Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Identity: CP-163,625-BV
Chemical name: (2-Benzydry1-1-aza-bicyclo[2.2.2]oct-3-y1)-benzyl-amine
Intended use: Pharmaceutical intermediate
Appearance: White powder
Storage conditions: Room temperature in the dark
Lot No: 38654-187-3
Purity: 97.2%
Date received: 20 May 1998

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly industrial (adaptation not specified)
Details on inoculum:
A sample of secondary effluent was collected on the day of the test from Thomdon Sewage
Treatment Works, a trickling-filter plant treating predominantly domestic waste. It was maintained
under aerobic conditions in the laboratory, then, immediately before use, vacuum-filtered through a
Whatman's GFC filter paper and the filtrate used as the inoculum for the test (1 ml filtrate/litre test
medium).
Duration of test (contact time):
ca. 28 d
Initial test substance concentration
Initial conc.:
ca. 3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Three groups of eighteen BOD bottles (277 ml) were filled with Mineral Salts Medium (MSM,
Appendix 1), inoculum and the test substance or sodium benzoate according to the schedule shown
below, care being taken to avoid the introduction of air bubbles. A fourth group of four bottles,
which was included to determine whether the test substance was inhibitory to the microbial inoculum
during five days of incubation, contained inoculated MSM, sodium benzoate and the test substance.

Group Contents
1 MSM + inoculum
2 MSM + inoculum + sodium benzoate (5 mg/L)
3 MSM + inoculum + CP-163,625-BV (3 mg/L)

Inhibition assay
4 MSM + inoculum + CP-163,625-BV (3 mg/L) + sodium benzoate (5 mg/L)

In a preliminary solubility test, CP-163,625-BV was found to be insufficiently soluble to allow the
preparation of an aqueous stock solution so appropriate nominal weights (0.83 mg) in glass ampoules
were added directly to BOD bottles: the ampoules were then broken before addition of mineral salts
medium, to allow the test material to disperse.
Stability information for the test article was not available at the time of the study conduct and was
not determined during the study.

The reference substance was added as an aqueous solution (1 g/L).
The concentrations of dissolved oxygen (DO) and the temperatures of the contents of duplicate
vessels from each group were measured, using a YSI dissolved oxygen meter fitted with a selfstirring DO/temperature bottle probe, at the start of the test and after incubation in darkness for five days (inhibition test) and also after 7, 11, 14, 18, 21, 25 and 28 days (defmitive test) at 22 ± 2°C.

The pH of each control, test and reference mixture was measured after oxygen and temperature
measurement.
The temperature of the incubator was measured at intervals during the test using a maximumminimum thermometer.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 2
Sampling time:
28 d

BOD5 / COD results

BOD5 / CODopen allclose all
Parameter:
BOD5
Value:
ca. 0.05 g O2/g test mat.
Parameter:
ThOD
Value:
ca. 2.42 g O2/g test mat.
Results with reference substance:
Sodium Benzoate calculated ThOD= 1.67 mgO2/mg.
Sodium benzoate had been degraded to 60% of its ThOD after five days of incubation and achieved a
maximum of 76% degradation during the 28 day test period. These results confirm that the inoculum
was exhibiting normal biodegradative activity and that the test was valid.

Any other information on results incl. tables

The temperatures of the contents of the BOD bottles and incubator ranged from 20.6 to 23.7°C and 22.4 to 23.1°C respectively during the test, and the pH of the mixtures ranged from 6.6 to 7.8.

Table 1 gives the measured oxygen levels and oxygen consumption (mg02/1) in test and control groups. Table 2 gives the mean level of oxygen consumption (mg02/mg) for mixtures containing the test or reference material and the extent of degradation expressed as a percentage of their ThOD values.

Sodium benzoate had been degraded to 60% of its ThOD after five days of incubation and achieved a maximum of 76% degradation during the 28 day test period. In the presence of CP-163,625-BV, benzoate had been degraded by 63% after five days. These results confirmed that the inoculum was viable and that CP-163,625-BV was not inhibitory to the microbial inoculum.

