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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:
2016-07-12 - 2016-08-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate issued by the "Department of Health of the Government of the United Kindom" (2015-06-17).
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present.
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
Study conducted in the dark at temperatures of between 21 and 24 °C. Duplicate test vessels were used for the test substance, procedure control and inoculum control. Additionally, vessels (in duplicate) were prepared containing mineral oil at the same concentration as in the test substance preparations. One toxicity control is measured. Samples were taken from the first vessels on days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second vessels were sampled on days 0 and 29.
Due to insolubility of the test substance in water, the test substance was added to the inoculum on a filter paper. To assure consistancy between the test and procedure control vessels, one filter paper with chloroform evaporated to dryness was added to each control vessel.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
Informations indicated that the test substance was most likely insoluble in water. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation. This work confirmed that the test substance was insoluble in water. A further study was conducted to ascertain the best method to emply the test substance in the biodegradation test. From this preliminary solubility work and following the recommendations of the International Standards Organisation (ISO, 1995) it was concluded that the best testable dispersion was found to be obtained when using the solvent on filter paper method of preparation.
Test performance:
The test substance attained 4% biodegradation after 28 days. The mineral oil samples showed 37% degradation during the test period. Correction of the biodegradation rate of the test substance for mineral oil degradation showed that the test substance achieved 0% biodegradation after 28 days.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
4
Sampling time:
28 d
Results with reference substance:
The reference material (sodium benzoat) is to 80% biodegradated after the test period of 28 days. The corrected value for the biodegradation of the reference material which includes any carry-over of CO2 detected in the CO2 absorber of the second vessel is 70%.

The reference material (sodium benzoat) is to 80% biodegradated after the test period of 28 days. The corrected value for the biodegradation of the reference material which includes any carry-over of CO2 detected in the CO2 absorber of the second vessel  is 70%.

Validation Criteria

The total CO2 evolution in the inoculum control vessels on Day 28 was 28.86 mg/L. The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content. The difference between the values for CO2 production at the end of the test for the replicate vessels was <20%.

Altogether, those findings fulfill the validation criterions given in the OECD Test Guidelines.

Biodegradation

Acidification of the test vessels on day 28 followed by the final analyses on day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore, any additional CO2 detected in the day 29 samples originated from dissolved CO2 that was present in the test vessels on day 28. Hence, the biodegradation value calculated from the day 29 analyses is taken as being the final biodegradation value for the test substance. The test item attained 4% biodegradation after 28 days.

The results obtained from the mineral oil vessels showed 37% degradation over the test period. Correction of the biodegradation rate of the test item for mineral oil degradation showed that the test item achieved 0% biodegradation after 28 days.

The toxicity control attained 40% biodegradation after 14 days and 38% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test. The slight decrease in biodegradation between days 14 and 28 was considered to be due to sampling/analytical variation.

Sodium benzoate attained 84% biodegradation after 14 days and 70% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The slight decrease in biodegradation between days 14 and 28 was considered to be due to sampling/analytical variation.

Table 1: Inorganic Carbon Values on Each Analysis Occasion.

