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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-04-07 to 2015-04-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, no deficiencies

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C8-16 (even numbered) and C18 unsaturated acyl] derivs.
EC Number:
940-422-0
Cas Number:
1591783-13-9
Molecular formula:
C15H31NO6 (C8 derivative) C17H35NO6 (C10 derivative) C19H39NO6 (C12 derivative) C21H43NO6 (C14 derivative) C23H47NO6 (C16 derivative) C25H51NO6 (C18 derivative) C25H49NO6 (C18 unsatd. derivative)
IUPAC Name:
D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C8-16 (even numbered) and C18 unsaturated acyl] derivs.
Test material form:
other: waxy
Details on test material:
- Molecular formula: C19H39NO6 (C12-Glucamide) and C21H43NO6 (C14-Glucamide)
- Molecular weight: 377.52 – 405.57 g/mol for C12- and C14-Glucamide
- Substance type: UVCB
- Physical state: waxy solid
- Stability under test conditions: Stable (in water and light at room temperature)
- Storage condition of test material: Room temperature, protected from light, in the tightly closed original container

- Other:
- Solubility (in water at RT): Slightly soluble
- pH value (in aqueous solution): Ca. 8-10 at concentration of 10.000 [mg/L]
- Melting point: 70 – 71 °C
- Boiling point: No boiling point, substance decomposes before boiling
- Vapour pressure: < 0.1 Pa

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The test item contents of all test item concentration and control solutions were analytically verified via LC-MS/MS at the beginning and the end of exposure. Separate replicates for the test item analysis at the beginning of the exposure were prepared without algae. For the test item analysis after 72 hours, samples were taken out of the test replicates.

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. Agitation until the solution is clear.


- Eluate: Dilution water
- Differential loading: 5 concentrations were tested in a geometrical series with a dilution factor of 3.16:
1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L
The concentrations are based on the results of a preliminary range-finding test (non-GLP).

- Controls: Six replicates (without test item) were incubated under the same test conditions as the test replicates.
- pH-Controls: None

Test organisms

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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No

Test conditions

Hardness:
not specified
Test temperature:
min.: 22.0 max.: 23.0, mean value: 22.5 °C
pH:
Nominal pH-value
test item concentration
[mg/L] Start; 0 hours End; 72 hours
100 7.91 7.79
31.6 7.86 7.98
10.0 8.04 8.24
3.16 8.06 8.46
1.00 8.03 8.66
Control 8.17 8.61
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
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 10^3 cells/mL, Actual: mean 4985 cells/mL
- Control end cells density: Mean 857036 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 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 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: 60.4 - 66.8 µE x m^-2 x s^-1, mean value: 63.4 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:
Chlorophyll-a-fluorescence:
Cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self fluorescence was found in the Range Finding Test at 100 mg/L.

Microscope evaluation:
Microscopic evaluation of the cells at the start and end of the exposure 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.


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

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
49 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 37.9-61.8
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 0.4-13.1
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 test item solutions were clear and have foam formation contingent on the concentrations over the period of exposure.
- 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 0.586
0.678 ± 0.414
95 % confidence interval 0.522 - 0.818 0.678 ± 0.414
Yield Inhibition
EyC50 0.401 0.319 ± 0.141
95 % confidence interval 0.285 - 0.469 0.319 ± 0.141

SD = Standard deviation
Reported statistics and error estimates:
EC-values of the growth rate were calculated by non-linear sigmoidal dose-response regression and yield inhibition by straight line regression..
The values for NOEC/LOEC were determined by calculation of statistical significance of inhibition of growth rate and yield. As standard methods, 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. 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.

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

4985

10096

10554

14304

 

2

4985

9410

13669

16902

 

3

4985

13577

15721

19808

 

Mean

4985

11028

13315

17005

 

   31.6

1

4985

13051

39522

221056

 

2

4985

12754

45541

175285

 

3

4985

13686

43067

209951

 

Mean

4985

13164

42710

202097

 

   10.0

1

4985

15556

74147

476474

 

2

4985

16998

104834

616056

 

3

4985

19379

116192

541076

 

Mean

4985

17311

98391

544535

 

      3.16

1

4985

13802

62845

509598

 

2

4985

17853

63913

397339

 

3

4985

21912

71108

511646

 

Mean

4985

17856

65955

472861

 

      1.00

1

4985

15697

89625

695653

 

2

4985

18202

102064

760696

 

3

4985

23526

109323

836379

 

Mean

4985

19142

100337

764243

 

Control

1

4985

19202

136982

959840

 

2

4985

17014

112009

875044

 

3

4985

23924

127924

942295

 

4

4985

17825

97294

647955

 

5

4985

19744

116538

944583

 

6

4985

22775

109568

772500

 

Mean

4985

20081

116719

857036

 

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

[mg/L]

Replicate

Growth rate

Growth rate inhibition

Yield

Inhibition of yield

No.

