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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:
2010-05-24 to 2010-06-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The concentration of the test item was analytically determined from all test concentrations and the control after 0 h (test start) and 72 h (test end).
- Sampling method: Separate samples at start (provided without alga) were prepared. Samples at the end of the test were taken directly from the test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis, if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 mg/L was freshly prepared with dilution water. The stock solution was stirred with approx. 1100 rpm for 2 h at room temperature.
- Eluate: Dilution water
- Differential loading: 0.100 - 0.320 - 1.00 - 3.20 - 10.0 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- 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 were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- 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
Post exposure observation period:
No
Hardness:
Not measured
Test temperature:
Mean: 22.8 °C, Min: 22.1 °C, Max: 23.5 °C
pH:
Geometric mean measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
9.52 7.64 7.48
2.87 7.68 7.49
0.917 7.68 7.89
0.289 7.83 7.90
0.0718 7.90 7.92
Control 7.90 7.94
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to "Any other information on materials and methods incl. tables"
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- 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: 4254 cells/mL
- Control end cells density: Mean 712901 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


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



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Tests (0-72 h)

Nominal
Test Item Concentration [mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
10 97 100
1 32 77
0.1 -9 -47
0.01 -8 -41







Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (1.87 - 2.61)
Remarks:
Confidence interval
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.323 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Confidence interval: 0.225 – 0.467 mg/L
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: Yes
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.614 (CI 0.551 - 0.679) mg/L
Yield Inhibition: 0.333 (CI 0.318 - 0.350) mg/L
Reported statistics and error estimates:
NOEC/LOEC was determined by calculation of statistical significance of growth rates 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. P-values for both Normality and Equal Variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05. Equal variance test failed for growth rate calculation. The data were transformed using Graph Pad Prism function Y = Y sqrt.
Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 %. These findings were marked “biologically not significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % are significant.

NOEC, LOEC and EC - values of the test item (0-72 h)

                   based on geometric mean measured test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

0.289

LOEC

0.917

ErC10

0.323 (0.225 – 0.467)

ErC20

0.656 (0.498 – 0.841)

ErC50

2.20 (1.87 – 2.61)

Inhibition of Yield

NOEC

0.0718

LOEC

0.289

EyC10

0.137 (0.0918 – 0.209)

EyC20

0.210 (0.151 – 0.281)

EyC50

0.437 (0.352 – 0.542)

Cell Densities

 

Geometric mean measured test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

9.52

1

4254

9736

8715

7268

 

2

4254

7620

9432

8588

 

3

4254

7177

11085

6911

 

Mean

4254

8178

9744

7589

 

2.87

1

4254

16677

29235

48686

 

2

4254

18346

29580

55094

 

3

4254

16177

25771

47357

 

Mean

4254

17067

28195

50379

 

0.917

1

4254

14578

68206

140247

 

2

4254

13721

52491

107599

 

3

4254

12778

55976

181587

 

Mean

4254

13692

58891

143144

 

0.289

1

4254

23840

135483

540489

 

2

4254

21536

107139

384234

 

3

4254

26017

119193

520231

 

Mean

4254

23798

120605

481651

 

0.0718

1

4254

23639

166093

659975

 

2

4254

21044

133322

671025

 

3

4254

27977

153064

856345

 

Mean

4254

24220

150826

729115

 

Control

1

4254

24180

190308

751450

 

2

4254

30137

140030

598541

 

3

4254

19178

194465

723189

 

4

4254

31113

176798

787924

 

5

4254

23987

160903

646593

 

6

4254

25927

145450

769708

 

Mean

4254

25754

167992

712901

 

Evaluation after 72 h

                   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

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

9.52

1

0.179

89.5

3014

 99.6

2

0.234

86.3

4334

 99.4

3

0.162

90.5

2657

 99.6

Mean

(+)

0.191

88.8

(+)

3335

 99.5

2.87

1

0.813

52.4

44432

 93.7

2

0.854

49.9

50840

 92.8

3

0.803

52.9

43103

 93.9

Mean

(+)

0.823

51.7

(+)

46125

 93.5

0.917

1

1.17

31.7

135993

 80.8

2

1.08

36.9

103345

 85.4

3

1.25

26.6

177333

 75.0

Mean

(+)

1.16

31.7

(+)

138890

 80.4

0.289

1

1.62

  5.32

536235

 24.3

2

1.50

12.0

379980

 46.4

3

1.60

  6.06

515977

 27.2

Mean

(+)*

1.57

  7.79

(+)

477397

 32.6

0.0718

1

1.68

  1.41

655721

   7.47

2

1.69

  1.09

666771

   5.91

3

1.77

 -3.68

852091

-20.2

Mean

(-)

1.71

 -0.39

(-)

