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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:
2016
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
Preparation of the saturated solution
The saturated solution (1.00 mg/L test item were weighed out) was prepared with demineralized water two days prior to the start of the exposure (0 h). The dispersion was stirred for 48 hours (1100 rpm, room temperature) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL filtrate were discarded. The filtration was interrupted for ca. 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. After the separation treatment the components of the dilution water were added to the saturated solution. The following filtrate, i.e. the saturated solution, was used in the test. During filtration the filter was always kept covered. 

Test concentration
The saturated solution was tested in a limit test.

Control
Six replicates (without test item) were tested under the same test conditions as the test replicates.

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 Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- 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:
yes
Total exposure duration:
72 h
Post exposure observation period:
No

Test conditions

Hardness:
Not specified
Test temperature:
min.: 21.5 max.: 22.5 mean value: 22.0
pH:
pH-value
Start; 0 hours End; 72 hours
Saturated solution with
a nominal loading of 1 mg/L 7.99 8.08
Control 8.08 8.24
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
No analytical monitoring
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 25 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 970173 cells/mL
- No. of vessels per concentration (replicates): 6
- 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: 71.8 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)

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 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
- 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. 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
NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As standard a t-test was used for NOEC/LOEC calculations. When running a t-test 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 were 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) was =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.

Any other information on results incl. tables

Cell Densities

 

Replicate

Cell density [cells/mL]

No.

0 hours

24 hours

48 hours

72 hours

Saturated solution with a nominal loading of
1 mg/L

1

4847

21265

169991

895381

2

4847

22751

174244

1174246

3

4847

23396

166220

1159862

4

4847

22535

151793

923619

5

4847

30876

185972

1192285

6

4847

25152

173416

1105888

Mean

4847

24329

170273

1075214

Control

1

4847

20216

109167

864098

2

4847

20976

137010

979069

3

4847

27264

119048

1101208

4

4847

22217

117725

870977

5

4847

20197

159822

919130

6

4847

27928

141556

1115639

Mean

4847

23133

130721

975020

         

Evaluation after 72 hours

 Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

 

 

Replicate

Growth rate

Inhibition of growth rate

Yield

Inhibition of yield

No.

[d-1]

[%]

[cells/mL]

[%]

Saturated solution with a nominal loading of 1 mg/L

1

 

1.73

2

 

890534

8

2

 

1.77

-4

 

1169399

-21

3

 

1.81

-3

 

1155015

-19

4

 

1.73

1

 

918772

5

5

 

1.75

-4

 

1187438

-22

6

 

1.81

-2

 

1101041

-13

Mean

(-)

1.77

-2

(-)

1070367

-10

Control

1

 

1.74

 

 

859251

 

2

 

1.83

 

 

974222

 

3

 

1.83

 

 

1096361

 

4

 

1.75

 

 

866130

 

5

 

1.84

 

 

914283

 

6

 

1.81

 

 

1110792

 

Mean

 

1.80

 

 

970173

 

 

 

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

1.43

1.69

2.07

1.73

0.322

18.7

16.7

2

1.47

1.88

1.97

1.77

0.267

15.1

3

1.73

1.47

2.22

1.81

0.382

21.1

4

1.52

1.67

2.00

1.73

0.246

14.2

5

1.43

2.07

1.75

1.75

0.321

18.3

6

1.75

1.62

2.06

1.81

0.227

12.5

 

 

 

Mean

1.77

 

 

 

 

 

SD ±

0.04

 

 

 

 

CV [%]

2.13

 

CV= Coefficient of variation

SD= Standard deviation

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study the test item 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 1.00 mg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 1.00 mg/L, respectively. All effect levels are given based on the saturated solution prepared with a nominal loading of 1 mg/L of the test item.
Executive summary:

The toxicity of the test item 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). 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 saturated solution with a nominal concentration of 1.00 mg/L was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

No analytical monitoring was carried out, since no suitable method could be established for the quantification of the test item. All effect values of the test item are given based on the nominal test item concentration.

NOEC, LOEC, EC - values of the test item (0-72 hours)

based on the saturated solution, prepared with 1 mg/L

                               

 

Growth Rate Inhibition

NOEC

  1.00

LOEC

> 1.00

ErC10

> 1.00

ErC20

> 1.00

ErC50

> 1.00

 

Inhibition of Yield

NOEC

  1.00

LOEC

> 1.00

EyC10

> 1.00

EyC20

> 1.00

EyC50

> 1.00