2-Propenoic acid, methyl ester, reaction products with mixed O,O-bis(branched and linear pentyl and iso-Bu) phosphorodithioates

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: screening tests

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-07-04 to 2002-08-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP Guideline study, with modification of method to use pre-adapted sewage sludge

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

yes

Remarks:

sludge pre-conditioned

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

yes

Remarks:

sludge pre-conditioned

Principles of method if other than guideline:

The test material was an insoluble, viscous liquid and therefore following the recommendations of the International Standards Organisation (IS0 1996) for the purpose of the definitive study the test material was dispensed onto a glass fibre filter paper (Whatman GF/A, 25 mm diameter) prior to addition to the test vessels. It was considered that this method of addition of the test material to the test vessels represented the most accurate method of addition of the relatively small amount of test material required to prepare the 50 mg/L test concentration and aided dispersion of the test material in the test medium thereby increasing the surface area of the test material exposed to the test organisms. A test concentration of 50 mg/L was selected for the biodegradation test in order minimise any possible inhibitory effects that the test material may exhibit on the activated sewage sludge micro-organisms.An amount of test material (25 mg) was dispensed onto the surface of a glass fibre filter paper (Whatman GF/A, 25 mm diameter) prior to addition of the filter paper to a final volume of 500 mL of inoculated culture medium to give the required test concentration of 50 mg/L.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

A mixed population of sewage sludge micro-organisms was obtained on 20 June 2002 from the final effluent stage of the Sevem Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.In an attempt to produce an acclimated population of bacteria that was capable of degrading the test material, the inoculum was pre-exposed to the test material. The pre-exposure regime was based on methods described by CONCAWE (1999) and the draft OECD Guideline No 302D (2001).The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at 2 1 °C ± 1 °C prior to use.The prepared inoculum was then supplemented with vitamin-free casamino acids (25 mg/L) and yeast extract (25 mg/L) and an aliquot (100 mL) of the supplemented inoculum added to a 2 litre conical flask prior to the addition of the following:Phosphate buffer solution 10 mLCalcium chloride solution 1 .0 mLMagnesium sulphate solution 1 .0 mLFerric chloride solution 1.0 mLAfter addition of these stock solutions, the volume was adjusted to 1 litre using deionised reverse osmosis water prior to addition of test material.Pre-conditioningOn Day 0 of the pre-exposure period, an amount of test material (87.7 mg) was dissolved in diethylether and the volume adjusted to 10 mL to give a 87.7 mg/10 mL solvent stock solution. An aliquot (1 .0 mL) of this solvent stock solution was dispensed onto the surface of a glass fibre filter paper (Whatman GF/A, 70 mm diameter) and the solvent allowed to evaporate to dryness (approximately 30 minutes) in a fume cupboard. It was considered appropriate to apply the test material to the filter papers using a solvent stock solution in order to enable accurate addition of a relatively small amount of test material.After the solvent had evaporated the filter paper containing the test material was placed in the conical flask containing the prepared inoculum to give a nominal carbon concentration of 4 mg C/L as test material.The flask was then incubated at 21±1°C with constant stirring via magnetic stirrer. On Days 7 and 11 of the pre-exposure period, any losses due to evaporation were made up using deionised water prior to addition of test material at a concentration of 8 mg C/L to the inoculum. The test material was added to the inoculum in the same manner as used on Day 0 of the preexposure period however a 175.4 mg/10 mL solvent stock solution was used in order to give a nominal carbon concentration of 8 mg C/L as test material.Assuming that no biodegradation of the test material occurred during the pre-exposure period, the concentration of test material present in the inoculum was 8.77 mg/L on Day 0, 26.31 mg/L on Day 7 and 43.85 mg/L on Day 11 of the pre-exposure period.Preparation of inoculum for the main testOn Day 14 of the pre-exposure period (Day 0 of the biodegradation test) the inoculum was homogenized (Kenwood Chef Mixer) for approximately 1 minute prior to filtration through glass wool. The filtrate was maintained on aeration prior to use in the biodegradation test.The prepared inoculum was used to inoculate all control, standard material. test material and toxicity control vessels. As a result of the addition of test material to the inoculum during the preexposure period it was inevitable that test material would be present in the inoculum used to prepare the control, standard material, test material and toxicity control vessels. However as the inoculum was homogenized prior to use, any degradation of the test material and associated oxygen consumption would be consistent between all vessels prepared and hence would not affect the calculation of percentage degradation values for the test and standard materials from the BOD values obtained.

Duration of test (contact time):

28 d

Initial conc.:

50 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS- Composition of medium: Prepared following the OECD Guideline- Additional substrate: No- Solubilising agent (type and concentration if used):The test material was an insoluble, viscous liquid and therefore following the recommendations of the International Standards Organisation (IS0 1996) for the purpose of the definitive study the test material was dispensed onto a glass fibre filter paper (Whatman GF/A, 25 mm diameter) prior to addition to the test vessels. It was considered that this method of addition of the test material to the test vessels represented the most accurate method of addition of the relatively small amount of test material required to prepare the 50 mg/L test concentration and aided dispersion of the test material in the test medium thereby increasing the surface area of the test material exposed to the test organisms. A test concentration of 50 mg/L was selected for the biodegradation test in order minimise any possible inhibitory effects that the test material may exhibit on the activated sewage sludge micro-organisms.An amount of test material (25 mg) was dispensed onto the surface of a glass fibre filter paper (Whatman GF/A, 25 mm diameter) prior to addition of the filter paper to a final volume of 500 mL of inoculated culture medium to give the required test concentration of 50 mg/L.Control vessels were prepared as above using glass fibre filter papers without the addition of test material in order to maintain consistency between the control and test material vessels.- Test temperature: 21 ±0.8°C.- pH: 7.5 - 8.3- pH adjusted: no- CEC (meq/100 g):- Aeration of dilution water: Filtrate aerated until use in the study- Continuous darkness: yesTEST SYSTEM- Culturing apparatus: 2L conical flask- Number of culture flasks/concentration: three- Method used to create aerobic conditions:- Test performed in open system: no, sealed vessels.- Details of trap for CO2 and volatile organics if used: ethanolamine CO2 trapSAMPLINGShimadzu TOC 5050A TOC analyser. Samples (27 or 13 µL) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of theTOC analyser. Total Carbon analysis was carried out at 680°C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involved conversion of the sample by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2C03) prepared in deionised water purified by reverse osmosis. Each analysis was carried out in triplicate.CONTROL AND BLANK SYSTEM- Inoculum blank: Yes, 4 replicates- Toxicity control: Yes, 2 replicates

Reference substance:

aniline

Preliminary study:

Not performed

Test performance:

Validity criteria were fulfilled.

Parameter:

% degradation (O2 consumption)

Value:

14

Sampling time:

7 d

Parameter:

% degradation (O2 consumption)

Value:

27

Sampling time:

14 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

47

Sampling time:

28 d

Details on results:

The test material attained 49% degradation after 28 days calculated from oxygen consumption values.As pre-exposed inoculum was used in this test, the criteria for ready biodegradability given in the OECD Guidelines do not apply, however it is considered that the test material has exhibited the potential for significant degradation to occur in the environment.The toxicity control attained 49% degradation after 14 days and 62% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.

Results with reference substance:

Aniline attained 72% degradation after 14 days and 76% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.Aniline attained 104% degradation after 28 days, calculated from the results of the DOC analyses performed on Days 0 and 28.The degradation value in excess of 100% was considered to be due to sampling and/or analytical variation. The degradation rate calculated from the results of the DOC analyses was higher than that calculated from oxygen consumption values. This was considered to be due to incorporation of aniline into the microbial biomass prior to degradation, and hence oxygen consumption, occurring.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

The test material attained 49% degradation after 28 days calculated from oxygen consumption values. As pre-exposed inoculum was used in this test, the criteria for ready biodegradability given in the OECD Guidelines do not apply, however it is considered that the test material has exhibited the potential for significant degradation to occur in the environment.

Executive summary:

Test Guideline

The study was performed to assess the biodegradability of the test material in an aerobic aqueous media. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301F, “Ready Biodegradability; Manometric Respirometry Test” referenced as method C.4-D of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC) and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 Paragraph (q), however the inoculum used in the test was pre-exposed to the test material in an attempt to produce an acclimated population of bacteria that was capable of degrading the test material.

Methods.

The test material at a concentration of 50 mg/L was exposed to pre-exposed sewage sludge with culture medium in sealed culture vessels in the dark at 21 5 0.8°C for 28 days. The degradation of the test material was assessed by the measurement of daily oxygen consumption values. Control solutions with inoculum and the standard material, aniline, together with a toxicity control were used for validation purposes. Pre-exposure of the inoculum to the test material was performed by the addition of test material at concentrations of 4, 8 and 8 mg carbon/L to the inoculum on Days 0, 7 and 11 of the pre-exposure period.

Results.

The test material attained 49% degradation after 28 days based on oxygen consumption values. As pre-exposed inoculum was used in this test, the criteria for ready biodegradability given in the OECD Guidelines do not apply, however it is considered that the test material has exhibited the potential for significant degradation to occur in the environment.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Biodegradability test of chemical substances by micro-organisms (Japanese notification, Yakusokuhatsu 0331 No. 7, Heisei 23.03.29 Seikyoku No.5, Kampokihatsu No. 110331009, March 31, 2011)

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Chemicals Evaluation and Research Institute, Japan- Laboratory culture: yes- Method of cultivation: Not stated- Storage conditions:- Storage length:- Preparation of inoculum for exposure:- Pretreatment:- Concentration of sludge:- Initial cell/biomass concentration:- Water filtered: yes/no- Type and size of filter used, if any:

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

BOD

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS- Composition of medium: In accordance with Japanese Indstrial Standards (JIS) K0102-1998, 21- Test temperature:- pH:- pH adjusted: yes/no- CEC (meq/100 g):- Aeration of dilution water:- Suspended solids concentration: 3900 mg/L (recovery test), 4050 mg/L (BOD measurement)- Continuous darkness: yes/no- Other:TEST SYSTEM- Culturing apparatus:- Number of culture flasks/concentration:- Method used to create aerobic conditions:- Method used to create anaerobic conditions:- Measuring equipment:- Test performed in closed vessels due to significant volatility of test substance:- Test performed in open system:- Details of trap for CO2 and volatile organics if used:- Other:SAMPLING- Sampling frequency:- Sampling method:- Sterility check if applicable:- Sample storage before analysis:- Other:CONTROL AND BLANK SYSTEM- Inoculum blank:- Abiotic sterile control:- Toxicity control:- Other:STATISTICAL METHODS:

Reference substance:

aniline

Preliminary study:

Not performed

Test performance:

All validity criteria were fulfilled

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

Observation of contents after the end of the BOD measurementObservations were made on growth of the activated sludge and colour of the contents in the test bottles using the inoculum blank as a reference. After the end of BOD measurement, observation on dissolution state of the test substance was also made.The contents in the activity control, the test suspensions and abiotic control bottles were white. Growth of the sludge was observed in the activity control in contrast with the inoculum blank, whereas no growth of the sludge was observed in the test suspensions. Undissolved test substance was visible in the abiotic control, but not obvious in the test suspensions.pH measurementAt the end of the BOD measurements the pH values were 7.1, 7.1 and 7.1 for the three test suspensions and 8.4 for the abiotic control. Degradability based on the BODThe BOD (theoretical value = 59.0 mg) in the test suspensions after 28-days (as corrected with the value in the inoculum blank) were -0.6, 0.0 and -0.3 mg, respectively, and the BOD in the abiotic control after 28 days was 0.4 mg.The degradabilities based on the BOD after 28 days were calculated to be 0% (calculated values were -1, 0 and -1%) for all of the test suspensions.Degradability based on the DOCThe DOC (theoretical value = 13.7 mg) in the test suspensions on Day 28 (as corrected with the value in the inoculum blank) were 1.8, 1.9 and 1.7 mg, respectively, and the DOC in the abiotic control on Day 28 was 0.9 mg.Since the DOC in the abiotic control on Day 28 was less than 90% of the theoretical value, the degradability based on the DOC on Day 28 was not calculated.Degradability based on the residual test substance amountThe residual amounts of the test substance (initial 30 mg) in the test suspensions and abiotic control on Day 28 were 28.7, 28.6, 28.7 and 29.3 mg, respectively.The degradabilities based on the residual amount of the test substance were calculated to be 2% for all of the test suspensions.Changes in peak shape were observed on the chromatograms of the test suspensions when compared to that of the standard test substance solution. The height of the peak shoulder at the retention time of approximately 9.3 min had risen, whereas the height of the peak top at the retention time of approximately 9.4 min had declined. No new peak was detected. Meanwhile, the chromatogram of the abiotic control showed no change in peak shape or appearance of any new peak.Confirmation of presence or absence of transformation productsChanges in peak shape were observed on the chromatograms of the test suspensions when compared to that of the standard test substance solution. The height of the peaks at the retention time of approximately 4.2 and 4.5 minutes had declined whereas the height of the peaks at the retention time between approximately 5.0 and 6.0 minutes had risen. No new peak was detected. Meanwhile the chromatogram of the abiotic control showed no change in peak shape or appearance of any new peak.

