Diesters of alcohols, C7-9-iso-, C8-rich, 2-ethylhexyl and nonanedioic acid

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 May - 25 June 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions: Analytical purity not stated.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

adopted in 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

adopted in 1992

Deviations:

no

GLP compliance:

yes

Remarks:

with the exception that carbon analysis was subcontratacted and not performed under GLP conditions

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Fresh activated sludge was obtained from a municipal sewage treatment plant: "Waterschap de Maaskant",
's- Hertogenbosch, The Netherlands.
- Preparation of inoculum for exposure: The sludge was kept under continuous aeration upon receipt. Before use, the sludge was allowed to settle for at least 30 minutes and the liquid decanted for use as inoculum at the amount of 10 mL/L of mineral medium.
- Concentration of sludge: The concentration of suspended solids was 4.4 g/L in the concentrated sludge (information obtained from the sewage treatment plant).
- Initial cell/biomass concentration: From the supernatant of the sludge the heterotrophic microbial colony count was determined (28000 cells/mL) This was done on agar plates, which contained 1:1 purified agar (Oxoid 18 g/L) and nutrient broth (Oxoid 25 g/L). Since 10 mL supernatant was added per litre of mineral medium, the test system contained 280 cells/mL

Duration of test (contact time):

28 d

Initial conc.:

ca. 12 mg/L

Based on:

other: TC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
Stock solution A: 8.50 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4 x 12H2O, 0.50 g NH4Cl, dissolved in 1 L Milli-Q water, pH 7.4 +/- 0.2
Stock solution B: 22.50 g MgSO4 x 7H2O dissolved in 1L Milli-Q water
Stock solution C: 36.40 g CaCl2 x 2H2O dissolved in 1L Milli-Q water
Stock solution D: 0.25 g FeCl3 x 6H2O dissolved in 1L Milli-Q water
1L mineral medium contained: 10 mL of solution A, 1 mL of solutions B,C and D and Milli-Q water
- Test temperature: 20-22°C
- pH: blank control, 7.5-7.9; positive control, 7.6-8.3; toxicity control, 7.6-8.5 and test substance vessels, 7.6-7.9
- Other: After addition of 109 mL Milli-Q water to each weighing bottle containing the test substance (Replicate A: 33.3; Replicate B: 33.6 and toxicity control: 33.7 mg), followed by thorough mixing, the resulting suspensions were quantitatively added to the test media. The test solutions were continuously stirred during the test.

TEST SYSTEM
- Culturing apparatus: 2L all-glass brown coloured bottles
- Number of culture flasks/concentration: 2 replicates except for toxicity and positive controls (1)
- Method used to create aerobic conditions: CO2-free air was led through a bottle, containing 0.5-1L 0.0125 M Ba(OH)2 solution to trap CO2 which
may be present in small amounts. The CO2-free air was sparged through the solutions at a constant rate of approx. 1-2 bubbles per second (ca. 30-100 mL/min)
- Details of trap for CO2 and volatile organics if used: Three CO2 absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle. The CO2 produced in these test bottles reacted with the barium hydroxide in the gas scrubbing bottle and precipitated as barium carbonate. The amount of CO2 was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl.

SAMPLING
- Sampling frequency: Days 2, 5, 7, 9, 14, 19, 23, 27 and 29
- Sampling method: Each time the CO2-absorber nearest to the test bottle was removed for titration and each of the remaining two absorbers were
moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. On day 28, the pH of the test suspensions was measured and 1 mL of HCl added to each bottle. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Positive control: 1
- Toxicity control: 1

Reference substance:

acetic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

81.3

Sampling time:

28 d

Remarks on result:

other: average of two replicates (73.2 and 89.3%)

Details on results:

The test substance reached biodegradation values of 89.3% (replicate 1) and 73.2% (replicate 2) after a test period of 28 days. Furthermore,
degradation over 60% took place within the 10 d-window period. Therefore, the test substance can be judged as readily biodegradable. In the toxicity control more than 25% degradation occurred within 14 days. Therefore, the test substance is assumed to be not inhibitory.

Results with reference substance:

Sodium acetate reached a biodegradation value of 97.4% at the end of the test. Besides, degradation over 60% occurred within 14 days (86.1%).

Table 1. Biodegradation percentages for test substance, positive and toxicity controls

Day	% Test material degradation (Replicate 1)	% Test material degradation (Replicate 2)	% Degradation sodium acetate	% Degradation toxicity control
2	1.1	0	2.3	0
5	7.8	5.8	20.1	6.1
7	9.5	30	29.4	19.6
9	24.8	46.3	42.1	33.4
14	52.8	66.1	86.1	56.6
19	69.5	71.8	91.1	71.4
23	80.5	71.8	96.4	74.6
27	84.0	71.8	96.8	77.5
29	88.1	71.8	96.8	78.4
29	89.3	73.2	97.4	79.1
29	89.3	73.2	97.4	79.1

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

90 Sep 2014 - 27 Oct 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- A mixed population of activated sewage sludge micro-organisms was obtained on 23 September 2014 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- The activated sewage sludge sample was washed twice by settlement and resuspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 °C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.3 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

13.4 mg/L

Based on:

test mat.

