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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:
2009-12-11 to 2009-12-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to current OECD guideline and GLP

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): p-Nitro-o-anisidin
- Molecular formula: C7H8N2O3
- Molecular weight: 168.15 g/mol
- Physical state: Solid, yellow powder

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: Separate samples were prepared at test start. At end of the test samples 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 of 1000 mg/L was prepared with dilution water. The dispersion 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 2: 6.25 - 12.5 - 25 - 50 and 100 % of the saturated solution corresponding to initial measured concentrations of 6.34 – 12.5 – 24.8 – 49.9 – 97.8 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:
Concentrations and Recovery Rates of p-Nitro-o-anisidin
Initial measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
97.8 7.86 8.03
49.9 8.09 8.14
24.8 8.19 8.18
12.5 8.14 8.15
6.34 8.17 8.36
Control 8.20 8.43


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 690386 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)

Saturated solution [%] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
Centrifugation of the saturated solution
100 100 100
1 - 3 - 18
0.1 - 2 - 9
Filtration of the saturated solution
100 100 100
1 - 4 - 20
0.1 - 2 - 12
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
33 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (30.7 - 35.6)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
24.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
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: 0.600 (CI 0.545 - 0.653) mg/L
Yield Inhibition: 0.280 (CI 0.266 - 0.299) mg/L
Reported statistics and error estimates:
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 p-Nitro-o-anisidin(0-72 h)

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

                               

Rate-related Inhibition

NOEC

12.5

LOEC

24.8

ErC10

16.2 (14.4 – 18.1)

ErC20

20.4 (18.5 – 22.1)

ErC50

33.0 (30.7 – 35.6)

Inhibition of Yield

NOEC

6.34

LOEC

12.5

EyC10

11.6 (11.0 – 12.1)

EyC20

12.7 (12.1 – 13.3)

EyC50

17.0 (16.2 – 17.9)

Cell Densities

 

Initial measured test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

97.8

1

5074

3420

4180

6460

 

2

5074

4560

4940

7600

 

3

5074

4560

4560

7600

 

Mean

5074

4180

4560

7220

 

49.9

1

5074

5320

11780

23180

 

2

5074

6840

8360

19760

 

3

5074

6840

7980

11400

 

Mean

5074

6333

9373

18113

 

24.8

1

5074

12540

45600

180120

 

2

5074

12160

42560

143640

 

3

5074

14440

40660

146300

 

Mean

5074

13047

42940

156687

 

12.5

1

5074

27669

148340

587930

 

2

5074

28309

150554

565007

 

3

5074

30715

160960

547586

 

Mean

5074

28898

153285

566841

 

  6.34

1

5074

33684

211353

957557

 

2

5074

28178

205814

895635

 

3

5074

32191

230500

987262

 

Mean

5074

31351

215889

946818

 

Control

1

5074

31023

161510

674350

 

2

5074

26402

184183

655970

 

3

5074

30125

174880

698195

 

4

5074

31564

169103

708978

 

5

5074

33167

174248

633629

 

6

5074

30724

212022

771196

 

Mean

5074

30501

179324

690386

 

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]

[%]

97.8

1

0.080

95.1

1386

99.8

2

0.135

91.8

2526

99.6

3

0.135

91.8

2526

99.6

Mean

(+)

0.117

92.9

(+)

2146

99.7

49.9

1

0.506

69.1

18106

97.4

2

0.453

72.3

14686

97.9

3

0.270

83.5

6326

99.1

Mean

(+)

0.410

75.0

(+)

13039

98.1

24.8

1

1.19

27.3

175046

74.5

2

1.11

31.9

138566

79.8

3

1.12

31.6

141226

79.4

Mean

(+)

1.14

30.3

(+)

151613

77.9

12.5

1

1.58

  3.23

582856

15.0

2

1.57

  4.04

559933

18.3

3

1.56

  4.68

542512

20.8

Mean

(-)

1.57

  3.98

(+)

561767

18.0

  6.34

1

1.75

 -6.70

952483

-39.0

2

1.72

 -5.34

890561

-30.0

3

1.76

 -7.32

982188

-43.3

Mean

(-)

1.74

 -6.45

(+)*

941744

-37.4

Control

1

1.63

669276

2

1.62

650896

3

1.64

693121

4

1.65

703904

5

1.61

628555

6

1.68

766122

Mean

1.64

685312

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

1.81

1.65

1.43

1.63

0.192

11.7

16.1

2

1.65

1.94

1.27

1.62

0.337

20.8

3

1.78

1.76

1.38

1.64

0.223

13.6

4

1.83

1.68

1.43

1.65

0.199

12.1

5

1.88

1.66

1.29

1.61

0.296

18.4

6

1.80

1.93

1.29

1.68

0.338

20.2

Mean

1.64

 

SD ±

0.02

 

CV [%]

1.40

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study p-Nitro-o-anisidin was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h. The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 12.5 and 6.34 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 24.8 and 12.5 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 33.0 (30.7 – 35.6) and 17.0 (16.2 – 17.9) mg/L, respectively.
All effect levels are given based on initial measured concentrations of p-Nitro-o-anisidin.
Executive summary:

The toxicity of p-Nitro-o-anisidin to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

The study was conducted under static conditions with an initial cell density of approximately  2 – 5 x 103cells/mL. Based on a preliminary test, a definitive test with the saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor of 2, nominal: 6.25 - 12.5 - 25 - 50 - 100 % of the saturated solution corresponding to initial measured test item concentrations of 6.34 – 12.5 – 24.8 – 49.9 – 97.8 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 p-Nitro-o-anisidinwere analysed at all concentration levels after 0 and   72 h via HPLC analysis. At the end of the test p-Nitro-o-anisidingave recoveries of 100 – 102 % of the initial measured concentrations. All effect values are given based on initial measured concentrations of the test item.

NOEC, LOEC and EC - values of p-Nitro-o-anisidin(0-72 h)

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

                               

Rate-related Inhibition

NOEC

12.5

LOEC

24.8

ErC10

16.2 (14.4 – 18.1)

ErC20

20.4 (18.5 – 22.1)

ErC50

33.0 (30.7 – 35.6)

Inhibition of Yield

NOEC

6.34

LOEC

12.5

EyC10

11.6 (11.0 – 12.1)

EyC20

12.7 (12.1 – 13.3)

EyC50

17.0 (16.2 – 17.9)