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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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2011-12-02 to 2011-12-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to relevant guidelines and compliant to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
according to German Chemikaliengesetzt and directive 88/320/EEC

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: The limit test item concentration and control were analytically verified via HPLC-DAD after 0 hours (test start) and 72 hours (test end).
- Sampling method: Samples at test start were taken from additional replicates without algae. After 72 hours samples were taken directly from pooled test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample preparation, if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1 mg/L was freshly prepared with dilution water.The stock solution was stirred for 48 hours (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). Centrifugation for 10 min at 10000 rpm was found to be insufficient.
- Eluate: Dilution water
- Differential loading: None, saturated solution corresponding to a nominal test item concentration of 1.00 mg/L.
- Controls: 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 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:
yes
Total exposure duration:
72 h
Post exposure observation period:
No

Test conditions

Hardness:
Dilution water: 0.24 mmol Ca+mg/L.
Test temperature:
min.: 21.5 max.: 22.0, mean value: 21.8
pH:
pH-value
Start; 0 hours End; 72 hours
Saturated solution prepared
with 1 mg test item/L 7.85 8.51
Control 7.91 8.12
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
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): 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, Actual: mean 4910 cells/mL
- Control end cells density: 792206 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: 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: 69.2



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.



TEST CONCENTRATIONS
- Range finding study:
Results of the Range Finding Test (0-72 hours)

Nominal
test item concentration
[mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 -4 -22
10 -2 -8
1 -1 -5



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
Remarks on result:
other: Due to the very poor solubility of the test item, quantitative analytical determination was precluded (below LOQ)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Due to the very poor solubility of the test item, quantitative analytical determination was precluded (below LOQ)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Due to the very poor solubility of the test item, quantitative analytical determination was precluded (below LOQ)
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):
- Results with reference substance valid? Yes
EC50 Values of the Reference Item
based on nominal concentrations (mg/L), (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Rate-related inhibition
ErC50 0.488 0.699 ± 0.471
95 % confidence interval 0.466 – 0.512
Yield inhibition
EyC50 0.291 0.318 ± 0.147
95 % confidence interval 0.274 – 0.328


Reported statistics and error estimates:
NOEC/LOEC was determined by calculation of statistical
significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) was 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 test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

Any other information on results incl. tables

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

                   based on the saturated solution prepared with 1 mg test item/L

                  

Rate-related 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

Cell Densities

 

Replicate

Cell density [cells/mL]

No.

0 hours

24 hours

48 hours

72 hours

 

Saturated solution prepared with

1 mg test item/L

1

4910

27542

151737

633314

 

2

4910

23202

156456

627729

 

3

4910

24627

125747

578816

 

4

4910

24505

167816

862648

 

5

4910

26049

151595

679317

 

6

4910

27887

163422

626951

 

Mean

4910

25635

152796

668129

 

Control

1

4910

27183

143295

641574

 

2

4910

26059

153978

890710

 

3

4910

28533

168486

920227

 

4

4910

28232

159570

819489

 

5

4910

28814

143119

754531

 

6

4910

24262

140709

726707

 

Mean

4910

27181

151526

792206

 

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

Rate-related inhibition

Yield

Inhibition of yield

No.

[d-1]

[%]

[cells/mL]

[%]

Saturated solution prepared with

1 mg test item/L

1

1.62

  4.26

628404

20.2

2

1.62

  4.44

622819

20.9

3

1.59

  6.03

573906

27.1

4

1.72

-1.83

857738

   -8.95

5

1.64

  2.88

674407

14.3

6

1.62

  4.46

622041

21.0

Mean

(-)

1.64

  3.37

(-)

663219

15.8

Control

1

1.62

636664

2

1.73

885800

3

1.74

915317

4

1.71

814579

5

1.68

749621

6

1.67

721797

Mean

1.69

787296

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

1.71

1.66

1.50

1.62

0.111

6.84

4.34

2

1.67

1.78

1.76

1.73

  0.0569

3.28

3

1.76

1.78

1.70

1.74

  0.0412

2.36

4

1.75

1.73

1.64

1.71

  0.0609

3.57

5

1.77

1.60

1.66

1.68

  0.0845

5.04

6

1.60

1.76

1.64

1.67

  0.0827

4.97

Mean

1.69

 

SD ±

0.05

 

CV [%]

2.66

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study no inhibition on growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item. The NOEC for inhibition of specific growth rate and yield after 72 hours was 1.00 mg/L. All effect levels are given based on the nominal concentration 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 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from 2011-12-02 to 2011-12-09, with the definitive exposure phase from 2011-12-06 to 2011-12-09. 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 5 x 103 cells/mL. Based on a preliminary range finding the saturated solution* was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

The saturated solution* and the control were analysed by HPLC-DAD after 0 h (fresh media) and 72 h (old media).

The concentration (fresh media, 0 h) of the test item in the freshly prepared saturated solution* and in the old media (72 h) was , therefore all effect values of the test item are given based on the saturated solution prepared with 1 mg/L.

Table 1:   NOEC, LOEC, EC - values of the test item (0-72 hours)

                   based on the saturated solution prepared with 1 mg test item/L

                  

Rate-related 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