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Environmental fate & pathways

Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideling Study
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
according to guideline
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
according to guideline
EPA OPPTS 835.3110 (Ready Biodegradability)
Principles of method if other than guideline:
Official Journal of the European Communities. Part C:
Methods for the determination of ecotoxicity, (C.4-D:
Determination of Ready Biodegradability), Directive
92/69/EEC, 31 July 1992.

Organisation for Economic Co-operation and Development
(OECD), OECD guidelines for Testing of Chemicals, Section 3,
Degradation and Accumulation, Guideline No. 301 F
(Manometric Respirometry Test) adopted July 17, 1992.

United States Environmental Protection Agency, Series 835
Fate, Transport, and Transformation Test Guidelines, OPPTS
835.3110 (Ready Biodegradability), EPA 712-C-98-076,
January, 1998.
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Molecular formula:
cyclohexane-1,3-dicarbaldehyde; cyclohexane-1,4-dicarbaldehyde
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report):UNOXOL™ 3,4-Dialdehyde
- Physical state:liquid
- Lot/batch No.:200502771-6M
- Storage condition of test material: ambient

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): oxidation ditch bioreactor at the City of Midland Wastewater Treatment Plant (Midland, Michigan). This facility treats an excess of 3.0 x 106 gallons wastewater daily, of which > 95% is from domestic sources. The mixed liquor inoculum was collected on the day before the test was initiated, and was continuously aerated until used

- Preparation of inoculum for exposure: 10 liters of the sterile
mineral medium was inoculated with 275 mL of the activated sludge mixed liquor, to
yield a final MLSS concentration of approximately 30 mg/L. The initial pH of the
inoculated mineral medium was 7.55, and was adjusted to 7.36 using a concentrated
HCl solution.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
13.1 mg/L
Based on:
Initial conc.:
12.8 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
A series of biodegradation reaction mixtures was prepared in specially designed
1-liter 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 a large PTFE-coated magnetic stir bar. The reaction
vessels are also fitted with two 20 x 105 mm glass side baffles to facilitate complete
mixing/aeration of the stirred reaction mixtures. Inoculum
Blanks, containing the inoculated mineral medium without added test or reference
substances, were prepared in duplicate. These Inoculum Blanks were used to
determine mean values for O2 consumption and CO2 production in the absence of
added test or reference substances. Biodegradation of aniline was determined in
duplicate Positive Control mixtures to verify viability of the inoculum. These
reaction mixtures contained 101.5 mg/L aniline, which was added to the inoculated
mineral medium as a concentrated aqueous solution. Biodegradation of the test
substance was examined in duplicate Test Mixtures containing 23.5 mg/L of the
substance, giving 53.6 mg/L of theoretical oxygen demand (ThOD). These Test
Mixtures were prepared by adding a concentrated aqueous solution of the test
substance to the inoculated mineral medium. A Toxicity Control reaction mixture was
similarly prepared by combining these same concentrations of the test and reference
substances in 500 mL of the inoculated mineral medium. Lastly, a single Abiotic
Control mixture was prepared in the same manner as the Test Mixtures, with addition
of HgCl2 (250 mg/L final conc.) as a chemical sterilant.
After addition of test and reference substances to the appropriate vessels containing
inoculated mineral medium, the resulting reaction mixtures were stirred for 30
minutes to ensure their complete dissolution. A sample (25 mL) of the reaction
mixture in each vessel was removed to determine the initial concentrations of
dissolved organic carbon (DOC), nitrate, and nitrite. In addition, the initial pH of
each reaction mixture was determined. Prior to measurement of initial O2 and CO2
concentrations, the vessels were purged with ambient air, and the associated
headspace gas volume of each individual reaction vessel was determined by the
automated respirometer system. The biodegradation reactions were incubated in
darkness and continuously stirred by a magnetic stir bar at approximately 150 r.p.m.
Reference substance
Reference substance:

Results and discussion

% Degradation
% degradation (DOC removal)
ca. 82
Sampling time:
28 d
Details on results:
Points of degradation plot (test substance):
12.4 % degradation after 6 d
60.6 % degradation after 11 d
81.6 % degradation after 28 d

