Reaction mass of 2-methoxy-6-methylocta-1,5-diene and 2-methoxy-6-methylocta-2,5-diene

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 23 January 2018 and 20 February 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Deviations:

yes

Remarks:

The deviation is considered to have no effect on the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: PG-RAW-0004
Physical state/Appearance: clear colorless liquid
Storage Conditions: approximately 4 °C in the dark

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 January 2018 from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum
Upon receipt in the laboratory, the sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded).
In order to reduce the inorganic carbon (IC) content of the inoculum, the filtrate was sparged with CO2 free air* for approximately 1-Hour whilst maintaining its pH at 6.5 using concentrated orthophosphoric acid. After sparging, the pH was restored to its original value of 7.4 using 7 M sodium hydroxide and the inoculum allowed to settle for approximately 1 hour prior to removal of an aliquot (2 liters) of the supernatant for use in the test. The supernatant was maintained on aeration using CO2 free air until use.

Duration of test (contact time):

28 d

Initial conc.:

21 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Mineral Medium
The mineral medium used in this study was that recommended in the OECD and ISO Guidelines.
The deionized reverse osmosis water used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).

Experimental Design and Study Conduct
Preliminary Work
The test item was a poorly water soluble, volatile, non viscous liquid and hence following the recommendations of the International Standards Organisation (ISO 1995) for dealing with such compounds, for the purpose of the biodegradation test the test item was added directly to the test vessels using a high precision volumetric syringe. Using this method enabled relatively small amounts of test item to be accurately added to each test vessel.
Preliminary work conducted showed that a volume of 3.3 µL of test item injected into a test vessel using a gas tight micro syringe gave a measured weight of 2.7 mg, mean of 15 separate weighings.

Test Item Preparation
The test item was dispersed directly in mineral medium.
Aliquots (107 mL) of inoculated mineral medium were dispensed to each of 29 replicate test vessels and the test vessels sealed using Teflon lined silicon septa and aluminum crimp caps. After sealing, an aliquot (2.7 µL) of test item was injected through the septum of each vessel to give the required test concentration of 21.0 mg/L, equivalent to 16.35 mg carbon/L.
A test concentration of 20 mg carbon/L was instructed to be employed in the study following the recommendations of the test guidelines, however in error the test concentration employed was equivalent to 16.35 mg C/L. This was a deviation to the study plan but was considered to have no effect on the integrity of the study given that the Test Guidelines state that a test concentration of normally 20 mg C/L is employed and the degradation values for the test item and toxicity control vessels were re-calculated using the revised carbon concentration of 16.35 mg C/L for the test item vessels.

Reference Item Preparation
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 (137.2 mL) of this stock solution was dispersed with inoculum (400 mL) and mineral medium to a final volume 4 liters, to give a test concentration of 34.3 mg/L, equivalent to 20 mg carbon/L. Aliquots (107 mL) of the 34.3 mg/L test concentration were dispensed to each of 33 replicate test vessels and the vessels sealed using Teflon lined silicon septa and aluminum crimp caps.
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 study.
An aliquot (34.3 mL) of the reference item stock solution was dispersed with inoculum (100 mL) and mineral medium, final volume 1 liter, to give a reference item concentration of 34.3 mg/L, equivalent to 20 mg carbon/L. Aliquots (107 mL) of the 34.3 mg/L reference item concentration were dispensed to 9 replicate test vessels and each test vessel sealed using Teflon lined silicon septa and aluminum crimp caps. After sealing, an aliquot (2.7 L) of test item was injected through the septa of each vessel to give a test item concentration of 21.0 mg/L, equivalent to 16.35 mg carbon/L.
The final concentration in the toxicity control vessels was 21.0 mg test item/L plus 34.3 mg reference item/L, equivalent to 36.35 mg carbon/L.
All volumetric flasks were inverted several times to ensure homogeneity of the solution.

Preparation of Test System
The following test preparations were prepared and incubated in 125 mL glass Wheaton bottles (total volume when full 160 mL) each containing 107 mL of solution:
a) An inoculated control consisting of inoculated mineral medium, 33 replicate vessels.
b) The procedure control containing the reference item (sodium benzoate) in inoculated mineral medium, to give a final concentration of 20 mg carbon/L, 33 replicate vessels.
c) The test item in inoculated mineral medium, to give a final concentration of 16.35 mg carbon/L, 29 replicate vessels.
d) The test item plus the reference item in inoculated mineral medium, to give a final concentration of 36.35 mg carbon/L to act as a toxicity control, 9 replicate vessels.
Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.
Test media a to d were inoculated with the prepared inoculum at a final concentration of 100 mL/L.
Aliquots (107 mL) of the test media were dispensed into replicate vessels to give a headspace to liquid ratio of 1:2. Sufficient vessels were prepared to allow a single inorganic carbon determination per vessel with triplicate vessels for the inoculum control, procedure control, test item and toxicity control at each sampling occasion (five replicates for analysis on Day 28). Additional inoculum control and procedure control vessels were prepared to provide samples for Dissolved Organic Carbon (DOC) analyses on Days 0 and 28 (duplicate vessels per sampling occasion).
All vessels were sealed using Teflon lined silicon septa and aluminum crimp caps and incubated in darkness at 20 ±1 °C with constant shaking at approximately 125 rpm (INFORS Version Multitron® Incubator).

