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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Sept 2009 to 29 Oct2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was conducted according to accepted international guidelines for ready biodegradability screening tests and for Good Laboratory Practice, was well documented and properly controlled

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test) AND also :EU Commission Directive 92/69 EEC, C.4-D, Manometric Respirometry Test, 1992. EC No 440/2008 C.4-D, Manometric Respirometry Test 30 May 2008
Deviations:
no
Principles of method if other than guideline:
This study has been performed in compliance with the Swiss Ordinance relating to Good
Laboratory Practice, adopted May 18th , 2005 [SR 813.112.1]. This Ordinance is based on the
OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26th, 1997 by decision of the OECD Council [C(97) 186/Final]. The testing laboratory was inspected with respect to compliance with the Swiss Ordinance on Good Laboratory Practice 05th to 09th and 26th to 30th November 2007, with an acceptible result
GLP compliance:
yes (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MTDID 8191
- Substance type: UVCB material
- Physical state: Yellowish viscous liquid
- Concentration of oligomers: ≥99.0%
- Purity test date: 21 January 2010
- Lot/batch no.: MK 12/048
- Expiration date of the lot/batch: 27-Jan-2010
- Stability under test conditions: Not reported
- Storage condition of test material: In the refrigerator at about 4 °C

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): ARA Ergolz II, Füllinsdorf, Switzerland
- Preparation of inoculum for exposure: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter. During the holding period of three days prior to use, the sludge was aerated at room temperature. Prior to use, the sludge was first thoroughly mixed and then diluted with test water to a concentration of 1 g per liter (dry weight basis). Based on the determined dry weight of this diluted activated sludge, defined amounts were added to test water to obtain a final concentration
of 30 mg dry material per liter.
- Pretreatment: None
- Concentration of sludge: 30 mg/L dry weight basis in reactor
- Initial cell/biomass concentration: Not reported
- Water filtered: No
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
Stock solution 1
KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4 ∙ 2H2O 33.40 g/L
NH4Cl 0.50 g/L
Stock solution 2
MgSO4 ∙ 7H2O 22.50 g/L
Stock solution 3
CaCl2 ∙ 2H2O 36.40 g/L
Stock solution 4
FeCl3 ∙ 6H2O 0.25 g/L, stabilized with one drop of concentrated HCl per liter
10 mL of stock solution No. 1 and 1 mL each of stock solution Nos. 2, 3 and 4 were combined and made up to 1000 mL with purified water. The pH was adjusted from 7.7 to 7.4 with a diluted hydrochloric acid solution. This mixture was used for all dilutions and preparation of activated sludge.

- Additional substrate: None
- Solubilising agent (type and concentration if used): None
- Test temperature: 22 °C
- pH: 7.4 - 7.8
- pH adjusted: No
- CEC (meq/100 g): Not reported
- Aeration of dilution water: Sealed bottles stirred in a SAPROMAT D12 (Voith GmbH, Heidenheim,
Germany)
- Suspended solids concentration: 30 mg/L sludge solids dry weight
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: SAPROMAT D12 (Voith GmbH, Heidenheim, Germany), 500 mL reactor volume, 250 mL solution per reactor.
- Number of culture flasks/concentration: Two
- Method used to create aerobic conditions: Continuous stirring. Oxygen regenerated electrolytically from a copper sulfate solution.
- Measuring equipment: SAPROMAT D12 (Voith GmbH, Heidenheim, Germany)
- Details of trap for CO2 and volatile organics if used: Soda lime, used only to reduce pressure due to CO2

SAMPLING
- Sampling frequency: Once daily on working days, non-destructive testing
- Sampling method: Non-destructive, electrode-type manometer.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: No
- Toxicity control: Yes

STATISTICAL METHODS:

BOD = (mg O2 uptake/L by test and/or reference item) - (mg O2 uptake/L by inoculum control)
____________________________________________________________________
mg test and/or reference item per liter

% biodegradation = BOD (mg O2 /mg chemical)
________________________________
COD and/or ThOD (mg O /mg chemical)
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
None
% Degradation
Parameter:
% degradation (O2 consumption)
Value:
5 - 8
Sampling time:
28 d
Remarks on result:
other: SEE section "Details on Results"
Details on results:
REMARKS from Section "% Degradation of test substance" :
Measurements of oxygen consumption were taken once daily on working days (Table 1). The biochemical oxygen demand (BOD) of the test item MTDID 8191 in the test media was in the normal range found for the inoculum controls throughout the study period of 28 days.

DETAILS ON RESULTS:
The measured COD of the test substance was 1.79 mg O2/mg.

The biochemical oxygen demand (BOD) of the test item MTDID 8191 in the test media was in the normal range found for the inoculum controls throughout the study period of 28 days. Consequently, MTDID 8191 was not biodegradable under the test conditions within 28 days. Percent biodegradation calculated according to OECD TG301F are given in Table 2. pH values at beginning and end of test are given in Table 3.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Test substance is insoluble, and the test solution was made as homogeneous as possible by sonication.

