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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:
11 February to 28 March 2003
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)
Deviations:
yes
Remarks:
See 'principles of method if other than guideline'
Principles of method if other than guideline:
Deviations from the protocol:
• From 1 November 2002, the Biodegradation and Microbiology activities of the Department of Environmental Toxicology have been transferred to the Department of Biomolecular Sciences under management of Dr.ir. J.P. Groten.
• On request of the Sponsor the title was changed into:
'Tributylchlorostannane (CAS # 1461-22-9): Determination of the ready biodegradability in a Manometric Respiration Test'.
(OECD Guideline No. 301 F, EU C.4-D).
• The test was carried out in the laboratories of the Department of Biomolecular Sciences located in Zeist, the Netherlands.
• Three blanks with filter were tested instead of two blanks without filter and two blanks with filter.
• NaNO3 was added separately to the mineral medium instead of via nutrient stock solution a.
• The temperature was in the range 13.3 to 18.0 °C between 311 and 539 hours of incubation (13 to 23 days). This might have lead to a slightly lower biodegradation rate. However, inspection of the rates in that time period did not indicate that this was the case. At other time-points the temperature was within the limits given by the guideline.
• The measurements were temporarily (approximately 22 hours) stopped on the 22nd day of the incubation, because of measurements for other studies.
• The experiment was extended 11 days to further allow the oxygen consumption curve to reach the plateau phase.
• The oxygen measurements were every 5 hours instead of every four.
• The TNO test substance number was 02010E.
• The test substance number of the reference substance was CBS 33145.

These deviations did not affect the results of this study.
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
A sample of activated sludge was taken from an oxidation ditch situated in the municipality of Hazerswoude, the Netherlands, on February 11, 2003. The oxidation ditch is used to treat domestic waste water. The activated sludge was transported in a plastic flask and aerated until use. Before the start of the test, the dry weight of the sludge was determined to be 3.6 g.L^-1. In order to yield a concentration of solids of approximately 30 mg.L^-1, 2.5 mL sludge was added to 300 mL of mineral medium.

Duration of test (contact time):
39 d
Initial conc.:
27.3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:

Nutrient stock solutions:

a) KH2PO4 (potassium dihydrogen phosphate): 8.5 g
K2HPO4 (dipotassium hydrogen phosphate): 21.8 g
Na2HPO4.7H20 (disodium monohydrogen phosphate heptahydrate): 33.4 g
NH4C1(ammonium chloride): 0.5 g
Dissolved in and made up to 1000 mL with ultrapure water. The pH of this solution was set to 7.4 ± 0.1.

NaNO3 (sodium nitrate): 5.0 g
Dissolved in and made up to 500 mL with ultrapure water.

b) Nutrient stock solution b per 1000 mL ultrapure water: MgSO4.7H20 (magnesium sulphate heptahydrate): 22.5 g
Dissolved in and made up to 1000 mL with ultrapure water.

c) Nutrient stock solution c per 1000 mL ultrapure water: CaCl2.2H20 (calcium chloridedihydrate): 36.4 g
Dissolved in and made up to 1000 mL with ultrapure water.

d) Nutrient stock solution d per 1000 mL ultrapure water: FeCl3.6H20 (iron(III) chloridehexahydrate): 0.25g
Dissolved in and made up to 1000 mL with ultrapure water. This solution was prepared immediately before use.

One mL of each of the nutrient stock solutions was combined with ultrapure water to a final volume of one litre.

The mineral medium was prepared from concentrated stock solutions in ultrapure water, and was aerated vigorously before use. To prevent nitrogen limitation, additional NaNO3 was added to the mineral medium as specified in the Guideline. The nitrate and nitrite contents in the mineral medium were not confirmed by analysis.

