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Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-11-16 to 2013-04-18

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)

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from municipal sewage STP D-31137 Hildesheim. The sludge of that STP comprises mostly municipal sewage and hardly any industrial chemical waster.
- Laboratory culture: Not applicable.
- Method of cultivation: Not applicable.
- Storage conditions: No.
- Storage length: NA.
- Preparation of inoculum for exposure: The activated sludge was washed twice with chlorine free tap water. After the second washing the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration for 2 hours. Thereafter the sludge was homogenized with a blender. The supernatant was decanted and maintained in an aerobic condition by aeration with CO2 free air until test start. 20 mL/L of this mixture were used to initiate inoculation.
- Pretreatment: See above.
- Concentration of sludge: See below, biomass concentration.
- Initial cell/biomass concentration: 10^7 to 10^8 CFU/L under test conditions.
- Water filtered: See above.
- Type and size of filter used, if any: No data

Duration of test (contact time):

28 d

Initial conc.:

8.38 mg/L

Based on:

other: Organic Carbon

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

other: CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium according to OECD 301.
- Solubilising agent (type and concentration if used): None.
- Test temperature: 20 to 24 °C.
- pH adjusted: No.
- CEC (meq/100 g): No data.
- Aeration of dilution water: Aeration with CO2 free air.
- Suspended solids concentration: Not determined.
- Continuous darkness: Low light conditions.

TEST SYSTEM
- Culturing apparatus: Several bottles in series (reactor system): 5 L reactor connected to 3 x 250 mL gas-washing bottles (in series), filled with 0.0125 mol/L Ba (OH)2 adsorption solution.
- Number of culture flasks/concentration:
Reactors 1 and 2: Test concentration 8.38 mg C/L, in duplicate.
Reactors 3 and 4: Blank control, in duplicate.
Reactor 5: Reference substance sodium benzoate at 11.6 mg C/L.
Reactor 6: Toxicity control, i.e. 8.38 mg C/L test substance + 11.6 mg C/L reference substance).
- Method used to create aerobic conditions: Continuous aeration with CO2 free air, mixing of the test solutions by magnetic stirring.
- Method used to create anaerobic conditions: Not applicable since the test was conducted under aerobic conditions.
- Measuring equipment: Titration equipment (type not specified).
- Test performed in closed vessels due to significant volatility of test substance: No.
- Test performed in open system: Sealed vessels, but aerated with CO2 free air.
- Details of trap for CO2 and volatile organics if used: 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution.

SAMPLING
- Sampling frequency: days 1, 4, 6, 8, 11, 14, 18, 21, 25 and 28 after incubation initiation. the degradation was stopped on day 28 by acidification of the test solutions.
- Sampling method: Removal of aliquots from gas washing bottles and subsequent CO2 analysis. On day 29 residual CO2 in the inocculum was removed by purging the inoculum for 24 hours. Aliquots were removed for CO2 determination.
- Sterility check if applicable: NA

- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, two replicates
- Abiotic sterile control: No
- Toxicity control: Yes (one replicate)
- Reference substance: Yes (one replicate)
- Other:

STATISTICAL METHODS: NA

Reference substance:

benzoic acid, sodium salt

Test performance:

No events are reported, which might have affected the quality of the study.

Parameter:

% degradation (TOC removal)

Value:

27.5

Sampling time:

28 d

Details on results:

The substance was degraded within 28 days by average 27.5 % (26 and 29%). Trigger for ready biodegradation is 60%. Biodegradation of the reference substance reached 77% within 14 days of incubation. Trigger for validity: 60%. The substance was not toxic to the activated sludge, since 55% of of the reference substance was degraded within 14 days in the presence of the test substance (trigger is 25%).

Results with reference substance:

Biodegradation of the reference substance reached 77 % within 14 days of incubation.

Interpretation of results:

inherently biodegradable

Conclusions:

The substance is not readily biodegradable: Average biodegradation (27.5%) ist below the guideline trigger value of 60% . However biodegradation was observed: The 10% generic trigger value for biodegradation initiation was reached within 11 days after test initiation.

