Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

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:
2016-07-15 till 2017-03-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Concentration control analyses were not performed, because a sufficiently sensitive method to determine the test substance in test water up to its saturation limit was not available.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
yes
Remarks:
Concentration control analyses were not performed.
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
No 440/2008
Deviations:
yes
Remarks:
Concentration control analyses were not performed.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:The test substance is a mixture of sparingly water soluble components. Therefore the test solutions were prepared separately for each test concentration following general guidance provided in OECD 23 in order to achieve a water accommodated fraction (WAF) of the test substance using a liquid-liquid saturator technique [Ref. 6, 11]. For each test solution, a glass aspirator bottle (approx. 2 L) with a bottom side-outlet attached to a stopcock was filled with 2 L test media. Then test substance (considering density = 0.8166 g/cm³) was pipetted carefully on the water surface. The bottle was closed and the solution was stirred on a magnetic plate for about 3 days. Undissolved test substance was observed at the water surface as oily drops and the aqueous phase of the stock solution was clear. The required volume of the WAF (approx. 1 L per test solution) was removed from the bottom port. The exposure was started after separation of the undissolved material. The test vessels were conditioned with the test solution for approx. 24-h prior initiating exposure. The exposure was started after separation of the undissolved material by adding inoculum culture at a ratio of 1:100. The test solution after addition of algal inoculum is considered equivalent to the nominal concentration since no more than 1% dilution occurs.
- Controls: The control was treated in the same way as the test substance.
- Evidence of undissolved material: no
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata, formerly known as Selenastrum capricornutum, and currently renamed as Raphidocelis subcapitata KORSHIKOV
- Strain: Pseudokirchneriella subcapitata KORSHIKOV (SAG 61.81)
- Source: SAG (Collection of algal cultures in Göttingen, Germany)
- Age of inoculum: A stock algal culture is maintained continuously at the test facility. Before the exposure an inoculum culture is prepared from the stock culture and incubated for 4 days at 21 – 24 °C (max. temperature difference 2 °C). After this time, the
inoculum culture is in exponential growth phase and can be used to initiate the test (study day 0).

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions: same as test conditions
- 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:
no
Test temperature:
23.3 - 23.5 °C
pH:
7.9 - 8.2
Nominal and measured concentrations:
1 - 3.2 - 10 - 3.2 - 100 mg/L nominal concentrations based on test substance density without correction for purity
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks (nominal volume 250 mL) plugged with gas permeable silicone sponge caps
- Fill volume: 100 mL
- Initial cells density: 0.5 x 10000 cells/mL
- Control end cells density: 55.7 x 10000 cells/mL
- No. of vessels per concentration: 3 replicates
- No. of vessels per control: 6 replicates
- No. of vessels per background fluorescence correction: 1 replicate
- Test chamber: Vötsch Industrietechnik GmbH Bioline (VB1014) controlled climate cabinet

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM PARAMETERS
- Preparation of dilution water: according to OECD Guidline for Testing of Chemicals, No. 201 Algal growth inhibition test, Mar 2006, Annex 3
- pH: 8.1
- Sterilization: after preparation

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: permanent, type universal white (OSRAM L 25)
- Light intensity and quality: Average 6790 lux (within ± 15% variability) at a wave length of 400 - 700 nm
- Shaking rate: Continuous (approx. 85 rpm)

EFFECT PARAMETERS MEASURED:
- Determination of cell density: counted under a microscope (Neubauer-counting chamber)
- Chlorophyll measurement: Tecan Infinite 200Pro fluorometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- range finding test: The results of the 72 hour range finding test were:
EfluorescenceC50 = between 1 and 10 mg/L (4 mg/L estimated)
ErC50 = between 10 and 100 mg/L (16 mg/L estimated)
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.71 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.96 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.53 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: After 72 h at the concentrations above 10 mg/L no algal cells were observed, at 3.2 mg/L a low cell density was observed.
- Any stimulation of growth found in any treatment: yes, at concentrations of 32 and 100 mg/L the mean growth rate at 72 h was -0.22 and -0.35 respectively relative to the control.
Results with reference substance (positive control):
- Results with reference substance valid: yes
- EC50: 1.14 mg/L, Tested 2016-07-04

Table 1: Cell density after 0 h, 24 h, 48 h and 72 h

The measured fluorescence was corrected by the fluorescence of the test substance and converted into cell densities (cells x 10000/mL) by the following correlation:

cell density (cells x 10000/mL) = fluorescence (relative units) - fluorescence not inoculated sample / slope

slope = 946.89

The linear correlation was obtained from a calibration curve (cell density versus fluorescence).