- Test temperature: 20 ± 2 °C
- pH: 7.4 ± 0.2
- pH adjusted: Yes, if necessary, the pH was adjusted to 7.4 ± 0.2 with 0.1 M HCl or 0.4 M NaOH solution.
- Aeration of dilution water: Yes
- Suspended solids concentration: Approximately 30 mg.L^-1
- Continuous darkness: Yes


TEST SYSTEM
- Measuring equipment: The flasks were closed and placed in the incubator of the Manometric Respirator (Micro-Oxymax). The Micro-Oxymax measures the percentage oxygen in the air of the respective flasks and calculates, based on the earlier measurement, the resulting oxygen consumption in a certain time period. Based on these values the oxygen consumption per flask was derived.
- Number of culture flasks/concentration: 3
- Test performed in closed vessels due to significant volatility of test substance: Yes


SAMPLING
- Sampling frequency: Every 5 hours


CONTROL AND BLANK SYSTEM
- Inoculum blank: This series was completed with an inoculum blank, containing mineral medium only and a ethanol treated glass fibre filter.
- Toxicity control: Sodium acetate + test material

STATISTICAL METHODS:
Based on the empirical formula, the Theoretical Oxygen Demand without nitrification (ThODNH3) value of the test material was calculated to be 1.84 mg O2.mg-1. The degree of biodegradation was calculated with this ThODNH3 value, assuming that the purity of the test material was 100 %.

Calculations were performed as given in the test Guidelines. The oxygen consumption (in mg O2.L^-1) in each test flask was calculated based on the respiration rate (mg O2.flask^-1.hour^-1). The oxygen consumption due to the test or control substance at each time was calculated by subtracting the mean cumulative oxygen consumption in the blanks from that in the flask under consideration. These crude values were then converted to values per mg substance (BOD). The percentage biodegradation of the test substance was calculated as BOD/ThOD x 100.
Reference substance:
acetic acid, sodium salt
Preliminary study:
No data
Test performance:
These conditions of validity have been met as follows:
• The amount of oxygen consumed in the biodegradation test (test medium with test material) is so low, that this criteria has little relevance. However, the extreme replicate cumulative oxygen consumption values for the inoculum toxicity and activity control vary less than 20 % at the end of the test.
• The reference substance was degraded 98 % (ThOD) within 14 days.
• The degradation in the toxicity control was above 25 % during the test.
• The oxygen consumption in the blanks (excluding blank 1) was 5.67 to 6.91 mg per flask after 28 days of incubation, which is equal to 18.9-23.0 mg.L^-1, which is within the limits of validity.
• The pH remained within the valid range during the test in all test flasks.

Thus, the conditions for test validity were met.
Key result
Parameter:
% degradation (O2 consumption)
Value:
-17
Sampling time:
28 h
Key result
Parameter:
% degradation (O2 consumption)
Value:
-23
Sampling time:
39 d
Details on results:
Table 2 shows the relevant mean Biochemical Oxygen Demand (BOD) values and the percentage biodegradation of the test material at a concentration of 27.3 mg.L^-1, calculated from the ThODNH3 (1.84 mg O2.mg^-1) of the test material. The negative BOD is an indication that the test material caused an inhibition of the oxygen consumption compared with the control.
Tributylchlorostannane was not biodegraded after 28 or 39 days of incubation.
Key result
Parameter:
BOD5
Value:
-0.31 mg O2/g test mat.
Results with reference substance:
The inoculum activity was sufficient; the reference substance sodium acetate reached the 60 % pass level of degradation within 14 days.

Test conditions:

The pH of the medium in the test flasks decreased to 6.55-6.69 after 39 days of incubation. In the activity and the toxicity controls the pH at the end of the test was 7.51-7.59. The higher pH in these flasks is due to the higher CO2 production. The pH value was within the limits of the validity criteria. The average temperature in the flasks was 21.2 ± 0.7 °C during most of the incubation time (range 18.7-22.9 °C), with a negligible amount of measurements above 22 °C. However, between 13 and 23 days of incubation the temperature is in the range between 13.1 and 18 °C, due to technical malfunction of the incubator. In general a lower temperature will lead to lower degradation rates. Inspection of the oxygen consumption rate in that time period did not show significant changes. After 13 days of incubation the reference test, as well as the toxicity control are already in the plateau phase, so not much influence is expected there. The cumulative consumption rate in the test material flask was never above the blank value, indicating that even at higher temperature, no biodegradation will have occurred due to inhibiting effects of the test material. It was therefore decided that although the temperature was temporarily too low, it did not influence the results of the study.

One of the blanks (Blank/1) was overall considered outlier and not taken into account in calculations of mean values. The oxygen consumption in this blank was much higher than the other two blanks of this study and higher than the values for blanks in other recent studies carried out by TNO.

