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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From December 20th, 2001 to January 17th, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
The read across approach is detailed into the document attached to the IUCLID section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Version / remarks:
adopted on 17 July 1992
GLP compliance:
no
Remarks:
The study described in this report was conducted in accordance with SN EN 45001
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Inoculum: activated sludge from the aeration tank of a municipal biological waste water treatment plant, not adapted, not pre-conditioned.
- Source of inoculum: ARA Werdhölzli, CH-8048 Zürich, 20 December 2001;9.30 a.m.
- Pre-treatment: the activated sludge was used immediately after sampling from the treatment plant without adaptation. Prior to the test the sludge was washed twice with tap water.
- Concentration: 0.2 g/l dry matter in the final mixture.
- Feed: none, test substance or procedure control as sole organic carbon sources.
Duration of test (contact time):
ca. 28 d
Initial conc.:
84.5 mg/L
Based on:
DOC
Initial conc.:
321 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Test vessels: 1200 ml closed glass bottle.
- Volume: total volume of test solution of 600 ml. The test material was diluted with mineral salts medium to give a final DOC concentration of about 50 mg/l.
- Stirring: the test vessels were stirred at 100 r.p.m.
- Aeration: test vessels were aerated with CO2-free air.
- Temperature: temperature-controlled, 22 ± 0.5 °C.
- Oxygen concentration: higher than 6 mg/l.
- pH value. checked periodically and adjusted to pH 6.5-8.0 with NaOH or HCl, if necessary.
- Continuous darkness: yes.

TEST SYSTEM
- Number of vessels for concentration: test suspension (T) 2 replicates, containing activated sludge + test medium + test substance.
- Number of vessels for blank: inoculum blank (B) 2 replicas, containing activated sludge + test medium.
- Number of vessels for control: procedure control (R) 1 replicate, containing activated sludge + test medium + diethyleneglycol as ready biodegradable reference compound.
- Measuring equipment:
- Trap for CO2 and volatile organics: air leaving the individual vessels was passed through gas-absorption bottles filled with NaOH.

TEST MEDIUM
Composition of the mineral salts medium:
- Stock solution I (pH 7.4) of 10 mg/l
Ammonium chloride, NH4Cl: 0.50 g
Di-sodium hydrogenphosphate, Na2H PO4 x 2 H2O: 33.40 g
Potassium dihydrogenphosphate, KH2PO4: 8.50 g
Di-potassium monohydrogenphosphate, K2HPO4: 21.75 g
- Stock solution II of 1 ml/l: Calcium chloride, anhydrous, CaCl2: 27.50 g
- Stock solution III of 1 ml/l: H2O Magnesium sulfate, MgSO4 x7 H2O: 22.50 g
- Stock solution IV of 1 ml/l: lron (lll) chloride, FeCl3 x 6 H2O: 0.25 g
Reference substance:
diethylene glycol
Remarks:
45.3 mg/l, as DOC
Parameter:
% degradation (DOC removal)
Value:
8
Sampling time:
28 d
Parameter:
% degradation (CO2 evolution)
Value:
18
Sampling time:
28 d
Details on results:
Based on the data of the individual DOC determinations, no significant elimination of test item was obserued after 28 days.
No significant elimination of the test substance due to adsorption to the activated sludge, on the glass surface or other physico-chemical processes was found as determined by means of DOC measurements 3 h after the start of the test.

Some biodegradation (18 %)of the substance on CO2, evolution and calculated as percentage was observed. The higher degradation value as compared to the DOC elimination cannot be explained.
Based on these results, the substance is not inherently biodegradable under the conditions, since less than 20 % degradation was attained after 28 days of contact time.
Results with reference substance:
The positive control, diethyleneglycol, showed 99 % biodegradation, based on DOC removal, after 14 days of incubation, confirming the suitability of inoculum and test conditions. The test was considered valid, since the degradation of the reference compound reached more than 70 % within 14 days of incubation.

