Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

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:
06 September 2016 to 05 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
use of Student's t-test with no impact on integrity or validity of study (see below)
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic (adaptation not specified)
Details on inoculum:
TEST SYSTEM
- A mixed population of activated sewage sludge micro-organisms was obtained on 05 September 2016 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 Whatman GF/A (70 mm diameter) filter paper (rinsed three times with 20 mL deionized 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 deionized 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 3.2 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 2, attached).

PRELIMINARY SOLUBILITY WORK
- Information provided by the Sponsor indicated that test item was insoluble in water.
- Preliminary solubility/dispersibility work was therefore performed in order to determine the most suitable method of preparation (see Annex 3, 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 adsorbed onto silica gel prior to dispersion in mineral medium to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to the test organisms.
- An amount of test item (54.0 mg) was adsorbed onto the surface of 500 mg of granular silica gel (230-400 mesh Sigma Lot No BCBM6195V) prior to dispersal in approximately 400 mL of mineral medium with the aid of high shear mixing (7500 rpm, 5 minutes). The test item/silica gel/mineral mineral medium dispersion was then dispersed in inoculated mineral medium and the volume adjusted to 3 L to give a final concentration of 18.0 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 of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium with the aid of ultrasonication for approximately 10 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.
- Silica gel (500 mg) was also added to the procedure control vessels in order to maintain consistency between the test and procedure control vessels.

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 (54.0 mg) was adsorbed onto the surface of 500 mg of granular silica gel (230-400 mesh Sigma Lot No. BCBM6195V) prior to dispersal in approximately 400 mL of mineral medium with the aid of high shear mixing (7500 rpm, 5 minutes).
- The test item/silica gel/mineral medium dispersion was then dispersed in inoculated mineral medium and an aliquot (51.4 mL) of the sodium benzoate stock solution added. The volume was adjusted to 3 L to give a final concentration of 18.0 mg test item/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/L.

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 500 mg silica gel.

(b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 10 mg carbon/L.
(c) The test item, in duplicate, in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 10 mg carbon/L.
(d) The test item plus the reference item in inoculated mineral medium plus 500 mg silica gel to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
- Silica gel was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.
- 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 21 and 24 °C, in darkness.
- Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 28.1 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 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.

IC ANALYSIS
- 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.
- All samples were analysed for IC immediately.

IC/TC RATIO
- Samples (30 mL) were removed from the test item vessels on Day 0 prior to the addition of the test item 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.
- On Day 28, concentrated hydrochloric acid (1 mL) 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.
- The samples were analyzed for IC using either a Shimadzu TOC-VCSH TOC analyser or a Shimadzu TOC-LCSH TOC analyser. Samples (135 or 50 μL) were injected into the IC channel of the TOC analyzer. 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.

IC/TC RATIO
- Samples (30 mL) were removed from the test item vessels on Day 0 prior to the addition of the test item 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 item dispersions after dosing was not possible due to the insoluble nature of the test item in water.

CALCULATION OF CARBON CONTENT
- The test item contains 55.47 % 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 theoretical amount of carbon present in the reference item, sodium benzoate (C6H5COONa) = (No of C atoms * mol wt of C / mol wt of sodium benzoate) * 100.
- The calculation used was therefore (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.

STATISTICAL ANALYSIS
- Statistical analysis of the Day 29 IC values for the inoculum control and test item vessels was carried out using a Student’s t-test to determine any statistically significant differences between the test and control groups.
- All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

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 less than 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
Key result
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Details on results:
DEFINITIVE TEST
- Inorganic carbon values for the test item, 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 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 C02 evolution in the inoculum control vessels on Day 28 was 32.77 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 C02 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 C02 present in the test vessels. Therefore, any additional C02 detected in the Day 29 samples originated from dissolved C02 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 with the exception of procedure control replicates 1 and 2 and the toxicity control.
- Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of C02 into the second absorber vessels occurred.
- The test item attained 0 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301 B.
- Statistical analysis of the Day 29 IC values for the control and test item vessels showed there were no statistically significant differences (P≥ 0.05) between the control and the test item. The test item was therefore considered not to have a toxic effect on the sewage sludge micro-organisms used in the study and this was confirmed by the toxicity control results.
- The toxicity control attained 35 % biodegradation after 14 days and 52 % 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 82 % biodegradation after 14 days and 96 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item attained 0% 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:

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; C02 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 to 24 °C for 28 days. F Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handleyet al, 2002), the test item was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to the test organisms. 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 0% 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 0% 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:
under test conditions no biodegradation observed

Additional information

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; C02 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 to 24 °C for 28 days. F Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handleyet al, 2002), the test item was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to the test organisms. 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 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.