Soybean meal, protein extn. residue

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 February 2018 to 20 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Version / remarks:

(Paragraph (m))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO 10634, (1995)) it was concluded that the best testable dispersion was found to be obtained when using the high shear mixing method of preparation.
- The test material was dispersed directly in mineral medium.
- An amount of test material (58.5 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 litres to give a final concentration of 19.5 mg/L, equivalent to 10 mg carbon/L.
- A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source: A mixed population of activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed twice by settlement and resuspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C and used on the day of collection.
- Concentration of sludge: Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper (Rinsed three times with 20 mL deionised reverse osmosis water prior to drying in an oven) using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105 °C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 4.1 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

19.5 mg/L

Based on:

test mat.

Initial conc.:

10 mg/L

Based on:

other: Carbon

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: To 1 litre (final volume) of purified water (Reverse osmosis purified and deionised water, Elga Optima 15+ or Elga Purelab Option R-15 BP) was added 10 mL of Solution A and 1 mL each of Solutions B, C and D.
Solution A: 8.50 g/L KH2PO4, 21.75 g/L K2HPO4, 33.40 g/L Na2HPO4.2H2O and 0.50 g/L NH4Cl; pH = 7.4
Solution B: 27.50 g/L CaCl2
Solution C: 22.50 g/L MgSO4.7H2O
Solution D: 0.25 g/L FeCl3.6H2O (one drop of concentrated HCl/L was added as a preservative)
- Solubilising agent: No, the test material was dispersed directly into mineral medium
- Test temperature: 21 to 24 °C
- pH: 7.4 ± 0.2
- pH adjusted: Yes using diluted HCl or NaOH prior to the volume in all the vessels being adjusted to 3 litres by the addition of mineral medium which had been purged overnight with CO2 free air.
- Suspended solids concentration: 30 mg (ss)/ L (final concentration per vessel)
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 5 litre test culture vessels each containing 3 litres of solution
- Number of culture flasks/concentration: 2; the test material was added to inoculated mineral medium to give a final concentration of 10 mg carbon/L.
- Method used to create aerobic conditions: Approximately 24 hours prior to addition of the test and reference materials the vessels were filled with 2400 mL of mineral medium and 34.6 mL of inoculum and aerated overnight. On Day 0 the test and reference materials were added and the volume was then adjusted to 3 L by the addition of mineral medium that had been purged overnight with CO2-free air. The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer. The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
- Measuring equipment: Hach HQ40d Flexi handheld pH meter, Shimadzu TOC-VCSH TOC analyser or a Shimadzu TOC-LCSH TOC analyser.
- Details of trap for CO2 and volatile organics if used: The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.

SAMPLING
- Sampling frequency: Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29.
- On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.
- Sampling method: The samples were analysed for IC using either a Shimadzu TOC-VCSH TOC analyzer or a Shimadzu TOC-LCSH TOC analyser. Samples (50 or 135 μL) were injected into the IC channel of the TOC analyser. IC analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid or 2M HCl using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.
- Sample storage before analysis: All samples were analysed for IC immediately. The remainder of all samples were frozen for further analysis if required.

CONTROL AND BLANK SYSTEM
- Inoculum blank: In duplicate, consisting of inoculated mineral medium.
- Positive (procedure) control: In duplicate, containing the reference material in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
- Toxicity control: The test material plus the reference material in inoculated mineral medium to give a final concentration of 20 mg carbon/L (one vessel only). An amount of test item (58.5 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 liters to give a final concentration of 19.5 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

Inorganic Carbon (IC)/Total Carbon (TC) Ratio
- Samples (30 mL) were removed from the test material vessels on Day 0 prior to the addition of the test material in order to calculate the IC content in the test media. The samples were filtered through 0.45 μm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis. Samples (30 mL) were also removed from the inoculum control vessels on Day 0 and filtered through 0.45 μm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis.
- IC/TC analysis of the test material dispersions after dosing was not possible due to the insoluble nature of the test material in water.
- The samples were analysed for IC and TC using a Shimadzu TOC-VCPH TOC Analyser. Samples (50 μL) were injected into the TC and IC channels of the TOC analyser. TC analysis is carried out at 680 °C using a platinum based catalyst and zero grade air as the carrier gas. IC analysis involves conversion by orthophosphoric acid at ambient temperature.
Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in at least triplicate with 3 replicates being used in the calculation/reported.

STATISTICAL METHODS
- Calculation of Carbon Content
The test item contains 51.29 % carbon and so for a concentration of 10 mg C/L the total organic carbon present was 30 mg C.

The theoretical amount of carbon present in the reference material was calculated as follows:
[(No. of C atoms x mol wt of C) / mol wt of sodium benzoate] x 100
[(7 x 12.0110)/144.11] x100 = 58.34 %
Thus for a 10 mg C/L test concentration the total organic carbon present for sodium benzoate was 30 mg C.

- Percentage Biodegradation
The percentage biodegradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values into the following equation. The values of Replicates R1 and R2 are meaned for the inoculum control, test and reference materials before substitution into the following equation:
%ThCO2 (= % biodegradation*) = [(mg IC in test flask – mg IC in control flask)/ mg TOC added as test chemical] x100
*The conversion factor for carbon to carbon dioxide is 3.67

-Total CO2 Evolution
The total CO2 evolution in the inoculum control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation:
Total CO2 evolution (mg C/L) = mg IC in control x (100 / %C of CO2) x (1 / test volume)
= mg IC in control x (100 / 27.29) x (1 / 3)

Reference substance:

benzoic acid, sodium salt

Test performance:

The results of the test satisfied the validation criterion given in the OECD Test Guidelines.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

95

Sampling time:

28 d

Remarks on result:

other: Standard deviation not reported.

Details on results:

DEFINITVE TEST
- Percentage biodegradation values of the test and reference materials and the toxicity control are given in Table 1.

VALIDATION CRITERIA AND BIODEGRADATION
- The total CO2 evolution in the inoculum control vessels on Day 28 was 37.22 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
- The IC content of the test material suspension in the mineral medium at the start of the test was below 5 % of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
- The difference between the values for CO2 production at the end of the test for the replicate vessels was <20 % and hence satisfied the validation criterion given in the OECD Test Guidelines.
- 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 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test material.
- The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels.
- Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
- The test material attained 95 % biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
- The toxicity control attained 78 % biodegradation after 14 days and 95 % biodegradation after 28 days thereby confirming that the test material did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

Results with reference substance:

- Sodium benzoate attained 71 % biodegradation after 14 days with greater than 60 % degradation being attained in a 10-Day window. After 28 days 103 % biodegradation was attained. Biodegradation values in excess of 100 % were considered to be due to sampling/analytical variation. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

Table1: Percentage Biodegradation Values

Day	% Biodegradation
	Procedure control	Test material	Toxicity control
0	0	0	0
2	43	20	43
6	70	54	63
8	68	57	69
10	85	70	75
14	71	71	78
21	70	71	82
28	86	85	88
29*	103	95	95

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

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Under the conditions of this study the test material can be considered to be readily biodegradable.

Executive summary:

The ready biodegradability of the test material was determined in accordance with the standardised guidelines OECD 301B, EU Method C.4-C and US EPA OCSPP 835.3110 under GLP conditions using the CO2 evolution test.

The study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The test material, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 21 and 24 °C for 28 days.

The biodegradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference material, sodium benzoate, together with a toxicity control were used for validation purposes.

The test material attained 95 % biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Sodium benzoate attained 71 % biodegradation after 14 days with greater than 60 % degradation being attained in a 10-Day window. After 28 days 103 % biodegradation was attained. Biodegradation values in excess of 100 % were considered to be due to sampling/analytical variation. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

All other validation criterion were satisfied.

Under the conditions of this study the test material can be considered to be readily biodegradable.

Description of key information

Under the conditions of this study the test material can be considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

The ready biodegradability of the test material was determined in accordance with the standardised guidelines OECD 301B, EU Method C.4-C and US EPA OCSPP 835.3110 under GLP conditions using the CO2 evolution test. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The test material, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 21 and 24 °C for 28 days.

The biodegradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference material, sodium benzoate, together with a toxicity control were used for validation purposes.

The test material attained 95 % biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Sodium benzoate attained 71 % biodegradation after 14 days with greater than 60 % degradation being attained in a 10-Day window. After 28 days 103 % biodegradation was attained. Biodegradation values in excess of 100 % were considered to be due to sampling/analytical variation. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

All other validation criterion were satisfied.

Under the conditions of this study the test material can be considered to be readily biodegradable.