Day Inorganic Carbon (mg IC)
Inoculum Control Procedure Control -Sodium Benzoate Test Item Toxicity Control Mineral Oil Control
R1 R2 R1 R2 R1 R2 R1 R1 R2
Abs 1 Abs 2 Abs 1 Abs 2 Abs Abs Abs Abs Abs 1 Abs Abs 1 Abs Abs 1 Abs 2 Abs 1 Abs 2 Abs 1 Abs 2
0 1.75 1.75 1.75 1.75 1.75 1.75 1.98 1.75 1.75 1.75 1.75 1.75 1.75 1.75 1.75 1.75 1.75 1.75
2 6.73 - 6.50 - 20.77 - 20.42 - 4.29 - 5.45 - 18.44 - 6.03 - 4.99 -
6 13.26 - 12.92 - 35.06 - 36.10 - 8.42 - 12.57 - 35.98 - 14.30 - 11.19 -
8 13.19 - 13.30 - 34.63 - 32.68 - 8.03 - 11.70 - 29.13 - 14.10 - 12.27 -
10 14.48 - 16.65 - 36.82 - 35.57 - 9.23 - 15.39 - 35.57 - 18.47 - 14.02 -
14 17.79 - 18.47 - 43.97 - 42.73 - 12.01 - 18.70 - 42.16 - 20.97 - 20.17 -
21 19.38 - 18.25 - 37.07 - 40.11 - 19.15 - 19.15 - 40.33 - 24.11 - 23.21 -
28 24.08 - 23.18 - 48.05 - 47.49 - 24.08 - 26.10 - 45.25 - 29.12 - 30.80 -
29 23.94 2.78 24.94 2.78 41.53 2.78 49.43 2.78 24.94* 2.78 26.05 2.78 47.15 2.78 33.40 2.67 34.18 2.78

R1, R2: replicates 1 and 2; Abs: CO2 absorber vessels; *: Result from re-analysis of sample.

Table 2: Percentage Biodegradation Values.

Day % Biodegradation
Procedure Control (Sodium Benzoate) Test Item Toxicity Control Mineral Oil Control
0 0 0 0 0
2 47 0 20 0
6 75 0 38 0
8 69 0 34 0
10 69 0 33 3
14 84 0 40 10
21 66 1 36 19
28 80 5 36 25
29* 70 4 38 37

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2.

Validity criteria fulfilled:
yes
Remarks:
The test complies with criterions given in the OECD Test Guidelines.
Interpretation of results:
not readily biodegradable
Conclusions:
The report describes a valid guideline which agrees with GLP compliance. The test substance attained 4% biodegradation after 28 days, the results obtained from the mineral oil vessels showed 37% degradation over the test period. Correction of the biodegradation rate of the test item for mineral oil degradation showed that the test item achieved 0% biodegradation after 28 days. It cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
Executive summary:

A 28 day study was performed to assess the ready bioavailability of the test substance in an aerobic aquaeous medium. The method followed the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)) and was conducted in compliance with GLP criteria. For the test, non-adapted activated sludge was taken from a wastewater treatment plant which predominantly treats domestic sewage. Due to the fact that the test substance was insolubility in water, preliminary work determined that the best testable dispersion was obtained when the solvent on filter paper method was applied. As reference substance, sodium benzoate was used as procedure control. To assure consistancy between test and procedure control vessels, one filter paper with chloroform evaporarted to dryness was added to each procedure control. Also, additional vessels containing mineral oil at the same concentration as the test substance were prepared. Although the mineral oil was dispered in water directly, it was applied with a filter paper with chloroform evaporated to dryness, as well, to maintain consistancy. All samples were analyzed for IC using either Shimadzu TOC-VCPH TOC analyzer or a Shimadzu TOC-LCSH TOC analyzer immediately after they were taken.

All validation criteria given by the OECD Test Guidelines are fulfilled.

The test substance attained 4% biodegradation after 28 days. The results obtained from the mineral oil vessels showed 37% degradation over the test period. Correction of the biodegradation rate of the test substance for mineral oil degradation showed that the test substance achieved 0% biodegradation after 28 days. The test substance therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Description of key information

Biodegradation screening test, OECD 301B, aerobic, activated sludge: 4% degradation (CO2 evolution) after 28 days, not readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

A 28 day study key study (Bayliss, 2016) was performed to assess the ready bioavailability of the test substance in an aerobic aquaeous medium. The method followed the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)) and was conducted in compliance with GLP criteria.

The test substance attained 4% biodegradation after 28 days. The results obtained from the mineral oil vessels showed 37% degradation over the test period. Correction of the biodegradation rate of the test substance for mineral oil degradation showed that the test substance achieved 0% biodegradation after 28 days. The test substance therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.