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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:
from January 31, 2003 to May 30, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been conducted according to the appropriate OECD guideline, under GLP and analytical monitoring was carried out.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Remarks:
Cetrificate is not included in the study report.
Analytical monitoring:
yes
Details on sampling:
The test solution of 2.0 mL was collected from three vessels of each test area and mixed as analytical samples at the start of exposure (0 hour). At completion of exposure (72 hours), 2.0 mL of the test culture solution was collected from three test vessels of each test area and mixed, and the supernatant after centrifuge of alga (3000 rpm, 10 minutes) was used as analysis samples. Each analysis sample of 0.75 mL was added to equal amount of acetone and mixed, and the solution was analyzed by GC.
Vehicle:
no
Details on test solutions:
Test substance solution;
For the culture medium at preparation of the test solution, the temperature was set to be 23 ± 2°C in the thermostat bath or temperature controlled room.
Preparation of the stock solution of the test substance;
The test substance of 100 mg was sampled and added in the culture medium and made to constant volume of 100 mL with culture medium to give 1000 mg/L test substance concentration. This stock solution was prepared before the starting of exposure. The test solution of 100 mL was prepared in the test vessel for each test concentration using this stock solution. Three test vessels were prepared for one concentration area. The amount of the culture medium which deducted adding the amount of the stock solution was placed into each test vessel, and the stock solution of the test substance was added.
Control area;
Only culture medium was used for the control area.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
1) Japanese name: Muremikazukimo (green unicellular alga)
2) Scientific name: Selenastrum capricornutum
3) Cell line name: ATCC22662
4) Obtained from: American Type Culture Collection
5) Date of obtainment: June 20, 1996
6) Management after obtainment:
Aseptic subculture using Gorham’s medium. A bacteria test was periodically performed (about every six months) for confirmation of sterility.
7) Preculture: preculture period:
February 21 in 2003 to February 25 in 2003
During this time, logarithmic growth of alga was recognized (under the same environmental conditions as those in the study).
Test type:
static
Water media type:
not specified
Limit test:
yes
Total exposure duration:
72 h
Hardness:
Not reported.
Test temperature:
°C (Exposure period); 23°C (0 hour), 23°C (24 hours), 23°C (48 hours), 23°C (72 hours)
pH:
Control solution; 7.9 (0 hour), 10.2 (after 72 hours)
Test solution (100 mg/L); 7.8 (0 hour), 9.8 (after 72 hours)
Dissolved oxygen:
No description
Salinity:
No description
Nominal and measured concentrations:
Test concentration;
The test concentration in this study was determined as 100 mg/L (nominal value) (the limiting test).
Measured test substance concentration; see Table 2
Details on test conditions:
1. Testing conditions
1) Exposure method: Static (open system condition), continuous shaking cultivation (100 rpm)
2) Exposure period: 72 hours
3) Amount of the test solution: 100 mL (OECD medium)/vessel
4) Replicate number: 3 vessels/test area
5) The early cell concentration: precultured algae, 1 x 10E4 cells/mL
6) Test temperature: 23 ± 2°C
7) Lighting: continuous lighting with 4000 lux (± 20% of fluctuation, near the liquid level in the flask)
8) pH: pH of the test solution was not adjusted.
2. Culture medium
Both in the preculture and the study, the medium shown in the OECD Guideline for testing of chemicals was used after filter sterilization (0.22 μm).
3. Test vessel and algae cultivation apparatus, etc.
1) Test vessel: 300 mL glass erlenmeyer flask (IWAKI) (with a breathable silicone stopper)
2) Algae cultivation apparatus: Ito Corporation, AGP-150RL
3) Optical microscope: Nikon, ECLIPSE TE300
4) Particle counter: Sysmex, CDA-500
5) Electrolytic solution for the particle counter: Sysmex, CELLPACK
6) pH meter: TOA, desktop pH meter, HM-40V No.2
7) Thermometer: Tasco Japan, TNA-120
8) Illuminometer: TOPCON, IM-2D
4. Testing operation
The number of precultured cells of alga was directly counted using the particle counter (CDA-500), hemocytometer and the microscope, and the defined amount of the precultured solution was added to the vessel including the test solution so that the cell concentration to be 1 x 10E4 cells/mL.
Each test container was placed into the cultivation apparatus at 23 ± 2°C, the test was started and the cell concentration was measured after 24, 48 and 72 hours. The cell concentration was measured using the particle counter (CDA-500), after collecting 1.0 mL (0.5 mL for 72 hr) of the test culture solution from each test vessel and mixing with 9.0 mL (9.5 mL for 72 hr) of the electrolytic solution for the particle counter (CELLPACK).
Alga in the test culture solution was observed visually for its color tone at 0, 24 and 48 hours after the starting of exposure, and at completion of exposure visual observation was also performed for its color tone and microscopic observation performed for its cell morphology.
pH of the test solution at the starting of exposure and pH of the test culture solution at completion of exposure was measured at each test area. pH at the starting of exposure was measured with one spare vessel separately prepared from three vessels at each test area, and pH at completion of exposure was measured for one vessel (No.1) of the three vessels.
Reference substance (positive control):
yes
Remarks:
potassium bichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 98.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 98.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 98.9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 98.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Morphological observation of alga
As the result of the microscopic observation of the cell morphology at the completion of exposure no changes in cell morphology (contraction, swelling, rupture, etc.) or cell agglutination were noted in 100 mg/L concentration area, and no differences were noted compared with the control area.

