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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-04-28 to 2003-07-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
July 17, 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
EEC Publication No. L 383 A, December 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge (microorganisms) from a domestic waste water treatment plant in Groß-Zimmern, Germany

- Preparation of inoculum for exposure: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was resuspended in tap water and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 1.5 g dry material per litre were mixed with test water and then aerated until use.
Duration of test (contact time):
28 d
Initial conc.:
102.4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Reconstituted water
- Test temperature: 21 - 22 °C
- pH: 7.6 (measured at the start of the test)
- pH adjusted: no
- Aeration of dilution water: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks containing a volume of 500 mL.
- Number of culture flasks/concentration: 2
- Measuring equipment: BSB/BOD-Sensor-System
- Test performed in closed vessels: the flasks were closed gas-tight by a measuring head
- Test performed in open system: no

SAMPLING
- Sampling frequency: everyday of exposure period

CONTROL AND BLANK SYSTEM
- Test item: 2
- Inoculum blank: 2
- Abiotic sterile control: 1
- Toxicity control: 1
- Procedure control: 1
Reference substance:
aniline
Key result
Parameter:
% degradation (O2 consumption)
Value:
>= 3 - <= 5
Sampling time:
28 d
Details on results:
After correction for the mean biochemical oxygen demand of the inoculum controls at the end of the 28-day exposure period degradation rates of 5 % and 3 % were found.
Results with reference substance:
The reference item Aniline was sufficiently degraded to 79 % after 14 days and to 96 % after 28 days of incubation, based on ThODNH4. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.

Validity Criteria of the Study


 


Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 29.5 mg O2/L and thus not greater than 60 mg O2/L within 28 days.


pH-Value: The pH-value of the test item flasks at the end of the test was 7.9, and thus within the range of pH 6.0 - 8.5.


Reference Item: The percentage degradation of the reference item Aniline, based on ThODNH4, reached the level for ready biodegradability (about 60 %) within 8 days.


Test Item: The difference of duplicate values for the degradation of the test item at any time during the test period was less than 20 %.


 


Individual Results


Biodegradation of Test Item: After correction for the mean biochemical oxygen demand of the inoculum controls at the end of the 28-day exposure period degradation rates of 5 % and 3 % were found.


Biodegradation of Reference Item Aniline: The reference item Aniline was sufficiently degraded to 79 % after 14 days and to 96 % after 28 days of incubation, based on ThODNH4. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.


Biodegradation in the Toxicity Control: In the toxicity control containing both, the test item and the reference item Aniline, 31 % biodegradation was noted within 14 days and 38 % biodegradation was determined after 28 days of incubation, based on ThODNH4. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was > 25 % within 14 days.


Abiotic Control Oxygen Demand: In the abiotic control no oxygen demand was determined during the exposure period of 28 days.


 


Table 1: Cumulative biochemical oxygen demand (mg O2/L) in test flasks during the test period of 28 days


 








































































































































































































































































































Time (days)



Flask 1



Flask 2



Flask 3



Flask 4



Flask 5



Flask 6



Flask 7



1



1.5



0



2.9



0



0



0



0



2



2.9



0



5.9



0



0



0



0



3



5.9



1.5



7.4



2.9



0



0



0



4



11.8



2.9



8.8



5.9



11.8



0



8.8



5



10.3



5.9



8.8



7.4



54.6



0



31.0



6



10.3



7.4



10.3



11.8



143.0



0



124.0



7



13.3



10.3



11.8



13.3



159.0



0



139.0



8



14.7



10.3



11.8



16.2



171.0



0



149.0



9



19.2



11.8



16.2



16.2



182.8



0



160.8



10



23.6



13.3



13.3



14.7



188.7



0



168.2



11



25.1



13.3



14.7



16.2



194.6



0



174.1



12



28.0



14.7



17.7



19.2



202.0



0



181.4



13



29.5



19.2



16.2



20.6



206.4



0



185.9



14



31.0



21.6



19.2



22.1



213.8



0



190.3



15



32.4



23.6



20.6



22.1



218.2



0



193.2



16



32.4



24.7



22.1



22.1



224.1



0



196.2



17



32.4



25.1



20.6



23.6



225.6



0



196.2



18



35.4



27.3



22.1



23.6



231.5



0



200.6



19



35.4



29.5



22.1



22.1



237.4



0



205.0



20



36.9



31.0



22.1



23.6



240.3



0



206.5



21



38.3



32.4



23.6



25.1



243.3



0



208.0



22



39.8



32.4



25.1



26.5



246.2



0



212.4



23



41.4



35.4



25.1



26.5



249.2



0



215.4



24



41.3



35.4



29.5



28.0



255.1



0



218.5



25



41.3



35.4



28.0



28.0



258.0



0



221.6



26



41.3



36.9



28.0



29.5



259.5



0



224.8



27



42.8



36.9



28.0



29.5



265.4



0



228.6



28



42.8



36.9



29.5



29.5



265.4



0



232.4



flasks 1 and 2: Test item


flasks 3 and 4: Inoculum control


flask 5: Aniline (Procedure control)


