2-(1-(3',3'-dimethyl-1'-cyclohexyl)ethoxy)-2-methyl propyl propanoate

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:

1995-03-07 to 1995-04-04

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study is deemed scientifically valid although some deviations from the guideline were observed: pH was not reported and the validity criteria regarding the reference substance was barely meet.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

pH was not reported and the validity criteria regarding the reference substance was barely meet.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Theoretical TOC (mg TOC/mg ST 02 C 95): 0.7179
- Theoretical CO2 (mg CO,lmg ST 02 C 95): 2.630
- Storage condition of test material: Store in a closed, preferably full, container away from heat sources and protected from extreme temperatures.

Oxygen conditions:

aerobic

Inoculum or test system:

other: An acclimated microbial inoculum was obtained on the final day of the testing phase from duplicate SCAS units dosed with the test substance at a nominal concentration of 20 mg active/L.

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
The initial activated sludge was collected from the DRWPCC (Downingtown Regional Water Pollution Control Center) on 7 February 1995. The sludge was screened through a 2 mm sieve. The sludge was acclimated for 7 days (from 7 to 13 February 1995), then pre-adapted to the test substance for 7 days (from 14 to 20 February 1995). Finally the sludge was in contact with the test substance during the 14-day testing period (from 21 February to 7 March 1995). Therefore the activated sludge was pre-adapted for 28 days (Please refer to the study summary "Biodegradation in water and sediment: simulation tests, Weston, 1995" (study n°95-007) for further information).

- Storage conditions: no data
- Storage length: no data
- Preparation of inoculum for exposure:
The inoculum was prepared for addition to the CO2 flasks as follows: Approximately 300 mL of mixed liquor was collected from each of the duplicate units, composited and homogenized at medium speed in a blender for approximately 2 minutes. The homogenized sample was poured into a beaker and allowed to settle for approximately 30 minutes. The supernatant was decanted and added to the test flasks at a concentration of 1% v/v.

- Pretreatment: none
- Concentration of sludge: approximately 2.5 mg SS/L at the beginning of the SCAS test (study n°95-007)
- Initial cell/biomass concentration: On the same day the test was set up a standard plate count (SPC) was performed on the inoculum described above. The plates were incubated at test temperature. The result was 5.3 x 106 CFU/mL
- Water filtered: no

Duration of test (contact time):

ca. 28 d

Initial conc.:

ca. 10 mg/L

Based on:

test mat.

Initial conc.:

ca. 20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Parameter followed for biodegradation estimation:

DOC removal

Remarks:

(final only)

Details on study design:

TEST CONDITIONS
- Composition of medium: modified biochemical oxygen demand (BOD) water containing the following standard reagent
solutions per liter of water:
1 mL standard magnesium sulfate solution (2.25%) (VWR No. VW3388-1 or equivalent)
1 mL standard calcium chloride solution (2.75%) (VWR No. VW3308-l or equivalent)
2 mL standard phosphate buffer- (pH 7.2) (VWR No. VW3345-l or equivalent)
4 mL standard ferric chloride solution (0.025%) (VWR No. VW3318-l or equivalent)
1 mL a 4% (w/v) solution of (NH.h SO4)

- Test temperature: 22.4 - 23.3°C
- pH: assumed to be 7.2 (buffer). Measured but not reported.
- pH adjusted: unknown
- CEC (meq/100 g): no data
- Aeration of dilution water: no data
- Suspended solids concentration: no data
- Continuous darkness: not mentioned in the report but the protocol indicates to “avoid direct sunlight to the test vessels and [to] keep room lighting off except during daily maintenance.”
- Other:

TEST SYSTEM
- Scrubbing apparatus:
A scrubbing apparatus was prepared as follows:
One empty 1-liter plastic bottle to prevent backflow into the air line.
Five Hiter plastic bottles containing 700 mL 10 N NaOH (E.M. Science, Lot No. 34078).
One 1-liter Erlenmeyer flask filled with 700 mL 0.024 N Ba(OHh (E.M. Science,
Lot No. 27271350) to serve as a CO2 indicator trap.
One empty 1-liter plastic bottle to prevent liquid carryover into the test containers.

- Culturing apparatus: Four glass 4-liter Erlenmeyer flasks
- Number of culture flasks/concentration: 1
- Method used to create aerobic conditions: CO2-free air was provided to the test flasks through a scrubbing apparatus (see description above)

- Measuring equipment:
- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: yes
- Details of trap for CO2: Three 4 oz. CO2 absorber bottles (French squares) filled with 100 mL of 0.024 N Ba(OH)2 and connected in series to the exit air line of each test flask.

SAMPLING
- Sampling frequency: every 3-4 days
- Sampling method: the traps nearest the test flasks were removed for titration
- Sample storage before analysis: no data

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

STATISTICAL METHODS:
Non-linear regression analysis on the form:
y = a(1-e^(-b(x-c)) for x > c or 0 for x ≤ c
where:
a = asymptote of curve (%TCO2)
b = rate constant (day-1)
c = lag time before CO2 production occurs (day)
x = days
y = cumulative % TCO2

Reference substance:

other: D-glucose

Preliminary study:

No data

Test performance:

No data

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

(%TCO2)

Value:

ca. 20.7

Sampling time:

29 d

Remarks on result:

other: Test substance at 10 mg/L

Key result

Parameter:

% degradation (CO2 evolution)

Remarks:

(%TCO2)

Value:

ca. 16.4

Sampling time:

29 d

Remarks on result:

other: Test substance at 20 mg/L

Details on results:

The test substance at 10 mg/L achieved 20.7% degradation at day 29 and 16.4% degradation at day 29 when tested at 20 mg/L.
No lag phase was observed in any of the condition and a plateau was not reached at day 29 for the test substance (neither at 10 mg/L nor 20 mg/L).
The cumulative CO2 in the control flask at the end of the test was 11.8 mg/L.
The regression analysis constant is provided in Table 5.2.1/1 below.
Please refer to Table 5.2.1/2 below for detailed results.

Results with reference substance:

The reference substance achieved 65.4% biodegradation at the end of the test. Its degradation was 56.9% and 60.3% at day 13 and day 17, respectively. The degradation of test substance at day 14 was likely slightly below 60%.
Please refer to Table 5.2.1/2 below for detailed results.

