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Environmental fate & pathways

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:
From the 20th of January to the 18th February, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
read-across source
Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From the 20th of January to the 18th February, 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Justification for Read Across is detailed in the section summary and it is further detailed in the report attached to the IUCLID section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
modified Sturm test
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
yes (incl. certificate)
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: ’Waterschap de Maaskant’, ‘s-Hertogenbosch, the Netherlands.
The sludge was kept under continuous aeration until further treatment.
The concentration of suspended solids was 3.8 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant).
Before use, the sludge was allowed to settle for at least 30 minutes and the liquid decanted for use as inoculum at the amount of 10 ml/l of mineral medium.
Duration of test (contact time):
28 d
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST SYSTEM
Test vessels: 2 litre brown all glass bottles
Milli Q water: tap water purified by reverse osmosis and passes over activated carbon and ion-exchange cartridges.
Stock solution with minerals
MIneral medium
Barium hydroxide 0.0125 M
CO2 free air
Test concentration: in duplicate at 48 mg per 2 litres, corresponding to 12 mg TOC/L. The organic content was based on the molecular formula.
Temperature: 20.5 - 22 °C
pH: 7.6 - 8.1 (end of the test)
PREPARATION OF THE BOTTLES
Mineral components, Milli-Q water (ca. 80% total volume) and inoculum (1% final volume) were added to each bottle. This mixture was aerated with C02-free air overnight to purge the system of C02.
Type and number of bottles
test suspension: containing test substance and inoculum (2 bottles). Inoculum blank: containing only inoculum (2 bottles) Positive control: containing reference substance (ca. 40 mg/l sodium acetate (Merck art. 6268, batch 049 TA933768), TOC= 12 mgll) and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
Preparation: the test substance and positive control were added to the bottles. The volumes of suspensions were made up in all bottles by the addition of Milli-Q water previously aerated with C02-free air, resulting in the mineral medium described before.
Three C02-absorbers (bottles filled with 100 ml 0 0125 M Ba(0H)2) were connected in series to the exit air line of each test bottle.
Start of the incubation: the test was started by bubbling C02-free air through the solution at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).
DETERMINATION OF CO2
Experimental CO2 production: theoretical C02 production The CO produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl.
Measurement: titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day. Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein was used as pH-indicator. On the 28th day, the pH of the test suspensions was measured and 1 ml of concentrated HCl was added to each bottle. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29. The theoretical CO2 production was calculated from the molecular formula.
Reference substance:
other: The positive and the toxicity control contained 80.1 mg sodium acetate (ThC02= 1.07 mg C02/mg) resulting in a theoretical C02 production following complete degradation of 85.7 mg per 2 litres.
Preliminary study:
The theoretical C02 production following complete degradation was 88.0 mg per 2 litres for A and B.
The theoretical CO2 production following complete degradation of the substance plus sodium acetate was 173.7 mg per 2 litres (toxicty control).
Test performance:
The relative degradation values calculated from the measurements performed during the test period revealed no significant degradation of the substance.
In the toxicity control more than 25% degradation occurred within 14 days (based on ThC02).
Therefore, the test substance was assumed to be not inhibitory.
Parameter:
% degradation (CO2 evolution)
Value:
>= 0.7 - <= 1.6
Sampling time:
28 d
Details on results:
ACCEPTABILITY OF THE TEST
The positive control substance was degraded at least 60% within 14 days
The total CO2 release in the blank reached a total value of 35 mg CO2 per 2 litres of medium
The difference of duplicate values for % degradation was always less than 20.
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The substance was not readiiy biodegradable under the conditions of the modified Sturm test presently performed
Executive summary:

The substance was tested for its ready biodegradability in the carbon dioxide (C02) evolution test (modified Sturm test) at 48 mg per 2 litres, corresponding to 12 mg TOC/L.

The study procedure was based on EEC directive 92/69, C 4-C, December 1992, and OECD guideline No. 301 B July 17, 1992.

The Theoretical CO2 production (ThC02) was calculated to be 1.83 mg C02/mg.

The relative degradation values calculated from the measurements performed during the test period revealed no significant degradation of the substance.

In the toxicity control the substance was found to be not inhibitory. Since all acceptability criteria prescribed by the protocol were met, this study was considered to be valid.

In conclusion, the test item was not readily biodegradable under the conditions of the modified Sturm test presently performed.

