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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:
2011-09-13 to 2011-09-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well performed OECD study with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

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:
2,3-Quinoxalinedione, 6-amino-1,4-dihydro-7-methoxy-
EC Number:
615-027-9
Cas Number:
69904-10-5
Molecular formula:
C9H9N3O3
IUPAC Name:
2,3-Quinoxalinedione, 6-amino-1,4-dihydro-7-methoxy-
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Amino-C-Dion trocken
- Molecular formula: C9H9N3O3
- Molecular weight: 207.19 g/mol
- Physical state: Solid, yellow powder
- Expiration date of the lot/batch: 2013-05-04
- Stability under test conditions: Not applicable
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: The concentrations of the test item were analytically determined from all test concentrations and the control after 0 hours (test start) and 72 hours (test end).
- Sampling method: Samples at test start were taken from an additional replicate without algae. At end of the test samples were taken directly from the pooled test replicates.
- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C until sample preparation, if necessary. Prepared samples were stored at room temperature until 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 160 mg/L was freshly prepared with dilution water. The stock solution was treated with ultrasound for 15 min and stirred with approximately 1100 rpm for 1 h at room temperature. Thereafter the stock solution was treated with ultrasound for 15 min again.
- Eluate: Dilution water
- Differential loading: 10.0 - 20.0 - 40.0 - 80.0 - 160 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): 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): Same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
Not measured
Test temperature:
Mean: 21.5 °C, Min: 21 °C, Max: 22 °C
pH:
Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
160 7.65 7.79
80.0 7.64 7.86
40.0 7.69 8.02
20.0 7.72 8.11
10.0 7.71 8.20
Control 7.93 8.30
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to "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
- 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: 4910 cells/mL
- Control end cells density: Mean 992333 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: 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

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 h)
Results of the Range Finding Test (0-72 hours)

Nominal
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 33 80
10 - 1 - 5
1 3 12
0.1 2 11
0.01 3 13
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrations
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
89.4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (81.2 - 98.9)
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
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.488 (CI 0.466 - 0.512) mg/L
Yield Inhibition: 0.291 (CI 0.274 - 0.328) 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. Calculation of the confidence intervals. EC10-, EC20- and EC50-values were carried out using standard procedures.

Any other information on results incl. tables

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Amino-C-Dion trocken

                   (0-72 hours)

                   based on nominal test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

20.0

LOEC

40.0

ErC10

23.8 (19.8 – 28.5)

ErC20

37.9 (33.0 – 42.6)

ErC50

89.4 (81.2 – 98.9)

Inhibition of Yield

NOEC

10.0

LOEC

20.0

EyC10

12.8 ( 10.0 – 16.9)

EyC20

17.5 (14.0 – 21.4)

EyC50

29.5 (25.5 – 33.9)

Cell Densities

 

Nominal test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

 

160

1

4910

7736

10803

16419

 

2

4910

9872

11923

21364

 

3

4910

9181

12539

19079

 

Mean

4910

8930

11755

18954

 

  80.0

1

4910

14780

31966

90286

 

2

4910

18013

41400

96416

 

3

4910

17590

38614

88353

 

Mean

4910

16794

37327

91685

 

  40.0

1

4910

15609

59144

223371

 

2

4910

17184

87707

389723

 

3

4910

22038

87439

296500

 

Mean

4910

18277

78097

303198

 

  20.0

1

4910

16544

91840

610706

 

2

4910

19042

112224

790517

 

3

4910

21168

114008

812926

 

Mean

4910

18918

106024

738050

 

  10.0

1

4910

22901

110931

830460

 

2

4910

20860

150630

1050925

 

3

4910

21879

119810

919046

 

Mean

4910

21880

127124

933477

 

Control

1

4910

19181

138708

959696

 

2

4910

21279

143112

974522

 

3

4910

27914

147797

991718

 

4

4910

19898

134569

984610

 

5

4910

24224

163883

1073635

 

6

4910

21530

132155

969817

 

Mean

4910

22338

143371

992333

 

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

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

160

1

0.402

77.3

11509

98.8

2

0.490

72.3

16454

98.3

3

0.452

74.4

14169

98.6

Mean

(+)

0.448

74.7

(+)

14044

98.6

  80.0

1

0.971

45.2

85376

91.4

2

0.992

43.9

91506

90.7

3

0.963

45.6

83443

91.6

Mean

(+)

0.975

44.9

(+)

86775

91.2

  40.0

1

1.27

28.1

218461

77.9

2

1.46

17.6

384813

61.0

3

1.37

22.7

291590

70.5

Mean

(+)

1.37

22.8

(+)

298288

69.8

  20.0

1

1.61

  9.13

605796

38.7

2

1.69

  4.27

785607

20.4

3

1.70

  3.74

808016

18.2

Mean

(+)*

1.67

  5.71

(+)

733140

25.8

  10.0

1

1.71

  3.34

825550

16.4

2

1.79

 -1.09

1046015

 -5.93

3

1.74

  1.43

914136

  7.42

Mean

(-)

1.75

  1.23

(-)

928567

  5.96

Control

1

1.76

954786

2

1.76

969612

3

1.77

986808

4

1.77

979700

5

1.80

1068725

6

1.76

964907

Mean

1.77

987423

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

1.36

1.98

1.93

1.76

0.344

19.5

13.9

2

1.47

1.91

1.92

1.76

0.257

14.6

3

1.74

1.67

1.90

1.77

0.122

  6.87

4

1.40

1.91

1.99

1.77

0.321

18.2

5

1.60

1.91

1.88

1.80

0.174

  9.68

6

1.48

1.82

1.99

1.76

0.262

14.8

Mean

1.77

 

SD ±

0.01

 

CV [%]

0.76

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study Amino-C-Dion trocken was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 hours were 20.0 mg/L and 10.0 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 hours were 40.0 mg/L and 20.0 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 hours were 89.4 (81.2 – 98.9) and 29.5 (25.5 – 33.9) mg/L, respectively.
All effect levels are given based on nominal concentrations of Amino-C-Dion trocken.
Executive summary:

The toxicity of Amino-C-Dion trocken (batch number: 241)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 September 13 to 16, 2011. 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 test 5 concentration levels were tested in a geometrical series with a dilution factor of 2, nominal: 10.0 - 20.0 - 40.0 - 80.0 - 160 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of Amino-C-Dion trocken were analytically verified by HPLC-DAD at test start and test end of all test concentrations and the control. Details of the analytical method are presented in part 9. The analytical results are presented in Table 8.
The measured concentrations of Amino-C-Dion trocken at test start were in the range of 98 to 106 % and after 72 hours in the range of 80 to 106 % of the nominal values. Therefore, all effect values given are based on the nominal concentrations of the test item Amino-C-Dion trocken.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Amino-C-Dion trocken

                   (0-72 hours)

                   based on nominal test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

20.0

LOEC

40.0

ErC10

23.8 (19.8 – 28.5)

ErC20

37.9 (33.0 – 42.6)

ErC50

89.4 (81.2 – 98.9)

Inhibition of Yield

NOEC

10.0

LOEC

20.0

EyC10

12.8 ( 10.0 – 16.9)

EyC20

17.5 (14.0 – 21.4)

EyC50

29.5 (25.5 – 33.9)

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