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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:
1996-10-30 to 1996-11-27
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
C29 H24 O7

Study design

Oxygen conditions:
Inoculum or test system:
mixture of sewage, soil and natural water
Details on inoculum:
- Source of inoculum:
A mixed population of activated sewage sludge micro-organisms were obtained on 19 September 1996 from ten different sampling sites around the UK. The sampling sites were as follows:
a) City domestic sewage plants (x3)
i) Liverpool
ii) Belper, Derbyshire
iii) Bristol

b) lndustrial sewage plant (x1)
i) Derby

c) River samples (x3)
i) River Mersey
ii) River Trent
iii) River Severn

d) Lake Water (x1)
i) Allestree Lake, Derby

e) Sea Water Samples (x2)
i) Skegness
ii) Southport

The sample types and volumes sampled from each site were as follows:
a) City sewage: 1 litre of return sludge at each sewage disposal plant.
b) Rivers, lakes and sea: 1 litre of surface water and 1 litre of surface soil on the bank/beach which is in contact with the atmosphere.

The samples obtained from the sampling sites were mixed thoroughly and the mixture allowed to settle. The floating foreign matter was removed and the supernatant filtered through a Whatman CF/A filter paper. The filtrate was then mixed with approximately 2litres of supernatant removed from a previously established culture and transferred to a culture vessel. The pH of the culture mixture was adjusted to 7.0 + 1.0 with sodium hydroxide or phosphoric acid and constantly aerated via a narrow bore glass pipette at a temperature of 24 °C. The culture was allowed to settle daily for approximately 30 minutes and approximately 1/3 of the volume of the supernatant removed. An equal volume of 0.1 % synthetic sewage was added and the aeration re-started again. Synthetic sewage was prepared by dissolving glucose, peptone and monopotassium phosphate in deionised water at a concentration of 0.1 % (w/v). The pH of the synthetic sewage and culture was adjusted daily to within the range pH 7.0 + 1.0 with sodium hydroxide or phosphoric acid. The sludge was found to form a clear supernatant on settling and to have an active microflora including a variety of protozoa, including ciliates, flagellates and a large population of motile bacteria. The sludge formed cloudy flocs when on aeration.
- Concentration of sludge: approx. 30 mg suspended solids/L test solution
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
100 mg/L
Based on:
test mat.
Details on study design:
- Composition of medium: Deionised water was aerated and allowed to stand for about 24 hours at 24 °C to give a dissolved oxygen concentration of 8.40 mg O2/L.
To 1 litre of water was added 3 ml each of solutions 1-4
Solution 1:
K2HPO4 21.75 g/L
KH2PO4 8.50 g/L
Na2HPO4.12H2O 44.60 g/L
NH4CI 1.70 g/L
Solution 2: MgSO4.7H2O 22.50 g/L
Solution 3: CaCl2 27.50 g/L
Solution 4: FeCl3.6H2O 0.25 g/L
- Additional substrate: Synthetic sewage was prepared by dissolving glucose, peptone and monopotassium phosphate in deionised water at a concentration of 0.1 %. (w/v).
- Test temperature: 25 +/- 0.1 °C.
- pH: 7.0 +/- 1.0
- pH adjusted: Yes, the pH of the synthetic sewage and culture was adjusted daily to within the range pH 7.0 + 1.0 with sodium hydroxide or phosphoric acid.
- Aeration of dilution water: Yes, deionised water was aerated and allowed to stand for about 24 hours at 24 °C to give a dissolved oxygen concentration of 8.40 mg O2/L.
- Suspended solids concentration: 30 mg suspended solids/L test solution
- Continuous darkness: yes

- Culturing apparatus: C.E.S. Multi-Channel Aerobic Respirometer
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: deionised water used for test solution was aerated to
give a dissolved oxygen concentration of 8.40 mg O2/L
- Measuring equipment:
ln order to confirm whether the pH of the test preparations were changed, the pH was measured using a WTW pH 320 SET.
The DOC analyses were carried out using a Dohrmann DC-190 Carbon Analyser. The carbon analyses were by the method of high temperature catalytic conversion.

- Test performed in closed vessels due to significant volatility of test substance: yes

- Sampling frequency: sampling on day 0 and 28

- Inoculum blank: 1 replicate
- Abiotic sterile control: 1 replicate
- Toxicity control: no
- Other: reference control (1 replicate)

Results and discussion

% Degradation
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
Points of degradation plot (test substance):
0 % degradation after 7 d
0 % degradation after 14 d

BOD5 / COD results

Results with reference substance:
Points of degradation plot (reference substance aniline):
60 % degradation after 7 d;
66 % degradation after 14 d

Any other information on results incl. tables

Table 1: Degradation based on DOC

Sample descriptionTheoretical carbon content (%) (A)Day 0Day 28
DOC (mg C/L)DOC (mg C/L)
MeasuredCorrected for control values (B)% Theoretical carbon content (C)MeasuredCorrected for control valuesDegradation (%) (D)
Aniline + inoculum77.3866.3864.69841.43-0.1100

Test concentration of 100 mg/L was added to each test vessel

(A) : Theoretical carbon content : [(No. carbon atoms x atomic weight carbon)/molecular weight]

(B) : DOC corrected for control values : DOCmeasured - DOCcontrol

(C) : % Theoretical carbon content: [(corrected DOCDayfheoretical carbon content) x 100]%

(D) : % Degradation : [1-(corrected DOC Day28/corrected DOCDay 0)] x 100%


Table 2: results on days 7, 14, 21 and 28, calculated from oxygen consumption values and the compound specific analyses

IdentificationDegradation (%)
Oxygen consumption 
Day 7Day 14Day 21Day 28Compound specific analysis
100 mg test item/L plus inoculumR1-2-1-11-4

R1, R2, R3: Replicates 1, 2 and 3

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
In a MITI (I) Test according to OECD 301 C no biodegradation of the test item was observed.
Executive summary:

The ready biodegradability of the test item was examined in a GLP compliant study according to OECD TG 301 C. The test item was prepared as an aqueous dispersion at a final concentration of 100 mg/L, inoculated with microorganisms from a laboratory culture originating from 10 different sites throughout the UK and incubated in the dark at 25 °C for 28 days. The degradation of the test material was assessed by the measurements of oxygen consumption and compound specific analyses on days 0 and 28. Control solutions with inoculum and the standard material, aniline, were used for validation purposes. The test item attained 1, -3 and 0% with a mean of -1 % degradation calculated from oxygen consumption values after 28 days. The degradation rates from the residual test material analyses were -4, -4 and -7 % with a mean value of -5 %. The test material cannot therefore be considered to be readily biodegradable under the strict terms and conditions of OECD TG 301 C.