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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The definitive study was conducted between 1997-08-26 and 1997-09-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no, or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of Inspection 1996-01-22; Date of Signature 1996-02-27
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: A mixed population of activated sewage sludge micro-organisms was obtained on 26 August 1997 from the aeration stage of the Seven Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic sewage.
_________________________________________________________________________________________________________________

- Laboratory culture:

Culture medium

Solution a)
KH2PO4 8.5 g/L
K2HPO4 21.75 g/L
Na2HPO4.2H2O 33.40 g/L

NH4Cl 0.50 g/L

pH = 7.4


Solution b.)
CaCl2 27.50 g/L
Solution c.)
MgSO4.7H20 22.50 g/L
Solution d.)
FeCl3.6H2O 0.25 g/L

To 1 litre (final volume) of purified water* is added the following volumes of solutions a-d.


10 ml of Solution a
1 ml of Solution b
1 ml of Solution c
1 ml of Solution d

*Reverse Osmosis purified and deionised water (Elga Optima 15+)
____________________________________________________________________________________________________________________

- Preparation of inoculum for exposure: The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. A sub-sample of the washed sewage sludge was then removed and the suspended solids concentration determined.


Duration of test (contact time):
28 d
Initial conc.:
14.2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
Sampling and CO2 analysis

Samples (2 mL) were taken from the first CO2 Absorber vessel on days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16 ,18 ,20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on days 0 and 29.

The samples taken on days 0, 1, 2, 3, 6, 8, 12, 14, 20, 22, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on days 10, 16, 18 and 24 were stored deep frozen at -20 degrees Centigrade. However, these samples were not analyzed for CO2 as the results obtained from previous and subsequent analyses showed that the level of degradation of the test material did not significantly increase during this time and therefore additional analyses were considered to be unnecessary.

On day 28, 1mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were released, aerated overnight and the final samples taken from both absorber vessels on day 29.

The samples were analyzed for CO2 using an Ionics 1555B TOC analyzer and a Dohrmann DC-190 TOC analyzer. Samples (40 or 50µl) were injected into the IC (Inorganic Carbon) channel of the TOC analyzer. Inorganic carbon analysis occurs by means of the catalytic conversion of an aqueous sample by phosphoric acid using zero grade oxygen or high purity nitrogen as the carrier gas. Calibration was by a standard solution of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

On days 0 and 28, samples (20 mL) were removed from the culture vessels and centrifuged (3500 rpm, 15minutes) prior to Dissolved Organic Carbon (DOC) analysis.

The samples were analysed for DOC using a Dohrmann DC-190 TOC analyzer. Samples (100 µL) were injected into the TC (Total Carbon) and IC (Inorganic Carbon) channels of the TOC analyzer. Total carbon analysis is carried out at 900 degrees Centigrade using a platinum catalyst and zero grade oxygen as the carrier gas. Inorganic carbon analysis occurs by means of the catalytic conversion of the aqueous sample by phosphoric acid at ambient temperature. Calibration was by a standard solution of potassium hydrogen phthalate (KHP). Each analysis was carried out in triplicate.

Reference substance:
other: Sodium benzoate
Preliminary study:
Preliminary investigational work to assess any toxic effect of the test material on sewage sludge microorganisms following the method described in OECD Guidleine No 209 "Activated sludge respiration inhibition test" was not performed. Therefore a toxicity control (meta-xylenediamine and sodium benzoate) was included in the study to assess any toxic affect of the test material on the sewage sludge micro-organism used in the study.

In an initial experiement conducted at 20 mg C/L, the degradation rate obtained for the toxicity control after 6 days was considered to be insufficient to enable the day 14 validation criterion, whereby greater than 25 % degradation must be attained after 14 days, to be met and hence the study was repeated at a test concentration of 10 mg C/L.
Test performance:
The results of the degradation test are considered valid if in the same test the standard material yields >= 60 % degradation by day 14.
The test material may be considered to be readily biodegradable if >=60 % degradation is attained after 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10%. The toxicity control (Meta-xylenediamine and sodium benzoate) should attain >=25 % degradation by day 14 for the test material to be considered as non-inhibitory. The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the end of the test is less than 20 %.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
49
Sampling time:
28 d
Details on results:
Inorganic carbon values for the test material, standard material, sodium benzoate and control vessels at each analysis occasion are given in Table 1 ( attached). Percentage biodegradation of the test and standard material is given in Table 2 and the biodegradation curves for the test and standard materials are presented in Figure1 (attached).

In an initial experiment conducted at 20 mg C/L, the degradation rate obtained for the toxicity control after 6 days was considered to be insufficient to enable the day 14 validation criterion, whereby greater than 25 % degradation must be attained after 14 days, to be met and hence the study was repeated at a test concentration of 10 mg C/L.

The test material attained a 49 % degradation after 28 days, therefore, cannot be considered as readily biodegradable under the strict terms of the OECD guidelines.

The results of the inorganic carbon analysis of samples from the first absorber vessels on day 29 showed an increase in all replicate vessels. These increases are considered to be due to CO2 present in solution being driven off by the addition of Hydrochloric acid on day 28 and resulted in an increase in the percentage degradation value for the test material from 49 % on day 28 to 54 % on day 29.

