N,N'-ethylenebis[N-acetylacetamide]

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2008-10-09 to 2008-12-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study has been carried out recently, is according OECD Guideline 222, follows GLP principles and does not deviate from the Guideline.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

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 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Version / remarks:

DIN ISO 11268-2: 1998 (E), BBA Guideline (1994)

Deviations:

yes

Remarks:

Room temperature increased once to 23 ° C over 2 h, once to about 22.5 °C over 4 hours and decreased once to about 17.5 over 5 h. The soil moisture of all test vessels deviated by more than 10 % of the individual at the end of the test.

GLP compliance:

yes

Test material

Test material information

Constituent 1

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: September 2008
- 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: 152 °C
- Water solubility (under test conditions): Ca. 1 g/L
- log Pow: -0.1



OTHER PROPERTIES (if relevant for this endpoint)
- Other:
pH: 4 - 6 (1 g/L in water, 20 °C)

Sampling and analysis

Analytical monitoring:

no

Test substrate

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): A mixture comprised of 10 g quartz sand with the necessary quantity of test item was prepared per treatment. The mixture was thoroughly mixed with the pre-moistened artificial soil with a long hadled blender for at least three minutes.
- Controls: Artificial soil with addition of quartz sand

Test organisms

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida SAVIGNY 1826, subspecies Eisenia fetida fetida
- Source: Own breeding
- Age at test initiation (mean and range, SD): synchronized earthworm culture, not deviating more than 1 month
- Weight at test initiation (mean and range, SD): 0.54 +/- 0.04 g


ACCLIMATION
- Acclimation period: 2 days prior to test start
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): healthy

Study design

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Remarks:

Removement of adults after 28 days

Test conditions

Test temperature:

20 +/- 2°C

pH:

Table 1: pH-value of the Test Media
Application rate
[mg test item/kg soil DW] pH-value
day 0 day 56
Control 6.26 6.52
62.5 6.34 6.53
125 6.25 6.60
250 6.13 6.48
500 6.07 6.58
1000 6.05 6.53

Measurement of pH out of mixed samples of all replicates per treatment.

Moisture:

Table 1: Moisture of the Test Media
Application rate
[mg test item/kg soil DW] Moisture [%] of dry weight

day 0 day 56
Control 32.2 36.4*
62.5 31.5 36.3*
125 31.1 37.2*
250 30.8 34.2*
500 29.0 37.3*
1000 30.4 35.5*

Measurement of moisture content of test medium out of mixed samples of all replicates per treatment.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): round plastic boxes, ID: 15 cm, bottom surface area: 177 cm3, height: 14 cm, transparent and perforated lids enabled sufficient gas exchange and light penetration and prevented the test substrate from drying
- Amount of soil or substrate: 600 g DW
- No. of organisms per container (treatment): 10 (40)
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control:


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10 % peat, 20 % kaolin, 69 % air dried quartz sand, 0.4 % calcium carbonate
- Organic carbon (%): not determined
- Maximum water holding capacity (in % dry weigth): 68
- CEC: not determined
- Pretreatment of soil: none
- Storage (condition, duration): 8 days after preparation at test conditions in the dark



OTHER TEST CONDITIONS
- Photoperiod: 16 h light - 8 h dark
- Light intensity: 657 +/- 105 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Adult mortality, biomass, behaviour and pathological symptoms after 28 days
Reproduction (Number of juveniles) after 56 days

VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Nominal and measured concentrations:

Nominal application rates: 62.5 - 125 - 250 - 500 - 1000 mg test substance/kg soil dry weight (DW)

Reference substance (positive control):

yes

Remarks:

1.0 - 2.5 - 5.0 mg Carbendazim/ kg soil DW

Results and discussion

Effect concentrations

Duration:

56 d

Dose descriptor:

other: NOEC FOR DAED

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality at end of exposure period: No mortality was observed in any of the tested test item and control groups
- Total mass of adults at beginning of test: 0.54 +/- 0.4
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: Control: 15.0 +/- 5.09, 62.5 mg/kg soil DW: 24.3 +/- 2.82, 125 mg/kg soil DW: 21.8 +/- 4.33, 250 mg/kg soil DW: 20.4 +/- 3.44, 500 mg/kg soil DW: 26.4 +/- 4.25, 1000 mg/kg soil DW: 22.0 +/- 4.64
- No. of offspring produced: Control: 266 +/- 21.4, 62.5 mg/kg soil DW: 280 +/- 37.4, 125 mg/kg soil DW: 240 +/- 9.71, 250 mg/kg soil DW: 250 +/- 15.8, 500 mg/kg soil DW: 188 +/-31.4, 1000 mg/kg soil DW: 172 +/- 31.5
- No. of unhatched cocoons: Not determined
- Morphological abnormalities: None
- Behavioural abnormalities: None
- Other biological observations: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 2.5 mg Carbendazim/kg soil DW

Reported statistics and error estimates:

One Way Analysis of Variance (ANOVA) was carried out for the determination of statistically significant differences compared to the control. A Normality Test and an Equal Variance Test were conducted prior to running the ANOVA. P-values for both Normality and Equal Variance Test are 0.05. The a-value for ANOVA test (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05.

Any other information on results incl. tables

Adult mortality

Compared to control no mortality of adult earthworms was observed at all application rates of the test item.

Behaviour and pathological symptoms

No pathological symptoms or changes in the behaviour of the earthworms were observed at all application rates compared to the control after 28 days of exposure.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item did not induce mortality or pathological symptoms of adult earthworms after an exposure to the test item concentrations of 62.5, 125, 250, 500 and 1000 mg/kg soil dry weight for 28 days. Also, the biomass increase of the adult earthworms throughout the first 28 days was not statistically significantly reduced compared to the control at all test item concentrations. However, the earthworm reproduction was statistically significantly reduced at 500 and 1000 mg/kg soil dry weight compared to the control.

Executive summary:

Effects of Peractive P (Batch no. DEA 4 TM 0014) on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Savigny) were determined according to the guidelines DIN ISO 11268-2 (1998), OECD 222 (2004) and BBA (1994) from October 9^thto December 4^th, 2008 at Dr.U.Noack-Laboratorienin D-31157 Sarstedt. The study was conducted under static conditions over 8 weeks with five different application rates (62.5 – 125 – 250 – 500 - 1000 mg test item/kg soil dry weight), which were mixed into artificial soil containing 10 % peat. 40 test organisms, with an individual weight of 0.30 to 0.60 g, were divided into 4 replicate groups with 10 test organisms per replicate.

After 28 days no mortality and no pathological symptoms or changes in the behaviour of adult earthworms were seen in the control and at all tested application rates (Table 1). The earthworm biomass increased in the control and at the application rates of 62.5, 125, 250, 500 and 1000 mg/kg soil dry weight over 28 days without a statistically significant reduction compared to the control (Figure 1, Table 1). After further four weeks the reproduction rate (average number of juveniles) was 266 in the control and ranged between 172 and 280 at the test item application rates. Compared to the control, the reproduction rate did not statistically significantly differ at 62.5, 125 and 250 mg/kg soil dry weight, but was statistically significantly reduced at the application rates of 500 and 1000 mg/kg soil dry weight (Table 1).

Overall, the NOEC of Peractive P concerning mortality, biomass and reproduction of earthworms was determined to be at 250 mg/kg soil dry weight (Table 2).

All validity criteria recommended by the test guidelines were fulfilled.

Table 1:          Summary of All Observed Effects in the Tested Doses

Effects	Peractive P
mg test item/kg soil dry weight	62.5	125	250	500	1000
Mortality of adult earthworms	No	No	No	No	No
Reduction of body weight	No	No	No	No	No
Reduced reproduction rate   (Number of juveniles)	No	No	No	Yes	Yes
Summary of all effects	No	No	No	Yes	Yes

Table 2:                 NOEC, LOEC and EC₅₀ for Reproduction, Mortality and Body Weight

Peractive P
	Application rate
	mg test item/kg soil dry weight
NOECreproduction, mortality, body weight	250
LOECreproduction, mortality, body weight	500