N,N'-ethylenebis[N-acetylacetamide]

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Study period:

2008-07-11 to 2008-08-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study has been carried out recently, is according OECD Guideline 211, 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:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

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:

yes

Details on sampling:

- Concentrations: 62.5 - 125 - 250 - 500 - 1000 mg/L
- Sampling method: 100 mL of each concentration were filled into brown glass bottles
- Sample storage conditions before analysis: 6 +/- 2 °C

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions of 1000 mg/L in dilution water were sonificated for 15 min, stirred for 2 h at approx. 1100 rpm at room temperature and diluted to the test concentrations with dilution water
- Differential loading: 62.5 - 125 - 250 - 500 - 1000 mg/L
- Controls: Dilution water without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not observed

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline: Daphnia magna STRAUS is recommended in the guideline
- Source: Own breeding (Origin: Institut fuer Wasser- Boden- und Lufthygiene)
- Age of parental stock (mean and range, SD): > 14 days
- Feeding during test
- Food type: Mix of Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: 0.2 mg C/daphnia per day
- Frequency: daily


ACCLIMATION
- Acclimation period: 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: During acclimation the daphnids were not fed
- Feeding frequency: None
- Health during acclimation (any mortality observed): Healthy

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The parent animals were removed from the culture medium and the juveniles collected over a sieve and flushed into fresh medium

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Test conditions

Hardness:

Total Hardness [mg/L] as CaCO3
Nominal Test Item Concentration
[mg/L] I F I F I F I F
Day 0 Day 3 Day 5 Day 7 Day 10 Day 12 Day 17 Day 19
July 11. July 14. July 16. July 18. July 21. July 23. July 28. July 30.
1000 175 162 171 162 180 176 172 175
Control 180 143 168 163 173 176 164 170
The total hardness should be more than 140 mg/L as CaCO3.

Test temperature:

Temperature [°C]
Nominal Test Item Concentration
[mg/L] I F I F I F I F
Day 0 Day 3 Day 5 Day 7 Day 10 Day 12 Day 17 Day 19
July 11. July 14. July 16. July 18. July 21. July 23. July 28. July 30.
1000 22.0 21.4 22.0 21.7 21.8 21.3 21.5 21.7
Control 21.9 21.4 22.0 22.0 21.8 21.6 21.5 21.8
The temperature should be within the range 18-22 °C, ± 2 °C constant

pH:

pH-Values
Nominal Test Item Concentration
[mg/L] I F I F I F I F
Day 0 Day 3 Day 5 Day 7 Day 10 Day 12 Day 17 Day 19
July 11. July 14. July 16. July 18. July 21. July 23. July 28. July 30.
1000 7.14 7.38 7.25 7.30 7.17 7.11 7.28 7.18
Control 7.47 7.62 7.74 7.49 7.72 7.38 7.57 7.31
The pH-values should be in the range of 6 - 9.
Deviations < 1.5 units between the initial and final pH-values are regarded to be not significant.

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal Test Item Concentration
[mg/L] I F I F I F I F
Day 0 Day 3 Day 5 Day 7 Day 10 Day 12 Day 17 Day 19
July 11. July 14. July 16. July 18. July 21. July 23. July 28. July 30.
1000 8.20 9.41 8.02 8.15 8.25 8.18 7.92 8.22
Control 8.28 9.36 8.38 8.52 8.24 8.25 8.58 8.89
The dissolved oxygen concentration should be above 3 mg/L.

Salinity:

Not measured

Nominal and measured concentrations:

Nominal 62.5 - 125 - 250 - 500 - 1000 mg/L
Measured values between Day 0 and Day 21 have a recovery of 86-100% for the testing range 125 to 1000 mg/L. As this concentration range is the relevant one for the derivation of the NOEC the nominal concentrations were used.
For 62.5 mg/L recovery was 41-95% but this concentration is not of relevance for the derivation of the NOEC reproduction. An explanation for the low recoveries at 62.5 mg/L might be that TAED hydrolyzes and biodegrades rapidly.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: 100 mL glass beaker, filled with 50 mL test solution
- Aeration: No
- Renewal rate of test solution (frequency): 3 x per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, modified to a total hardness of 160 - 180 mg CaCO3/L
- Culture medium different from test medium: No
- Intervals of water quality measurement: once per week in fresh and old media


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark per day
- Light intensity: Max. 20 µE*m-2*s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Adult mortality: daily
- Number of juveniles: daily
- Stillborn juveniles and aborted eggs: daily
- Appearance of first brood
- Intrinsic rate of natural increase: test end
- Growth (total length and dry weight): test end


RANGE-FINDING STUDY
- Test concentrations: 100 - 500 - 1000 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

21 d

Dose descriptor:

other: NOEC FOR DAED

Effect conc.:

500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and intrinsic rate

Remarks on result:

other: EC50 (mortality) = 1000 mg/L

Details on results:

- Mortality of parent animals: The nominal test item concentration 1000 µg/L had a biologically significant effect on adult mortality (> 20 %) after 21 d of exposure. The EC50 for the adult mortality was determined to be 1000 mg/L.

