Reaction mass of stereoisomers of 4-METHYLENE-2-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-TETRAHYDRO-PYRAN and; 4-METHYL-6-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-3,6-DIHYDRO-2H-PYRAN and; 4-METHYL-2-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-3,6-DIHYDRO-2H-PYRAN

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:

supporting study

Study period:

11 April to 15 June 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study was performed according to EU Method C.4-E with GLP compliance, and is considered, in a first step, reliable with restrictions for the following reasons: - The substance is adequately identified, but some data on composition is missing. - Chloroform was used as solvent in this study, which could have been avoided considering the sufficiently high water solubility of the substance and the concentration used in this study. - The consumption of oxygen was increased in the solvent vessel compared to the blank vessels: 1,42 mg/L at the end of the test, which is close to the limit value for acceptance at 1.5 mg/L. And a slight increase compared to the test substance which is ambiguous. - The oxygen uptake in the test vessels was corrected due to the presence of chloroform. If not corrected, ca. 12% biodegradation was calculated by the assessor after 28 days.  - Regarding the analytical measurement, ca. 40% (39.7% exactly) of the test substance could not be recovered after 28 days. No explanation for this is given in the study report.

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

26 April 2007

Specific details on test material used for the study:

- Water solubilityy = 4,2 mg/L
- ThOD = 2,98 g/g

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

The inoculum was derived from surface waters collected in the near area to Pau, France receiving little but predominantly domestic sewage.
The freshly collected sample of surface water was previously pre-conditioned to the experimental conditions under aerobic conditions for 6 days in the mineral medium at the test temperature (20 +/- 1 °C). The pre-conditioned inoculum was further mixed with mineral medium at a level of 2 mL/L of medium. The suspended solids were measured and represented 5.7 µg/L.

Duration of test (contact time):

28 d

Initial conc.:

4.97 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Stock solutions and preparation of mineral medium as detailed in the OECD Guideline 301.
The mineral medium was strongly aerated for at least 20 minutes, and then allowed to stand for approximately 20 h at the test temperature before use.

TEST DESIGN
Parallel groups of 250 mL BOD bottles were prepared of sufficient number to allow assessments every 3-4 days throughout the 28-day incubation period for each of the following experimental series:
- Blank group: mineral medium alone with inoculum (duplicate determinations);
- Solvent group: mineral medium with the same amount of chloroform as for the introduction of the test substance, with inoculum (single determination);
- Test group: 2-5 mg/L test solutuon in mineral medium with inoculum (duplicate determinations);
- Reference group: 2-5 mg/L aniline solution in mineral medium with inoculum (duplicate determinations);
- Toxic control group: solution containing 2-5 mg/L of each the test substance and aniline in mineral method, with inoculum (single determination).

PREPARATION OF THE BOTTLES
The 250 mL BOD bottles were thoroughly cleaned before use using 5-10 mL of a wash solution (2.5 g iodine plus 12.5 g potassium iodide per litre of 1% w/v sulphuric acid). The bottles were shaken to coat the bottle walls and left to stand for 15 min. The wash solution was poured off and the bottles were thoroughly rinsed with tap water and finally demineralised water.

PREPARATION OF THE EXPERIMENTAL SOLUTIONS
The solubility of the test substance in water is low (4.2 mg/L) and chloroform was used as solvent for the preparation of the test solution. For that purpose, a stock solution was prepared using 0.7735 g of test substance for 10 mL of chloroform. The test concentration (4.97 mg/L) was achieved using 20 µL of the stock solution for 300 mL of mineral medium.
The test substance is a volatile substance and care was taken to avoid undue agitation of the test solution before the BOD bottles were stoppered. For that purpose, the BOD bottles were individually prepared: 250 mL of mineral medium were added with 20 µL of the test substance stock solution and stirred for a few seconds. The volume was added with 50 mL of mineral medium and 0.6 mL of preconditioned medium, resulting in a 4.97 mg/L solution.
The pH of the resulting solution was measured at 7.42.
The other groups were prepared in the same way.
In every case, care was taken to avoid undue appearance of air bubbles within the aqueous volumes. The bottles were kept open for approximately 30 minutes and then stoppered (each BOD bottle was fitted with a glass stopper and incubated at dark at 20 +/- 1 °C).

