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Environmental fate & pathways

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was conducted under GLPs, and was a valid study with no deviations.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The microbial inoculum consisted of activated sludge mixed liquor, collected from a Midland Municipal Wastewater Treatment Plant that treats an excess of 11 million liters of wastewater per day, of which > 90% is from domestic sources. The activated sludge was collected one day prior to initiation of the test, and was continuously aerated until used. Prior to use, the activated sludge was screened through 500μm nylon mesh, and briefly homogenized. The mixed liquor suspended solids (MLSS) content of the homogenized sludge was determined gravimetrically to be 1270 mg/L. Based on this determination, 435 mL of the homogenized activated sludge was added to 18 liters of the sterilized mineral medium to yield a final MLSS concentration of approximately 30 mg/L.
Duration of test (contact time):
28
Initial conc.:
30.1 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
The biodegradation reaction mixtures were prepared in specially 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. All reaction vessels were labeled using a color coded system for vessel identification. The various reaction mixtures which comprised the experimental design for this study along with dose concentrations are listed in Table 2. Inoculum Blanks, containing the inoculated mineral medium with unamended silica gel and without added test or reference material, were prepared in duplicate. These Inoculum Blanks were used to determine mean values for cumulative O2 consumption, CO2 evolution, and DOC concentration in the absence of added test material. Biodegradation of the Reference material, aniline, was determined in duplicate Positive Control mixtures to verify the viability of the inoculum. Biodegradation of the test material in the Test Suspensions was determined by adding test material-amended silica gel to the inoculated mineral medium (500 mL). A single Abiotic Control mixture was prepared by adding mercuric chloride (sterilizing agent) to inoculated mineral medium containing the test material-amended silica gel. This Abiotic Control was used to determine the amount of O2 consumption, CO2 evolution, and DOC formation or removal measured in the Test Suspensions which is attributed to abiotic reactions. A portion of unamended silica gel was added to each of the Inoculum Blank and Positive Control mixtures to replicate the associated particle surface area resulting from addition of test material-amended silica gel to the Test Suspensions.
After addition of test material, aniline, and sterilizing agent to the appropriate vessels; the pH of the reaction mixtures were measured and adjusted as necessary to 7.4 ± 0.2, then stirred for 30 minutes to homogenize their contents prior to initiation of the test. Samples (30 mL) of the Inoculum Blank, Test Suspensions, Positive Control, and Abiotic Control reaction mixtures were collected for initial analyses of dissolved organic carbon (DOC)
and nitrate/nitrite analysis. Prior to measurement of initial oxygen and CO2 concentrations, the headspace volume of each individual reaction vessel was determined by the automated respirometer system. Other specific operating parameters for the respirometer system are described in detail in the study file. A temperature reference vessel containing 500 mL water was incubated and stirred alongside the biodegradation reaction mixtures in this test. The water temperature in this vessel was recorded by a submerged NIST-traceable thermocouple. The biodegradation reaction mixtures were
incubated in the dark at a temperature between 20 and 24 °C, and maintained within ± 1 °C of the average incubation temperature. The reaction mixtures were continuously stirred by a PTFE-coated magnetic stir bar rotating at a setting of 150 rpm.
Reference substance:
aniline
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Parameter:
% degradation (DOC removal)
Value:
0
Sampling time:
28 d
Details on results:
Biological oxygen demand (BOD) is used as the primary indicator of biodegradation in the OECD 301F: Manometric Respirometry test. These measurements of BOD indicated that cumulative oxygen demand in the Test Suspensions was substantially lower than that recorded in the Inoculum Blanks. Therefore, the test material appeared to be toxic or inhibitory to the inoculum under these test conditions, and no biodegradation of the test material was evident over 28 days.

Two of the OECD tests for ready biodegradability utilize measurements of CO2 evolution to indicate the extent of test material mineralization. The pass criterion for these tests is60% of theoretical carbon dioxide evolution. While measurement of CO2 evolution is not a requirement of OECD Guideline No. 301F, these supplemental measurements of CO2 evolution confirmed that no biodegradation of the test material components occurred
under the conditions of this test, and that the test material was toxic or inhibitory to the inoculum under the tested conditions .

