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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10. April 2019 - 03. May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Duration of test (contact time):
28 d
Initial conc.:
96 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
other: ThOCD
Reference substance:
ethylene glycol
Preliminary study:
-
Test performance:
-
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Remarks on result:
not determinable
Parameter:
ThOD
Value:
0 mg O2/g test mat.
Remarks on result:
not determinable
Results with reference substance:
Both test item replicates did not reach 10% level (beginning of biodegradation) within the 28 day period of the study. The mean biodegradation on day 28 was 0%.
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable, not fulfilling specific criteria
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 December 2002 to 15 January 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed to GLP and in line with the standardised guidelines OECD 301-F and EU method C.4-D with no deficiencies thought to influence the accuracy of the presented results. The study was reported to a high standard.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
yes
Remarks:
100 mg (dry weight) of suspended solids, as no biodegradation occurred, this was not considered to affect the results
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
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: The activated sludge was obtained from an oxidation ditch situated in the municipality of Hazerswoude, the Netherlands (3 Dec 2002).
- Preparation of inoculum for exposure: 8 mL of sludge was added to 300 mL of mineral medium.
- Concentration of sludge: 100 mg of solids per litre
Duration of test (contact time):
755 h
Initial conc.:
23.7 mg/L
Based on:
test mat.
Initial conc.:
50.5 mg/L
Based on:
ThOD/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
PREPARATION OF THE TEST SUBSTANCE
The test substance was thoroughly mixed (homogenisation was not possible). Portions with and average weight of 0.00712 g of the test substance (average of all bottles containing test substance, including the toxicity control bottles) were dosed directly on glass fibre filters. The filters were placed into the test flask containing 300 mL of the medium.

TEST CONDITIONS
- Composition of medium: 1 mL of the following nutrient stock solutions were added to ultrapure water with a final volume
Stock solution a) Dissolved in and made up to 1000 mL ultrapure water: 8.5 g KH2PO4, 21.8 g K2HPO4, 33.4 g Na2HPO4.7H2O, 0.5 g NH4Cl and 100 g NaNO3. (pH 7.4 ± 0.1)
Stock solution b) Dissolved in and made up to 1000 mL ultrapure water: 22.5 g MgSO4.7H20
Stock solution c) Dissolved in and made up to 1000 mL ultrapure water: 36.4 g CaCL2.2H2O
Stock solution d) 0.25 g FeCl3.6H2O
To prevent nitrogen limitation, additional NaNO3 was added to the mineral medium.
- Test temperature: 20 ± 2 °C
- pH: 7.3-7.4
- pH adjusted: no
- Suspended solids concentration: 100 mg/L

TEST SYSTEM
- Number of culture flasks/concentration: 3 flasks containing test substance and 3 flasks containing a blank filter.
- Measuring equipment: A Micro-Oxymax respirometer. The respirometer measured the percentage oxygen in the air of the respective flasks and calculated, 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. Oxygen concentration was determined every 5 hours.

CONTROL AND BLANK SYSTEM
- Inoculum activity control: 3 flasks containing mineral medium only and 2 flasks containing 100 mg/L sodium acetate
- Toxicity control: 2 flasks containing 100 mg/L of sodium acetate and 23.7 mg/L test substance.