- No. of offspring produced per day per female: The mean number of offspring alive produced per parent animal surviving at the end of the test was 133 juveniles in the control group. The mean number of juveniles of the surviving daphnids of the treatment groups were compared to the control by One Way Analysis of Variance. There was no statistically significant difference at any of the tested concentration levels when compared to the control.

- Body length and weight of parent animals: The mean body weight in the concentration levels 62.5 to 1000 mg/L ranged from 0.68 to 1.03 mg per daphnid. The mean body weight in the control was 0.85 mg per daphnid. The mean value of the body length in the tested concentrations was in the range of 5.05 to 5.45 mm per daphnid and 5.33 mm per daphnid in the control group.

- Type and number of morphological abnormalities: None
- Type and number of behavioural abnormalities: None
- Number of males and females (parental): No males occurred

- Time to first brood release or time to hatch: The first brood was released till day 9 in the control group and in the tested concentration levels 62.5 to 1000 mg/L by all daphnids.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.75 mg/L

Reported statistics and error estimates:

The NOEC and LOEC for the adult mortality were determined directly from the results. Significant deviations were determined in comparison to the control group using statistical standard procedures as Normality Test, Equal Variance Test and Analysis of Variance. For the determination of significant deviations for the reproduction rates and the body lengths One Way Analysis of Variance, p = 0.05 was used. One Way Analysis of Variance, DUNNETT’S Method, p = 0.05 was used for the determination of significant deviations for intrinsic rates of natural increase. Statistical evaluation of the age of first reproduction and the occurrence of stillborn juveniles and aborted eggs could not be carried out because Normality Test failed. The coefficients of variation around the mean number of living offspring produced per parent in the control and test groups were evaluated.

Any other information on results incl. tables

Reproductive output:

Number of Juveniles in the Control and Test Groups after 21 Days

Nominal conc. [mg/L]												No.	Mean No. of Juveniles			Comparison
	Number of Juveniles in Replicate No.										Total	of			CV	versus
											No.	Parents	per Parent			Control
												prod. Juv.	producing Juveniles			Red.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD		[%]	[%]
1000	133	131	159	--	--	--	--	--	148	115	686	5	137 ±	17	12	-3*	no
500	134	126	143	171	129	148	159	123	150	180	1463	10	146 ±	19	13	-10*	no
250	128	155	147	162	159	130	128	161	121	95	1386	10	139 ±	22	16	-5*	no
125	144	137	127	126	149	165	149	118	119	150	1384	10	138 ±	16	11	-4*	no
62.5	129	116	128	140	140	133	127	129	149	141	1332	10	133 ±	9	7	0	no
Control	152	111	132	127	121	132	119	139	144	156	1333	10	133 ±	15	11	—	—

Nominal conc.                = Nominal concentration

N                                 = Number of parents producing juveniles alive

MV ± SD                      = Mean value ± standard deviation

CV                              = Coefficient of variation

Red.                            = Reduction of reproduction

Stat.                            = Statistical significance (One Way Analysis of Variance, p = 0.05)

no                               = Statistically not significant

--                                = Not applicable, due to mortality of the parent animal

*)                               = Negative value means increase of reproductive output in comparison to the control group

Intrinsic rate of natural increase:

The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and offspring numbers were used for calculation of population growth and maintenance. The IR of the surviving daphnids of the test groups were compared to the control by One Way Analysis of Variance, Dunnett’s Method (p = 0.05).There was a statistical significance only at the concentration level 1000 mg/L when compared to the control, but no significant effect on the reproductive output.

