2-(2,6-diethyl-4-methyl-phenyl)propanediamide

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:

17 October 2003 to 19 November 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: Buckland Sewage Treatment Works, Newton Abbot, Devon, UK
- Laboratory culture: The activated sludge was kept aerated at room temperature and the pH maintained at 7.0 ± 1.0.
- Preparation of inoculum for exposure: Five days prior to the test the activated sludge was centrifuged, washed and resuspended in the test medium and the solids concentration determined. This sludge was then diluted in medium, added to test bottles and stirred until required for use.
- Concentration of sludge: 30 mg/L

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: According to the OECD and EU guidelines and contained 85 mg of KH2PO4, 217.5 mg of K2HPO4, 334 mg of Na2HPO4.2H2O, 5 mg of NH4Cl, 22.5 mg of MgSO4.7H2O, 36.4 mg of CaCl2.2H2O, 0.25 mg of FeCl3.6H2O and 0.40 mg of EDTA (disodium salt) per litre of deionised water.
- Test temperature: 22 ± 2 °C
- pH: 7.4 ± 0.2
- pH adjusted: yes

TEST SYSTEM
- Culturing apparatus: dark glass 500 mL bottles
- Number of culture flasks/concentration: three for the test material, blank and toxic control, six for the reference substance, and one for the additional pH measurement.
- Measuring equipment: Manometric biochemical oxygen demand (BOD) apparatus
- Details of trap for CO2: Potassium hydroxide solution (KOH) placed in a sealed cup in the neck of each bottle.

OBSERVATIONS
- Frequency: Day 5, 9, 15, 20 and 28
- Method: Manometer readings

CONTROL AND BLANK SYSTEM
- Inoculum blank: A control set up with no test material or reference substance
- Toxicity control: Test material and the reference substance at nominal 100 and 200 mg/L respectively.

Reference substance:

acetic acid, sodium salt

Test performance:

The measured COD value of sodium acetate was 0.80 g O2 g/L of substance. The COD obtained for sodium acetate is high compared with its expected maximum oxygen demand of 0.68 g O2 g/L. A high measured COD makes degradation harder to achieve, despite this > 60 % biodegradation was still observed within the 10 day window. The sludge was considered sensitive enough and the test valid.

Parameter:

% degradation (O2 consumption)

Value:

< 5

Sampling time:

5 d

Parameter:

% degradation (O2 consumption)

Value:

< 5

Sampling time:

9 d

Parameter:

% degradation (O2 consumption)

Value:

< 5

Sampling time:

15 d

Parameter:

% degradation (O2 consumption)

Value:

< 5

Sampling time:

20 d

Parameter:

% degradation (O2 consumption)

Value:

< 5

Sampling time:

28 d

Details on results:

The BOD value for the test material after 28 days indicated negligible biodegradation (< 5 %), and therefore the test substance can be classified as not readily biodegradable.
The results obtained from the toxicity control bottles showed that the test material did not inhibit the biodegradation of sodium acetate, as the mean oxygen consumption in the toxicity control bottles was similar to the mean oxygen consumption of the sodium acetate.
At day 0 the pH of the controls and additional test material bottle were between 7.22 and 7.26. At the end of the 28 day test period, the pH values ranged from 7.12 to 7.18 in the inoculum blank bottles, from 7.13 to 7.29 in the sodium acetate bottles, from 7.26 to 7.30 in the test material bottles and from 7.41 to 7.31 in the toxicity control bottles.
Temperature measurements recorded in several of the test bottles on days 0 and 28 indicated the temperature was within the range 22 ± 2 °C.

Results with reference substance:

More than 60 % degradation was achieved within the 10 day window as expected for a biodegradable substance, confirming the activated sludge contained viable organisms.

Table 2: Biochemical oxygen demand results – inoculum blanks and reference substance

Bottle contents	Bottle number	BOD
		Day 5	Day 9	Day 15	Day 20	Day 28
Inoculum blank	11	4.0	6.0	5.5	5.0	4.0
	13	4.0	4.0	4.0	4.0	4.0
	17	3.0	5.5	6.0	6.0	6.0
Mean^a(mg O2/L)		3.5	5.0	5.0	5.0	4.5
Sodium acetate (200 mg/L)	4	90	90	90	90	75
	12	110	115	120	120	110
	14	100	110	105	100	85
	15	95	105	105	100	90
	20	100	100	100	100	75
	21	100	105	105	105	95
Sodium acetate (200 mg/L) - inoculum blank mean	4	87	85	85	85	71
	12	107	110	115	115	106
	14	97	105	100	95	81
	15	92	100	100	95	86
	20	97	95	95	95	71
	21	97	100	100	100	91
BOD^b(g O2 g/L)	4	0.44	0.43	0.43	0.43	0.36
	12	0.54	0.55	0.58	0.58	0.53
	14	0.49	0.53	0.50	0.48	0.41
	15	0.46	0.50	0.50	0.48	0.43
	20	0.49	0.48	0.48	0.48	0.36
	21	0.49	0.50	0.50	0.50	0.46
% Biodegradation (BOD/COD x 100)^c	4	55	54	54	54	45
	12	68	69	73	73	66
	14	61	66	63	60	51
	15	58	63	63	60	54
	20	61	60	60	60	45
	21	61	63	63	63	58
Mean % biodegradation		61	63	63	62	53
Manometer readings are quoted to the nearest 0.5 mg O2/L for the inoculum blanks and 5 mg O2/L for the reference and test substances. All mean values are quoted to the nearest 0.5 mg O2/L ^aMean inoclulm blank BOD is normally in the range 4 to 15 mg O2/L and less than 60 mg O2/L after 28 days for manometric respirometry tests carried out at this laboratory ^bBOD (g/O2 g/L test substance) = BOD blank corrected (mg O2/L)/test substance concentration (mg/L) ^cThe chemical oxygen demand (COD) of sodium acetate was measured as 0.80 g O2 g/L

