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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:
supporting study
Study period:
27 July 1992 to 11 December 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
Cited as 84/449/EEC, C.5 modified Sturm test
Deviations:
yes
Remarks:
see "Principles of method if other than guideline" for details
Principles of method if other than guideline:
Deviations: The volume of the test solution was reduced from 3.0 l to 1.5 l. The CO2 formed by biodegradation was absorbed with NaOH and determined on a carbon analyzer. Due to the poor solubility of the test substance in water, an emulsifier was used to acheive a better distribution in the medium. The test substance was added directly into the medium without preparing a stock solution.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Bacteria collected from activated sludge of the sewage treatment plant of CH-4153 Reinach on 07/08/92. The pH after collection was 7.6.
The preparation was carried out according to the method described in the guideline.
Duration of test (contact time):
28 d
Initial conc.:
11.7 mg/L
Based on:
test mat.
Initial conc.:
22.7 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
DESIGN AND PROCEDURE
Vessels: 2 Liter flasks (dark brown glass) equipped with gas inlet and magnetic stirrer.
Water: The test medium was prepared according to the method described in the guideline
Temperature: 22 ± 2°C
Duration: 28 days
Air: ~ 25 ml/min purified from carbon dioxide.

TEST CONCENTRATIONS
Test substance: 1200 ml of the mineral solution with the inoculum were aerated for 24 hours in the test vessel. In 300 ml mineral solution 15 ml Polyoxyethylen-Sorbitan-Monooleate (TWEEN 80) (FLUKA 93781) (solution of 300 mg in 1000 ml bidist. water) and 17.6 rsp. 34.1 mg of test substance were added and homogenized. This solution was given to the test vessel which was immediately connected to the CO2 traps.

Reference substance : 20 mg/l

Blank : Water with inoculum containing 15 ml of the Polyoxyethylen-Sorbitan-Monooleate (TWEEN 80) (FLUKA 93781) solution.

THEORETICAL CARBON DIOXIDE EVOLUTION
Test substance: 11.7 mg/l = 34.53 mg CO2/1.5 liter.
Test substance: 22.7 mg/l = 66.91 mg CO2 /1.5 liter.

This calculation is based on the chemical formula: C66H129S5Sn2O10

MEASUREMENTS
Determination of the initial CO2 of the 0.05 N sodium hydroxide and the CO2 , absorbed in the absorbers filled with 200 ml 0.05 N sodium hydroxide on the days 0, 3, 6, 9, 13, 16, 20, 23, 27 and 28.

CALCULATIONS/STATISTICAL ANALYSIS
The biodegradation was calculated on the basis of the theoretical carbon content of the test substance and the cumulative quantities of carbon dioxide determined on the days of measurements. For the calculation the formula given in the guideline was used.

CONTROL AND BLANK SYSTEM
- Inoculum blank: water with inoculum containing 15 ml of the Polyoxyethylen-Sorbitan-Monooleate (TWEEN 80) (FLUKA 93781) solution
Reference substance:
aniline
Parameter:
% degradation (CO2 evolution)
Value:
28 - 40
Sampling time:
28 d
Remarks on result:
other: Biodegradation calculated as percentage of measured amount of carbon dioxide over the theoretical organic carbon (ThOC)

Measured CO2 evolution values (cumulative, by day sampled), in mg:

[Data are reported as mean blank, mean reference (aniline), test substance 1 (11.7 mg/l), test substance 2 (22.7 mg/l)]

3 d 3.4 3.8 3.3 3.6
6 d 4.8 47.2 4.9 5.5
9 d 6.3 64.0 6.9 9.9
13 d 7.7 71.7 12.0 14.1
16 d 8.1 75.8 4.9 17.5
20 d 9.3 78.9 18.7 22.0
23 d 10.0 80.0 20.6 25.6
27 d 21.4 93.2 35.3 42.5
28 d 11.5 86.6 25.5 30.1

Measured values from 27th data are not reliable due tp instrumental defect and not take for calculations and the biodegradation curve

Biodegradation in relation to ThCO2 evolution (cumulative, by day sampled), in %:

[Data are reported as mean reference (aniline), test substance 1 (11.7 mg/l), test substance 2 (22.7 mg/l)]

3 d 0.0 0.0 0.0
6 d 50.0 0.0 1.0
9 d 68.0 2.0 5.0
13 d 75.0 12.0 10.0
16 d 80.0 19.0 14.0
20 d 82.0 27.0 19.0
23 d 82.0 31.0 23.0
27 d 84.0 40.0 32.0
28 d 88.0 40.0 28.0