Reference substance:

aniline

Preliminary study:

No data

Test performance:

The test was performed using a 4.97 mg/L test solution.
The test was terminated after 28 days of incubation.
The test was considered as valid on the basis of the following fulfilled conditions:
- Mean oxygen uptake in the blank vessels < 1.5 mg/L at the end of the test;
- The residual concentration of oxygen in the test vessels did not fall below 0.5 mg/L at any time;
- Differences of extremes of replicate values of the removal of the test item was less than 20% at the end of the test;
- Percentage biodegradation of the reference compound (aniline) had reached the pass-level by day 14;
- In the toxic control series, more than 25% biodegradation of aniline had occured on day 14.

Key result

Parameter:

% degradation (O2 consumption)

Value:

2.74

Sampling time:

28 d

Remarks on result:

other: Not readily biodegradable

Parameter:

% degradation (test mat. analysis)

Value:

39.7

Sampling time:

28 d

Remarks on result:

other: Not readily biodegradable

Details on results:

OXYGEN UPTAKE AND RELATED BIODEGRADATION:
See tables 5.2.1/1, /2 and /3 in "Any other information on results incl. tables".
Mean oxygen uptake in the blank vessels was 0.75 mg/L at the end of the test. The consumption of oxygen was slightly higher in the solvent vessel (1.4 mg/L at the end of the test) than that in the standard control.
As a consequence, the oxygen uptake in the test vessels was corrected so that the consumption of oxygen due to the presence of chloroform was excluded:
Corrected oxygen uptake Test = Oxygen uptake in the test vessel - Oxygen uptake in the solvent control vessel
Oxygen uptake in the toxic reference vessels was corrected so that the BOD of Aniline was solely accounted for:
Corrected oxygen uptake Aniline = Oxygen uptake in the Aniline vessel - Mean oxygen updake in the test vessels

The biodegradation of the test substance remained almost zero throughout the test period. Mean biodegradation of the reference substance, Aniline, was 74% at the end of test, and the pass-level for 60% biodegradation was reached on day 10 of the incubation period. In the toxic control series, the biodegradation of Aniline had reached 47% on day 14 and was thus higher than the threshold value of 25%: the test substance was therefore not considered as inhibitory.

ACTIVE INGREDIENT CONTENT:
The measured initial concentration of the test substance represented 96 - 103% of the nominal value (4.97 mg/L). The treatment application was considered as valid. At the end of the test, 60% of the initial concentration was recovered in the test vessels. The reason why the test substance was not fully recovered is not reported. See table 5.2.1/4 in "Any other information on results incl. tables".

Results with reference substance:

See tables 5.2.1/1, /2 and /3 in "Any other information on results incl. tables".

Table 5.2.1/1: Time evolution of dissolved oxygen in the reacting vessel

Days	Blank		Solvent	Test		Toxic	Aniline
	Rep. 1	Rep. 2		Rep. 1	Rep. 2		Rep. 1	Rep. 2
0 3 7 10 14 17 21 24 28	9.06 8.90 8.61 8.61 8.61 8.66 8.48 8.36 8.28	9.09 8.82 8.68 8.59 8.66 8.51 8.42 8.37 8.37	9.04 7.84 7.51 7.60 7.56 7.88 7.81 7.87 7.62	9.11 8.24 7.82 7.60 7.38 7.05 7.07 6.87 6.60	9.01 8.18 7.57 7.33 7.32 6.86 6.91 7.07 6.37	9.03 8.14 7.81 4.02 4.57 3.55 3.29 4.44 4.06	9.09 8.78 5.67 5.17 5.25 4.45 4.35 4.20 4.76	9.04 8.79 5.97 5.07 5.12 5.02 3.17 3.81 4.52

Table 5.2.1/2: Measured oxygen uptake (mg/L) throughout the 28 -day incubation period

Days	Blank	Solvent	Test		Toxic	Aniline		Corrected Test*		Corrected Toxic**
	Mean		Rep. 1	Rep. 2		Rep. 1	Rep. 2	Rep. 1	Rep. 2
3 7 10 14 17 21 24 28	0.22 0.43 0.48 0.44 0.49 0.63 0.71 0.75	1.20 1.53 1.44 1.48 1.16 1.23 1.17 1.42	0.87 1.29 1.51 1.73 2.06 2.04 2.24 2.51	0.83 1.44 1.68 1.69 2.25 2.10 1.94 2.64	0.89 1.22 5.01 4.46 5.48 5.74 4.59 4.97	0.31 3.42 3.92 3.84 4.64 4.74 4.89 4.33	0.25 3.07 3.97 3.92 4.02 5.87 5.23 4.52	-0.33 -0.24 0.07 0.25 0.90 0.81 1.07 1.09	-0.37 -0.09 0.24 0.21 0.99 0.87 0.77 1.22	0.04 -0.15 3.42 2.75 3.38 3.67 2.50 2.40

