(E)-3-methyl-5-cyclopentadecen-1-one

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 2 June 2000 to 14 July 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed according to OECD Guideline 302C under GLP compliance. No Certificate of Analysis was provided in the study report but the purity and isomers composition were provided by the Sponsor. Thus this study can be considered as reliable without restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Appearance: Colorless transparent liquid
- Melting point: -20°C
- Boiling point: 316.8 - 339.8 °C (988 mbar)
- Vapor pressure: 0.04 Pa (25°C)
- Density: 0.925 - 0.935 g/cm3
- Solubility: Water 0.07 mgIL (20°C)

Oxygen conditions:

aerobic

Inoculum or test system:

mixture of sewage, soil and natural water

Details on inoculum:

- Sludge sampling sites: On-site sludge sampling was carried out at the following 10 locations in Japan.
Fushikogawa city sewage plant (Sapporo-shi, Hokkaido)
Kashima industrial sewage plant (Kashima-gun, Ibaragi)
Nakahama city sewage plant (Osaka-shi, Osaka)
Ochiai city sewage plant (Shinjuku-ku, Tokyo)
Kitakami River (Ishinomaki-shi, Miyagi)
Shinano River (Nishikanbara-gun, Niigata)
Yoshino River (Tokushima-shi, Tokushima)
Lake Biwa (Otsu-shi, Shiga)
Hiroshima Bay (Hiroshima-shi, Hiroshima)
Dookai Bay (Kitakyushu-shi, Fukuoka)
- Sludge sampling date: March 2000
- Sludge sampling, city sewage: return sludge from sewage plants were collected.
- Sludge sampling, rivers lake and sea: surface water and surface soil which was in contact with the atmosphere were collected.
- Preparation of activated sludge: Activated sludge was prepared as follows to maintain its uniformity. The filtrate (5 L) ofthe supernatant of the activated sludge (the activated sludge cultivated the mixed filtrate (10 L) of the supernatant of sludge collected at the ten locations) cultivated about for 3 months was mixed with the mixed filtrate (5 L) ofthe supernatant of a sludge collected newly at each location. The mixed filtrate (10 L) was aerated (prefiltered open air was used) after the pH value of the mixture was adjusted to 7.0±1.0.
- Synthetic sewage: Glucose, peptone and potassium dihydrogenphosphate were dissolved in dechlorinated water to obtain 50 g/L ofthe solution for each component. The pH of the solution was adjusted to 7.0±1.0 with sodium hydroxide
- Cultivation: Roughly 30 minutes after ceasing aeration of the sludge mixture, supernatant corresponding to about 1/3 of the whole volume was removed. Dechlorinated water was added to the remaining portion so that the total volume reached 10 L. This mixture was aerated, and then a predetermined amount of synthetic sewage was added to the mixture so that the concentration of the synthetic sewage was 0.1 wt% in the volume of dechlorinated water added. This procedure was repeated once every day. Cultivation was carried out at 25±2 °C.
- Control and use: During cultivation, the appearance ofthe supernatant, sedimentation ofthe sludge, formation of flock, pH, dissolved oxygen concentration in the solution and temperature were checked to maintain a normal state of sludge. It was confirmed that these were within the scope of the control standard stipulated in the "Testing Methods for New Chemical Substances", and these results were stored as raw data. Microflora in the activated sludge was microscopically observed and sludge with no abnormal symptoms was used for the test.
- Inspection of activity: Activity of the sludge was assessed using a reference substance

Duration of test (contact time):

28 d

Initial conc.:

