Diallylamine

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:

The study was conducted between 11 November 2015 and 23 December 2015

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

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:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

A mixed population of sewage treatment micro-organisms was obtained on 23 November 2015 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum:
The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at 21 ± 1 ºC prior to use.

Mineral Medium:
The mineral medium used in this study was that recommended in the OECD Guidelines.

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Experimental Preparation:
Test item:
The test item was dissolved directly in mineral medium.

A nominal amount of test item (200 mg) was dissolved in mineral medium with the aid of ultrasonication for approximately 10 minutes and the volume adjusted to 1 liter to give a 200 mg/L stock solution. The pH of the 200 mg/L stock solution was 9.4 and adjusted to 7.5 using dilute HCl. The pH values were measured using a Hach HQ40d Flexi handheld meter. An aliquot (50 mL) of this stock solution was diluted with mineral medium (445 mL) and inoculum (5 mL) to give the final test concentration of 20 mg/L. The volumetric flasks containing the stock solution and the test concentration were inverted several times to ensure homogeneity.

A test concentration of 20 mg/L giving a Theoretical Oxygen Demand value of 65.8 mg O2/L was selected for use in the study following the recommended Test Guideline of at least 50-100 mg ThOD/L.

Reference Item:
A reference item, aniline (C6H5NH2), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium with the aid of ultrasonication for approximately 10 minutes. An aliquot (50 mL) of this stock solution was diluted with mineral medium (445 mL) and the inoculum (5 mL), to give the test concentration of 100 mg/L. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity.

Toxicity Control:
A toxicity control, containing the test item and aniline, was prepared in order to assess any toxic effect of the test item on the sewage treatment micro-organisms used in the test.

An aliquot (50 mL) of the 200 mg/L test item stock solution and an aliquot (50 mL) of the 1000 mg/L aniline stock solution was diluted with mineral medium (395 mL) and inoculum (5 mL) to give the test concentration of 20 mg test item/L and 100 mg aniline/L.

Preparation of Test System:
The following test preparations were prepared and inoculated in 500 mL bottles:

a) Three replicate bottles containing inoculated mineral medium to act as the inoculum control.
b) Two replicate bottles containing inoculated mineral medium and the reference item, aniline, at a concentration of 100 mg/L.
c) Three replicate bottles containing inoculated mineral medium and the test item at a concentration of 20 mg/L.
d) Two replicate bottles containing inoculated mineral medium, the reference item, aniline, at a concentration of 100 mg/L and the test item at a concentration of 20 mg/L to act as toxicity control vessels.

All vessels were inoculated with the prepared inoculum at a rate of 1% v/v.

On Day 0 the test item and reference item were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter prior to the addition of the inoculum and the volume in all the vessels being adjusted to 500 mL by the addition of mineral medium.

In order to confirm that the aniline and test item stock solutions were prepared correctly, a diluted, 100 mg/L stock solution (in reverse osmosis water) was also sampled for DOC analysis.

All inoculum control, test item, procedure control and toxicity control vessels were placed in a CES Multi-Channel Aerobic Respirometer.

The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the test with a magnetically coupled stirrer.

As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into the ethanolamine solution (50% v/v) causing a net reduction in gas pressure within the sample flask. The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data generated from the respirometer’s own battery backed memory was collected on the hard disk drive of a non-dedicated computer.

The test was conducted in diffuse light at temperatures of between 22 and 23 ºC.

On Day 28, two inoculum control, one procedure control, two test item and one toxicity control vessel were sampled for pH and analysis.

The remaining vessels which were not sampled were discarded and are not reported. Additional replicate vessels were prepared and incubated in order that in the event of a leak in the test system a replicate vessel could be discarded without jeopardizing the integrity of the test.


Evaluations:
Oxygen Consumption Measurements:
The daily Biological Oxygen Demand (BOD) values for the inoculum control, test item, procedure control and the toxicity control were measured and recorded.

Physico-chemical Measurements:
The temperature of the water bath was recorded daily.

pH Measurements:
In order to confirm whether the pH of the test preparations changed, the pH was measured using a Hach HQ40d Flexi handheld meter on Days 0 and 28

Data Evaluation:
Calculation of Theoretical Oxygen Demand:
ThOD was calculated based on the test items molecular formula.

In the assessment of the biodegradability of a test item errors can occur if the oxygen consumption due to nitrification is not taken into account. For test items that do not contain nitrogen the errors due to not considering nitrification are marginal, even if oxidation of the ammonium-nitrogen in the mineral medium occurs erratically between the test and control vessels. However for test items containing nitrogen errors can arise if the observed oxygen consumption is not corrected for the amount of oxygen used in oxidising ammonium to nitrite and nitrate.

As the test item attained less than 10% degradation at the end of the study it was not necessary to conduct nitrate/nitrite analysis.

Percentage Biodegradation:
The percentage biodegradation in terms of oxygen consumption was calculated as follows:

% degradation = (BOD - B / ThOD) x 100

Where:

BOD = Biological Oxygen Demand of the test item or reference item (mgO2/L)
B = Oxygen consumption in basal mineral medium to which inoculum is added (control) (mgO2/L)
ThOD = Theoretical oxygen demand to completely oxidize the reference and/or test item (mgO2/L)

Reference substance:

aniline

Test performance:

The mean BOD of the inoculated mineral medium (control) was 30.20 mg O2/L after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The pH of the inoculated test item vessels on Day 28 were 7.6 and hence satisfied the validation criterion given in the OECD Test Guidelines.