* Oxygen uptake in the test vessel minus oxygen uptake in the solvent vessel

** Oxygen uptake in the toxic reference vessel minus mean oxygen uptake in the test vessels.

Table 5.2.1/3: Measured percent biodegradation

Days	Test*			Toxic*	Aniline
	Rep. 1	Rep. 2	Mean		Rep. 1	Rep. 2	Mean
0 3 7 10 14 17 21 24 28	0 -3.69 -4.53 -2.74 -1.29 2.77 1.25 2.44 2.30	0 -3.96 -3.52 -1.59 -1.56 3.38 1.66 0.41 3.18	0 -3.82 -4.02 -2.16 -1.42 3.08 1.45 1.42 2.74	0 -3.53 -11.60 59.32 46.61 58.21 61.44 36.11 33.19	0 1.92 60.33 69.51 68.60 83.73 83.02 84.34 72.23	0 0.71 53.26 70.51 70.21 71.22 105.82 91.20 76.06	0 1.31 56.80 70.01 69.41 77.48 94.42 87.77 74.15

* Based on corrected values for oxygen uptake.

Table 5.2.1/4: Measured concentrations of the test substance

	Replicate samples	Replicate injections	Test item (mg/L)	% recovery
Test initiation	1	1 2	4.78 4.78	96.2% 96.2%
	2	1 2	5.10 5.11	102.8% 103.0%
	Mean		4.94	99.6%
End of test	1	1 2	3.05 3.14	61.3% 63.1%
	2	1 2	2.84 2.88	57.2% 58.0%
	Mean		2.98	59.9%

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

No or little mineralisation of the test substance occurred during the 28 days of testing (mean of 2.74% after 28 days). Based on analytic assessment, recovery of the test substance at the end of test was approximately 60%. The conclusion was that the test substance is not easily biodegradable.

Executive summary:

This study was performed according to EU Method C.4-E with GLP compliance, to determine the biodegradability of the test substance using the Closed Bottle Test.

The test substance (prepared with chloroform as a solvent and which remained in the study after initiation) was put in contact with surface water collected from a river at a nominal concentration of 4.97 mg/L (14.80 mg ThOD/L) with culture medium in closed bottles in the dark at 20 +/- 1°C for 28 days. The degradation was followed by analysis of dissolved oxygen over a 28-day period. The amount of oxygen taken up by the microbial population during biodegradation of the test substance, corrected for uptake by the blank inoculum run in parallel, was expressed as a percentage of the theorical oxygen demand (ThOD). Additional analytical assessments were performed at test initiation and at test completion in the test vessels for quantification of the test item, so as to determine the primary degradation throughout the test period.

The biodegradation of the test substance remained almost zero throughout the test period (mean of 2.74% after 28 days). Mean biodegradation of the reference substance (Aniline) was 74% at the end of test, and the pass-level for 60% biodegradation was reached on day 10 of the incubation period. In the toxic control series, the biodegradation of Aniline had reached 47% on day 14 and was thus higher than the threshold value of 25%: the test substance was not considered as inhibitory by the Study Director. Nevertheless, basal respiration in the test solutions containing test substance (and chloroform) was observed to be lower than the control (containing chloroform) until day 14 of the study, suggesting ambiguity on this point.

Regarding the analytical assessment, the measured initial concentration represented 96 -103% of the nominal value (4.97 mg/L). The treatment application was considered as valid. At the end of the test, 60% of the initial concentration was still recovered in the test vessels, so ca. 40% of the substance was not recovered. One probable explanation for this is that primary degradation occurred. This would suggest that the parent substance can be considered as not persistent in the environment.

This study is considered valid with restrictions for the following reasons:

- The substance is adequately identified, but some data on composition is missing.

- Chloroform was used as solvent in this study, which could have been avoided considering the sufficiently high water solubility of the substance and the concentration used in this study.

