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Environmental fate & pathways

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:
2017-05-22 to 2017-09-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Batch no.: 201701151
- Purity: 99.2 area-%
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Remarks:
Activated sludge from a municipal sewage plant
Details on inoculum:
- Source of inoculum/activated sludge: Waste water sewage plant Mannheim, Germany
- Preparation of inoculum for exposure: A suitable aliquot of the suspension was sieved by finely woven mesh with a mesh size of about 1 mm. To reduce the content of inorganic carbon in the blank control, the activated sludge suspension was aerated with carbon dioxide free air for about 48 hours at 22 +- 2 °C. At the day of exposure, the aeration was stopped and the sludge was allowed to settle. The supernatant was discarded and the residue of the sludge suspension was filled up with drinking water.
- Concentration of sludge: The sludge of the suspension was adjusted to a concentration of 800 mg/L of dry weight.
- Initial cell/biomass concentration: Aliquots of 0.5 mL were added to the test vessels to obtain a sludge concentration of 4 mg/L dry substance in the test assays.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
TOC
Remarks:
equivalent to approximately 33 mg/L test substance
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: in compliance with OECD Guideline 310
- Additional substrate: no
- Solubilising agent: none
- Test temperature: 20 +- 2 °C
- pH: 6.7 to 7.2 after 28 days
- pH adjusted: pH value of the mineral medium was adjusted to 7.3 with 1 molar sulphuric acid; no addititional pH adjustment conducted
- Other: agitation by orbital shaker with a speed of about 150 r/min

TEST SYSTEM

- Number of culture flasks/concentration:
29 blank control assays
31 test substance assays
29 inhibition control test assays
17 reference substance assays
5 assays for abiotic control

- Method used to create aerobic conditions: oxygen available in the headspace of the bottles
- Measuring equipment: TOC-analyzer equipped with an auto sampler (Shimadzu TOC-5000A and/or TOC-L CSH/CSN).
- Test performed in closed vessels due to significant volatility of test substance: yes, 160 mL incubation bottles filled up to a volume of 100 mL; closed gas tight with butyl rubber septa
- Test performed in open system: no

SAMPLING
- Sampling method:
At the sampling days three bottles of each test assay were sacrificed. 1 mL 7 molar sodium hydroxide solution was injected with a syringe trough the septum in each test bottle. The bottles were shaken for about one hour. The carbon dioxide in the headspace, evolved by biological processes, is solved in the alkaline solution. For the measurement at the end of exposure five test substance assays were prepared. The single values of these test assays were used for mean value calculation of the biodegradation degree with a limit of confidence limit of 95 %. The inorganic carbon content of the sodium hydroxide solution used for the alkalization of the test assays was checked and considered at the evaluation. The bottles were opened and the amount of Total Inorganic Carbon (TIC) was determined with a carbon analyzer.
- Sample storage before analysis: no

CONTROL AND BLANK SYSTEM
- blank control
- inhibition control
- positive control (with reference substance)
- abiotic control

EVALUATION OF TEST RESULTS
The measured TIC-values were compared with the added amount of TOC and expressed as biodegradation degree and plotted by a biodegradation curve. At the end of exposure, the mean value of biodegradation degree was calculated with a confidence limit of 95 %.
Reference substance:
aniline
Preliminary study:
Yes.
Test performance:
VALIDITY OF THE TEST

- TIC-value in the blank control at the end of exposure (mean value): 1.2 mg/L
- The deviation of the degradation degree of the test substance at end of exposure should be <20 % between highest and lowest single value: yes
- Degree of biodegradation of the reference substance should be > 60% TIC/TOC after 14 days: yes
- Degree of biodegradation in the inhibition control should be > 25 % TIC/TOC after 28 days: yes
- TIC-value in the blank control at the end of exposure should be <15% of the amount of test substance added initially (mg TOC/L): yes
- Test temperature in the range of 20 ± 2 °C: yes

The test is valid.
Key result
Parameter:
% degradation (inorg. C analysis)
Value:
>= 44 - <= 50
Sampling time:
28 d
Details on results:
Freshly preprared sodium hydoxide solution contains traces of carbon dioxide. These values were subtracted form each TIC value of the test vessels. The mean values were calculated from the single values of each measurement.

