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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to guideline
Guideline:
other: Japanese national standard
Version / remarks:
Testing method relating to the new chemical substances, etc. (Yakushokuhatsu No. 1121002, Heisei 15-11-13 Seikyoku No. 2, Kanpokihatsu No. 031121002 on November 21, 2003).
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Principles of method if other than guideline:
To assess the toxicity of the test substance by conducting algal growth inhibition test in Pseudokirchneriella subcapitata for notification of the new chemical substance under the Chemical Substance Control Law (CSCL) in Japan.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
At the start of exposure and 72 hours after the start of exposure, test substance concentrations in the test solution of all test groups and in the test culture solution were analyzed by high performance liquid chromatography-mass spectrometry (LC/MS).

At the start of exposure
Test solution: An aliquot of the test solution was sampled from all test vessels at each test group and mixed.
Sampling, 1.5 mL of the test solution (appropriately diluted with purified water)

LC/MS measurement
At 24, 48 and 72 hours after the start of exposure
Test culture solution: An aliquot of the test solution was sampled from all test vessels at each test group and mixed.
Centrifuge (3000 rpm, 10 minutes, equipment; Hitachi Koki Co., Ltd., Type CR21E)
Supernatant (appropriately diluted with purified water*) 1.5 mL
LC/MS measurement
*: Diluted when the estimated concentration was over the range of the calibration curve.
Vehicle:
no
Details on test solutions:
Preparation of the stock solution
Sampling volume = 100 mg
Test water = Medium (22°C)
Final volume = 1000 mL
Vessel = Volumetric flask
Concentration = 100 mg/L
Mixing method = Stirring by stirring bar (for 3 minutes), tightly sealed.

Preparation of the test solution
The stock solution was collected as shown in below table and diluted with test water, and used as the test solution.
Final volume = 0.10 L
Vessel = 300 mL glass erlenmeyer flask (IWAKI), (with breathable silicone rubber stopper)
Mixing method = Hand shaking
Common ratio for concentration = 2.15
Replication = 6 vessels/control group, 3 vessels/concentration group
(The following concentration is shown to match minimum digits number.)
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Single-cell green algae
- Strain: ATCC22662
- Source (laboratory, culture collection): American Type Culture Collection
- Method of cultivation: Asceptically subcultured with Gorham's medium. Sterility was periodically (approximately every 6 month) confirmed by bacteriological examination.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed:No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Test temperature:
22°C
Nominal and measured concentrations:
0, 1.0, 22, 4.6, 10, 22, 46, 100 mg/L (Nominal)
0, 0.896, 1.97, 3.96, 8.46, 19.3, 39.8 and 85.1 mg/L (Time weight mean average)
Details on test conditions:
TEST SYSTEM
- Test vessel: 300mL glass erlenmeyer flask with breathable silicone rubber stopper
- Type (delete if not applicable): open
- Fill volume: 100 mL/vessel
- Renewal rate of test solution (frequency/flow rate): not applicable (static test)
- Initial cells density: 5 x 10^3 cells/mL:
- No. of vessels per concentration (replicates): 3 vessels
- No. of vessels per control (replicates): 6 vessels

GROWTH MEDIUM
- Standard medium used: yes (as per guideline)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: not adjusted
- Photoperiod: continuous illumination
- Light intensity and quality :65-75 μE/m2/s, continuous illumination with white fluorescent light (around the surface of test solution)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Measurement of biomass
At 24, 48 and 72 hours after the start of exposure, 1.0 mL of test culture solution (0.5 mL at 72 hours) was collected from each test vessel, mixed with 9.0 mL of electrolyte solution for particle counter (9.5 mL at 72 hours), and then biomass was measured using particle counter.

Colour observation of the test culture solution and cell morphology observation
At the start of exposure, 24, 48 and 72 hours after the start of exposure, color of test culture solution was observed by comparison with the reserved vessel. At 72 hours after the start of exposure, cell morphology observation was also conducted using microscope.

Measurement of test environment
pH at the start of exposure was measured by collecting aliquots of the test solution from reserved vessel of each test group, and pH at 72 hours after the start of exposure was measured by test culture solution from a vessel (No. 1) of each test group. Temperature, light intensity and number of rotation in the incubator were measured once daily during exposure period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Justification for using less concentrations than requested by guideline: not applicable

Range finding study:
- Test concentrations: 0, 0.1, 1, 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate (performed every 6 month to confirm sensitivity of the test system)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 85.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
8.46 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
From the results of colour observation of the test culture solution, tendency of increasing degree of green over time was observed at all test concentration groups.

From the results of cell morphology observation by microscope at 72 hours after the start of exposure, change of cell morphology (contraction, swelling, rupture and etc.,) and cell aggregation were not observed, and there was no difference compared with the control group.
Results with reference substance (positive control):
- Results with reference substance valid? Yes

Reported statistics and error estimates:
NOEC were determined using one-way analysis of variance (1-way ANOVA, = 0.05) and Williams multiple comparison ( = 0.05, two-sided) after the homogeneity of variance was confirmed by Bartlett s test ( = 0.01).

Table 1: Growth Inhibition (%)

 Test group 

 Nominal Concentration    

[mean measured conc.]

    Growth rate
 mg/L  Vessel no.  Rate (0 -72h)

 Inhibition (%)

(0 -72h)

 Control  -

 1

2

3

4

5

6

Average

SD

CV

0.0881

0.0855

0.0856

0.0838

0.0821

0.0861

0.0852

0.0021

2.5 

 
 1

 1.0

[0.896]

1

2

3

Average

SD

0.0872

0.0858

0.0834

0.0855

0.0019

 

-0.4

 2

 2.2

[1.97]

1

2

3

Average

SD

 0.0875

0.0855

0.0829

0.0853

0.0023

 

-0.1

 3

 4.6

[3.96]

 1

2

3

Average

SD

 0.0849

0.0795

0.0853

0.0832

0.0032

 

2.3

 4

 10

[8.46]

 1

2

3

Average

SD

 0.0820

0.0819

0.0823

0.0821

0.0002

 

3.6

 5

 22

[19.3]

1

2

3

Average

SD 

0.0747

0.0753

0.0755 

0.0752

0.0004

 

11.7**

 6

 46

[39.8]

1

2

3

Average

SD 

0.0707

0.0688

0.0729

0.0708

0.0021 

 

16.9**

 7

 100

[85.1]

1

2

3

Average

SD

0.0660

0.0633

0.0660

0.0651

0.0016

23.6**

** significant difference (a = 0.01) from control

Validity criteria fulfilled:
yes
Conclusions:
The test item is of low toxicity to aquatic algae under the condition of the test. EC50 value was greater than the maximum tested concentration.

Description of key information

In an GLP-compliant study conducted with analytical monitoring in accordance with a standard method equivalent to the OECD guideline, the toxicity of the test item to algae was assessed. It was not possible to determine the 72h EC50 value as this was greater than the highest concentration tested (100 mg/L - nominal; 85.1 mg/L - measured Time Weighted Average). The 72h NOEC (growth rate) was determined to be 8.46 mg/L. This value will be used in the Chemical Safety Assessment

.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
8.46 mg/L

Additional information