- Inhibitory concentration in comparison of the area under the growth curves
50% effective concentration for growth inhibition, EbC50 (0-72 h): > 98.9 mg/L (95% confidence limits: cannot be calculated)
Maximum no observed effect concentration, NOECb (0-72 h): > 98.9 mg/L

- Inhibitory concentration in comparison of the growth rates
50% effective concentration for growth inhibition, ErC50 (24-48 h): > 98.9 mg/L (95% confidence limits: cannot be calculated)
Maximum no observed effect concentration, NOECr (24-48 h): > 98.9 mg/L
50% effective concentration for growth inhibition, ErC50 (24h-72 h): > 98.9 mg/L (95% confidence limits: cannot be calculated)
Maximum no observed effect concentration, NOECr (24-72 h): > 98.9 mg/L
Results with reference substance (positive control):
A growth inhibition study was periodically (about every six months) conducted with standard material (potassium bichromate, special grade chemicals) to examine the sensitivity of the test living organism. The 72-hour 50% growth inhibition concentration (EbC50) since November 1996 is shown as follows;
Mean ± Standard deviation = 0.433 ± 0.0802 mg/L, n = 12
(Minimum to maximum values = 0.285 to 0.543 mg/L)

Table 3. Cell densities of Selenastrum capricornutum during the 72 hours exposure

Nominal Concentration

[Measured Conc. at 0 hour]

(mg/L)

Vessel No.

Cell Densities (cells/mL) (x 100)

0 Hour

24 Hours

48 Hours

72 Hours

Control

1

100

499

3927

22490

2

100

527

4027

29890

3

100

583

4497

30190

Average

SD

100

0

536

43

4150

304

27523

4362

100

[98.9]

1

100

469

3767

26490

2

100

500

3787

26690

3

100

521

4137

27890

Average

SD

100

0

497

26

3897

208

27023

757

SD: Standard deviation

Table 4. Growth inhibition (%) of Selenastrum capricornutum

Nominal Concentration

[Measured Conc. at 0 hour]

(mg/L)

Vessel No.

Area under the growth curve (x 1000)

Area

(0-72 hours)

Inhibition* (%)

(0-72 hour)

Control

1

37010

2

46198

3

47820

Average

SD

43676

5830

---

100

[98.9]

1

41354

2

41717

3

44047

Average

SD

42373

1461

3.0

*: Values are the growth inhibition (%) relative to the control

Validity criteria fulfilled:
yes
Conclusions:
A 72 h EC50 value of >99 mg/l has been determined for the effects of the test substance on growth rate and biomass of the freshwater algae Selenastrum capricornutum (new name: Pseudokirchnerella subcapitata). In the same study a 72 h NOEC value of ≥99 mg/l has been determined. The results are based on geometric mean measured concentration.

Description of key information

72 h, EC50 >98.9 mg/L growth rate, Selenastrum capricornutum (new name: Pseudokirchneriella subcapitata), measured.

72 h, NOEC >=98.9 mg/L growth rate, Selenastrum capricornutum (new name: Pseudokirchneriella subcapitata), measured.

Key value for chemical safety assessment

Additional information

A 72 h EC50 value of >99 mg/L has been determined for the effects of the test substance on growth rate and biomass of the freshwater algae Selenastrum capricornutum (new name: Pseudokirchneriella subcapitata). In the same study a 72 h NOEC value of ≥99 mg/l has been determined. The results are based on geometric mean measured concentration.

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