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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 August and 2 September 2004.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Deviations:
yes
Remarks:
Deviation was not considered to affect the integrity of the study or the validity of the conclusions drawn
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
The test substance was received at Brixham Environmental Laboratory on 19 May 2004 and assigned the Brixham test substance number 04-0169. The test substance (Batch Sample Ref SP04027/GLP) was supplied as a white/cream solid.

The sample was stored, at ambient temperature, in the container in which it was received until required for testing, when appropriate subsamples were provided for the test operators.
Oxygen conditions:
not specified
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Four treatments were prepared. Three of the treatments served as controls; the biological control (containing inoculum only), the procedural control (containing 300 mg C L-1 aniline, as the reference substance, plus inoculum) and a chemical control (containing 10 mg r1 ceftazidime dihydrochloride with no inoculum). The remaining treatment, termed the biotic exposure, contained 10 mg r1 ceftazidime dihydrochloride plus inoculum . After being made up to volume (3000 ml}, the pH of each treatment was measured and adjusted to between 6.5 and 8.0 using 2 M sodium hydroxide or 2 M hydrochloric acid as appropriate. Each treatment was then divided equally between two replicate flasks, providing an initial test volume of 1500 ml. The flasks were covered with aluminium foil to exclude light, provided with gentle aeration and stirred (magnetic stirrer) for 14 days at a nominal 20 to 25°C. Prior to sampling, the volume in each flask was measured and evaporative losses were corrected as necessary by the addition of deionised water. The pH of each flask was recorded on weekdays during the experimental period and adjusted to between 6.5 and 8.0 as necessary.
Duration of test (contact time):
ca. 14 d
Initial conc.:
ca. 10 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
TOC removal
Details on study design:
Principle of the test method
The concentration of ceftazidime dihy
lnoculum
Activated sludge was obtained from Buckland Sewage Treatment Works Newton Abbot, Devon, UK. This works treats sewage of predominantly domestic origin. At the laboratory the activated sludge was kept aerated at room temperature and the pH maintained at approximately 7. The day before the test the activated sludge was allowed to settle, the supernatant liquid decanted and the remaining sludge washed with test nutrient solution. Finally the supernatant was decanted and the total solids concentration determined on the remaining sludge. The activated sludge used in the test was not adapted to the test or reference substances.

Nutrient solution
The test medium was made up according to the OECD 302 B guideline and contained approximately the following nutrients per litre of deionised water: 85 mg of KH2P04, 217.5 mg of K2HP04, 334 mg of Na2HP04.2H20, 5 mg of NH4Cl, 22.5 mg of MgS04.7H20, 36.4 mg of CaCl2.2H20, 0.25 mg of FeCl3.6H20 and 0.40 mg of EDTA (disodium salt).

Test and reference substance solutions
A 100 mg L-1 stock solution of ccftazidirne dihydrochloride was prepared by dissolving a known quantity in deionised water and stirred for 15 minutes.
A 3000 mg C L-1 (ie expressed as mg of carbon per litre) stock solution of aniline was prepared by dissolving a known quantity in deionised water .
Both stock solutions were observed to be clear and colourless.

Experimental design
Four treatments were prepared as shown in Table 1. Three of the treatments served as controls; the biological control (containing inoculum only), the procedural control (containing 300 mg C L-1 aniline, as the reference substance, plus inoculum) and a chemical control (containing 10 mg L-1 ceftazidime dihydrochloride with no inoculum). The remaining treatment, termed the biotic exposure, contained 10 mg L-1 ceftazidime dihydrochloride plus inoculum . After being made up to volume (3000 ml}, the pH of each treatment was measured and adjusted to between 6.5 and 8.0 using 2 M sodium hydroxide or 2 M hydrochloric acid as appropriate. Each treatment was then divided equally between two replicate flasks, providing an initial test volume of 1500 ml. The flasks were covered with aluminium foil to exclude light, provided with gentle aeration and stirred (magnetic stirrer) for 14 days at a nominal 20 to 25°C. Prior to sampling, the volume in each flask was measured and evaporative losses were corrected as necessary by the addition of deionised water.