30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

- Addition of test substance or aniline:
(a) Test solution (water + test substance) (n=2): In one test vessel, 9 mg of the substance supplied by the sponsor was accurately weighed and added to 300 mL of purified water, so that the concentration ofthe test substance reached 30 mg/L.
(b) Test solution (sludge + test substance) (n=3, Vessel No.1, 2 and 3): In each test vessel, 9 mg of the substance supplied by the sponsor was accurately weighed and added to the basal culture medium (the volume was less than 300 mL by the volume of activated sludge inoculated, 6.25 mL), so that the concentration of the test substance reached 30 mg/L.
(c) Test solution (sludge + aniline) (n=1, Vessel No.6): In one test vessel, 29.5 µL [30 mg = 29.5 µL x 1.022 g/cm3 (density)] of aniline was added into the basal culture medium (the volume was less than 300 mL by the volume of activated sludge inoculated, 1.88 mL), so that the concentration reached 100 mg/L.
(d) Test solution (control blank) (n=2, Vessel No.4 and 5): In one test vessel, nothing was added to the basal culture medium (the volume was less than 300 mL by the volume of activated sludge inoculated, 6.25 mL).

- Inoculation of activated sludge: The activated sludge was added to each test vessel, (b) and (d), so that the concentration of the suspended solid reached 100 mg/L, and test vessel (c), so that the concentration of the suspended solid reaches 30 mg/L.

- Cultivation temperatire: 25 +/- 1°C
- Stirring method: Each test solution is stirred by a magnetic stirrer

- Observation of test solution: The appearance of the test solution was observed periodically and conditions of the instruments were checked properly.
- Measurement of biochemical oxygen demand (BOD): During the test period, the change in BOD of the test solutions was measured by autorecording using a data sampler. Cultivation temperature was measured and recorded once a day.

Reference substance:

aniline

Key result

Parameter:

% degradation (test mat. analysis)

Value:

100

Sampling time:

28 d

Remarks on result:

other: GC analysis

Key result

Parameter:

% degradation (O2 consumption)

Value:

85

Sampling time:

28 d

Remarks on result:

other: BOD

Details on results:

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

Results with reference substance:

Percentage biodegradations of aniline calculated by the BOD values were 69 % and 75 % after 7 and 14 days, respectively. It was concluded that this test conditions were valid

Table 5.2.1/1: Appearances of test solutions

	Test solution	Appearance
At the start of cultivation	Water + test substance	The test substance was not dissolved
	Sludge + test substance	The test substance was not dissolved
At the termination of cultivation	Water + test substance	Insoluble compound was observed
	Sludge + test substance	Insoluble compound except the sludge could not be observed. Growth of the sludge was observed.

Table 5.2.1/2: Analytical results of test solutions after 28 days

		Water + test substance		Sludge + test substance			Theoretical amount
		1	2	Vessel-1	Vessel-2	Vessel-3
BOD*	mg	-	-	23.0	25.3	21.5	27.5
Residual amount and percentage residue of test substance (GC)	mg	8.8	8.7	0.0	0.0	0.0	9.0
	%	97	96	0	0	0	-

* The value of control blank was substracted from the values of the test solutions (sludge + test substance)

Table 5.2.1/3: Percentage biodegradation after 28 days

Method	Percentage biodegradation (%)
	Vessel-1	Vessel-2	Vessel-3	Average
BOD	94	92	78	85
GC	100	100	100	100

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The test substance was biodegraded by microorganisms under the present test conditions.

Executive summary:

This test was performed to evaluate the biodegradability of the test substance by microorganisms according to OECD Guideline 302C with GLP statement.

Conditions of cultivation:

- Concentration of test substance: 30 mg/L

- Concentration of activated sludge (as the concentration of suspended solid): 100 mg/L

- Volume of test solution: 300 mL

- Cultivation temperature: 25 +/- 1°C

- Cultivation duration: 28 days

Measurement and analysis:

- Measurement of biochemical oxygen demand (BOD) by means of a closed system oxygen consumption measuring apparatus

- Determination of test substance by means of gas chromatography (GC)

Results:

- Percentage biodegradation by BOC: 84%, 92%, 78%, average 85%

- Percentage biodegradation by GC: 100%, 100%, 100%, average 100%

In conclusion the test substance was biodegraded by microorganisms under the present test conditions.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 21 MAR 1996 to 04 JUN 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study follows the OECD 301F guideline and is GLP compliant. No Certificate of Analysis was provided in the study report but the purity and isomers composition were provided by the Sponsor. The report lacks information on the ageing and the handling of the inoculum. Thus the results reported are reliable but with restrictions.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 22 January 1996. Signed on 27 February 1996

