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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:
2016-02-22 to 2016-02-25
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Determination of the test item
Analytical evaluation of the limit concentration of Genamin DMG 75 and the control was carried out via LC-MS/MS after 0 (start of the exposure) and 72 hours (end of the exposure) with algae.
Vehicle:
no
Details on test solutions:
Stock solution, preparation of the test media
A stock solution with a concentration of 100 mg/L was freshly prepared with dilution water and agitated until the test item was completely dissolved.

Test concentrations
A limit concentration level of 100 mg/L was tested.

Control
Six replicates (without test item) were tested under the same conditions as the test concentration.
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:
yes
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
not specified
Test temperature:
min.: 21.0 max.: 22.0, mean value: 21.5 °C
pH:
Nominal test item concentration
[mg/L] pH-value
Start; 0 hours End; 72 hours
100 8.76 8.47
Control 7.96 8.43
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 10^3 cells/mL, Actual: mean 4847 cells/mL
- Control end cells density: Mean 756300 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 - 120 µE x m^-2 x s^-1, mean value: 70.5 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, 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.


Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
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):
The toxicity of potassium dichromate (SIGMA, batch number MKBQ9179V, purity 100.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Desmodesmus subspicatus was determined over a period of 72 hours from 2015-10-19 to 2015-10-22. For results see Table 10. The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.685 0.671 ± 0.401
95 % confidence interval 0.603 - 0.794
Yield Inhibition
EyC50 0.362 0.320 ± 0.139
95 % confidence interval 0.328 - 0.398
SD = Standard deviation

Reported statistics and error estimates:
EC-values and statistical analyses
EC-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (Limit test design).

NOEC- and LOEC-values
The NOEC / LOEC was determined by calculation of statistical significance of biomass integrals, growth rate and yield. The following statistical tests were conducted: Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01. Levene’s test on variance homogeneity was done with a significance level of 0.01. Two-sample t-test was done with a significance level of 0.05.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
• Excel, MICROSOFT CORPORATION
•SigmaPlot, SPSS INC.
•ToxRat Pro Version 3.2.1

Cell Densities

 

Nominal test item concentration
[mg/L]

Replicate

Cell density [cells/mL]

No.

0 hours

24 hours

48 hours

72 hours

100

1

4847

21029

131060

790526

2

4847

25633

142947

855661

3

4847

28072

127062

787977

4

4847

24964

133446

747232

5

4847

23699

143173

814194

6

4847

24291

127697

812895

Mean

4847

24615

134231

801414

Control

1

4847

20490

142071

873075

2

4847

22078

128376

632886

3

4847

26807

123690

690035

4

4847

29606

129535

690949

5

4847

25094

135101

819919

6

4847

35769

136770

830935

Mean

4847

26641

132591

756300

         

 


 

 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

Inhibition of growth rate

Yield

Inhibition of yield

No.

[d-1]

[%]

[cells/mL]

[%]

100

1

 

1.70

-1

 

785679

-5

2

 

1.73

-3

 

850814

-13

3

 

1.70

-1

 

783130

-4

4

 

1.68

0

 

742385

1

5

 

1.71

-2

 

809347

-8

6

 

1.71

-2

 

808048

-8

Mean

(-)

1.70

-1

(-)

796567

-6

Control

1

 

1.73

 

 

868228

 

2

 

1.62

 

 

628039

 

3

 

1.65

 

 

685188

 

4

 

1.65

 

 

686102

 

5

 

1.71

 

 

815072

 

6

 

1.72

 

 

826088

 

Mean

 

1.68

 

 

751453

 

 

 

Section-by-Section and Average Specific Growth Ratesof 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

1.44

1.94

1.82

1.73

0.258

14.9

10.6

2

1.52

1.76

1.60

1.62

0.125

7.67

3

1.71

1.53

1.72

1.65

0.107

6.49

4

1.81

1.48

1.67

1.65

0.168

10.1

5

1.64

1.68

1.80

1.71

0.0828

4.84

6

2.00

1.34

1.80

1.72

0.338

19.7

 

 

 

Mean

1.68

 

 

 

 

 

SD ±

0.04

 

 

 

 

CV [%]

2.57

 

CV= Coefficient of variation

SD= Standard deviation

Validity criteria fulfilled:
yes
Conclusions:
In this study, Genamin DMG 75 was found not to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-value of the test item for both inhibition of growth rate and inhibition of yield after 72 hours was 100 mg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were both > 100 mg/L, respectively. All effect levels are given based on the nominal test item concnetartion of Genamin DMG 75.
Executive summary:

The toxicity of Genamin DMG 75  to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011) and Council Regulation (EC) No.761/2009 Method C.3 (2009) at the test facility from
2016-02-22 to 2016-02-25.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 4847 cells/mL. A stock solution with a nominal concentration of 100 mg/L was tested. Sixr eplicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of the test item Genamin DMG 75 in the limit concentration and the control were analytically verified via LC/MS-MS at the start and the end of exposure. The measured concentration of the test item at the start of the exposure (0 hours) was 106 % of the nominal value at the tested limit concentration. The measured concentration in the old media (72 h) was 109 % of the nominal value.

All effect values of Genamin DMG 75 are given based on the nominal concentration.

 

NOEC, LOEC, ECx-values of Genamin DMG 75 (0-72 hours)

              based on the nominal test item concentrations [mg/L]

 

 

Growth Rate Inhibition

NOEC

  100

LOEC

> 100

ErC10

> 100

ErC20

> 100

ErC50

> 100

 

Inhibition of Yield

NOEC

  100

LOEC

> 100

EyC10

> 100

EyC20

> 100

EyC50

> 100

 

Description of key information

In this study, Genamin DMG 75  was found not to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-value of the test item for both inhibition of growth rate and inhibition of yield after 72 hours was 100 mg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were both > 100 mg/L, respectively. All effect levels are given based on the nominal test item concnetartion of Genamin DMG 75.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information