Since components of the test material are insoluble in water, the extent of its biodegradation could not be determined from analyses of dissolved organic carbon (DOC) removal. However, DOC analyses were performed on the Test Suspensions, Positive Controls, and Abiotic Controls to determine if biodegradation of the test material resulting in formation of any measurable, persistent, soluble degradation products. The initial DOC concentration (mean ± 1 SD) in the Test Suspensions was 2.53 ± 0.0 mg/L, which indicated that the test material components were not completely dissolved in the inoculated mineral medium. The mean DOC concentration in the Test Suspensions after 28 days was 2.83 ± 0.1 mg/L. Similarly, the DOC concentration in the Abiotic Control mixture was initially 2.64 mg/L, and was 2.39 mg/L at the end of this test.
Results with reference substance:
The inoculum used in this test produced > 60% biodegradation of the reference material, aniline, within the required 10-day window prior to day 14 of the test. The 60% DO2 pass level was exceeded after 5.7 days, and biodegradation based on O2 consumption, CO2 production and DOC removal reached 119%, 82.5% and 98.0%, respectively, at the end of the test.
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test material was not biodegraded under the stringent conditions of this OECD ready biodegradation test, due to apparent toxicity or inhibition of the microbial inoculum caused by the test material.
Executive summary:

The ready biodegradability of BADGE MXDA (#42) was evaluated using the OECD Guideline No. 301F: Manometric

Respirometry Test. Due to the low aqueous solubility of some components, the test material was coated onto silica gel

to facilitate its dispersion in the biodegradation reaction mixtures at approximately 12 mg/L. No biodegradation of the

test material occurred over the duration of this test. However, cumulative oxygen demand in duplicate Test

Suspensions containing the test material was substantially lower than that in unamended Inoculum Blanks; indicating

that the test material was toxic or inhibitory to the inoculum under these test conditions, and that no biodegradation of

the test material occurred over the duration of this test. Therefore, the results of this test indicate that BADGE MXDA

(#42) does not meet OECD criteria for “ready biodegradability” in the Manometric Respirometry test. However,

because the test material was toxic/inhibitory to the inoculum under these conditions, further testing would be

necessary to assess the intrinsic biodegradability of this material. Biodegradation of a reference material (aniline, 101 mg/L) exceeded 60% by 5.7 days, verifying the viability of the activated sludge inoculum in duplicate Positive Control reaction mixtures. The results of this test met or exceeded each of the OECD-specified criteria for validation of the ready biodegradability tests. These include parameters such as viability of the inoculum, control of pH and temperature, and precision in percentage biodegradation recorded among replicate Positive Control test mixtures.

Description of key information

Ready biodegradability of BADGE MXDA (#42) was evaluated using the OECD Guideline No. 301F: Manometric Respirometry Test.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The ready biodegradability of BADGE MXDA (#42) was evaluated using the OECD Guideline No. 301F: Manometric

Respirometry Test. Due to the low aqueous solubility of some components, the test material was coated onto silica gel

to facilitate its dispersion in the biodegradation reaction mixtures at approximately 12 mg/L. No biodegradation of the

test material occurred over the duration of this test. However, cumulative oxygen demand in duplicate Test

Suspensions containing the test material was substantially lower than that in unamended Inoculum Blanks; indicating

that the test material was toxic or inhibitory to the inoculum under these test conditions, and that no biodegradation of

the test material occurred over the duration of this test. Therefore, the results of this test indicate that BADGE MXDA

(#42) does not meet OECD criteria for “ready biodegradability” in the Manometric Respirometry test. Biodegradation of a reference material (aniline, 101 mg/L) exceeded 60% by 5.7 days, verifying the viability of the activated sludge.

The material is considered as persistent based on the screening criteria for the purpose of the PBT assessment though it is expected to slowly degrade at not inhibitive concentrations. No further testing is deemed relevant to further assess the biodegradation potential in line with Annex XIII REACH.