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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:
Jan 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with complete documentation.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Principles of method if other than guideline:
Ready biodegradability was followed based on enhanced O2 consumption due when compared to a control without test material. In order to account for the poor water solubility , the test material was coated onto silica before adding to the reactors. Test material specific was performed at selected time points during the exposure and an attempt was made to identify metabolites.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
A mixed population of sewage treatment micro-organisms was obtained on
21 January 2010 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
other: coated to silica gel (50 mg/L)
Initial conc.:
33.2 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
test mat. analysis
Reference substance:
aniline
Parameter:
% degradation (O2 consumption)
Value:
5
Sampling time:
28 d
Parameter:
% degradation (test mat. analysis)
Value:
10
Sampling time:
7 d
Parameter:
% degradation (test mat. analysis)
Value:
37
Sampling time:
14 d
Parameter:
% degradation (test mat. analysis)
Value:
46
Sampling time:
21 d
Parameter:
% degradation (test mat. analysis)
Value:
82
Sampling time:
28 d
Results with reference substance:
Aniline attained 98% degradation after 28 days, calculated from the results of the DOC analyses performed on Days 0 and 28. The degradation rate calculated from the results of the DOC analyses was higher than that calculated from oxygen consumption values. This is considered to be due to incorporation of the test material/aniline into the microbial biomass prior to degradation, and hence oxygen consumption, occurring.

Based on the chemical oxygen demand for the test material, the test material attained 5% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301F.

The compound specific analyses conducted on Day 0 using gas chromatography analysis showed the measured concentrations in the test material plus inoculum vessels to range from 87% to 90% of the nominal value.  In the abiotic test material vessels the measured concentrations ranged from 92% to 99% of the nominal value.  Examination of the chromatograms from Day 0 of the test period showed one peak attributable to the test material in all vessels containing the test material.

The compound specific analyses conducted on Day 28 showed measured concentrations in the test material plus inoculum vessels to range from 15% to 16% of nominal values.  Two new peaks in the chromatogram were identified as the mono-diol and di-diol of the test material using liquid chromatography mass spectrometry.  In the abiotic vessels the measured concentration of the test material was 9% of the nominal value with two additional peaks which were identified as the mono-diol and di-diol of the test material using liquid chromatography mass spectrometry.  

Although the results from chemical analysis on Day 28 indicated that there was an approximate 85% loss of test material from the inoculated test material vessels, this loss was considered not to be due to biological degradation as a similar loss was observed in the abiotic vessels. The two additional peaks shown in the chromatograms of the test material and abiotic control vessels analysed using liquid chromatography mass spectrometry on Day 28 confirmed that hydrolysis of the test material occurred over the test period.

The oxygen consumption values from the inoculated test material vessels confirmed no biological degradation of the test material occurred over the test period.

Table 1: BOD and Biodegradation Values

Sample Description

ThOD/COD

(mg O2/l)

Day 7

Day 14

Day 28

BOD

(mg O2/l)

Degradation (%)

BOD

(mg O2/l)

Degradation (%)

BOD

(mg O2/l)

Degradation (%)

Mean Biodegradation (%)

Control

R1

-

7.24

-

12.82

-

27.04

-

-

R2

-

7.38

-

13.62

-

31.24

-

Aniline

 

309

96.80

29

211.16

64

248.70

71

-

Test Material

R1

33.2

6.28

-3

12.74

-1

30.36

4

5

R2

33.2

7.38

0

14.36

3

30.90

5

Toxicity Control

 

342.2

7.74

0

115.38

30

225.88

57

-

Abiotic Control

R1

33.2

2.34

-

3.88

-

6.82

-

-


R1– R2= Replicates 1 and 2

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
No biodegradation observed. However, significant hydrolysis occurred eliminating 82 % over 28 days.
Executive summary:

The material has been tested for biodegradation in an enhanced ready test following the OECD 301F (oxygen depletion) design. Enhancement consisted of coating the material to silica gel to address the low solubility and the high viscosity of the test material. For a series of similar materials (epoxydes), respiration rates in test vessels were significantly lower than in the controls. This is taken as a sign of inhibition of the inoculum that is more relevant than the toxicity control. Based on these findings, test material concentration was reduced to 20 mg/l. After 28 days of exposure, 5% of the test material has mineralised. Test material specific analysis revealed a substantial elimination of the diepoxide with the mono- and di-diol as relevant products formed. At termination of the study (day 28) 18% of the initial measured concentration could be recovered. The same elimination was observed in the inhibited reactions mixtures (sodium azide) indicating that this is not due to a biological process (Harlan, 2010). The pseudo first order rate of elimination in activated sludge at 20 °C is - 0.0565 [1/d].

Description of key information

Not readily biodegradable. Significant hydrolysis to form mono- and di-diol from the di-epoxides at a rate of -0.0565 [1/d] at 20°C.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

The material has been tested for biodegradation in an enhanced ready test following the OECD 301F (oxygen depletion) design. Enhancement consisted of coating the material to silica gel to address the low solubility and the high viscosity of the test material. For a series of similar materials (epoxydes), respiration rates in test vessels were significantly lower than in the controls. This is taken as a sign of inhibition of the inoculum that is more relevant than the toxicity control. Based on these findings, test material concentration was reduced to 20 mg/l. After 28 days of exposure, 5% of the test material has mineralised. Test material specific analysis revealed a substantial elimination of the diepoxide with the mono- and di-diol as relevant products formed. At termination of the study (day 28) 18% of the initial measured concentration could be recovered. The same elimination was observed in the inhibited reactions mixtures (sodium azide) indicating that this is not due to a biological process (Harlan, 2010). The pseudo first order rate of elimination in activated sludge at 20 °C is - 0.0565 [1/d].

Testing according to the modified Sturm test (OECD 301B) at 10 and 20 mg DOC per Litre (approx 13 and 27 mg test material/L) indicated only limited mineralisation of 6 and 12%, respectively, which is attributed to the low solubility of the test material (Ciba-Geigy Limited 1985). Likewise, BOD testing at 10 mg/L ThOD (approx 4 mg/L test material) showed no decrease in Oxygen at day 5, 10 and 20 (TDCC, 1975).