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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 December 2013 to 2 January 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: New study performed to GLP and to internationally accepted guidelines
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
- Source of inoculum: Worlingworth sewage treatment works (Suffolk, UK), the sludge was sieved (1 mm2)
- Preparation of inoculum for exposure: after transportation to the laboratory and it was left to stand for approximately 30 minutes to allow the sewage solids to settle. A portion of the supernatant was removed and the sludge aerated until required.
- Concentration of sludge:30 mg/L in test cultures
Duration of test (contact time):
28 d
Initial conc.:
50 other: mgO2/L (oxygen demand)
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:mineral salts medium
- Test temperature:22 ± 2ºC
- pH: 7.45
- pH adjusted: no
- Continuous darkness: yes

Kalama® K-Flex® PG was added to two bottles containing mineral salts medium inoculated with activated sludge (30 mg solids/L) to give a nominal test concentration of 50 mgO2/L. Two control cultures contained inoculated mineral salts medium alone. Two cultures contained inoculated mineral salts medium plus the reference substance sodium benzoate (50 mgO2/L), of which one also contained Kalama® K-Flex® PG (50 mgO2/L) in order to
assess the potential inhibitory effects of the test substance on the microbial inoculum. The test system comprised of an automated system for oxygen (O2) generation and the cultures were stirred and held in a thermostatically-controlled water bath.
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
7
Sampling time:
1 d
Parameter:
% degradation (O2 consumption)
Value:
32
Sampling time:
2 d
Parameter:
% degradation (O2 consumption)
Value:
60
Sampling time:
7 d
Key result
Parameter:
% degradation (O2 consumption)
Value:
81
Sampling time:
28 d
Results with reference substance:
The blank-corrected oxygen demanded by the culture containing the reference substance had achieved 16.04 mgO2/500 mL or 64% of the ThOD (25 mgO2/500 mL) after 3 days of incubation and 25.8 mgO2/500 mL or 103% by Day 28. Although slightly in excess of that theoretically available (100%) this was not considered significant as it is within the level of experimental error for this study type. In the presence of Kalama® K-Flex® PG, degradation of sodium benzoate had achieved 63% by Day 5. Cumulative levels of oxygen consumption by the controls after 28 days (17.95 and 19.63 mgO2/500 mL, equivalent to 35.90 and 39.26 mgO2/L) were considered to be acceptable for this assay system. These results confirm that Kalama® K-Flex® PG was not inhibitory to the activity of the microbial inoculum and that the test was valid.

The pH of mixtures ranged between 7.39 and 7.49 at the start of the test and 7.38 and 7.60 at the end. The temperature of the water bath was measured daily ranged from 21.9 to 22.9˚C.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Mean oxygen consumption in mixtures containing Kalama® K-Flex® PG was 7% of the theoretical value (25 mgO2/500 mL) after 1 day, 32% after 2 days, 60% after 7 days and 81% at the end of the test (Day 28). Therefore, Kalama® K-Flex® PG was considered to be readily biodegradable under the conditions of this test.
Executive summary:

Mean oxygen consumption in mixtures containing Kalama® K-Flex® PG was 7% of the theoretical value (25 mgO2/500 mL) after 1 day, 32% after 2 days, 60% after 7 days and 81% at the end of the test (Day 28). Therefore, Kalama® K-Flex® PG was considered to be readily biodegradable under the conditions of this test.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 April 1997 - 17 May 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with OECD, EC, and US EPA test guidelines, and in compliance with GLP.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
Note that the version of this guideline cited in the study report was dated January 1998 (after the testing period but prior to the report issue date).
Deviations:
no
Principles of method if other than guideline:
A preliminary test using the closed bottle method was undertaken prior to the definitive test which followed the modified Sturm procedure.
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): A sample of activated sludge was collected on the day before the test from Oakley STW a sewage treatment works that treats predominantly domestic waste.
- Storage conditions: Not specified.
- Storage length: Not more than one day (refer to above comment)
- Preparation of inoculum for exposure: The solids content of the sludge was determined by filtering the sludge through pre-weighed filters, then drying and re-weighing the filters. The appropriate volume of inoculum was added to the control and test vessels to give a final suspended solids concentration of 30 mg/L.
- Initial cell/biomass concentration: 30 mg/L.
- Water filtered: yes - Ultrapure water was used.
- Type and size of filter used, if any: Whatman GFC filter paper
Duration of test (contact time):
29 d
Initial conc.:
10 other: mgC/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Remarks:
for modified Sturm
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
for preliminary closed bottle assessment
Details on study design:
For definitive modified Sturm test

TEST CONDITIONS
- Composition of medium: Consistent with the medium described in OECD test guideline 301B
- Solubilising agent (type and concentration if used): Solution in acetone was prepared, but the acetone was removed by evaporation under a stream of nitrogen.
- Test temperature: 21.1 - 22.9°C
- pH: 7.6 at the start, 7.3 and 7.5 at the end of the test.
- pH adjusted: no
- Aeration of dilution water: Air flow rate was between 30 to 46 mL/minute
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: Glass bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Continuous flushing with treated air through an air inlet tube reaching approximately 10 cm under the liquid surface.
- Measuring equipment: CO2 evolution measured by trap system described below.
- Test performed in open system: An air outlet was located just below the stopper of each test flask.
- Details of trap for CO2 and volatile organics if used: Three Drechsel bottles in series, each containing 100 mL of 0.025N Barium hydroxide.


