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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-06-22 to 2009-06-25
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

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:
N,N'-ethylenebis[N-acetylacetamide]
EC Number:
234-123-8
EC Name:
N,N'-ethylenebis[N-acetylacetamide]
Cas Number:
10543-57-4
Molecular formula:
C10H16N2O4
IUPAC Name:
N,N'-ethylenebis[N-acetylacetamide]
Details on test material:
- Name of test material (as cited in study report): Peractive P
- Physical state: solid (powder)
- Analytical purity: 99.1 (certified)
- Composition of test material, percentage of components: Tetraacetylethylenediamine (TAED), 99.1 %
- Purity test date: 2007-09-25
- Lot/batch No.: DEA 4 TM 0014
- Expiration date of the lot/batch: 2009-09-25
- Storage condition of test material: Room temperature, protected from moisture and light

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 125 - 250 - 500 - 1000 mg/L
- Sampling method: 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). 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: 1000 mg/L, freshly prepared with dilution water anduUltrasound for 15 min, room temperature
- Eluate: Dilution water
- Differential loading: 125 - 250 - 500 - 1000 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): 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): 3 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 °C, Min: 21 °C, Max: 23 °C
pH:
Geometric mean measured a.i. concentrations pH-value
[mg/L] Start; 0 h End; 72 h
824 7.53 8.48
376 7.58 8.59
153 7.58 8.76
44.8 7.97 8.96
Control 8.02 9.03
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Concentrations and Recovery Rates of Tetraacetylethylenediamine (TAED),
Active Ingredient of Peractive P

Sampling date 2009-06-22
0 h 2009-06-25
72 h
Start of analysis 2009-06-22 2009-06-25
Nominal concentration Tetraacetylethylenediamine (TAED)
Geometric mean measured
Test item

[mg/L] Active ingredient
[mg/L] Measured
conc.
[mg/L] RR

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

[%] Active ingredient
[mg/L]
1000 991 942 95 720 73 824
500 496 478 96 296 60 376
250 248 231 93 101 41 153
125 124 121 98 16.6 13 44.8
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 1206687 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)

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 concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
655 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 473-824
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:
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.

Any other information on results incl. tables

EC - values and 95 % Confidence Intervals of Peractive P (0-72 h)

                   based on geometric mean meaured a.i. concentrations [mg/L]

                               

Rate-related Inhibition

ErC10

655 (473 – > 824)

ErC20

> 1000

ErC50

> 1000

Inhibition of Yield

EyC10

83.0 (49.1 – 124)

EyC20

214 (168 – 265)

EyC50

> 1000

Cell Densities

 

Geometric mean measured a.i. concentrations

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

824

1

4878

34325

163933

614637

 

2

4878

35841

217107

609351

 

3

4878

36071

189451

629184

 

Mean

4878

35412

190164

617724

 

376

1

4878

39364

257540

834250

 

2

4878

39269

210110

850646

 

3

4878

34520

186630

875709

 

Mean

4878

37718

218093

853535

 

153

1

4878

34387

183554

1014858

 

2

4878

36877

205174

982794

 

3

4878

34260

221272

1049261

 

Mean

4878

35175

203333

1015638

 

44.8

1

4878

30478

193224

1118592

 

2

4878

35632

263876

1095101

 

3

4878

31959

258752

1183707

 

Mean

4878

32690

238617

1132467

 

Control

1

4878

38821

241412

1135702

 

2

4878

30044

222001

1229444

 

3

4878

38104

247285

1042552

 

4

4878

45582

253595

1203065

 

5

4878

44246

306716

1261929

 

6

4878

39228

267944

1367430

 

Mean

4878

39338

256492

1206687

 

Evaluation after 72 h

                              

Geometric mean measured a.i. concentrations

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

824

1

1.612

12.18

609759

49.26

2

1.609

12.34

604473

49.70

3

1.620

11.76

624306

48.05

Mean

1.614

12.09

612846

49.00

376

1

1.714

6.64

829372

30.99

2

1.720

6.28

845768

29.63

3

1.730

5.76

870831

27.54

Mean

1.721

6.23

848657

29.39

153

1

1.779

3.08

1009980

15.96

2

1.769

3.66

977916

18.63

3

1.790

2.47

1044383

13.10

Mean

1.779

3.07

1010760

15.90

44.8

1

1.812

1.31

1113714

7.33

2

1.805

1.70

1090223

9.28

3

1.831

0.28

1178829

1.91

Mean

1.816

1.10

1127589

6.17

Control

1

1.817

1130824

2

1.843

1224566

3

1.788

1037674

4

1.836

1198187

5

1.852

1257051

6

1.879

1362552

Mean

1.836

1201809

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

2.074

1.828

1.549

1.817

0.2630

14.5

15.6

2

1.818

2.000

1.712

1.843

0.1458

7.91

3

2.056

1.870

1.439

1.788

0.3164

17.7

4

2.235

1.716

1.557

1.836

0.3545

19.3

5

2.205

1.936

1.414

1.852

0.4020

21.7

6

2.085

1.921

1.630

1.879

0.2304

12.3

Mean

1.836

 

SD ±

0.03

 

CV [%]

1.68

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study Peractive P was found to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 h.
All effect values are given based on geometric mean measured concentrations of Tetraacetylethylenediamine (TAED), active ingredient of Peractive P
Executive summary:

The toxicity of Peractive P (batch number DEA 4 TM 0014) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany , from June 22 to 25, 2009. 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. Four concentration levels were tested in a geometrical series with a dilution factor of 2, nominal: 125 - 250 - 500 - 1000 mg test item/L, corresponding to geometric mean measured concentrations of 44.8 – 153 – 376 – 824 mg a.i./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 the active ingredient Tetraacetylethylenediamine (TAED) were analysed at all concentration levels after 0 and 72 h via HPLC analysis. The measured concentrations of Tetraacetylethylenediamine (TAED) at test start were in the range of 93 – 98 % of the nominal values. TAED hydrolyses to Diacetylethylenediamine (DAED) via TriAED. DAED itself is stable to hydrolysis and shows no inhibition of growth rate nor inhibition of yield to algae at 1000 mg/L (see Report SSO12974). At the end of the test Tetraacetylethylenediamine (TAED) gave recoveries of 13 – 73 % of the nominal values due to partial hydrolysis. All effect values are given based on geometric mean measured concentrations of Tetraacetylethylenediamine (TAED), active ingredient of Peractive P