- The consumption of oxygen was increased in the solvent vessel compared to the blank vessels: 1,42 mg/L at the end of the test, which is close to the limit value for acceptance at 1.5 mg/L. And a slight increase compared to the test substance which is ambiguous.

- The oxygen uptake in the test vessels was corrected due to the presence of chloroform. If not corrected, ca. 12% biodegradation was calculated by the assessor after 28 days.

- Regarding the analytical measurement, ca. 40% (39.7% exactly) of the test substance could not be recovered after 28 days. No explanation for this is given in the study report.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 04 July 2019 to 05 November 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The substance is adequately identified, but some data on composition is missing, so validation applies with restrictions. On Day 55 of the test the temperature in the water bath was recorded as being 19.4 ºC. This was a deviation from the Study Plan which states the test will be conducted at a temperature of between 20 to 24 °C with a maximum deviation of ± 1ºC. With the exception of Day 55, the test was run at temperatures of between 20 and 21 °C.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

On Day 55 of the test the temperature in the water bath was recorded as being 19.4 ºC, so not within the range 20 to 24°C with a maximum deviation of ± 1ºC. With the exception of Day 55, the test was run at temperatures of between 20 and 21 °C.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

On Day 55 of the test the temperature in the water bath was recorded as being 19.4 ºC, so not within the range 20 to 24°C with a maximum deviation of ± 1ºC. With the exception of Day 55, the test was run at temperatures of between 20 and 21 °C.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 21/08/2018; Date of issue: 19/11/2018

Specific details on test material used for the study:

- Physical state/Appearance: Pale yellow liquid
- Storage conditions: Approximately 4°C, in the dark
- Molecular Formula: C15H24O
- Molecular Weight: 220 g/mol
- Water Solubility: 44.4 mg/L at 25 ºC (iSafeRat ® v1.8)
- Vapour Pressure: 0.313 Pa at 20 ºC (OECD 104/EEC A4, effusion method, Laus 2011)
- Log Kow: 3.75 at 20 °C (EEC A8, Shake-Flask Method, Phytosafe 2009)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of sewage treatment micro-organisms was obtained on 02 September 2019 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 for exposure: 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 temperatures of approximately 21°C prior to use.

Duration of test (contact time):

60 d

Initial conc.:

100 mg/L

Based on:

test mat.

Initial conc.:

298 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

The BOD values for the inoculum control, test item, procedure control and the toxicity control were measured daily.

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral medium used in this study was that recommended in the OECD 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).

TEST SYSTEM
The following test preparations were prepared and inoculated in 500 mL amber glass bottles:
a) Five replicate bottles containing inoculated mineral medium to act as the inoculum control.
b) Two replicate bottles containing inoculated mineral medium and the reference item, sodium benzoate, at a concentration of 100 mg/L to act as the procedure control.
c) Five replicate bottles containing inoculated mineral medium and the test item at a concentration of 100 mg/L.
d) Two replicate bottles containing the test item at a concentration of 100 mg/L in inoculated mineral medium plus the reference item, sodium benzoate, at a concentration of 100 mg/L to act as toxicity control vessels.
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 study.
All vessels were inoculated with the prepared inoculum at a rate of 1% v/v.
On Day 0, the test and reference items were added to the mineral medium and the pH of all vessels was measured using a Hach HQ40d Flexi handheld meter prior to the addition of inoculum and adjusting to the final volume of 500 mL with mineral medium.
On Day 0, two inoculum control and test item vessels were sampled for compound specific analysis. In order to confirm that the sodium benzoate stock solution was prepared correctly, a diluted 100 mg/L stock solution (in reverse osmosis water) was also sampled for Total Organic Carbon (TOC) analysis.
All remaining 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 19 and 21 °C.
From those samples prepared, on Day 60, two inoculum controls, one procedure control, two test item and one toxicity control vessel were sampled for compound specific analysis and/or pH analysis. As none of the vessels showed errorenous results, the first vessel(s) in each
series were selected for reporting.
The remaining inoculum control and test item vessels which were not sampled were stored frozen for further analysis if required. The other vessels were discarded.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes

TEST ITEM PREPARATION
A nominal amount of test item (50 mg) was dispersed in mineral medium (350 mL) and subjected to high shear mixing (approximately 7500 rpm, 15 minutes) prior to allowing to cool to temperatures of between 21 and 22 °C and measurement of the pH values using a Hach HQ40d Flexi handheld meter and the addition of inoculum (5 mL). The volume was adjusted to 500 mL with mineral medium to give the test concentration of 100 mg/L. The inoculum control vessels were prepared by inoculating mineral medium (495 mL) with inoculum (5 mL). The pH values were measured using a Hach HQ40d Flexi handheld meter prior to the addition of inoculum. A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guidelines.