SAMPLING
- Sampling frequency: Samples taken on days 2, 3, 4, 6, 10, 12, 14, 18, 24, and 28. A Sample was taken on day 29, in the abiotic phase of the test (test and control mixtures were acidified on day 28 of the test to kill the microbial inoculum and to drive off any residual carbon dioxide).
- Sampling method: The nearest of the three dechsel bottles was removed and the second connected in its place. The residual barium hydroxide
concentration in the removed bottle was determined by duplicate titrations with hydrochloric acid (0.05N) using phenolphthalein indicator.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes (performed in duplicate)
- Abiotic sterile control: No
- Toxicity control: No
Reference substance:
benzoic acid, sodium salt
Remarks:
Both both preliminary and definitive tests
Preliminary study:
Sodium benzoate was degraded to 54% of its ThOD (Theoretical Oxygen Demand) after five days of incubation which indicates that the inoculum was viable and exerting normal biodegradive activity. In the presence of the test material at 10 mg C/L, degradation of sodium benzoate was reduced to 10% of its ThOD
inhibited. This indicated that DPGDB was inhibitory to the inoculum under the conditions of this test.
Oxygen consumption by mixtures containing DPGDB alone at 10 mg C/L was equivalent to 6% of its ThOD which indicated that it was not readily biodegradable under the conditions of this preliminary test.
The level of oxygen consumption in bottles containing inoculated mineral salts medium alone at five days (0.3 mgO2/L) was considered to be acceptable for this assay system.
Parameter:
% degradation (CO2 evolution)
Value:
6
Sampling time:
2 d
Parameter:
% degradation (CO2 evolution)
Value:
62
Sampling time:
12 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
85
Sampling time:
28 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
87
Sampling time:
29 d
Remarks on result:
other: Includes residual CO2 released following acidification of medium.
Key result
Parameter:
BOD5
Value:
0.12 g O2/g test mat.
Results with reference substance:
The results obtained for the degradation of sodiurn benzoate (63% of its TC02 after 6 days and 79% after 29 days) and for cumulative CO2 production by the control mixtures (69.9 and 65.7 mg) fulfil the validity criteria for this test.

Although DPGDB appeared to be inhibitory to the inoculum in the preliminary test, it did not inhibit the biodegradation of benzoic acid in the definitive test (modified Sturm)
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Substances are considered to be readily degradable in the modifed Sturm test if CO2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. DPGDB met these criteria, so may be considered to be readily biodegradable.

Although DPGDB appeared to be inhibitory to the inoculum in the preliminary test, it did not inhibit the biodegradation of benzoic acid in the definitive test (modified Sturm).
Executive summary:

Key data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is presented as an attachment included in Section 13 of the IUCLID dossier.

A ready biodegradability test was performed to determine the biodegradability of the test material DPGDB. The study was conducted according to EC, OECD, and US EPA test guidelines, and in compliance with GLP.

The modified Sturm test DPGDB was found to have degraded by 6% after 2 days, 62% after 12 days, and by 85% at the end of the 28-day biotic phase of the test. The postive control substance, sodium benzoate, which was analysed contemporaneously degraded rapidly (63% degradation after 6 days), and confirmed that the inoculum was viable and that the test was valid.

Substances are considered to be readily degradable in this test if CO2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. In the Modified Sturm test, DPGDB met these criteria, so may be considered to be readily biodegradable.

Description of key information

Key data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is provided as an attachment included in Section 13 of the IUCLID dossier.

Mean oxygen consumption in mixtures containing Kalama® K-Flex® PG (PGDB) was 7% of the theoretical value (25 mgO2/500 mL) after 1 day, 32% after 2 days, 60% after 7 days and 81% at the end of the test (Day 28).Therefore, Kalama® K-Flex® PG was considered to be readily biodegradable under the conditions of the OECD Guideline 301 F, Ready Biodegradability: Manometric Respirometry Test (Huntingdon Life Sciences, 2014c; Klimisch score = 1).       

In a modified Sturm test DPGDB (Huntingdon Life Sciences, 1998a; Klimisch score = 1) was found to have degraded by 6% after 2 days, 62% after 12 days, and by 85% at the end of the 28-day biotic phase of the test.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Key data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is provided as an attachment included in Section 13 of the IUCLID dossier.

Kalama® K-Flex® PG was added to two bottles containing mineral salts medium inoculated with activated sludge (30 mg solids/L) to give a nominal test concentration of 50 mgO2/L. Two control cultures contained inoculated mineral salts medium alone. Two cultures contained inoculated mineral salts medium plus the reference substance sodium benzoate (50 mgO2/L), of which one also contained Kalama® K-Flex® PG (50 mgO2/L) in order to assess the potential inhibitory effects of the test substance on the microbial inoculum. The test system comprised of an automated system for oxygen (O2) generation and the cultures were stirred and held in a thermostatically-controlled water bath.

Mean oxygen consumption in mixtures containing Kalama® K-Flex® PG was 7% of the theoretical value (25 mgO2/500 mL) after 1 day, 32% after 2 days, 60% after 7 days and 81% at the end of the test (Day 28). Therefore, Kalama® K-Flex® PG was considered to be readily biodegradable under the conditions of this test (Huntingdon Life Sciences, 2014c; Klimisch score = 1).

In a modified Sturm test DPGDB was found to have degraded by 6% after 2 days, 62% after 12 days, and by 85% at the end of the 28-day biotic phase of the test. The positive control substance, sodium benzoate, which was analysed contemporaneously degraded rapidly (63% degradation after 6 days), and confirmed that the inoculum was viable and that the test was valid (Huntingdon Life Sciences, 1998a; Klimisch score = 1).

 

Substances are considered to be readily degradable in this test if CO2production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. In the Modified Sturm test, DPGDB met these criteria, so may be considered to be readily biodegradable. The BOD / COD studies (Huntingdon Life Sciences, 1999a, 1998b; Klimisch score = 1) show DPGDB to be inherently biodegradable and the anaerobic study showed it to be anaerobically biodegradable (Huntingdon Life Sciences, 1998c; Klimisch score = 1).