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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:
24 January 2018 to 28 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
yes
Remarks:
(see "principles of method if other then guideline' for further information)
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
yes
Remarks:
(see "principles of method if other then guideline' for further information)
Principles of method if other than guideline:
The suspended solids concentration in the inoculum blank control vessels was 35 mg/L in the final volume. Although this is a deviation to the guideline which states the concentration should not be greater than 30 mg/L, it is not believed this had an adverse effect on the outcome of the study as it represents a worse-case result. Inoculum blank controls containing 35 mg/L suspended solids would likely contain more bacteria than inoculum blank controls containing 30 mg/L suspended solids and therefore have a higher oxygen uptake; this would therefore make the blank corrected BOD results lower than if the blank controls contained 30 mg/L suspended solids.
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Inoculum or test system:
sewage, predominantly domestic, adapted
Details on inoculum:
TEST ORGANISMS
- Source of inoculum/activated sludge:
Activated sludge was obtained from Totnes Sewage Treatment Works, Totnes, Devon, UK on 24 January 2018. This works treats sewage of predominantly domestic origin. At the laboratory, the activated sludge was kept aerated at room temperature and the pH maintained at 7.0 ± 1.0.
Seven days prior to the exposure start the activated sludge was centrifuged, washed and re suspended in the mineral medium and the solids concentration determined. This sludge was then diluted in medium, added to test bottles and stirred until required for use. The seeded mineral medium was pre-conditioned for seven days to reduce the blank oxygen uptake readings in the test.

MINERAL MEDIUM
The mineral medium was made up according to the OECD and EC guidelines and contained the following nutrients per litre of reverse osmosis (RO) water:
- KH2PO4: 85.0 mg/L
- K2HPO4: 217.5 mg/L
- Na2HPO4.2H2O: 334.0 mg/L
- NH4Cl: 5.0 mg/L
- CaCl2.2H2O: 36.4 mg/L
- MgSO4.7H2O: 22.5 mg/L
- FeCl3.6H2O: 0.25 mg/L
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Details on study design:
TEST APPARATUS
The measurement of oxygen uptake was conducted in the Oxitop™ respirometers (Wissenschaftlich-Technische Werkstätten, GmbH, Germany). Each individual unit consisted of a dark glass 500 mL bottle with an Oxitop™ bottle top containing a piezoresistive electronic pressure sensor. Bottles were situated on a magnetic stirrer in a constant temperature incubator. Carbon dioxide produced by microbial respiration was absorbed by potassium hydroxide solution (KOH) placed in a seal cup in the neck of each bottle, and the oxygen taken up was measured as a decrease in pressure. The Oxitop™ controller collected the pressure values from the measuring tops and calculated the BOD (as mg/L).

METHODOLOGY
All test bottles contained the prepared activated sludge inoculum in mineral medium. Following the pre-conditioning the test bottles were set up for the test according to the experimental design shown in Table 1. Inoculum blanks contained no test or reference substance, in order to demonstrate there was no other carbon source in the medium. Positive controls contained the reference substance, sodium benzoate at 100 mg/L, to demonstrate the viability of the inoculum. The test material bottles contained test material at 100 mg/L, to determine the biodegradation of the test material. Toxicity controls contained the test and reference substances, both at 100 mg/L, and were used to show if there had been any inhibition of the inoculum by the test material. Each set of bottles was prepared in triplicate.
The bottles containing test material were dosed from a primary stock solution containing 1000 mg/L prepared by dissolving test material in RO water to give a clear and colourless solution. Sodium benzoate was dosed as a 1000 mg/L stock solution, prepared by dissolving sodium benzoate in RO water to give a clear and colourless solution. Both solutions were prepared on the day of exposure start.
Oxygen uptake was recorded automatically every 112 minutes during the 28-day experimental period. Oxygen uptake values were corrected for the inoculum blank and the biodegradation was calculated as a percentage of the theoretical oxygen demand for the substance under test and as a percentage of the theoretical oxygen demand for the reference substance.

WATER QUALITY MEASUREMENTS
The pH of the seeded test medium was measured and adjusted as necessary to 7.4 ± 0.2 prior to the acclimation phase. On day 0 the pH was measured in a single bottle from the inoculum blanks, positive controls, test materal and toxicity control bottles.
The study was performed in an incubator to ensure the test bottle contents were maintained within the range 22 ± 1 °C. The temperature of the solutions in a selection of test bottles was measured on days 0 and 28. The temperature of the incubator was continuously monitored throughout the study.
Reference substance:
benzoic acid, sodium salt
Remarks:
(100 mg/L)
Test performance:
The validity requirements of the OECD guideline state:
- the difference between extremes of replicate biodegradation values should be less than 20 % at the end of 10-day window, at the plateau or at the end of the test;
- the positive control should achieve > 60 % biodegradation by Day 14.
- the oxygen consumption of the inoculum blank should not exceed 60 mg/L in 28 days.