Table 5.2.1/1: Final results

				Computer regression analysis constants
Substance	Testing concentration (mg/L)	Final % TCO2	Final SOC (mg C/L)	Asymptote %TCO2	Rate (Day^-1)	Lag (days)
Blank Control	-	-	0.8	-	-	-
D-glucose	20	65.4	0.9	64.4 +/- 0.7	0.19 +/- 00.1	0.4 +/- 0.2
Test substance	10	20.7	5.1	20.3 +/- 1.3	0.11 +/- 0.03	0.0 +/- 1.0
Test substance	20	16.4	10.5	17.2 +/- 0.3	0.11 +/- 0.01	0.0 +/- 0.3

Table 5.2.1/2: Summary of cumulative CO2 production

Date	Day	mL os standard 0.05 N HCl/100mL Ba(HO)2					Cumulative mg CO2 Control Corrected			% of TCO2			Cumulative mg CO2 Control per Liter
		Flask 13 Blank Control	Flask 14 D-glucose (20 mg/L)	Flask 15 Test substance (10 mg/L)	Flask 16 Test substance (20 mg/L)	Initial	Flask 14 D-glucose (20 mg/L)	Flask 15 Test substance (10 mg/L)	Flask 16 Test substance (20 mg/L)	Flask 14 D-glucose (20 mg/L)	Flask 15 Test substance (10 mg/L)	Flask 16 Test substance (20 mg/L)
03/09/1995	2	42.4	33.5	39.5	39.4	47.4	9.8	3.2	3.3	16.7	6.1	3.1	2.8
03/11/1995	4	44.7	36.7	43.4	41.9	47.7	18.6	4.6	6.4	31.7	8.7	6.1	4.5
03/14/1995	7	45.4	36.7	43.8	42.1	47.6	28.2	6.4	10.0	48.1	12.1	9.5	5.7
03/16/1995	9	45.8	43.4	45.8	44.8	47.4	30.8	6.4	11.1	52.5	12.1	10.6	6.6
03/20/1995	13	45.5	43.1	44.9	44.0	46.9	33.4	7.1	12.8	56.9	13.5	12.2	7.4
03/24/1995	17	46.1	44.3	44.7	43.9	47.8	35.4	8.6	15.2	60.3	16.3	14.4	8.3
03/28/1995	21	46.1	44.9	45.4	44.9	47.7	36.7	9.4	16.5	62.5	17.9	15.7	9.2
03/31/1995	24	46.2	45.7	45.7	45.9	47.3	37.3	10.0	16.8	63.6	19.0	16.0	9.8
04/04/1995	28*	45.3	44.4	44.7	45.0	47.7	38.3	10.7	17.1	65.3	20.3	16.3	11.1
04/05/1995	29	46.4	46.3	46.2	46.2	47.6	38.4	10.9	17.3	65.4	20.7	16.4	11.8
* Test flasks were acidified after the traps were sampled for titration

Validity criteria fulfilled:

yes

Remarks:

even though the reference substance achieved slightly less than 60% degradation at day 14.

Interpretation of results:

not readily biodegradable

Conclusions:

The test substance did not meet the criteria for ready biodegradability according to OECD 301 B guideline.

Executive summary:

Introduction

This study was performed to assess the potential for ready biodegradability of the test substance. It was conducted according to the OECD Guideline 301 B (Ready Biodegradability: CO₂Evolution Test) with no major deviation.

Methods

The test material at a concentration of 10 and 20 mg/L was exposed to an acclimated microbial inoculum was obtained from duplicate SCAS units of a previous study. CO₂-free air was provided to the test flasks through a scrubbing apparatus and CO₂production was measured using three CO₂absorber bottles filled with 100 mL of 0.024 N Ba(OH)₂and connected in series to the exit air line of each test flask.

The degradation of the test material was assessed by the determination of CO₂produced on Days 0, 4, 7, 9, 13, 17, 21, 24, 28 and 29. D-glucose was used as a reference substance.

Results

After 29 days the test material attained 20.7% and 16.4% degradation at nominal concentration of 10 and 20 mg/L, respectively.

The reference substance attained slightly less than 60% at day 14, whereas the validity criteria is that at the reference substance degradation should attain the pass level by day 14. However this does not impair the study since the degradation of test substance is well below the pass level at day 29.

 

Conclusion

In this study the test substance did not meet the criteria for ready biodegradability according to OECD 301 B guideline.

Reference 2

Endpoint:

biodegradation in water: screening tests

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 7 February 1995 to 7 March 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

This Semi-Continuous Activated Sludge (SCAS) Removability study was not performed according to international guideline but according to internal standard protocol, with GLP statement.

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study was performed according to internal standard protocol.
In this study, activated sludge is exposed to a specified concentration of the test substance and the soluble organic carbon is analyzed after a specific time interval to determine the percent soluble carbon removal.
The procedure described in this test method is based on the Soap and Detergent Association's "A Procedure and Standards for the Determination of Biodegradability of Alkyl Benzene Sulfonate and Linear Alkylate Sulfonate". The Association's method is limited to the determination of primary removability because of its reliance on a specific analytical method. By measuring soluble carbon, the method can be expanded to measure ultimate removability because the fate of the entire organic molecule is followed. In addition, the method can be extended to test substances for which there are no analytical methods.

GLP compliance:

yes

Specific details on test material used for the study:

- Theoretical TOC (mg TOC/mg ST 02 C 95): 0.7179
- Theoretical CO2 (mg CO,lmg ST 02 C 95): 2.630
- Storage condition of test material: Store in a closed, preferably full, container away from heat sources and protected from extreme temperatures.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

The activated sludge used to initiate the study was obtained from a municipal treatment plant receiving predominantly domestic waste. The sludge was screened through a 2 mm sieve to remove large clumps. The total suspended solids (TSS) level was determined to be 3,280 mg/L and based on this reading the sludge was distributed among the SCAS units such that when the volume in each unit was adjusted to 1.5 L with tap water the suspended solids level was 2,500 mg/L. The units were set up the same day the sludge was collected.

Duration of test (contact time):

14 d

Initial conc.:

20 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

other: Average Soluble Organic Carbon (SOC) removal

Key result

Parameter:

other: Average Soluble Organic Carbon (SOC) removal

Value:

25

St. dev.:

1.6

Sampling time:

14 d

Details on results:

The average percent SOC removal with 95% confidence limits for the test substane was 25.0 +/- 1.6.
See table 5.2.1/1 in "Any other information on results incl. tables".

