[No public or meaningful name is available]

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 December 2016 to 08 February 2017

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

Qualifier:

according to guideline

Guideline:

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

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:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

INOCULUM
- A mixed population of activated sewage sludge micro-organisms was obtained on 09 January 2017 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
- 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 °C 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 pre-weighed 70 mm diameter Whatman GF/A filter paper (rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven) using a Buchner funnel. Filtration was 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 dried in an oven at approximately 105 °C for at least 1 hour and allowed to cool before weighing. The process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.6 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

other: carbon

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

MEDIUM
- The mineral medium used in this study was that recommended in the OECD guidelines (see Annex 3, attached).

PRELIMINARY SOLUBILITY WORK
- Information provided by the Sponsor indicated that the test item was slightly soluble in water.
- Preliminary solubility/dispersibility work was performed in order to determine the most suitable
method of preparation (see Annex 4, attached).

TEST ITEM PREPARATION
- Following preliminary solubility work and the recommendations of the International Standards Organisation (ISO, 1995) and in the published literature (Handley et al, 2002) the test item was dissolved in an auxiliary solvent prior to adsorption onto Whatman GF/A (70 mm diameter) filter paper. High shear mixing was also applied to break up the filter paper containing the test item. Using this method, the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.
- A nominal amount of test item (1000 mg) was dissolved in 10 mL of chloroform to give a 1000 mg/10 mL solvent stock solution. An aliquot (537 µL) of this solvent stock solution was dispensed onto a filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to inoculated mineral medium. The volume was then adjusted to 3 liters to give a final concentration of 17.9 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution.
- A Whatman GF/A (70 mm diameter) filter paper was added to each control vessel in order to maintain consistency between the test and procedure control vessels. Chloroform (537 µL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to each vessel.
- A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.
- As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This was an exception with regard to GLP and was been reflected in the GLP compliance statement.

REFERENCE ITEM PREPARATION
- A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium with the aid of ultrasonication for approximately 5 minutes. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium and the volume 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.
- A Whatman GF/A (70 mm diameter) filter paper was added to each vessel in order to maintain consistency between the test and procedure control vessels. Chloroform (537 μL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to each vessel.

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 aliquot (537 μL) of the test item solvent stock solution was dispensed onto a Whatman GF/A (70 mm diameter) filter paper and the solvent allowed to evaporate for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to the test vessel containing inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume was adjusted to 3 L to give a final concentration of 17.9 mg test item/L plus 17.1 mg sodium benzoate/L equivalent to a total of 20 mg carbon/L.

MINERAL OIL CONTROL PREPARATION
- At the Sponsors request additional vessels were prepared containing mineral oil at the same concentration as in the test item preparations; 7.6 mg/L.
- A nominal amount of mineral oil (1000 mg) was dissolved in 10 mL of chloroform to give a 1000 mg/10 mL solvent stock solution.
- An aliquot (228 µL) of this solvent stock solution was dispensed onto a filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes.
- The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to inoculated mineral medium.
- The volume was then adjusted to 3 L to give a final concentration of 7.6 mg/L, equivalent to 6.6 mg carbon/L.
- The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution.

PREPARATION OF THE TEST SYSTEM
- Test preparations were prepared and inoculated in 5 L test culture vessels each containing 3 L of solution.
(a) An inoculated control, in duplicate, consisting of inoculated mineral medium plus a Whatman GF/A (70 mm diameter) filter paper.
(b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium plus a Whatman GF/A (70 mm diameter) filter paper to give a final concentration of 10 mg carbon/L.
(c) The test item on a Whatman GF/A (70 mm diameter) filter paper, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
(d) The test item on a Whatman GF/A (70 mm diameter) filter paper 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).
(e) The mineral oil on a Whatman GF/A (70 mm diameter) filter paper, in duplicate, in inoculated mineral medium to give a final concentration of 7.6 mg carbon/L
- A filter paper with chloroform evaporated to dryness was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.
- 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 21 and 24 °C, for 28 days.
- Approximately 24 hours prior to addition of the test and reference items 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 items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter. If necessary, the pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 L by the addition of mineral medium which 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.
- 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.

OBSERVATIONS
- The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27.

MEASUREMENT of pH
- The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter.

