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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:
16th September to 17th October 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and documented study following a guideline and conducted according to GLP
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
yes
Principles of method if other than guideline:
Deviations from the protocol:
- The dilution with tap water represents a protocol deviation because settled sludge effluent is specified as the diluent in the protocol. This unintentional deviation did not impact the study because tap water is a major component of municipal wastewater.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
The test system was activated sludge collected on 16 September 1996 from the Downingtown Regional Water Pollution Control Center (DRWPCC) in Downingtown, Pennsylvania. The sludge was screened through a 2 mm sieve prior to determining the total suspended solids (TSS). Based on the TSS result approximately 1.5 liters of the sludge was adjusted to a target solids level of 2500 mg/L by diluting with tap water. The adjusted sludge was aerated in a semi-continuous activated sludge (SCAS) unit until used in the preparation of the inoculum added to all flasks.
The inoculum was prepared for addition to the CO2 flasks as follows:
Activated sludge was homogenized in a blender for ~2 minutes. The homogenized sample was poured into a beaker and allowed to settle for ~30 minutes. The supernatant was decanted and added to the flasks at a concentration of 1% v/v. On the same day, a standard plate count (SPC) was performed on the inoculum. The plates were incubated at test temperature. The result was 2.1 x 10ˆ5 CFU/mL.
- Storage conditions: Store the stock solution under refrigeration for a maximum of three days prior to test initiation.

Duration of test (contact time):
28 d
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
The test medium was modified BOD water containing the following standard reagent solutions per liter of water:
1 mL magnesium sulfate solution (2.25%), VWR, (Lot - 9502168)
1 mL calcium chloride solution (2.75%), VWR, (Lot - 9502027)
2 mL phosphate buffer (pH 7.2), VWR, (Lot - 9502056)
4 mL ferric chloride solution (0.025%), VWR, (Lot 9503395)
1 mL of a 4% (w/v) solution of (NH4)2SO4, EM Science, (Lot - 35065517)

- Solubilising agent (type and concentration if used): The test substance has limited solubility in water and was hence added to the appropriate test flasks at a concentration of 10 mg C/L by direct weight addition
- Test temperature: 21.6°C-23.4°C
- Aeration of dilution water: Yes, overnight aeration
- Suspended solids concentration: Based on the TSS result approximately 1.5 liters of the sludge was adjusted to a target solids level of 2500 mg/L by diluting with tap water.


TEST SYSTEM
- Culturing apparatus:
CO2-free air was provided to the test flasks through a scrubbing apparatus prepared as follows:
One empty 1-liter plastic bottle to prevent backflow into the air line.
Five 1-liter plastic bottles containing 700 mL 10 N NaOH (EM Science, Lot - 36051).
One 1-liter flask containing 700 mL 0.024 N Ba(OH)2 (to serve as a CO2 indicator trap).
One empty 1-liter plastic bottle to prevent liquid carryover into the test containers.

Six glass 4-liter Erlenmeyer flasks were used as the test vessels for this study. Duplicate flasks were required for the test substance (test suspension) and for the blank control. Single flasks were used for the reference substance and for the toxicity control. The flasks were identified by test substance identification number (TSIN), test concentration, study number, flask number, flask content code, and replicate number where applicable.


CONTROL AND BLANK SYSTEM
- Inoculum blank: Duplicate blank controls containing test medium and inoculum were included to monitor background CO2 production.
- Toxicity control: A single flask receiving sodium benzoate at a concentration of 10 mg C/L (reference substance) was included in the test


Reference substance:
benzoic acid, sodium salt
Test performance:
The CO2 production from the reference chemical exceeded the 60% of theoretical necessary to consider the test valid. The %TCO2 result for the toxicity flask (IC) was > 25 as of day 12, therefore, the test substance is considered to be non-inhibitory at the concentration tested.
Parameter:
% degradation (CO2 evolution)
Value:
4.7 - 10.8
Sampling time:
28 d
Details on results:
Breakdown products were not noted or identified.
Results with reference substance:
Reference (sodium benzoate): 88.1% (0.45 day lag period)

Based on the results the test substance would not be considered readily biodegradable under the European Economic Community (EC) criteria, which requires 60% biodegradation within 28 days, achieved within 10 days of reaching 10% biodegradation.

The reference substance, sodium benzoate, degraded 88.1% in the same test period and the toxicity test showed the test substance to be non-inhibitory, so the test was valid.
Validity criteria fulfilled:
yes
Interpretation of results:
other: Not readily biodegradable
Conclusions:
The test substance may not be considered readily biodegradable under the conditions tested since the pass level of 60% TCO2 was not achieved. However since tests of ready biodegradability provide limited opportunity for biodegradation to occur, a negative result does not necessarily mean that the test substance will not be biodegraded under relevant environmental conditions.
Executive summary:

This test was designed to determine the rate and extent of the ultimate biodegradation of the test substance under aerobic conditions. The test was conducted according to the EU method: "Carbon Dioxide (CO2) Evolution (Modified Sturm Test)" Method C.4-C under conditions of GLP.

The test substance showed approximately 4.7% to 10.8% degradation over the course of the study and based on these results the test substance would not be considered readily biodegradable under the European Economic Community (EC) criteria, which requires 60% biodegradation within 28 days, achieved within 10 days of reaching 10% biodegradation.

