Eldew APS-307

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 9 April 2009 and 11 May 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

In view of difficulties associated with the evaluation of the biodegradeablity of organic compounds with low water solubility, a modification to the standard method of preparation of the test concentration was performed. An approach endorsed by the Internal Standards Organisation (ISO 1995) is to adsorb the test material onto an inert support prior to dispersion in the test vessels. Using this method the test material is evenly distributed throughout the test medium and the surface area of the test material exposed to the test organisms is increased thereby increasing the potential for biodegradation.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 19th August 2008 Date of Signature: 04/03/2009

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Eldew APS-307
- Substance type: Pale yellow waxy solid block
- Physical state: Solid
- Lot/batch No.: 809114
- Date received : 22 December 2008
- Storage condition of test material: Room temperature in the dark.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
A mixed population of activated sewage sludge micro-organisms was obtained on 8 April 2009 from the aeration stage of the Severn Trent Water PIC sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

- Preparation of inoculum for exposure:
The activated sewage sludge sample was washed three times by settlement and resuspension in culture 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 2I °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 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 deionised 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.

- Concentration of sludge: 2.8g/L prior to use
- Initial cell/biomass concentration:30 mg suspended solids (s/s)/L
- Water filtered: yes
- Type and size of filter used, if any:
pre-weighed GF/A filter paper using a Buchner funnel

Duration of test (contact time):

29 d

Initial test substance concentration

Initial conc.:

10 other: mg Carbon/L

Based on:

DOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Composition of Medium:

The culture medium used in this study was that recommended in the OECD Guidelines and is detailed as follows:

Solution a KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4.2H2O 33.40 g/L
NH4Cl 0.50 g/L

pH = 7.4

Solution b CaCl2 27.50 g/L
Solution c MgSO4.7H2O 22.50 g/L
Solution d FeCl3.6H2O 0.25 g/L

To 1 litre (final volume) of purified water was added the following volumes of solutions a – d.

10 mL of Solution a
1 mL of Solution b
1 mL of Solution c
1 mL of Solution d

- Test temperature: 21 °C
pH Measurement:
The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

- pH adjusted: No (7.8 - 7.9)

- Suspended solids concentration: 30 mg suspended solids (ss)/L
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus:
The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:

a) A control, in duplicate, consisting of inoculated culture medium plus a filter paper*.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium plus a filter paper* to give a final concentration of 10 mg carbon/L.
c) The test material weighed onto a filter paper*, in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/L.
d) The test material weighed onto a filter paper* plus the standard material in inoculated culture medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).

- Number of culture flasks/concentration: 7
- Method used to create aerobic conditions:
Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 ml of culture medium and 32.1 ml of inoculum and aerated overnight. On Day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium. The culture vessels were sealed and C02-free air bubbled through the solution at a rate of approximately 40 mL/minute 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 granules.

- Measuring equipment:
The samples were analysed for C02 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-VcsH TOC analyser. The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser.

-Test performed in closed vessels due to significant volatility of test substance: yes

- Details of trap for CO2 and volatile organics if used:
The C02 produced by degradation was collected in two 500 ml Dreschel bottles containing 350 ml of 0.05 M NaOH. The C02 absorbing solutions were prepared using purified de-gassed water.


SAMPLING & ANALYSIS

CO2 Analysis:
Samples (2 mL) were taken from the control, standard and test material first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 and from the toxicity control first C02 absorber vessel on Days 0, 2, 6, 8, 10 and 14. The second absorber vessel was sampled on Days 0 and 29 for the control, tandard and test material and on Day 0 for the toxicity control.

The samples taken on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 were analysed for C02 immediately.

On Day 28, 1 ml of concentrated hydrochloric acid 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 analysed for C02 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-VcsH TOC analyser. Samples (300 or 50 µL) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. lnorganic carbon analysis occurs by means of the conversion of an aqueous sample to C02 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by standard solutions of sodium carbonate Ca2C03). Each analysis was carried out in triplicate.


DOC Analysis
Dissolved Organic Carbon (DOC) Analysis:
Samples (20 mL) were removed from the test material and toxicity control vessels on Day 0 prior to the addition of the test material in order to calculate the lnorganic Carbon content in the test media. The samples were filtered through Gelman 0.45 µm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis.