Table 5.2.1/1: Soluble Organic Carbon Data

Days	Reps.	Control, unit SOC (mg C/L)	Test substance, unit SOC (mg C/L)	Test unit Carbon removal (%)
1	1	7.8	17.0	32.29
	2	6.7	17.1	31.60
2	1	9.1	21.6	9.37
	2	8.0	19.8	21.88
3	1	8.7	18.9	25.69
	2	7.7	18.1	31.25
4	1	7.6	17.5	30.56
	2	7.4	17.2	32.64
5	1	7.5	19.4	15.97
	2	7.1	18.8	20.14
6	1	6.4	17.6	22.22
	2	6.4	17.2	25.00
7	1	6.5	16.6	28.12
	2	6.0	17.6	21.18
8	1	6.4	16.3	30.21
	2	6.1	16.5	28.82
9	1	6.2	17.6	20.49
	2	6.1	18.1	17.01
10	1	6.5	16.4	31.25
	2	6.5	17.2	25.69
11	1	5.8	17.7	16.32
	2	5.5	16.6	23.96
12	1	5.3	15.6	28.12
	2	5.2	16.2	23.96
13	1	5.2	15.5	27.78
	2	5.0	15.8	25.69
14	1	5.6	16.3	24.65
	2	5.3	16.0	26.74

Validity criteria fulfilled:

not applicable

Interpretation of results:

other: 25% SOC removal

Conclusions:

The average percent (%) SOC removal with 95% confidence limits for the test substance after fourteen days was 25.0 +/- 1.6.

Executive summary:

This study was performed according to internal standard protocol. In this study, activated sludge is exposed to a specified concentration of the test substance and the soluble organic carbon (SOC) is analyzed after a specific time interval to determine the percent soluble carbon removal.

The test apparatus consisted of four SCAS aeration chambers containing 1.5L of activated sludge. The suspended solids level in each unit was adjusted to 2,500 mg/L. The units were aerated at a rate adequate to maintain solids suspension.

Following a sludge-acclimation period, the test substance was added by direct weight to duplicate units incrementally for a seven-day acclimation period. The final nominal test substance concentration was 20 mg active/L. Two additional units did not receive any test substance and served as controls. Throughout the study all units were fed synthetic sewage daily.

During the testing period effluents were withdrawn from each unit and analyzed for SOC. The testing period was extended from seven to fourteen days in an attempt to obtain seven consecutive days of steady-state SOC data. This did not occur; therefore, the average percent (%) SOC removal with 95% confidence limits for the test substance after fourteen days was 25.0 +/- 1.6.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Remarks:

(Q)SAR assessment of target substance constituents; within acceptable limits of the structural domain of the model and satisfying reliability criteria within a weight-of-evidence approach. Specifically, the predictions are to be used in a qualitative manner to compare the similarity of primary biodegradability and transformation pathways of each constituent. The predictions are supporting reliable GLP study on the primary biodegradation of the major isomeric constituent which are used for quantitative assessment of the substance’s overall biodegradability.

Justification for type of information:

1. SOFTWARE
OASIS CATALOGIC v.5.11.17

2. MODEL (incl. version number)
CATALOGIC Kinetic 301C v.08.12 (June, 2015)

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Major C6 diastereomer: CCC(=O)OCC(C)(C)OC(C)C1CCCC(C)(C)C1
Major C7 diastereomer: CCC(=O)OCC(C)(C)OC1CC(C)(C)CCCC1C
See details presented in test material information.

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

5. APPLICABILITY DOMAIN
See attached QPRF

6. ADEQUACY OF THE RESULT
See attached QPRF

Qualifier:

according to guideline

Guideline:

other: Reach Guidance on QSAR - R.6

Deviations:

no

Principles of method if other than guideline:

CATALOGIC Kinetic 301C v.08.12 Ready Biodegradability - expressed as; BOD - decimal fraction ; Ready degradability – dichotomous (Ready/Not ready) ; Primary half-life – hours, days, months, or years ; Ultimate half-life – hours, days, months, or years ; Metabolites – qualitative (metabolites 2D structure and metabolic tree) and Metabolites quantitative distribution – quantity of generated metabolites (in mol) per mol parent (decimal fraction). Model accounts for a number of mitigating factors, such as molecular size, metabolism of parent chemical, water solubility and ionization. BOD[28] output is the oxygen used by aerobic microorganisms to mineralize the test substance after a given time (28 days), corrected for oxygen uptake by the blank inoculum control after the same time, and related to the theoretical oxygen demand needed for full mineralization of the substance.

GLP compliance:

no

Oxygen conditions:

other: Not applicable (QSAR)

Inoculum or test system:

other: Not applicable (QSAR)

Details on inoculum:

Not applicable (QSAR)

Duration of test (contact time):

28 d

Details on study design:

Not applicable (QSAR)

Preliminary study:

No data

Test performance:

No data

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

prediction given as BOD[28] and as % degradation under OECD TG 301C

Value:

ca. 11

Remarks on result:

other: Concomitant predictions: Primary Half Life for the C6 isomer = 22.97 days; Primary Half Life for the C7 isomer = 22.93 days. See 'Any other information results incl. tables' for more information on the prediction(s)

Details on results:

See "Any other information on results incl. tables"

Results with reference substance:

Not applicable (QSAR)

1. Defined Endpoint:

QMRF 2. Environmental Fate Parameters

QMRF 3. Persistence: Biodegradation: 2.3.a Ready Biodegradability

 

Reference to type of model used and description of results:

CATALOGIC Kinetic 301C v.08.12, June 2015

Platform version: OASIS Catalogic v5.11.17

 

2. Description of results and assessment of reliability of the prediction:

The outcome of the model for all constituents is considered suitable to support the assessment of the primary biodegradability of the multi-constituent substance. The predictions are to be used in a qualitative manner to compare the similarity of primary biodegradability and transformation pathways of each diastereoisomer. Full reliable GLP studies on the primary biodegradation of the major C6 isomer are used for quantitative assessment of the substance’s overall biodegradability. This is reported in parallel to the predictions below where appropriate.

Summary:

1. C6 isomer: 2-{(1RS)-1-[(1SR)-3,3-dimethylcyclohexyl]ethoxy}-2-methylpropyl propionate

Predicted BOD [28]= 11±3 %

Measured BOD [28] = 19% (OECD TG 301C, 2009)

28-days Primary Degradation (measured) = > 98%

Concomitant predictions

Primary Half Life = 22.97 days

Ultimate Half Life: 5m 17d

2. C7 isomer: 2-methyl-2-{[(1RS,2RS)-2,6,6-trimethylcycloheptyl]oxy}propyl propionate

Predicted BOD [28]= 11±2 %

Measured BOD [28] = Not available.

28-days Primary Degradation (measured) = Not available.