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 a nominal amount of the reference item (500 mg) directly in mineral medium (500 mL). An aliquot (50 mL) of
this stock solution was diluted with mineral medium (350 mL) prior to measurement of the pH value and then addition of inoculum (5 mL) and adjusting to a final volume of 500 mL with mineral medium, to give the test concentration of 100 mg/L. The volumetric flask
containing the stock solution was inverted several times to ensure homogeneity. The pH of the reference item stock solution was 7.4. The pH value was measured using a Hach HQ40d Flexi handheld meter.

TOXICITY CONTROL PREPARATION
A nominal amount of test item (50 mg) was dispersed in mineral medium (350 mL) and subjected to high shear mixing (approximately 7500 rpm, 15 minutes) prior to allowing to cool to temperatures of approximately 21 °C and then the addition of an aliquot (50 mL) of the 1000 mg/L aniline stock solution (see Section 3.6.3) and measurement of the pH value using a Hach HQ40d Flexi handheld meter. The inoculum (5 mL) was then added prior to adjusting to a final volume of 500 mL with mineral medium to give the test concentration of 100 mg test item/L and 100 mg aniline/L.

Reference substance:

benzoic acid, sodium salt

Remarks:

Purity >99.5%; Batch SLBT3039

Preliminary study:

Information provided by the Sponsor indicated that the water solubility of the test item was 44.4 mg/L at 25 ºC (iSafeRat ® v1.8). Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation. From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO 10634, 1995) it was concluded that the best testable dispersion was found to be obtained when using the high shear mixing method of preparation.

Key result

Parameter:

% degradation (O2 consumption)

Value:

2

Sampling time:

28 d

Remarks on result:

other: Not readily biodegradable

Key result

Parameter:

% degradation (O2 consumption)

Value:

3

Sampling time:

60 d

Details on results:

The test item attained 2% biodegradation after 28 days and 3% biodegradation after 60 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.
Chemical analysis of the 100 mg/L test preparations containing inoculum at 0 hours showed measured concentrations of 90 and 86% of nominal were obtained. A decline in measured test concentration was observed on Day 60 to 5% of nominal for both replicates, (calculated to be a 95 % loss over the test duration assuming 100% recovery on Day 0).
The losses observed by chemical analysis were higher than those observed by oxygen consumption. An hypothesis would be the result of a primary biodegradation of the test item. Indeed, insertion of one oxygen atom between a carbon and hydrogen atom into all the isomers in the test item would result in 2 to 3% biodegradation and result in a total loss of the parent compound.

The toxicity control attained 29% biodegradation after 14 days, 31% biodegradation after 28 days and 33% biodegradation after 60 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Results with reference substance:

TOC of the diluted sodium benzoate stock solution confirmed that it had been prepared correctly.
Sodium benzoate (procedure control) attained 86% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 96% biodegradation was attained with 101% biodegradation after 60 days.