CALCULATION OF CARBON CONTENT
- The test item contains 55.95 % carbon (data supplied by the Sponsor) and so for a concentration of 10 mg C/L the total organic carbon present was 30 mg C.
- The mineral oil contains 86.20% carbon (data supplied by the Sponsor) and so for a concentration of 6.6 mg C/L the total organic carbon present was 19.5 mg C.
- The theoretical amount of carbon present in the reference item, sodium benzoate (C6H5COONa) was calculated using the equation Theoretical amount of carbon = [No of C atoms * mol wt of C / mol wt of sodium benzoate] * 100 = [7 * 12.011 / 144.11] * 100 = 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 Carbo Dioxide (ThCO2) produced was calculated by substituting the inorganic carbon values (see Table 1, attached) into the equation % ThCO2 = (mg IC in test flask - mg IC in control flask) / (mg TOC added as test chemical) x 100
- The mean values of replicates R1 and R2 were obtained for the inoculum, control, test and reference items before substitution into the equation.
- The % ThCO2 result equals % biodegradation where 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 was calculated using 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)
- The mean inorganic carbon values for replicates R1 and R2 on Day 28 were obtained before substitution into the equation.

VALIDATION CRITERIA
- The results of the degradation test are considered valid if in the same test the reference item yields ≥ 60 % degradation (in a 10-day window) by Day 14.
- The test item may be considered to be readily biodegradable if ≥ 60 % degradation is attained within 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10 %.
- The toxicity control (test item and sodium benzoate) should attain ≥ 25 % degradation by Day 14 for the test item to be considered non-inhibitory.
- The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the time the plateau is reached, at the end of the test or at the end of the 10-day window, is < 20 %.
- The total CO2 evolution in the inoculum control vessels on Day 28 of the test should not normally exceed 40 mg/L medium (= 120 mg/3 L corresponding to 33 mg C per flask). However, values up to 70 mg/L are acceptable. Data from studies where values in excess of 70 mg/L are obtained should be critically examined.
- The IC content of the test item suspension in the mineral medium at the beginning of the test should be < 5 % of the TC.

MAJOR COMPUTERISED SYSTEMS
- TOC measurement: Shimadzu TOC
- Building management: Delta control system

Reference substance:

benzoic acid, sodium salt

Remarks:

Sigma Aldrich (Batch SLBM8408V; Purity > 99.5 %; Expiry date 11 May 2017; Stored at room temperature over silica gel)

Key result

Parameter:

% degradation (CO2 evolution)

Value:

25

Sampling time:

28 d

Details on results:

DEFINITIVE TEST
- Inorganic carbon values for the test item, the mineral oil control, procedure control, toxicity control and inoculum control vessels at each analysis occasion are given in Table 1 (attached).
- Percentage biodegradation values of the test and reference items, mineral oil control and the toxicity control are given in Table 2 (attached).
- The biodegradation curves are presented in Figure 1 (attached).
- Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3 (attached).
- The pH values of the test preparations on Days 0 and 28 are given in Table 4 (attached).
- Observations made on the contents of the test vessels are given in Table 5 (attached).

VALIDATION CRITERIA
- The total CO2 evolution in the inoculum control vessels on Day 28 was 33.04 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 (see Table 3, attached) 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.

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 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 test item attained 25 % biodegradation after 28 days. The test item therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
- The results obtained from the mineral oil vessels showed 24 % biodegradation over the test period.
- The toxicity control attained 48 % biodegradation after 14 days and 54 % 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.

Results with reference substance:

- Sodium benzoate attained 67 % biodegradation after 14 days and 74 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 25 % biodegradation after 28 days and results obtained from the mineral oil vessels showed 24 % biodegradation over the test period. The test item therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B. 

Executive summary:

GUIDELINE

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. 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)).

 

METHODS

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 21 and 24 °C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 1995), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in test media. Using this method, the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.

The test item was a complex mixture containing 42.2% mineral oil. At the Sponsors request additional vessels were prepared containing mineral oil at the same concentration as in the test item preparations; 7.6 mg/L.

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.

RESULTS

The test item attained 25 % biodegradation after 28 days and results obtained from the mineral oil vessels showed 24 % biodegradation over the test period. The test item therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B. 

Description of key information

Two biodegradation studies were conducted. The first test item containing mineral oil attained 25 % biodegradation after 28 days and the second test item without oil attained 33 % biodegradation after 35 days (OECD 301B and EU Method C.4-C). The substance is therefore considered not readily biodegradable (OECD 301 B, EU Method C4 -C and OCSPP 835.3110).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Key study 1

Ready biodegradability of the test item was investigated in an aerobic aqueous medium. 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)).

 

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 21 and 24 °C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 1995), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in test media. Using this method, the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.

The test item was a complex mixture containing 42.2% mineral oil. At the Sponsors request additional vessels were prepared containing mineral oil at the same concentration as in the test item preparations; 7.6 mg/L.

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 25 % biodegradation after 28 days and results obtained from the mineral oil vessels showed 24 % biodegradation over the test period. The test item therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Key study 2

Ready biodegradability of the test item was assessed in an aerobic aqueous medium. 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)).

 

The test item, at a concentration of 20 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 20 and 25 °C for 35 days. The test was extended to 35 days at the request of the Sponsor.

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 33 % biodegradation after 35 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.