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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 March 20th to April 17th, 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Justification for Read Across is explained and detailed in the report attached to the IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Version / remarks:
adopted on 17 July 1992
GLP compliance:
no
Remarks:
the study was conducted in accordance with ISO/EC 17025
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: activated sludge from the aeration tank of a municipal biological waste water treatment plant, ARA Werdhölzli, CH-8048 Zürich.
- Pre-conditioning: not pre-conditioned.
- Adaptation: the activated sludge was used immediately after sampling from the treatment plant without adaptation.
- Preparation of inoculum for exposure: prior to the test, the sludge was washed twice with tap water.
Duration of test (contact time):
28 d
Initial conc.:
50 mg/L
Based on:
test mat.
Initial conc.:
83.3 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
DOC removal
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: aerobic mineral salts medium prepared with double distilled water (conductivity: < 1.5 µS/cm; DOC: < 0.3 mg/l).
Stock solution I, 10 ml/l (pH 7.4): Ammonium chloride 0.50 g, Di-sodium hydrogenphosphate 33.40 g, Potassium dihydrogenphosphate 8.50 g, Di-potassium monohydrogenphosphate 21.75 g
Stock solution Il, 1 ml/l: Calcium chloride, anhydrous 27.50 g
Stock solution III, 1 ml/l: Magnesium sulfate 22.50 g
Stock solution IV, 1 ml/l: Iron (III) chloride 0.25 g
- Oxygen: it was assured that during the test the oxygen concentration was greater than 6 mg/I.
- Test temperature: 22 ± 0.5 °C
- Additional substrate: none, test substance or procedure control as sole organic carbon source.
- Continuous darkness: yes.
- pH: the pH-value was checked periodically and adjusted to pH 6.5-8.0 with NaOH or HCI, if necessary.

TEST SYSTEM
- Test suspension: two replicates containing activated sludge, test medium and the test substance.
- Stirring: the test vessels were stirred (100 r.p.m.)
- Aeration: the test vessels were areated with synthetic CO2-free air for the test period.
- Inoculum: 0.2 g/l dry metter of activated sludge in final mixture.
- Test unit: 1200 ml closed glass bottle containing a total volume of test solution of 600 ml; aerated with CO2-free air and fitted to gas-absorption bottles containing 120 ml of 0.5 M NaOH.

SAMPLING
- Sampling: the elimination of the test material was followed by DOC determinations at regular intervals. First samples were analyzed at the beginning of and 3 h after starting the test.
- Air control: the air leaving the individual vessels was passed through gas-absorption bottles filled with NaOH. The trapped CO2 was determined as inorganic carbon (IC).

CONTROL AND BLANK SYSTEM
- Inoculum blank: two replicates containing activated sludge and test medium
- Reference compound: one replicate containing activated sludge, the test medium and the diethylene glycol as ready biodegradable reference compound.
Reference substance:
diethylene glycol
Parameter:
% degradation (DOC removal)
Value:
94
Sampling time:
28 d
Remarks on result:
other: water elimination
Parameter:
% degradation (CO2 evolution)
Value:
-21
Sampling time:
28 d
Remarks on result:
other: ThCO2
Details on results:
DOC removal at 1 day: 99 %
No biodegradation of test item based on CO2 evolution and calculated as % ThCO2 was observed. The negative mineralization values even showed that the test substance had some toxic effects on the activated sludge microorganisms.
Results with reference substance:
DOC removal at 28 days: 100 %
DOC removal at 14 days: 99 %
The extent of mineralization of the procedure control with the reference substance was 92 % based on ThCO2.

