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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
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:
2009
Report Date:
2009

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): Aminoanisoylaminobenzamid Hydrochlorid
- Molecular formula: C15H16ClN3O3
- Molecular weight: 321.77 g/mol
- Analytical purity: 94.7 % (w/w)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: One loading level of 100 mg/L was tested as limit test
- Sampling method: THe limit concentration and the control were analytically verified at the beginning and the end of the test via HPLC analysis. Separate replicates for the test item analysis after 0 h were prepared. At the 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 if necessary

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 200 mg/L, freshly prepared with dilution water and dispersion treatment was ultrasound for 30 minutes at 40 °C.
- Eluate: Dilution water
- Differential loading: 100 mg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

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:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
Not measured
Test temperature:
Mean: 22 °C, Min: 21 °C, Max: 23 °C
pH:
Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
100 7.89 8.16
Control 7.93 8.46
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Concentrations and Recovery Rates of Aminoanisoylaminobenzamid Hydrochlorid

Sampling date 2009-05-04
Test start 2009-05-07
Test end
Start of analysis 2009-05-04
2009-05-07

Nominal concentration Aminoanisoylaminobenzamid Hydrochlorid
Test item

[mg/L] Main component
[mg/L] Meas.
conc.
[mg/L] RR

[%] Meas.
conc.
[mg/L] RR

[%]
100 94.7 85.1 90 84.3 89
Control < LOQ — < LOQ —
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: 4878 cells/mL
- Control end cells density: Mean 900551 cells/mL
- No. of vessels per concentration (replicates): 6
- 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)

Nominal
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition
[%] Yield Inhibition

[%]
100 -6 -31
10 -2 -7
1 -4 -19

Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrations
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and biomass
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: Yes
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.695 (CI 0.657 - 0.729) mg/L
Yield Inhibition: 0.320 (CI 0.294 - 0.358) mg/L
Reported statistics and error estimates:
All calculations were based on the fluorescence values of each replicate.

Any other information on results incl. tables

Effect Levels of Aminoanisoylaminobenzamid Hydrochlorid (0-72 h)

                                based on nominal concentration of the test item [mg/L]

                               

Yield inhibition

NOEC

< 100

LOEC

   100

EyC10

100

EyC20

> 100

EyC50

> 100

Rate-related inhibition

NOEC

   100

LOEC

> 100

ErC10

> 100

ErC20

> 100

EyC50

> 100

Cell Densities

 

Nominal test item
concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

100

1

4878

29534

296480

790655

 

2

4878

30160

273552

559950

 

3

4878

31667

285337

749187

 

4

4878

33180

274535

700925

 

5

4878

30224

289765

760880

 

6

4878

32906

297884

770990

 

Mean

4878

31279

286259

722098

 

Control

1

4878

23274

216936

926281

 

2

4878

37375

209157

905675

 

3

4878

36378

248663

912077

 

4

4878

38009

208145

877627

 

5

4878

37788

220862

771701

 

6

4878

37219

241583

1009943

 

Mean

4878

35007

224224

900551

 

Evaluation after 72 h

                                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]

[%]

100

1

1.696

2.43

785777

12.27

2

1.581

9.05

555072

38.03

3

1.678

3.47

744309

16.90

4

1.656

4.74

696047

22.29

5

1.683

3.17

756002

15.59

6

1.688

2.92

766112

14.47

Mean

(+)*

1.664

4.30

(+)

717220

19.93

Control

1

1.749

921403

2

1.741

900797

3

1.744

907199

4

1.731

872749

5

1.688

766823

6

1.778

1005065

Mean

1.738

895673

Section-by-Section and Average Specific Growth Rates of the Control Group

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

1.563

2.232

1.452

1.749

0.4223

24.2

20.3

2

2.036

1.722

1.466

1.741

0.2858

16.4

3

2.009

1.922

1.300

1.744

0.3870

22.2

4

2.053

1.700

1.439

1.731

0.3082

17.8

5

2.047

1.766

1.251

1.688

0.4037

23.9

6

2.032

1.870

1.430

1.778

0.3114

17.5

Mean

1.738

 

SD ±

0.03

 

CV [%]

1.68

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study the effects of the test item Aminoanisoylaminobenzamid Hydrochlorid on the growth of the freshwater green alga Desmodesmus subspicatus were tested. The EC50-values for specific growth rate inhibition (ErC50) and inhibition of yield (EyC50) after 72 h were > 100 mg/L.
Executive summary:

The toxicity of Aminoanisoylaminobenzamid Hydrochlorid to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorien in 31157 , from May 04 to 07, 2009. The aim of the study was to assess the effects on growth rate and yield production over a period of 72 h in a limit test.
The study was conducted under static conditions with an initial cell density of approximately 2 - 5 x 103cells/mL. One limit concentration of 100 mg/L was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.
The concentration of Aminoanisoylaminobenzamid Hydrochlorid was analysed at test start and end via HPLC analysis. Measured concentrations at test start and end well met the nominal ranges.

Effect Levels of Aminoanisoylaminobenzamid Hydrochlorid (0-72 h)

                                based on nominal concentration of the test item [mg/L]

                               

Yield inhibition

NOEC

< 100

LOEC

   100

EyC10

100

EyC20

> 100

EyC50

> 100

Rate-related inhibition

NOEC

   100

LOEC

> 100

ErC10

> 100

ErC20

> 100

EyC50

> 100

Aminoanisoylaminobenzamid Hydrochlorid caused no significant inhibiting effects > 20 % on algal growth tested at the limit concentration of 100

mg/L.