BOD5 / COD results

Results with reference substance:
The percent biodegradation of the reference item sodium benzoate was calculated based on the theoretical oxygen demand of 1.67 mg O2/mg. In the procedure controls, the reference item was degraded by an average of 90% by Exposure Day 14, thus confirming suitability of the activated sludge.

In the toxicity control, the course of biodegradation over the 28-day exposure period was similar to the two procedure controls, containing only the reference item. Within 14 days of exposure, biodegradation amounted to 72%. Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 30 mg/L because biodegradation in the toxicity control was >25% within 14 days.

Any other information on results incl. tables

Table 1, Oxygen consumption in test flasks
Time
(days)		 Cumulative oxygen consumption (mg/L)
Test
substance		 Inoculum
control		 Procedure
control		 Toxicity
control
Replicate Replicate Replicate Replicate
1 2 1 2 1 2 1
0 0 0 0 0 0 0 0
1 0 0 0 0 3 2 6
2 0 2 0 0 95 95 94
3 1 3 0 1 120 121 126
4 3 4 6 2 128 129 134
5 4 5 6 2 135 136 141
6 4 5 6 3 138 140 145
7 5 6 6 3 141 143 149
8 6 6 6 4 144 145 152
9 ND1 ND ND ND ND ND ND
10 ND ND ND ND ND ND ND
11 7 9 6 4 150 151 160
12 8 9 6 5 152 153 161
13 8 10 6 5 153 155 163
14 9 10 6 5 154 156 164
15 10 11 6 6 155 157 165
16 ND ND ND ND ND ND ND
17 ND ND ND ND ND ND ND
18 10 12 7 6 158 159 168
19 10 12 7 6 158 159 168
20 10 12 7 7 159 160 168
21 10 12 7 7 159 160 169
22 11 13 9 8 160 162 170
23 ND ND ND ND ND ND ND
24 ND ND ND ND ND ND ND
25 11 13 9 8 160 162 170
26 11 13 9 8 160 162 171
27 11 13 9 8 160 162 171
28 11 13 9 8 160 162 171
1. ND, not determined

Table 2, Biodegradation in test flasks
Time
(days)		 Percent biodegradation
Test
substance		 Procedure
control		 Toxicity
control
Replicate Replicate Replicate
1 2 1 2 1
0 0 0 0 0 0
1 0 0 2 1 3
2 0 4 57 57 43
3 1 5 72 72 57
4 -2 0 74 75 59
5 0 2 78 79 62
6 -1 1 80 81 64
7 1 3 82 83 65
8 2 2 83 84 66
9 ND1 ND ND ND ND
10 ND ND ND ND ND
11 4 7 87 87 70
12 5 6 88 88 70
13 5 8 88 90 71
14 6 8 89 90 72
15 7 9 89 90 72
16 ND ND ND ND ND
17 ND ND ND ND ND
18 6 10 91 91 73
19 6 10 91 91 73
20 6 9 91 92 73
21 6 9 91 92 73
22 5 8 91 92 73
23 ND ND ND ND ND
24 ND ND ND ND ND
25 5 8 91 92 73
26 5 8 91 92 73
27 5 8 91 92 73
28 5 8 91 92 73
Mean
(Day 28)		 6 91 N/A
1. ND, not determined

Table 3, pH Values at the Start and at the End of the Test
Replicate No. Identification pH
    Start End
1 Test item 7.5 7.5
2 Test item 7.5 7.4
1 Inoculum control 7.5 7.4
2 Inoculum control 7.5 7.4
1 Procedure control 7.5 7.8
2 Procedure control 7.5 7.8
1 Toxicity control 7.5 7.8

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test substance is not readily biodegradable. The test substance is not inhibitory to biodegradation.
Executive summary:

Ready biodegradability of the test substance (CAS# 1101874-33-2) was assessed according to OECD TG301F. The study also was based on regulations 92/69 EEC, C.4-D, Manometric Respirometry Test (1992) and EC No 440/2008 C.4-D, Manometric Respirometry Test (30 May 2008). All dilutions were made in standard minimal salts medium (MSM). Activated sludge of predominantly domestic sewage was washed, diluted to 4 g/L dry weight and aerated 3 days prior to test start. Sludge was diluted to 30 mg/L dry weight in for the test. Test substance (30 mg/L final) was sonicated in MSM to obtain the most homogeneous suspension possible. Sodium benzoate (100 mg/L final) was used as a reference substance and in a toxicity control. Test substance, inoculum control (no chemical) and the reference substance were tested in duplicate, while only one toxicant control flask was tested. Evolved CO2 was trapped in soda lime. Oxygen consumption was tested by manometric respirometry. Percent biodegradation for the reference substance was 90% within 14 days. Percent biodegradation in the toxicity control was 73% within 28 days. Percent biodegradation for the test substance was ca. 6 %, but oxygen consumption for the test substance was within the range as the inoculum controls. Therefore, the substance was not biodegraded during the study, and is not readily biodegradable. However, it is not inhibitory to biodegradation at the concentration tested.

This study is a direct assessment of ready biodegradability. It was conducted according to accepted international guidelines for ready biodegradability screening tests and for Good Laboratory Practice, was well documented and properly controlled. Therefore, this study is relevant to this endpoint and is classified as acceptable.