[d-1]

[%]

[cells/mL]

[%]

100

1

 

0.35

79

 

9319

99

2

                                                                                        

0.41

76

 

11917

99

3

 

0.46

73

 

14823

98

Mean

(+)

0.41

76

(+)

12020

99

31.6

1

 

1.26

26

 

216071

75

2

 

1.19

31

 

170300

80

3

 

1.25

27

 

204966

76

Mean

(+)

1.23

28

(+)

197112

77

10.0

1

 

1.52

11

 

471489

45

2

 

1.61

6

 

611071

28

3

 

1.56

9

 

536091

37

Mean

(+)

1.56

9

(+)

539550

37

3.16

1

 

1.54

10

 

504613

41

2

 

1.46

15

 

392354

54

3

 

1.54

10

 

506661

41

Mean

(+)

1.52

12

(+)

467876

45

1.00

1

 

1.65

4

 

690668

19

2

 

1.68

2

 

755711

11

3

 

1.71

0

 

831394

2

Mean

(-)

1.68

2

(-)

759258

11

Control

1

 

1.75

 

 

954855

 

2

 

1.72

 

 

870059

 

3

 

1.75

 

 

937310

 

4

 

1.62

 

 

642970

 

5

 

1.75

 

 

939598

 

6

 

1.68

 

 

767515

 

Mean

 

1.71

 

 

852051

 

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

1.35

1.97

1.95

1.75

0.351

20.0

18.7

2

1.23

1.89

2.06

1.72

0.437

25.4

3

1.57

1.68

2.00

1.75

0.223

12.8

4

1.27

1.70

1.90

1.62

0.318

19.6

5

1.38

1.78

2.09

1.75

0.359

20.5

6

1.52

1.57

1.95

1.68

0.237

14.1

 

 

 

Mean

1.71

 

 

 

 

 

SD ±

0.05

 

 

 

 

CV [%]

3.02

 

CV = Coefficient of variation

SD = Standard deviation

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, Glucamide CC was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values: The NOEC-value of the test item for both inhibition of growth rate and inhibition of yield after 72 hours was 1.00 mg/L, the LOEC for both parameters was 3.16 mg/L. The EC50-values with 95 % confidence intervals of the test item for inhibition for growth rate (ErC50) and yield (EyC50) after 72 hours were determined to be 60.1 (46.8 – 76.3) mg/L and 8.18 (5.56 – 11.8) mg/L. All effect levels are given based on the nominal concentrations of Glucamide CC.


Executive summary:

The toxicity of Glucamide CC (batch number:EK13/177) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011) and Council Regulation (EC) 440/2008/Method C.3 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from 2015-04-07 to 2015-04-10.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 4985 cells/mL. A stock solution with a nominal concentration of 100 mg/L was tested. Five concentrations were tested in a geometrical series with a dilution factor of 3.16: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L.Three replicates were tested for the test item concentrations and six for the control. Environmental conditions were determined to be within the acceptable limits.

The test item concentration in the test and control solutions were analysed by LC-MS/MS at the start and the end of exposure. The recovery rate of the test item of the fraction C12-Glucamide was in the range of 97 – 118 % of the nominal concentration at the test start and between 102 and 115 % at the test end. The recovery rate of the test item of the fraction C14-Glucamide was in the range of 94 – 114 % of the nominal concentration at the test start and between 89 and 112 % at the test end.
Alleffect values
of Glucamide CC are given based onthe nominal test item concentrations.

 

NOEC, LOEC, EC-values of Glucamide CC (0-72 hours)

               based on nominal test item concentrations [mg/L]

                               

 

Growth Rate Inhibition
Nominal test item concentration [mg/L]

NOEC

1.00

LOEC

3.16

ErC10

8.88 (3.80 – 16.2)

ErC20

22.1 (15.2 – 27.8)

ErC50

60.1 (46.8 – 76.3)

 

Inhibition of Yield

Nominal test item concentration [mg/L]

NOEC

1.00

LOEC

3.16

EyC10

<1.00

EyC20

<1.00

EyC50

13.3 (7.49 – 21.3)