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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:
2016-02-23 to 2016-02-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(2006)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 6.25 - 12.50 - 25.0 - 50.0 - 100 mg/L
- Sampling method: The concentrations of the test item were analytically verified from all test concentrations and the control after 0 hours (test start) and 72 hours (test end). Samples at test start were taken from an additional replicate without algae. At end of the test samples were taken directly from the pooled test replicates.
- Sample storage conditions before analysis:All samples were stored at room temperature before preparation and before analysis, if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Stock solution: A stock solution of 100 mg/L was freshly prepared with dilution water.

Media preparation: direct addition of the test item and shaking until the test item was completely dissolved.

Test concentrations: 5 concentrations were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Pseudokirchneriella subcapitata
- Strain: HINDÁK, SAG 61.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE·m-2·s-1 for 24 hours per day.
- Culture medium: nutrient medium Z according to LÜTTGE et al. (1994)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
0.24 mmol Ca+Mg/l
Test temperature:
Nominal range: 21 - 24 °C, controlled at ± 2 °C
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
Nominal: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 5 x E03 - E04 cells/mL, Actual: mean 6305 cells/mL
- Control end cells density: Mean 1592351 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
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  2 H2O 1 x 10-5
NaHCO3 50
pH 8.1  0.2


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, mean value: 75.3

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.

TEST CONCENTRATIONS

Results of the Range Finding Test (0-72 hours)
Nominal Test Item Concentration [mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 12 45
10.0 -1 -5
1.00 -8 -56















Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
45.7 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.:
12.5 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.:
25.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: 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):
- Results with reference substance valid? Yes
EC50-Values of the Reference Item based on nominal concentrations [mg/L], (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.564 0.797 ± 0.428
95 % confidence interval 0.546 - 0.582

Yield inhibition
EyC50 0.322 0.415 ± 0.275
95 % confidence interval 0.239 - 0.342

SD = Standard deviation



Reported statistics and error estimates:
NOEC/LOEC was determined by calculation of statistical significance of growth rate and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05. Detection of statistically significant differences was considered negligible when inhibition values were ≤ 10 %. These findings were classified as “biologically not significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % are significant.
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals
EC10-, EC20- and EC50-values were carried out using standard procedures.

Cell densities

Nominal test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

100

1

6305

5242

5620

4526

2

6305

5682

5902

6412

3

6305

5327

6302

4574

Mean

6305

5417

5941

5171

 50.0

1

6305

10438

23955

76118

2

6305

11393

25181

70370

3

6305

10942

22875

43975

Mean

6305

10924

24004

63488

 25.0

1

6305

14260

76718

715064

2

6305

17215

127094

1084155

3

6305

15565

82689

838113

Mean

6305

15680

95500

879111

 12.5

1

6305

16896

142925

1092415

2

6305

18937

171369

1530995

3

6305

18263

134434

1160071

Mean

6305

18032

149576

1261160

   6.25

1

6305

17747

152092

1176703

2

6305

22655

204520

1560651

3

6305

26946

234571

1637356

Mean

6305

22449

197061

1458237

Control

1

6305

28186

229091

1505934

2

6305

27639

245740

1751356

3

6305

24217

205761

1567670

4

6305

22892

164440

1353990

5

6305

29145

248587

1594338

6

6305

30777

224022

1780815

Mean

6305

27143

219607

1592351

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal test item concentration

Replicate

Growth rate

Inhibition of growth rate

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

100

1

 

-0.111

100

 

-1779

100

2

 

0.006

100

 

107

100

3

 

-0.107

100

 

-1731

100

Mean

(+)

-0.071

100

(+)

-1134

100

 50.0

1

 

0.830

55

 

69813

96

2

 

0.804

56

 

64065

96

3

 

0.647

65

 

37670

98

Mean

(+)

0.761

59

(+)

57183

96

 25.0

1

 

1.58

14

 

708759

55

2

 

1.72

7

 