Executive summary:

In a valid GLP study according to OECD 301B the test item was aerobically exposed in duplicate at 20 mg/L test substance (8.38 mg C/L, 1.54 ThCO2/mg test item) to standard OECD mineral medium and non-adapted, activated sludge obtained from a domestic STP. Final concentration of bacteria in test: 10^5 x 10^6 CFU/L. Exposure was for 28 days at low light and under stirring conditions, and between 20 and 24°C. Six brown glass reactors with a volume of 5 L were stocked with 3 L volume each, which was constantly aerated with CO2 free air. Each reactor had received inocculum and mineral medium and was equipped with 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution. Determination of CO2 was carried out by titration subsequent to complete adsorption of the released CO2 in an alkaline solution (0.0125 mol/L Ba(OH)2). Five test groups were incubated: Test item (8.38 mg C/L, two replicates), blank control (two replicates), reference substance (11.6 mg C/L of sodium acetate, one replicate) and a toxicity control (8.38 mg C/L test substance plus 11.6 mg C/L reference substance, one replicate). Biodegradation was determined on days 1, 4, 6, 8, 11, 14, 18, 21, 25 and 28. In addition residual CO2 in the inoculum was determined on day 28 after release of CO2 from the inocculum by HCL and measurement of trapped CO2 after purging the inoculum for 24 hours. The results show that all validity criteria of the test guideline were met. Hence the results obtained for the test item are valid. Test item results show that average 27.5 % (26% and 29%) were degraded within 28 days of which 10% were reached within 9 days. Hence the substance is not readily biodegradable (trigger of 60% not met). However biodegradation was observed, since the generic 10% trigger value for biodegradation initiation was reached within 11 days after test initiation.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-05-28 to 2014-07-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Fresh samples of activated sludge are withdrawn on June 10th, 2014 from the sewage treatment plant Ruhrverband Kläranlage, Sunthelle 6, 57392 Schmallenberg, Germany.
- Laboratory culture: Not applicable.
- Method of cultivation: Not applicable.
- Storage conditions: Aerobic until use.
- Storage length: Not specified.
- Preparation of inoculum for exposure: Since it was not necessary, the samples were not washed with mineral medium after the arrival.
- Pretreatment: No data.
- Concentration of sludge: The concentration used in the test was 29.6 mg dry mass/litre mineral medium (7.39 mg dry mass/250 mL mineral medium).
- Initial cell/biomass concentration: See above.
- Water filtered: For the test deionized water, free from inhibitory concentrations of toxic substances (e.g. Cu2+ ions) was used.
- Type and size of filter used, if any: No data

Duration of test (contact time):

28 d

Initial conc.:

127 mg/L

Based on:

test mat.

Initial conc.:

67 mg/L

Based on:

ThOD/L

Initial conc.:

384 mg/L

Based on:

test mat.

Initial conc.:

203 mg/L

Based on:

ThOD/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium according to OECD 301.
- Solubilising agent (type and concentration if used): None.
- Test temperature: 21 to 24°C.
- pH adjusted: No. pH was 7.2 to 7.6 at test initiation and between 7.2 and 7.9 throughout the test (all test groups).
- Continuous darkness: Yes.

TEST SYSTEM
- Culturing apparatus: The measurement and recording of the oxygen demand was carried out continuously using a SAPROMAT respirometer (VOITH Inc.).
- Number of culture flasks/concentration:
Test suspension A: 3 vessels containing test item (384 mg/L, corresponding to 203 mg/L ThOD) and inoculum.
Test suspension B: 3 vessels containing test item (127 mg/L, corresponding to 67 mg/L ThOD) and inoculum.
Inoculum blank: 2 vessels containing only inoculum.
Abiotic control: 2 vessels containing test item (384 mg/L) and a sterilising agent.
Procedural control: 2 vessels containing reference item (100 mg/L) and inoculum.
- Method used to create aerobic conditions: Continuous air supply (The suspension was aerated during the whole test).
- Test performed in closed vessels (due to significant volatility of test substance): Yes.
- Test performed in open system: Sapromat Respirometer.

SAMPLING
- Sampling frequency: Continuously, almost every two hours during 28 days.
- Sampling method: None, oxygen consumption was measured electrochemically inside the respirometer.

- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, two replicates.
- Abiotic sterile control: Yes, 2 replicates.
- Toxicity control: No.
- Reference substance: Yes (two replicates).
- Other:

STATISTICAL METHODS: NA

Reference substance:

benzoic acid, sodium salt

Test performance:

Due to a calculation error in the pipetting scheme, the target concentrations of 300 and 100 mg test item per liter were not achieved. The actual test item concentrations at test start were 384 and 127 mg/L. Whereas the value of 203 mg O2 per mg represented at ThOD value above the required range of 50 to 100 mg O2 per liter (OECD 301 F), the value of 67 mg/L (2nd test concentration) was within this range. The deviation is without influence on the integrity of the study, since the test concentration 2 (67 mg O2/L) was used to characterize the biodegradation behavior of the substance.

Parameter:

% degradation (O2 consumption)

Value:

86

Sampling time:

28 d

Remarks on result:

other: 67 mg/L ThOD test group, one replicate

Parameter:

% degradation (O2 consumption)

Value:

62

Remarks on result:

other: 67 mg/L ThOD test group, two replicates

Parameter:

% degradation (O2 consumption)

Value:

52

St. dev.:

4.2

Sampling time:

28 d

Remarks on result:

other: 203 mg/L ThOD test group, three replicates

Details on results:

The biodegradation of 127 mg test item per litre (67 mg/L ThOD) in the static test was found to be at average 62,8 % (similar replicates, 61.3% and 64.3%): The third replicate showed a biodegradation of 86.8%. Trigger for ready biodegradation is 60%. A plateau was not yet reached on day 28. As expected due to the high ThOD of the test material, concentration (2) (384 mg/L) showed a lower biodegradation, i.e. maximum average 52.1±4.2% (mean of 3 replicates) on day 28. A plateau was not yet reached after 28 days. The abiotic sterile control showed maximum 9.9% degradation, indicating slight abiotic decomposition.

Results with reference substance:

Biodegradation of the reference substance reached 93,7 % within 14 days of incubation.

The difference of extremes of replicate values of the removal of the test item at the end of the 10-day-window is less than 20 % in both test assays. At the end of the test it is less than 20 % for 384 mg/L. For 127 mg/L, it is 26 %. However, the great variance is due to

one replicate showing clearly higher degradation than the other ones. With at mean 63 % degradation and a difference of extremes of replicate values of 3 % at test end the two replicates still surpass the threshold value. Therefore, the test can be considered as valid.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

In conclusion, the substance is readily biodegradable: Average biodegradation of 62.8% based on O2 consumption is above the guideline trigger value of 60%. The 10% generic trigger value for biodegradation initiation was typically reached within latest 6 days after test initiation. The 10-day window should not be applied in this case, as the test material is a mixture of structurally similar chemicals, and not a monoconstituent substance.

Executive summary:

At the Fraunhofer Institute for Molecular Biology and Applied Ecology the biodegradation of IS-2014-535 was investigated over a 28-day period in a Manometric Respirometry Test according to EC method C.4-D (440/2008/EEC) and OECD guideline 301 F (1992). Standard OECD 301 test medium was inoculated with 29.6 mg dry mass/L non-adapted microorganisms from a digester of a sewage treatment plant mainly fed with municipal wastewater (STP: Ruhrverband Kläranlage, Sunthelle 6, 57392 Schmallenberg, Germany). The test solutions were stirred in closed flasks at 22 °C ± 1 °C for 28 days. The rate of degradation was monitored by measuring the quantity of oxygen required to maintain a constant gas volume in the respirometer flasks over a 28-d period. For that purpose a SAPROMAT respirometer (VOITH Inc.) was used. The amount of oxygen taken up by the microbial population during biodegradation of the test item was expressed as a percentage of the ThOD (Theoretical Oxygen Demand). The following test concentrations were used: (1) 384 mg/L (ThOD = 203 mg/L, in triplicate) and (2) 127 mg/L (ThOD = 67 mg/L, in triplicate), respectively, corrected for uptake by the blank inoculum run in duplicate, The second concentration was chosen for the following reason: Some substances with high ThOD values have gently inclined degradation curves, even when they are completely degradable. However, for these substances it is hard to reach the threshold value within the 10-day window. Therefore, an additional assay with 127 mg test item (67 mg ThOD) was added. This concentration meets the required concentration of 50 - 100 mg ThOD/L acc. to OECD 301 F and is considered more realistic than concentration (1). In order to check the procedure, sodium benzoate (in duplicate) was used as a degradable reference item at a concentration of 100 mg/L. Furthermore, an abiotic control in duplicate was used. The pH values ranged from 7.2 to 7.9 throughout the study. The oxygen uptake of the inoculum blank was 60 mg/L in 28 days and the pH value was inside the range of 6.0 - 8.5. The degradation of the reference substance sodium benzoate had reached average 91.6 % within the first 14 days, i.e. the pass level of 60%. With 3 and 8 % the difference of extremes of replicate values of the removal of the test item at the end of the test was less than 20 % for test concentrations 127 mg/L (2 of 3 replicates) and 384 mg/L, respectively. Hence, the test is valid according to the validty criteria of the guideline. The abiotic sterile control showed maximum 9.9% degradation, indicating slight abiotic decomposition. The biodegradation of 127 mg test item per litre (67 mg/L ThOD) was found to be at average 62,8% (mean of 61.3% and 64.3%): the third replicate showed a biodegradation of 86.8%. As expected, due to the high ThOD of the test material (see above), concentration (2) (384 mg/L) showed a lower biodegradation, i.e. maximum average 52.1±4.2% (mean of 3 replicates) on day 28. A plateau was not yet reached after 28 days for both test concentrations. Considering test material concentration 127 mg/L, corresponding to 67 mg/L ThOD, which is more realistic than test concentration 384 mg/L, the test substance can be considered readily biodegradable, since the test substance degradation was average 62.8% (worst case), exceeding the trigger value of 60%. The 10-day window should not be applied in this case, as the test material is a mixture of structurally similar chemicals, and not a monoconstituent substance.

Description of key information

The results of the key study, which is appropriate for the assessment of ready biodegradability, show s that the substance is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

The result of the key study is supported by one other ready biodegradation study which shows that 63% of the test material was biodegraded within 28 days, based on DOC and maximum 66.4% based on CO2 evolution.

Four further ready biodegradation experiments confirm that the substance is well biodegradable under OECD 301 testing conditions: Within 28 days of incubation, total maximum 48% to 54% of the test substance were biologically degraded, depending on the test design and the sludge which was used to run the tests. One of the studies shows a biodegradation of 28%.

All findings are summarized below:

Table – Summary of Ready Biodegradation results

OECD301F Simon (2014)	127 mg/L (test item) 67 mg/L (ThOD) BOD (Sapromat) Biodegradation based on ThOD (based on elementary analysis	Schmallenberg (Germany)	86,8% (one replicate) 62,8% Average if 64,3% and 61,3%
	348 mg/L (test item) 203 mg/L (ThOD) BOD (Sapromat) Biodegradation based on ThOD (based on elementary analysis		52,1% ± 4.2 Average of 47,3%, 53,7% and 55,2%
OECD 301B Häner (2013)	128 mg/L (test item) 20 mg/L (TOC) CO2evolution (IC measurement). Biodegradation evaluation based on TOC	Weidhölzli (Zürich, Switzerland)	60% Average of 66.4% and 53.1%
	DOC (Day 28)		63.0%
OECD301F Häner (2013)	175 mg/L (test item) 100 mg/L (COD) BOD (OxiTop). Biodegradation evaluation based on COD (ThOD unknown)	Weidhölzli (Zürich, Switzerland)	53,8% Average of 51.7% and 55.8%
	DOC (day 28)		49.0%
OECD301F Fiebig (2014)	35 mg/L (test item) 50 mg O2/L (oxygen demand) BOD (OxiTop). Biodegradation based on ThODNH3	Hildesheim (Germany)	47,5% Average of 39%and 56%
OECD 301B Fiebig (2014)	20 mg/L (test item) 8,38 mg C/L CO2evolution (titration). Biodegradation evaluation based on ThCO2	Hildesheim (Germany)	27,5% Average of 26% and 29%

Pass level for surfactants: 60%. The 10 -day window should not be applied in this case, as the test material is a mixture of structurally similar chemicals, and not a monoconstituent substance.