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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:
1 October - 5 November 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
not specified
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
14C aniline, was used as the reference substance. Lot#209-033-060, specific activity = 60 mCi/mmol., radiopurity = 99.6%.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
This study was conducted using an inoculum prepared with an activated sludge collected from the Madison Metropolitan Sewage District, 1610 Mooreland Road, Madison, Wisconsin, U.S.A., a sewage treatment facility that receives primarily domestic sewage. An appropriate volume of activated sludge was collected on September 29, 1997, and delivered to Covance.
Duration of test (contact time):
29 d
Details on study design:
Preparation of Inoculum
Upon arrival at Covance, the activated sludge was homogenized in a blender for approximately 2 minutes. After the solids were allowed to settle for 60 minutes, the supernatant was filtered though glass wool. The amount of suspended solids was then determined by filtering a small amount of the solution, drying the filter paper, and calculating the weight of the filtered solids. The concentration of suspended solids remaining in the inoculum was 40.0 mg/mL. The supernatant was then aerated overnight prior to the inoculation of the systems. The inoculum was analyzed at Covance for microorganisms. The results of the microbial analysis confirmed that the solution was viable.

Preparation of Fortification Solution
The fortification solution was prepared by diluting 10 uL of 14C-RH-573 test material at a concentration of 44.73 ug/uL (17.89 mg of test material/400 uL of solution) with water to a volume of 5.0 mL with a total concentration of 0.0895 ug/uL based on theoretical ug/mL. Liquid scintillation counting analysis of duplicate predose (50 uL) and postdose (25 uL) aliquots of the fortification solution verified that the solution was homogeneous.

The 14C-aniline-HCI reference material received weighed 155 ug and contained 100 uCi of radioactivity. A solution of 14C-aniline and nonradiolabeled aniline was prepared by mixing the 100 uCi/155 ug of 14C-aniline-HCI (containing 100 uCi/111 ug of 14C-aniline equivalent-Inventory # 1973) with 1.0 g of nonradiolabeled aniline. 200 mg of isotopically diluted aniline was then dissolved in 7.5 mL of water to prepare the fortification solution. The fortification solution concentration based on LSC analysis was 22.4 ug/uL. LSC analysis of duplicate 50-uL aliquots of the fortification solution verified that the solution was homogeneous.

Preparation of Systems
Twelve systems were prepared by placing 300 mL of test medium in flasks and inoculating each with 3 mL of a solution prepared from activated sludge. Mercuric chloride (HgCI2) was added to three of the flasks, designated as abiotic controls, at a concentration of 83 ppm. While being constantly stirred, all systems were aerated overnight in a dark, temperature-controlled room at 22°C ± 2° with carbon dioxide (C02)-free air to remove all CO2 from the flasks. The CO2-free air was generated by passing a stream of air through a CO2 trap (Chromatography Research Systems). After aeration, duplicate flasks were fortified at nominal concentrations of 0.1, 0.03, and 0.01 ppm by adding 335 ulL, 100 uL, and 35 uL of the 14C-RH-573 fortification solution, respectively, and were designated as test systems. The three abiotic controls flasks were dosed at nominal concentrations of 0.1, 0.03, and 0.01 ppm by adding 335 uL, 100 uL, and 35 uL of the 14C-RH-573 fortification solution, respectively. Duplicate flasks were dosed at a nominal concentration of 13 ppm by adding 180 uL of the 14C-aniline fortification solution and were designated as reference control systems. The remaining flask received no test or reference material and was designated as a blank control system.

After dosing, the pH of each system was determined to be pH 7.4 ± 0.2. The systems were placed in a dark, temperature-controlled room at 22°C ± 2°. Each flask was connected to a series of traps (two 2 N sodium hydroxide traps and one ethylene glycol trap) containing approximately 25 to 100 mL of trapping media to collect volatile components. A continuous airflow through the flasks to the traps was maintained by vacuum. The air flow rate was adjusted and monitored to maintain a rate of 30 to 100 mL/minute .

Sample Collections
The trapping media from each system were collected and replaced with fresh media on Days 2, 5, 7, 9, 14, 19,24, and 29. On Day 28, 0.5 mL of concentrated HCI was added to each flask to drive off CO2 from the systems. Prior to acidification on Day 28, aliquots were removed from all of the test systems, to determine the microbial population at the end of the study.