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 activated sewage treatment micro-organisms was obtained on 21 March 1996 from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, U.K., which treats predominantly domestic sewage
- Storage conditions: no data
- Storage length: no data
- Preparation of inoculum for exposure: 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 and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. A sub-sample of the sewage sludge was removed and the suspended solids concentration determined.
- Pretreatment: none
- Concentration of sludge: 30 mg/L of total suspended solids
- Initial cell/biomass concentration: no data

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: as recommended in the guideline.
- Additional substrate: none
- Solubilising agent (type and concentration if used): none
- Test temperature: 21°C thermostatic bath
- pH: from 6.7 to 8.0
- pH adjusted: no data
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes (darkened bottles)

TEST SYSTEM
- Culturing apparatus: BOD bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: no data
- Measuring equipment: oxymeter and Yellow Springs BOD Probe
- Test performed in closed vessels due to significant volatility of test substance: yes
- Test performed in open system: no

SAMPLING
- Sampling frequency: daily
- Sampling method: BOD determination
- Sterility check if applicable: none
- Sample storage before analysis: room temperature

CONTROL AND BLANK SYSTEM
- Inoculum blank: duplicate
- Abiotic sterile control: none
- Toxicity control: one vessel

STATISTICAL METHODS: no data

Reference substance:

aniline

Remarks:

at 100 mg/L

Preliminary study:

None

Test performance:

No unusual observations during the test.

Key result

Parameter:

% degradation (O2 consumption)

Value:

61

Sampling time:

28 d

Remarks on result:

other: 10d window validation criteria was not met (only ca. 52% biodegradation at the 10d window)

Details on results:

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

Results with reference substance:

92% biodegradation after 28 days (O2 consumption).

None

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

The test substance biodegraded up to 61% after 28 days but failing 10-day window. The substance is not considered readily biodegradable under the strict conditions of OECD Guideline.

Executive summary:

The readily biodegradability of the test substance was assessed in a GLP compliant OECD 301F “Manomteric Respirometry Test” study.

Briefly, 100 mg/L (1 000 times the water solubility) of test substance was put into contact with non adapted activated sludge from a domestic sewage treatment plant for 28 days in a closed bottle system. Daily, O2 consumption from bacteria as a proxy for bacterial activity was monitored and compared to the theoretical oxygen demand. Aniline was used a reference substance and a control vessel with only inoculum was set up. In addition, a toxicity control was set up. The test group and the toxicity control was duplicated.

After 28 days, 61% of biodegradation was calculated in the test group with a 10-day window starting at day 6 (12%). On day 16 biodegradability rate did not reach 60% but 52%. Thus criteria were not met to fulfill the 10-day window. The reference substance degraded at 92% after 28 days meeting the 10-day window showing evidence of appropriate conditions for the test. The toxicity control did not show evidence of toxicity to bacteria. All validity criteria were met in this study.

In conclusion, the test substance biodegraded up to 61% after 28 days, but failing the 10-day window. The substance is not considered readily biodegradable under the strict conditions of OECD Guideline.

Reference 3

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This Semi Continuous Activated Sludge (SCAS) study was performed according to OECD Guideline 302A under GLP compliance. The test duration was shorter than recommended but measured results confirm this shortening. No Certificate of Analysis was included in the study report but the purity and isomers composition were provided by the Sponsor. Thus this study can be considered as reliable without restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 A (Inherent Biodegradability: Modified SCAS Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Specific details on test material used for the study:

- Molecular formula: C16H28O
- Molecular weight: 236.37
- Solubilty in water < 7 x 10-5 g/L at 20°C

