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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 March 10, 1997 to April 09, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge micro-organisms were obtained on March 10, 1997, from the aeration stage of the Severn Trent Water plc sewage treatment plant at Belper, Derbyshire, treating predominantly domestic sewage.
- Preparation of inoculum for exposure: 100 mL samples were filtered through Whatman GF/A paper and dried at 105 °C.
- Pretreatment: Sample washed 3 times by settlement and resuspension in culture medium prior to suspended solids determination. Samples were maintained on aeration at 21°C prior to use.
- Concentration of sludge: 30 mg TSS/L
- Initial cell/biomass concentration:
- Water filtered: Yes
- Type and size of filter used, if any: Whatman GF/A paper
Duration of test (contact time):
ca. 28 d
Initial conc.:
20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Standard culture medium according to OECD 301B.
- Test temperature: 22±2°C
- pH: 7.4
- Aeration of dilution water: Yes, see below
- Suspended solids concentration: 30 mg TSS/L
- Continuous darkness: Yes, in darkness sheilded from laboratory lighting


TEST SYSTEM
- Culturing apparatus: 5 L glass culture vessels
- Number of culture flasks/concentration: Two for every conc.
- Method used to create aerobic conditions: Using CO2 free air at 30-100 mL/min
- Method used to create anaerobic conditions: Not applicable
- Other:
- Method of preparation of test solution: Direct dispersion in culture medium.
-Agitation: Yes, using magnetic stirrer


SAMPLING
- Sampling frequency: On Day 0, 1, 2, 3, 6, 8, 10, 14, 16, 20, 22, 24, 27, 28 and 29.
- Sampling method: 2 mL of samples were taken on the days specified above for IC analysis.
- Sample storage before analysis: Samples taken on Days 12 & 18 were stored at -20°C


CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: No
- Toxicity control: No, since a preliminary study performed on Empilan CME indicated that, the test material is non-toxic and does not inhibit the respiration of sewage sludge micro-organisms at the concentration employed in the test.
- Reference control: Yes


Reference substance:
benzoic acid, sodium salt
Remarks:
10 mg C/L (ThCO2 =58.34%; TOC = 30 mg C; total carbon = 51.4 mg)
Preliminary study:
The oxygen consumption rates (mg O2/L) for the inoculum blank was 0.43 for both replicates; for the test material it was 0.46 and 0.42 at 10 and 20 mg C/L respectively and for reference material it was 0.38 and 0.11 at 3.2 and 32 mg/L respectively. Hence, toxicity control was omitted from the study.
Test performance:
- The decrease of percentage degradation value for the test material from 99% on Day 28 to 98% on Day 29 due to the increase in inorganic carbon (caused by addition of HCl) in the control absorber vessels was greater than that in the standard material absorber vessels and this decrease is considered to be due to biological variation between the activated sewage sludge in the control and standard material vessels.
- Inorganic carbon analysis of samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
- Sodium benzoate attained 97% degradation after 28 d thereby confirming the suitability of the inoculum and test conditions.
- Toxicity control was omitted from the study, in light of results of a preliminary investigation of the test material. These results carried out using Activated Sludge Respiration Inhibition test method (OECD Guideline No 209), indicated that Empilan CME did not inhibit the respiration of sewage sludge micro-organisms at the concentration employed in the test.
- Low DOC values obtained for the samples taken from the test material culture vessels on day 0 are due to physical removal of the test material by the suspended solids during centrifugation. This effect is considered to be due to the test material being a fine homogenous and not a true solution at the concentrations employed in the study.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 24
Sampling time:
3 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 62
Sampling time:
8 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 99
Sampling time:
28 d
Details on results:
- Percent degradation values: 0, 3, 9, 24, 54, 62, 69, 72, 92, 97, 98, 96, 99, 99 and 98 after 0, 1, 2, 3, 6, 8, 10, 14, 16, 20, 22, 24, 27, 28 and 29 d.
- The measured concentrations of DOC was found to be 56% of nominal on Day 0 and 17 to 26% of nominal on Day 28.
Results with reference substance:
The reference material met the validity criteria for the test, greater than 60% biodegradation within 14 d, and within the 10 d window (Day 2 = 22%, Day 10 = 64%, Day 28 = 97% ThCO2).
- The increase in inorganic carbon in the first absorber vessels on Day 29 resulted in an increase in the percentage degradation value for the standard material from 97% on Day 28 to 98% on Day 29.

None.