STATISTICAL METHODS: The oxygen consumption in each flask was calculated based on the respiration rate (mg O2/flask/hour). 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.
Based on the empirical formula, the Theoretical Oxygen Demand (ThOD) without nitrification (ThODNH3) value of the test substance was calculated to be 2.13 mg O2/mg. The degree of biodegradation was calculated with this ThODNH3 value, assuming a purity of 100 %.
Reference substance:
acetic acid, sodium salt
Test performance:
The pH of the test medium was fairly constant during the test with the final values in the range of 7.2-7.8 after 39 days incubation. The higher pH values were found in the activity and toxicity control vessels were CO2 production was highest.
The temperature during the test was recorded as between 20.1-22.3 °C.
On day 32, oxygen measurements were stopped for a short period to allow recalibration. In a later stage all data after day 31 were considered unreliable and were not reported. Between 661 and 675 h a data point was missing as it was out of range.
Very low negative values of oxygen consumption and high values that are not part of a peak are attributed to possible leakages of the system. These occurred mainly after 755 h and between 581 and 675 h and are related to a technical failure of the oxygen sensor, these results were therefore disregarded.
Parameter:
% degradation (O2 consumption)
Value:
1.5
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
1.9
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
1.9
Sampling time:
31 d
Details on results:
The average percent degradation of the test substance after 28 days was approximately 2 % with a concentration of 23.7 mg/L. In two flasks, negligible biodegradation occurred, in the third approximately 8 % biodegradation was observed.
The oxygen consumption in the blanks (with and without filter) was 1.21 to 2.96 mg per flasks after 28 days incubation, equivalent to 4.0-9.9 mg/L.
Figure 1 depicts the oxygen consumption in the control and test flasks over time
Figure 2 depicts the biological oxygen demand (BOD)
Figure 3 depicts the percentage biodegradation based on the ThODNH3
Results with reference substance:
>25 % biodegradation was observed in the toxicity controls, demonstrating the the test substance is not toxic to the inoculum.
The reference substance was sufficiently degraded within 14 days of incubation (>60%).

Table 1: Results with test substance

Time (days)

BOD mg O2/mg

Biodegradation ThODNH3%

14

1.4

1.7

28

1.8

2.1

31

1.7

2.0

 

Table 2: Results with control vessels

Time (days)

Inoculum blank (mg O2/flask)

Inoculum blank with filter (mg O2/flask(

Inoculum activity control

Toxicity control

mg O2/flask

Biodegradation ThOD %1

mg O2/flask

Biodegradation ThOD %1

14

1.4

1.7

21.6

99

21.5

56.0

28

1.8

2.1

23.1

105

23.2

56.6

31

1.7

2.0

23.0

105

23.1

59.6

All values are expressed as the mean of all the replicates in that test series

1Corrected for corresponding blank

 

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The average percentage degradation of the test substance after 28 days was approximately 2 % under the conditions of the test. In two of the three flasks containing the test substance, no significant biodegradation was observed. In the third flask approximately 8 % biodegradation was observed. The test substance was not toxic to the inoculum.
Executive summary:

The ready biodegradability of the test substance was assessed in a Manometric Respirometry test conducted in accordance with OECD guideline 301F and EU method C.4 -D and to GLP. Under the conditions of the test, the test substance degraded approximately 2 % within 28 days of incubation with activated sludge. Two of the flasks performed with the test substance gave negligible oxygen consumption, whilst the third gave 8 % within 28 days. All controls performed as part of the test were demonstrated that the test system was operating correctly and the test substance exhibited no toxic effect on the inoculum. All validity criteria of the guideline were met. Therefore, under the conditions of the test, the test substance can be categorised as not readily biodegradable.

Description of key information

Key studies:

- Maischak (2019), OECD 302B (adapted acc. to OECD 301F); Not inherently biodegradable

within the 28 day period of the study

- Hanstveit (2003), OECD 301-F and EU method C.4-D; Not readily biodegradable, approximately 2 % biodegradation occurred within 28 days.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Two studies show no biodegradability for the test item (DOTO)

- Maischak study (2019), OECD 302B (adapted acc. to OECD 301F):

Both test item replicates did not reach 10% level (beginning of biodegradation) within the 28 day period of the study. The mean biodegradation on day 28 was 0%. The validity criteria of the guideline are fulfilled.

The test item is classified as not inherently biodegradable within the 28 day period of the study.

- Hanstveit study (2003), OECD 301F:

The validy criteria of the gudeline are fulfilled. Under the conditions of the test, approximately 2 % biodegradation of the test substance during 28 days of incubation occurred, it can be concluded from the results of this study that the test substance is not readily biodegradable.