Pentanediamide, N1,N5-dibutyl-2-[(2-ethyl-1-oxohexyl)amino]-, (2S)-

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 August 2009 - 3 September 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

The inoculum: Activated sludge, microorganisms from a domestic waste water treatment plant.
Origin: The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
Conditioning: The activated sludge used for this study was washed by centrifugation (for 10 min.) and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged (for 10 min.). This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 4 g dry material per litre were mixed with test water and then aerated until use. Before use the sludge was filtered through cotton wool.

Duration of test (contact time):

28 d

Initial conc.:

2.2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Stock solutions
In deionised water analytical grade salts were added to prepare the following stock solutions:
a) Solution: KH2PO4 2.125g
K2HPO4 5.4375g
Na2HPO4 x 12H2O 16.795g
NH4Cl 0.125g
Deionised water ad 250 mL
b) Solution: MgSO4 x 7 H2O 5.625g
Deionised water ad 250 mL
c) Solution: CaCl2 x 2 H2O 9.10 g
Deionised water ad 250 mL
d) Solution: FeCl3 x 6 H2O 0.25 g
Deionised water ad 1000 mL

Ratio of ingredients
1 mL of the stock solutions a) - d) were combined and filled to a final volume of 1000 mL with deionised water. The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration was about 8.4 mg/L at about 22 °C.

TEST UNITS
Type and Size: BOD bottles (300 ml) with special neck and glass stoppers.
Identification: Each test flask was uniquely identified with study code, test group, days of measurement and replicate number.

TEST CONDITIONS
The test was carried out in an incubator and controlled environment room (during the formulation and oxygen measuring) at a temperature of 22 ± 2 °C according to guideline. The test flasks were placed into an incubator and kept at 22.0 - 22.9 °C in the dark. The temperature was measured daily during the experiment.
The oxygen concentration of test water was 8.4 mg/L at the start of the test.
The pH value of the test water was checked prior study start. The pH of the test water was 7.42.
The test conditions were measured with suitable instruments.

PREPARATION OF THE TEST SOLUTIONS
The components were applied in the following ratio in the test flasks:
The preparation of the respective test solutions with the test material was performed according to the following:
The respective amount of test material was weighed in directly to reach the required test item concentration of 2.2 mg/L.
During the performance of the test the test solutions were ultrasonicated under continuous cooling for < 10 min. to ensure a good dispersion.
The chosen test item concentration was based on the theoretical oxygen demand (ThODNO3): 2.79 mg O2/mg test item and on the performed 14-d preliminary test.

1.) Test Item (flasks 1a and 1b)
Based on the theoretical oxygen demand (ThODNO3) of 2.79 mg O2/mg test item, 7.6 mg of test material was thoroughly mixed into 3.46 litres of aqueous test medium (corresponding to 2.2 mg/L test item, respectively a ThODNO3 of about 6.138 mg O2/L).
2.) Procedure Control: Sodium benzoate (flasks 2a and 2b)
Based on the theoretical oxygen demand (ThODNH4) of Sodium benzoate (1.67 mg O2 per mg) (details on calculation are given in the guidelines), stock solution* corresponding to 12.096 mg of Sodium benzoate was mixed into 3.36 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThODNH4 of about 6.012 mg O2/L).
* The concentration of the stock solution was 360 mg/L.
3.) Inoculum Control (flasks 3a and 3b)
Only filtered inoculum was added to 3.40 litres of aqueous test medium.
4.) Toxicity Control (flasks 4a and 4b)
7.6 mg of test material and reference item stock solution* (34.6 mL) were mixed into 3.46 litres of aqueous test medium corresponding to 2.2 mg/L test item (ThODNO3 of 6.138 mg O2/L) and 3.6 mg/L reference item (ThODNH4 of 6.012 mg O2/L).
* The concentration of the reference item stock solution was: 360 mg/L.

Microbial inoculum (2.0 ml per litre) was added to each preparation bottle.

Preparation of Test Flasks
Sufficient number of BOD flasks was cleaned with 5 - 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing standing for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly closed with glass stopper.

Test Bottles
The number of test bottles was as follows:
10 bottles containing the test item and inoculum
10 bottles containing the reference item and inoculum (procedure control)
10 bottles containing only inoculum (inoculum control)
10 bottles containing the test item, reference item and inoculum (toxicity control)

Measurement of Oxygen
The incubation period of the closed bottle test was 28 days.
The oxygen concentrations were measured with oxygen meter with a stirring O2 electrode. Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28.