Measured values from the 27th day are not reliable due to instrumental defect and not taken for calculations and the biodegradation curve

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
MOTE is not readily biodegradable according to this test.
Executive summary:

Study: determination of the biodegradability by measurement of the carbon dioxide formation in per cent of THCO2 (Theoretical Carbon Dioxide) from the ThOC (Theoretical organic carbon) or TOC (Total organic carbon)

Test system: bacteria collected from a sewage treatment plant

DurationL 28 days

Guideline: 84/449/EEC C.5 Biotic degradationn: modified Sturm test

Deviations: The volume of the test solution was reduced from 3.0 l to 1.5 l. The CO2 formed by biodegradation was absorbed with NaOH and determined on a carbon analyzer. Due to the poor solubility of the test substance in water, an emulisifer was used to achieve a better distribution in the medium. The test substance was added directly into the medium without preparing a stock solution.

Results: The biodegradation calculated as percentage of measured amount of carbon dioxide over the theory was:

11.7 mg test substance/1 = 40% in 28 days

22.7 mg test substance/1 = 28% in 28 days

Conclusion: MOTE is not readily biodegradable in this test.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 November 1999 to 20 December 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Qualifier:
according to guideline
Guideline:
ISO DIS 9408 (Ultimate Aerobic Biodegradability - Method by Determining the Oxygen Demand in a Closed Respirometer)
Principles of method if other than guideline:
The Manometric Respirometry Test is a static method for testing the ultimate aerobic biodegradability of a test substance in water. Mixtures of the test substance, a defined inorganic medium and an inoculum, which is not pre-adapted (e.g. activated sludge or effluent of a municipal or laboratory waste water treatment plant), are incubated and aerated in a respirometer (Sapromat) at room temperature up to 28 days regularly. The test period may be expanded if biodegradation processes are still ongoing after 28 days. The oxygen used for the biodegradation of the test substance (biochemical oxygen demand, BOD) is continuously produced and measured by the test apparatus.

In addition to the assays with test substance (in triplicate) the following investigations are required: blank values (BC), biodegradation of a reference substance (RS), inhibition of the inoculum (IH) and abiotic elimination (PC).
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Municipal activated sludge from the waste water treatment plant of Mannheim, stored for 12 days in laboratory waste water treatment plant (WWTP) No. 1 which was fed with municipal waste water. 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 to obtain a sludge concentration of 30 mg/l dry solids.
Duration of test (contact time):
39 d
Initial conc.:
50 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Evaluation of Test Results

The biochemical oxygen demand gives unequivocal evidence for biodegradation. The measured BOD is compared with the calculated theoretical oxygen demand (ThOD) or the measured chemical oxygen demand (COD) of the test substance and indicated as biodegradation degree in per cent. Since the micro-organisms oxidize only part of the test substance and incorporate the rest as biomass, substances with a degree of >60 % biodegradation are considered to be sufficiently biodegradable. The relation BOD to ThOD or COD is considered by the author as follows:
>60 % in 10 days - "readily biodegradable (OECD criteria)"
>60% - "biodegradable"
20-60% - "in this test primarily biodegradable"
<20% - "in this test poorly biodegradable"

One of the OECD criteria for ready biodegradable substances is the threshold value of 60% biodegradation within a period of 10 days during a total test period of 28 days. If a test substance is biodegradable in this test, it can be assumed that it will be biodegraded in the environment, especially in wastewater treatment plants and surface waters.
Reference substance:
aniline
Test performance:
No unusual observations
Parameter:
% degradation (TOC removal)
Value:
>= 50 - <= 60
Sampling time:
39 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: -40, 20, -60, -20
4d: 580, 60, 20, 79
7d: 1620, 79, 119, 139
10d: 1880, 179, 238, 258
13d: 2140, 278, 317, 317
16d: 2600, 397, 437, 397
19d: 2820, 516, 595, 516
22d: 2900, 556, 734, 595
25d: 2940, 635, 853, 655
28d: 3020, 694, 972, 774
*39d: 3120, 1052, 1409, 1151
* - incubation time was increased to 39 days as biodegradation of the test substance was shown to be a comparatively slow process

BOD of blank (after 39 days) = 16 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: -2, 1, -3, -1
4d: 24, 3, 1, 4
7d: 68, 4, 5, 6
10d: 79, 8, 11, 12
13d: 89, 13, 14, 14
16d: 109, 18, 20, 18
19d: 118, 23, 27, 23
22d: 121, 25, 33, 27
25d: 123, 29, 39, 30
28d: 126, 32, 44, 35
*39d: 130, 48, 64, 52
* - incubation time was increased to 39 days as biodegradation of the test substance was shown to be a comparatively slow process

The reference substance (aniline) was degraded more than 60% in 10 days and the BOD of the blank after 39 days was 16 mg/L (within the test method criteria of 60 mg/L).