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): from the DRWPCC sample on 7 February 1995
- Preparation of inoculum for exposure: Approximately 200 mL of mixed liquor was collected composited and homogenized at medium speed in a blender for ca 2 min. The homogenized sample was poured into a beaker and allowed to settle for ca 30 min. the supernatant was decanted to the test flasks at a concentration of 1%v/v.
- Pretreatment: The sludge was screened through a 2 mm sieve to remove large clumps. The units were aerated for 23.5 hours (+- 0.5 hr.) at a rate adequate to maintain solids suspension. At the end of this period, the air was turned off and the sludge was allowed to settle for - 30 minutes. One liter of effluent was drawn off and replaced with one liter of influent consisting of 10 mL synthetic sewage and 990 mL tap water to bring the volume back to 1.5 liters. The air was turned on and this process was repeated on a daily basis
- Concentration of sludge: 3 280 mg of total suspended solid/L adjusted to a final concentration of 2 500 mg of total suspended solid/L.
- Initial cell/biomass concentration: 2.4x106 CFU/mL

Duration of test (contact time):

7 d

Parameter followed for biodegradation estimation:

other: soluble organic carbon

Details on study design:

see details below, in "Any other information on materials and methods incl. tables"

Reference substance:

not required

Key result

Parameter:

other: Soluble Organic Carbon removal

Value:

99.9

St. dev.:

1.6

Sampling time:

7 d

Details on results:

During the seven-day testing period, the effluents withdrawn from each unit were analyzed for SOC. On day five (26 February 1995) the SOC result for the test substance (2) was 59.8 mg/L. The same sample was repurged and reanalyzed. The result was 57.9 mg/L. A fresh aliquot was taken from the stored effluent collected on day five. The sample was prepared and submitted for SOC analysis to confirm the original results. The result of this final analysis was 47.5 mg/L. Based on the amount of carbon originally added to the test units and all other SOC values, this result is considered an outlier which most probably occurred due to a decrease in air flow to the unit. The value was not used in the average %SOC removal calculation.

Table 5.2.1/1: Soluble Organic Carbon Data

Day of testing period	Control (1) (mg C/L)	Control (2) (mg C/L)	Test substance (1) (mg C/L)	Test substance (2) (mg C/L)
1	7.8	6.7	6.5	6.1
2	9.1	8.0	10.2	9.1
3	8.7	7.7	7.1	6.7
4	7.6	7.4	6.8	6.7
5	7.5	7.1	7.4	58.9 ; 57.8 ; 47.5*
6	6.4	6.4	7.3	7.7
7	6.5	6.0	7.5	6.8

* Values were found to be outstanding values and were not used in calculation of SOC removal

Validity criteria fulfilled:

not applicable

Interpretation of results:

inherently biodegradable

Conclusions:

The average SOC removal was 99.9% ± 1.6% for the test substance. Thus the test substance can be regarded as ultimately biodegradable.

Executive summary:

The inherent biodegradability of the test substance was assayed following an OECD 302A, semi continuous activated sludge (SCAS) removability test under GLP compliance. Degradability of the test substance was monitored in the test system by measurements of soluble organic carbon (SOC).

The activated sludge used was obtained from a municipal treatment plant receiving predominantly domestic waste. It was aged properly to eliminate any residual organic carbon from the sludge during the sludge acclimation period. Then during the 7-day test substance acclimation period, the test substance was added daily increasing the concentration by 20% each day to reach a final concentration of 20 mg/L (water solubility is 0.9 mg/L). The volume in each vessel was then adjusted to 1.5L using tap water. The inoculum was thus adapted to the test substance within just 7 days and the test was commenced.

For the following 7 days, in duplicate, 1L from each test and control vessels were sampled for soluble carbon analysis after centrifugation, acidification and purged from nitrogen. Daily SOC measures from control were then subtracted from the test group and divided by the theoretical organic carbon content. On day 5 after repeating analysis three times, in only one replicate of the test group, values ten times the one in other vessels were measured. Those values were considered as outliners and were used in calculation or statistical analysis. An ANOVA was performed to test difference between groups and time and concluded no significant difference between control and test groups were observed.