Intrinsic Rates of Natural Increase

Nominal Test Item Concentration	Intrinsic Rate of Natural Increase in Replicate No.										Mean IR		CV
[mg/L]	1	2	3	4	5	6	7	8	9	10	MV ± SD		[%]
1000	0.49	0.49	0.50	--	--	--	--	--	0.51	0.48	0.49 ±	0.01*	3
500	0.52	0.53	0.50	0.51	0.51	0.52	0.52	0.50	0.50	0.52	0.51 ±	0.01	2
250	0.51	0.55	0.57	0.54	0.52	0.55	0.53	0.54	0.53	0.47	0.53 ±	0.03	5
125	0.53	0.54	0.51	0.51	0.52	0.57	0.54	0.53	0.54	0.52	0.53 ±	0.02	4
62.5	0.51	0.50	0.52	0.51	0.51	0.58	0.57	0.52	0.52	0.50	0.52 ±	0.03	5
Control	0.58	0.51	0.58	0.52	0.53	0.52	0.56	0.56	0.53	0.51	0.54 ±	0.03	5

IR                = Intrinsic rate of natural increase

MV ± SD     = Mean value ± Standard deviation

CV             = Coefficient of variation

--                = Not applicable, due to mortality of the parent animal

*                 = Statistically significant (One Way Analysis of Variance, Dunnett’s Method, p = 0.05)

Appearance of first brood:

The first day of appearance of juveniles at all test item and control groups producing juveniles was between day 7 and day 9.

First Appearance of Living Juveniles in the Individual Groups

Nominal Test Item Concentration	Day of First Appearance of Living Juveniles										First
	in Replicate No.										Appearance
[mg/L]	1	2	3	4	5	6	7	8	9	10	Mean Day
1000	8	8	8	--	--	--	--	--	8	8	8.0
500	8	8	8	8	8	8	8	8	8	8	8.0
250	7	7	7	8	8	8	8	7	7	9	7.6
125	7	8	8	8	8	7	7	8	7	8	7.6
62.5	8	8	8	8	8	7	7	8	8	8	7.8
Control	7	8	7	8	8	8	7	7	8	8	7.6

  --       = Not applicable, due to mortality of the parent animal

Mortality:

At the concentration level 1000 mg/L a biologically significant effect on adult mortality (> 20 %) was observed after 21 d of exposure. The EC₅₀ for adult mortality was determined directly from the results to be 1000 mg/L.

Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure (n = 10)

Nominal Test Item Concentration	Adult Mortality [%]
[mg/L]	7 days	14 days	21 days
1000	0	0	50*
500	0	0	0
250	0	0	0
125	0	0	0
62.5	0	0	0
Control	0	0	0

*)       = Biologically significant effect, mortality > 20 %

Growth:

Total Length and Dry Weight of the Parent Animals

Nominal Test Item Concentration [mg/L]	Total Length of the Parent Animals [mm]										MV^2)	N^1)	Dry Weight [mg]
	Replicate No.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
1000	5.00	5.00	5.25	--	--	--	--	--	5.00	5.00	5.05	5	3.4	0.68
500	5.25	5.00	5.25	5.25	5.00	5.50	5.75	5.00	5.50	6.00	5.35	10	8.6	0.86
250	5.00	5.75	5.25	5.50	5.50	5.50	5.00	5.50	5.00	5.00	5.30	10	10.0	1.00
125	5.75	5.25	5.25	5.00	6.00	5.50	5.50	5.50	5.25	5.50	5.45	10	10.3	1.03
62.5	5.25	5.00	5.00	5.50	5.50	5.50	5.00	5.25	5.50	5.50	5.30	10	9.3	0.93
Control	5.50	5.50	5.25	5.00	5.25	5.25	5.00	5.50	5.25	5.75	5.33	10	8.5	0.85

      ¹⁾N       = Number of parent animals alive

      ²⁾MV    = Mean value

      --          = Not applicable, due to mortality of the parent animal

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) after 21 days based on adult mortality and intrinsic rate of natural increase as the most sensitive effects were determined directly from the results. Mortality of > 20 % is seen to be significant and given as EC50 adult mortality. All effect values are given based on the nominal test item concentrations.