1077850

32

3

 

1.63

12

 

831808

48

Mean

(+)

1.64

11

(+)

872806

45

 12.5

1

 

1.72

7

 

1086110

32

2

 

1.83

1

 

1524690

4

3

 

1.74

6

 

1153766

27

Mean

(-)

1.76

4

(-)

1254855

21

   6.25

1

 

1.74

5

 

1170398

26

2

 

1.84

0

 

1554346

2

3

 

1.85

-1

 

1631051

-3

Mean

(-)

1.81

2

(-)

1451932

8

Control

1

 

1.83

 

 

1499629

 

2

 

1.88

 

 

1745051

 

3

 

1.84

 

 

1561365

 

4

 

1.79

 

 

1347685

 

5

 

1.84

 

 

1588033

 

6

 

1.88

 

 

1774510

 

Mean

 

1.84

 

 

1586046

 

Section-by-Section and Average Specific Growth Rates of the Control Group (0 - 72 hours)

 

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 hours)

SD

±

CV
[%]

Mean CV [%]

section-by-section

0 - 24 hours

24 - 48 hours

48 - 72 hours

Control

1

1.50

2.10

1.88

1.83

0.303

16.6

19.0

2

1.48

2.19

1.96

1.88

0.362

19.3

3

1.35

2.14

2.03

1.84

0.430

23.4

4

1.29

1.97

2.11

1.79

0.439

24.5

5

1.53

2.14

1.86

1.84

0.307

16.6

6

1.59

1.99

2.07

1.88

0.260

13.8

 

 

 

Mean

1.84

 

 

 

 

 

SD ±

0.03

 

 

 

 

CV [%]

1.83

 

 

SD = Standard deviation CV = Coefficient of variation

Validity criteria fulfilled:
yes
Conclusions:
In this study, the test substance was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 45.7 (43.2 – 47.9) mg/L and 26.1 (22.6 – 31.7) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 12.5 mg/L. All effect level are based on nominal concentration which had been analytically verified.
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. 761/2009/Method C.3.The aim of the study was the determination of NOEC, LOEC, EC10-, EC20- and EC50-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 6305 cells/mL. A stock solution of 100 mg/L was prepared by direct addition of the test item to the dilution water and shaken until the test item was completely dissolved. Five concentrations were tested in a geometrical series with a dilution factor of 2.0 (nominal): 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditionswere within the acceptable limits.

The concentrations of the test subsance were determined in the fresh media (0 h) and old media (72 h) of all tested concentration levels and the control via LC-MS/MS.
The measured concentrations in the fresh media (0 h) were in the range of 111 to 118% of the nominal values at tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 106 to 120% of the nominal values of all concentrations. Therefore, all effect values given are based on the nominal test item concentrations.
In this study, the test substance was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 45.7 (43.2 – 47.9) mg/L and 26.1 (22.6 – 31.7) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 12.5 mg/L.

Description of key information

The toxicity of the test substanceto the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 761/2009/Method C.3.The aim of the study was the determination of NOEC, LOEC, EC10-, EC20- and EC50-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 6305 cells/mL. A stock solution of 100 mg/L was prepared by direct addition of the test item to the dilution water and shaken until the test item was completely dissolved. Five concentrations were tested in a geometrical series with a dilution factor of 2.0 (nominal): 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditionswere within the acceptable limits.

The concentrations of the test subsance were determined in the fresh media (0 h) and old media (72 h) of all tested concentration levels and the control via LC-MS/MS.
The measured concentrations in the fresh media (0 h) were in the range of 111 to 118% of the nominal values at tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 106 to 120% of the nominal values of all concentrations. Therefore, all effect values given are based on the nominal test item concentrations.
In this study, the test substance was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 45.7 (43.2 – 47.9) mg/L and 26.1 (22.6 – 31.7) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 12.5 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
45.7 mg/L
EC10 or NOEC for freshwater algae:
12.5 mg/L

Additional information