The extent of mineralization of the procedure control with diethyleneglycol was 94 % based on ThCO2.

DOC concentrations of test suspension, inoculum blank and procedure control and calculation of degradation dala.

Time (days) Inoculum blank* Procedure control with diethylene glycol Test suspension with test item*
DOC (mg/l) DOC (mg/l) DOC net (mg/l) Degradation (%) DOC (mg/l) DOC net (mg/l) Degradation (%)
0 0.1 45.5 45.4 - 77.1 77.0 -
0.125 1.0 45.2 44.3 0 73.9 72.9 0
1 1.5 44.7 43.2 2 69.6 68.1 7
4 2.1 34.3 32.2 27 74.5 72.4 1
7 1.8 2.5 0.6 99 72.7 70.8 3
11 2.1 2.5 0.5 99 72.8 70.7 3
14 1.6 2.1 0.6 99 73.3 71.7 2
15 1.8 2.1 0.3 99 72.5 70.6 3
18 1.6 1.8 0.2 100 70.0 68.4 6
21 1.7 1.7 0.0 100 71.8 70.1 4
25 1.6 2.0 0.4 99 71.3 68.7 6
27 1.8 2.1 0.4 99 69.6 67.9 7
28 1.5 1.6 0.1 100 68.8 67.3 8

*Mean of duplicate

IC concentrations, calculated from the concentrations in the gas absorption bottles, of test suspension, inoculum blank and procedure control and corresponding degradation data.

Time (days) Test suspension IC (mg/l) Procedure control IC (mg/l) Inoculum blank IC (mg/l) Biodegradation Test suspension (% ThCO2) Biodegradation rocedure contro (% ThCO2)
0 nd nd nd - -
7 36.8 65.1 28.4 10 81
14 49.7 76.8 36.7 15 89
21 54.6 82.7 40.5 17 93
28 60.1 87.4 44.9 18 94
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Neither readily nor inherently biodegradable.
Executive summary:

The biodegradability of test substance exposed to activated sludge of a municipal sewage trealment plant was investigated under aerobic static conditions. Based on the data of the individual DOC determinations no significant elimination of test item was observed after 28 days. No significant elimination of the test substance due to adsorption was found, as determined by means of DOC measurements 3 h after the start of the test. The positive control, diethyleneglycol, showed 99 % biodegradation after 14 days of incubation thus confirming suitability of inoculum and test conditions. The test was considered valid, since the degradation of the reference compound reached more lhan 70 % within 14 days of incubation. Some biodegradation (18 %) of test item, based on CO2 evolution and calculated as percentage, was observed. The higher degradation value as compared to the DOC elimination cannot be explained. The extent of mineralization of the procedure control with diethyleneglycol was 94 % based on ThCO2. Based on these results the substance is not inherently biodegradable under the test conditions, since less than 20 % degradation was attained after 28 days of contact time.

Conclusion

Neither readily nor inherently biodegradable.

Description of key information

Neither readily nor inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Direct Orange 118 is a carbocyclic azo dye. The substance is not expected to be ready biodegradable because of its chemical structure and its specific function. Commonly, dyes undergo a primary transformation, i.e. discolourization due to the interrupting the conjugation. Nevertheless, the degradation process involves more steps and take more time.

There are no experimental information about the biodegradability potential of Direct Orange 118, thus the available data on structural analogues Similar Substance 01 have been taken into account. The read across approach can be considered as reliable and suitable for the purpose; details and explanations are detailed in the report attached to the IUCLID section 13.

The biodegradability of the Similar Substance 01 was investigated under aerobic static conditions, according to the OECD guideline 302B. No significant elimination of test item was observed after 28 days, based on the DOC removal; no significant elimination of the test substance due to adsorption was found. Some biodegradation (18 %) of test item, based on CO2 evolution and calculated as percentage, was observed. However, on the basis of the obtained results, the substance resulted to be not inherently biodegradable under the test conditions, since less than 20 % degradation was attained after 28 days of contact time.