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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August - September 1994
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was performed in accordance with an appropriate test guideline and in compliance with the GLP regulations.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Principles of method if other than guideline:
Not relevant.
GLP compliance:

Test material

Constituent 1
Reference substance name:
Constituent 2
Reference substance name:
Ro 31-9373/000
Ro 31-9373/000
Details on test material:
The test substance PTH-Decahydroamide, batch No. 25927, purity 99.8 %, was received from F. Hoffmann-La Roche Ltd, Switzerland, on 28 April 1994. The test substance was stored at room temperature as specified by the sponsor.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not relevant.

Study design

Oxygen conditions:
Inoculum or test system:
other: Secondary (biologically treated) effluent obtained at an STP receiving sewage/waste waters of predominantly domestic origin.
Details on inoculum:
A fresh sample of secondary effluent was obtained on Day 0 (05 August 1994). On arrival at tbe laboratory, the effluent sample was aerated with compressed air for 20 minutes, then left to settle for 1.5 hours, before it was used to inoculate the test medium.
Duration of test (contact time):
35 d
Initial test substance concentration
Initial conc.:
2.056 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The test design comprised a blank control, two series of reference vessels: one dosed with sodium acetate (2.038 mg/L) and the other dosed with 'Dobanic Acid 103' (2.097 mg/L), and a series of test vessels containing PTH-Decahydroamide dosed at 2.056 mg/L. In addition, the test design also included two further series of toxicity control vessels containing PTH-Decahydroamide applied in combination with either sodium benzoate or 'Dobanic Acid 103'.

[The justification for the unconventional use of two reference compounds is not reported, but the reason is that sodium acetate in general degrades too easily with microorganisms fortuitously present in the test medium to provide a robust confirmation of the activity of the added inoculum - see OECD Environment Monograph No 98, Detailed Review Paper on Biodegradability Testing, p 167. 'Dobanic Acid 103' (linear alkyl benzene sulphonate) is not named as a reference substance in the 301D test guideline, but was selected by the test laboratory to give a more robust indication of the activity of the test inoculum].

The incubation took place in sealed glass vessels completely filled with their respective test media, allowing no headspace, and measurements of residual dissolved oxygen (DO) were made in pairs of vessels sacrificed at test initiation (t0) and at intervals over the course of the test duration. Mean measured oxygen demand (the decline in DO between measurements at time t and t0), corrected for contemporary mean blank uptake was compared to the theoretical oxygen demand (ThOD) required for total oxidation of the test or reference substance. The ratio of measured oxygen uptake to ThOD was expressed as percentage dgradation. Achievement of 60% degradation or more is considered to equate to complete mineralisation.

All test media were prepared in the same manner; 1 mL of each of nutrient solutions I to 4 was added to aerated MilliQ purified water for every litre of final volume of test solution. The test and reference substances were added to the respective media preparations, either in the solid or liquid form, and dispered as the media were made up to final volume. The media were inoculated at a rate of 5 mL secondary effluent/L .

Test vessels were incubated in the dark at 22 ± 2 °C for a period of up to 35 days in an LKB Bromma 2021 Maxicoldlab incubator cabinet. A record of daily temperatures was maintained using a Haenni strip-chart temperature recorder, while independent readings were taken, for comparison, using a standard mercury thermometer.

Residual DO was measured at Day 0, and following incubation, on Day 3, 7, 14, 21, 28, and for certain samples Day 35, also. Dissolved oxygen was not determined on Day 3 for both sets of toxicity controls (Test Substance plus Reference Substance 1, Test Substance plus Reference Substance 2).

Reference substanceopen allclose all
Reference substance:
acetic acid, sodium salt
Reference substance:
other: 'Dobanic Acid 103', a proprietary linear alkyl sulphonate from Shell Detergents

Results and discussion

Preliminary study:
Not relevant
Test performance:
Closed Bottle Test method
% Degradationopen allclose all
% degradation (O2 consumption)
Sampling time:
3 d
% degradation (O2 consumption)
Sampling time:
7 d
% degradation (O2 consumption)
Sampling time:
14 d
% degradation (O2 consumption)
Sampling time:
21 d
% degradation (O2 consumption)
Sampling time:
28 d
Remarks on result:
other: PTH-Decahydroamide did not meet the requirement for classification as readily biodegradable (measured O2 uptake equal to or greater than 60% of the theoretical maximum) within 28 d. Final degradation following test extension to 35d was 1.46%.
Details on results:
Using the Closed Bottle Test method, biodegradability is calculated upon the depletion of oxygen determined in the lest system and compared with the level of oxygen depletion experienced in the inoculum blank, versus compound concentration and the theoretical oxygen demand (ThOD) of
the chemical. ThOD values were calculated using the following equation;
16[2c + l/2(h-cl-3n) + 3s + 5/2p + 1/2 na-o]
ThOD = ·Molecular Weight
This equation calculates the ThOD based upon the assumption that no nitrification effects occur, and that any nitrogen that the test substance contains will be mineralised to ammonium salts. In the case of compounds undergoing biodegradation and mineralisation via a nitrification pailiway
the consumption of oxygen is increased relative to transformation via ammonium. In such circumstances the following equations should be employed in the calculation ofThOD, dependent upon the stage of nitrification.

