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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:
supporting study
Study period:
From February 03, 2005 to September 28, 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
not specified
Remarks:
Toxicity control
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of activated sludge: Activated sludge taken from a laboratory-scale unit receiving synthetic wastewater
- Concentration of suspended solids in reaction mixture: 30 mg/L
Duration of test (contact time):
ca. 28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineralised medium
- Suspended solids concentration: 30 mg/L


TEST SYSTEM
- Culturing apparatus: Closed respirometer flask
- Number of culture flasks/concentration: Two
- Measuring equipment: Closed WTW OxiTop OC 110 respirometer for BOD determination, WPiB P/19-S-BS4


SAMPLING
- Sampling frequency: 3, 7, 10, 14, 16, 18, 21, 23, 25 & 28 d


CONTROL AND BLANK SYSTEM
- Inoculum blank: Containing only inoculum but without test substance
- Reference control: Yes


STATISTICAL METHODS: Calculations and the graphs were performed using SigmaPlot 9.0 software of SYSTAT Software, Inc. USA purchased from GAMBIT CoiS Ltd. Poland
Reference substance:
acetic acid, sodium salt
Remarks:
100 mg/L; fine, white powder, provided by Chempur, Piekary Slaskie Poland
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 71.1
Sampling time:
28 d
Details on results:
Test substance:
- ThOD (NH4) = 2.57 mgO2/mg substance
- COD = 2.04 mgO2/mg substance

For more details, see the following in the attached PDF:
- Figure on page 14/15: Rokamid KAD- Ultimate aerobic biodegradability testing with manometric respirometry method.
- Tables on page 13/15:
- Sample oxygen uptake: biodegradability without the correction for nitrification reaction
- Correction for nitrification reaction (oxygen uptake in nitrification reaction)
Key result
Parameter:
COD
Value:
ca. 2.04 g O2/g test mat.
Results with reference substance:
% degradation after 28 d: Approximately 80%

None.

Validity criteria fulfilled:
not specified
Remarks:
Toxicity control not available
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 assess the ready biodegradability of the test substance, C8-18 and C18-unsatd. DEA, according to EU Method C.4-D (Manometric respirometry), in compliance with GLP. The test (100 mg/L) and reference (acetic acid, sodium salt; 100 mg/L) substances were inoculated with activated sludge, kept in closed respirometer flasks and observed for degradation by measurement of the dissolved O2 concentration over a 28 d period. The degradation of the test and reference substances was equivalent to 71.1 and 80%, respectively, after 28 d. Under the study conditions, the test substance was therefore considered to be readily biodegradable (Zielinski, 2005).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 1, 2008 to September 4, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: 21-22 °C
- Aeration of dilution water: 50-100 mL/min (2.7 - 5.5 bubbles/second)
- Continuous darkness: no, diffuse light
- 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. 92.5
Sampling time:
28 d
Details on results:
The mean degradation extent of the test material was 92.5% within 28 days 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 (see table 3 of the attached study report). On day 1 the calculated mean degradation extent of the test material was for the first time higher than 10% (mean value: 12.8 %). Thus the end of the 10-d-window was on day 11. The calculated degradation extent on day 11 was 75.5 % (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 days.