Validity criteria fulfilled:
yes
Remarks:
However, the data on replicate difference and the biodegradation data for control were not documented.
Interpretation of results:
readily biodegradable
Conclusions:
Under the study conditions, the test substance was considered to be readily biodegradable.
Executive summary:

A study was conducted to determine the ready biodegradability of the test substance, C8-18 and C18-unsatd. MEA, according to OECD Guideline 301B (CO2 evolution test). The test (20 mg C/L) and reference (sodium benzoate) substances were incubated with activated sludge and observed for degradation by measurement of the theoretical amount of carbon dioxide (ThCO2) over a 28 d period. The reference substance, sodium benzoate reached 10% biodegradation after Day 2 and > 60% after Day 10. It met the validity criteria established in the guideline for a reference substance. Biodegradation of the test substance on Day 28 equalled 99%. Under the study conditions, the test substance was considered to be readily biodegradable (Mead, 1997).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 29, 2008 to June 06, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Municipal wastewater treatment plant Breisgauer Bucht, sampling date of activated sludge was Aug. 4, 2008. Dry solid of the activated sludge was determined as 5.9 g/L by weight measurements after 3.5 h drying at 105°C (mean of triplicate measurements).
- Preparation of inoculum for exposure: The activated sludge was washed twice by settling the sludge, decanting the supernatant and resuspending the sludge in aerated tap water.
- Concentration of sludge: 30 mg dry solids per litre
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Composition of medium:
Mineral medium:
A. KH2PO4 8.50 g
K2HPO4 21.75 g
Na2HPO4*2H2O 33.40 g
NH4Cl 0.50 g
Demineralised water q.s. 1 litre
B. CaCl2*2H2O 36.4 g
Demineralised water q.s. 1 litre
C. MgSO4*7H2O 22.5 g
Demineralised water q.s. 1 litre
D. FeCl3*6H2O 0.25 g
Demineralised water q.s. 1 litre and stabilised with one drop of concentrated HCl
For preparation of the mineral medium 10 mL of solution (A) was mixed with 800 mL demineralised water, 1 mL each of solutions (B), (C) and (D) were added and the volume was made up to 1 litre.
- Test temperature: 20-22°C
- Aeration of dilution water: 50-100 mL/min (2.7 - 5.5 bubbles/second)
- Continuous darkness: No
- Other: The reactors were kept mixed with magnetic stirrers. The aeration rate was determined visually daily on working days, the determination by counting the gas bubbles over a defined period using a stop watch was made at day 6 and 28. The CO2-free air production system, the air-tightness of the whole experimental set-up, the aeration of the absorber flasks and the magnetic stirrers were controlled daily on working days.


TEST SYSTEM
- Culturing apparatus: Gas wash bottles (2000 mL volume) with lateral connecting pieces for butyl rubber septums were used as reactors.
- Number of culture flasks/concentration: Three reactors each for the test material, inoculum (blank) reference substance
- Method used to create aerobic conditions: The CO2-free air was passed on to an air distributor with two input and 22 output channels and through PE-tubes.
- Measuring equipment: IC measurement was performed with a total carbon analyser (TOC-5000A Shimadzu with an autosampler ASI-5000A) by purging the inorganic carbon with H3PO4 (25 %) using a non dispersive infrared (NDIR) detector.
- Test performed in closed vessels: Yes
- Details of trap for CO2: The vials were immediately closed with sealing film in order to avoid CO2 uptake from the air.
- Other: 4.82 mL of the stock solution of the test material (10 g/L) was added into the three test vessels, corresponding to a TOC concentration of 20 mg/L. The reference compound (5.15 ml of a 10 g/l stock solution) was added to the reference vessels.


SAMPLING
- Sampling frequency: At the beginning of the study, 3rd, 6th, 10th, 14th, 21st and 28th day
- Sampling method: Sampling was performed through the lateral connecting pieces through the butyl rubber septum using 5 mL PE syringes.
- Other: 4 mL NaOH from the first of two CO2-absorber flasks connected in line was sampled and the IC's were determined. On the 28th day 1 mL concentrated hydrochloric acid (HCl) was added into each reactor to release the CO2 dissolved in water. On day 29 the IC was determined in both CO2-absorber flasks in line.


CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
Reference substance:
benzoic acid, sodium salt
Remarks:
Roth, Lot 26461087, Molecular weight: 144.1 g/mol, Storage-conditions: Room temperature, Durability: Nov. 3, 2009, Solubility in water: Soluble, C-content: 0.583 mg/mg (calculation by Hydrotox), ThCO2: 2.137 mg/mg (calculation by Hydrotox)
Key result
Parameter:
% degradation (inorg. C analysis)
Value:
ca. 88.3
Sampling time:
28 d
Details on results:
The mean degradation extent of the test material was 88.3% within 28 d after acidification (mean value of three test vessels). For finding the exact position of the 10 d window the degradation extents of the days without measurement were calculated by interpolation. On Day 1 the calculated mean degradation extent of the test material was for the first time higher than 10% (mean value: 11.7%). Thus the end of the 10 d window was on Day 11. The calculated degradation extent on Day 11 was 85.5% (mean value). Therefore the test material reached the pass level for ready biodegradability (60% ThCO2 and 10 d window).
Results with reference substance:
The reference substance reached the pass levels for ready biodegradability within 3 d.