Concomitant predictions

Primary Half Life = 22.93 days

Ultimate Half Life: 5m 21d

The performance of the model for the C7 isomer, which has marked structural similarity to the C6 isomer, is therefore expected to provide reliable information on the primary and ultimate biodegradation of the molecule. It is therefore concluded that the primary biodegradation of the C7 isomer is equivalent to that of the C6 isomer, despite the uncertainty associated with the structural domain of the prediction for the constituents of this substance. In addition to the predictions on the parent molecules, model output biodegradation predictions on a mixture of the primary degradation products: C6 (75 %) & C7 (10.3 %) are also consistent with non-GLP experimental biodegradation data to OECD TG 301F; available and submitted by the applicant.

n-Propionic acid, which was assumed to be completely mineralised in the OECD 301C study (2009) as the substance could not be seen in supporting chemical specific analysis, and based on the known rapid degradability of this substance (ECHA Substance Information Portal). The same conclusion is reached from the model output. C6 and C7 primary transformation products achieved 0-4 % degradation in the non-GLP OECD 301F study (2013), a result that is corroborated by the model output in the above table. The model output therefore supports the conclusions reached from non-GLP laboratory testing that the primary degradation products may meet the persistence criteria.

Full details of the constituents are provided in the attached ‘QPRF Title: Reaction mass of 2-{(1RS)-1-[(1SR)-3,3-dumethylcyclohexyl]ethoxy}-2-methylpropyl propionate and 2-{(1RS)-1-[(1RS)-3,3-dimethylcyclohexyl]ethoxy}-2-methylpropyl propionate and 2-methyl-2-{[(1RS,2RS)-2,6,6-trimethylcycloheptyl]oxy}propyl propionate using the model CATALOGIC Kinetic 301C v.08.12 within LMC OASIS CATALOGIC v5.11.17 for the endpoint: Ready Biodegradability’ version 1.0 prepared 21 April 2016.

 

Assessment of the substance within the applicability domain as documented within the corresponding QMRF named ‘QMRF Title: Catalogic Kinetic 301C v08.12 model for predicting biodegradability of chemicals’; updated QMRF: June 2015 – section 5; indicates:

That for all constituents:

a. Descriptors domain:

(i) Falls within the Log Kow domain of: Min -7.51 to Max 24.8; (ii) Molecular weight: Min 26.0 to Max 1350 Daltons and; (iii) Water Solubility: Min 0.0 to Max 1000000 mg/L.

Input descriptors are summarised by the applicant in the attached QPRF.

b. Structural domain (Atom Centred Fragments, ACFs):

The substance constituents are generally within the model structural domain (85 – 90%) however not entirely within the total structural domain. The high percentage of correctly predicted atom centred fragments (> 80%) allows that the conclusion that the predictions are relevant. Based on expert judgement and considering the regulatory purpose the predictions are considered reliable as supporting evidence for qualitative information to support experimental biodegradation data to compare the similarity of primary biodegradability and transformation pathways of each constituent.

 

3. Uncertainty of the prediction and mechanistic domain:

The training set is embedded in the software of the model; refer to the QMRF which is available in OASIS Catalogic v5.11.17. The training set data is proprietary and not made publicly available by the model developers. It is noted that the software model itself determines if the test item falls within the general properties requirements [lipophilicity (log KOW), molecular weight (MW) and water solubility (WS)] domain; then the appropriate structural and mechanistic/metabolic domains. Full details are provided of the methodology in the corresponding QMRF (dated: 30 April 2014, updated June 2015) for model CATALOGIC Kinetic 301C v.08.12. Although the constituents of the substance are considered out of the interpolation structure space of the structural fragment domain, unknown fragments constitute less than or equal 15% which is not considered to be significant given the regulatory purpose of the predictions presented in the report. Similarly, the variation of the model outcomes are small ranging from BOD = 0.00 – 0.03 for both the parent substances and transformation products (refer to tables in the attached QPRF presented by the applicant), indicating that the model outputs were not subject to significant uncertainty despite there being less than or equal to 15 % unknown fragments. The predictions are to be used in a qualitative manner to compare the similarity of primary biodegradability and transformation pathways of each isomer which is not considered to be significant given the regulatory purpose of the predictions presented in the attached QPRF presented by the applicant.

Validity criteria fulfilled:

yes

Interpretation of results:

other: Primarily degradation

Conclusions:

The results are adequate as contributing information for the regulatory purpose.
The C6 and C7 isomers of the registered substance achieve complete primary biodegradation. The transformation products may meet the persistence criteria.

Executive summary:

OASIS Catalogic v5.11.17 - CATALOGIC Kinetic 301C v.08.12, Ready Biodegradability, June 2015

Summary:

BOD [28]: C6 isomer = 11±3 %; C7 isomer = 11±2 %; C6 prediction is consistent with reliable measured data.

Concomitant predictions:

Primary Half Life: C6 isomer = 22.97 days; C7 isomer = 22.93 days

Ultimate Half Life: C6 isomer = 5m 17d; C7 isomer = 5m 21d

The outcome of the model for all constituents is considered suitable to support the assessment of the primary biodegradability of the multi-constituent substance. The C6 and C7 isomers of the multi-constituent substance achieve complete primary biodegradation, but the transformation products may meet the persistence criteria. It is concluded that the primary biodegradation of the C7 isomer is equivalent to that of the C6 isomer, despite the uncertainty associated with the structural domain of the prediction for the constituents of this substance. This uncertainty was by expert judgement considered acceptable on the basis of the regulatory purpose of the predictions as supporting information.

Adequacy of the QSAR:

Adequacy of assessment of the (Q)SAR results:

1) QSAR model is scientifically valid; 2) The substance falls within acceptable limits of the applicability domain of the QSAR model and satisfying reliability criteria within a weight-of-evidence approach and 3) The prediction is adequate as contributing data for the Classification and Labelling or risk assessment of the substance as indicated in REACH Regulation (EC) 1907/2006: Annex XI Section 1.3

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1994-04-29 to 1994-05-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study is deemed scientifically valid although the preparation of test material is questionable since a stock solution was prepared above the water solubility of the test substance. In addition, no information on batch number tested and isomers composition.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Storage condition of test material: +4°C in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): effluent from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, treating predominantly domestic sewage
- Laboratory culture: none
- Storage conditions: the sample was allowed to settle for 1 hour, the supernatent was removed and maintained on aeration at 21°C prior to use
- Concentration of sludge: not determined
- Initial cell/biomass concentration: not determined
- Water filtered: no

Duration of test (contact time):

ca. 28 d

Initial conc.:

ca. 2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium:
#solution a:
KH2PO4: 8.5 g/L
K2HPO4: 21.75 g/L
Na2HPO4,2H20: 33.40 g/L
NH4Cl: 0.50 g/L
#solution b:
MgSO4,7H2O: 22.5 g/L
#solution c:
CaCl2: 27.5 g/L
#solution d:
FeCl3,6H2O: 0.25 g/L

1 mL of each of solutions a-d were added to each liter of aerated reverse osmosis purified and deionised water.