Finally the average SOC removal was 99.9% ± 1.6% for the test substance. This level of removal was already noted from day 1 of the study thus the microorganisms had completely adapted to the test substance within a period of just 8 days.

Thus the test substance can be regarded as ultimately biodegradable and would be expected to completely mineralise within a WasteWater Treatment Plant (WWTP).

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 28-02-1995 to 28-08-1995

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

unsuitable test system

Remarks:

This study indicates it followed an OECD 301B and is GLP compliant. No Certificate of Analysis was provided in the study report but the purity and isomers composition were provided by the Sponsor. The reference substance (D-Glucose) is not the one recommended by the guidelines, and results indicate higher biodegradability of the test substance than for the reference substance. However the inoculum was taken after an OECD 302 A (SCAS) on the same test substance was conducted. Under such conditions, the inoculum is regarded as adapted and the conditions for a stringent OECD 301B are not respected. Thus this study cannot conclude on the readily biodegradability of the substance but only on its ultimate biodegradability. Therefore this study can be considered as not reliable and cannot be used for assessing the readily biodegradation of the test substance.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

. However an adapted inoculum was used.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Specific details on test material used for the study:

- Molecular formula: C16H28O
- Molecular weight: 236.37
- Solubilty in water < 7 x 10-5 g/L at 20°C

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): from the DRWPCC sample on 7 February 1995
- Preparation of inoculum for exposure: Approximately 200 mL of mixed liquor was collected composited and homogenized at medium speed in a blender for ca 2 min. The homogenized sample was poured into a beaker and allowed to settle for ca 30 min. the supernatant was decanted to the test flasks at a concentration of 1%v/v.
- Pretreatment: The sludge was screened through a 2 mm sieve to remove large clumps. The units were aerated for 23.5 hours (+- 0.5 hr.) at a rate adequate to maintain solids suspension. At the end of this period, the air was turned off and the sludge was allowed to settle for - 30 minutes. One liter of effluent was drawn off and replaced with one liter of influent consisting of 10 mL synthetic sewage and 990 mL tap water to bring the volume back to 1.5 liters. The air was turned on and this process was repeated on a daily basis
- Concentration of sludge: 3 280 mg of total suspended solid/L adjusted to a final concentration of 2 500 mg of total suspended solid/L.
- Initial cell/biomass concentration: 2.4x106 CFU/mL

Duration of test (contact time):

>= 28 d

Initial conc.:

10 mg/L

Based on:

act. ingr.

Initial conc.:

20 mg/L

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium:
1 mL magnesium sulfate solution, 2.25% (w/v)
1 mL calcium chloride solution, 2.75%
2 mL phosphate buffer pH = 7.2
4 mL ferric chloride solution, 0.025%
1 mL of a 4% (w/v) solution of ammonium sulfate
- Additional substrate: none
- Solubilising agent (type and concentration if used): none
- Test temperature:22.4°C-23.3°C
- pH: no data
- pH adjusted: no data
- Aeration of dilution water: no data
- Suspended solids concentration: no data
- Continuous darkness: yes/no no data

TEST SYSTEM
- Method used to create aerobic conditions: aeration 110 rpm
- Measuring equipment: titration
- Details of trap for CO2 and volatile organics if used: 3 absorber bottles were filled of 0.024 N Ba(OH)2 and connected in series to the exit air line of each test flask.

SAMPLING
- Sampling frequency: on day 2, 3, 6, 8, 10, 13, 16, 20, 24, 28, 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 1980 mL of dilution water + 20 mL of inoculum
- Abiotic sterile control: none
- Toxicity control: none

STATISTICAL METHODS: regression model to fit best curve

Reference substance:

other: D-glucose

Preliminary study:

none

Key result

Parameter:

% degradation (CO2 evolution)

Value:

96.4

Sampling time:

28 d

Remarks on result:

other: within the 10d window / test concentration: 10 mg/L

Key result

Parameter:

% degradation (CO2 evolution)

Value:

93.4

Sampling time:

28 d

Remarks on result:

other: within the 10d window / test concentration: 20 mg/L

Details on results:

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

Table 5.2.1/2: Summary of cumulative CO2 production 

	%ThCO2
day	D-Glucose (20 mg/L)	Test substance (10 mg/L)	Test substance (20 mg/L)
2	26.4	16.6	12.3
3	38.9	35.3	34.4
6	48.2	56.1	53.9
10	51.6	68.5	65.1
13	54.9	76.1	73.1
16	58.5	83.1	80.8
20	60.8	88.1	85.2
24	64.2	92.0	88.8
28*	67.5	95.7	93.2
29	68.2	96.4	93.4

* Test flasks were acidified after the traps were sampled for titration

Validity criteria fulfilled:

yes

Interpretation of results:

other: ultimately biodegradable

Conclusions:

In conclusion, because of the use of an adapted inoculum, this study cannot state on the readily biodegradation of the test substance. According to these experimental conditions the test substance can be regarded as ultimately biodegradable.

Executive summary:

The readily biodegradability of the test substance was assessed according with OECD 301B “CO2 evolution” guidelines and under GLP compliance.

Briefly, two concentrations of the test substance (10 and 20 mg/L) was put into contact with an inoculums of aged activated sludge for up to 28 days in closed vessels. As the substance was biodegraded, bacteria evolved respiratory CO2 which was then captured in BaOH vessels and CO2 analysed by titration. The activated sludge originated from a waste water treatment plant receiving predominantly domestic sewage. The inoculum was aged and handled so the only carbon source available for biomass was the test substance. However, the inoculum was adapted in an OECD 302A (SCAS) test. D-Glucose was used as reference substance et 20 mg/L. It is not a recommended substance of reference however it is the primary source of carbon of aerobic bacteria. CO2 evolution was analysed on day 2, 3, 6, 8, 10, 13, 16, 20, 24, and 28. On day 29 all test vessels were acidified to measure remaining carbon. The percentage of biodegradation was calculated as the difference between the theoretical CO2 production and the ones analysed in test flasks. A blank vessel was used to delete blank results from test and reference vessel analyses.

After 28 days, at 10 mg/L and 20 mg/L, 96.4% and 93.4% respectively of the test substance were degraded and 68.2% of the reference substance was degraded. The final carbon content in the blank control was < 0.5 mg C/L and 0.7 mg C/L and 1.0 mg C/L in the test vessels. All validity criteria were thus met and the test substance met also the 10-day window. No bacteria toxicity was assumed according to results.

In conclusion, because of the use of an adapted inoculum, this study cannot sate on the readily biodegradation of the test substance. According to these experimental conditions the test substance can be regarded as ultimately biodegradable.

Reference 5

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 9-08-1999 to 7-01-2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study followed guidelines and compliant with GLP and scientific purposes due to the test substance. It met all validity criteria and results were well reported. No Certificate of Analysis was provided in the study report but the purity and isomers composition were provided by the Sponsor. The 10d window cannot be verified based on the choice of time of measurement. However, based on the graph present in the study report, the replicat 1 and the mean fulfilled the 10d validation criteria. Thus this study can be considered as reliable without restrictions.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

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): Activated sludge was collected from Prospect Bay Wastewater Treatment Facility, GrasonviIle, Maryland on August 11, 1999
- Pretreatment: The sludge was sieved using a 2 mm screen and then aerated for four hours. After the aeration period, an aliquot of the sludge was homogenized in a blender at medium speed for approximately two minutes and then allowed to settle for approximately 30 minutes. The supernatant was poured into 4-250 ml centrifuge bottles and centrifuged for 5 minutes at 1000 rpm. The supernatant was then poured off and used as the inoculum. A total suspended solids measurement and standard plate count were performed on the inoculum. Plates were incubated at 20±3 °C for approximately 48 hours.
- Concentration of sludge: 83 mg/L of total suspended solid
- Initial cell/biomass concentration: 8.4 x 10^2 CFU/mL
- Water filtered: no