 

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
modified Sturm test
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Description Brown powder
Test substance storage At room temperature in the dark
Stability under storage conditions Not indicated
Expiry date 29 August 2002 (allocated by NOTOX, 1 year after)

Study design

Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: ’Waterschap de Maaskant’, ‘s-Hertogenbosch, the Netherlands.
The sludge was kept under continuous aeration until further treatment.
The concentration of suspended solids was 3.8 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant).
Before use, the sludge was allowed to settle for at least 30 minutes and the liquid decanted for use as inoculum at the amount of 10 ml/l of mineral medium.
Duration of test (contact time):
28 d
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST SYSTEM
Test vessels: 2 litre brown all glass bottles
Milli Q water: tap water purified by reverse osmosis and passes over activated carbon and ion-exchange cartridges.
Stock solution with minerals
MIneral medium
Barium hydroxide 0.0125 M
CO2 free air
Test concentration: in duplicate at 48 mg per 2 litres, corresponding to 12 mg TOC/L. The organic content was based on the molecular formula.
Temperature: 20.5 - 22 °C
pH: 7.6 - 8.1 (end of the test)
PREPARATION OF THE BOTTLES
Mineral components, Milli-Q water (ca. 80% total volume) and inoculum (1% final volume) were added to each bottle. This mixture was aerated with C02-free air overnight to purge the system of C02.
Type and number of bottles
test suspension: containing test substance and inoculum (2 bottles). Inoculum blank: containing only inoculum (2 bottles) Positive control: containing reference substance (ca. 40 mg/l sodium acetate (Merck art. 6268, batch 049 TA933768), TOC= 12 mgll) and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
Preparation: the test substance and positive control were added to the bottles. The volumes of suspensions were made up in all bottles by the addition of Milli-Q water previously aerated with C02-free air, resulting in the mineral medium described before.
Three C02-absorbers (bottles filled with 100 ml 0 0125 M Ba(0H)2) were connected in series to the exit air line of each test bottle.
Start of the incubation: the test was started by bubbling C02-free air through the solution at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).
DETERMINATION OF CO2
Experimental CO2 production: theoretical C02 production The CO produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl.
Measurement: titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day. Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein was used as pH-indicator. On the 28th day, the pH of the test suspensions was measured and 1 ml of concentrated HCl was added to each bottle. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29. The theoretical CO2 production was calculated from the molecular formula.
Reference substance
Reference substance:
other: The positive and the toxicity control contained 80.1 mg sodium acetate (ThC02= 1.07 mg C02/mg) resulting in a theoretical C02 production following complete degradation of 85.7 mg per 2 litres.

Results and discussion

Preliminary study:
The theoretical C02 production following complete degradation was 88.0 mg per 2 litres for A and B.
The theoretical CO2 production following complete degradation of the substance plus sodium acetate was 173.7 mg per 2 litres (toxicty control).
Test performance:
The relative degradation values calculated from the measurements performed during the test period revealed no significant degradation of the substance.
In the toxicity control more than 25% degradation occurred within 14 days (based on ThC02).
Therefore, the test substance was assumed to be not inhibitory.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
>= 0.7 - <= 1.6
Sampling time:
28 d
Details on results:
ACCEPTABILITY OF THE TEST
The positive control substance was degraded at least 60% within 14 days
The total CO2 release in the blank reached a total value of 35 mg CO2 per 2 litres of medium
The difference of duplicate values for % degradation was always less than 20.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The substance was not readiiy biodegradable under the conditions of the modified Sturm test presently performed
Executive summary:

The substance was tested for its ready biodegradability in the carbon dioxide (C02) evolution test (modified Sturm test) at 48 mg per 2 litres, corresponding to 12 mg TOC/L.

The study procedure was based on EEC directive 92/69, C 4-C, December 1992, and OECD guideline No. 301 B July 17, 1992.

The Theoretical CO2 production (ThC02) was calculated to be 1.83 mg C02/mg.

The relative degradation values calculated from the measurements performed during the test period revealed no significant degradation of the substance.

In the toxicity control the substance was found to be not inhibitory. Since all acceptability criteria prescribed by the protocol were met, this

study was considered to be valid.

In conclusion, the test item was not readily biodegradable under the conditions of the modified Sturm test presently performed.