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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-18 to 2010-10-25
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 concentrations of the test item were analytically determined from all test concentrations and the control after 0 h (test start) and 72 h (test end).
- Sampling method: Samples at test start were taken from an additional replicate. At end of the test samples were taken directly from the test replicates.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution: 320 mg/L, freshly prepared with dilution water
- Eluate: Dilution water
- Differential loading: test concentrations (nominal): 0.320 - 1.00 - 3.20 - 10.0 - 32.0 - 100 - 320 mg/L (factor √10)
- 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:
Dilution water: 0.24 mmol Ca+mg/L.
Test temperature:
min.: 23, max.: 24, mean value: 23.5
pH:
Nominal test item concentration, pH-value [mg/L], Start (0 h), End (72 h)
320 8.02 8.11
100 8.02 8.20
32.0 8.01 8.25
10.0 8.01 8.48
3.20 7.97 8.52
1.00 7.98 8.51
0.320 7.96 8.12
Control 8.07 8.33
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
Please refer to "Any other information on materials and methods incl. tables"
The measured concentrations of Hostapon SG dried (C12 and C14 fraction) at test start were in the range of 81 - 117% and after 72h in the range of 2 % – 111 % of the nominal values. Recoveries for the samples at test start indicate the accurate preparation of the test solutions.
At test end reduced recoveries (< 80% of nominal value) for the C12 fraction were obtained in the exposure groups with 32 and 100 mg/L test item. Analogous to these findings recoveries were < 80% for the C14 fraction in these exposure groups. These reduced recoveries were not a result of an inaccurate analytical method as confirmed by duplicate analysis (results not shown here but documented in the raw data!) but rather caused by the biotic test system.
All effect values were based on the nominal product concentrations.
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, current: approximately 4 x 103 cells/mL
- Control end cells density: Mean 706945 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: 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, mean value: 71.8



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter
Cell density was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. The pH-value at the beginning of the test was measured out of one additional replicate from each dilution level and the control. At the end it was measured from one pool per concentration and control. The room temperature was measured continuously by a hygro-thermograph. Light intensity was measured prior to test start. Microscopic evaluation of the cells at the start and end of the incubation 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
- Range finding study: Yes
The results of the preliminary range finding test are presented below:

Results of the Range Finding Test (0-72 h)

Nominal
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition
[%] Yield Inhibition

[%]
100 48 93
10 8 36
1 3 14
0.1 3 13
0.01 4 17



Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
21.9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
68.5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
21.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 17.7-26,4 (95% CI)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
61 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 57.3-65.9 (95% CI)
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape, Aggregation of algal cells: At the end of the test the cells of the concentrations 32 - 320 mg/L were agglutinated and partly enlarged and at test concentration of 10 mg/L cells were partly enlarged.
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: 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 h)

Current Study Valid Range (average ± 3 x SD)
Rate-related inhibition
ErC50 0.589 0.738 ± 0.437
95 % confidence interval 0.535 – 0.652
Yield inhibition
EyC50 0.263 0.328 ± 0.146
95 % confidence interval 0.252 – 0.274
SD = Standard deviation

Reported statistics and error estimates:
NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. One Way Analysis of Variance was carried out for the determination of statistically significant differences compared to control replicates. When running a One Way Analysis of Variance a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests were 0.05. The a-value for ANOVA was a=0.05. Normality test failed for yield calculation. No suitable transformation could be found. Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 % acc. to HEGER et al (1998). These findings were marked “biologically not significant”.
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Hostapon SG dried (0-72 h)

based on nominal test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

32.0

LOEC

100

ErC10

31.7 (25.9 – 38.6)

ErC20

47.2 (40.8 – 53.5)

ErC50

89.9 (84.3 – 96.2)

Inhibition of Yield

NOEC

1.00

LOEC

3.20

EyC10

6.27 (3.29 – 12.3)

EyC20

11.2 (6.55 – 17.9)

EyC50

30.5 (21.5 – 43.4)

Cell Densities

 

Nominal test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

320

1

4069

5085

3898

4217

 