The pH of each flask was recorded on weekdays during the experimental period and adjusted to between 6.5 and 8.0 as necessary.

Total organic carbon (TOC) analysis
During the course of the test, measured aliquots were removed from the biological and procedural controls for TOC analysis (Ref 2). Prior to analysis the samples were centrifuged, at approximately 13 800 G for 15 minutes, to remove particulate matter. This was performed 0, 1, 3 and 8 hours after the start of the test and on days 1, 4, 7, 12 and 14 .

Specific analysis by HPLC
During the course of the test measured aliquots were removed from the biological and chemical controls and the biotic exposure for specific ceftazidime dihydrochloride analysis. Prior to analysis the samples were centrifuged, at approximately 13 800 G for 15 minutes, to remove the particulate matter. These were taken 0, 1, 3 and 8 hours after the start of the test and on days 1, 4, 7, 12 and 14 for the biotic exposure. The biological and chemical controls were sampled at O hours, plus days 1 and 14. The supernatant was analysed using the high performance liquid chromatography.
Reference substance:
aniline
Remarks:
Aniline (300 mg C L-1)
Key result
Parameter:
% degradation (TOC removal)
Value:
ca. 0
Sampling time:
0 h
Key result
Parameter:
% degradation (TOC removal)
Value:
< 5
Sampling time:
8 h
Key result
Parameter:
% degradation (TOC removal)
Value:
ca. 8
Sampling time:
1 d
Key result
Parameter:
% degradation (TOC removal)
Value:
ca. 29
Sampling time:
4 d
Key result
Parameter:
% degradation (TOC removal)
Value:
ca. 46
Sampling time:
7 d
Key result
Parameter:
% degradation (TOC removal)
Value:
ca. 61
Sampling time:
12 d
Key result
Parameter:
% degradation (TOC removal)
Value:
ca. 65
Sampling time:
14 d
Key result
Parameter:
% degradation (TOC removal)
Value:
ca. 0
Sampling time:
1 h
Key result
Parameter:
% degradation (TOC removal)
Value:
ca. 0
Sampling time:
3 h
Results with reference substance:
The results for the procedural control show that a minimum of 70% aniline biodegradation had been achieved within 14 days, confirming the viability of the activated sludge
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
Analysis of the biotic exposure at the end of the 14 day test period showed mean degradation of 65%. Analysis of the chemical control at the end of the 14 day test period showed mean degradation of 31 %. It is, therefore, considered that the degradation of ceftazidime dihydrochloride in this study was partially an abiotic process.
Executive summary:

Subject: Determination of inhere~t biodegradability (Zahn-Wellens test)

Guideline: OECD Guidelines for the Testing of Chemicals (1993). Test Guideline 302 B, Zahn-Wellens/EMPA Test. Adopted 17 July 1992

Test substance concentration: 10mg L-1

Duration of test: 14 days

Reference substance: Aniline (300 mg C L-1)

Time             Ceftazidime dihydrochloride              Chemical control              Reference substance

0 h                                   -                                                 -                                         -

1 h                                   -                                                 -                                         -

3 h                                   -                                                 -                                         -

8 h                                  <5                                               -                                        <5

1 d                                   8                                              <5                                       <5

4 d                                   29                                              -                                         34

7 d                                   46                                              -                                          93

12 d                                 61                                              -                                          94

14 d                                 65                                            31                                          98

Comments: Analysis of the biotic exposure at the end of the 14 day test period showed mean degradation of 65%. Analysis of the chemical control at the end of the 14 day test period showed mean degradation of 31 %. It is, therefore, considered that the degradation of ceftazidime dihydrochloride in this study was partially an abiotic process.

Description of key information

Analysis of the biotic exposure at the end of the 14 day test period showed mean degradation of 65%. Analysis of the chemical control at the end of the 14 day test period showed mean degradation of 31 %. It is, therefore, considered that the degradation of ceftazidime dihydrochloride in this study was partially an abiotic process.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable
Type of water:
freshwater

Additional information