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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:
2012-12-10 to 2012-12-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study, no deficiencies
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1.00 - 3.16 - 10.0 - 31.6 - 100 - 316 mg/L
- Sampling method: The concentrations of the test item were analytically determined from all test concentrations and the control at the start of the exposure (0 h) and at the end of the exposure (72 h). Samples at the start of the exposure were taken from an additional replicate without algae. At the end of the exposure samples were taken directly from the pooled test replicates.
- Sample storage conditions before analysis:
All original samples were stored at room temperature until sample preparation. Prepared samples were stored at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 316 mg/L was freshly prepared with dilution water.
- Eluate: DIlution water
- Differential loading: 1.00 - 3.16 - 10.0 - 31.6 - 100 - 316 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): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.
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:
min.: 22.0 max.: 23.0, mean value: 22.5 °C
pH:
Nominal test item concentration pH-value
Test item [mg/L] Active ingredient [mg/L a.i.] Start; 0 hours End; 72 hours
316 193 7.73 7.90
100 61.0 7.73 8.01
31.6 19.3 7.76 8.14
10.0 6.10 7.79 8.29
3.16 1.93 7.83 8.38
1.00 0.610 7.89 8.54
Control 7.97 8.71
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
See window "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, 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 2 - 5 x 103 cells/mL, Actual: mean 4332 cells/mL
- Control end cells density: Mean 717179 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: 67.9



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 56 95
10.0 13 48
1.00 2 9
0.1 3 13
0.01 1 8













Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
117 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
is UVCB means 100% act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 99.7-138 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
11.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
is UVCB means 100% act.ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 6.75-16.5 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: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes

Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.631 0.682 ± 0.452
95 %
confidence
interval 0.583 - 0.687
Yield Inhibition
EyC50 0.256 0.314 ± 0.144
95 %
confidence
interval 0.247 - 0.265
SD = Standard deviation


Reported statistics and error estimates:
NOEC/LOEC were 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 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.

Cell Densities

 

Nominal concentration

Replicate

Cell density [cells/mL]

Test item [mg/L]

Active ingredient [mg/L a.i.]

No.

0 hours

24 hours

48 hours

72 hours

 

316

193

1

4332

9329

7797

7492

 

2

4332

7747

8332

10219

 

3

4332

8511

7036

7851

 

Mean

4332

8529

7722

8521

 

100

  61.0

1

4332

10254

19431

78554

 

2

4332

10757

22157

84787

 

3

4332

10552

20069

76038

 

Mean

4332

10521

20552

79793

 

  31.6

  19.3

1

4332

10493

30973

159493

 

2

4332

9395

33876

186408

 

3

4332

9448

31350

197636

 

Mean

4332

9779

32066

181179

 

  10.0

  6.10

1

4332

13531

81365

641408

 

2

4332

14748

90193

544070

 

3

4332

16622

85042

546272

 

Mean

4332

14967

85533

577250

 

    3.16

  1.93

1

4332

16088

124647

600491

 

2

4332

16553

102739

558286

 

3

4332

17739

95706

533817

 

Mean

4332

16793

107697

564198

 

    1.00

  0.610

1

4332

21777

107248

622003

 

2

4332

22598

138151

731638

 

3

4332

25721

137742

794129

 

Mean

4332

23365

127714

715923

 

Control

1

4332

17506

119822

636187

 

2

4332

19148

137245

789380

 

3

4332

21604

108771

774253

 

4

4332

18852

113425

648673

 

5

4332

16056

123602

699543

 

6

4332

22925

129389

755037

 

Mean

4332

19349

122042

717179

 

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

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

Nominal concentration

Replicate

Growth rate

Growth rate inhibition

Yield

Inhibition of yield

Test item [mg/L]

Active ingredient [mg/L a.i.]

No.

[d-1]

[%]

[cells/mL]

[%]

316

193

1

0.183

89.3

3160

 99.6

2

0.286

83.2

5887

 99.2

3

0.198

88.4

3519

 99.5

Mean

(+)

0.222

86.9

(+)

4189

 99.4

100

  61.0

1

0.966

43.3

74222

 89.6

2

0.991

41.8

80455

 88.7

3

0.955

43.9

71706

 89.9

Mean

(+)

0.971

43.0

(+)

75461

 89.4

  31.6

  19.3

1

1.20

29.4

155161

 78.2

2

1.25

26.3

182076

 74.5

3

1.27

25.2

193304

 72.9

Mean

(+)

1.24

27.0

(+)

176847

 75.2

  10.0

  6.10

1

1.67

  2.12

637076

 10.6

2

1.61

  5.34

539738

 24.3

3

1.61

  5.26

541940

 24.0

Mean

(+)1

1.63

  4.24

(+)

572918

 19.6

    3.16

  1.93

1

1.64

  3.41

596159

 16.4

2

1.62

  4.84

553954

 22.3

3

1.61

  5.71

529485

 25.7

Mean

(+)1

1.62

  4.65

(+)

559866

 21.5

    1.00

  0.610

1

1.66

  2.72

617671

 13.4

2

1.71

 -0.46

727306

  -2.03

3

1.74

 -2.07

789797

-10.8

Mean

(-)

