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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:
2009-10-09 to 2009-10-16
Reliability:
1 (reliable without restriction)

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
Details on test material:
- Name of test material (as cited in study report): p-Nitrobenzoylaminobenzamid trocken
- Molecular formula: C14H11N3O4
- Molecular weight: 285.26 g/mol
- Physical state: Solid, white powder
- Expiration date of the lot/batch: 2010-10-01
- Storage condition of test material: Room temperature, protected from moisture and light.

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 66 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 2: 6.25 - 12.5 - 25 - 50 and 100 % of the saturated solution corresponding to initial measured concentrations of 0.0157 – 0.0314 – 0.0712 – 0.153 – 0.304 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: 22.5 °C, Min: 22 °C, Max: 23 °C
pH:
Initial measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
0.304 8.09 8.72
0.153 8.17 8.34
0.0712 8.13 8.36
0.0314 8.07 8.23
0.0157 8.16 8.43
Control 7.97 8.49
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 873651 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 h)

Dilution level of the saturated solution
[%] Rate Related Inhibition[%] Yield Inhibition[%]
100 29 68
10 -2 -8
1 1 3
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrations
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.304 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and inhibition of 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
- 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:
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures.
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-Nitrobenzoylaminobenzamid trocken (0-72 h)

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

                               

Rate-related Inhibition

NOEC

0.304

LOEC

> 0.304

ErC10

> 0.304

ErC20

> 0.304

ErC50

> 0.304

Inhibition of Yield

NOEC

0.304

LOEC

> 0.304

EyC10

> 0.304

EyC20

> 0.304

EyC50

> 0.304

Cell Densities

 

Initial measured test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

0.304

1

5074

28858

207909

925614

 

2

5074

21790

170427

897132

 

3

5074

23864

195671

1091558

 

Mean

5074

24837

191336

971435

 

0.153

1

5074

28444

223420

948956

 

2

5074

28952

179422

937676

 

3

5074

30617

197827

1029516

 

Mean

5074

29338

200223

972049

 

0.0712

1

5074

22860

186282

896820

 

2

5074

26201

181313

921387

 

3

5074

31031

210714

1085682

 

Mean

5074

26697

192770

967963

 

0.0314

1

5074

24754

145192

837169

 

2

5074

27341

172916

940592

 

3

5074

28526

182383

935893

 

Mean

5074

26874

166830

904551

 

0.0157

1

5074

27522

150722

870014

 

2

5074

25480

167327

976901

 

3

5074

32232

216585

1077290

 

Mean

5074

28411

178211

974735

 

Control

1

5074

23532

184998

824193

 

2

5074

26312

182768

824656

 

3

5074

27788

204904

1002731

 

4

5074

24615

171239

781946

 

5

5074

30383

181964

843692

 

6

5074

23942

183375

964687

 

Mean

5074

26095

184875

873651

 

Evaluation after 72 h

                              

Initial measured test item concentration

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

0.304

1

1.74

-1.20

920540

-5.98

2

1.73

-0.60

892058

-2.70

3

1.79

-4.41

1086484

       -25.1

Mean

1.75

-2.07

966361

       -11.3

0.153

1

1.74

-1.69

943882

-8.67

2

1.74

-1.46

932602

-7.37

3

1.77

-3.27

1024442

       -17.9

Mean

1.75

-2.14

966975

       -11.3

0.0712

1

1.73

-0.59

891746

-2.67

2

1.73

-1.11

916313

-5.50

3

1.79

-4.30

1080608

       -24.4

Mean

1.75

-2.00

962889

       -10.9

0.0314

1

1.70

0.75

832095

4.20

2

1.74

-1.52

935518

-7.71

3

1.74

-1.42

930819

-7.17

Mean

1.73

-0.73

899477

-3.56

0.0157

1

1.72

0.00

864940

0.42

2

1.75

-2.25

971827

       -11.9

3

1.79

-4.15

1072216

       -23.5

Mean

1.75

-2.13

969661

       -11.6

Control

1

1.70

819119

2

1.70

819582

3

1.76

997657

4

1.68

776872

5

1.71

838618

6

1.75

959613

Mean

1.72

868577

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

1.53

2.06

1.49

1.70

0.317

18.7

13.4

2

1.65

1.94

1.51

1.70

0.220

13.0

3

1.70

2.00

1.59

1.76

0.212

12.0

4

1.58

1.94

1.52

1.68

0.228

13.6

5

1.79

1.79

1.53

1.71

0.148

8.67

6

1.55

2.04

1.66

1.75

0.254

14.5

Mean

1.72

 

SD ±

0.03

 

CV [%]

1.92

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study p-Nitrobenzoylaminobenzamid trocken was found not 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 > 0.304 mg/L, respectively. All effect levels are given based on initial measured concentrations of p-Nitrobenzoylaminobenzamid trocken.
Executive summary:

The toxicity of p-Nitrobenzoylaminobenzamid trocken to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201.

The study was conducted under static conditions with an initial cell density of approximately  2 – 5 x 103cells/mL. 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 0.0157 – 0.0314 – 0.0712 – 0.153 – 0.304 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-Nitrobenzoylaminobenzamid trocken were analysed at all concentration levels after 0 and 72 h via HPLC analysis. The measured concentrations of p-Nitrobenzoylaminobenzamid trocken at test end were in the range of 99 – 120 % of the initial measured values. The concentration of the lowest dilution level was below the LOQ of 0.03 mg/L. The lowest concentration was calculated from the second lowest test item concentration with the dilution factor of 2. All effect values are given based on initial measured concentrations of the test item.

NOEC, LOEC and EC - values of p-Nitrobenzoylaminobenzamid trocken (0-72 h)

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

                               

Rate-related Inhibition

NOEC

0.304

LOEC

> 0.304

ErC10

> 0.304

ErC20

> 0.304

ErC50

> 0.304

Inhibition of Yield

NOEC

0.304

LOEC

> 0.304

EyC10

> 0.304

EyC20

> 0.304

EyC50

> 0.304