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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:
July 2018 - April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Details on sampling:
At the start of the exposure (0 hours), samples of the fresh media were taken after preparation of each concentration and analysed. At the end of the exposure (72 hours), samples of the old media were taken from the test vessels for analyses.
Vehicle:
no
Details on test solutions:
A saturated solution with a nominal loading of 116 mg/L was prepared once 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out and transferred with an appropriate amount of the dilution water into a glass flask. This dispersion was stirred on a magnetic stirrer at approx. 1100 rpm for 24 ± 1 hour at room temperature. After completion of stirring, the solution was centrifuged for 15 minutes with 14000 rpm at 20 °C, to remove undissolved particles, and used for testing. After centrifugation, the supernatant was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Age of inoculum: A four days old preculture, prepared in dilution water and incubated under test conditions, was used as inoculum.
- Method of cultivation: Nutrient medium Z according to LUTTGE et al. (1994)

ACCLIMATION
- Acclimation period: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22 - 24 °C
pH:
7.85 - 8.81
Nominal and measured concentrations:
Nominal concentrations: 0.316, 1.00, 3.16, 10.0, 31.6, 100% of the saturated solution
Geometric mean measured test item concentrations: 0.0181, 0.0448, 0.148, 0.588, 2.32, 8.48 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile glass Erlenmeyer flasks
- Type: closed (sealed with cotton wool plugs)
- Material, size, fill volume: glass, 25 mL, 10 mL
- Aeration: no
- Initial cells density: 6442 cells/mL
- Control end cells density: 1984933 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: Dilution water (OECD TG 201 medium)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: 5484 Lux

EFFECT PARAMETERS MEASURED
The cell density was measured daily via Chlorophyll a- fluorescence, excitation at 436 nm, emission at 685 nm.
Microscopic evaluation of the cells at the start and the end of the incubation period was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10
- Range finding study
- Test concentrations: 1, 10, 100 % saturated solution
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 8.48 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
8.48 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: 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 that might cause a difference between measured and nominal values: no
Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2018-09-25 to 2018-09-28. The reference item toxicity is in the valid range following test facility SOPs. EC50 (growth rate): 0.799 (0.707-1.3), EC50 (biomass): 0.607 (0.404-0.694)

Table 1: Cell Densities

Geometric mean measured test item concentration

[mg/L]

Replicate

No.

Cell density [cells/mL]

0 hours

24 hours

48 hours

72 hours

8.48

1

6442

16332

104017

1303653

2

6442

16970

110290

1342131

3

6442

17765

118472

1414918

Mean

6442

17022

110926

1353567

2.32

1

6442

25222

206514

1920263

2

6442

27571

216161

1828312

3

6442

23082

176277

1845143

Mean

6442

25292

199651

1864573

0.588

1

6442

29673

237369

1875377

2

6442

28514

228129

1922098

3

6442

29601

234822

1671117

Mean

6442

29263

233440

1822864

0.148

1

6442

27484

231394

1960487

2

6442

28730

216692

1877373

3

6442

30284

215198

1909397

Mean

6442

28833

221095

1915752

0.0448

1

6442

26925

229800

1992000

2

6442

16782

228514

1886295

3

6442

29514

235326

1873535

Mean

6442

24407

231213

1917277

0.0181

1

6442

27595

221265

1987087

2

6442

28499

206072

1943246

3

6442

29097

219215

1824335

Mean

6442

28397

215517

1918223

Control

1

6442

27034

244191

1919710

2

6442

28861

237369

1910822

3

6442

30123

225389

2056744

4

6442

32118

221079

2039466

5

6442

30887

218899

2015435

6

6442

33772

248853

1967423

Mean

6442

30466

232630

1984933

 

Table 2: Inhibition of Growth rate and Yield: Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Geometric mean measured test item concentration

[mg/L]

Replicate

No.

Growth Rate

[d-1]

Inhibition of Growth Rate [%]

Yield

[cells/mL]

Inhibition of Yield [%]

8.48

1

1.77

7

1297211

34

2

1.78

7

1335689

32

3

1.80

6

1408476

29

Mean

(+) 1.78

7

(+) 1347125

32

2.32

1

1.90

1

1913821

3

2

1.88

1

1821870

8

3

1.89

1

1838701

7

Mean

(-) 1.89

1

(-) 1858131

6

0.588

1

1.89

1

1868935

6

2

1.90

1

1915656

3

3

1.85

3

1664675

16

Mean

(+)11.88

2

(+)21816422

8

0.148

1

1.91

0

1954045

1

2

1.89

1

1870931

5

3

1.90

1

1902955

4

Mean

(-) 1.90

1

1909310

4

0.0448

1

1.91

0

(-) 1985558

0

2

1.89

1

1879853

5

3

1.89

1

1867093

6

Mean

(-) 1.90

1

(-) 1910835

3

0.0181

1

1.91

0

1980645

0

2

1.90

0

1936804

2

3

1.88

1

1817893

8

Mean

(-) 1.90

1

(-) 1911781

3

Control

1

1.90

 