DOC analysis of the test material dispersions after dosing was not possible due to the insoluble nature of the test material in water.

On Days 0 and 28 samples (20 mL) were removed from the control and standard material vessels and filtered through Gelman 0.45 µm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis.

The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser. Samples (27 or 13 µL) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680°C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2C03) in deionised water. Each analysis was carried out in
triplicate.


CONTROL AND BLANK SYSTEM

Evaluation of DATA:
see section on any other information on materials and methods incl. tables.

Reference substance

Reference substance:

other: sodium benzoate

Results and discussion

Preliminary study:

Not conducted.

Test performance:

The total C02 evolution in the control vessels on Day 28 was 29.08 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The IC content of the test material suspension in the mineral medium at the start of the test (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 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.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

64

Sampling time:

29 d

Details on results:

The test material attained 64% degradation after 28 days.

Despite attaining in excess of 60% degradation the test material failed to satisfy the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. Therefore the test material cannot be considered to be readily biodegradable under the strict terms and conditions of OECD guideline No 301B. However, the test material has exhibited the potential for rapid degradation.

Analysis of the test media taken from the standard material culture vessles on Days 0 and 28 for dissolved Organic Carbon (DOC), gave percentage degradation values of 99% and 97% respectively for replicate R1 and R2. The degradation rates calculated from the results of the DOC analyses were similar to those calculated from inorganic carbon analysis.

BOD5 / COD results

Results with reference substance:

The toxicity control attained 74% degradation after 14 days and thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test.

Sodium benzoate attained 88% degradation after 14 days and 89% degradation after 28 days thereby confirming the suitability of the innoculum and test conditions.

Any other information on results incl. tables

Following the recommendations of the International Standards Organisation (IS0 1995) the test material was dispensed onto filter paper prior to dispersion in the test medium in order to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.

lnorganic carbon values for the test material, standard material, toxicity control and control vessels at each analysis occasion are given in Table 1 Percentage biodegradation values of the test and standard materials and the toxicity control are given in Table 2 and the biodegradation curves are presented in Figure 1. Total and lnorganic Carbon values in the culture vessels on Day 0 are given in Table 3, and the results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given in Table 4. The pH values of the test preparations on Day 28 are given in Table 5.

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 material.

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels. lnorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carryover of CO2 into the second absorber vessels occurred.

Observations on Day 0 (see Table 6), showed the contents of the control test vessels to be light brown dispersions containing a piece of filter paper and the contents of the standard material test vessels to contain light brown dispersions containing a piece of filter paper with no undissolved standard material visible. On Days 5, 11, 18 and 25, the filter paper in both control and standard test vessels had broken into pieces.

On Day 0, the contents of the test material test vessels were light brown dispersions containing a piece of filter paper with test material visible adhered to the filter paper. On Days 5, 11, 18 and 25, the filter paper in the test material vessels had broken into pieces in a cloudy light brown dispersion and no undissolved test material was visible.

On Day 0, the contents of the toxicity control vessel was a light brown dispersion containing a piece of filter paper with test material visible adhered to the filter paper. No undissolved standard material was visible. On Days 5, and 11 the filter paper had broken into pieces in a cloudy light brown dispersion and no undissolved test or standard material was visible.

Please see attachment 1 for tables 1 -6, figure 1 and appendix 1.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

The test material attained 64% degradation after 28 days.

Despite attaining in excess of 60% degradation the test material failed to satisfy the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. Therefore the test material cannot be considered to be readily biodegradable under the strict terms and conditions of OECD guideline No 301B. However the test material has exhibited the potential for rapid degradation.

Executive summary:

Introduction: A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The method followed that described in the OECD guidelines for testing 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 OPPTS 835.3110 (Paragraph (m)).

Methods: The test material, at a concentration of 10mg Carbon/L, was exposed to activated sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 1995) the test material was adsorbed onto filter paper prior to dispersion in the test medium in order to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.

The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with innoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

Results: The test material attained 64% degradation after 28 days. Despite attaining in excess of 60% degradation the test material failed to satisfy the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. Therefore the test material cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B. However, the test material has exhibited the potential for rapid degradation.

Conclusion: The test material attained 64% degradation after 28 days.

Despite attaining in excess of 60% degradation the test material failed to satisfy the 10 -Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. Therefore the test material cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B. However, the test material has exhibited the potential for rapid degradation.