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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-11-20 to 2012-11-23
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Sorbitan monocaprylate
- Physical state: Viscous liquid, brown, clear
- Analytical purity: 99.7 % (w/w)
- Purity test date: 2011-09-21
- Lot/batch No.: DEG4149186
- Expiration date of the lot/batch: 2013-07-06
- Stability under test conditions: Stable in water and light at room temperature
- Storage condition of test material: Room temperature, protected from light, in the original container

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L
- Sampling method: All test item concentrations and the control were analytically verified via analysis of organic carbon (TOC, according to DIN EN 1484) at the start and at the end of the test. Separate replicates for the test item analysis after 0 and 72 hours were prepared without algae and incubated under test conditions.
- Sample storage conditions before analysis: None, direct analysis

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 mg/L was freshly prepared with dilution water and agitated.
- Eluate: Dilution water
- Differential loading: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test vessels.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus CHODAT SAG 86.81
- 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): Not the same, standard growth medium
- Any deformed or abnormal cells observed: No

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None

Test conditions

Hardness:
Not measured
Test temperature:
Mean: 22.5 °C, Min: 21.5 °C, Max: 23.5 °C
pH:
Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
100 7.49 8.04
50.0 7.55 8.06
25.0 7.60 8.14
12.5 7.66 8.22
6.25 7.76 8.32
Control 7.98 8.40
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to "Any other information on materials and methods"
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): sealed with 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: 4332 cells/mL
- Control end cells density: Mean 714201 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:
According to the guideline (OECD)
Composition of Dilution Water

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-value 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



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 Test (0-72 hours)

Nominal Test Item Concentration
[mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 72 98
10 1 5
1 -7 -43

















Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
79.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI (74.7-84.5 mg/L)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
16.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI (13.1 - 19.3 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: The stock solution was slightly turbid. After 24 hours a concentration-related agglutination of the test item was observed in the two highest concentration levels.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.631 (CI 0.583 - 0.687) mg/L
Yield Inhibition: 0.256 (CI 0.247 - 0.265) mg/L
Reported statistics and error estimates:
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. The calculation of the confidence intervals for the EC10-, EC20- and EC50-values were carried out from the best fit values, the standard error and the t-distribution.
NOEC/LOEC were determined by calculation of statistical
significance of growth rate and yield. As a 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 a=0.05.
The Normality Test failed for yield calculation. Transformation (y = Log (y)) was carried out to pass the Normality Test.

Any other information on results incl. tables

Cell Densities

Nominal test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

100

1

4332

9722

21588

27592

2

4332

12713

24390

33121

3

4332

8838

24585

32419

Mean

4332

10424

23521

31044

50.0

1

4332

8983

38257

156411

2

4332

9247

31360

132390

3

4332

10040

33379

144479

Mean

4332

9423

34332

144427

25.0

1

4332

11839

66577

338255

2

4332

12911

74377

296930

3

4332

12087

71182

291866

Mean

4332

12279

70712

309017

12.5

1

4332

13185

80422

426944

2

4332

12961

72874

428705

3

4332

12785

66118

433769

Mean

4332

12977

73138

429806

  6.25

1

4332

13367

106110

680179

2

4332

13260

131037

730688

3

4332

15298

129880

706409

Mean

4332

13975

122342

705759

Control

1

4332

18107

146920

877434

2

4332

15509

122741

640584

3

4332

16518

136214

750565

4

4332

17437

133069

724209

5

4332

19396

124741

652189

6

4332

13851

104396

640222

Mean

4332

16803

128014

714201

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), non 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

0.617

63.7

23260

96.7

2

0.678

60.1

28789

95.9

3

0.671

60.5

28087

96.0

Mean

(+)

0.655

61.4

(+)

26712

96.2

50.0

1

1.20

29.7

152079

78.6

2

1.14

32.9

128058

82.0

3

1.17

31.2

140147

80.3

Mean

(+)

1.17

31.3

(+)

140095

80.3

25.0

1

1.45

14.5

333923

53.0

2

1.41

17.1

292598

58.8

3

1.40

17.4

287534

59.5

Mean

(+)

1.42

16.3

(+)

304685

57.1

12.5

1

1.53

   9.96

422612

40.5

2

1.53

   9.88

424373

40.2

3

1.54

   9.65

429437

39.5

Mean

(+)*

1.53

   9.83

(+)

425474

40.1

  6.25

1

1.69

   0.83

675847

   4.79

2

1.71

 -0.58

726356

 -2.32

3

1.70

   0.09

702077

   1.10

Mean

(-)

1.70

   0.11

(-)

701427

   1.19

Control

1

1.77

873102

2

1.67

636252

3

1.72

746233

4

1.71

719877

5

1.67

647857

6

1.67

635890

Mean

1.70

709869

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

1.43

2.09

1.79

1.77

0.332

18.8

20.3

2

1.28

2.07

1.65

1.67

0.397

23.8

3

1.34

2.11

1.71

1.72

0.386

22.5

4

1.39

2.03

1.69

1.71

0.320

18.8

5

1.50

1.86

1.65

1.67

0.182

10.9

6

1.16

2.02

1.81

1.67

0.448

26.9

Mean

1.70

 

SD ±

0.04

 

CV [%]

2.43

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, Sorbitan monocaprylate, was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus over the period of 72 hours. The nominal EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 79.8 (74.7 – 84.5) and 17.5 (15.4 – 19.7) mg test item/L, respectively. The nominal NOEC-values for inhibition of growth rate and yield after 72 hours were 12.5 and 6.25 mg test item/L, respectively. For other endpoints, please refer to Table1.
All effect values given are based on nominal test item concentrations of Sorbitan monocaprylate.
Executive summary:

The toxicity of Sorbitan monocaprylate (batch number: DEG4149186)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 2012-11-20 to 2012-11-23. 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, 5 concentrations were tested in a geometrical series with a dilution factor of 2, 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. Environmental conditions were within the acceptable limits.

All test concentrations of Sorbitan monocaprylate, and the control were analytically verified via analysis of organic carbon (TOC) at the start and the end of the exposure.

All effect values given are based on nominal test item concentrations of Sorbitan monocaprylate. The growth inhibition results of the test item are summarized in Table 1.

Table 1:    NOEC, LOEC, EC - values and 95 % Confidence Intervals of Sorbitan monocaprylate
(0-72 hours)

                   based on nominal test item concentrations [mg/L]

Inhibition of Growth Rate

NOEC

12.5

LOEC

25.0

ErC10

16.3 (13.1 – 19.3)

ErC20

31.5 (28.5 – 34.8)

ErC50

79.8 (74.7 – 84.5)

Inhibition of Yield

NOEC

6.25

LOEC

12.5

EyC10

7.33 (6.46 – 8.13)

EyC20

9.00 (8.12 – 10.1)

EyC50

17.5 (15.4 – 19.7)