Table 5.2.1/1: Biological Oxygen Demand Values

Day	BOD (mg O2/L)
	Inoculum Control		Procedure Control	Test item		Toxicity Control
	R1	R2		R1	R2
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60	0.00 0.00 0.00 0.92 1.42 2.00 3.04 3.04 3.62 4.74 4.78 5.00 6.38 7.16 7.84 7.84 8.20 8.42 8.74 9.38 9.66 10.12 11.58 12.08 12.08 13.24 13.92 14.82 15.58 16.08 16.08 16.08 16.54 17.32 18.04 19.04 19.50 19.50 19.50 20.50 21.00 21.54 22.40 22.78 23.24 23.28 23.70 24.62 25.32 25.32 25.44 25.78 26.40 27.28 28.08 28.32 28.90 29.20 29.20 29.20 29.44	0.00 0.00 0.00 1.00 1.46 2.00 3.08 3.08 3.84 4.96 5.08 5.24 6.70 7.46 8.08 8.24 8.70 9.08 9.50 10.6 10.62 11.20 12.62 13.28 13.32 14.58 15.24 16.36 17.12 17.66 17.66 17.78 18.46 19.28 20.04 20.94 21.50 21.54 21.74 22.86 23.32 23.94 24.90 25.28 25.82 26.08 26.62 27.54 28.20 28.32 28.62 28.98 29.82 30.70 31.48 31.78 32.44 32.66 32.66 32.70 33.36	0.00 0.00 48.02 82.88 109.62 122.50 130.36 135.12 139.20 142.66 144.36 145.74 148.28 150.06 151.48 153.32 155.18 156.94 158.98 160.76 163.22 165.10 167.52 169.06 169.76 171.80 172.56 174.72 175.88 176.48 177.10 177.76 178.80 179.88 180.92 182.02 182.72 183.46 184.00 185.34 185.92 186.72 188.22 188.68 189.38 190.22 190.88 191.96 192.68 193.00 193.80 194.34 195.50 196.42 197.22 197.58 198.38 198.68 198.68 199.18 199.92	0.00 1.38 1.58 3.16 3.80 4.24 5.24 5.58 6.42 7.50 7.74 7.88 9.46 10.28 10.66 11.28 11.74 12.16 12.62 13.16 13.78 14.42 15.82 16.70 16.86 18.40 18.96 20.58 21.54 22.12 22.32 22.70 23.36 24.04 24.86 25.62 26.12 26.58 26.90 27.90 28.32 28.90 30.02 30.40 31.08 31.70 32.12 33.08 33.78 33.98 34.56 35.02 36.02 36.82 37.36 37.70 38.36 38.56 38.56 38.94 39.56	0.00 1.30 1.74 3.34 3.80 4.46 5.50 5.66 6.58 7.74 7.96 8.20 9.82 10.66 11.24 11.66 12.24 12.70 13.24 13.86 14.46 15.12 16.66 17.46 17.54 18.96 19.54 20.94 21.74 22.32 22.40 22.90 23.74 24.62 25.58 26.62 27.20 27.54 27.86 28.98 29.48 30.16 31.28 31.70 32.36 32.78 33.16 34.16 34.86 35.06 35.40 35.82 36.82 37.70 38.40 38.78 39.48 39.82 39.82 39.98 40.74	0.00 1.08 36.02 76.26 90.76 102.20 112.58 122.04 129.94 134.24 136.98 139.44 142.20 143.56 144.48 145.40 146.20 146.94 147.64 148.32 149.40 150.24 151.94 153.24 153.78 155.56 156.28 158.14 159.32 159.98 161.18 162.18 163.44 164.72 165.94 167.06 167.72 168.60 169.10 170.30 170.84 171.68 172.98 173.38 174.10 174.80 175.26 176.30 177.02 177.38 177.98 178.52 179.60 180.56 181.14 181.68 182.52 183.02 183.02 183.84 184.76

R= Replicate

Table 5.2.1/2: Percentage Biodegradation values

Day	Biodegradation (%)
	Procedure Control	Test item			Toxicity Control
		R1	R2	Mean
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60	0 0 29 49 65 72 76 79 81 83 83 84 85 85 86 87 88 89 90 90 92 92 93 94 94 95 95 95 96 96 96 96 97 97 97 97 97 98 98 98 98 98 99 99 99 99 99 99 99 100 100 100 100 100 100 100 100 100 100 101 101	0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3	0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3	0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3	0 0 8 16 19 22 24 26 27 28 28 29 29 29 29 30 30 30 30 30 30 30 30 30 30 30 30 31 31 31 31 31 31 31 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 33 33 33 33 33 33 33 33 33 33

R= Replicate

Table 5.2.1/3: pH values of the test preparations on days 0 and 60

Test Vessel	pH
	Day 0	Day 60
Inoculum ControlR1	7.4	7.7
Inoculum Control R2	7.4	7.7
Procedure Control	7.4	8.2
Test Item + Inoculum R1	7.4	7.7
Test Item + Inoculum R2	7.4	7.7
Toxicity Control	7.4	8.2

 R = Replicate

Table 5.2.1/4: Analytical measurements

Time point (day)	Nominal concentration of test item in test sample (mg/L)	Sample dilution factor F	Determined concentration of test item in test sample (mg/L)	Percentage of nominal concentration (%)
0	Control – R1 Control – R2 100 – R1 100 – R2 P. Rec 1 – 101 P. Rec 2 – 101	0.1 0.1 0.1 0.1 0.1 0.1	Not detected Not detected 89.7 85.9 105 104	- - 90 86 103 103
60	Control – R1 Control – R2 100 – R1 100 – R2 P. Rec 1 – 100 P. Rec 2 – 100	0.1 0.1 0.1 0.1 0.1 0.1	Not detected Not detected 4.76 5.04 97.8 99.2	- - 5 5 98 10

R = Replicate

- = Not applicable

P. Rec = Procedural recovery

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 2% biodegradation after 28 days and 3% biodegradation after 60 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.