 

Table 3: Biochemical oxygen demand results with the test material

Bottle contents	Bottle number	BOD
		Day 5	Day 9	Day 15	Day 20	Day 28
Test material (100 mg/L)	7	0	0	0	0	0
	8	0	0	0	0	0
	18	10	10	10	5	0
Test material (100 mg/L) - inoculum blank mean	7	<5	<5	<5	<5	<5
	8	<5	<5	<5	<5	<5
	18	7	5	5	<5	<5
BOD^a (g O2 g/L)	7	<0.05	<0.05	<0.05	<0.05	<0.05
	8	<0.05	<0.05	<0.05	<0.05	<0.05
	18	0.07	0.05	0.05	<0.05	<0.05
% Biodegradation (BOD/COD x 100)^b	7	<5	<5	<5	<5	<5
	8	<5	<5	<5	<5	<5
	18	<5	<5	<5	<5	<5
Mean % biodegradation		<5	<5	<5	<5	<5
Manometer readings are quoted to the nearest 0.5 mg O2/L for the inoculum blanks and 5 mg O2/L for the reference and test substances. All mean values are quoted to the nearest 0.5 mg O2/L. The mean of values including a 'less than' figure is calculated as the mean of those figures, expressed as a 'less than' figure. ^aBOD (g/O2 g/L test substance) = BOD blank corrected (mg O2/L)/test substance concentration (mg/L) ^bThe chemical oxygen demand (COD) of the test material was measured as 2.06 g O2 g/L

 

Table 4: Toxicity control BOD comparison

Contents	BOD (mg O2/L)
	Day 5	Day 9	Day 15	Day 20	Day 28
Toxicity control	85	90	90	90	70
	95	100	100	100	90
	100	110	110	110	110
Toxicity control - inoculum blank	82	85	85	85	66
	92	95	95	95	86
	97	105	105	105	106
Mean	90	95	95	95	86
Sodium acetate - inoculum blank	87	85	85	85	71
	107	110	115	115	106
	97	105	100	95	81
	92	100	100	95	86
	97	95	95	95	71
	97	100	100	100	91
Mean	96	99	99	98	84

 

Table 5: pH values in bottles

Bottle contents	Bottle number	pH
		Day 0	Day 28
Inoculum blank	11	7.23	7.18
	13	-	7.12
	17	-	7.15
200 mg/L sodium acetate	4	7.26	7.13
	12	-	7.15
	14	-	7.21
	15	-	7.20
	20	-	7.29
	21	-	7.20
100 mg/L test material	7	-	7.30
	8	-	7.26
	18	-	7.27
100 mg/L test material (toxic control)	2	-	7.15
	5	-	7.14
	16	-	7.31
100 mg/L test material (extra vessel pH only)	22	7.22	-

Validity criteria fulfilled:

yes

Remarks:

Although the values for the reference substance are higher than the validity criteria, examination of the data indicate this does not impact on the validity of the test.

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Under the conditions of the test, the BOD value for the test material after 28 days indicated negligible biodegradation (< 5 %) and therefore the test substance can be classified as not readily biodegradable. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.

Executive summary:

The ready biodegradability of the test material was determined in accordance with the standardised guidelines OCED 301 F and EU Method C.4-D by the measurement of biochemical oxygen demand (BOD) in a respirometric test. The BOD value for the test material after 28 days indicated negligible biodegradation (< 5 %), and therefore the test substance can be classified as not readily biodegradable.

The results obtained from the toxicity control bottles showed that the test material did not inhibit the biodegradation of sodium acetate, as the mean oxygen consumption in the toxicity control bottles was similar to the mean oxygen consumption of the sodium acetate.

At day 0 the pH of the controls and additional test material bottle were between 7.22 and 7.26. At the end of the 28 day test period, the pH values ranged from 7.12 to 7.18 in the inoculum blank bottles, from 7.13 to 7.29 in the sodium acetate bottles, from 7.26 to 7.30 in the test material bottles and from 7.41 to 7.31 in the toxicity control bottles. Temperature measurements recorded in several of the test bottles on days 0 and 28 indicated the temperature was within the range 22 ± 2 °C.

Description of key information

Not readily biodegradable (<5 % degradation after 28 days), OECD 301 F, EU method C.4-D, Maynard 2003

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The ready biodegradability of the test material was determined in accordance with the standardised guidelines OCED 301 F and EU Method C.4-D by the measurement of biochemical oxygen demand (BOD) in a respirometric test. The BOD value for the test material after 28 days indicated negligible biodegradation (<5%) at 22 °C, and therefore the test substance can be classified as not readily biodegradable.

The results obtained from the toxicity control bottles showed that the test material did not inhibit the biodegradation of sodium acetate, as the mean oxygen consumption in the toxicity control bottles was similar to the mean oxygen consumption of the sodium acetate. The available data is considered complete and the conclusion, not readily biodegradable, was taken forward for risk assessment.

The inherent biodegradation of the test substance was assessed in a guideline study according to GLP over 28 days in an inorganic salts medium inoculated with microorganisms collected from 10 sites in Nanjing district. The biodegradation was assessed based on the actual biological oxygen demand (BOD) compared to the theoretical oxygen demand (ThOD), and on residue analysis based on the measured amounts of the test item after 28 days. The BOD results showed that inherent biodegradation of the test substance was less than 1% after 28 days based on the calculation of the ThODNH3. Based on the residue analysis, inherent biodegradation of the test substance was 1.22% during the testing period. Thus, the study on inherent biodegradation confirmed that the substance is not readily biodegradable.