Duration of test (contact time):

29 d

Initial conc.:

10 mg/L

Based on:

ThIC

Parameter followed for biodegradation estimation:

CO2 evolution

Parameter followed for biodegradation estimation:

DOC removal

Remarks:

at test termination

Details on study design:

The test chambers were ambered 4-liter gas-washing-bottles .The air entering the chambers was passed through Drierite to remove ambient moisture and then through Ascarite to produce CO2 free air. The air exiting the test-chambers was passed through a series of three gas washing bottles each containing approximately 100 mL of 0.5 N KOH to trap the C02 that had evolved within the chamber. An additional set of gas washing bottles that were not connected to a chamber were maintained concurrently with the traps connected to the chambers. These bottles contained approximately 100 mL of 0.5 N KOH and the amount of CO2 detected in the KOH solution was subtracted from the CO2 in the blank control traps to determine the amount of CO2 produced by the inoculum in the blank control.

Reference substance:

benzoic acid, sodium salt

Remarks:

10 mg C/L

Preliminary study:

None

Test performance:

no unusual observation during the test was reported.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

78.8

St. dev.:

8.8

Sampling time:

29 d

Remarks on result:

other: mean value; met 10-day window

Parameter:

% degradation (CO2 evolution)

Value:

77.1

St. dev.:

7.9

Sampling time:

26 d

Remarks on result:

other: mean value

Details on results:

See tables in "Any other information on results incl. tables" for further details.

Results with reference substance:

Reference substance degraded at 88.8% and 91.1% of theoretical CO2 after 26 and 29 days, respectively; and the 10-day window started on day 7 and was over 60% by day 17.

Table 5.2.1/1: Dissolved Organic Carbon Concentration and pH of Test Solutions at Test Termination

Test Chamber	DOC mg C/L	pH
Control Rep A	<1.0	6.6
Control Rep B	<1.0	6.6
Sodium Benzoate Rep A (10 mg CL)	<1.0	6.3
Sodium Benzoate Rep B (10 mg CL)	<1.0	5.5
Test substance Rep A (10 mg C/L)	<1.0	6.4
Test substance Rep B (10 mg C/L)	<1.0	5.9
Test substance Killed control (10 mg C/L)	1.4	5.8
Test substance Toxicity control (10 mg C/L)	<1.0	6.1

Table 5.2.1/2: Cumulative Percent of Theoretical Carbon Dioxide Evolved

Date	Day	Control Rep A	Control Rep B	Benzoate Rep A	Benzoate Rep B	Test substance Rep A	Test substance Rep B	Test substance Killed control	Test substance Toxicity control
13-August-99	1	N/A	N/A	0.5	0.3	0.5	0.4	0.3	0.3
16-August-99	4	N/A	N/A	0.6	39.3	1.9	1.1	-0.5	23.3
19-August-99	7	N/A	N/A	60.5	63.4	3.2	4.5	-3.7	43.6
23-August-99	11	N/A	N/A	62.9	75.2	47.9	35.1	-1.5	52.2
26-August-99	14	N/A	N/A	63.1	79.7	59.2	49.2	-1.7	55.5
30-August-99	18	N/A	N/A	62.9	82.5	69.4	56.6	-2.6	58.5
02-September-99	21	N/A	N/A	71.1	86.5	76.0	63.7	-0.9	60.4
07-September-99	26	N/A	N/A	89.0	88.6	82.7	71.5	-1.5	62.9
10-September-99	29	N/A	N/A	90.2	92.0	85.0	72.6	-2.3	64.8
Mean Std deviation					91.1 1.3		78.8 8.8

 

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The mean biodegradation rate of the test substance was 77.1% +/- 7.9% and 78.8% +/- 8.8% after 26 and 29 days, respectively, within the 10-day window. The substance is considered readily biodegradable.

Executive summary:

The ready biodegradability of the test substance was assessed in an OECD 301B “CO2 evolution test”, GLP compliant study.