Blank: The highest mean CO2-evolution of the blank flasks in both test series was 24.9 mg/L within 28 days after acidification (see table 2 of the attached study report). Before adding the test material, the IC in the reactor was determined, but insignificant amounts of IC were found (0.1 mg/L). The IC-concentration of the NaOH in the second CO2-absorber flasks in line, used as protective flasks, was below 8 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) 
7 Test flasks  0 37,4  65,6  72,7  78,0  85,1  77,2  85,2 
8   0 40,6  65,4  74,0  79,2  91,9  97,4  93,1 
9   0 37,3  57,0  74,7  84,0  94,8  100,0  99,3 
4 Reference flasks  0 62,8  86,4  94,9  92,4  101,4  105,2  103,9 
5 0 75,8  86,6  93,6  91,4  100,6  98,8  94,4 
6 0 67,3  83,3  93,8  86,2  96,8  90,8  93,9 
Validity criteria fulfilled:
yes
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 biodegradability of the test substance, C8-18 and C18-unsatd. DEA, according to OECD Guideline 301B (modified Sturm test), in compliance with GLP. A solution of the test substance in a mineral medium, corresponding to 10-20 mg total organic carbon (TOC)/L, was inoculated with activated sludge (30 mg dry substance (d.s.)/L). The test vessels were incubated under aerobic conditions for 28 d. Degradation was followed by determining the CO2 produced on Days 3, 6, 10, 14, 21 and 28. 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 of the test substance was 92.5% within 28 d of acidification. Under the study conditions, the test substance was considered to be readily biodegradable (Kronenberger-Schäfer, 2008).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
September 14 1995 to January 23 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the section 13 for details on the category justification.
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum: Sewage treatment plant, Hochdahl
- Concentration of sludge: 1 mL/L
Duration of test (contact time):
ca. 28 d
Initial conc.:
2 mg/L
Based on:
act. ingr.
Initial conc.:
5 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Inoculum and test material in a mineral culture medium were placed in closed flasks and kept at a constant tempetarture of 20 +/- 1°C either in the dark or under diffuse light. O2 consumption was measured iodometrically.
Biodegradability was estimated by comparing actual and the theoretical O2 consumption.
Negative (no test substance) and positive (sodium benzoate) controls were run in parallel.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 84
Sampling time:
28 d
Remarks on result:
other: at 2 mg/L of test substance.
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 71
Sampling time:
28 d
Remarks on result:
other: at 5 mg/L test substance. Insufficient O2 left in the flask.
Details on results:
Plateau for ready biodegradability of the test substance reached within 14 d of time point when 10 % degradation occured. The 14 d window was therefore respected.

Results with reference substance:
The degradation of sodium benzoate was equivalent to 89 % within 28 d. 73 % degradation was reached within7 d

None.

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

A study was performed to assess the ready biodegradability of the read across substance, C12-18 and C18-unsatd. DEA, according to OECD Guideline 301D (Closed bottle test), in compliance with GLP. The test substance at 2 and 5 mg/L was incubated with sludge from a domestic wastewater treatment plant and O2 consumption was followed over a period of 28 d. A total of 84% O2 consumption was obtained at 2 mg/L and the plateau for ready biodegradability was reached within 14 d, at which point there was 10% degradation. At 5 mg/mL, 71% O2 consumption was obtained, although there was insufficient O2 left in the flask at the end of the test. Under the study conditions, the test substance was considered to be readily biodegradable (Werner, 1996). Based on the results of the read across study, the test substance is also expected to have similar biodegradability rate in water.

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 biodegradability of the test substance, C8-18 and C18-unsatd. DEA, according to OECD Guideline 301B (modified Sturm test), in compliance with GLP. A solution of the test substance in a mineral medium, corresponding to 10-20 mg total organic carbon (TOC)/L, was inoculated with activated sludge (30 mg dry substance (d.s.)/L). The test vessels were incubated under aerobic conditions for 28 d. Degradation was followed by determining the CO2 produced on Days 3, 6, 10, 14, 21 and 28. 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 of the test substance was 92.5% within 28 d of acidification. Under the study conditions, the test substance was considered to be readily biodegradable (Kronenberger-Schäfer, 2008).

A study was conducted to assess the ready biodegradability of the test substance, C8-18 and C18-unsatd. DEA, according to EU Method C.4-D (Manometric respirometry), in compliance with GLP. The test (100 mg/L) and reference (acetic acid, sodium salt; 100 mg/L) substances were inoculated with activated sludge, kept in closed respirometer flasks and observed for degradation by measurement of the dissolved O2 concentration over a 28 d period. The degradation of the test and reference substances was equivalent to 71.1 and 80%, respectively, after 28 d. Under the study conditions, the test substance was therefore considered to be readily biodegradable (Zielinski, 2005).

A study was performed to assess the ready biodegradability of the read across substance, C12-18 and C18-unsatd. DEA, according to OECD Guideline 301D (Closed bottle test), in compliance with GLP. The test substance at 2 and 5 mg/L was incubated with sludge from a domestic wastewater treatment plant and O2 consumption was followed over a period of 28 d. A total of 84% O2 consumption was obtained at 2 mg/L and the plateau for ready biodegradability was reached within 14 d, at which point there was 10% degradation. At 5 mg/mL, 71% O2 consumption was obtained, although there was insufficient O2 left in the flask at the end of the test. Under the study conditions, the test substance was considered to be readily biodegradable (Werner, 1996).