flask 6: Abiotic control


flask 7: Toxicity control


 


Table 2: Percentage biodegradation (BOD/ThODNH4) of test item, of Aniline and of the toxicity control


 



























































































































































































































Time


(days)



 



Percentage Biodegradation



 



Test item



Aniline



Toxicity control



flask 1



flask 2



flask 5



flask 7



1



0



0



-1



0



2



0



-1



-1



-1



3



0



-1



-2



-1



4



2



-2



2



0



5



1



-1



19



4



6



0



-1



54



21



7



0



-1



60



23



8



0



-1



64



25



9



1



-2



68



27



10



3



0



71



29



11



3



-1



73



29



12



3



-1



75



30



13



4



0



77



31



14



4



0



79



31



15



4



1



80



32



16



4



1



82



32



17



4



1



83



32



18



4



2



85



33



19



5



3



88



34



20



5



3



89



34



21



5



3



89



34



22



5



2



90



35



23



6



3



91



35



24



4



2



92



35



25



5



3



94



36



26



4



3



94



36



27



5



3



97



37



28



5



3



96



38



ThODNH4 of Test item: 2.77 mg O2/mg test item


ThODNH4 of Aniline: 2.41 mg O2/mg Aniline


 


Table 3: pH-values at the end of the test


 














































Flask No.



Treatment



pH-value



1



Test item



7.9



2



Test item



7.9



3



Inoculum control



7.7



4



Inoculum control



7.7



5



Procedure control (Aniline)



7.8



6



Abiotic control



7.8



7



Toxicity control



7.9


Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The degradation rate of the test material did not reach 60 % within the 10-day window and after 28 days of incubation. Thus, the test item is not readily biodegradable.
Executive summary:

The ready biodegradability of the test item in a Manometric Respirometry Test according to OECD TG 301 F and EU Method C.4-D was determined with aerobic activated sludge from a domestic wastewater treatment plant for 28 days under GLP. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item aniline was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. The oxygen concentration was measured using a BSB/BOD-Sensor-System and the flasks were closed gas-tight by a measuring head. The validity criterion was fulfilled. The reference item Aniline was sufficiently degraded to 79 % after 14 days and to 96 % after 28 days of incubation, based on ThODNH4. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item Aniline, 31 % biodegradation was noted within 14 days and 38 % biodegradation was determined after 28 days of incubation, based on ThODNH4. The test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was > 25 % within 14 days. After correction for the mean biochemical oxygen demand of the inoculum controls at the end of the 28-day exposure period degradation rates of 5 % and 3 % were found. The degradation rate of the test material did not reach 60 % within the 10-day window and after 28 days of incubation. Thus, the test item is not readily biodegradable.

Description of key information

The degradation rate of the test material did not reach 60 % within the 10-day window and after 28 days of incubation. Thus, the test item is not readily biodegradable (reference 5.2.1-1).

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable
Type of water:
freshwater

Additional information

The ready biodegradability of the test item in a Manometric Respirometry Test according to OECD TG 301 F and EU Method C.4-D was determined with aerobic activated sludge from a domestic wastewater treatment plant for 28 days under GLP. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item aniline was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. The oxygen concentration was measured using a BSB/BOD-Sensor-System and the flasks were closed gas-tight by a measuring head. The validity criterion was fulfilled. The reference item Aniline was sufficiently degraded to 79 % after 14 days and to 96 % after 28 days of incubation, based on ThODNH4. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item Aniline, 31 % biodegradation was noted within 14 days and 38 % biodegradation was determined after 28 days of incubation, based on ThODNH4. The test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was > 25 % within 14 days. After correction for the mean biochemical oxygen demand of the inoculum controls at the end of the 28-day exposure period degradation rates of 5 % and 3 % were found. The degradation rate of the test material did not reach 60 % within the 10-day window and after 28 days of incubation. Thus, the test item is not readily biodegradable.