16[2c + 1/2(h-cl) + 3s + 3/2n + 5/2p + 1/2na -o]


Molecular Weight

16[2c + l/2(h-cl) + 3s + 5/2n + 5/2p + 112na-o]
Molecular Weight Utilising the initial equation we anive at the following ThOD values; 2.487 ppm 0,/mg PTHDecahydroamide;
2.475 ppm 0,/mg Dobanic Acid 103; 0.780 ppm 0,/mg Sodium Acetate (see
calculations and relevent information in Appendix II, pg. 31).

The ThOD of the test substance was calculated on the basis of the first equation since little
biodegradation, if any, was observed during the study proceedings (see Section 5.3, and Table

3.2 ).
Using the values calculated for ThOD, and from dissolved oxygen levels determined in samples,
the percentage biodegradability of a substance can be calculated using the following equation;

(So -S,) (B0 -B,) x 100
% Biodegdn.
(at DayJ ThOD of Substance x Concn. of Substance

Where, -S0 , S, B0 , and B, are the mean values of two replicate samples obtained for
dissolved oxygen for a chemical substance, and in the inoculated blank
control, respectively, at Day 0 and Day x in ppm 0 2 •

-ThOD is the calculated theoretical oxygen demand of the chemical substance· in mg
0,/mg substance.

-Concn. is the concentration of chemical substance in the prepared test system in mg/L.

BOD5 / COD results

Results with reference substance:
Mean degradation in the reference vessels containing sodium acetate reached 72.50% by the time of the first measurement made after 3 days. Mean degradation in the reference vessels containing 'Dobanic Acid 103' reached 60.08% on Day 14. Both reference compounds were rapidly and completely degraded under the conditions of the test. The performance of the reference compounds confirmed theactivity of the inoculum present in the test system.

Similar degradation of the reference substances (both in terms of rate and extent) occurred in the toxicity control vessels where sodium acetate or 'Dobanic Acid 103' were each co-dosed with PTH-Decahydroamide. These findings show that the test substance had no inhibitory impact on the microbial degradation of either reference compound, and indicate that the failure of PTH-Decahydroamide to biodegrade in the test vessels in which it was dosed alone was not the result of toxicity of the test substance toward the inoculum.

Any other information on results incl. tables

Control Blank -Oxygen depletions are observed throughout the 35 day period that readings were taken. Oxygen depletions are lowest within this system, as would be expected.

PTH-Decahydroamide -Throughout the course of the study rates of oxygen depletion in the test system containing PTH-Decahydroamide demonstrated very close resemblence to the pattern of oxygen depletion observed in the Control Blank with Inoculum test system , until Day 28, when an apparent mean degradation of 6.1 % was observed. Based on this result, and despite the fact that PTH-Decahydroamide had already failed the test for classification of "readily biodegradable", the study was continued for a further 7 day period to assess the situation, utilising incubated contingency samples. Results derived from the subsequent Day 35 samples demonstrated little difference from the Control Blank with Inoculum samples, and suggested a mean biodegradation of only 1.5 %. On account of no apparent biodegradation taking place, sub-samples of incubated samples taken for potential NO,/NO, determinations were not analysed, as ThOD correction was not deemed necessary under these circumstances. The test substance did not appear to exhert any inhibitory effects toward the microbial population (see below).

PTH-Decahydroamide plus Dobanic Acid 103 -Oxygen depletion registered after 7 days incubation represents an apparent degradation of 49.1 %, 62.6 % at Day 14, 82.9 % at Day 21, and 76.4 % at Day 28. These values are calculated based on only the ThOD of Dobanic Acid 103, since PTH-Decahydroamide demonstrated no biodegradation when incubated alone. This test system does however demonstrate a slightly more rapid rate of degradation than Dobanic Acid 103 incubated alone, indicating that degradation of PTH-Decahydroamide may be taking place in this test system, possibly due to a flurry of activity, and an increase in microbial population experienced within the test system due to the breakdown of Dobanic Acid 103.

PTH-Decahydroamide plus Sodium Acetate -This test system demonstrated a similar pattern of oxygen depletion as the reference system containing Sodium Acetate only (based on Sodium Acetate ThOD only), again indicating that PTH-Decahydroamid did not act as a direct inhibitor of microbial activity. Following a period of 7 days incubation, there was apparently 82.2 % degradation, at 14 days this value was calculated to be slightly less (77.0 %), at 21 days represented 105.8 %, and 28 days 105.8 %, also (all values calculated on the ThOD of Sodium Acetate only).

Dissolved oxygen determined in a sub-sample of aerated MilliQ dilution water on Day 0 was found to contain 7. 85 mg 0,/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
Based on the outcome of a test conducted according to the Closed Bottle procedure (OECD TG 301D), PTH-Decahydroamide is not readily biodegradable.
Executive summary:
PTH-Decahydroamide (Ro 31 -9373/000) was tested for ready biodegradability according to the Closed Bottle test procedure at a concentration of 2.056 mg/L, in parallel with a 'soft' reference substance (sodium acetate) and a more challenging reference substance: "Dobanic Acid 103" (= linear alkyl benzene sulphonate). Slight (6%) apparent degradation was recorded on Day 28 and further confirmatory measurements were made at Day 35, however no further biodegradation occurred during the final week of incubation. Both reference substances were extensively biodegraded within 28 days, confirming the activity of the inoculum, and toxicity controls containing mixtures of PTH-Decahydroamide and each of the reference substances showed no evidence of microbial toxicity that might suggest that auto-inhibition had contributed to the failure of the test substance to degrade. Based on the outcome of this study, PTH-Decahydroamide is not readily biodegradable.