BOD5 / COD results

Results with reference substance:
Points of degradation plot (reference substance):
45.8 % degradation after 3 d
64.6 % degradation after 5 d
96.1 % degradation after 11 d

Applicant's summary and conclusion

Interpretation of results:
readily biodegradable
The results of this study demonstrate that the UNOXOL Dial substance meets both
the pass level (> 60% DO2) and 10-day window criteria for ready biodegradability in
the Manometric Respirometry test. Whereas this commercial substance is derived
from a reaction mixture of constitutional and stereo- isomers (i.e., cis - and transisomers
of 1,3- and 1,4-cyclohexanedicarboxaldehyde), the extent of degradation
observed in this test indicates that all isomers were fully biodegraded. Thus, the
UNOXOL Dial components can be regarded as readily biodegradable, and as such
can be expected to rapidly and completely biodegrade in a variety of aerobic
Executive summary:

The biodegradability of UNOXO 3,4-Dialdehyde was evaluated using the OECD Guideline

301F- Manometric Respirometry test. This test employed biodegradation reaction mixtures

(500 mL) containing a defined mineral medium, which was inoculated with 30 mg/L (dry solids)

activated sludge collected from the City of Midland Wastewater Treatment Plant (Midland,

Michigan). Duplicate biodegradation Test Mixtures were prepared by adding the substance

(23.5 mg/L) to this inoculated mineral medium, and duplicate Positive Control mixtures were

similarly prepared for a readily biodegradable reference substance (aniline, 101.5 mg/L). A

single Toxicity Control mixture, which contained both substances at these same concentrations,

was prepared to determine whether UNOXOL™ 3,4-Dialdehyde was toxic or inhibitory to the

inoculum used in the test. In addition, an Abiotic Control mixture, in which the test substance

was combined with chemically-sterilized (250 mg/L HgCl2) inoculated mineral medium, was

used to determine the e xtent to which the substance was degraded in the absence of microbial

activity. These reaction mixtures were incubated in darkness at 20.9 ± 0.1°C (mean ± 1 Std.

Dev.), with continual stirring at 150 rpm. Measurements of biological oxygen demand (BOD)

and carbon dioxide evolution (mineralization) were performed at 6 hour intervals over the entire

28-day test period, using a MicroOxymax respirometer system. These measurements were

corrected for oxygen consumed and carbon dioxide evolved in the Inoculum Blanks, which

contained only the inoculated mineral medium. The percentage biodegradation based on

removal of dissolved organic carbon (DOC) was also determined after 28 days.

The onset of UNOXOL™ 3,4-Dialdehyde biodegradation (i.e. BOD > 10% of theoretic al oxygen

demand, ThOD) occurred after 6.1 days in the Test Mixtures, and biodegradation exceeded the

pass level of 60% ThOD consumption within 5 days thereafter. At the end of the 28-day test, the

extent of biodegradation based on BOD, CO2 evolution, and DOC removal reached 81.6 ± 12.5

%, 65.2 ± 6.2 %, and 96.7 ± 1.0 % (mean ± 1 Std. Dev.), respectively. Thus, biodegradation

exceeded both the pass level and 10-day window criteria for classification as “readily

biodegradable in the Manometric Respirometry Test”. In addition, the recorded extents of CO2

evolution, and DOC removal exceeded the pass levels specified for similar ready

biodegradability tests which are based on these measurements. The Toxicity Control mixture

showed no apparent toxic or inhibitory effects of UNOXOL™ 3,4-Dialdehyde on the inoculum

employed in this test, and no DOC removal or net consumption of oxygen was observed in the

Abiotic Control mixture.

All test parameters met the guideline-specified validation criteria for ready biodegradability tests.

Biodegradation of aniline exceeded the pass level within 14 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. Thus, UNOXOL™ 3,4-Dialdehyde can be classified as

readily biodegradable, and is expected to rapidly biodegrade in a variety of aerobic