Briefly, 10 mg C/L equivalent of test substance was put into contact with an activated sludge sample from a predominantly domestic sewage treatment plant. Bacteria will transform organic carbon of the test substance into CO2. CO2 evolved will be trapped in bottles filled with KOH and then analysed by titration. Biodegradation is calculated from the percentage of CO2 evolved relatively to the theoretical carbon content in the test substance. Sodium benzoate at 10 mg C/L was used as reference substance. Control, test and reference substance was duplicated. An abiotic (killed control) and a toxicity control vessel were added to the test system.

Each vessel was analysed for CO2 at day 1, 4, 7, 11, 14, 18, 21, 26, and 29. The abiotic control gave evidence for no CO2 evolution in the absence of inoculums. The reference substance was degraded at 88.8% and 91.1% after 26 and 29 days, respectively, and meeting the 10-day window showing evidence the inoculum was activated. The average biodegradation rates of the test substance after 26 and 29 days, respectively, were 77.1% ± 7.9% and 78.8% ± 8.8%, and the 10 -day window started between day 7 and day 11 and passed the 60% level at day 18 (mean value). The standard deviation between test replicates during the 10 -day window was less than 20%. The toxicity control did not reveal any toxicity to bacteria and the control vessels evolved an average of 13.6 mg C/L. Thus all validity criteria were met to rely on the reported results.

In conclusion, the test substance was readily biodegradable meeting the 10-day window.

Description of key information

OECD Guideline 301B, GLP, key study, validity 1:
77.1% and 78.8 % biodegradation after 26 and 29 days, respectively, within the 10-day window.
Readily biodegradable.

OECD Guideline 302A, GLP, key study, validity 1:
99.9% removal
Ultimately biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

The biodegradability of the substance has been tested in six GLP compliant studies and following OECD guidelines.

Three valid studies tested readily biodegradability: 301B “CO2 evolution test” (Wildlife International, 2000), 301D “Closed bottle test” (Safepharm, 1995), 301F (Safepharm, 1996) and two valid studies tested inherent biodegradability: 302A (Weston, 1995a) and 302C (Kurume Lab, 2000). One last study (Weston, 1995b) followed a 301B guideline, however an adapted inoculum was used thus readily biodegradability from this study could not be concluded and it is considered not reliable.

The 301B (Wildlife International, 2000) and 302A (Weston, 1995a) studies were assessed as key studies. The first key study showed ready biodegradability fulfilling the 10-day window criteria (Wildlife International, 2000). The second key study allows to conclude that the 99.9% of removal of the substance (observed in this study) is also likely to occur in real WasteWater Treatment Plants (WWTP). According to the strict terms of the OECD 302A guideline, the test does not simulate the conditions experienced in a WWTP based on the long detention period and the intermittent addition of nutrients which can promote the adaptation of microorganisms. However, almost total and consistent loss of the TOC was observed even after day 1 of the study (after 7 days of preadatation of the sludge to increasing concentrations of the substance). Throughout the entire test period the substance was totally degraded within 24 hours of addition. As according to the REACH guidance this study can be used to refine the PEC and as the loss was so extensive and consistent over the study that it is concluded that the results from this test can be considered as reasonable evidence that the substance would be completely removed from a WWTP. While it is recognised that some of this substance may have been lost due to volatility, this would also be expected to occur in a WWTP and therefore the result of 99.9% is considered to account for "Total removal" in the STP and not just biodegradation in the STP .

For the other valid studies, the substance is not considered readily biodegradable. The biodegradation rate did not reach a plateau after 28 days (Safepharm, 1995) or the 10-day window was failed (Safepharm, 1996). In addition, in the 302C test (Kurume Lab, 2000), the substance is inherently biodegradable, and support the key study (Weston, 1995a).

 

In conclusion, a rapid biodegradation of the substance is proved based on the key OECD 301B study (readily biodegradable, 10d-window fulfilled after only 17 days) and the total degradation observed in the OECD 302A study after only 1 day.