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

Time Blank* Positive substance Positive substance
DOC (mg/l) DOC (mg/l) DOC net. (mg/l) Degradation (%) DOC (mg/l) DOC net. (mg/l) Degradation (%)
0 0.2 48.0 47.8 - n.d. - -
0.125 0.9 48.2 47.3 2 n.d. 83.3 0
1 0.9 45.5 44.6 7 1.6 0.7 99
4 0.9 20.6 19.7 59 4.0 3.1 96
7 1.2 1.8 0.6 99 4.2 3.0 96
11 1.3 2.2 0.8 98 1.3 3.0 96
14 1.8 2.1 0.3 99 4.2 2.4 97
17 1.5 1.8 0.3 99 4.9 3.4 96
21 1.3 1.8 0.5 99 4.6 3.3 96
25 1.4 2.0 0.6 99 5.6 4.3 95
27 1.4 1.5 0.1 100 6.2 4.8 94
28 1.3 1.5 0.2 100 5.9 4.6 94

*Average of two replicates

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

Time Test subspension IC (mg/l) Procedure control IC (mg/l) Inoculum blank IC (mg/l) Biodegrad. Test suspension ( % ThCO2) Biodegrad. Procedure control ( % ThCO2)
0 n.d. n.d. n.d. - -
7 9.5 48.2 17 -9 66
14 10.5 59.7 22.5 -14 79
21 9.2 67.4 24.3 -18 91
28 8.2 96.3 25.8 -21 92
Validity criteria fulfilled:
yes
Remarks:
the degradation of the reference compound was more than 70 % within 14 days of incubation
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The substance resulted eliminated from water mostly by physico-chemical adsorption. Based on the mineralization potential, the test item resulted as not inherently biodegradable.
Executive summary:

The biodegradation potential of the test substance was examined in the Zahn-Wellens/EMPA test, according to internationally accepted test guidelines OECD 302B. The test substance was incubated with activated sludge of municipal sewage treatment plant (non-adapted) under aerobic conditions, for 28 days. The initial TOC content was 83.3 mg/l.

94 % DOC removal was observed after 28 days. 99 % elimination was observed after one day of incubation. However, the DOC concentration subsequently increased again up to the incubation period.

No biodegradation of test item, based on CO2 evolution and calculated as % ThCO2, was observed. The negative mineralization values even showed that the test substance had some toxic effects on the activated sludge microorganisms. The extent of mineralization of the procedure control with the reference substance was 92 % based on ThCO2.

Discussion and conclusion

Based on the DOC removal, the substance resulted eliminated from water mostly by physico-chemical adsorption, thus a precise distinction between biodegradation (or partial degradation) and adsorption can not be drawn. On the contrary, on the basis of the mineralization (CO2 evolution), calculated as % ThCO2, the test item resulted to be not 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

The biodegradation potential of Basic Blue 140 (BBl140) was examined in the Zahn-Wellens/EMPA test, according the OECD guideline 302B. The test substance was incubated with activated sludge from a municipal wastewater treatment plant with aeration and at room temperature, for 28 days. 67 % of DOC removal was observed after 3 hours of exposure and 91 % DOC removal was observed after 28 days of exposure. During the test BBl140 was eliminated from water mostly by physicochemical adsorption, nevertheless a precise distinction between biodegradation (or partial degradation) and adsorption can not be drawn and a conclusion about biodegradability potential can not be reached.

In order to assess the biodegradation potential, the Zahn-Wellens test performed on the Similar Substance 01, has been taken into account. 94 % DOC removal was observed after 28 days and 99 % elimination was observed after one day of incubation. No biodegradation of test item, based on CO2 evolution and calculated as % ThCO2, was observed. The negative mineralization values even showed that the test substance had some toxic effects on the activated sludge microorganisms. The extent of mineralization of the procedure control with the reference substance was 92 % based on ThCO2.

Based on the DOC removal, as in the case of BBl140, Similar Substance 01 resulted eliminated from water mostly by physicochemical adsorption, thus a precise distinction between biodegradation and adsorption can not be drawn. On the contrary, on the basis of the mineralization (CO2evolution), calculated as % ThCO2, the Similar Substance 01 resulted to be not inherently biodegradable. The same behaviour is expected also for BBl140.

BBl140 and Similar Substance 01 share the same phthalocyaninic scaffold structure and, in both cases, they present a zwitterionic moiety formed by sulphonic acid, which conducts an acid function, and a tertiary amine on the sulphonaminic chain, characterized by a basic function. BBl140 presents a further sulphonaminic functionalization, of which chain ends with a tertiary amine that, based on the stechiometrical ratio reported in the analytical characterization, in most of the cases can be salified with acetic acid.

It is expected that this difference does not impact on the biodegradability potential. The read across approach has been further detailed in the report attached to the IUCLID section 13.