Fatty acids, tall-oil, compds. with ethanolamine

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:

28 June 2016 to 27 July 2016

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:

yes

Remarks:

temperature variation not considered to have affected the integrity of the study (see below)

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

temperature variation not considered to have affected the integrity of the study (see below)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

yes

Remarks:

temperature variation not considered to have affected the integrity of the study (see below)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

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:

INOCULUM
- A mixed population of activated sewage sludge microorganisms was obtained on 27 June 2016 from the aeration stage of the Severn Trent Water plc sewage treatment plant.
- The treatment plant is located in Loughborough, Leicestershire, UK, and 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 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 using a Buchner funnel. The filter paper had been rinsed three times with 20 mL deionised reverse osmosis water prior to drying in an oven.
- Filtration was continued for a further 3 minutes after rinsing of 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 one hour and allowed to cool before weighing.
- The process was repeated until a constant weight was attained.
- The suspended solids concentration was equal to 3.0 g/L prior to use.

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 the test item was dispersible in water.
- 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 published literature, the test item was dissolved in an auxiliary solvent prior to adsorption onto Whatmann 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 was evenly distributed throughout the test medium and the surface area exposed to the test organisms was increased thereby increasing the potential for biodegradation.
- A nominal amount of test item (1000 mg) was dissolved in 10 mL of acetone to give a 1000 mg/10 mL solvent stock solution.
- An aliquot (432 µL) of the solvent stock solution was dispensed onto a filter paper and the solvent was 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 for 5 minutes) prior to addition of inoculated mineral medium.
- The volume was then adjusted to 3 L to give a final concentration of 14.4 mg/L (equivalent to 10 mg carbon/L).
- The volumetric flask containing 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.
- Acetone (432 µL) was disepensed 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 for 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 guideline.
- 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 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 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.
- 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.
- Acetone (432 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 for 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 toxici effect of the test item on the sewage sludge microorganisms used in the test.
- An aliquot (432 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 for 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 14.4 mg test item/L plus 17.1 mg sodium benzoate/L (equivalent to a total of 20 mg carbon/L).

PREPARATION OF TEST SYSTEM
- Test preparations a, b, c and d 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 with acetone evaporated off.
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 with acetone evaporated off, to give a final concentration of 10 mg carbon/L.
c) The test item on a Whatman GF/A (70 mm diameter) filter paper with acetone evaporated off, 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 with acetone evaporated off, 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).
- A filter paper with acetone evaporated to dryness was added to the inoculum control and procedure control vessels in order to maintain consistency between those 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 between 20 and 25 °C for 28 days, 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 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 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 was 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 test preparations was determined using a Hach HQ40d Flexi handheld meter on Day 0 and on Day 28 prior to acidification with hydrochloric acid.

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. The remainder of all samples with the exception of the Day 0 samples were frozen for further analysis if required.
- 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 were taken from both absorber vessels on Day 29.
- The samples were analysed for IC using a Shimadzu TOC-LCSH TOC analyser.
- Samples (135 µL) were injected into the IC channel of the TOC analyser.
- IC analysis occurs by means of 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 addition of the test item in order to calculate the IC content in the test media.
- Samples were filtered through 0.45 µm Gelman AcroCap filters prior to DOC analysis (first approximate 5 mL discarded in order to precondition the filter).
- Samples (30 mL) were also removed from inoculum control vessels on Day 0 and filtered through 0.45 µm Gelam AcroCap filters prior to DOC analysis (first approximate 5 mL discarded in order to precondition the filter).
- IC/TC analysis of the test item dispersions after dosing was not possible due to the insoluble nature of the test material in water.
- 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 triplicate.

CALCULATION OF CARBON CONTENT
- According to data supplied by the sponsor, the test item contained 69.32 % carbon and, for a concentration of 10 mg carbon/L, the total organic carbon present was 30 mg.
- The theoretical amount of carbon present in the reference item, sodium benzoate (C6H5COONa) was calculated using the equation (No of C atoms x mol wt of C) / (mol wt of sodium benzoate) x 100. The result obtained was 58.34 % given by (7 x 12.011) / 144.11 x 100.
- Thus for a 10 mg carbon/L test concentration, the total organic carbon present for sodium benzoate was 30 mg.

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 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: TOC measurement
- Building management: Delta control system

Parameter:

% degradation (CO2 evolution)

Value:

37

Sampling time:

28 d

Results with reference substance:

- Sodium benzoate attained 70 % biodegradation after 14 days and 68 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
- The slight decrease in biodegradation between Days 14 and 28 was considered to be due to sampling/analytical variation.

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 are given in Table 2 (attached) together with values for the toxicity control.

- 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 30.38 mg/L and therefore satisfied the validation criteria 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 criteria 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 criteria given in the OECD test guidelines.

 

BIODEGRADATION

- Acidification of the test vessels on Day 28 followed by the final analysis on Day 29 was conducted according to the methods specified in the test guidelines.

- This acidification effectively kills the microorganisms 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 a decrease in all replicate vessels except for inoculum control R1 and test item R2. This decrease was considered to be due to sampling/analytical variation.

- 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 item attained 37 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301B.

- The toxicity control attained 55 % biodegradation after 14 days and 58 % biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment microorganisms used in the test.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 37 % biodegradation after 28 days and therefore cannot be considered 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 microorganisms with mineral medium in sealed culture vessels in the dark at temperatures between 21 and 25 °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 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.

 

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

 

RESULTS

The test item attained 37 % biodegradation after 28 days and therefore cannot be considered readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Description of key information

The test item attained 37 % biodegradation after 28 days and cannot be considered readily biodegradable (OECD 301B, EU Method C4 -C and US EPA OCSPP 835.3110).

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; 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 microorganisms with mineral medium in sealed culture vessels in the dark at temperatures between 21 and 25 °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 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.

 

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

 

RESULTS

The test item attained 37 % biodegradation after 28 days and therefore cannot be considered readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.