 

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2nd April to 4th May 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study following GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
not specified
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Activated sludge was collected from the Prospect Bay Wastewater Treatment Facility on April 01, 1998. The sludge was sieved using a 2 mm screen and then aerated for approximately four hours. The sludge was homogenized in a blender at medium speed for approximately 2 minutes and then was allowed to settle for approximately 30 minutes. The supernatant was used as the inoculum for this study and was used the same day that it was prepared. A total suspended solids measurement and standard plate count were performed on the inoculum using procedures based on Standard Methods. The plates were incubated at 20 ± 3 °C for approximately 48 hours and then the number of colony forming units was enumerated.
Duration of test (contact time):
28 d
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
The test medium was a modified biochemical oxygen demand (BOD) test dilution water and was prepared using high quality water as described in the Protocol.
- Test temperature: 20 ± 3ºC.
- pH: The pH values ranged from 5.2 to 6.5.
- Aeration of dilution water: The sludge was sieved using a 2 mm screen and then aerated for approximately four hours.
- Suspended solids concentration: The average measured total suspended solids (TSS) concentration of the inoculum was 78.6 mg/L. The solids concentration of the test solution was 0.786 mg/L and was within the acceptable limit specified in the test guideline.

Concentration of test chemical: An amount of test substance necessary to deliver 10 mg Carbon/L was added to the treatment group by direct
weight addition.


TEST SYSTEM
- Culturing apparatus:
The test chambers were ambered 4-liter 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 CO2 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. The test was conducted at 20 ± 3 °C. Test chambers were identified by project number, test substance ID, test concentration, and vessel number. Magnetic stir bars and stir plates were used to mix the contents of the test chambers. Stir plate motors were cycled on and off approximately every 15 minutes to prevent the heating of the stirrer motors.


SAMPLING
- Sampling frequency:
The CO2 traps were removed for analysis on days 1, 4, 8, 11, 14, 19, 21, 25, and 29. The CO2 trap nearest the chamber was removed and analyzed for inorganic carbon. The two remaining traps were placed one position closer to the test chamber and a new trap was placed on the end of the series.
- Sampling method:
On the 28th day of the test, an aliquot of the contents of each test chamber was removed and the pH determined. The contents of each chamber then were acidified by the addition of 3 mL of concentrated hydrochloric acid to drive off inorganic carbonate. The chambers were aerated overnight after which the trapping solutions closest to the test chambers were analyzed for inorganic carbon.
- Sample storage before analysis: The test substance was stored under ambient conditions.


CONTROL AND BLANK SYSTEM
- Inoculum blank: The blank control was used to measure the background CO2 production of the inoculum and was not dosed with a carbon source.
- Toxicity control: The reference chambers were used to check the viability of the inoculum and were dosed with sodium benzoate, a substance known to be biodegradable, at a concentration of 10 mg Carbon/L.

Reference substance:
benzoic acid, sodium salt
Test performance:
The viability of the inoculum and validity of the test were supported by the results of the reference substance, sodium benzoate, from which 99.3% of theoretical CO2 was evolved. An average percent biodegradation of greater than 60% was achieved within 7 days, thereby fulfilling the criteria for a valid test by reaching the pass level by day 14
Parameter:
% degradation (CO2 evolution)
Value:
13.4
Sampling time:
28 d
Details on results:
The average cumulative percent of theoretical carbon dioxide produced by the test substance at 10 mg C/L was 13.4%.
Results with reference substance:
Reference (sodium benzoate) – 99.3% (28d). An average percent biodegradation of >60% was achieved within 7 days, thereby fulfilling the criteria for a valid test reaching the pass level by day 14.

Test material degraded 13.4% in 28 days. The reference substance, sodium benzoate, reached a level of 88.8% in the same test period.

Validity criteria fulfilled:
yes
Interpretation of results:
other: Not readily biodegradable
Conclusions:
The test substance may not be considered readily biodegradable under the conditions tested since the pass level of 60% TCO2 was not achieved. However since tests of ready biodegradability provide limited opportunity for biodegradation to occur, a negative result does not necessarily mean that the test substance will not be biodegraded under relevant environmental conditions.
Executive summary:

In a study on the ready biodegradability of the test substance in a CO2 Evolution Test (WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 331E-114) the test substance can be described as not readily biodegradable under the conditions of the test and only 13.4% degradation was seen in 28 days.

OECD guideline 301 B (Ready Biodegradability: CO2Evolution Test) was followed to conduct this study under GLP conditions.

Evidence of ready biodegradability in a Carbon Dioxide Evolution Test is 60% TCO2 within the 28-day test period. In addition, the pass value must be reached within 10 days of achieving 10% TCO2. The test substance may not be considered readily biodegradable under the conditions tested since the pass level of 60% TCO2 was not achieved. However since tests of ready biodegradability provide limited opportunity for biodegradation to occur, a negative result does not necessarily mean that the test substance will not be biodegraded under relevant environmental conditions.

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The key studies for Biodegradation are considered to have a reliability rating of 1.

       

There are 2 studies for this endpoint which can be considered key studies: 

    

Edwards et al, 1996 (WESTON report number: 96-043) conducted a CO2 evolution test on a test substance. 4.7 - 10.4% degradation was seen in 28 days. Based on these results the test substance would not be considered readily biodegradable.

    

In a study on the ready biodegradability of the test substance in a CO2 Evolution Test by Schafefer et al, 1998 (WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 331E-114) the test substance can be described as not readily biodegradable under the conditions of the test and 13.4% degradation was seen in 28 days.