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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:
25 June 2002 to 28 June 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese MAFF Alga Growth inhibition test (2-7-3) (12 Nousan No. 8147)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- The concentration of the test material in test solution was measured at the start of the exposure and the end of the exposure. At the start of exposure, sample of test solution was taken out from the vessel for measurement of pH and temperature. At the end of exposure, it was taken out from one of 3 vessels in each level and then the solution which alga was removed from by centrifugation (3,000 rpm, 10 min) was used for the measurement.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Desired amount of the test material which was calculated by its density [2.26(25 "C)] was taken and dissolved in medium to prepare the stock solutions of 200 mg/L. The necessary amount of the stock solution was mixed with medium in test vessel and then the mixed solution was continuously gyrated for about 24 hours in order to equilibrate the vapor phase and liquid phase of the test material in the test vessel. The preparation of the stock solution and the test solutions was performed the day before the inoculum of the test organisms.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name:
- Strain: ATCC 22662
- Source: Originally from the American Type culture collection. The algae used in the test had been cultured in the testing laboratory.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72
Test temperature:
23 ± 2 °C
pH:
pH values at the start of the exposure ranged from 7.2-7.5 and from 7.5-10.2 at the end of the exposure.
Nominal and measured concentrations:
- Nominal concentrations: 1, 2, 4, 8 and 16 mg/L
- Mean measured cocentration at 72 h: 0.404, 0.817, 1.57, 3.27 and 6.19 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterilised 500 mL Erlenmeyer flask
- Type: closed
- Material, size, headspace, fill volume: 100 mL green algae
- Aeration: continuous shaking by rotation at about 100 rpm was used
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 99.8 x 10^4 cells/mL
- No. of vessels per concentration: Three replicates per concentration
- No. of vessels per control: Three

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous lighting
- Light intensity and quality: Uniform and continuous fluorescent illumination at 4000 lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Cell concentration was counted with an electronic particle counter, plus cell condition was observed under the microscope.
- Cell densities were determined at 24 hour intervals.
- At 72, microscopic observations were conducted to detect any morphological effects.
- Measurement of pH and temperature in each test solution were recorded at 0, 72 and 96 hours.
Reference substance (positive control):
yes
Remarks:
K2Cr2O7
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.69 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 1.16-2.46 mg/L
Dose descriptor:
EC50
Effect conc.:
2.55 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 24-72 h
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.846 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Dose descriptor:
NOEC
Effect conc.:
1.63 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 24-72 h
Details on results:
- Exponential growth in the control : yes
- Observation of abnormalities : Cells in the 6.37 and 3.43 mg/L groups were swollen. Those in other exposure levels were the same as the control.
- Colour differences: At initiation test solutions were colourless and clear. At the end of the test the 6.37 mg/L solution was colourless and clear, the 3.43 mg/L solution was slightly light green and other solutions showed a green colour due to algal growth.
Results with reference substance (positive control):
An algal growth inhibition test of a reference substance [K2Cr2O7(Reagent chemical), Wako Pure Chemical Industries, Ltd.] with the test organisms was conducted to confirm the reproducibility of the test system. The EbC50(0-72 h) of the reference substance was 0.431 mg/L and this value was within the range of normal data that had been obtained in this laboratory.

Table 2: Area under the growth curve (biomass)

Parameter measured

Treatment (mg test material (mean measured)/L)

0

0.404

0.817

1.57

3.27

6.19

Area under the growth curve (mean)

2140

2480

1980

1180

87.4

51.1

% Inhibition relative to the control

-

-15.7

7.72

45.1

95.9

97.6

A negative value represents an increase relative to the control

 
Table 3: Growth rate

Parameter measured

Treatment (mg test material (mean measured)/L)

0

0.404

0.817

1.57

3.27

6.19

Growth rate (mean) 24- to 48-h

0.0825

0.0826

0.0759

0.0650

0.00891

0.00460

% Inhibition relative to the control

-

-0.131

7.96

21.2

89.2

94.4

Growth rate (mean) 48- to 72-h

0.0615

0.0685

0.0672

0.0653

0.00790

0.00314

% Inhibition relative to the control

-

-11.4

-9.16

-6.10

87.2

94.9

A negative value represents an increase relative to the control

Table 4: Cell concentration

Cell concentration (x 104cells/mL)

