Reaction mass of methyl 2-({(E)-[4-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(E)-[3-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(Z)-[4-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(Z)-[3-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate

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:

Experimental start date 03 October 2017 Experimental completion date 31 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Kl1: The result is reliable and adequate for this purpose

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Lyrame
Batch: SM16066125
Purity: 77% (represents the sum of the typical concentrations of the four isomers as shown in Annex 2)
Physical state/Appearance: yellow viscous liquid
Expiry Date: 07 June 2019
Storage Conditions: Room temperature in the dark, under nitrogen from 20 January 2017 to 26 January 2017, thereafter approximately 4 ºC in the dark under nitrogen

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

A mixed population of sewage treatment micro organisms was obtained on 03 October 2017 from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Duration of test (contact time):

28 d

Initial conc.:

19.6 mg/L

Based on:

other: Carbon

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preparation of Inoculum
Upon receipt in the laboratory, the sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and the pH measured using a Hach HQ40d Flexi handheld meter to be 7.7.
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 7.7 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.

Mineral Medium
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 Solubility Work
Information provided by the Sponsor indicated that the water solubility of the test item was 1.28 mg/L. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation.

Test Item Preparation
Following preliminary solubility work and the recommendations of the International Standards Organisation (ISO, 1995) and in the published literature (Handley et al, 2002) the test item was dissolved in an auxiliary solvent prior to adsorption onto filter paper**.

A nominal amount of test item (1164 mg) was dissolved in 10 mL of acetone to give a 1164 mg/10 mL solvent stock solution. An aliquot (25 µL) of this solvent stock solution was dispensed onto a filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was added to inoculated mineral medium (107 mL) to give a final concentration of 27.2 mg/L, equivalent to 19.6 mg carbon/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution. The test vessels were then sealed using Teflon lined silicon septa and aluminum crimp caps.

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 19.6 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
19.6 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 with the aid of ultrasonication for approximately 10 minutes. An aliquot (171.5 mL) of this stock solution was dispersed with inoculum (500 mL) and mineral medium to a final volume 5 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.

Aliquots (107 mL) of the 34.3 mg/L reference item concentration were dispensed to 9 replicate test vessels.

An aliquot (25 µL) of the 1164 mg/10 mL test item solvent stock solution was dispensed separately on to 9 filter papers and the solvent allowed to evaporate to dryness for approximately 15 minutes.

The final concentration in the toxicity control vessels was 27.2 mg test item/L plus 34.3 mg reference item/L, equivalent to 39.6 mg carbon/L.

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, plus a filter paper, to give a final concentration of 19.6 mg carbon/L, 29 replicate vessels.
d) The test item plus the reference item in inoculated mineral medium, plus a filter paper*, to give a final concentration of 39.6 mg carbon/L to act as a toxicity control, 9 replicate vessels.

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 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 approximately 20°C with constant shaking at approximately
125 rpm (INFORS Version 2 Multitron® Incubator).

Reference substance:

other: Sodium benzoate

Test performance:

The toxicity control attained 44% 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.
Sodium benzoate attained 64% biodegradation after 14 days and 68% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

26

Sampling time:

28 d

Details on results:

The mean TIC in the inoculum control vessels on Day 28 was 1.35 mg/L; equivalent to 7% of the organic carbon added initially as test item to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.

The test item attained 26% biodegradation of the ThIC yield 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 44% 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.

Sodium benzoate attained 64% biodegradation after 14 days and 68% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

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.

DOC analyses conducted on samples taken from the reference item vessels on Days 0 and 28 (see Table 3) showed that the replicate reference item vessels attained 93% and 92% 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.

Results with reference substance:

Sodium benzoate attained 64% biodegradation after 14 days and 68% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Percentage Biodegradation Values

Day	% Biodegradation
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
2	43	0	-
6	57	6	-
8	61	15	39
10	60	14	-
14	64	20	44
16	64	21	-
21	65	20	-
28	68	26	-

-       =      No value determined

Validity criteria fulfilled:

yes

Remarks:

The mean TIC in the inoculum control vessels on Day 28 was 1.35 mg/L; equivalent to 7% of the organic carbon added initially as test item to the test vessels and therefore satisfied the validation criterion given in the Test Guideline.

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 26% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 310.

Executive summary:

The ready biodegradability of Lyrame was investigated in a study conducted in accordance with OECD TG 310 (CO2 Headspace Test) and GLP.

The concentration tested was 27.2 mg/L, equivalent to 19.6 mg carbon/L.

The test substance biodegrades for 26% and it was not toxic to the inoculum. (68% biodegradation of the reference substance).

Description of key information

The ready biodegradability of Lyrame was investigated in a study conducted in accordance with OECD TG 310 (CO2 Headspace Test) and GLP.

The concentration tested was 27.2 mg/L, equivalent to 19.6 mg carbon/L.

The test substance biodegrades for 26% and it was not toxic to the inoculum. (68% biodegradation of the reference substance).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater