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:
16 January 2018 to 12 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
yes
Remarks:
concentration of suspended solids lower than planned with no impact on results or integrity of the study (see below)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
TEST SYSTEM
- Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Treatment: The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium. After treatment, the concentration of suspended solids (SS) was determined to be 3 g/L in the concentrated sludge as used for the test. The sludge was used as inoculum at a concentration of 1 mL per litre of mineral medium, leading to a final concentration SS of 3 mg/L.
Duration of test (contact time):
28 d
Initial conc.:
30.5 mg/L
Based on:
other: ThODNO3 of 80 mg O2/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
DEFINITIONS
- Biochemical oxygen demand (BOD) is the amount of oxygen consumed by microorganisms when metabolizing a test item; also expressed as mg oxygen uptake per mg of test item.
- Readily biodegradable are those test items giving a result of at least 60% biodegradation within 28 days. This pass level must be reached within the 10 days immediately following the attainment of 10% biodegradation (10-day window).
- Suspended solids (SS) are the solids removed from activated sludge by filtration and dried to a constant mass, generally expressed in grams per litre.
Theoretical oxygen demand (ThOD) is the total amount of oxygen required to oxidise a chemical completely; it is calculated from the molecular formula and is also expressed as mg oxygen required per mg test compound.

REFERENCE ITEM CONCENTRATION AND PREPARATION OF TEST SOLUTIONS
- The reference item was added directly to the test medium of the procedure and the toxicity control. Using a positive displacement pipette, aliquots of 11.8 µL and 8.0 µL of 1-octanol were added to the test medium of the procedure control (final volume 360 mL) and toxicity control (final volume 244 mL), respectively. The final test concentration of 1-octanol was 27 mg/L, corresponding to a ThOD of 80 mg O2/L.

TEST ITEM CHARACTERISATION
- The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item.
- A Certificate of Analysis or equivalent document was provided to the Test Facility.

RESERVE SAMPLES
- For each batch (lot) of test item, a reserve sample (about 0.5 gram) was collected and maintained under the appropriate storage conditions by the Test Facility and destroyed after the expiration date.

TEST CONCENTRATION AND PREPARATION OF TEST SOLUTIONS
- The test item was a clear colourless liquid with a purity of 84.1%. The test item was tested in duplicate at a target concentration of 30.5 mg/L, corresponding to a ThODNO3 of 80 mg O2/L.
- The ThODNO3 was based on the results of elemental composition analysis and was confirmed by TOC analysis.
- Volumes of test item, reference item, and mineral medium added to the test vessels can be found in Table 1 (below).
- Test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms.
- Test duration was 28 days.
- Any residual volumes were discarded.

TEST PROCEDURE AND CONDITIONS
- Test duration: 28 days. During the test period, the test media were aerated and stirred continuously.
- Test vessels: 500 mL brown coloured glass bottles. The end volume of medium was 360 mL or 244 mL, in order to obtain the headspace-to-volume ratio required for the associated ThOD.
- Milli-RO water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.
- Stock solutions of mineral components: See table below.
- Mineral medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water.
- CO2 absorber: Sodium hydroxide pellets (Merck, Darmstadt, Germany).
- Illumination: The test media were excluded from light.

PREPARATION OF BOTTLES
- Inoculation: At the start of the test (day 0) mineral medium was inoculated with activated sludge.
- Type and number of bottles: Test suspension containing test item and inoculum (2 bottles); Inoculum blank containing only inoculum (2 bottles); Procedure control containing reference item and inoculum (1 bottle); Toxicity control containing test item, reference item and inoculum (1 bottle).
- Preparation: At the start of the test (day 0), test and/or reference item were added to the respective bottles. The volumes of suspensions were made up to 360 mL or 244 mL using inoculated mineral medium. Rubber gaskets containing two pellets of sodium hydroxide were inserted into each bottle. Subsequently, each bottle was sealed by screwing an automated respirometer head on top.

DETERMINATION OF BIODEGRADATION
- Experimental CO2 production: The CO2 produced in each test bottle reacted with the CO2
absorbent in the rubber gaskets. As gaseous O2 was converted into gaseous CO2 that was absorbed, the gas pressure in the test system slowly decreased. This decrease in air pressure was measured by the respirometer heads and automatically converted into oxygen consumption (mg O2/L).
- Measurements: Measurements were recorded on day 0, 4, 7, 11, 14, 19, 21, 25 and 28. After recording the oxygen consumption on day 28, the pH was determined in all test vessels.
- Theoretical Oxygen Demand: The ThODNO3 was calculated from the molecular composition.

MEASUREMENTS AND RECORDINGS
- pH: At the start of the test (day 0) and on the last day (day 28), after the last measurement.
- Respirometer: Lovibond BD600-GLP manometric respirometry system, equipped with an inductive stirring system.
- Temperature of medium: Continuously in a vessel with Milli-RO water in the same climate controlled incubator.

ACCEPTABILITY OF THE TEST
- The reference item was biodegraded by at least 60% (65%) within 14 days, which increased to 68% at the end of the test.
- The difference of duplicate values for %-degradation of the test item was always less than 20 (18% on day 7 and 13% at the end of the test).
- The total oxygen uptake in the blank at the end of the test did not exceed 30 mg O2/L (11 mg O2 per litre).
- The pH value in the test item bottles at the end of the test was within the range 6 to 8.5 (7.7).
- Since all criteria for acceptability of the test were met, this study was considered to be valid.
- All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

ANALYSIS
- ThODNO3, expressed as mg O2 required for full biodegradation of one mg of test item, was
calculated from the elemental composition of the test item.
- For test item CcHhXxNnNanaOoPpSs, with Xx being all halogens and assuming full nitrification, ThODNO3 was calculated using the equation ThODNO3 = [16(2c + ½(h-x) + 5/2n + 3s + 5/2p + 1/2na - o) / molecular weight] mg O2/mg test item.
- The first step in calculating the amount of O2 consumed was to correct for background O2
consumption. Thus, the amount of O2 consumed for biodegradation of the test item (BOD)
was determined using the equation BOD = O2 uptake by test item (mg O2/L) – O2 uptake by blank (mg O2/L).
- Relative biodegradation values were then calculated using the ThODNO3 of the added amount of test item, expressed as mg O2/L, using the equation % degradation = [BOD (mg O2/L) / ThODNO3 (mg O2/L)] * 100
- The relative biodegradation values were plotted versus time together with the relative biodegradation in the procedure control and the toxicity control. If not clear from the
experimental data the number of days was calculated from the attainment of 10% biodegradation until 60% biodegradation. Should this period be ≤ 10 days (10-day window), then the test item is designated as readily biodegradable.
- Toxicity control: if less than 25% biodegradation (based on combined ThODNO3 of the test item and reference item) occurred within 14 days, the test item was assumed to be inhibitory.

COMPUTERISED SYSTEMS
- Critical computerised systems used in the study are listed below.
- All computerised systems used in the conduct of this study were validated.
- When a particular system did not satisfy all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.
- Temperature, relative humidity and/or atmospheric pressure monitoring: REES Centron (version SQL 2.0).
- System control, data acquisition and processing: Shimadzu TOC-Control V (version 2.10).
Reference substance:
other: octan-1-ol
Remarks:
VWR International, Fontenay-sous-Bois, France (Batch 17B174008; Purity 98%; Expiry date 31 January 2022; Room temperature storage conditions)
Parameter:
% degradation (O2 consumption)
Value:
>= 47 - <= 59
Sampling time:
28 d
Remarks on result:
other: based on ThODNO3
Details on results:
THEORETICAL OXYGEN DEMAND
- The ThODNO3 of the test item was calculated to be 2.62 mg O2/mg based on the results of elemental composition analysis.
- The Carbon and hydrogen content (wt%) of the test item were determined directly, after which oxygen content was determined by difference (i.e. total should add up to 100%).
- According to the elemental composition analysis the carbon content of the test item was 69.6%. TOC analysis indicated an organic carbon content of 72.14%, which was considered an acceptable error margin.
- The ThOD of 1-octanol was calculated to be 2.95 mg O2/mg.

BIODEGRADATION
- The results of O2 consumption and biodegradation in blank bottles, procedure and toxicity control, and each test bottle are listed in Table 3 to 7 (attached).
- Table 8 (attached) contains the comparison of biodegradation of test item in bottles A and B.
- Figure 1 (attached) shows the curves for biodegradation of the two bottles with test item, the procedure control and the toxicity control.
- The relative biodegradation values calculated from the measurements performed during the test period revealed 59% and 47% biodegradation of test item based on ThODNO3, for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.
- In the toxicity control, more than 25% biodegradation occurred within 14 days (69%, based on ThODNO3, which increased to 79% at the end of the test). Therefore, the test item was assumed not to inhibit microbial activity.
- Functioning of the test system was checked by testing the reference item 1-octanol, which showed a normal biodegradation curve.

MONITORING OF TEMPERATURE AND pH
- The temperature recorded in a vessel with water in the same incubator varied between 22 and 24 °C.
- The pH values of the different test media are presented in Table 2 (below).

TABLE 2 – pH VALUES OF DIFFERENT TEST MEDIA

Test medium

Day 0

Day 28

Blank control (A)

7.7

7.7

Blank control (B)

7.7

7.7

Procedure control

7.7

7.7

Test Item (A)

7.7

7.7

Test Item (B)

7.7

7.7

Toxicity control

7.7

7.7

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item was not readily biodegradable under the conditions of the Manometric Respirometry Test presently performed. However, since the results of the present test indicate that the pass level criterion was almost fulfilled the results can be used to support inherent biodegradability.
Executive summary:

GUIDELINE

The objective of the study was to evaluate the test item for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge; Manometric Respirometry Test. Study procedures were conducted in compliance with OECD Guideline 301F, Section 3. Degradation and Accumulation. "Ready Biodegradability: Manometric Respirometry Test", (adopted July 17, 1992).

 

METHODS

The test item was a clear colourless liquid with a purity of 84.1%. The Theoretical Oxygen Demand with full nitrification (ThODNO3) of test item was calculated to be 2.62 mg O2/mg. The test item was tested in duplicate at a target concentration of 30.5 mg/L, corresponding to a ThODNO3 of 80 mg O2/L. The ThODNO3 was based on the results of elemental composition analysis and was confirmed by TOC analysis. The study consisted of six bottles: 2 inoculum blanks (no test item); 2 test bottles (test item); 1 procedure control (1-octanol) and; 1 toxicity control (test item plus 1-octanol).

 

The test item was added directly to the inoculated mineral medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Test duration was 28 days. In order to transfer test item to the test vessels, the required amounts were pipetted directly into the test bottles containing medium with microbial organisms and mineral components.

 

RESULTS

The relative biodegradation values calculated from the measurements performed during the test period revealed 59% and 47% biodegradation of test item based on ThODNO3, for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met. In the toxicity control, test item was found not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid.

 

CONCLUSION

The test item was not readily biodegradable under the conditions of the Manometric Respirometry Test presently performed. However, since the results of the present test indicate that the pass level criterion was almost fulfilled the results can be used to support inherent biodegradability.

Description of key information

The test item was not readily biodegradable under the conditions of the Manometric Respirometry Test presently. However, since the results indicated that the pass level criterion was almost fulfilled the results can be used to support inherent biodegradability (OECD 301 F).

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

GUIDELINE

The objective of the study was to evaluate the test item for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge; Manometric Respirometry Test. Study procedures were conducted in compliance with OECD Guideline 301 F, Section 3. Degradation and Accumulation. "Ready Biodegradability: Manometric Respirometry Test", (adopted July 17, 1992).

 

METHODS

The test item was a clear colourless liquid with a purity of 84.1%. The Theoretical Oxygen Demand with full nitrification (ThODNO3) of test item was calculated to be 2.62 mg O2/mg. The test item was tested in duplicate at a target concentration of 30.5 mg/L, corresponding to a ThODNO3 of 80 mg O2/L. The ThODNO3 was based on the results of elemental composition analysis and was confirmed by TOC analysis. The study consisted of six bottles: 2 inoculum blanks (no test item); 2 test bottles (test item); 1 procedure control (1-octanol) and; 1 toxicity control (test item plus 1-octanol).

 

The test item was added directly to the inoculated mineral medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Test duration was 28 days. In order to transfer test item to the test vessels, the required amounts were pipetted directly into the test bottles containing medium with microbial organisms and mineral components.

 

RESULTS

The relative biodegradation values calculated from the measurements performed during the test period revealed 59% and 47% biodegradation of test item based on ThODNO3, for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met. In the toxicity control, test item was found not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid.

 

CONCLUSION

The test item was not readily biodegradable under the conditions of the Manometric Respirometry Test presently performed. However, since the results of the present test indicate that the pass level criterion was almost fulfilled the results can be used to support inherent biodegradability.