Oxygen consumption in bottles containing CP-163,625-BV alone was, at most, 0.05 mg02/mg or 2% of its ThQD on Day 5. Substances are considered to be readily degradable in this test if oxygen consumption is equal to or greater than 60% of the theoretical value within ten days of the level first exceeding 10%. CP-163,625-BV cannot, therefore, be considered to be readily degradable.

Oxygen consumption by Day 28 in control bottles containing inoculated medium (maximum = 0.82 mg02/1 on Day 18) was acceptable for this assay system (recommended maximum value = 1.5 mg02/1).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The five-day inhibition assay was conducted to determine whether CP-163,625-BV, at a
concentration of 3 mg/I, inhibited the normal degradative activity of the microbial inoculum on the
reference material, sodium benzoate. The presence of test substance at this concentration was not
inhibitory to the microbial inoculum.
Oxygen consumption in bottles containing CP-163,625-BV was, at most, equivalent to 2% of its
ThOD. Substances are considered to be readily degradable in this test if oxygen consumption is
equal to or greater than 60% of the 'ThOD in ten days after the level exceeds 10%. CP-163,625-BV
cannot, therefore, be considered to be readily degradable.
Sodium benzoate had been degraded to 60% of its ThOD after five days of incubation and achieved a
maximum of 76% degradation during the 28 day test period. These results confirm that the inoculum
was exhibiting normal biodegradative activity and that the test was valid.
Oxygen consumption in control bottles containing inoculated medium (maximum = 0.82 mg02/1 on
Day 18) was acceptable for this assay system (recommended maximum value 1.5 mg02/1).
Owing to the stringency of this type of test, substances that fail to achieve the required rate and level
of degradation are not necessarily non-degradable under environmental conditions. Degradation of
poorly soluble materials such as CP-163,625-BV may occur at concentrations within its limit of
aqueous solubility and under more favourable conditions, such as incubation with a larger
concentration of sewage inoculum over a longer period of time.
Executive summary:

The ready biodegradability of CP-163,625-BV was assessed in the Closed Bottle Test (Procedure CA-E of the Annex to Directive 92/69/EEC; OECD Procedure 301D). Eighteen bottles were filled with mineral salts medium inoculated with sewage effluent (1 m1/1), and CP-163,625-BV was added at a nominal concentration of 3 mg/l. A further four bottles were established for a concurrent five-day microbial inhibition assay, in which the degradation of the readily degradable reference material sodium benzoate was examined in the presence of the test substance. Groups of control and reference bottles were filled with inoculated mineral salts medium, with and without sodium benzoate (5 mg/1). The concentrations of dissolved oxygen (DO) in duplicate bottles from each group were measured at the start of the test and after five days of incubation at 22 ± 2°C in darkness. DO concentrations in the control and reference groups and in bottles containing CP-163,625-BV alone were measured also after 7, 11, 14, 18, 21, 25 and 28 days. Sodium benzoate had been degraded to 60% of its ThOD (1.67 mg02/mg) after five days of incubation and achieved a maximum of 76% degradation during the 28 day test period. In the presence of CP-163,625-BV, benzoate had been degraded by 63% after five days. These results confirm that the inoculum was viable and that the test substance was not significantly inhibitory to the microbial inoculum. Oxygen consumption by Day 28 in control bottles containing inoculated medium (at most, 0.82 mg02/1 on Day 18) was acceptable for this assay system (recommended maximum value 1.5 mg02/1). Oxygen consumption in bottles containing CP-163,625-BV alone was negligible and was, at most, 0.05 mg02/mg or 2% of its ThOD (2.42 mg02/mg) on Day 5. Substances are considered to be readily degradable in this test if oxygen consumption is equal to or greater than 60% of the theoretical value within ten days of the level exceeding 10%. CP-163,625-BV cannot, therefore, be considered to be readily degradable.