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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:
Not applicable
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to test guidelines and in accordance with GLP.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):Not applicable
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):The microbial inoculum consisted of activated sludge mixed liquor, collected from the oxidation ditch bioreactor at the Midland Municipal Wastewater Treatment Plant (Midland, Michigan) on November 17, 2009. This facility treats an excess of 11 million liters of wastewater per day, of which > 90% is from domestic sources. The activatedsludge was collected one day prior to initiation of the test, and was continuously aerated until used.- Laboratory culture: not applicable- Method of cultivation:not specified in the report- Storage conditions:continuously aerated until used- Storage length: one day prior to the initiation of the test- Preparation of inoculum for exposure: Prior to use, the activated sludge was screened through 500 μm nylon mesh, and briefly homogenized in a Waring blender (Waring Products Inc., Torrington, Connecticut). The mixed liquor suspended solids (MLSS) content of the homogenized sludge was determined gravimetrically to be 1,987 mg/L. Based on this determination, 245 mL of the homogenized activated sludge was added to 16 liters of the sterilized mineral medium to yield a final MLSS concentration of 30 mg/L.- Pretreatment:not specified in the report- Concentration of sludge:30 mg/L- Initial cell/biomass concentration:not applicable- Water filtered: yes- Type and size of filter used, if any:
Duration of test (contact time):
28 d
Initial conc.:
11.1 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS- Composition of medium:A defined mineral medium was prepared as specified in OECD Guideline 301F, by dissolving appropriate volumes of concentrated mineral stock solutions in Milli-Q water.- Additional substrate:not applicable- Solubilising agent (type and concentration if used):not applicable- Test temperature:20 and 24°C.- pH:7.2 to 7.6- pH adjusted: not specified in the report- CEC (meq/100 g):- Aeration of dilution water:- Suspended solids concentration:The initial blank-corrected DOC concentration in these Test Suspensions (mean ± 1 SD) was 5.86 ± 0.1 mg/L, thus indicating complete dissolution of test substance in the biodegradation reaction mixtures.- Continuous darkness:yesTEST SYSTEM- Culturing apparatus:designed 1-liter glass reaction vessels, each containing a 500-mL portion of the inoculated mineral medium. The reaction vessels are designed with flat glass bottoms to accommodate stirring with large PTFE-coated magnetic stir bars. These vessels are also fitted with 20 x 105 mm glass side baffles to facilitate complete mixing/aeration of the stirred reaction mixtures.- Number of culture flasks/concentration:one- Method used to create aerobic conditions:mixing/aeration of the stirred reaction mixtures- Method used to create anaerobic conditions:not applicable- Measuring equipment:Columbus Micro-Oxymax automated respirometry system- Test performed in closed vessels due to significant volatility of test substance:yes- Test performed in open system:no- Details of trap for CO2 and volatile organics if used:SAMPLING- Sampling frequency: oxygen and CO2 in the headspace of each reaction vessel were recorded at six-hour intervals over the entire 28-day test period- Sampling method:Columbus Micro-Oxymax automated respirometry system- Sterility check if applicable:not specified in the report- Sample storage before analysis:not specified in the reportCONTROL AND BLANK SYSTEM- Inoculum blank: yes- Abiotic sterile control:yes- Toxicity control: yesSTATISTICAL METHODS:
Reference substance:
aniline
Preliminary study:
NOt applicable
Test performance:
Several criteria are specified by the OECD for validating the results of its tests for ready biodegradability. These criteria are based on parameters such as inoculum viability, precision among replicate reaction mixtures, maintenance of temperature, and pH of the reaction mixtures.The results indicate that the procedures used to prepare, incubate, and analyze the biodegradation reaction mixtures resulted in sufficient precision in the test results.
Parameter:
% degradation (O2 consumption)
Value:
89.3
St. dev.:
10.8
Sampling time:
28 d
Parameter:
% degradation (CO2 evolution)
Value:
90.9
St. dev.:
2.9
Sampling time:
28 d
Parameter:
% degradation (DOC removal)
Value:
98.1
St. dev.:
1.7
Sampling time:
28 d
Details on results:
Refer below to the other information section
Results with reference substance:
The complete biodegradation of aniline indicated from measurements of BOD and CO2 evolution was confirmed by analyses of DOC removal. DOC was removed by 98.6% in both replicate Positive Control mixtures.

Biological Oxygen Demand (BOD): The time required for biodegradation to exceed 10% DO2 (i.e., the lag period) was 7.9 days and the 60% DO2 level was exceeded after 9.6 days. By the end of the 28- day test, biodegradation of the test substance reached 89.3 ± 10.8% DO2 (mean ± 1SD).

CO2 Evolution: Biodegradation of the substance exceeded 10% DCO2 after 6.9 days and after 28 days reached 90.9 ± 2.9% DCO2 (mean ± 1SD). Therefore, the rates and extents of biodegradation determined from CO2 evolution closely reflected those determined from the BOD values.

DOC Analyses DOC analyses also indicated that biodegradation of the test substance occurred under the conditions of this test. The initial DOC concentration (mean ± 1 SD) in the Test Suspensions was 5.86 ± 0.1 mg/L, which indicated that the test substance components were completely dissolved in the inoculated mineral medium. The DOC concentration after 28 days was 0.110 ± 0.1 mg/L, which equates to a 98.1% reduction of the initial DOC concentration.

The complete biodegradation of aniline indicated from measurements of BOD and CO2 evolution was confirmed by analyses of DOC removal. DOC was removed by 98.6% in both replicate Positive Control mixtures.

The recorded values from the temperature reference vessel averaged 21.9 ± 0.4°C (± 1SD, n=17), and the minimum and maximum recorded temperatures were 21.0 and 22.8°C, respectively, over the entire duration of this test.

The pH of the biodegradation reaction mixtures remained within the required range of 7.2 to 7.6 over the duration of this test. The pH of the Test Suspensions decreased by no more than 0.29 pH units from their initial values over 28 days, and showed only a 0.06 pH unit (maximum) difference relative to the Inoculum Blanks at the end of the test. This minimal variation in pH indicates that the mineral medium contained adequate buffering capacity for the inoculum and test substances evaluated in this test.

The Toxicity Control mixtures containing 100 mg/L aniline and 10.9 mg/L of the test substance showed no evidence for inhibition of the microbial inoculum by the test substance. The onset and rate of biodegradation in the Toxicity Control mixture closely matched that in the Positive Controls further indicating that the test substance did not cause toxicity to or inhibition of the microbial inoculum.

A single Abiotic Control mixture was included in the experimental design to determine the extent to which abiotic processes may result in degradation of the test substance. The mixture contained 11.0 mg/L of test substance in the inoculated mineral medium, which was chemically sterilized by addition of 250 mg/L HgCl2. The Abiotic Control mixture exhibited no O2 consumption or CO2 production over the duration of the 28-day test (data not shown). Therefore, the O2 consumption and CO2 production in the Test Suspensions was solely attributed to biodegradation of the test substance.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The AMP (2-AMINO-2-METHYL-1-PROPANOL) test substance exceeded 60% biodegradation within 9.6 days of the start of the test and within the 10-day window detailed in OECD 301F guideline. The results of this test demonstrated that AMP can be classified as “readily biodegradable”, according to the OECD 301F: Manometric Respirometry Test.
Executive summary:

The ready biodegradability of AMP (2-amino-2-methyl-1-propanol) was evaluated using the OECD Guideline No. 301F: Manometric Respirometry Test. The test employed biodegradation reaction mixtures (500 mL) containing a defined mineral medium, which was inoculated with 30 mg/L activated sludge (dry solids) collected from the City of Midland Wastewater Treatment Plant (Midland, Michigan). Biodegradation of AMP was evaluated at a concentration of 11.1 mg/L, which was equivalent to 21.8 mg/L theoretical oxygen demand (ThOD). The onset of AMP biodegradation (i.e. BOD ≥ 10% of theoretical oxygen demand, ThOD) occurred after 7.9 days in the Test Suspensions, and biodegradation exceeded the pass level of 60% ThOD consumption within 1.7 days thereafter. At the end of the 28-day test, the extent of biodegradation based on BOD, CO2 evolution, and dissolved organic carbon (DOC) removal reached 89.3 ± 10.8%, 90.9± 2.9%, and 98.1 ± 1.7% (mean ± 1 Std. Dev.), respectively. Thus, biodegradation exceeded both the pass level and 10-day window criteria for classification as “readily biodegradable”. The Toxicity Control mixture showed no apparent toxic or inhibitory effects of the AMP substance on the inoculum employed in this test; and no net consumption of oxygen or DOC removal was observed in the Abiotic Control mixture. All test parameters met the OECD-specified validation criteria for ready biodegradability tests. Biodegradation of the reference compound aniline exceeded the pass level within 6.6 days, and thus confirmed the viability of the inoculum. Other experimental parameters, such as reaction mixture pH, incubation temperature, and total oxygen consumption in the Inoculum Blanks, fell within the ranges required by the OECD guideline. Therefore, the results of this test indicate that AMP does meet OECD criteria for “ready biodegradability” in the Manometric Respirometry test.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-08-18 to 2004-12-23
Reliability:
2 (reliable with restrictions)
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
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: municipal wastewater treatment plant Breisgauer Bucht, Germany
- Laboratory culture: no
- Method of cultivation: not applicable
- Storage conditions: not mentioned
- Storage length: 2 days (test 1) resp. 1 day (test 2)
- 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.
- Pretreatment: aerated with CO2-free air at a rate of 50-100 ml/min overnight
- Concentration of sludge: Dry solid of the activated sludge was determined as 2.96 g/l (test 1) and 3.9 g/l (test 2) by weight measurements after 2 h drying at 105 °C (mean of triplicate measurements)
- Initial cell/biomass concentration: 30 mg dry solid/L
- Water filtered: not mentioned
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: according to the guideline
- Additional substrate: none
- Solubilising agent (type and concentration if used): not used
- Test temperature: mean 21.0 ¿ 22.1 (first test) and 20.0 - 22.0 °C (second test)
- pH: not determined
- Aeration of dilution water: not mentioned
- Suspended solids concentration: 30 mg/L
- Continuous darkness: no, diffuse light during test


TEST SYSTEM
- Culturing apparatus: Gas wash bottles (2000 ml volume) with lateral connecting pieces for butyl rubber septum were used as reactors. The liquid volume was fixed as 1500 ml each. Mixing was performed by a magnetic stirrer with 2 cm stir bars.
- Number of culture flasks/concentration: three vessels per concentration
- Method used to create aerobic conditions: aerated by passage of CO2-free air (2.7 - 5.5 bubbles/second)
- Measuring equipment: Inorganic carbon measurement with total carbon analyzer TOC-5000A, Shimadzu
- Details of trap for CO2: The CO2 produced in the reactors was absorbed in two 250 mL gas wash bottles in series, each filled with 200 mL 0.2 M NaOH.


SAMPLING
- Sampling frequency: days 4, 7, 11, 14, 21, 28 and 28 after acidification
- Sampling method: Sampling was performed through the lateral connecting pieces through the butyl rubber septum using 5 ml PE syringes.
- Sample storage before analysis: not mentioned
- Other: none


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 3 vessels (without test substance with inoculum)
- Abiotic sterile control: not performed
- Toxicity control: yes, 1 vessel
- Other: none


STATISTICAL METHODS:
Reference substance:
benzoic acid, sodium salt
Remarks:
20 mg/L DOC
Preliminary study:
not performed
Test performance:
The CO2-free air production system consisted of an air compressor, two 1000 ml gas wash bottles filled with dry soda lime, followed by one bottle filled with 0.1 M NaOH (sodium hydroxide) and one gas wash bottle filled with demineralised water. The CO2-free air was passed on to an air distributewith two input and 22 output channels and through PE-tubes. Gas wash bottles (2000 ml volume) with lateral connecting pieces for butyl rubber septums were used as reactors. The liquid volume was fixed as 1.500 ml each. Mixing was performed by a magnetic stirrer with 2 cm stir bars. In both tests 7.1 mL of a stock solution (10 g/L) of the test item were added into the three test vessels and the vessel for toxicity control, corresponding to a TOC concentration of 20 mg/l TOC. From the reference compound 5.15 mL of a stock solution of 10 g/L were added to the reference vessels and the vessels for toxicity control in both tests. The CO2 produced in the reactors was absorbed in two 250 ml gas wash bottles in series, each filled with 200 ml 0.2 M NaOH. Sampling was performed through the lateral connecting pieces through the butyl rubber septum using 5 ml PE syringes. The amount of CO2 released from the reactors is calculated through IC-measurements in the CO2-absorber while considering the amount of CO2 removed for IC-measurement.
Parameter:
% degradation (CO2 evolution)
Value:
99.8
Sampling time:
28 d
Remarks on result:
other: Result of second test
Details on results:
The degradation of the toxicity control was 94.0% within 14 days and 100.2% within 28 days (after acidification) in test 2, so there was no inhibition of the inoculum caused by the test item.
Results with reference substance:
The reference compound reached the pass level for ready biodegradablity (60% ThCO2) within 4 days (test 2).

Table #1: Ultimate biodegradation in % ThCO2 (first test)

 days biodegradation of test flasks biodegradation of reference flasks toxicity control 
   #1  #2  #3  mean  #1  #2  #3  mean  
 4  2.7  -1.2  36.0  12.5  54.5  32.4  67.2 51.4   33.3
 7  84.0  89.5  78.5 84.0   72.7  72.2  87.7  77.5  88.7
 11  104.2  103.0  96.4  101.2  84.4  83.8  98.0  88.7  100.2
 14  93.6  107.5  95.8  99.0  84.9  86.7  99.2  90.3  105.3
 21  108.0  103.8  106.5  106.1  91.1  93.2  97.2  93.8  109.7
 28  123.4  118.4  91.7  111.2  91.2  94.8  98.7  94.9  108.7
 28 (after acidification)  136.4  113.1  95.6  115.0  92.2  101.5  96.3  96.7  116.2

Table #2: Ultimate biodegradation in % ThCO2 (second test)

 days biodegradation of test flasks biodegradation of reference flasks toxicity control 
   #1  #2  #3  mean  #1  #2  #3  mean  
 4  45.4  34.0  58.0  45.8  71.6  66.2  65.1  67.6  38.6
 7  74.3  79.5  78.6  77.5  82.6  78.1  75.5  78.7  71.5
 11  87.9  91.4  95.0  91.4  84.5  86.6  78.3  83.1  87.2
 14  101.1  100.9  104.3  102.1  91.8  96.2  92.7  93.6  94.0
 21  101.1  112.6  106.9  106.9  101.2  102.4  92.5  98.7  98.2
 28  116.1  113.2  105.4  111.6  91.1  91.5  93.1  91.9  93.1
 28 (after acidification)  94.2  108.0  97.1  99.8  101.1  105.5  102.4  103.0  100.2
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test was repeated for consideration of the degradation extent data of the first test, because the test item showed a too high degradation extents > 100%.
In the repetition the test item showed again high degradation extents (> 100%), but the degradation extents of the single test vessels were more parallel and there were no outlier like in the first test.
In principle the result of the first test was confirmed.

Description of key information

PTSA was tested in a CO2 evolution test and showed >99% biodegrdation in 28 days.The extent of biodegradation of AMP based on BOD, CO2 evolution, and dissolved organic carbon (DOC) removal reached 89.3 ± 10.8%, 90.9± 2.9%, and 98.1 ± 1.7% (mean ± 1 Std. Dev.).

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Based on the information on the components the substance is expected to be readily biodegradable.

The rationale for read-across can be found in the document attached in section 13.