The percentage degradation of the test material, relative to the ThODNH3 of 1.84 mg O2.mg-1 after 28 days of incubation did not exceed 0 % in a manometric respiration test at a test material concentration of 27.3 mg.1-1. The biodegradation of the test material did not exceed the pass level of 60 % ThODNH3 within 28 days and, therefore, is classified as not readily biodegradable.

The test material inhibited the degradation of the reference substance and also inhibited the oxygen consumption in the biodegradability test. Therefore any biodegradability of the test material could not be demonstrated in this test due to its inhibiting effect to the inoculum, although the test material is not toxic according to the definitions given by the guidelines.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The percentage degradation of the test material, relative to the ThODNH3 of 1.84 mg O2.mg^-1 after 28 days of incubation did not exceed 0 % in a manometric respiration test at a test material concentration of 27.3 mg.L^-1. The biodegradation of the test material did not exceed the pass level of 60 % ThODNH3 within 28 days and, therefore, is classified as not readily biodegradable.
The test material inhibited the degradation of the reference substance and also inhibited the oxygen consumption in the biodegradability test. Therefore any biodegradability of the test material could not be demonstrated in this test due to its inhibiting effect to the inoculum, although the test material is not toxic according to the definitions given by the guidelines.
Executive summary:

The biodegradability of the test material (TNO study number: V4100/01) was determined as described under 'Manometric Respirometry Test' in the OECD Guideline 301F, for testing of chemicals: 'Ready Biodegradability', using oxygen consumption as test criterion in a 39 day test. This method is in agreement with the EU Test Guideline C.4-D. The only deviation of the guideline is that additional NaNO3 was added to the mineral medium to prevent nitrogen limitation to occur. The study was carried out in accordance with the OECD Principles of Good Laboratory Practice.

The test material (a clear liquid) was only slightly soluble in water. Therefore, glass fibre filter carriers were used to introduce the test material into the inoculated medium according to the International Standard ISO 10634. One concentration of 27.3 mg.L-1 (based on the test material as received) was tested, corresponding to a ThODNH3 of 50 mg O2.L-1. An inoculum was prepared from activated sludge taken from an oxidation ditch used to treat domestic sewage (30 mg (d.w.).L-1).

The test was incubated at the target temperature of 20 ± 2 °C and the test duration was prolonged to 39 days. The test met the conditions of validity given by the guidelines. The inoculum activity was sufficient; the reference substance sodium acetate reached the 60 % pass level of degradation within 14 days. In a toxicity control test with 27.3 mg.L-1 of test material and 100 mg.L-1 sodium acetate, a slight inhibition of the degradation of sodium acetate was found. However, the test material is not toxic to the inoculum as defined by the guidelines.

The test was prolonged to 39 days. The test material was not degraded after 28 or 39 days. In this study, the 60 % degradation criterion after 28 days was not met and therefore the test substance was considered not readily biodegradable. However, the results of the biodegradability test indicated that any biodegradability of the test material could not be demonstrated in this test due to its inhibiting effect to the inoculum.

Description of key information

The biodegradability of the test material (TNO study number: V4100/01) was determined as described under 'Manometric Respirometry Test' in the OECD Guideline 301F, for testing of chemicals: 'Ready Biodegradability', using oxygen consumption as test criterion in a 39 day test.

The percentage degradation of the test material, relative to the ThODNH3 of 1.84 mg O2.mg-1 after 28 days of incubation did not exceed 0 % in a manometric respiration test at a test material concentration of 27.3 mg.1-1. The biodegradation of the test material did not exceed the pass level of 60 % ThODNH3 within 28 days and, therefore, is classified as not readily biodegradable.

The test material inhibited the degradation of the reference substance and also inhibited the oxygen consumption in the biodegradability test. Therefore any biodegradability of the test material could not be demonstrated in this test due to its inhibiting effect to the inoculum, although the test material is not toxic according to the definitions given by the guidelines.

Key value for chemical safety assessment

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

Additional information

Ir. R. Hanstveit (2003) was used as the key study for this endpoint as the study was performed according to OECD 301F and in compliance with GLP. The study was deemed to be of high reliability, and adequate for use as a stand alone study.

A reliability rating of 1 was assigned to this study, according to the criteria of Klimisch, 1997.