- Test temperature: 21°C
- pH: 7.4
- pH adjusted: no data
- CEC (meq/100 g): no data
- Aeration of dilution water: The 'dilution water' was left at room temperature (approximately 21°C) under gentle aeration for 24 hours prior to use.
- Suspended solids concentration: not measured
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 250-300 ml BOD bottles (darkened glass) with ground glass stoppers
- Number of culture flasks/concentration: 2
- Measuring equipment: Yellow Springs BOD probe (Model 54)
- Test performed in closed vessels: yes

SAMPLING
- Sampling frequency: 0, 3, 6, 9, 12, 15, 18, 21, 24 and 28 days
- Sampling method: dissolved oxygen concentrations were determined in duplicate
- Sample storage before analysis: not mentioned

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes, test substance (1 mg/L) and sodium benzoate (1.5 mg/L)

STATISTICAL METHODS: none

Reference substance:

benzoic acid, sodium salt

Preliminary study:

No data

Test performance:

The method of preparation of test material is questionable since a stock solution of 100 mg/L was prepared and used while the water solubility of test substance is less than 10 mg/L.
The oxygen depletion in the inoculated control series were within the prescribed limits (< 1.5 mg/L 02/L after 28 days).

Key result

Parameter:

% degradation (O2 consumption)

Value:

ca. 17

Sampling time:

28 d

Details on results:

The test substance attained 17% of degradation by day 28. No particular kinetics was observed.
In the toxicity control around 36% degradation was observed, therefore the test material is not considered to be inhibitory to the inoculum.
Dissolved Oxygen Measurement are given in Table 5.2.1/1 below. Oxygen depletion and percentage Biodegradation values are given in Table 5.2.1/2 below.
The biodegradation curves ar shown in Figure 1 below.

Results with reference substance:

Sodium Benzoate achieved more than 80% degradation by day 14. Therefore the test meets the validity criteria attached to reference substance.

Table 5.2.1/1: Dissolved Oxygen Measurement

Test series		Dissolved Oxygen (mg 02/L)
		Day
		0	3	6	9	12	15	18	21	24	28
(a) Culture medium with inoculum	R1	8.90	8.60	8.50	8.40	8.30	8.30	8.30	8.25	8.15	8.10
	R2	8.90	8.75	8.50	8.55	8.40	8.30	8.30	8.25	8.20	8.10
(b) Standard material, Sodium benzoate (3 mg/L), with inoculum	R1	8.90	6.10	6.00	4.35	4.25	4.10	4.40	4.15	4.20	4.00
	R2	8.90	6.05	5.85	4.45	4.20	4.30	4.20	4.25	4.05	3.70
(c) Test material (2 mg/L) with inoculum	R1	8.90	8.15	8.40	8.30	7.90	7.90	7.80	7.50	7.25	7.20
	R2	8.90	8.30	8.05	8.00	7.70	7.80	7.55	7.50	7.30	7.20
(d) Test material (1 mg/L) plus sodium benzoate (1.5 mg/L) with inoculum	R1	8.90	7.10	6.90	6.70	6.50	6.40	6.40	6.35	6.25	6.20
	R2	8.90	6.95	6.90	6.65	6.50	6.30	6.40	6.30	6.30	6.30

R1 and R2 = Replicates 1 and 2

Table 5.2.1/2: Oxygen depletion and percentage Biodegradation values

Test series		Day
		3	6	9	12	15	18	21	24	28
(a) Culture medium with inoculum		0.225	0.400	0.425	0.550	0.600	0.600	0.650	0.725	0.800
(b) Standard material, Sodium benzoate (3 mg/L), with inoculum	R1	2.575	2.500	4.125	4.100	4.200	3.900	4.100	3.975	4.100
	R2	2.625	2.650	4.025	4.150	4.000	4.100	4.000	4.125	4.400
	mean	52	52	81	83	82	80	81	81	85
(c) Test material (2 mg/L) with inoculum	R1	0.525	0.100	0.175	0.450	0.400	0.500	0.750	0.925	0.900
	R2	0.375	0.450	0.475	0.650	0.500	0.750	0.750	0.875	0.900
	mean	9	6	6	11	9	12	14	18	17
(d) Test material (1 mg/L) plus sodium benzoate (1.5 mg/L) with inoculum	R1	1.575	1.600	1.775	1.850	1.900	1.900	1.900	1.925	1.900
	R2	1.725	1.600	1.825	1.850	2.000	1.900	1.950	1.875	1.800
	mean	32	31	35	36	38	37	38	37	36

R1 and R2 = Replicates 1 and 2

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

After 28 days the test material attained 17% degradation, therefore it cannot be considered as readily biodegradable under the strict criteria of OECD Guideline 301D.

Executive summary:

Introduction

This study was performed to assess the potential for ready biodegradability of the test substance. It was conducted according to the OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test) without deviation.

Methods

The test material was exposed to an inoculum obtained from a secondary effluent with predominantly domestic sewage. The test substance (2 mg/L), sodium benzoate (reference substance), and the test substance plus sodium benzoate (toxicity control) where exposed to this inoculum for 28 days. Oxygen depletion was measured every 3 days to assess the biodegradation of material, expressed as % of ThOD NO₃).

Results

After 28 days the test material attained 17% degradation, therefore it cannot be considered as readily biodegradable under the strict criteria of OECD Guideline 301D.

The reference substance attained more than 80% at day 14, demonstrating the efficacy of the testing conditions.

The test material did not exhibit inhibitory effect since the toxicity control attained 36% degradation after 28 days.

  

 Conclusion

In this closed bottle test the test substance did not meet the criteria for ready biodegradability according to OECD guideline 301 D.

Reference 5

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Remarks:

Experimental study on the C6 isomers of the registered substance.

Adequacy of study:

key study

Study period:

2009-05-25 to 2009-08-24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

This study is deemed scientifically reliable although some data were not reported such as the origin and preparation of inoculum, the composition of mineral medium and whether the bottles were placed in darkness during the test period. The test substance is the C6 isomer of the registered substance.