724861

  -2.29

Control

1

1.73

747196

2

1.65

594287

3

1.71

718935

4

1.74

783670

5

1.68

642339

6

1.73

765454

Mean

1.71

708647

Section-by-Section and Average Specific Growth Rates of the Control Group

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 h)

SD

±

CV
[%]

Mean CV [%]

section-by-section                            

0 - 24 h

24 - 48 h

48 - 72 h

Control

1

1.74

2.06

1.37

1.73

0.345

20.0

16.9

2

1.96

1.54

1.45

1.65

0.271

16.4

3

1.51

2.32

1.31

1.71

0.532

31.1

4

1.99

1.74

1.49

1.74

0.248

14.2

5

1.73

1.90

1.39

1.68

0.261

15.6

6

1.81

1.73

1.67

1.73

0.071

  4.10

Mean

1.71

 

SD ±

0.04

 

CV [%]

2.12

 
Validity criteria fulfilled:
yes
Conclusions:
In this study the test item was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h. The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 0.289 and 0.0718 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 0.917 and 0.289 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 2.20 (1.87 – 2.61) and 0.437 (0.352 – 0.542) mg/L, respectively. The EC10-values for inhibition of specific growth rate (ErC10) and yield (EyC10) after 72 h were 0.137 (0.0918 – 0.209) and 0.323 (0.225 – 0.467) mg/L, respectively.
All effect levels are given based on geometric mean measured concentrations of the test item.
Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 .  

The study was conducted under static conditions with an initial cell density of approximately 2 – 5 x 103cells/mL. Based on a preliminary test 5 concentration levels were tested in a geometrical series with a dilution factor of √10, nominal: 0.100 - 0.320 - 1.00 - 3.20 - 10.0 mg/Lcorresponding to geometric mean measured test item concentrations of 0.0718 - 0.289 - 0.917 - 2.87 - 9.52 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of the test item were analysed at all concentration levels after 0 and 72 h via LC-MS/MS analysis. At test start the recoveries of the test item were in the range of 102 to 111 % of the nominal values and at test end in the range of 47 to 86 % of the nominal values. All effect values are given based on geometric mean measured concentrations of the test item.

NOEC, LOEC and EC - values of the test item (0-72 h)

                   based on geometric mean measured test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

0.289

LOEC

0.917

ErC10

0.323 (0.225 – 0.467)

ErC20

0.656 (0.498 – 0.841)

ErC50

2.20 (1.87 – 2.61)

Inhibition of Yield

NOEC

0.0718

LOEC

0.289

EyC10

0.137 (0.0918 – 0.209)

EyC20

0.210 (0.151 – 0.281)

EyC50

0.437 (0.352 – 0.542)

Description of key information

Toxicity to aquatic algae - OECD 201

The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 0.289 and 0.0718 mg/L, respectively.

The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 0.917 and 0.289 mg/L, respectively.

The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 2.20 (1.87 – 2.61) and 0.437 (0.352 – 0.542) mg/L, respectively.

The EC10-values for inhibition of specific growth rate (ErC10) and yield (EyC10) after 72 h were 0.137 (0.0918 – 0.209) and 0.323 (0.225 – 0.467) mg/L, respectively.

All effect levels are given based on geometric mean measured concentrations of the test item.

PARAD consortium, NOACK, 2010, D. Scheerbaum)

Key value for chemical safety assessment

EC50 for freshwater algae:
2.2 mg/L
EC10 or NOEC for freshwater algae:
0.323 mg/L

Additional information

Toxicity to aquatic algae - OECD 201 – EC50: 2.2 mg/l, 72h - PARAD consortium, NOACK, 2010, D. Scheerbaum)

The toxicity of the test item (batch number: S700040031) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from May 24 to June 09, 2010 with the definitive exposure phase of the study from May 25 to 28, 2010. 

The study was conducted under static conditions with an initial cell density of approximately 2 – 5 x 103cells/mL.Based on a preliminary test 5 concentration levels were tested in a geometrical series with a dilution factor of √10, nominal: 0.100 - 0.320 - 1.00 - 3.20 - 10.0 mg/L corresponding to geometric mean measured test item concentrations of 0.0718 - 0.289 - 0.917 - 2.87 - 9.52 mg/L.Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of the test item were analysed at all concentration levels after 0 and 72 h via LC-MS/MS analysis. At test start the recoveries of the test item were in the range of 102 to 111 % of the nominal values and at test end in the range of 47 to 86 % of the nominal values. All effect values are given based on geometric mean measured concentrations of the test item.The ERC50 after 72 h was determined to be: ERC50: 2.2 mg/l and the ERC10 after 72 h was determined to be: ERC10: 0.323 mg/l

(based on geometric mean measured test item concentrations [mg/L])

 

Evaluation: valid without restrictions, Klimisch-I , Key study

Assessment: The test item is toxic to algae.