Treatment (mg test material (mean measured)/L)

0

0.404

0.817

1.57

3.27

6.19

24 hours

5.06

4.88

4.29

2.99

2.07

1.72

48 hours

36.8

35.2

26.6

14.2

2.56

1.91

72 hours

99.8

131

108

68.6

3.02

1.99

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this 72-hour toxicity study with Pseudokirchneriella subcapitata, the EbC50 (72-h), ErC50 (24 – 72h) were 1.69 and 2.55 mg/L, respectively. The corresponding NOEC values were 0.846 and 1.63 mg/L, respectively.
Executive summary:

The toxicity of the test material to algae was investigated in accordance with the standardised guidelines OECD 201, EU Method C.3 and JMAFF No. 12 Nousan 8147, under GLP conditions.

Algae were exposed to a geometric series of five concentrations and a negative (OECD test medium) control. Nominal test concentrations of 1, 2, 4, 8 and 16 mg/L were selected. 300 mL of green alga, Pseudokirchneriella subcapitata in exponential growth (1 x 104cells/mL) were added to each vessel (3/treatment). Cultures were exposed for a total of 72 hours. Cell densities were determined at 24 hour intervals and at 72 hours, microscopic observations were conducted to detect any morphological effects. Measurement of pH and temperature in each test solution were recorded at 0, 72 and 96 hours. 

The concentration of the test material in the test solutions was measured at the start and end of exposure. The mean measured concentrations at 72 hours were: 0.404, 0.817, 1.57, 3.27 and 6.19 mg/L at the nominal concentrations of 1, 2, 4, 8 and 16 mg/L, respectively. Mean measured concentrations as % of nominal concentrations were: 40.4, 40.8, 39.3, 40.8 and 38.7, respectively.

Under the conditions of this 72-hour toxicity study with Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), the EbC50(72-h), ErC50 (24 – 72h) were 1.69 and 2.55 mg L, respectively. The corresponding NOEC values were 0.846 and 1.63 mg/L, respectively.

Description of key information

Under the conditions of this 72-hour toxicity study withPseudokirchneriella subcapitata(formerly known as Selenastrum capricornutum), the EbC50(72-h), ErC50 (24 – 72h) were 1.69 and 2.55 mg L, respectively. The corresponding NOEC values were 0.846 and 1.63 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
1.69 mg/L
EC10 or NOEC for freshwater algae:
0.846 mg/L

Additional information

The toxicity of the test material to algae was investigated in accordance with the standardised guidelines OECD 201, EU Method C.3 and JMAFF No. 12 Nousan 8147, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Algae were exposed to a geometric series of five concentrations and a negative (OECD test medium) control. Nominal test concentrations of 1, 2, 4, 8 and 16 mg/L were selected. 300 mL of green alga,Pseudokirchneriella subcapitatain exponential growth (1 x 104cells/mL) were added to each vessel (3/treatment). Cultures were exposed for a total of 72 hours. Cell densities were determined at 24 hour intervals and at 72 hours, microscopic observations were conducted to detect any morphological effects. Measurement of pH and temperature in each test solution were recorded at 0, 72 and 96 hours. 

The concentration of the test material in the test solutions was measured at the start and end of exposure. The mean measured concentrations at 72 hours were: 0.404, 0.817, 1.57, 3.27 and 6.19 mg/L at the nominal concentrations of 1, 2, 4, 8 and 16 mg/L, respectively. Mean measured concentrations as % of nominal concentrations were: 40.4, 40.8, 39.3, 40.8 and 38.7, respectively.

Under the conditions of this 72-hour toxicity study with Pseudokirchneriella subcapitata (formerly known asSelenastrum capricornutum), the EbC50(72-h), ErC50 (24 – 72h) were 1.69 and 2.55 mg L, respectively. The corresponding NOEC values were 0.846 and 1.63 mg/L, respectively.