Qualifier:

according to guideline

Guideline:

other: Biodegradability Test of Chemical Substances by Microorganisms (Yakushokuhatsu No. 1121002, Heisei 15.11.13 Seikyoku No.2, Kanpokihatsu No. 031121002, November 21, 2003; the latest revision, November 20, 2006)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Storage condition of test material: sealed under nitrogen atmosphere and stored in a dessicator at room temperature in the dark for the duration of the study

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, industrial (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Chemicals Evaluation and Research Institute, Japan
- Storage conditions: no data
- Storage length: no data
- Preparation of inoculum for exposure: no data
- Pretreatment: no data
- Concentration of sludge (Mixed liquor suspended solid): 2450 mg/L
- Initial cell/biomass concentration: no data
- Water filtered: no data

Duration of test (contact time):

ca. 28 d

Initial conc.:

ca. 100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: not available
- Test temperature: 25 +/- 1°C
- pH: 6.4 to 8.2 (measured)
- pH adjusted: no data
- Stirring: Continuous stirring with magnetic stirrer
- Test volume: 300 mL
- Concentrations: test substance (bottles (3-6): 100 mg/L; aniline (bottle 1): 100 mg/L; activated sludge (bottles 1-5): 30 mg/L
- CEC (meq/100 g): no data
- Water: purified water Grade A4, Japanese Industrial Standards (JIS) K0557
- Aeration of dilution water: no data
- Suspended solids concentration (Mixed liquor suspended solid): 2450 mg/L
- Continuous darkness: no data

TEST SYSTEM
- Culturing apparatus: not described
- Number of culture flasks/concentration: 6 flasks in total
Flask 1: Activated sludge + Aniline (30 mg) (reference substance)
Flask 2: Control blank
Flask 3: Activated sludge + test substance (replicate n°1)
Flask 4: Activated sludge + test substance (replicate n°2)
Flask 5: Activated sludge + test substance (replicate n°3)
Flask 6: water + test substance (abiotic control)
- Method used to create aerobic conditions: not described
- BOD measurement:
#Method: The BOD was measured for 28 days. The test solutions were observed and proper operation of the apparatus was confirmed once a day except holidays throughout the exposure period. After the measurement, the contents in the test bottles were observed.
#Apparatus: OM-2001, Ohkura Electric Co. (ID code:G)
- DOC measurement:
#Apparatus: TOC analyzer TOC-5000A, Shimadzn Co.
#Conditions:
- Furnace temperature : 680°C (TC)
- Air flow rate : 150 mL/min
- Sensitivity : x 5
- Injection volume : 0.030 mL
- Repeated number : n=3 (adopt mean value)
#Calibration curve:
The following standard solutions were injected into the TOC analyzer. The calibration curve was prepared by the data processor of the analyzer.
Standard solutions:
TC (total carbon): 40 and 80 mg C/L aqueous solutions of potassium hydrogen phthalate.
IC (inorganic carbon): 0 mg C/L purified water;10 mg C/L aqueous solution of sodium hydrogen carbonate and sodium carbonate.
- Test performed in closed-system oxygen consumption measuring apparatus

SAMPLING
- Sampling frequency: day 7, 14, 31 and 28
- Sampling method: no available
- Sample storage before analysis: no data

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: none
- Toxicity control: none

STATISTICAL METHODS:
the equations for calculation of degradability used was identical to those described in the OECD guideline 301C

Reference substance:

aniline

Preliminary study:

No data

Test performance:

No data

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

based on BOD measurments

Value:

ca. 19

St. dev.:

0

Sampling time:

28 d

Key result

Parameter:

% degradation (test mat. analysis)

Remarks:

degradability based on the residual test substance amount

Value:

> 98

St. dev.:

0

Sampling time:

28 d

Details on results:

- Degradability based on the BOD
The BOD in bottles 3, 4, and 5 (as corrected with the value in bottle 2) were all 14.7 mg, and the BOD in bottle 6 was 0.8 mg (Maximum theoretical value = 79.3 mg).
The degradabilities based on the BOD measurement were calculated to be all 19% for bottles 3, 4, and 5 (See Table 5.2.1/1).

- Degradability based on the DOC
The DOC in bottles 3, 4 and 5 (as corrected with the value in bottle 2) were 16.0, 16.3 and 16.5 mg, respectively, and the DOC in bottle 6 (abiotic control) was 1.2 mg (initial amount = 21.5 mg).
The degradability based on the DOC was not calculated since the test substance was hardly soluble in water and the DOC in bottle 6 is less than 90% of initial amount (See Table 5.2.1/1.b) and Table 5.2.1/2).

- Degradability based on the residual test substance amount
The amounts of residual test substance were all less than 0.4 mg in bottles 3, 4 and 5, and 27.7 mg in bottle 6 (initial amount = 30 mg).
The degradabilities based on the residual test substance amount were calculated to be all >98% for bottles 3, 4 and 5 (See Table 5.2.1/1.b)).

- Relevant transformation product amount
The amounts of the relevant transformation product were 19.3, 18.8, 19.0 and 0.3 mg in bottles 3, 4, 5 and 6, respectively. Formation rate of the relevant transformation product were calculated to be 80, 78, 79 and 1% for bottles 3, 4, 5 and 6, respectively (See Table 5.2.1/1.b)).

- Propionic acid amount
The amounts of propionic acid were all <0.1 mg in bottles 3,4 and 5, and 0.1 mg in bottle 6. Detection rate of propionic acid were calculated to be all <2% for bottles 3, 4 and 5, and 1% for bottle 6 (See Table 5.2.1/1.b)).

Results with reference substance:

The observed degradation in flask with activated sludge + Aniline was 63.6% at day 7, 90.2% at day 14, 93.9% at day 21 and 95.0% at day 28. These results fulfill the criteria for validity regarding degradation of reference substance.

- Observation of the contents after the exposure period

The colour of the solution and growth of the sludge in the test bottles were observed in contrast with the control (bottle 2).

The solution in bottle 1 was yellow, and the solutioninbottles 3, 4, 5 were white, and the solution in bottles 6 was slightly white.

Growth of the sludge was observed in bottles 1, 3, 4 and 5.

Table 5.2.1/1 Summary of the test results

a) BOD measurement and pH

Bottle n°	Sample description	BOD (mg)				pH
		day 7	day 14	day 21	day 28	day 0	day 28
1	Activated sludge + Aniline	60.8	86.7	92.6	93.6	-	8.2
2	Control blank	3.4	5.3	7.0	7.9	7.1	7.3
3	Activated sludge + test substance (N°1)	14.8	19.6	22.1	22.6	-	7.2
4	Activated sludge + test substance (N°2)	14.4	19.3	21.9	22.6	-	7.1
5	Activated sludge + test substance (N°3)	14.5	19.5	22.0	22.6	-	7.2
6	Water + test substance	0.0	0.2	0.7	0.8	6.4	8.0

b) Measured values at day 28

		Activated sludge + test substance			Water + test substance	Theoretical value
		N°1	N°2	N°3
		Bottle N°3	Bottle N°4	Bottle N°5	Bottle N°6
BOD	mg	14.7*	14.7*	14.7*	0.8	79.3
DOC	mg	16.0*	16.3*	16.5*	1.2	21.5
Test substance (GC)	mg	<0.4	<0.4	<0.4	27.7	30.0
	%	<2	<2	<2	92	-
Relevant transformation product (GC)	mg	19.3	18.8	19.0	0.3	24.1
	%	80	78	79	1	-
Propionic acid (HPLC)	mg	<0.1	<0.1	<0.1	0.1	7.8
	%	<2	<2	<2	1	-