1.70

  0.06

(-)

711591

   0.18

Control

1

1.66

631855

2

1.74

785048

3

1.73

769921

4

1.67

644341

5

1.70

695211

6

1.72

750705

Mean

1.70

712847

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 h)

SD

±

CV
[%]

Mean CV [%]

section-by-section                           

0 - 24 hours

24 - 48 hours

48 - 72 hours

Control

1

1.40

1.92

1.67

1.66

0.264

15.8

12.7

2

1.49

1.97

1.75

1.74

0.242

13.9

3

1.61

1.62

1.96

1.73

0.203

11.7

4

1.47

1.80

1.74

1.67

0.174

10.4

5

1.31

2.04

1.73

1.70

0.367

21.7

6

1.67

1.73

1.76

1.72

0.0497

2.89

Mean

1.70

 

SD ±

0.03

 

CV [%]

1.81

 

Validity criteria fulfilled:
yes
Conclusions:
In this study Sodium methyl oleyl taurate was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-values of the test item for inhibition of growth rate and inhibition of yield after 72 hours were 10.0 mg/L and 1.00 mg/L, respectively. The LOEC-values of the test item for inhibition of growth rate and inhibition of yield after 72 hours were 31.6 mg/L and 3.16 mg/L, respectively. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 hours were 117 (99.7 – 138) and 19.5 (14.6 – 24.9) mg/L, respectively.
The NOEC-values of the active ingredient for inhibition of growth rate and inhibition of yield after 72 hours were 6.10 mg/L a.i. and 0.610 mg/L a.i., respectively. The LOEC-values of the active ingredient for inhibition of growth rate and inhibition of yield after 72 hours were 19.3 mg/L a.i. and 1.93 mg mg/L a.i., respectively. The EC50-values of the active ingredient for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 hours were 71.6 (60.8 – 84.3) and 11.9 (8.90 – 15.2) mg/L a.i., respectively.
All effect levels are given based on nominal concentrations of Sodium methyl oleyl taurate.
Executive summary:

The toxicity of Sodium methyl oleyl taurate(batch number: DEB4003485)to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2006) at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from 2012-12-10 to
2012-12-13
. The aim of the study was to assess 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 4332 cells/mL. Based on a preliminary test 6 concentration levels were tested in a geometrical series with a nominal dilution factor of : 1.00 - 3.16 - 10.0 - 31.6 - 100 - 316 mg/L corresponding to active ingredient (a.i.) concentrations of 0.610 - 1.93 - 6.10 - 19.3 - 61.0 - 193 mg a.i./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 Sodium methyl oleyl taurate (C18:1-part) were analytically verified by LC-MS/MS at the start and the end of the exposure of all test concentrations and the control.
The measured concentrations of the
test item Sodium methyl oleyl taurateat the start of the exposure were in the range of 93 to 103 % and after 72 hours in the range of 82 to 98 % of the nominal values. Therefore, all effect values given are based on the nominal test item and active ingredient (a.i.) concentrations of Sodium methyl oleyl taurate.

IMPORTANT NOTE TO AVOID CONFUSION:

As the registration substance is a UVCB this means that the test item is 100% active ingredient.

Table 1:    NOEC, LOEC, EC - values and 95 % Confidence Intervals of Sodium methyl oleyl taurate
(0-72 hours)

                   based on nominal test item and active ingredient (a.i.) concentrations [mg/L]

                               

Growth Rate Inhibition

Nominal [mg/L]

NOEC

10.0

LOEC

31.6

ErC10

11.1 (6.75 – 16.5)

ErC20

29.2 (22.6 – 36.3)

ErC50

117 (99.7 – 138)

Inhibition of Yield

NOEC

1.00

LOEC

3.16

EyC10

2.55 ( – 9.81)

EyC20

9.28 (5.65 – 14.4)

EyC50

19.5 (14.6 – 24.9)

Description of key information

In this study Sodium methyl oleyl taurate was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-values of the test item for inhibition of growth rate and inhibition of yield after 72 hours were 10.0 mg/L and 1.00 mg/L, respectively. The LOEC-values of the test item for inhibition of growth rate and inhibition of yield after 72 hours were 31.6 mg/L and 3.16 mg/L, respectively. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 hours were 117 (99.7 – 138) and 19.5 (14.6 – 24.9) mg/L, respectively.

The NOEC-values of the active ingredient for inhibition of growth rate and inhibition of yield after 72 hours were 6.10 mg/L a.i. and 0.610 mg/L a.i., respectively. The LOEC-values of the active ingredient for inhibition of growth rate and inhibition of yield after 72 hours were 19.3 mg/L a.i. and 1.93 mg mg/L a.i., respectively. The EC50-values of the active ingredient for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 hours were 71.6 (60.8 – 84.3) and 11.9 (8.90 – 15.2) mg/L a.i., respectively.

All effect levels are given based on nominal concentrations of Sodium methyl oleyl taurate.

Key value for chemical safety assessment

EC50 for freshwater algae:
117 mg/L
EC10 or NOEC for freshwater algae:
11.1 mg/L

Additional information