1913268

 

2

1.92

 

1904380

 

3

1.92

 

2050302

 

4

1.92

 

2033024

 

5

1.90

 

2008993

 

6

1.91

 

1960981

 

Mean

1.91

 

1978491

 

1: Since inihibtion values were <5% and showed no concentration dependent correlation, growth rate inhibition is considered to be not significant

2: statistically significant, but with a higher concentration level showing no harmful effect/statistical significance compared to control (as definition of NOEC/LOEC).

Table 3: Measured Concentration of the test item in the Definitive Test with Algae

Sampling date

Fresh Media, 0 hours

Old Media, 72 hours

 

Dilution of a saturated Solution [%]

Test item

Meas. conc. [mg/L]

Meas. conc. [mg/L]

%

Geometric mean

measured concentration* [mg/L]

100

9.19

7.83

85

8.48

31.6

2.68

2.00

75

2.32

10.0

0.897

0.386

43

0.588

3.16

0.251

0.0867

35

0.148

1.00

0.0845

0.0237

28

0.0448

0.316

0.0274

0.0119

43

0.0181

Control

< LOQ

< LOQ

 

Meas. conc.= measured concentration of the test item, dilution factors taken into account

% = percent of the initial measured concentration of the test item

LOQ = limit of quantification of the analytical method (0.0200 mg/L of the test item)

*= formula for the calculation of geometric mean measured concentration (GM =\jyl * y2)

GM = Geometric mean ; y1 = Meas. cone, of the fresh media ; y2 = Meas. cone, of the old media

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201. The results based on the geometric mean measured test item concentrations were: NOEC: 2.32 mg/L, EC50 > 8.48 mg/L (for growth rate and biomass).
Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6442 cells/mL. A saturated solution with a nominal loading of 116 mg/L, corresponding to 100 mg active substance/L was prepared once 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out and transferred with an appropriate amount of the dilution water into a glass flask. This dispersion was stirred on a magnetic stirrer at approx. 1100 rpm for 24 ± 1 hour at room temperature. After completion of stirring, the solution was centrifuged for 15 minutes with 14000 rpm at 20 °C, to remove undissolved particles, and used for testing. After centrifugation, the supernatant was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. Six concentration levels were tested in a geometrical series with a spacing factor of: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 % of the saturated solution, corresponding to the geometric mean measured test item concentrations: 0.0181 - 0.0448 - 0.148 - 0.588 - 2.32 - 8.48 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. At the end of exposure after 72 hours, the measured concentrations were in the range of 28 to 85% of the initially measured concentrations. All effect values given are based on the geometric mean measured test item concentrations of the test item. The results based on the geometric mean measured test item concentrations were: NOEC: 2.32 mg/L, LOEC: 8.48 mg/L, EC50 > 8.48 mg/L (for growth rate and biomass).

Description of key information

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201. The results based on the geometric mean measured test item concentrations were: NOEC: 2.32 mg/L, EC50 > 8.48 mg/L (for growth rate and biomass).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
2.32 mg/L

Additional information

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 (reference 6.1.5 -1). The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6442 cells/mL. A saturated solution with a nominal loading of 116 mg/L, corresponding to 100 mg active substance/L was prepared once 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out and transferred with an appropriate amount of the dilution water into a glass flask. This dispersion was stirred on a magnetic stirrer at approx. 1100 rpm for 24 ± 1 hour at room temperature. After completion of stirring, the solution was centrifuged for 15 minutes with 14000 rpm at 20 °C, to remove undissolved particles, and used for testing. After centrifugation, the supernatant was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. Six concentration levels were tested in a geometrical series with a spacing factor of: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 % of the saturated solution, corresponding to the geometric mean measured test item concentrations: 0.0181 - 0.0448 - 0.148 - 0.588 - 2.32 - 8.48 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. At the end of exposure after 72 hours, the measured concentrations were in the range of 28 to 85 % of the initially measured concentrations. All effect values given are based on the geometric mean measured test item concentrations ofthe test item. The resultsbased on the geometric mean measured test item concentrations were: NOEC: 2.32 mg/L, LOEC: 8.48 mg/L, EC50 > 8.48 mg/L (for growth rate and biomass).