Mean oxygen uptake of the inoculum blank was below 60 mg/L, as required in the OECD guideline.
The difference between replicate test material extremes was < 20 % after the 10-day window, at plateau, and on day 28. Sodium benzoate reached a mean 64 % biodegradation by Day 14. The average of the mean oxygen consumed in the inoculum blanks was 14.0 mg/L after 28 days. Therefore, this test satisfied all the validity criteria
Key result
Parameter:
other: BOD: ThOD ratio
Value:
77
Sampling time:
28 d
Details on results:
- Theoretical oxygen demand (ThOD) and Biochemical oxygen demand (BOD)
The ThOD of the test material was calculated as 1.39 g O2/g.
The test material attained a mean level of biodegradation (based on the BOD: ThOD ratio) of 77 % after 28 days and the results showed good replication.
Greater than 60 % degradation was achieved within the 10-day window, therefore, the test material can be classified as readily biodegradable

The mean toxicity control degradation achieved on day 14 was 64 % (based on ThOD), as this is > 25 % the test material is assumed not to be inhibitory at this concentration.

- pH and temperature measurements
At the end of the 28-day test period, the pH values ranged from 7.2 to 7.3 in the inoculum blank bottles, from 6.7 to 6.9 in the sodium benzoate bottles, from 6.6 to 6.7 in the test material bottles and were 6.4 in the toxicity control bottles.
Temperature measurements recorded in several of the test bottles on days 0 and 28 indicated the temperature was within the range 22 ± 1 °C. Continuous monitoring of the incubator temperature showed it to have remained within the range 22 ± 1 °C throughout the study.
Key result
Parameter:
ThOD
Value:
1.39 g O2/g test mat.
Key result
Parameter:
BOD5*100/ThOD
Value:
77
Results with reference substance:
The ThOD of sodium benzoate was calculated as 1.67 g O2/g of substance.
Sodium benzoate attained a mean level of biodegradation (based on the BOD:ThOD ratio) of 66 % after 28 days, and the results showed good replication.
Greater than 60 % degradation was achieved by day 14 as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms.

Table 2: Mean percentage biodegradation

    Test material     Reference substance
 Day  Mean % biodegradation  Day  Mean % biodegradation
 5  60  5  56
 10  70  10  62
 15  73  15  64
 20  75  20  64
 25  76  25  66
 28  77  28  66
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Under the conditions of the study, the test material attained a maximum mean level of biodegradation (based on the BOD:ThOD ratio) of 77 % after 28 days, and the results showed good replication. Greater than 60 % degradation was achieved within the 10-day window, hence the test material can be considered to be readily biodegradable.
Executive summary:

The biodegradability of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 301F and EU Methe C.4-D, under GLP conditions.

Under the conditions of the study, the test material attained a maximum mean level of biodegradation (based on the BOD:ThOD ratio) of 77 % after 28 days, and the results showed good replication. Greater than 60 % degradation was achieved within the 10-day window, hence the test material can be considered to be readily biodegradable.

Description of key information

Scymaris, 2018 - OECD 301F, EU Method C.4-D

Under the conditions of the study, the test material attained a maximum mean level of biodegradation (based on the BOD:ThOD ratio) of 77 % after 28 days, and the results showed good replication. Greater than 60 % degradation was achieved within the 10-day window, hence the test material can be considered to be readily biodegradable.

Suflita and Mormile (1993)

The anaerobic degradation of the test material was investigated in the terrestrial subsurface.

Under the conditions of the study:

- The acclimation period was determined to be 5 days.

- The rate of anaerobic biodegradation in aquifer slurries was determined to be 7.3 ± 3.6 ppm C/day

- The methane recovery was 109% theoretical.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Scymaris, 2018 - OECD 301F, EU Method C.4-D

The biodegradability of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 301F and EU Methe C.4-D, under GLP conditions. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997) and considered the key study.

Under the conditions of the study, the test material attained a maximum mean level of biodegradation (based on the BOD:ThOD ratio) of 77% after 28 days, and the results showed good replication. Greater than 60 % degradation was achieved within the 10-day window, hence the test material can be considered to be readily biodegradable.

Supporting information is available from a literature reference (Suflita, 1993). The study reported was conducted in accrodance with sound scientific principles and was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Suflita and Mormile (1993)

The anaerobic degradation of the test material was investigated in the terrestrial subsurface.

During the study, sediment and groundwater were collected from a methanogenic portion of a shallow anoxic aquifer polluted by municipal landfill leachate. Slurries were prepared by placing 50 g of sediment and 75 mL of groundwater in sterile 160-mL serum bottles. The bottles were sealed and incubated overnight in the dark. The test material was then added to the incubation mixture at an initial substrate concentration of 50 ppm C. Pressure increases resulting from biogas formation were monitored with an automated pressure transducer system. The acclimation period was estimated as the amount of time where no significant pressure differences were measured between substrate-amended and unamended controls. At the end of the incubation period, the depletion of the parent substrate (or lack thereof) and the formation of methane over background controls were confirmed with a Varian 3300 gas chromatograph equipped with a flame ionisation detector. A 1.8 m X 0.32 cm 801 100 porapak Q column or a 0.2 % Carbowax 1500 on Carbopack C column were used for headspace methane analyses and oxygenate determinations, respectively. Nitrogen (30 mL/min) served as the carrier gas for both analyses. Autoclaved controls were similarly assayed and were uniformly unable to exhibit methane formation or oxygenate disappearance. The rate of substrate depletion was determined in incubations receiving a subsequent addition of the oxygenate.

Under the conditions of the study:

- The acclimation period was determined to be 5 days.

- The rate of anaerobic biodegradation in aquifer slurries was determined to be 7.3 ± 3.6 ppm C/day

- The methane recovery was 109 % theoretical.