Sample Analysis
The total amounts of volatile radioactivity in the traps were quantitated by LSC analysis of duplicate I-mL aliquots of each trapping solution, (Hionic-Fluor was added to the NaOH trap aliquots and Ultima Gold XR plus 1 mL of Megapure water was added to the ethylene glycol trap aliquots to facilitate analysis). In order to confirm that 14C_RH_573 did not volatilize during the study, the radioactivity in the NaOH traps of the test systems that was >1.0% of applied radioactivity was precipitated with barium chloride as 14C-barium carbonate. After precipitation, the samples were centrifuged, and the supernatants were analyzed by LSC to determine whether there was any remaining radioactivity that did not precipitate. The radioactivity that remained in the supernatant was assumed to be 14C-RH_573 and was subtracted from the total radioactivity in the traps.

Determination of the Amount of 14C02 Produced
The mean cumulative amount of 14C02 trapped from the duplicate systems was calculated.

The rate of biodegradation was calculated using the cumulative percent of 14C02 evolved over time using the following formula:

% Biodegradation = [(Mean dpm/mL x Total Volume of Solution)/(dpm Applied)] x 100

The percent of 14C02 calculated at each sampling interval was added to the previous results to determine the cumulative 14C02 released during the study.

Statistical Treatment of Data
The statistical treatment of data was limited to the calculation of mean values, and deviation from the mean values, as appropriate.
Reference substance:
aniline
Remarks:
14C aniline, was used as the reference substance. Lot#209-033-060, specific activity = 60 mCi/mmol., radiopurity = 99.6%.
Parameter:
% degradation (CO2 evolution)
Value:
54.1
Sampling time:
29 d
Parameter:
% degradation (CO2 evolution)
Value:
55.8
Sampling time:
29 d
Parameter:
% degradation (CO2 evolution)
Value:
47.6
Sampling time:
29 d
Details on results:
The mean cumulative recovery of 14C CO2 from the taps was 54.1%, 55.8%, and 47.6% of the applied radioactivity at the end of the study for the 0.1, 0.03, and 0.01 ppm test systems, respectively. The mean cumulative recovery of 14C CO2 for the reference control was 93.9%.

In the 0.01 ppm test system, the variation between replicates observed in early sampling intervals was probably attributable to a low initial microbial activity and the low concentration of test material. In later sampling intervals, as the microbial activity increased in replicate 2, the degradation patterns were similar in both replicates.

The 10-day window for the 0.1 and 0.03 ppm test systems started at Day 1 and ended at Day 11. The 10-day window for the 0.01 ppm test system started at Day 2 and ended at Day 12. The 10-day window for the reference control system started at approximately Day 1 and ended at Day 10. During the 10-day window, 37%, 30%, and 30% of the applied 14C-RH-573 degraded to CO2 at test substance concentrations of 0.1, 0.03, and 0.01 ppm, respectively.
Results with reference substance:
The mean cumulative recovery of 14C CO2 for the reference control was 93.9%.

The reference control degraded 80% of the applied radioactivity during the 10-day window.

The microbial population in the test systems was monitored by microbial analysis of the flasks at the beginning and end of the study. The results of microbial analysis showed that those flasks containing 14C-RH-573 had reduced microbial population when

compared to the reference flasks, as shown in Table I.

Table I. Microbial Population at Day 28

Description   Microbial Population(CFU/mL)a
 14C-RH-573 0.1 ppm  60,000,000
 14C-RH-573 0.03 ppm  54,000,000
 14C-RH-573 0.01 ppm  70,000,000
 Reference control  126,000,000
 Abiotic  <10
 Blank Control  127,000,000

Note: Aerobic microbial counts of preconditioned inoculum at beginning of the study were 5,600,000 CFU/mL.

a Microbial population shown is the mean of two flasks.