Octyltin tris(EHMA) was degraded 32-44% after 28 days and was determined to be not readily biodegradable.

Kinetic of test substance (in %):
= 8 ... 12 after 10 day(s)
= 32 ... 44 after 28 day(s)
= 48 ... 64 after 39 day(s)
Kinetic of control substance (in %):
= ... 79 after 10 day(s)
> ... 100 after 39 day(s)
Degradation products: not measured
Value:
> 30 - < 40 other: % biodegradation at 28 days, % BOD/ThOD
Value:
> 50 - < 60 other: % biodegradation at 39 days, % BOD/ThOD
Results with reference substance:
Degradation degree of the reference substance was >60% after 14 days and therefore met the validity criteria. Degradation degree of the inoculum control was also <60% after 14 days.

No pre-adapted inoculum used

Test duration (days): 39

Duration of adaptation phase (days): 10

Duration of degradation phase (days): >29

Degradation of the test substance at the end of the ten-day window (% BOD/ThOD): --

Degradation degree of the test substance after 28 days (% BOD/ThOD): 30 -40

Degradation degree of the test substance at the end of the test (% BOD/ThOD): 50 -60

Degree of degradation of the test substance in the abiotic control after 20 days of the test (main study) (% BOD/ThOD): 3*

Reference substance: aniline

Degradation degree of the reference substance after 14 days (% BOD/ThOD): 90 -100

Degradation degree in the inhibition control after 14 days (% BOD/ThOD): 40-50

Remarks: *Increasing BOD values of the abiotic test assay (PC) after 20 days might be due to microbial biodegradative activities. Therefore this assay was validated for a test period of 20 days only. Further abiotic test assays did not show any significant degradation during the usual test period of 28 days.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Test substance was not biodegradable (e.g. BOD >60% of ThOD) within a 10 day window within the 28 day period, as required by OECD 301 to be considered "readily biodegradable." However, biodegradation continued after 28 day and thus measurements continued to 39 day. At 39 day, biodegradation ranged 50-60%. Thus the compound may be considered somewhat biodegrable, but not "ready biodegradable".
Executive summary:

Determination of Biodegradability of MOTE in the Manometric Respirometry Test according to Annex of EEC-Directive 92/69; corresponds to OCED Guideline 301 F and ISO Standard 948.

Test result

Biodegradation degree (BOD of ThOD) after 28 days: 30 -40%

Biodegradation degree (BOD of ThOD) after 39 days: 50 -60%

Evaluation of the test result

The test substance is not primarily biodegradable (according to OCED criteria).

Concentration of the test substance: 50 mg/l

Test temperature: 22±1ºC

Inoculum: municipal activated sludge from the waste water treatment plant of Mannheim, stored for 12 days in laboratory waste water treatment plant (WWTP) No. 1 which was fed with municipal waste water. One day before the start of the test sludge from laboratory WWTP No. 1 was washed and aerated for 24 hours and then added to the test vessels to obtain a sludge concentration of 30 mg/l dry solids.

Description of key information

The substance was not found to be readily biodegradable in the two available guideline studies.

Key value for chemical safety assessment

Additional information

The Beimborn, 2000, study is a Manometric Respirometry Test performed according to Annex of EEC-Directive 92/69 and corresponds to OCED Guideline 301 F and ISO Standard 948. The test was performed at 22 ± 1ºC with a concentration of 50 mg/l. The inoculum was collected 12 days before the study initiation from the municipal waste water treatment plant of Mannheim, 1 day before the study initiation the inoculum was supplimented with sludge from the waste water treatment plant of the testing laboratory in order to obtain a concentration of 30 mg/l dry solids.

The test produced the following results:

Biodegradation degree (BOD of ThOD) after 28 days: 30 -40%

Biodegradation degree (BOD of ThOD) after 39 days: 50 -60%

Based upon these results the registered substance is considered to be not readily biodegradable (according to OCED criteria) .

The Baumann, 1992, study is presented as supporting information only. The percentage biodegradation results of the study were as follows: 11.7 mg test substance/l = 40% in 28 days and 22.7 mg test substance/1 = 28% in 28 days. Based upon these results it is considered that the substance does not meet the EU criteria for classification as a readily biodegradable substance.