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Environmental fate & pathways

Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
Cited as Directive 92/69/EEC, C.4-D
according to guideline
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
according to guideline
ISO DIS 9408 (Ultimate Aerobic Biodegradability - Method by Determining the Oxygen Demand in a Closed Respirometer)
GLP compliance:
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Municipal activated sludge from the waste water treatment plant of Mannheim
- Storage conditions: stored in laboratory waste water treatment plant (WWTP) No. 1 which was fed with municipal waste water.
- Storage length: stored for 5 days
- Preparation of inoculum for exposure: One day before the Start of the test the sludge from laboratory WWTP No. 1 was washed and aerated for
24 hours and then added to the test vessels .
- Concentration of sludge: a sludge concentration of 30 mg/l dry solids
Duration of test (contact time):
28 d
Initial conc.:
50 mg/L
Based on:
test mat.
Initial conc.:
103 mg/L
Based on:
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Composition of medium:
- Test temperature: 22 ± 1 °C
- pH: 6 to 8.5
- pH adjusted: yes/no no
- Aeration of dilution water: aerated in a respirometer

Reference substance:
Preliminary study:
Summary Parameters TOC DOC C* H* O* S* ThOC* ThOD*
[mg/g] __ __ 507 81 131 128 507 2061
(*) calculated by: 0 summary formula at -% purity
Theoretical oxygen demand (ThOD) was calculated assuming no Change in the Oxidation state of Sn.
Test performance:
Methyltin tris(2 -EHMA) was prepared in triplicate (@ 50 mg/L) in aqueous solutions consisting of a defined inorganic medium and a non-adapted inoculum. A blank assay, reference substance assay, inoculum inhibition assay, and an abiotic elimination assay were prepared and tested concurrently. Activated sewage sludge was collected the week prior to the test from a municipal wastewater treatment plant (Mannheim). The sludge was washed and aerated for 24 hours prior to the test and was added to all test vessels in appropriate amounts to obtain a final sludge concentration of 30 mg/L (dry solids). Biochemical oxygen demand (BOD) was measured daily and was compared to the calculated theoretical oxygen demand (ThOD) in order to determine the degree of biodegradation (in percent).
% degradation (O2 consumption)
4 - 5
Sampling time:
3 d
% degradation (O2 consumption)
37 - 42
Sampling time:
4 d
Remarks on result:
other: onset of biodegradation
% degradation (O2 consumption)
79 - 90
Sampling time:
14 d
Remarks on result:
other: 10 days after the onset of biodegradation
Key result
% degradation (O2 consumption)
90 - 100
Sampling time:
28 d
Details on results:
Calculated specific BOD values (cumulative, by day sampled), in mg/g:
[data are reported as reference (aniline), followed by each test substance replicate]
1d: -20, 20, 20, 20
4d: 880, 758, 874, 780
7d: 1980, 1270, 1280, 1360
10d: 2100, 1516, 1524, 1640
13d: 2140, 1598, 1646, 1800
16d: 2160, 1639, 1728, 1940
19d: 2200, 1680, 1789, 2020
22d: 2220, 1721, 1850, 2100
25d: 2240, 1762, 1890, 2140
28d: 2220, 1783, 1911, 2160

BOD of blank (after 28 days) = 17 mg/L

Biodegradation in relation to ThOD (cumulative, by day sampled), in %:
[data are reported as reference (aniline), followed by each test substance replicate]
1d: -1, 1, 1, 1
4d: 37, 37, 42, 38
7d: 83, 62, 62, 66
10d: 88, 74, 74, 80
13d: 89, 78, 80, 87
16d: 90, 80, 84, 94
19d: 92, 82, 87, 98
22d: 93, 84, 90, 102
25d: 94, 85, 92, 104
28d: 93, 87, 93, 105

The reference substance (aniline) was degraded more than 60% in 10 days and the BOD of the blank after 28 days was 17 mg/L (within the test method criteria of 60 mg/L).

Methyltin tris(2-EHMA) was degraded 87-105% (mean = 95%) in 28 days and was determined to be readily biodegradable in accordance with OECD criteria (i.e., >60% degradation in 10 days).

Kinetic of test substance (in %):
= 74 ... 80 after 10 day(s)
>= 87 ... 100 after 28 day(s)
Kinetic of control substance (in %):
= ... 88 after 10 day(s)
= ... 93 after 28 day(s)
Degradation products: not measured
Results with reference substance:
The reference substance (aniline) was degraded more than 60 % in 10 days
Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
Methyltin tris(2-EHMA) is considered "readily biodegradable" in accordance with OECD criteria. The test substance exhibited 87-105 % (mean = 95 %, standard deviation = 9.2 %) degradation in 28 days and greater than 60 % degradation within 10 days after the onset of the biodegradation.
Executive summary:

The ready biodegradability of the test substance was determined using the Manometric Respirometry Test according to Annex of EEC-Directive 92/69; corresponds to OECD Guideline 301 F and ISO Standard 9408. This test employed biodegradation reaction mixtures containing a non-adapted activated sludge inoculum, which was suspended in a defined mineral medium, at a concentration of 30 mg/L (dry solids). The test substance was added into each reaction mixture at a concentration of 50 mg/L (103 mg/L theoretical oxygen demand).

Biodegradation of the substance began at Day 4, and reached 95 ± 9.2 % (mean ± 1 standard deviation) based on biological oxygen demand (BOD), at the end of the 28-day test. The test substance met the 10-day window criteria as the biodegradation reached 83.3 ± 5.9 % (mean ± 1 standard deviation) based on BOD at Day 14 since the onset of the biodegradion at Day 4. Since the pass level of 60 % biodegradation was achieved within the 28-day test period and 10-day window criteria was satisfied, the substance meets OECD criteria for “ready biodegradability” in the Manometric Respirometry test. The results of this test met or exceeded each of the OECD-specified criteria for validation of its ready biodegradability tests. These include parameters such as viability of the inoculum, control of pH and temperature, and precision in percentage biodegradation recorded among replicate test mixtures.

Biodegradation of a reference substance (aniline) reached 90 - 100 % in 14 days, and thus verified the viability of the inoculum employed in this test. An Inhibition (Toxicity) Control mixture, containing both aniline (50 mg/L) and the test substance, showed no evidence for inhibition of the microbial inoculum. An Abiotic Control mixture, containing the test substance and a chemical sterilant (HgCl2), verified that the observed test substance degradation was due solely to biological activity. Thus, the test substance is considered “readily biodegradable” according to OECD criteria. Therefore, the substance is expected to rapidly biodegrade in a variety of environments.

Description of key information

Readily biodegradable in OECD 301F- Manometric respirometry test. Biodegradation equivalent to 95 ± 9.2 % (mean ± 1 Std. Dev.) consumption of theoretical oxygen demand (ThOD) was observed after 28 days, and 83.3 ± 5.9 % (mean ± 1 Std. Dev.) biodegradation was observed within the 10-day window.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The test substance is considered readily biodegradable according to OECD definition; therefore, the test substance is expected to be rapidly and ultimately biodegradable in various environmental media.