2

4069

5301

3215

2507

 

3

4069

5309

3349

2484

 

Mean

4069

5232

3487

3069

 

100

1

4069

8812

22143

33606

 

2

4069

9473

23453

44453

 

3

4069

9047

21205

39671

 

Mean

4069

9111

22267

39243

 

  32.0

1

4069

10397

67699

418643

 

2

4069

11747

85011

440259

 

3

4069

14440

86283

459831

 

Mean

4069

12195

79664

439578

 

  10.0

1

4069

9999

78784

498107

 

2

4069

15527

80618

473943

 

3

4069

16193

88817

549963

 

Mean

4069

13906

82740

507338

 

    3.20

1

4069

15165

80545

591179

 

2

4069

16353

100627

612683

 

3

4069

17907

109559

665547

 

Mean

4069

16475

96910

623136

 

    1.00

1

4069

16025

117267

777239

 

2

4069

16969

138038

749323

 

3

4069

19455

130231

903267

 

Mean

4069

17483

128512

809943

 

    0.320

1

4069

14911

118973

817839

 

2

4069

17755

119950

695395

 

3

4069

18063

140779

744199

 

Mean

4069

16910

126567

752478

 

Control

1

4069

14146

108310

658295

 

2

4069

16462

149479

857571

 

3

4069

19699

127661

778835

 

4

4069

17377

112093

664091

 

5

4069

16540

104530

626571

 

6

4069

20572

124934

656307

 

Mean

4069

17466

121168

706945

 

Evaluation after 72 h

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

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

320

1

 0.012

  99.3

148

100

2

-0.161

100

-1562

100

3

-0.165

100

-1585

100

Mean

(+)

-0.105

100

(+)

-1000

100

100

1

 0.704

  59.0

29537

  95.8

2

 0.797

  53.6

40384

  94.3

3

 0.759

  55.8

35602

  94.9

Mean

(+)

 0.753

  56.1

(+)

35174

  95.0

  32.0

1

 1.55

  10.0

414574

  41.0

2

 1.56

    9.07

436190

  37.9

3

 1.58

    8.22

455762

  35.2

Mean

(+)*

 1.56

    9.11

(+)

435509

  38.0

  10.0

1

 1.60

    6.67

494038

  29.7

2

 1.59

    7.64

469874

  33.2

3

 1.64

    4.75

545894

  22.3

Mean

(+)*

 1.61

    6.35

(+)

503269

  28.4

    3.20

1

 1.66

    3.35

587110

  16.5

2

 1.67

    2.65

608614

  13.4

3

 1.70

    1.05

661478

  5.89

Mean

(-)

 1.68

    2.35

(-)1

619067

  11.9

    1.00

1

 1.75

   -1.97

773170

 -10.0

2

 1.74

   -1.26

745254

   -6.03

3

 1.80

   -4.88

899198

 -27.9

Mean

(-)

 1.76

   -2.70

(-)

805874

 -14.7

    0.320

1

 1.77

   -2.95

813770

 -15.8

2

 1.71

    0.19

691326

    1.64

3

 1.74

   -1.12

740130

   -5.30

Mean

(-)

 1.74

   -1.29

(-)

748409

   -6.48

Control

1

 1.70

654226

2

 1.78

853502

3

 1.75

774766

4

 1.70

660022

5

 1.68

622502

6

 1.69

652238

Mean

 1.72

702876

*) biologically not significant ( 10 %)

1) biologically significant

negative inhibitions = increase of growth

Validity criteria fulfilled:
yes
Remarks:
The cell growth increased 174-fold after 72 h in the control (required: at least 16-fold). The temperature during the test was in the range of 21 – 24 °C. The difference from the mean was within ± 2 °C: min.: 23 °C, max.: 24 °C mean value: 23.5 °C.
Conclusions:
In this study Hostapon SG dried 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 32.0 and 1.00 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 100 and 3.20 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 h were 89.9 (84.3 – 96.2) and 30.5 (21.5 – 43.4) mg/L, respectively.
All effect levels are given based on nominal concentrations of Hostapon SG dried.Values based on a.i. basis are given in the result section.
Executive summary:

The toxicity of Hostapon SG dried (batch number: EK 10/101) 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 October 18 to 25, 2010 with the definitive exposure phase from October 18 to 21, 2010. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

The study was conducted under static conditions with an initial cell density of approximately 4 x 103cells/mL. Based on a preliminary test 7 concentration levels were tested in a geometrical series with a dilution factor of 10, nominal: 0.320 - 1.00 - 3.20 - 10.0 - 32.0 - 100 - 320 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 Hostapon SG dried were analysed at all concentration levels after 0 and 72 h via LC-MS/MS analysis. The measured concentrations of Hostapon SG dried at test start were in the range of 105 – 117 % (C12-chain) and 81 – 110 % (C14-chain) of the nominal values. At the end of the test Hostapon SG dried gave recoveries of 2 - 119 % (C12-chain) and 31 – 106 % (C14-chain) of the nominal values. All effect values are given based on the nominal concentrations of the test item as it contains different C-chains of the N-Cocoyl glycine sodium salt, and toxicity cannot be attributed to any single compound. All effect values are given based on nominal concentrations of the test item.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Hostapon SG dried(0-72 h)

                   based on nominal test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

32.0

LOEC

100

ErC10

31.7 (25.9 – 38.6)

ErC20

47.2 (40.8 – 53.5)

ErC50

89.9 (84.3 – 96.2)

Inhibition of Yield

NOEC

1.00

LOEC

3.20

EyC10

6.27 (3.29 – 12.3)

EyC20

11.2 (6.55 – 17.9)

EyC50

30.5 (21.5 – 43.4)

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2010-10-18 to 2010-10-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Sodium Lauroyl Glycinate (SLG) (Chapter 13)
Reason / purpose for cross-reference:
read-across source
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 concentrations of the test item were analytically determined from all test concentrations and the control after 0 h (test start) and 72 h (test end).
- Sampling method: Samples at test start were taken from an additional replicate. At end of the test samples were taken directly from the test replicates.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution: 320 mg/L, freshly prepared with dilution water
- Eluate: Dilution water
- Differential loading: test concentrations (nominal): 0.320 - 1.00 - 3.20 - 10.0 - 32.0 - 100 - 320 mg/L (factor √10)
- 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:
Dilution water: 0.24 mmol Ca+mg/L.
Test temperature:
min.: 23, max.: 24, mean value: 23.5
pH:
Nominal test item concentration, pH-value [mg/L], Start (0 h), End (72 h)
320 8.02 8.11
100 8.02 8.20
32.0 8.01 8.25
10.0 8.01 8.48
3.20 7.97 8.52
1.00 7.98 8.51
0.320 7.96 8.12
Control 8.07 8.33
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
Please refer to "Any other information on materials and methods incl. tables"
The measured concentrations of Hostapon SG dried (C12 and C14 fraction) at test start were in the range of 81 - 117% and after 72h in the range of 2 % – 111 % of the nominal values. Recoveries for the samples at test start indicate the accurate preparation of the test solutions.
At test end reduced recoveries (< 80% of nominal value) for the C12 fraction were obtained in the exposure groups with 32 and 100 mg/L test item. Analogous to these findings recoveries were < 80% for the C14 fraction in these exposure groups. These reduced recoveries were not a result of an inaccurate analytical method as confirmed by duplicate analysis (results not shown here but documented in the raw data!) but rather caused by the biotic test system.
All effect values were based on the nominal product concentrations.
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, current: approximately 4 x 103 cells/mL
- Control end cells density: Mean 706945 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: 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, mean value: 71.8



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter
Cell density was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. The pH-value at the beginning of the test was measured out of one additional replicate from each dilution level and the control. At the end it was measured from one pool per concentration and control. The room temperature was measured continuously by a hygro-thermograph. Light intensity was measured prior to test start. Microscopic evaluation of the cells at the start and end of the incubation 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
- Range finding study: Yes
The results of the preliminary range finding test are presented below:

Results of the Range Finding Test (0-72 h)