NOECadult mortality, intrinsic rate : 500 mg/L
LOECadult mortality, intrinsic rate : 1000 mg/L
EC50 adult mortality : 1000 mg/L

Executive summary:

T he Daphnia magna Reproduction Test (Semi-Static, 21 d) of Peractive P (batch No. DEA 4 TM 0014) was conducted according to OECD 211 (1998) from 2008-07-11 to 2008-08-01 at Dr.U.Noack-Laboratorien in Sarstedt, Germany, . Test system was Daphnia magna STRAUS (Clone 5). 10 test organisms, individually held, were used per concentration level and control. At test start they were 2 to 24 hours old. The test method was semi-static. The test solutions were renewed 3 times per week. Aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects on parameters such as occurrence of aborted eggs and stillborn juveniles, time of production of first brood, adult mortality, intrinsic rate, dry body weight and length of the parent animals. Based on the results of the preliminary range finding test the study was performed with 5 test item concentrations ranging from 62.5 to 1000 mg/L set up in a geometrical series with a dilution factor of 2. The active ingredient Tetraacetylethylenediamine (TAED) and its main metabolite Diacethylethylenediamine (DAED) of all concentration levels and the control were analytically verified via HPLC of samples on days 3, 10, 17 (fresh media, 0 h) and on days 5, 12, 19 (old media, 48 h). The mean recoveries of the active ingredient TAED were in the range of 81 to 94 % of the nominal values in fresh and old media. Recoveries of the main metabolite DAED remained below LOQ in all samples. Analytical results are given in Table 23 and Table 24. All effect values are based on the nominal test item concentrations. In the Daphnia magna Reproduction Test with the test item Peractive Ponly significant effects on adult mortality occurred at the concentration level 1000 mg/L. Significant effects on reproductive output could not be observed.

·   The adult mortality after 21 days amounted to 50 % at the concentration level 1000 mg/L. In the control as well as in the tested concentration levels 62.5 to 500 mg/L all daphnids survived until the end of the study. The EC₅₀ for the adult mortality was determined directly from the results to be 1000 mg/L.

·   The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and number of offspring were used for calculation of population growth and maintenance.

·   The average number of juveniles per parent in the control group was 133 after 21 days. The reproductive output was not statistically significant reduced at any of the tested concentrations.

·   The coefficient of variation of the mean number of living offspring produced per parent in the control group was 11 %.      

A summary of all test item related effects is given in Table 1. The effect levels (NOEC and LOEC) are given based on the adult mortality and intrinsic rates of natural increase as the most sensitive effects. The EC₅₀ for the adult mortality was determined directly from the results.

Table 1:        Test Item Related Effects, NOEC, LOEC and EC₅₀

Effects	Nominal Test Item Concentration [mg/L]
	Control	62.5	125	250	500	1000
Adult Mortality after 21 Days [%]	0	0	0	0	0	50^1)
Mean Intrinsic Rates	0.54	0.52	0.53	0.53	0.51	0.49^2)
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)	133 ± 15	133 ± 9	138 ± 16	139 ± 22	146 ± 19	137 ± 17
Appearance of First Brood [day]	7.6	7.8	7.6	7.6	8.0	8.0
Parent Animals: Mean Dry Weight [mg]	0.85	0.93	1.03	1.00	0.86	0.68
Parent Animals: Total Length [mm]	5.33	5.30	5.45	5.30	5.35	5.05
NOEC^3)	500 mg/L
LOEC^3)	1000 mg/L
EC50 ^4) with Confidence Interval (CI) p = 95 %	1000 mg/L (Not applicable)

SD     = Standard deviation

¹⁾           = Biological significant effect, mortality > 20 %

²⁾           = Statistically significant (One Way Analysis of Variance, Dunnett’s Method, p = 0.05)

³⁾           = based on the most sensitive parameters adult mortality & intrinsic rate of natural increase

⁴⁾           = based on adult mortality

·   No stillborn juveniles and aborted eggs were produced by the control group. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was ≤ 1 % at all concentration levels.

·   The first brood was released till day 9 in the control group and in the tested concentration levels 62.5 to 1000 mg/L by all daphnids.

·   The mean body weight in the concentration levels 62.5 to 1000 mg/L ranged from 0.68 to 1.03 mg per daphnid. The mean body weight in the control was 0.85 mg per daphnid. The mean value of the body length in the tested concentrations was in the range of 5.05 to 5.45 mm per daphnid and 5.33 mm per daphnid in the control group.

No males and ephippia (winter eggs) occurred in control or test groups.

Water quality parameters as pH-value, dissolved oxygen, water hardness and temperature were determined to be within the acceptable limits.

·          In order to prove the validity of the test system and test conditions at the test facility, an acute immobilization test according to DIN 38412 L 11 was carried out with potassium dichromateas reference item once per month.
The EC₅₀ of the reference item at 1.75 mg/L after 24 hours was within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS P 9/2 (05/1996) for daphnids clone 5 cultured in Elendt M4 medium. The EC₅₀-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.