Reference substance:

benzoic acid, sodium salt

Remarks:

Test concentration of 34.3 mg/L

Test performance:

The mean TIC in the inoculum control vessels on Day 28 was 1.6 mg/L; equivalent to 9.8% of the organic carbon added initially as test item to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

46

Sampling time:

28 d

Details on results:

The test item attained 46% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310.
The toxicity control attained 59% biodegradation after 14 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro organisms used in the test.
Variation in the biodegradation rates obtained on different sampling days was considered to be the result of normal biological variation between the respiration rates of replicate vessels. Due to the sacrificial nature of the study design, the biodegradation rates obtained on each sampling occasion were for individual replicate vessels and not the result of cumulative biodegradation values determined from a single vessel sampled on numerous occasions and as such variation in biodegradation rates on different sampling days was to be expected.

Results with reference substance:

Sodium benzoate attained 83% biodegradation after 14 days and 87% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
DOC analyses conducted on samples taken from the reference item vessels on Days 0 and 28 showed that the replicate reference item vessels attained 96% biodegradation for each replicate vessel. The biodegradation rates for the reference item were higher than those determined by IC analyses. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to biodegradation and hence CO2 evolution occurring.

Inorganic Carbon Values on Each Analysis Occasion

Day	Inorganic Carbon (mg IC)
	Inoculum Control					Procedure Control					Test Item					Toxicity Control
	R1	R2	R3	R4	R5	R1	R2	R3	R4	R5	R1	R2	R3	R4	R5	R1	R2	R3	R4	R5
0	0.14	0.16	0.14	-	-	0.13	0.13	0.13	-	-	0.09	0.10	0.10	-	-	0.10	0.10	0.10	-	-
2	0.13	0.17	0.15	-	-	1.47	1.47	1.43	-	-	0.10	0.11	0.10	-	-	-	-	-	-	-
6	0.16	0.14	0.16	-	-	1.82	1.75	1.84	-	-	0.65	0.69	0.58	-	-	-	-	-	-	-
8	0.14	0.16	0.16	-	-	1.93	1.70	1.82	-	-	0.85	0.81	0.77	-	-	2.35	2.38	2.19	-	-
10	0.15	0.17	0.16	-	-	2.08	1.96	2.07	-	-	0.68	0.76	0.68	-	-	-	-	-	-	-
14	0.16	0.16	0.16	-	-	1.94	1.91	1.97	-	-	0.78	0.77	0.76	-	-	2.50	2.44	2.40	-	-
16	0.16	0.18	0.17	-	-	2.05	2.00	2.19	-	-	0.91	0.77	0.91	-	-	-	-	-	-	-
21	0.16	0.17	0.18	-	-	2.13	2.01	2.04	-	-	0.91	0.99	0.84	-	-	-	-	-	-	-
28	0.19	0.18	0.18	0.18	0.14	2.03	2.06	1.99	2.00	2.10	0.87	0.90	1.04	1.03	1.07	-	-	-	-	-

R   = Replicate

-    = No measurement made

Percentage Biodegradation Values

Day	Biodegradation (%)
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
2	61	0	-
6	77	28	-
8	78	38	56
10	88	31	-
14	83	35	59
16	89	39	-
21	88	42	-
28	87	46	-

-    = No measurement made

Dissolved Organic Carbon Values on Day 0 and Day 28

DOC Concentration
Test Vessel		Day 0			Day 28
		mg C/L	mg C/L Corrected for Mean Control Value	Nominal Carbon Content (%)	mg C/L	mg C/L Corrected for Mean Control Value	Biodegradation (%)
Inoculum Control	R1	1.36	-	-	1.33	-	-
	R2	1.34	-	-	1.03	-	-
Procedure Control	R1	21.23	19.88	99	2.07	0.89	96
	R2	21.61	20.26	101	2.03	0.85	96

R   = Replicate

-    = Not applicable

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Remarks:

The test item attained 46% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310.

Conclusions:

The substance showed PG-RAW-0004 biodegradation in an OECD TG 310 test and is considered to be not readily biodegradable.

Executive summary:

The ready biodegradability of PG-RAW-0004 was investigated in a study conducted in accordance with OECD TG 310 and GLP. The concentration tested was 21.0 mg/l test substance, with an activated sludge concentration of 16.35 mg/l. The test item attained 46% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310. Sodium benzoate attained 83% biodegradation after 14 days and 87% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Description of key information

 The ready biodegradability of PG-RAW-0004 was investigated in a study conducted in accordance with OECD TG 310 and GLP. The concentration tested was 21.0 mg/l test substance, with an activated sludge concentration of 16.35 mg/l. The test item attained 46% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310. Sodium benzoate attained 83% biodegradation after 14 days and 87% 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

Type of water:

freshwater