Nominal
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition
[%] Yield Inhibition

[%]
100 48 93
10 8 36
1 3 14
0.1 3 13
0.01 4 17



Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
21.9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
68.5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
21.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 17.7-26,4 (95% CI)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
61 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 57.3-65.9 (95% CI)
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape, Aggregation of algal cells: At the end of the test the cells of the concentrations 32 - 320 mg/L were agglutinated and partly enlarged and at test concentration of 10 mg/L cells were partly enlarged.
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: 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 h)

Current Study Valid Range (average ± 3 x SD)
Rate-related inhibition
ErC50 0.589 0.738 ± 0.437
95 % confidence interval 0.535 – 0.652
Yield inhibition
EyC50 0.263 0.328 ± 0.146
95 % confidence interval 0.252 – 0.274
SD = Standard deviation

Reported statistics and error estimates:
NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. One Way Analysis of Variance was carried out for the determination of statistically significant differences compared to control replicates. When running a One Way Analysis of Variance a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests were 0.05. The a-value for ANOVA was a=0.05. Normality test failed for yield calculation. No suitable transformation could be found. Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 % acc. to HEGER et al (1998). These findings were marked “biologically not significant”.
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Hostapon SG dried (0-72 h)

based on nominal test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

32.0

LOEC

100

ErC10

31.7 (25.9 – 38.6)

ErC20

47.2 (40.8 – 53.5)

ErC50

89.9 (84.3 – 96.2)

Inhibition of Yield

NOEC

1.00

LOEC

3.20

EyC10

6.27 (3.29 – 12.3)

EyC20

11.2 (6.55 – 17.9)

EyC50

30.5 (21.5 – 43.4)

Cell Densities

 

Nominal test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

320

1

4069

5085

3898

4217

 

2

4069

5301

3215

2507

 

3

4069

5309

3349

2484

 

Mean

4069

5232

3487

3069

 

100

1

4069

8812

22143

33606

 

2

4069

9473

23453

44453

 

3

4069

9047

21205

39671

 

Mean

4069

9111

22267

39243

 

  32.0

1

4069

10397

67699

418643

 

2

4069

11747

85011

440259

 

3

4069

14440

86283

459831

 

Mean

4069

12195

79664

439578

 

  10.0

1

4069

9999

78784

498107

 

2

4069

15527

80618

473943

 

3

4069

16193

88817

549963

 

Mean

4069

13906

82740

507338

 

    3.20

1

4069

15165

80545

591179

 

2

4069

16353

100627

612683

 

3

4069

17907

109559

665547

 

Mean

4069

16475

96910

623136

 

    1.00

1

4069

16025

117267

777239

 

2

4069

16969

138038

749323

 

3

4069

19455

130231

903267

 

Mean

4069

17483

128512

809943

 

    0.320

1

4069

14911

118973

817839

 

2

4069

17755

119950

695395

 

3

4069

18063

140779

744199

 

Mean

4069

16910

126567

752478

 

Control

1

4069

14146

108310

658295

 

2

4069

16462

149479

857571

 

3

4069

19699

127661

778835

 

4

4069

17377

112093

664091

 

5

4069

16540

104530

626571

 

6

4069

20572

124934

656307

 

Mean

4069

17466

121168

706945

 

Evaluation after 72 h

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

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

320

1

 0.012

  99.3

148

100

2

-0.161

100

-1562

100

3

-0.165

100

-1585

100

Mean

(+)

-0.105

100

(+)

-1000

100

100

1

 0.704

  59.0

29537

  95.8

2

 0.797

  53.6

40384

  94.3

3

 0.759

  55.8

35602

  94.9

Mean

(+)

 0.753

  56.1

(+)

35174

  95.0

  32.0

1

 1.55

  10.0

414574

  41.0

2

 1.56

    9.07

436190

  37.9

3

 1.58

    8.22

455762

  35.2

Mean

(+)*

 1.56

    9.11

(+)

435509

  38.0

  10.0

1

 1.60

    6.67

494038

  29.7

2

 1.59

    7.64

469874

  33.2

3

 1.64

    4.75

545894

  22.3

Mean

(+)*

 1.61

    6.35

(+)