Executive summary:

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 Guideline 301F, EU Method C.4 -D and EPA OPPTS 835.3110, with GLP statement.

The test item at a concentration of 100 mg/L was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at temperatures of between 19 and 21 ºC for 60 days. At the request of the Sponsor the study was extended from 28 to 60 days in order to assess if any further degradation would occur. The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values andcompound specific analyses on Days 0 and 60. Control solutions with inoculum and the reference item, sodium benzoate, and a toxicity control were used for validation purposes.

The test item attained 2% biodegradation after 28 days, and 3% biodegradation after 60 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Sodium benzoate (procedure control) attained 86% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 96% biodegradationwas attained with 101% biodegradation after 60 days.

The toxicity control attained 29% biodegradation after 14 days, 31% biodegradation after 28 days and 33% biodegradation after 60 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Chemical analysis of the 100 mg/L test preparations containing inoculum at 0 hours showed measured concentrations of 90 and 86% of nominal were obtained. A decline in measured test concentration was observed on Day 60 to 5% of nominal for both replicates (calculated to be a 95% loss over the test duration assuming 100% recovery on Day 0).  The losses observed by chemical analysis were higher than those observed by oxygen consumption. An hypothesis would be the result of a primary biodegradation of the test item. Indeed, insertion of one oxygen atom between a carbon and hydrogen atom into all the isomers in the test item would result in 2 to 3% biodegradation and result in a total loss of the parent compound.

Description of key information

OECD Guideline 301F, EU Method C.4 -D, EPA OPPTS 835.3110, GLP, key study, validity 2:

2% biodegradation after 28 days and 3% biodegradation after 60 days.

The registered substance is not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

To assess the biodegradation potential of the registered substance, two experimental studies are available.

The most recent study (Covance, 2020), assessed as the key study, was performed on the registered substance to assess the ready biodegradability of the test item in an aerobic aqueous media, according to OECD Guideline 301F, EU Method C.4 -D and EPA OPPTS 835.3110, with GLP statement. The test item at a concentration of 100 mg/L was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at temperatures of between 20 and 22 ºC for 60 days. The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values and compound specific analyses on Days 0 and 60.  The test substance attained 2% biodegradation after 28 days, and 3% biodegradation after 60 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F. Chemical analysis of the 100 mg/L test preparations containing inoculum at 0 hours showed measured concentrations of 90 and 86% of nominal were obtained. A decline in measured test concentration was observed on Day 60 to 5% of nominal for both replicates (calculated to be a 95% loss over the test duration assuming 100% recovery on Day 0). The losses observed by chemical analysis were higher than those observed by oxygen consumption. An hypothesis would be the result of a primary biodegradation of the test item. Indeed, insertion of one oxygen atom between a carbon and hydrogen atom into all the isomers in the test item would result in 2 to 3% biodegradation and result in a total loss of the parent compound.

The other study (Phytosafe, 2017) was also performed on the registered substance according to the Closed Bottle test EU Method C.4-E with GLP statement. The test substance (prepared with chloroform as a solvent and which remained in the study after initiation) was put in contact with surface water collected from a river at a nominal concentration of 4.97 mg/L (14.80 mg ThOD/L) with culture medium in closed bottles in the dark at 20 +/- 1°C for 28 days. The degradation was followed by analysis of dissolved oxygen over a 28 -day period. Additional analytical assessments were performed at test initiation and at test completion in the test vessels for quantification of the test item, so as to judge upon the primary degradation throughout the test period. The biodegradation of the test substance remained almost zero throughout the test period (mean of 2.74% after 28 days). Regarding the analytical assessment, the measured initial concentration represented 96 -103% of the nominal value (4.97 mg/L). At the end of the test, 60% of the initial concentration was still recovered in the test vessels, so ca. 40% of the substance was not recovered. One probable explanation for this is that primary degradation occurred. This would suggest that the parent substance can be considered as not persistent in the environment. This result supports the key study.