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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:
2014-11-10 to 2014-11-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed in accordance with standard test protocols (OECD 201 resp. EU C.3) in a quality controlled laboratory. The study is valid according to criteria mentioned in the test protocols.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1 - 100 mg/L
Vehicle:
no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular freshwater green alga
- Genus: Desmodesmus
Species subspicatus
SAG Strain Number 86.81
Taxonomic position Chlorophyta - Chlorophyceae
- Source (laboratory, culture collection): MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen)
- Method of cultivation: permanent culture

ACCLIMATION
- Acclimation period: 72 h
- 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
Test temperature:
21.6 - 23.6 °C
pH:
8.3-8.4
Nominal and measured concentrations:
Nominal concentrations: 1.0 / 3.2 / 10 / 32 / 100 mg/L
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: closed glass flasks total volume 65 mL
- Initial cells density: 5390 cells/mL
- Control end cells density: 352865 cells/mL
- No. of vessels per concentration (replicates): three replicates for each treatment
- No. of vessels per control (replicates): six replicates for the control
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: To avoid a strong pH increase and CO2 shortage in the closed system, additional 6 mL sodium hydrogen carbonate solution (50 g/L) was added to 900 mL nutrient medium. Then, pH was adjusted to 7.0 (± 0.2) and the solution was filled up to 1000 mL with nutrient medium
- Photoperiod:continuous lightening
- Light intensity and quality: 5800 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: photometric measurement of optical density every 24 h
- Chlorophyll measurement: no


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: no
- Test concentrations: 1.0, 3.2, 10, 32, 100 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (CAS No. 7778-50-9)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
37 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 25 - 55
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
11 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 9.8 - 17
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 2.7 - 11
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 3.3 - 7.9
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
Parameter: 72h ErC50 Value: 0.99 mg/L 95%-confidence-interval: 0.94 – 1.1 mg/L Range of interlaboratory test (EU C.3):0.60 – 1.03 mg/L
Parameter: 72h EyC50 Value: 0.55 mg/L 95%-confidence-interval: 0.52 – 0.58 mg/L Range of interlaboratory test (EU C.3): none stated
Reported statistics and error estimates:
The values for the Yield and the growth rate were used for the assessment of observed effect levels.
In order to select a suitable test for significance, it was checked whether equality of variance was given.
Equality of variance was tested using the following equation:
F = s1^2 / s2^2
with s1 being the greater variance and s2 the smaller one.
The calculated value F was compared with the F-test table (level of significance 95%). If the calculated value was smaller than the tabular value, equality of variance was given.
If equality of variance was given, the t-test was used; else, the WEIR test was used.

Cell Numbers Main Study

Nom. Conc. in mg/L

Repl.

Cell Number/mL

 

 

0 h

24 h

48 h

72 h

0

1

5390

25872

88396

323400

2

5390

28028

91630

336336

3

5390

31262

100254

369754

4

5390

26950

99176

403172

5

5390

28028

98098

315854

6

5390

29106

93786

368676

Mean

5390

28208

95223

352865

SD

0

1857

4707

33398

1

1

5390

29106

95942

350350

2

5390

29106

74382

194040

3

5390

33418

86240

299684

Mean

5390

30543

85521

281358

SD

0

2490

10798

79750

3.2

1

5390

26950

97020

366520

2

5390

28028

108878

413952

3

5390

29106

98098

269500

Mean

5390

28028

101332

349991

SD

0

1078

6557

73631

10

1

5390

30184

116424

211288

2

5390

33418

118580

215600

3

5390

31262

111034

210210

Mean

5390

31621

115346

212366

SD

0

1647

3887

2852

32

1

--

--

--

--

2

5390

21560

20482

25872

3

5390

20482

19404

24794

Mean

5390

21021

19943

25333

SD

0

762

762

762

100

1

5390

16170

16170

17248

2

5390

15092

16170

26950

3

5390

18326

20482

20482

Mean

5390

16529

17607

21560

SD

0

1647

2490

4940

SD = Standard Deviation


Validity criteria fulfilled:
yes
Remarks:
All validity criteria were met.
Conclusions:
The study was performed using five concentrations of 1.0 / 3.2 / 10 / 32 / 100 mg/L nominal concentration. Significant inhibition of algal growth was observed at the following concentrations: 100 / 32 / 10 mg/L
Because in the two lowest concentrated treatments no significant inhibition of algal growth could be observed, the lowest test item concentration was not used for evaluation of the results.
At the start and at the end of the test, the content of the test item in the test solutions was determined using HPLC. The measured concentrations in the treatments 3.2 / 10 / 32 / 100 mg/L lay between 96% and 103% of the nominal concentration at the beginning of the test and between 88% and 98% of the nominal concentration at the end of the test.
Therefore, the determination of the results was based on the nominal concentrations.
The EC50 values of potassium dichromate were determined in a separate reference test. For the estimation of the EC50 values of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the normal range of the laboratory.
All validity criteria were met.
No observations were made which might cause doubts concerning the validity of the study outcome.
Executive summary:

The study was performed using five test item concentrations of 1.0 / 3.2 / 10 / 32 / 100 mg/L (nominal concentration). Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the absorption of the corresponding solution at 440 nm every 24 hours with a spectral photometer. The cell density of the cultures was calculated based on the correlation curve between the adsorption and the cell density of the cultures determined with a cell counter. Growth rate µ and yield[1] were determined from the cell densities at the respective observation times.

Significant inhibition of algal growth was observed at the following test item concentrations:

100 / 32 / 10 mg/L

Because in the two lowest concentrated treatments no significant inhibition of algal growth could be observed, the lowest test item concentration was not used for evaluation of the results.

At the start and at the end of the test, the content of the test item in the test solutions was determined by HPLC. The measured concentrations in the treatments 3.2 / 10 / 32 / 100 mg/L lay between 96% and 103% of the nominal concentration at the beginning of the test and between 88% and 98% of the nominal concentration at the end of the test.

Therefore, the determination of the results was based on the nominal concentrations.

The EC50 values of potassium dichromate were tested in a separate reference test. The values lay within the normal range of the laboratory.

The following results for the test item nicotine were determined:

Endpoint

NOEC

LOEC

EC10

EC50

Growth Rate

3.2 mg/L

10 mg/L

5.2 mg/L

37 mg/L

Yield

3.2 mg/L

10 mg/L

5.3 mg/L

11 mg/L

[1]Yield (according to OECD Guideline 201) is defined as the difference between the biomass at the end and the biomass at the start of the exposure period.

Description of key information

One experimental study available in Desmodesmus subspicatus. The overall quality of the study is considered very high (Klimisch 1). The study is GLP compliant and appropriate for risk assessment, therefore no further testing is needed.

Key value for chemical safety assessment

EC50 for freshwater algae:
11 mg/L
EC10 or NOEC for freshwater algae:
5.2 mg/L

Additional information