Table 2. Biodegradation of 14C-RH-573

 Sample Interval              Mean Cumulative Recovery      
 (Day)  0.1 ppm RH-573  0.03 ppm RH-573  0.01 ppm RH-573  Reference substance (aniline)  0.1 ppm Abiotic System 0.03 ppm Abiotic System 0.01 ppm Abiotic System 
 2  15.2  11.5  7.0  37.6  0.0  0.0  0.0
 5  30.7  23.0  16.4  75.7  0.0  0.0  0.0
 7  34.4  27.0  20.0  78.4  0.0  0.0  0.0
 9  36.5  29.9  22.7  79.7  0.0  0.0  0.0
 14  40.9  35.3  36.4  83.0  0.0  0.0  0.0
 19  47.9  38.3  41.0  86.7  0.0  0.0  0.0
 24  52.0  45.4  44.7  90.7  0.0  0.0  0.0
 29  54.1  55.8  47.6  93.9  0.0  0.0  0.0
Validity criteria fulfilled:
yes
Interpretation of results:
other: 50 % biodegraded within 29 days and fails the 10 day window
Conclusions:
Although extensive metabolism occurs over the 29 day interval, the test material does not meet the requirements for readily biodegradable.
Executive summary:

The ready biodegradability of the test material was evaluated in the Modified Sturm test. Although extensive metabolism occurs over the 29 day interval, the test material does not meet the requirements for readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-01-30 to 2002-02-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without restriction.
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Duration of test (contact time):
28 d
Initial conc.:
7.7 mg/L
Based on:
ThOD/L
Initial conc.:
10 mg/L
Based on:
test mat.
Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
The test material Acticide M 50, i.e its active substance MIT, did not reach the pass level of a biodegradation > 60% neither in the 10 day-window nor after 28 days. It did not even reach the level of 10% (beginning of biodegradation) throughout the study.
In the 4 incubation sets, pH varied between 6.99 and 7.19 at the start of the study and between 6.95 and 7.38 at the end of the study.
The concentration is given for the test substance. It has to be corrected for 
the content of the a.s. of 50.50 %.
A kinetic of the test substance or the active substance is not given as there was no biodegradation within the 28-d-window.
Validity criteria fulfilled:
yes
Remarks:
Validity criteria fulfilled except oxygen depletion in inoculum blank excceded 1.5 mg diss. oxygen after 28 d.
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test material Acticide M 50, i.e its active substance MIT, has to be assessed as not readily biodegradable.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2003-03-31 to 2003-05-06
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: non-GLP study, scientific verified data
Qualifier:
according to
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Duration of test (contact time):
36 d
Initial conc.:
10 mg/L
Based on:
test mat.
Initial conc.:
20 mg/L
Based on:
test mat.
Initial conc.:
2.11 mg/L
Based on:
other: TOC
Initial conc.:
4.22 mg/L
Based on:
other: TOC
Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (DOC removal)
Value:
5 - 12
Sampling time:
36 d
Remarks on result:
other: 10 mg/L test mat.
Parameter:
% degradation (DOC removal)
Value:
9
Sampling time:
36 d
Remarks on result:
other: 20 mg/L test mat.
Details on results:
The test material at the test concentrations of 10 mg/L and 20 mg/L reached the level of 10% (begin of biodegradation) after a long lasting adaptation phase of 26 days. The 70% level was not passed neither in the 10 d-window nor after 36 days. After 29 days, the biodegradation rate came to a maximum of 12 - 17%.
The percentage degradation of the functional control reached the pass level of > 70% after 2 days. After 14 days, a degradation rate of 98% was reached.
In the toxicity control containing both test and reference substance, the biodegradation came to 94% after 14 days. The test material was not inhibitory at a concentration of 10 mg/L in the test.
Validity criteria fulfilled:
yes
Remarks:
Validity criteria according to OECD Guideline 301 A fulfilled.
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test material Acticide M 50, i.e its active substance MIT, must be regarded to be not readily biodegradable in the 10 d-window.

Description of key information

The follwoing 3 studies were identified as key studies -

OECD 301 B - Although extensive metabolism occurs over the 29 day interval, the test material did not meet the requirements for readily biodegradable.

OECD 301D - The test material Acticide M 50, i.e its active substance MIT, was observed to be not readily biodegradable in the 10 d-window.

OECD 301A - The test material Acticide M 50, i.e its active substance MIT, was observed to be not readily biodegradable in the 10 d-window.

Key value for chemical safety assessment

Additional information