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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 301F, EPA OPPTS 835.3110 and EEC Part C.4-D and in accordance with the Principles of Good Laboratory Practices (GLP).
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,4 Cyclohexanedimethanol Reaction Products with Epichlorohydrin
- Analytical purity: The purity of the test material was determined to be 9.5 area % 1,4-Cyclohexanedimethanol monoglycidiyl ether, 45.0 area %, 1,4-Cyclohexanedimethanol diglycidyl ether, and 25.0 area % of the reaction product of 1,4-cyclohexanedimethanol with 3 equivalents of epichlorohydrin by gas chromatography/flame ionization detector with tentative identification of the major components by nuclear magnetic resonance and gas chromatography mass spectrometry
- Impurities (identity and concentrations): no information available
- Lot number : Lot # 1H1050NZP1
- Expiration date of the lot/batch: March 05, 2015
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
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 19 June 2013. This facility 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 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 1,193 mg/L. Based on this determination, 465 mL of the homogenized activated sludge was added to 18 liters of the sterilized mineral medium to yield a final MLSS concentration of 30 mg/L.
Duration of test (contact time):
34 d
Initial test substance concentrationopen allclose all
Initial conc.:
51.1 mg/L
Based on:
test mat.
Initial conc.:
111 mg/L
Based on:
ThOD/L
Parameter followed for biodegradation estimationopen allclose all
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. Inoculum Blanks, containing the inoculated mineral medium 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 dissolved organic carbon (DOC) concentration in the absence of added test material. Biodegradation of a 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 to the inoculated mineral medium (500 mL). A single Toxicity Control reaction mixture was similarly prepared by combining the test material and aniline in the inoculated mineral medium. The rate and extent of aniline degradation in the Toxicity Control reaction mixture was used to assess whether the test material was inhibitory to the microbial inoculum. A single Abiotic Control mixture was prepared by adding mercuric chloride (sterilizing agent, 252 mg/L) to inoculated mineral medium
containing the test material. This Abiotic Control was used to determine the amount of O2 consumption, CO2 evolution, and DOC removal measured in the Test Suspensions which could be attributed to abiotic reactions.
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. After initiation of the test a 30 mL aliquot was taken from the Inoculum Blank, Positive Control, and Toxicity Control reaction mixtures for analyses of DOC and nitrate/nitrite. Prior to measurement of initial oxygen and CO2 concentrations, the headspace volume of each individual reaction vessel was allowed to equilibrate in the respirometer system. The water temperature in the water bath 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.
Reference substance
Reference substance:
aniline
Remarks:
Manufacturer: ACROS, lot no.: A0300558, purity: 99+%

Results and discussion

Preliminary study:
not applicable
Test performance:
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 6.8 days, and biodegradation based on O2 consumption, CO2 production and DOC removal reached 109 ± 21%, 94.6 ± 23.2% and 95.7 ± 0.0%, respectively, at the end of the test.

% Degradationopen allclose all
Parameter:
other: BOD
Value:
19.1
Sampling time:
34 d
Parameter:
% degradation (DOC removal)
Value:
16.6
Sampling time:
34 d
Details on results:
For the Test Suspensions and Positive Controls, the extent of biodegradation recorded for replicate reaction mixtures must not differ by more than 20% DO2 at the end of the 10-day window, plateau of degradation, or the end of the test. In this test, the percentage of test material biodegradation in the replicate Test Suspensions differed by < 4.3% DO2 over all sample intervals of the 34-day test. The results indicate that the procedures used to prepare, incubate, and analyze the biodegradation reaction mixtures resulted in sufficient precision in the test results.

The recorded values as read from the temperature reference vessel thermometer averaged 22.5 ± 0.4°C (± 1 std. dev.), and the minimum and maximum recorded temperatures were 22.0 and 23.0°C, respectively, over the entire duration of this test. The pH of the biodegradation reaction mixtures remained within the required range of 6.0 to 8.5 over the duration of this test. The pH of the Test Suspensions decreased by no more than 0.20 pH units from their initial values over 34 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 materials evaluated in this test.

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 material. The mixture contained test material (104 mg/L as ThOD) in the inoculated mineral medium, which was chemically sterilized by addition of 252 mg/L HgCl2. The Abiotic Control mixture exhibited no O2 consumption or CO2 production over the duration of the 34-day test (data not shown). Therefore, the O2 consumption and CO2 production in the Test Suspensions was solely attributed to biodegradation of the test material.

BOD5 / COD results

Results with reference substance:
The Toxicity Control mixtures containing aniline (237 mg/L as ThOD) and test material (110 mg/L as ThOD) showed no evidence for inhibition of the microbial inoculum by the test material.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
other: partial biodegradation observed
Conclusions:
Biodegradation of 1,4-CHDM DGE in the Test Suspension indicated a total of 19.1% biodegradation over the 34-day test period (as determined by BOD). This value indicates that some biodegradation did occur under the conditions of the test. Validity criteria specified by the OECD for viability of the inoculum, pH, and control of temperature were met in this study.
Executive summary:

The ready biodegradability of 1,4-cyclohexanedimethanol, reaction products with epichlorohydrin (1,4-CHDM DGE) was determined using the OECD Guideline No. 301F: Manometric Respirometry Test. The test material, 1,4-CHDM DGE, is a mixture of 1,4- cyclohexanedimethanol diglycidyl ether, 1,4-cyclohexanedimethanol monoglycidyl ether and the reaction product of 1,4-cyclohexanedimethanol with 3 equivalents of epichlorohydrin. This study employed a series of biodegradation reaction mixtures containing a non-adapted activated sludge inoculum, which was suspended in a defined mineral medium at a concentration of 30 mg/L (dry solids). Biodegradation of the test material (51.1 mg/L equivalent to 111 mg/L as theoretical oxygen demand [ThOD]) reached 19.1% based on biological oxygen demand (BOD) at the end of this 34-day test. These results show that some biodegradation occurred under the conditions of the test.

The results of this test met or exceeded each of the OECD-specified criteria for validation of the ready biodegradability test. These include parameters such as viability of the inoculum, control of pH and temperature, and precision in percentage biodegradation recorded among replicate test mixtures. Biodegradation of a reference material (aniline, 98.4 mg/L equivalent to 237 mg/L as ThOD) exceeded 60% by 6.8 days, verifying the viability of the activated sludge inoculum. A Toxicity Control mixture, containing both aniline (98.4 mg/L equivalent to 237 mg/L as ThOD) and the test material (50.6 mg/L equivalent to 110 mg/L as ThOD), showed no evidence for inhibition of the microbial inoculum by the test material. Oxygen consumption and CO2 evolution observed in the reaction mixtures could be attributed solely to biological activity, as no net O2 consumption or CO2 production was observed in an Abiotic Control mixture containing the test material and a chemical sterilant (HgCl2, 252 mg/L). The results of this test indicate that the test material, 1,4-CHDM DGE, can undergo partial biodegradation.