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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:
2009-10-16 to 2009-10-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP and guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
GLP compliance statement and certificate included in full study report.

Test material

Reference
Name:
Unnamed
Type:
Constituent

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 h (test start) and 72 h (test end).
- Sampling method: Samples at start and end of the test were taken directly from the test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis, if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution* was prepared. A dispersion of 1440 mg/L prepared in dilution water was shaken with 20 rpm for 48 h at ambient temperature and filtrated with 0.45 µm RC filter.
- Eluate: Dilution water
- Differential loading: The saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor √10: 1.0 - 3.2 - 10 - 32 and 100 % of the saturated solution corresponding to initial measured concentrations of 0.106 – 0.323 – 0.974 – 3.26 – 9.92 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
Post exposure observation period:
No

Test conditions

Hardness:
Not measured
Test temperature:
Mean: 23.5 °C, Min: 23 °C, Max: 25 °C
pH:
Initial measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
9.92 8.12 8.13
3.26 8.12 8.35
0.974 8.10 8.32
0.323 8.09 8.33
0.106 8.06 8.34
Control 7.96 8.34
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: 5074 cells/mL
- Control end cells density: Mean 492829 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
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.26 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
9.92 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.46 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (3.26 - 9.92)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.26 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
9.92 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: CI: 3.26 - 9.92
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: No
- Colour differences: Not reported
- Flocculation: No data
- Adherence to test vessels: No
- Aggregation of algal cells: yes
- Other: Not reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not reported
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: Rate-related inhibition: 0.600 (CI 0.545 - 0.653) mg/L
Yield Inhibition: 0.280 (CI 0.266 - 0.299) mg/L
Reported statistics and error estimates:
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by linear dose-response regression. Calculation of the confidence intervals were estimated empirically.

NOEC, LOEC NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. One Way Analysis of Variance (ANOVA) and DUNNETT’s test 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. P-values for both Normality and Equal Variance test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) was a=0.05.

Any other information on results incl. tables

NOEC, LOEC and EC - values of 1-Chlor-2,5-dimethoxy-4-nitrobenzol trocken (0-72 h)

                   based on initial measured test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

3.26

LOEC

9.92

ErC10

4.12 (3.26 – 9.92)

ErC20

4.60 (3.26 – 9.92)

ErC50

6.46 (3.26 – 9.92)

Inhibition of Yield

NOEC

3.26

LOEC

9.92

EyC10

5.40 (3.26 – 9.92)

EyC20

5.77 (3.26 – 9.92)

EyC50

7.10 (3.26 – 9.92)

Cell Densities

 

Initial measured test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

9.92

1

5074

9391

7915

14906

 

2

5074

11245

8735

7481

 

3

5074

13602

7058

5988

 

Mean

5074

11413

7903

9458

 

3.26

1

5074

19170

127885

882978

 

2

5074

18657

127344

729392

 

3

5074

18776

141100

795743

 

Mean

5074

18868

132110

802704

 

0.974

1

5074

17116

91646

602272

 

2

5074

16144

99460

599824

 

3

5074

21310

119710

578033

 

Mean

5074

18190

103605

593376

 

0.323

1

5074

16628

87816

641997

 

2

5074

20478

76283

531317

 

3

5074

18079

131387

587877

 

Mean

5074

18395

98495

587064

 

0.106

1

5074

15254

80707

515889

 

2

5074

15898

75533

536282

 

3

5074

17784

99665

578701

 

Mean

5074

16312

85302

543624

 

Control

1

5074

18538

79173

455381

 

2

5074

16402

73470

461560

 

3

5074

19826

88862

599549

 

4

5074

17727

86135

466550

 

5

5074

17747

76611

463257

 

6

5074

17919

101695

510678

 

Mean

5074

18027

84324

492829

 

Evaluation after 72 h

                  Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

               

Initial measured test item concentration

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

9.92

1

0.359

 76.4

9832

 98.0

2

0.129

 91.5

2407

 99.5

3

0.055

 96.4

914

 99.8

Mean

(+)

0.181

 88.1

(+)

4384

 99.1

3.26

1

1.72

-12.9

877904

-80.0

2

1.66

  -8.68

724318

-48.5

3

1.69

-10.6

790669

-62.1

Mean

(-)

1.69

-10.7

(+)*

797630

-63.5

0.974

1

1.59

  -4.50

597198

-22.4

2

1.59

  -4.41

594750

-21.9

3

1.58

  -3.60

572959

-17.5

Mean

(-)

1.59

  -4.17

(-)

588302

-20.6

0.323

1

1.61

  -5.89

636923

-30.6

2

1.55

  -1.75

526243

  -7.89

3

1.58

  -3.97

582803

-19.5

Mean

(-)

1.58

  -3.87

(-)

581990

-19.32

0.106

1

1.54

  -1.11

510815

  -4.73

2

1.55

  -1.96

531208

  -8.91

3

1.58

  -3.62

573627

-17.6

Mean

(-)

1.56

  -2.23

(-)

538550

-10.4

Control

1

1.50

450307

2

1.50

456486

3

1.59

594475

4

1.51

461476

5

1.51

458183

6

1.54

505604

Mean

1.52

487755

Section-by-Section and Average Specific Growth Rates of the Control Group
               (0 – 72 h)

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 h)

SD

±

CV
[%]

Mean CV [%]

section-by-section                           

0 - 24 h

24 - 48 h

48 - 72 h

Control

1

1.30

1.45

1.75

1.50

0.231

15.4

17.5

2

1.17

1.50

1.84

1.50

0.332

22.1

3

1.36

1.50

1.91

1.59

0.284

17.9

4

1.25

1.58

1.69

1.51

0.228

15.2

5

1.25

1.46

1.80

1.51

0.276

18.4

6

1.26

1.74

1.61

1.54

0.246

16.0

Mean

1.52

 

SD ±

0.04

 

CV [%]

2.33

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study 1-Chlor-2,5-dimethoxy-4-nitrobenzol trocken was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 6.46 (3.26 – 9.92) mg/L and 7.10 (3.26 – 9.92) mg/L, respectively. All effect levels are given based on initial measured concentrations of 1-Chlor-2,5-dimethoxy-4-nitrobenzol trocken.
Executive summary:

The toxicity of 1-Chlor-2,5-dimethoxy-4-nitrobenzol trocken (batch number DALA 053552) 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 October 16 to 23, 2009 with the definitive exposure phase being conducted from October 20 to 23, 2009. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h. Due to the poor solubility of the test substance the test solutions were prepared according to OECD (diffucult substances).

All effect values are given based on initial measured concentrations of the test item.

NOEC, LOEC and EC - values of 1-Chlor-2,5-dimethoxy-4-nitrobenzol trocken (0-72 h)

                   based on initial measured test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

3.26

LOEC

9.92

ErC10

4.12 (3.26 – 9.92)

ErC20

4.60 (3.26 – 9.92)

ErC50

6.46 (3.26 – 9.92)

Inhibition of Yield

NOEC

3.26

LOEC

9.92

EyC10

5.40 (3.26 – 9.92)

EyC20

5.77 (3.26 – 9.92)

EyC50

7.10 (3.26 – 9.92)