Inorganic carbon analysis of the samples from the second absorber vessels on day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

The toxicity control attained 26 % degradation after 14 days and 60 % after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study at the reduced test concentration of 10mg C/L. The increase in inorganic carbon in the control first absorber vessel and hence resulted in a decrease in the percentage degradation value for the toxicity control from 60 % on day 28 to 59 % on day 29.

Analysis of the test media from the test material culture vessels on days 0 and 28 for dissolved organic carbon (DOC) gave percentage degradation values of 100 % and 100 % respectively for replicates R1 and R2 and 100 % for the toxicity control.

The degradation rates calculated from the results of the DOC analyzer were higher than that calculated from oxygen consumption values. This is considered to be due to incorporation of the test material into the microbial biomass prior to degradation, and hence oxygen consumption occurring.

Sodium benzoate attained 94 % degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

The increase in inorganic carbon in the first absorber vessel on day 29 was not sufficient to increase the percentage degradation value for the standard material from 94 %.

Results with reference substance:
The toxicity control attained 26 % degradation after 14 days and 60 % after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study at the reduced test concentration of 10mg C/L. The increase in inorganic carbon in the control first absorber vessels on day 29 was greater than that in the toxicity control first absorber vessel and hence resulted in a decrease in the percentage degradation value for the toxicity control from 60 % on day 28 to 59 % on day 29.

Inorganic carbon values for the test material, standard material, sodium benzoate and control vessels at each analysis occasion are given in Table 1 (attached). Percentage biodegradation of the test and the standard materials is shown below and the biodegradation curves for the test and standard material are presented in Figure 1 (attached).

Percentage Biodegradation Values

Day

% Degradation

Sodium Benzoate

% Degradation

meta-xylenediamine

% Degradation

Test Material plus Sodium Benzoate Toxicity Control

0

0

0

0

1

14

6

7

2

49

1

10

3

48

4

14

6

58

8

18

8

69

15

19

12

75

16

20

14

80

29

26

20

93

30

40

22

96

37

47

27

94

47

60

28

94

49

60

29*

94

54

59


* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

The test material attained 49 % degradation after 28 days, therefore, cannot be considered as readily biodegradable under the strict terms and conditions of the OECD Guidelines.

 

The results of the inorganic carbon anlaysis of samples from the first absorber vessels on day 29 showed an increase in all replicate vessels. These increases are considered to be due to CO2 present in solution being driven off by the addition of hydrochloric acid on day 28 and resulted in an increase in the percentage degradation value for the test material from 48 % on day 28 to 54 % on day 29.

 

Inorganic carbon analysis of the samples from the second absorber vessels on day 29 confirmed that no significant carry-over of CO2into the second absorber vessels occurred.

 

Analysis (see Table 3, attached) of the test media from the test material culture vessels on days 0 and 28 for dissolved organic carbon (DOC) gave percentage degradation values of 100 % and 100 % respectively for Replicates R1and R2and 100 % for the toxicity control.

 

The degradation rates calculated from the results of the DOC analyses were higher than that calculated from oxygen consumption values. This is considered to be due to incorporation of the test material into the microbial biomass prior to degradation, and hence oxygen consumption occurring.

 

Sodium benzoate attained 94% degradation after 28 days thereby confiming the suitability of the inoculum and test conditions.

 

The increase in organic carbon in the first absorber vessels on day 29 was not sufficient to increase the percentage degradation value for the standard material from 94 %.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test material attained 49 % degradation after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No 301B
Endpoint:
biodegradation in water: screening test, other
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Testing was conducted between 1982-10-26 to 1983-06-16
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
Qualifier:
according to guideline
Guideline:
other: Degradation test of chemical substances by microorganisms specified in Test Methods for Novel Chemical Substances Kanpogyo: Bureau certified No 5, Yakuhatsu: Bureau certified No 615 and 49, Kikyoku: Bureau certified No 392
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
No data
Duration of test (contact time):
28 d
Initial conc.:
30 other: ppm
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Sampling for analysis

300 g of the test substance was dissolved in 90 mL of water and, after adjustment of pH 7.0 with hydrochloric acid, the 3,000 ppm solution was made up to 100 mL. A 3 mL aliquot of this solution was then added to 297 mL of culture liquid or water.
Reference substance:
aniline
Preliminary study:
No data
Parameter:
% degradation (O2 consumption)
Value:
22
Remarks on result:
other: See Figure 1 (attached)
Parameter:
% degradation (TOC removal)
Value:
6
Remarks on result:
other: See Table 1 and Figure 2 (attached)
Parameter:
other: Liquid Chromatography
Value:
21
Remarks on result:
other: See Table 2 and Figure 3 (attached)
Value:
other: No data
Results with reference substance:
The degradation level of aniline on the 7th day was 67 % (see Figure 1, attached)
Validity criteria fulfilled:
not specified
Conclusions:
Based on oxygen consumption, degradation of the test substance was 22 % after seven days.

Description of key information

GLP study demonstrating 49 % biodegradation after 28 days (OECD 301B)

Non-GLP study showing 22 % degradation after 7 days (OECD 302C)

Key value for chemical safety assessment

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

Additional information

The test substance is not readily biodegradable. A ready biodegradation study showed that 49% degradation was achieved after 28 days and, based on oxygen consumption, a supporting study reported degradation of the test substance as 22 % after seven days.