The difference between extremes of replicate BOD values at the end of the test was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.

Parameter:

% degradation (O2 consumption)

Value:

6

Sampling time:

28 d

Remarks on result:

other: not readily biodegradable

Details on results:

Daily BOD values for the test item, procedure control, toxicity control and inoculum control vessels are given in Table 1. Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2. The pH values of each individual vessel on Days 0 and 28 are given in Table 3.

The test item attained 6% biodegradation after 28 days, calculated from the oxygen consumption values, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

The toxicity control attained 55% biodegradation after 14 days and 56% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Results with reference substance:

Aniline (procedure control) attained 69% biodegradation after 14 days and 78% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Table1     Biological Oxygen Demand (BOD) Values

Day	BOD (mg O2/L)
	Inoculum Control		Procedure Control	Test Item		Toxicity Control
	R1	R2		R1	R2
0	0.00	0.00	0.00	0.00	0.00	0.00
1	0.08	0.04	0.20	0.00	0.00	0.00
2	1.04	1.08	1.20	1.46	0.00	0.00
3	4.28	4.04	6.38	4.24	2.38	3.24
4	6.04	5.24	61.14	5.88	3.24	72.68
5	7.92	7.04	117.46	7.78	4.46	158.86
6	8.92	7.96	164.02	8.92	5.04	177.10
7	10.16	9.24	191.64	10.58	7.04	183.60
8	10.74	10.28	201.00	11.88	8.50	188.50
9	12.04	11.54	207.62	13.42	10.08	203.16
10	13.16	12.58	212.96	14.74	11.42	213.00
11	14.28	13.62	217.58	16.28	12.54	217.12
12	15.58	14.74	221.74	17.90	13.78	221.12
13	17.04	16.12	226.58	19.46	15.20	223.50
14	19.08	18.12	232.82	21.90	17.24	226.24
15	19.32	18.70	238.32	22.28	18.58	227.40
16	20.16	19.74	242.74	23.12	19.86	229.20
17	21.20	20.82	246.20	24.04	21.24	230.82
18	22.16	21.62	249.56	24.90	22.20	231.86
19	22.86	22.32	252.06	25.70	23.16	232.54
20	23.70	23.12	254.32	26.62	24.04	233.20
21	24.54	23.94	256.44	27.86	25.28	233.86
22	25.66	24.94	258.82	29.48	26.66	234.94
23	26.62	25.82	261.10	30.70	27.66	235.82
24	27.36	26.54	263.02	31.58	28.48	236.36
25	28.32	27.32	265.10	32.52	29.44	237.08
26	29.48	28.36	267.06	33.94	30.70	237.94
27	30.24	29.08	268.60	34.90	31.82	238.52
28	30.82	29.58	269.76	35.56	32.52	238.98

 

R₁– R₂= Replicates 1 and 2

Table2     Percentage Biodegradation Values

Day	Procedure Control	Test Item			Toxicity Control
		R1	R2	Mean
0	0	0	0	0	0
1	0	0	0	0	0
2	0	1	-2	0	0
3	1	0	-3	0	0
4	18	0	-4	0	18
5	36	0	-5	0	40
6	50	1	-5	0	45
7	59	1	-4	0	46
8	62	2	-3	0	47
9	63	2	-3	0	51
10	65	3	-2	1	53
11	66	4	-2	1	54
12	67	4	-2	1	55
13	68	4	-2	1	55
14	69	5	-2	2	55
15	71	5	-1	2	56
16	72	5	0	3	56
17	73	5	0	3	56
18	74	5	0	3	56
19	74	5	1	3	56
20	75	5	1	3	56
21	75	6	2	4	56
22	76	6	2	4	56
23	76	7	2	5	56
24	76	7	2	5	56
25	77	7	2	5	56
26	77	8	3	6	56
27	77	8	3	6	56
28	78	8	4	6	56

 

R₁– R₂= Replicates 1 and 2

Table3     pH Values of the Test Preparationson Days 0 and 28

 

Test Vessel	pH
	Day 0	Day 28
Inoculum Control R1	7.4	7.7
Inoculum Control R2	7.4	7.7
Procedure Control	7.4	7.9
Test Item R1	7.5	7.6
Test Item R2	7.5	7.6
Toxicity Control	7.5	7.7

 R₁– R₂    =      Replicates 1 and 2

 

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 6% biodegradation after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Executive summary:

Introduction:

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301F, “Ready Biodegradability; Manometric Respirometry Test” referenced as method C.4-D of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (q)).

Methods:

The test item at a concentration of 20 mg/L was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at temperatures of between 22 and 23 ºC for 28 days. 

 

The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values on Days 0 to 28. Control solutions with inoculum and the reference item, aniline, together with a toxicity control were used for validation purposes.

Results…….

The test item attained 6% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Description of key information

Introduction:

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301F, “Ready Biodegradability; Manometric Respirometry Test” referenced as method C.4-D of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (q)).

Methods:

The test item at a concentration of 20 mg/L was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at temperatures of between 22 and 23 ºC for 28 days. 

 

The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values on Days 0 to 28. Control solutions with inoculum and the reference item, aniline, together with a toxicity control were used for validation purposes.

Results…….

The test item attained 6% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

The toxicity control attained 55% biodegradation after 14 days and 56% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

 

Aniline (procedure control) attained 69% biodegradation after 14 days and 78% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed