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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:

20 March 2020 - 11 November 2020

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 07 September 2020 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic sewage.

Preparation of 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. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection. 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 a pre-weighed Whatman GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC 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 3.0 g/L prior to use.

Duration of test (contact time):

ca. 28 d

Initial conc.:

ca. 15.7 mg/L

Based on:

test mat.

Remarks:

equivalent to 10 mg carbon/L

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preliminary Solubility Work
Information provided by the Sponsor indicated that the test item was insoluble in water. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation.

Test Item Preparation
From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO 10634, 1995) and Handley et al (2002) it was concluded that the best testable dispersion was found to be obtained when using the high shear mix method of preparation. An amount of test item (47.1 mg) was dispersed directly in approximately 400 mL of mineral medium, in duplicate, with the aid of high shear mixing (approximately 7500 rpm, 15 minutes). It was then allowed to cool to temperatures of 23 °C prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 L to give a final concentration of 15.7 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.

Reference Item Preparation
A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution was prepared by dissolving a nominal amount of the reference item (1000 mg) directly in mineral medium and the volume adjusted to 1 L to give a 1000 mg/L stock solution. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium prior to the volume being adjusted to 3 L to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Toxicity Control
A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test. An amount of test item (47.1 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to allowing to cool to a temperature of 23 °C 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 L to give a final concentration of 15.7 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

Preparation of Test System
The following were prepared and inoculated in 5 L test culture vessels each containing 3 L of preparation:
a) An inoculated control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).

Data from the inoculum control and procedure control vessels was shared with similar concurrent studies. Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at temperatures of between 22 °C and 24 °C for 28 days, in darkness. The temperature was recorded daily from a vessel incubated alongside the test containing 3 L of reverse osmosis water.

Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 30 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter. The pH values of the medium were adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid prior to the volume in all of the vessels being adjusted to 3 L by the addition of mineral medium, which had been purged overnight with CO2-free air. The inoculum control vessels were prepared in a similar manner without the addition of test item or reference item.

In order to confirm that the sodium benzoate solution was prepared correctly, a diluted 100 mg/L stock solution (in reverse osmosis water) was sampled for TOC analysis. The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/minute per vessel and stirred continuously by magnetic stirrer. The CO2-free air was produced by passing compressed air through a moisture trap prior to being passed through a glass column containing self-indicating soda lime (Carbosorb®) granules.

The CO2 produced by degradation was collected in two Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using deionized reverse osmosis water.

Reference substance:

other: Sodium benzoate

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 40

Sampling time:

28 d

Details on results:

Definitive Test
The IC values for the test item, procedure control, toxicity control and inoculum control vessels at each analysis occasion are given in Table 'Inorganic Carbon Values on Each Analysis Occasion'. Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 'Percentage Biodegradation Values'. TC and IC values in the culture vessels on Day 0 are given in Table 'Total and Inorganic Carbon Values in the Culture Vessels on Day 0'.


Validation Criteria and Biodegradation
The total CO2 evolution in the inoculum control vessels on Day 28 was 17.31 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines. The IC content of the test item suspension in the mineral medium at the start of the test, before dosing (see Table 3), 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 item.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels. The IC 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 item attained 40% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
The toxicity control attained 47% biodegradation after 14 days and 63% 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. Sodium benzoate attained 67% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines. After 28 days 75% biodegradation was attained.

Total Organic Carbon Confirmation
Total organic carbon of the diluted sodium benzoate stock solution confirmed that it had been
prepared correctly

Conclusion
The test item attained 40% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No.301B.

Results with reference substance:

Sodium benzoate attained 67% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window.

Percentage Biodegradation Values 

Day	Biodegradation (%)
	Procedure Control	Test item	Toxicity control
0	0	0	0
2	52	4	29
6	69	16	38
8	70	21	49
10	68	23	51
14	67	28	47
21	78	40	60
28	77	40	60
29*	75	40	63

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

 

Inorganic Carbon Values on Each Analysis Occasion

Day	Inorganic Carbon (mg IC)
	Inoculum Control				Procedure Control				Test Item				Toxicity Control
	R1		R2		R1		R2		R1		R2		R1
	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	2.45	3.03	2.33	2.1	2.1	2.1	2.1	2.1	2.1	2.22	2.1	6.53	2.1	2.45
2	3.37		4.52		19.49		19.37		5.22		5.11		21.23
6	6.34		7.15		26.76		28.26		11.88		11.3		29.52
8	7.11		8.6		26.95		30.62		14.45		13.87		37.04
10	7.52		8.55		27.7		28.96		14.25		15.39		38.65
14	9.07		8.84		27.54		30.37		16.89		17.68		37.17
21	11.49		11.49		34.36		35.49		23.32		23.43		47.77
28	14.45		13.89		35.95		38.3		26.1		26.43		50.4
29	15.36	1.74	15.36	1.39	36.85	1.39	38.97	1.74	27.16	1.04	27.5	1.04	52.75	1.74

 

R = Replicate
Abs = CO2 absorber vessel

 

Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test Vessel	Total Carbon∗ (mg/L)	Inorganic Carbon* (mg/L)	IC Content (% of TC)
Test Item 10 mg C/L R1	9.73**	-0.17	0
Test Item 10 mg C/L R2	10.07**	0.12	1

 

R = Replicate
∗ Corrected for control values. Negative values are due to measured concentrations being less than
control values.
** TC value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 40% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No.301B.

Executive summary:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium when exposed to sewage sludge micro-organisms under defined conditions. Carbon dioxide (CO2) evolution from the test item was followed by means of Inorganic Carbon (IC) analysis over a period of 28 days when exposed to sewage sludge micro-organisms under defined conditions. The method followed was designed to be compatible with 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)). Study conducted under GLP conditions.

 

The test item, 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 22°C and 24 °C for 28 days. The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

 

The test item attained 40% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No.301B.

Description of key information

The test item attained 40% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No.301B.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium when exposed to sewage sludge micro-organisms under defined conditions. Carbon dioxide (CO2) evolution from the test item was followed by means of Inorganic Carbon (IC) analysis over a period of 28 days when exposed to sewage sludge micro-organisms under defined conditions. The method followed was designed to be compatible with 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)). Study was conducted under GLP conditions. 

 

The test item, 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 22°C and 24 °C for 28 days. The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

 

The test item attained 40% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No.301B.