503269

  28.4

    3.20

1

 1.66

    3.35

587110

  16.5

2

 1.67

    2.65

608614

  13.4

3

 1.70

    1.05

661478

  5.89

Mean

(-)

 1.68

    2.35

(-)1

619067

  11.9

    1.00

1

 1.75

   -1.97

773170

 -10.0

2

 1.74

   -1.26

745254

   -6.03

3

 1.80

   -4.88

899198

 -27.9

Mean

(-)

 1.76

   -2.70

(-)

805874

 -14.7

    0.320

1

 1.77

   -2.95

813770

 -15.8

2

 1.71

    0.19

691326

    1.64

3

 1.74

   -1.12

740130

   -5.30

Mean

(-)

 1.74

   -1.29

(-)

748409

   -6.48

Control

1

 1.70

654226

2

 1.78

853502

3

 1.75

774766

4

 1.70

660022

5

 1.68

622502

6

 1.69

652238

Mean

 1.72

702876

*) biologically not significant ( 10 %)

1) biologically significant

negative inhibitions = increase of growth

Validity criteria fulfilled:
yes
Remarks:
The cell growth increased 174-fold after 72 h in the control (required: at least 16-fold). The temperature during the test was in the range of 21 – 24 °C. The difference from the mean was within ± 2 °C: min.: 23 °C, max.: 24 °C mean value: 23.5 °C.
Conclusions:
In this study Hostapon SG dried 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 32.0 and 1.00 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 100 and 3.20 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 h were 89.9 (84.3 – 96.2) and 30.5 (21.5 – 43.4) mg/L, respectively.
All effect levels are given based on nominal concentrations of Hostapon SG dried.Values based on a.i. basis are given in the result section.
Executive summary:

The toxicity of Hostapon SG dried (batch number: EK 10/101) 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 October 18 to 25, 2010 with the definitive exposure phase from October 18 to 21, 2010. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

The study was conducted under static conditions with an initial cell density of approximately 4 x 103cells/mL. Based on a preliminary test 7 concentration levels were tested in a geometrical series with a dilution factor of 10, nominal: 0.320 - 1.00 - 3.20 - 10.0 - 32.0 - 100 - 320 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 Hostapon SG dried were analysed at all concentration levels after 0 and 72 h via LC-MS/MS analysis. The measured concentrations of Hostapon SG dried at test start were in the range of 105 – 117 % (C12-chain) and 81 – 110 % (C14-chain) of the nominal values. At the end of the test Hostapon SG dried gave recoveries of 2 - 119 % (C12-chain) and 31 – 106 % (C14-chain) of the nominal values. All effect values are given based on the nominal concentrations of the test item as it contains different C-chains of the N-Cocoyl glycine sodium salt, and toxicity cannot be attributed to any single compound. All effect values are given based on nominal concentrations of the test item.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Hostapon SG dried(0-72 h)

                   based on nominal test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

32.0

LOEC

100

ErC10

31.7 (25.9 – 38.6)

ErC20

47.2 (40.8 – 53.5)

ErC50

89.9 (84.3 – 96.2)

Inhibition of Yield

NOEC

1.00

LOEC

3.20

EyC10

6.27 (3.29 – 12.3)

EyC20

11.2 (6.55 – 17.9)

EyC50

30.5 (21.5 – 43.4)

Description of key information

The toxicity to aquatic algae of the registration substance Hostapon SLG was evaluated based on the read-across approach using the toxicity to aquatic algae data of Hostapon SG.

In this study Hostapon SG dried 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 32.0 and 1.00 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 100 and 3.20 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 h were 89.9 (84.3 – 96.2) and 30.5 (21.5 – 43.4) mg/L, respectively.

All effect levels are given based on nominal concentrations of Hostapon SG dried.Values based on a.i. basis are given in the result section.

Key value for chemical safety assessment

EC50 for freshwater algae:
61.6 mg/L
EC10 or NOEC for freshwater algae:
21.7 mg/L

Additional information