*Value of is corrected with value of Control blank.

c) Degradabilities (%)

	Activated sludge + test substance			Average
	N°1	N°2	N°3
	Bottle N°3	Bottle N°4	Bottle N°5
BOD (%)	19	19	19	19
DOC (%)	NA**	NA**	NA**	-
Test substance (%)	>98	>98	>98	>98

**Degradability is not calculated because the test substance is insoluble in water.

Table 5.2.1/2: Result of DOC measurement

mgC/L	Bottle 2		Bottle 3		Bottle 4		Bottle 5		Bottle 6
	TC	IC	TC	IC	TC	IC	TC	IC	TC	IC
Measure 1	1.868	0.000	55.54	0.000	55.93	0.000	56.54	0.000	4.336	0.000
Measure 2	1.861	0.000	54.28	0.000	56.06	0.000	56.84	0.000	4.005	0.000
Measure 3	1.876	0.000	55.84	0.000	57.18	0.000	58.07	0.000	3.516	0.000
Mean	1.868	0.000	55.22	0.000	56.39	0.000	57.15	0.000	3.952	0.000

Validity criteria fulfilled:

yes

Interpretation of results:

other: Primarily degradation

Conclusions:

The test substance is not readily degradable. All of the test substance disappeared and the relevant transformation product was formed at a rate of 79% (see Figure 1).

Executive summary:

Introduction

This study was performed to assess the potential for ready biodegradability of the C6 isomers of the registered substance. It was conducted according to a guideline similar to the OECD Guideline 301 C (MITI) without deviation.

Methods

The test material was exposed to an inoculum obtained from an industrial activated sludge provided by Chemicals Evaluation and Research Institute, Japan. The test substance (100 mg/L) and Aniline (reference substance) where exposed to this inoculum for 28 days. BOD was measured every 7 days to assess the biodegradation of material, expressed as % of ThOD).

The residual parent compound and degradation products were monitored. The anticipated degradation products were a relevant transformation product and propionic acid. The registered substance and the corresponding relevant transformation product (C6 isomers) were analysed through GC while propionic acid was analysed using an HPLC method.

Results

After 28 days the overall degradability observed was 19%. The residual test substance amount was < 2% in all test bottles, while the observed relevant transformation product formation rate was 79% on average. However the detection rate of propionic acid was less than 2%.

Therefore, the C6 isomers of the registered substance cannot be considered as readily biodegradable under the strict criteria of OECD Guideline 301C.

Propionic acid is considered to have been mineralized because the BOD value, 14.7 mg, was greater than the ThOD of anticipated propionic acid, 11.8 mg.

 

The reference substance attained more than 40% after 7 days and more than 65% at day 14, demonstrating the efficacy of the testing conditions.

 

Conclusion

The test substance is not readily biodegradable, but primarily degraded within 28 days. All of the test substance disappeared and a relevant transformation product was formed at a rate of 79%. The degradation product propionic acid is considered to have been mineralised.

Reference 6

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Remarks:

Experimental study performed on the transformation product (C6 and C7 isomers) of the registered substance.

Adequacy of study:

supporting study

Study period:

From 2013-01-09 to 2013-03-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study was performed according to international guidelines and was well conducted. However, this study is not GLP.

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Specific details on test material used for the study:

- Storage conditions: stored at 4°C (refrigerator) in the dark under N2 conditions.

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

- Test system: A mixed population of activated sewage sludge micro-organisms was obtained on January 9th, 2013 from the secondary treatment stage of the sewage treatment plant at Villette (STEP Villette, avenue de Thônex 105, 1226 THONEX (Geneva, Switzerland)), which treats predominantly domestic sewage.

- Preparation if inoculum: The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement (centrifuge: Heittich rotenta 460 RS) and suspension in culture medium. To remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present, the solution was stirred and maintained on with pure oxygen at room temperature. Determination of dry weight is made to inoculate final solution with 30mg/L dry weight activated sludge.

Duration of test (contact time):

28 d

Initial conc.:

104.5 mg/L

Based on:

test mat.

Initial conc.:

99.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Remarks on result:

other: Test 1 (104.5 mg/L)

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

60 d

Remarks on result:

other: Test 1 (104.5 mg/L)

Key result

Parameter:

% degradation (O2 consumption)

Value:

2

Sampling time:

28 d

Remarks on result:

other: Test 2 (99.5 mg/L)

Key result

Parameter:

% degradation (O2 consumption)

Value:

4

Sampling time:

60 d

Remarks on result:

other: Test 2 (99.5 mg/L)

Details on results:

See tables below in "Any other information on results incl. tables".

Table 1: Percentage Biodegradation in Test 1

days	blanc 1	blanc 2	average	test1	BOD (mgO2)	BOD(mgO2/mg test)	% degradation
							NH3
0	0	0	0	0	0.0	0.0	0
2	6.2	6.2	6.2	6.2	0.0	0.0	0
4	12.4	12.4	12.4	12.4	0.0	0.0	0
6	12.4	18.6	15.5	18.6	0.6	0.0	1
8	18.6	18.6	18.6	18.6	0.0	0.0	0
10	24.8	18.6	21.7	18.6	-0.6	0.0	-1
12	24.8	24.8	24.8	18.6	-1.2	-0.1	-2
14	24.8	18.6	21.7	24.8	0.6	0.0	1
16	24.8	24.8	24.8	24.8	0.0	0.0	0
18	31	24.8	27.9	24.8	-0.6	0.0	-1
20	24.8	24.8	24.8	24.8	0.0	0.0	0
22	31	24.8	27.9	24.8	-0.6	0.0	-1
24	24.8	24.8	24.8	24.8	0.0	0.0	0
26	31	24.8	27.9	24.8	-0.6	0.0	-1
28	24.8	24.8	24.8	24.8	0.0	0.0	0
30	24.8	31	27.9	24.8	-0.6	0.0	-1
32	31	24.8	27.9	24.8	-0.6	0.0	-1
34	31	24.8	27.9	24.8	-0.6	0.0	-1
36	31	31	31	24.8	-1.2	-0.1	-2
38	31	24.8	27.9	24.8	-0.6	0.0	-1
40	24.8	24.8	24.8	18.6	-1.2	-0.1	-2
42	31	31	31	31	0.0	0.0	0
44	31	24.8	27.9	24.8	-0.6	0.0	-1
46	31	24.8	27.9	18.6	-1.9	-0.1	-3
48	31	31	31	24.8	-1.2	-0.1	-2
50	37.2	31	34.1	31	-0.6	0.0	-1
52	31	24.8	27.9	24.8	-0.6	0.0	-1
54	31	31	31	31	0.0	0.0	0
56	37.2	31	34.1	31	-0.6	0.0	-1
58	31	31	31	31	0.0	0.0	0
60	31	31	31	31	0.0	0.0	0