Measurement of Temperature
Temperature was measured continuously and registered on weekdays.

Calculation of BOD
The BOD (mg O2 per mg test item) expected after each period was calculated as follows:

(mg O2/L of T.i. and/or R. i. -mg O2/L of i.control) / (mg T.i. and/or R.i./L in flask)

where:
T.i. = test item
R.i. = reference item,
i.control = inoculum control

Calculation of Biodegradation %
The percentage biodegradation of the test item and the reference item was calculated as follows:
[BOD(mg O2/mg T.i. or R.i.)]/[ThODNH3(mg O2/mg T.i. or ThODNH4(mg O2/mg R.i.)]x100

where:
T.i. = test item
R.i. = reference item

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

21.1

Sampling time:

28 d

Details on results:

The test item was investigated for its ready biodegradability in a Closed Bottle Test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.
The test system was a microbial inoculum of good quality, collected from the sewage plant for domestic sewage. The sludge was washed by centrifugation filtered through cotton wool and aerated until use. Based on the determined dry weight content, the washed sludge suspension contained 4 g dry material per litre.
Under the test conditions the percentage biodegradation of the test material reached a mean of 21.1 % after 28 days based on its ThODNO3.
The test item can therefore be considered to be not ready biodegradable.
According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThODNO3.
The reference item Sodium benzoate was sufficiently degraded to a mean of 80.8 % after 14 days, and to a mean of 87.5 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used activated sludge inoculum.
In the toxicity control containing both the test item and the reference item Sodium benzoate, a mean of 38.3 % biodegradation was noted within 14 days and 40.2 % biodegradation after 28 days of incubation.
According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 2.2 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days.

Results with reference substance:

The reference item Sodium benzoate was sufficiently degraded to a mean of 80.8 % after 14 days, and to a mean of 87.5 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used activated sludge inoculum.

Table 1: Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment	Concentration [mg/L]	Flask No.	Percent of biodegradation after n days of exposure
			7	14	21	28
Test item	2.2	1a	1.6	26.0	19.5	22.8
		1b	0.00	22.8	17.9	19.5
		Mean	0.8	24.4	18.7	21.1
Reference item	3.6	2a	80.0	80.0	81.7	86.7
		2b	78.3	81.7	85.0	88.3
		Mean	79.2	80.8	83.3	87.5
Toxicity control	Test item: 2.2 Ref. item: 3.6	4a	35.6	38.6	38.6	41.0
		4b	36.3	37.9	37.1	39.4
		mean	35.9	38.8	37.9	40.2

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the test conditions the percentage biodegradation of the test material reached a mean of 21.1 % after 28 days based on its ThODNO3.
The test item can therefore be considered to be not ready biodegradable.
According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThODNO3.
The reference item Sodium benzoate was sufficiently degraded to a mean of 80.8 % after 14 days, and to a mean of 87.5 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used activated sludge inoculum.
In the toxicity control containing both the test item and the reference item Sodium benzoate, a mean of 38.3 % biodegradation was noted within 14 days and 40.2 % biodegradation after 28 days of incubation.
According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 2.2 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days.

Executive summary:

In an assessment of ready biodegradability study, closed bottle test (09/106-322AN), the test material was determined to not be readily biodegradable according to EU test method C.4-E given that the degradation rates calculated from ThOD values were less than 60 %.

Description of key information

In an assessment of ready biodegradability study using a closed bottle test method, the percentage biodegradation of the test material reached a mean of 21.1 % after 28 days based on its ThODNO3.

The test item can therefore be considered to be not ready biodegradable.

According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThODNO3.

The reference item Sodium benzoate was sufficiently degraded to a mean of 80.8 % after 14 days, and to a mean of 87.5 % after 28 days of incubation, based on ThODNH4, thus confirming the suitability of the used activated sludge inoculum.

In the toxicity control containing both the test item and the reference item Sodium benzoate, a mean of 38.3 % biodegradation was noted within 14 days and 40.2 % biodegradation after 28 days of incubation.

According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 2.2 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days.

Additional information

In an assessment of ready biodegradability study, closed bottle test (09/106-322AN), the test material was determined to not be readily biodegradable according to EU test method C.4-E given that the degradation rates calculated from ThOD values were less than 60 %.