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Diss Factsheets

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:
2009-06-22 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
Reference substance name:
Diacetylethylenediamine (DAED)
IUPAC Name:
Diacetylethylenediamine (DAED)
Details on test material:
- Name of test material (as cited in study report): N,N'-Diacetylethylenediamine (DAED)
- Physical state: Solid, powder
- Analytical purity: 99.9 %
- Lot/batch No.: 14-016
- Expiration date of the lot/batch: 2010-05-21
- 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: A stock solution of 1000 mg/L was freshly prepared with dilution water
- 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.
- 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): 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
Post exposure observation period:
No

Test conditions

Hardness:
Not measured
Test temperature:
Mean: 22.5 °C, Min: 22 °C, Max: 23 °C
pH:
Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
1000 7.63 9.48
500 7.67 9.54
250 7.88 9.11
125 8.03 8.84
Control 8.02 9.03
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Concentrations and Recovery Rates of N,N’-Diacetylethylenediamine (DAED)

Sampling date 2009-06-22
0 h 2009-06-25
72 h
Start of analysis 2009-06-22 2009-06-25
Nominal concentration N,N’-Diacetylethylenediamine (DAED)
Test item
Measured
conc. RR
Measured
conc. RR

[mg/L] [mg/L] [%] [mg/L] [%]
1000 1063 106 1098 110
500 548 110 553 111
250 278 111 283 113
125 131 105 132 106
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 1215537 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 -1 -3
10 1 6


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

Results and discussion

Effect concentrations
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 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

EC - values N,N’-Diacetylethylenediamine (DAED) (0-72 h)

                   based on nominal test item concentrations [mg/L]

                               

Rate-related Inhibition

ErC10

> 1000

ErC20

> 1000

ErC50

> 1000

Inhibition of Yield

EyC10

> 1000

EyC20

> 1000

EyC50

> 1000

Cell Densities

 

Nominal test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

1000

1

4878

36800

277980

1289606

 

2

4878

42169

270151

1261041

 

3

4878

43033

325505

1402432

 

Mean

4878

40667

291212

1317693

 

500

1

4878

45889

336771

1370575

 

2

4878

40018

281800

1260823

 

3

4878

43741

310764

1474435

 

Mean

4878

43216

309778

1368611

 

250

1

4878

37939

256056

1164207

 

2

4878

39906

288877

1394278

 

3

4878

39517

268679

1315283

 

Mean

4878

39121

271204

1291256

 

125

1

4878

37759

229556

1253138

 

2

4878

30915

245282

1140386

 

3

4878

42287

262434

1281249

 

Mean

4878

36987

245757

1224924

 

Control

1

4878

38821

241412

1135702

 

2

4878

30044

222001

1229444

 

3

4878

38104

247285

1042552

 

4

4878

45582

253595

1256165

 

5

4878

44246

306716

1261929

 

6

4878

39228

267944

1367430

 

Mean

4878

39338

256492

1215537

 

Evaluation after 72 h

                              

Nominal test item concentration

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

1000

1

1.859

-1.14

1284728

-6.12

2

1.852

-0.73

1256163

-3.76

3

1.887

-2.66

1397554

-15.44

Mean

1.866

-1.51

1312815

-8.44

500

1

1.879

-2.24

1365697

-12.81

2

1.852

-0.73

1255945

-3.74

3

1.904

-3.57

1469557

-21.38

Mean

1.878

-2.18

1363733

-12.64

250

1

1.825

0.72

1159329

4.24

2

1.885

-2.55

1389400

-14.76

3

1.866

-1.50

1310405

-8.24

Mean

1.859

-1.11

1286378

-6.25

125

1

1.850

-0.62

1248260

-3.11

2

1.818

1.09

1135508

6.21

3

1.857

-1.02

1276371

-5.43

Mean

1.842

-0.18

1220046

-0.78

Control

1

1.817

1130824

2

1.843

1224566

3

1.788

1037674

4

1.850

1251287

5

1.852

1257051

6

1.879

1362552

Mean

1.838

1210659

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

0.263

14.5

15.4

2

1.818

2.000

1.712

1.843

0.146

7.91

3

2.056

1.870

1.439

1.788

0.316

17.7

4

2.235

1.716

1.600

1.850

0.338

18.3

5

2.205

1.936

1.414

1.852

0.402

21.7

6

2.085

1.921

1.630

1.879

0.230

12.3

Mean

1.838

 

SD ±

0.03

 

CV [%]

1.71

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study N,N’-Diacetylethylenediamine (DAED) caused no inhibition on growth of the freshwater green alga Desmodesmus subspicatus after 72 h.
All effect levels are given based on nominal concentrations of N,N’-Diacetylethylenediamine (DAED)
Executive summary:

The toxicity of N,N’-Diacetylethylenediamine (DAED) (batch number 14-016)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 , 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/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 N,N’-Diacetylethylenediamine (DAED) were analysed at all concentration levels after 0 and 72 h via HPLC analysis. The measured concentrations of N,N’-Diacetylethylenediamine (DAED) at test start were in the range of 105 – 111 % of the nominal values. At the end of the test N,N’-Diacetylethylenediamine (DAED) gave recoveries of 106 – 113 % of the nominal values. All effect values are given based on nominal concentrations of the test item.

EC - values of N,N’-Diacetylethylenediamine (DAED) (0-72 h)

                   based on nominal test item concentrations [mg/L]

                               

Rate-related Inhibition

ErC10

> 1000

ErC20

> 1000

ErC50

> 1000

Inhibition of Yield

EyC10

> 1000

EyC20

> 1000

EyC50

> 1000