N,N'-ethylenedi(diacetamide)

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-07-14 to 2009-07-16

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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-ethylenedi(diacetamide)

EC Number:

212-811-9

EC Name:

N,N'-ethylenedi(diacetamide)

Cas Number:

871-78-3

Molecular formula:

C6H12N2O2

IUPAC Name:

N-(2-acetamidoethyl)acetamide

Constituent 2

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: Limit concentration 1000 mg/L
- Sampling method: The limit concentration and control were analytically verified via HPLC after 0 hours (fresh media) and after 48 hours (old media). For the old media, separate replicates without test organisms were prepared and incubated under test conditions.
- 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: The stock solution (1000 mg/L test item were weighed out) was freshly prepared with dilution water prior to application.
- Eluate: Dilution water
- Differential loading: 1000 mg/L
- Controls: Dilution water without test item tested under the same conditions as the test group.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation (mean and range, SD): 2 to 24 h
- Method of breeding: In 2-3 L glass vessels with appr. 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µExm-2 x s-1
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

No

Test conditions

Hardness:

Dilution water at test start: 274 (mg CaCO3/L)

Test temperature:

21.7 °C

pH:

Water Quality Parameters at Test Start (0 h)
(measured in one additional replicate of the limit concentration and control)
Nominal
Test Item Concentration
[mg/L] pH-Value
1000 7.69
Control 7.62

Water Quality Parameters at the Test End (48 h)
(measured in all replicates)
Nominal
Test Item Concentration
[mg/L] pH-Values
Replicates
1 2 3 4
1000 7.65 7.60 7.59 7.61
Control 7.68 7.76 7.71 7.67

Dissolved oxygen:

Water Quality Parameters at Test Start (0 h)
(measured in one additional replicate of the limit concentration and control)
Nominal
Test Item Concentration
[mg/L] Dissolved
O2-Concentration

[mg/L]
1000 7.62
Control 8.02

Water Quality Parameters at the Test End (48 h)
(measured in all replicates)
Nominal
Test Item Concentration
[mg/L] Dissolved O2 –Concentration [mg/L]
Replicates
1 2 3 4
1000 8.06 7.46 8.02 8.15
Control 8.11 8.16 8.18 8.01

Salinity:

Freshwater

Nominal and measured concentrations:

Concentrations and Recovery Rates of N,N’-Diacetylethylenediamine (DAED)
Sampling date 2009-07-14
Test start (0 h) 2009-07-16
Test end (48 h)
Start of analysis 2009-07-14 2009-07-16
Nominal Concentration N,N’-Diacetylethylenediamine (DAED)
Test Item
[mg/L] Active Ingredient
[mg/L] Meas. conc.
[mg/L] RR
[%] Meas. conc.
[mg/L] RR
[%]
1000 999 1110 111 1085 109
Control < LOQ < LOQ

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, 50 mL
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): One application at test start, static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Acc. to Directive 92/69/EEC Method C.2, Annex 1
- Conductivity: Dilution water at test start: 640 µS/cm
- Culture medium different from test medium: Elendt M4 acc. to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: At test start and test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :


TEST CONCENTRATIONS
- Spacing factor for test concentrations: None, limit test
- Range finding study
- Test concentrations: 10 - 100 - 1000 mg/L
- Results used to determine the conditions for the definitive study:
Immobilization Rates of the Preliminary Range Finding Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item Concentration
[mg/L] IMMOBILIZATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
1000 0 0 0 0 0 0
100 0 0 0 0 0 0
10 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: None
- Mortality of control: 0%
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50/LC50: 1.29 (CI 1.15 - 1.45) mg/L

Reported statistics and error estimates:

The effects of the limit concentration of 1000 mg/L were determined directly from the observation data.

Any other information on results incl. tables

Percentage of Daphnids Incapable of Swimming after 24 and 48 h of Exposure

                  (n = 20, divided into 4 replicates with 5 daphnids each)

IMMOBILIZATION [%]
Nominal Test Item Concentration [mg/L]	24 h					48 h
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
1000	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

At the limit concentration 1000 mg/L (nominal) of the test item N,N’-Diacetylethylenediamine (DAED) no effect was observed.

Executive summary:

In the acute immobilization test to Daphnia magna (STRAUS) the effects of the limit concentration 1000 mg/L of the test item N,N’-Diacetylethylenediamine (DAED) (batch number: 14-016), was determined according to OECD 202 (2004). The limit test was conducted under static conditions over a period of 48 h from 2009-07-14 to 2009-07-16 at Dr. U. Noack-Laboratorien, Käthe-Paulus-Str. 1, D-31157 Sarstedt, Germany .

20 test organisms were exposed to the limit concentration and the control. The limit concentration and the control were analysed via HPLC after 0 hours (fresh media) and 48 hours (old media). The recoveries of the test item in the limit concentration were 111 % of the nominal value in the fresh media at test start (0 hours) and 109 % of the nominal value in the old media at test end (48 hours). All effect values are given based on the nominal test item concentration. Water quality parameters (pH-value and dissolved oxygen concentration), measured in the fresh (0 hours) an old media (48 hours), were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

EC-Values, NOEC and LOEC

(based on the nominal test item concentration)

	Test Duration [h]	Nominal Test Item Concentration [mg/L]	Confidence Interval p = 95% [mg/L]
EC50	24	> 1000	not applicable
	48	> 1000	not applicable
EC100	24	> 1000	---
	48	> 1000	---
NOEC	48	1000	---
LOEC	48	> 1000	---

At the limit concentration 1000 mg/L (nominal) of the test item
N,N’-Diacetylethylenediamine (DAED),
no effect was observed.