Initial conc.:

10 mg/L

Based on:

other: carbon

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

- Test Item: Following the recommendations of the International Standards Organisation (ISO, 1995) and in the published literature (Handley et al, 2002) the test item was adsorbed onto silica gel prior to dispersion in mineral medium to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to the test organisms.
An amount of test item (40.2 mg) was adsorbed onto the surface of 500 mg of granular silica gel prior to dispersal in approximately 400 mL of mineral medium with the aid of high shear mixing (7500 rpm, 15 minutes). The test item/silica gel/mineral mineral medium dispersion was then dispersed in inoculated mineral medium and the volume adjusted to 3 liters to give a final concentration of 13.4 mg/L, equivalent to 10 mg carbon/L.
A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

- Reference Item: A reference item, sodium benzoate (C6H5COONa), 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. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium and the volume adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

- Toxicity Control: A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
An amount of test item (40.2 mg) was adsorbed onto the surface of 500 mg of granular silica gel prior to dispersal in approximately 400 mL of mineral medium with the aid of high shear mixing (7500 rpm, 15 minutes). The test item/silica gel/mineral medium dispersion was then dispersed in inoculated mineral medium and an aliquot (51.4 mL) of the sodium benzoate stock solution added. The volume was adjusted to 3 liters to give a final concentration of 13.4 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

- Preparation of Test System: The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:
a) An inoculated control, in duplicate, consisting of inoculated mineral medium plus 500 mg silica gel
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
Silica gel was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels. Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at 20 to 22 °C, in darkness. Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 39.1 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter. The pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2 free air. The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb ) granules. The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.
- Observations: The appearance of the test preparations was recorded on Days 0, 5, 12, 19 and 26
- pH Measurements: The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter or a Hach 160d handheld meter.

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

72

Sampling time:

28 d

Details on results:

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an decrease in all replicate vessels. This decrease was considered to be due to sampling/analytical variation. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
The test item attained 72% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

The toxicity control attained 70% biodegradation after 14 days and 83% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect an the sewage treatment micro-organisms used in the test.

Results with reference substance:

Sodium benzoate attained 86% biodegradation after 14 days and 96% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

 Table. Percentage Biodegradation Values

Day	% Biodegradation
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
1	41	1	27
5	72	33	53
8	90	55	76
10	97	63	79
14	86	72	70
21	98	76	86
28	99	75	87
29*	96	72	83

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

 

 Table. Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel	Total Carbon** (mg/L)	Inorganic Carbon* (mg/L)	IC Content (')/0 of TC)
Test Item 10 mg C/L R1	10.14	-0.02	0
Test Item 10 mg C/L R2	9.54	-0.02	0

R1 - R2 = Replicates 1 and 2

* Corrected for control values. Negative values are due to measured concentrations being less than control values

** Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item

 

Table. pH Values of the Test Preparations an Days 0 and 28

Test Vessel	pH
	Day 0 Pre-Adjustment	Day 0 Post-Adjustment	Day 28
Inoculum Control R1	7.8	7.6	7.5
Inoculum Control R2	7.8	7.6	7.5
Procedure Control R1	7.8	7.5	7.5
Procedure Control R2	7.8	7.5	7.5
Test Item R1	7.8	7.6	7.5
Test Item R2	7.8	7.6	7.5
Toxicity Control	7.8	7.5	7.5

 

 

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item attained 72% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Description of key information

In a CO2 evolution test C9-11 branched alcohols, C10 rich diesters with nonanedioic acid attained 72% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B (Roulstone 2014).

Bis(2-ethylhexyl) azelate reached biodegradation values of 89.3% (replicate 1) and 73.2% (replicate 2) after a test period of 28 days in a CO2 evolution test. Furthermore, degradation over 60% took place within the 10 d-window period. Therefore, this substance can be judged as readily biodegradable. In the toxicity control more than 25% degradation occurred within 14 days. Therefore, test substance is assumed to be not inhibitory towards sludge (Desmares 1998).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

The results of the biodegradation test with both analogue substances allow the conclusion that diesters of alcohols, C7-9-iso-, C8-rich, 2-ethylhexyl and nonanedioic acid is expected to readily biodegradable based on the presence of similar components (and starting materials) (see also document on read-across rationale).