N,N'-ethylenedi(diacetamide)

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-05-29 to 2008-06-02

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity 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 %
- Composition of test material, percentage of components: 99.9 % (w/w) DAED; 0.05 % (w/w) water; 0.09 % (w/w) acetic acid; < 0.01 % (w/w) ethylene diamine
- (Purity test date) Date ofcertificate of analysis: 2008-05-21
- Lot/batch No.: 14-016
- Expiration date of the lot/batch: 2010-05-21
- Stability under test conditions: Stable (ca. 0.1 % aqueous solution)
- Storage condition of test material: Room temperature, protected from moisture and light

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: 170 - 172 °C
- Water solubility (under test conditions): Soluble, > 1 g/L
- log Kow: -1 (measured)
- Stability in water: stable (ca. 0.1 % aqueous solution)


OTHER PROPERTIES (if relevant for this endpoint)
- Density: 400 - 500 g/L (bulk density, 20 °C)
- pH: 4-6 (1 g/L, 20 °C)
- TOC: 50 %

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1000 mg/L
- Sample storage conditions before analysis: 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: The test medium (1000 mg/L) was mixed with an ultraturrax (1 min, 12000 rpm).
- Differential loading: 1000 mg/L
- Controls: Dilution water without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The test item was clearly dissolved.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Danio rerio (zebrafish)
- Source: AQUARIUM AM AEGI, Volgersweg 6, D-30175 Hannover
- Length at study initiation (mean): 2.71 cm
- Weight at study initiation (mean): 0.19 g
- Method of breeding: Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (0.1-10 µmol photons x m-2 x s-1, natural photoperiod). Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine. Nominal water parameters: Total hardness: 10 - 250 mg CaCO3/L; pH-value: 6.0 - 8.5. The water is analysed biannual acc. to German tap water regulation. The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value.
- Feeding during test: no feeding during the test

ACCLIMATION
- Acclimation period: Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within these days before the study starts were used in the test.
- Acclimation conditions (same as test or not): same
- Type and amount of food: 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Food: Stör perlets; SERA GMBH, D-52518 Heinsberg
- Feeding frequency: 3 times per week
- Health during acclimation (any mortality observed): No disease treatments were administered throughout holding and testing.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

No

Test conditions

Hardness:

Total hardness of the water was determined at the beginning out of the test control to be 54 mg/L as CaCO3.

Test temperature:

During the test the room temperature was recorded continuously with a thermohygrograph. Temperature was measured in all vessels at the beginning of the test and every 24 h to be:
Day Concentration [mg/L] Temperature [°C]

0 1000 24.0
Control 24.0
1 1000 24.0
Control 24.0
2 1000 23.2
Control 23.2
3 1000 23.5
Control 23.9
4 1000 23.7
Control 24.0

pH:

pH-value was measured in all vessels at the beginning of the test and every 24 h to be:
Day Conc. pH-Value
[mg/L]
0 1000 7.55
Control 7.61
1 1000 7.67
Control 7.68
2 1000 7.71
Control 7.69
3 1000 7.65
Control 7.64
4 1000 7.38
Control 7.35

Dissolved oxygen:

Oxygen saturation was measured in all vessels at the beginning of the test and every 24 h to be:
Day Conc. Oxygen-Saturation
[mg/L] [%]
0 1000 100
Control 100
1 1000 100
Control 100
2 1000 100
Control 100
3 1000 99
Control 95
4 1000 95
Control 96

Salinity:

Nor measured

Nominal and measured concentrations:

1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open, loosely covered by glass tops
- Material, size, headspace, fill volume: Glass-aquaria
- Aeration: Gentle aeration
- Renewal rate of test solution (frequency): One application at test start
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: less than 1 g fish per liter test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin, pH-value: 6.0 - 8.5.

- Total organic carbon: 10 - 250 mg CaCO3/L
- Chlorine: The dilution water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine
- Culture medium different from test medium: No
- Intervals of water quality measurement: The water is analysed biannual acc. to German tap water regulation.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: natural photoperiod
- Light intensity: 0.1 - 10 µmol photons x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Observations were made after 24, 48, 72 and 96 h. Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: Not observed
- Mortality of control: 0 %
- Other adverse effects control: Not observed
- Abnormal responses: Not observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not observed
- Effect concentrations exceeding solubility of substance in test medium: No

Reported statistics and error estimates:

The concentration leading to 0 % mortality (LC0) after 96 h was determined directly from the test results. LC50-values have not to be calculated in a limit test.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Observations in the Test Vessels:

Nominal test item Concentration [mg/L]	Effect*)	Test Duration [h]
		24	48	72	96
1000	(1)	7/7	7/7	7/7	7/7
Control	(1)	7/7	7/7	7/7	7/7

*)  The number in brackets correspond to the following observation: (1) = normal behaviour                                

Cumulative Mortality [%] in the Test Vessels:

Nominal test item Concentration [mg/L]	Test Duration [h]
	24	48	72	96
1000	0	0	0	0
Control	0	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study N,N' -Diacetylethylenediamine (DAED) did not cause any effects to zebrafish after 96 h when tested with a nominal test item concentration of 1000 mg/L. The NOEC (0 – 96 h) is laid down as 1000 mg/L.

Executive summary:

The acute toxicity of the test item N,N' -Diacetylethylenediamine (DAED)(Batch no.: 14-016) to fish (zebrafish) was determined according to OECD-Guideline for Testing of Chemicals No. 203 (1992) from 2008-05-29 to 2008-06-02 at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany. A static limit test was conducted with a single test item concentration of 1000 mg/L. Duration of the test was 96 h. 7 test organisms were exposed to the test concentration and control. Water quality parameters pH-value, temperature and oxygen-saturation measured after 0, 24, 48, 72 and 96 h were determined to be within the acceptable limits. The limit concentration of N,N' -Diacetylethylenediamine (DAED) and control were analysed at test start and test end via HPLC analysis. The acute toxicity of the test item N,N' -Diacetylethylenediamine (DAED) is summarised in the following table:

Table 1: LC-Values with 95 % Confidence Intervals (nominal concentration)

Test duration [h]	LC50		p = 95 %
24	> 1000 mg/L		not applicable
48	> 1000 mg/L		not applicable
72	> 1000 mg/L		not applicable
96	> 1000 mg/L		not applicable
LC100 =		> 1000 mg/L
Lowest test item concentration
with 100 % mortality after 96 h
LC0 =		1000 mg/L
Highest test item concentration
with 0 % mortality after 96 h
NOEC (0-96 h) = No observed effect concentration		1000 mg/L