Blank: The highest mean CO2-evolution of the blank flasks in both test series was 34.6 mg/L within 28 d after acidification (see table 2 of the attached study report). Before adding the test material, the IC in the reactor was determined, but no IC was found. The IC concentration of the NaOH in the second CO2-absorber flasks in line, used as protective flasks, was below 5 ppm and was not considered in the data processing, because CO2 absorption from room air was its source.


Criteria met for the validity of the study:


- The IC content in the test vessel was less than 5% of the TOC introduced with the test material.


- The CO2 evolution in the inoculum blank at the end of the test was below 40 mg/L.


- The difference of extremes of replicate values at the end of the 10-d-window and at the end of the test was less than 20 %.


- The biodegradation of the reference compound reached the pass level of 60 % ThCO2 by day 14.


 


Table 1: Ultimate biodegradation after x days [% of ThCO2]


























































































Reactor 



Day 



0



3



6



10



14



21



28



28 (after acidification) 



16



Test flasks 



0



40,5



70,4



83,2



90,1



96,5



91,2



84,5



17



 



0



21,6



73,0



81,1



85,5



91,6



88,9



84,7



18



 



0



43,4



74,3



86,9



96,2



101,5



100,5



95,6



4



Reference flasks 



0



73,9



91,4



96,5



102,3



117,0



107,6



108,3



5



 



0



66,5



90,6



95,9



99,5



106,8



89,2



91,5



6



 



0



68,4



93,8



99,4



102,6



109,7



102,3



95,8



 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the study conditions, the test substance was considered readily biodegradable.
Executive summary:

A study was conducted to determine the ready biodegradability of the test substance, C8-18 and C18-unsatd. MEA, according to OECD Guideline 301B (CO2 evolution test), in compliance with GLP. A mineral medium, corresponding to 10-20 mg total organic carbon (TOC)/L, was inoculated with activated sludge (30 mg d.s./L). The test vessels were aerated by the passage of CO2-free air and incubated under aerobic conditions for 28 d. Degradation was followed by determining the CO2 produced and absorbed to sodium hydroxide via IC-measurement on Days 3, 6, 10, 14, 21 and 28 of the study. The reference substance used was sodium benzoate at a concentration of 20 mg/L organic carbon. The reference substance reached the pass levels for ready biodegradability within 3 d. The mean degradation extent of the test substance was 88.3% within 28 d after acidification (mean value of three test vessels). Under the study conditions, the test substance was considered readily biodegradable (Kronenberger-Schäfer, 2008).

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

A study was conducted to determine the ready biodegradability of the test substance, C8-18 and C18-unsatd. MEA, according to OECD Guideline 301B (CO2 evolution test), in compliance with GLP. A mineral medium, corresponding to 10-20 mg total organic carbon (TOC)/L, was inoculated with activated sludge (30 mg d.s./L). The test vessels were aerated by the passage of CO2-free air and incubated under aerobic conditions for 28 d. Degradation was followed by determining the CO2 produced and absorbed to sodium hydroxide via IC-measurement on Days 3, 6, 10, 14, 21 and 28 of the study. The reference substance used was sodium benzoate at a concentration of 20 mg/L organic carbon. The reference substance reached the pass levels for ready biodegradability within 3 d. The mean degradation extent of the test substance was 88.3% within 28 d after acidification (mean value of three test vessels). Under the study conditions, the test substance was considered readily biodegradable (Kronenberger-Schäfer, 2008).

A study was conducted to determine the ready biodegradability of the test substance, C8-18 and C18-unsatd. MEA, according to OECD Guideline 301B (CO2 evolution test). The test (20 mg C/L) and reference (sodium benzoate) substances were incubated with activated sludge and observed for degradation by measurement of the theoretical amount of carbon dioxide (ThCO2) over a 28 d period. The reference substance, sodium benzoate reached 10% biodegradation after Day 2 and > 60% after Day 10. It met the validity criteria established in the guideline for a reference substance. Biodegradation of the test substance on Day 28 equalled 99%. Under the study conditions, the test substance was considered to be readily biodegradable (Mead, 1997).