 

time(h)

flask No.

nominal concentration [mg/L]

0 (control)

1

3.2

10

32

100

0

1

0.445

0.436

0.439

0.431

0.402

0.373

2

0.377

0.441

0.402

0.407

0.420

0.423

3

0.470

0.373

0.404

0.455

0.391

0.409

4

0.398

 

 

 

 

 

5

0.448

 

 

 

 

 

6

0.455

 

 

 

 

 

mean

0.432

0.417

0.415

0.431

0.404

0.402

24

1

1.94

2.24

1.27

0.933

0.248

0.167

2

1.90

2.04

1.41

0.796

0.319

0.129

3

2.15

2.05

1.57

0.949

0.387

0.127

4

2.00

 

 

 

 

 

5

2.47

 

 

 

 

 

6

2.55

 

 

 

 

 

mean

2.17

2.11

1.42

0.893

0.318

0.141

48

1

10.6

8.00

1.53

0.381

0.152

0.039

2

8.67

8.59

1.90

0.696

0.098

0.048

3

11.6

9.36

1.85

0.628

0.152

0.045

4

10.8

 

 

 

 

 

5

9.02

 

 

 

 

 

6

12.7

 

 

 

 

 

mean

10.6

8.6

1.76

0.569

0.134

0.044

72

1

53.5

29.4

2.36

0.609

0.200

0.131

2

43.7

39.3

3.61

0.563

0.227

0.156

3

47.9

31.5

3.03

0.715

0.204

0.133

4

46.6

 

 

 

 

 

5

69.8

 

 

 

 

 

6

72.9

 

 

 

 

 

mean

55.7

33.4

3.00

0.629

0.210

0.140

negative calculated growth rates are represented as 0.000

mean = mean value

Tabel 2: Mean specific growth rate and Yield in Pseudokirchneriella subcapitata as dependent on concentration of the test

item and time; Mean: arithmetic mean

Treatment [mg/L]

Replicate No.

Growth rate [d-1]

Inhibition of growth rate [%]

Yield [cells/mL]

Inhibition of yield [%]

100

1

-0.349

100.00

-0.242

100.00

2

-0.332

100.00

-0.267

100.00

3

-0.374

100.00

-0.276

100.00

Mean

-0.352

 

-0.262

 

32

1

-0.234

100.00

-0.203

100.00

2

-0.205

100.00

-0.193

100.00

3

-0.217

100.00

-0.187

100.00

Mean

-0.219

 

-0.194

 

10

1

0.116

92.84

0.178

99.68

2

0.108

93.28

0.156

99.72

3

0.151

90.67

0.260

99.53

Mean

0.125

 

0.198

 

3.2

1

0.561

65.26

1.923

96.52

2

0.732

54.66

3.213

94.19

3

0.671

58.42

2.625

95.25

Mean

0.655

 

2.587

 

1

1

1.403

13.05

28.954

47.64

2

1.496

7.30

38.863

29.72

3

1.479

8.38

31.129

43.71

Mean

1.460

 

32.982

 

Control

1

1.597

 

53.096

 

2

1.584

 

43.279

 

3

1.542

 

47.444

 

4

1.587

 

46.193

 

5

1.683

 

69.350

 

6

1.692

 

72.446

 

Mean

1.614

 

55.301

 

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 (2011) and Council Regulation (EC) No 761/2009 C.3 at the test facility. After 72 hours the NOEC was determined to <1 mg/L, the ErC10 was determined to be 0.96 mg/L and the ErC50 was determined to be 2.71 mg/L.
Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 440/2008/Method C.3 with the definitive exposure phase from 2016-07-22 to 2016-07-25 at BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany. The aim of the study was to determine the NOEC, LOEC and ECx-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 0.5 x 10000 cells/mL. Five concentrations were tested in a geometrical series with a dilution factor of 3.2 (nominal): 1.00 - 3.2 - 10.0 - 32 - 100 mg/L. Three replicates for each test item concentration and six replicates for the control were used. The environmental conditions were within the acceptable limits.

Concentration control analyses were not performed, because a sufficiently sensitive method to determine the test substance in test water up to its saturation limit was not available.

After 72 hours the NOEC based on growth rate was determined to be <1 mg/L, the ErC10 was determined to be 0.96 mg/L and the ErC50 was determined to be 2.71 mg/L.

Description of key information

Key Study:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 (2011) and Council Regulation (EC) No 761/2009 C.3 at the test facility. After 72 hours the NOEC was determined to <1 mg/L, the ErC10 was determined to be 0.96 mg/L and the ErC50 was determined to be 2.71 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
2.71 mg/L
EC10 or NOEC for freshwater algae:
0.96 mg/L

Additional information

Key Study:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 440/2008/Method C.3 with the definitive exposure phase from 2016-07-22 to 2016-07-25 at BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany. The aim of the study was to determine the NOEC, LOEC and ECx-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 0.5 x 10000 cells/mL. Five concentrations were tested in a geometrical series with a dilution factor of 3.2 (nominal): 1.00 - 3.2 - 10.0 - 32 - 100 mg/L. Three replicates for each test item concentration and six replicates for the control were used. The environmental conditions were within the acceptable limits.

Concentration control analyses were not performed, because a sufficiently sensitive method to determine the test substance in test water up to its saturation limit was not available.

After 72 hours the NOEC based on growth rate was determined to be <1 mg/L, the ErC10 was determined to be 0.96 mg/L and the ErC50 was determined to be 2.71 mg/L.