Table 2: Percentage biodegradation in Test 2

days	blanc 1	blanc 2	average	test2	BOD (mgO2)	BOD(mgO2/mg test)	% degradation
							NH3
0	0	0	0	0	0.0	0.0	0
2	6.2	6.2	6.2	12.4	1.2	0.1	2
4	12.4	12.4	12.4	18.6	1.2	0.1	2
6	12.4	18.6	15.5	24.8	1.9	0.1	3
8	18.6	18.6	18.6	24.8	1.2	0.1	2
10	24.8	18.6	21.7	24.8	0.6	0.0	1
12	24.8	24.8	24.8	31	1.2	0.1	2
14	24.8	18.6	21.7	31	1.9	0.1	3
16	24.8	24.8	24.8	31	1.2	0.1	2
18	31	24.8	27.9	31	0.6	0.0	1
20	24.8	24.8	24.8	31	1.2	0.1	2
22	31	24.8	27.9	31	0.6	0.0	1
24	24.8	24.8	24.8	31	1.2	0.1	2
26	31	24.8	27.9	37.2	1.9	0.1	3
28	24.8	24.8	24.8	31	1.2	0.1	2
30	24.8	31	27.9	37.2	1.9	0.1	3
32	31	24.8	27.9	37.2	1.9	0.1	3
34	31	24.8	27.9	31	0.6	0.0	1
36	31	31	31	31	0.0	0.0	0
38	31	24.8	27.9	31	0.6	0.0	1
40	24.8	24.8	24.8	31	1.2	0.1	2
42	31	31	31	37.2	1.2	0.1	2
44	31	24.8	27.9	31	0.6	0.0	1
46	31	24.8	27.9	24.8	-0.6	0.0	-1
48	31	31	31	37.2	1.2	0.1	2
50	37.2	31	34.1	37.2	0.6	0.0	1
52	31	24.8	27.9	37.2	1.9	0.1	3
54	31	31	31	37.2	1.2	0.1	2
56	37.2	31	34.1	43.4	1.9	0.1	3
58	31	31	31	43.4	2.5	0.1	4
60	31	31	31	43.4	2.5	0.1	4

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item (test 1 and 2) attained respectively 0% and 2% degradation after 28 days, and 0% and 4% degradation after 60 days. Therefore the test material cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline

Executive summary:

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed that described in the OECD Guideline 301F (1992) and EU Method C.4-D (2008).

The test item, at a concentration of 104.5 mg/L in test 1 and at a concentration of 99.5 mg/L in test 2 was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in diffused light at 22°C ± 1°C for 28 days.

 The degradation of the test item was assessed by the measurement of daily oxygen consumption between days 0 and 28. Control solutions with inoculum and the reference material, sodium benzoate, together with a toxicity control were used for validation purposes.

The test item (test 1 and 2) attained respectively 0% and 2% degradation after 28 days, and 0% and 4% degradation after 60 days. Therefore the test material cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline

Description of key information

Constituent approach:

- C6 isomers of the registered substance (parent): experimental study (OECD 301C). 19% biodegradation after 28 days. Not readily biodegradable, but totally primarily degraded within 28 days to form a transformation product (C6 isomer) at 79% and Propionic acid at ca. 20%, but mineralised. 

- Registered substance (C6 and C7 isomers): QSAR prediction (Catalogic 301C v.08.12). 11% biodegradation after 28 days. Not readily biodegradable, but primarily degraded to form a transformation product (C6 and C7 isomers).

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Type of water:

freshwater

Additional information

Experimental and QSAR data are available to assess the biodegradability of the registered substance.

 

The first readily biodegradation study (Yokohama Lab, 2009), assessed as a key study, was performed on the C6 isomers of the registered substance according to a guideline similar to the OECD Guideline 301C. After 28 days, the overall degradability observed was 19%. Therefore, the test substance is not readily degradable. However, all of the substance disappeared and a transformation product C6 was formed at a rate of 79%. The other transformation product Propionic acid is considered to have been mineralised, suggesting that the parent compound was totally primarily degraded within 28 days. The primary biodegradation of the C6 isomer of the registered substance was confirmed with a QSAR prediction (Catalogic Kinetic 301C v.08.12) and the same primary biodegradation was observed for the C7 isomer of the registered substance, using the same QSAR model.

The second ready biodegradation study (Weston, 1995a), performed on the commercial grade of the registered substance according to the OECD Guideline 301B, was assessed as a supporting study. After 29 days the substance attained 20.7% and 16.4% degradation at nominal concentration of 10 and 20 mg/L, respectively. Based on this result, the substance is not considered readily biodegradable.

The third readily biodegradation study (Safepharm, 1994), performed also on the commercial grade of the registered substance but according to OECD Guideline 301 D, was assessed as a supporting study. After 28 days the substance attained 17% degradation, therefore the substance is not considered readily biodegradable.

In addition, one other supporting study is available (Weston, 1995b). This study was performed on the commercial grade of the registered substance according to internal standard protocol. In this study, activated sludge was exposed to a specified concentration of the substance and the soluble organic carbon (SOC) was analyzed after a specific time interval to determine the percent soluble carbon removal. The average percent (%) SOC removal with 95% confidence limits for the test substance after fourteen days was 25.0 +/- 1.6.

 

In conclusion, the registered substance is not considered readily biodegradable but the key studies demonstrate that the registered substance is entirely biotransformed and therefore is not persistent. Indeed, total primary biodegradation of the parent substance is demonstrated. To conclude on the persistence of the relevant transformation product, a screening non-GLP biodegradation study (Firmenich, 2013) was performed according to OECD Guideline 301F and EU Method C.4 -D on the C6 and C7 isomers of the transformation product. In this study, 0 to 4% biodegradation was observed after 28 days and after prolonging to 60 days, respectively. This result is confirmed with the QSAR prediction (Catalogic Kinetic 301C v.08.12). Indeed, in addition to the predictions on the parent molecules, model output biodegradation predictions on a mixture of the primary transformation products: C6 (75 %) & C7 (10.3 %) are also consistent with the above non-GLP experimental biodegradation study (Firmenich, 2013). Therefore, the transformation product of the registered substance can be considered as potentially persistent and in the absence of simulation testing, may be considered as meeting Persistency criteria.