The mean value of degree of biodegradation was calculated by the single values of five test assays at the end of exposure with a limit of confidence of 95 %.

The required pass level for ready biodegradability was not reached.

Evaluation was conducted in accordance to criteria of the OECD Test Guideline 310.
Results with reference substance:
The degree of biodegradation of the reference substance after 14 days was 81 % TIC/TOC (mean value) and 56 % TIC/TOC (mean value) after 28 days.

Table 1: Measurement of total inorganic carbon

Test Duration [days] values TIC values [mg/L]
NaOH BC BC1 BC2 BC3 RS1 RS2 RS3 IH1 IH2 IH3 TS1 TS2 TS3
2 a 1.0 1.3 1.4 1.5 1.6 2.1 1.5 1.7 1.8 2.0 1.8 2.0 3.1
b 1.0 1.3 1.3 1.4 1.5 1.6 1.5 1.7 1.7 1.9 1.7 2.1 3.1
5 a 0.7 1.3 1.4 1.4 12.9 12.9 13.6 14.2 13.7 13.9 2.0 1.9 1.7
b 0.8 1.4 1.4 1.4 13.1 12.7 13.4 13.7 13.7 13.8 1.8 1.8 1.9
9 a 0.9 1.4 1.6 1.5 16.0 16.2 18.5 17.7 20.1 21.4 3.3 3.3 3.2
b 0.9 1.5 1.5 1.4 15.9 16.1 18.5 17.5 19.7 21.3 3.3 3.4 3.7
13 a 0.7 1.6 1.7 1.6 17.5 17.8 17.7 22.8 20.9 21.1 10.7 9.5 10.2
b 0.9 1.7 1.6 1.6 17.6 17.8 17.7 22.7 20.9 20.8 10.5 9.9 10.1
14 a 0.9 1.7 1.6 1.7 18.6 17.4 17.2 24.9 24.0 23.0 12.0 13.0 11.6
b 1.1 1.8 1.7 1.7 18.7 17.7 17.1 24.7 23.5 23.4 11.8 13.1 11.6
19 a 0.6 1.6 1.7 1.7 27.4 26.9 27.5 10.9 10.9 13.1
b 0.6 1.6 1.9 1.7 27.4 27.3 27.5 10.8 10.5 12.6
23 a 0.9 2.0 2.0 2.0 27.4 28.6 28.9 16.3 16.5 16.8
b 1.0 2.0 1.8 2.9 27.4 28.6 28.9 16.0 16.5 16.9
26 a 0.9 1.8 2.0 2.0 32.7 29.9 30.9 15.5 10.2 10.9
b 0.9 1.9 2.0 2.0 31.9 29.3 30.2 16.2 10.0 11.0
Test Duration [days) values NaOH BC BC1 BC2 BC3 PC1 PC2 PC3 IH1 IH2 IH3 TS1 TS2 TS3
a 0.7 1.9 1.9 1.9 1.1 1.1 1.2 31.6 31.6 31.0 11.2 11.9 11.0
b 0.8 1.9 1.9 1.9 1.0 1.1 1.2 31.3 31.9 30.9 11.2 11.9 10.6
TS4 TS5 pH values in the reference substance assays after 14 days
RS4: 6.8
RS5: 6.8
a 10.5 11.6
b 10.5 11.5

pH-values in the test assays at the end of exposure after 28 days:

Test assay BC4: 7.2

Test assay BC5: 7.2

Test assay TS6: 6.9

Test assay TS7: 6.9

Test assay PC4: 7.1

Test assay PC5: 7.1

Test assay IH4: 6.7

Test assay IH 5: 6.7

Abbreviations:

BC: Blank control assay

TS: Test substance assay

RS: Reference substance assay

PC: Abiotic control assay

IH: Inhibition control assay

Table 2: Degree of biodegradation in the treatment groups

Test duration [days] Degree of biodegradation [%]
RS1 RS2 RS3 RS mv IH1 IH2 IH3 ICH mv TS1 TS2 TS3 TS mv
2 1 3 1 2 1 1 2 1 2 4 9 5
5 58 57 61 59 31 31 31 31 3 2 2 2
9 73 74 85 77 40 46 50 45 9 10 10 10
13 80 81 80 80 53 48 48 50 45 40 43 43
14 85 80 78 81 58 55 54 56 51 57 50 49
19 64 64 65 64 46 45 56 49
23 64 57 68 66 a71 a73 a74 a73
26 76 69 72 72 a70 41 45 43
Test duration [days] PC1 PC2 PC3 PC mv IH1 IH2 IH3 IH mv TS1 TS2 TS3 TS 4
28 2 2 3 2 74 75 73 74 47 50 45 43
TS5 TS mv
48 47

aRemark: regarded as outlier values and therefore not evaluated

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable

Description of key information

The test item is not readily biodegradable (by OECD criteria), but moderately biodegradable (47% degradation).

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The ready biodegradability of the test item was assessed by measurement of the Total Inorganic Carbon (TIC) formed by mineralization of the organic carbon of the test compound (according to OECD Guideline 310, in compliance with GLP criteria) (BASF SE, 2017). The selected test concentration of 20 mg/L TOC corresponds to approximately 33 mg/L test substance. It is anticipated that the selected test concentration causes no toxic effects to the microorganisms. The selected test concentration was tested in an additional inhibition control test assay and no toxic effects to the microorganisms were observed. Municipal activated sludge was collected from the aeration tank of the wastewater treatment plant of Mannheim, Germany. A suitable aliquot of the suspension was sieved by a finely woven mesh with a mesh size of about 1 mm. To reduce the content of inorganic carbon in the blank control, the activated sludge suspension was aerated with carbon dioxide free air for about 48 hours at 22 ± 2° C. At the day of exposure, the aeration was stopped and the sludge was allowed to settle. The supernatant was discarded and the residue of the sludge suspension was filled up with drinking water. The sludge of the suspension was adjusted to a concentration 800 mg/L of dry weight. Aliquots of 0.5 mL were added to the test vessels to obtain a sludge concentration of 4 mg/L dry substance in the test assays. The following test assays were prepared: Blank control assays (BC, 29 replicates), reference item assays (RS, 17 replicates) with aniline for the check of the biological activity of the inoculum, inhibition control assays (IH, 29 replicates) for the check of the toxic effect of the test item at the selected test concentration, test item assays (TS, 31 replicates) with the required amount of test item for a test concentration of 20 mg/L TOC and a abiotic control (PC, 5 replicates), test assays without inoculum, inhibited with mercury chloride for the check of physical-chemical elimination processes. The TIC-analyses were performed as repeat determination, using a TOC-analyzer equipped with an auto sampler The samples were analyzed on the day of sampling without further treatment. At the end of exposure, the mean value of biodegradation degree was calculated with a confidence limit of 95 %. In result, the degree of biodegradation of the test substance at the end of exposure (mean value of five test assays, limit of confidence 95%) was 47 ± 3% TIC/TOC. The degree of elimination of the test substance at physical chemical control at the end of exposure was <10 %TIC/TOC (mean value). The degree of biodegradation of the reference substance after 14 days was 81 % TIC/TOC (mean value). The deegree of biodegradation in the inhibition control after 28 days was 56 % TIC/TOC (mean value). In conclusion, the test item was not readily biodegradable in this test, since the required pass level for ready biodegradability was not reached. Based on determined rate of biodegradation at the end of exposure, the test substance can be evaluated as moderately or partly biodegradable. The results in this study are consistent with all validity criteria and the test is valid according to the guideline of this study. No deviations from the test guidelines or other incidents occurred during the course of the reported test which may have influenced the results.