im(0-72hr)
-----------------------------------------------------------
Control         1.43            ---
1,000           1.38           0.03246
-----------------------------------------------------------


- Growth Curves: During the test period algae grew almost
linearly(log scale) in each concentration.


Validity criteria fulfilled:
yes
Conclusions:
NOECb = 1000 mg/L; NOECr = 1000 mg/mL
Executive summary:

The test was conducted under the OECD 201 Guideline "Alga Growth Inhibition Test" (1984).

Inhibition concentration by comparison of the area under the growth curve:

- 50% growth inhibition concentration EbC50 (0 -72): > 1000 mg/L

- NOECb (0 -72): 1000 mg/L

Inhibition concentration by comparison of growth rate

- 50% growth inhibition concentration ErC50 (24 -48): > 1000 mg/L

- NOECr (24 -48): 1000 mg/L

- 50% growth inhibition concentration ErC50 (24 -72): > 1000 mg/L

- NOECr (24 -72): 1000 mg/L

Description of key information

An OECD 201 Guideline "Algal Growth Inhibition Test" (1984) resulted in a NOEC of 1000 mg/L and an EC50>1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
1 000 mg/L

Additional information

A test is available for MMB which was conducted under the OECD 201 Guideline "Alga Growth Inhibition Test" (1984). Results were obtained as follows:

50% growth inhibition concentration EbC50 (0 -72): > 1000 mg/L

50% growth inhibition concentration ErC50 (24 -48): > 1000 mg/L

50% growth inhibition concentration ErC50 (24 -72): > 1000 mg/L