Results with reference substance:

The activity control showed measured BOOD values of 63.3 mg on Day7; 67.6 mg on Day 14, 68.8 mg on Day 21 and 69.5 mg on Day 28. The test system was shown to be valid.

From the average degradability after 28 days of 0% and 2% based on the BOD and the residual amount of test material, respectively, the test substance was found not to be readily biodegradable under the conditions of the study.

Although changes in the height of the peak shoulder and peak top were observed onthe GPC chromatograms of the test suspensions when compared to that of the standard test solutions, no shift in the time range of detected peaks had occurred. No new peak was detected, either.

Additional analyses with HPLC and LC/MS were performed to detect the presence or absence of transformation products. Although changes in the height of some peaks similar to those observed in the GPC chromatograms were also seen on the HPLC chromatograms, no shift in the time range of detected peaks had occurred. No new peak was detected either. Furthermore, mass spectra of peaks on the LC/MS total ion chromatograms (TIC) corresponding to peaks detected in the HPLC analysis were identified as being attributable to either the test substance or inoculum blank, but not to any transformation product.

Meanwhile, no change in peak shapes was observed on the GPC and HPLC chromatograms of the abiotic control when compared to those of the standard test substance solution. No new peak was detected either. Furthermore, no mass spectrum attributable to any transformation product was identified on the LC/MS TIC.

The results suggest that the changes in the peak height observed on the GPC and HPLC were due to alteration of the relative proportion of the components of the test substance, and that no new transformation product had been formed. The analysis of the standard solution of monodisperse polystyrene with known molecular weight confirmed that the retention time of the test substance peak corresponded to that of a MW < 1000.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Based on the average degradability after 28 days of 0% and 2% based on the BOD and the residual amount of test material, respectively, the test substance was found not to be readily biodegradable under the conditions of the study.

Executive summary:

Test Guideline

Biodegradability test of chemical substances by micro-organisms (Japanese notification, Yakusokuhatsu 0331 No. 7, Heisei 23.03.29 Seikyoku No.5, Kampokihatsu No. 110331009, March 31, 2011)

Method

The ready biodegradability of the test material was determined by exposing activated sewage sludge to a test concentration of 100 mg/L over a 28-day period. The rate of degradation was measured using biological oxygen demand (BOD) and residual test substance. The study comprised of three replicate test suspensions, an activity control using a reference substance, aniline, and an abiotic control (test material, reference substance and inoculum). Confirmation of transformation products was performed using HPLC.

Results

Observation of contents after the end of the BOD measurement

Observations were made on growth of the activated sludge and colour of the contents in the test bottles using the inoculum blank as a reference. After the end of BOD measurement, observation on dissolution state of the test substance was also made.

The contents in the activity control, the test suspensions and abiotic control bottles were white. Growth of the sludge was observed in the activity control in contrast with the inoculum blank, whereas no growth of the sludge was observed in the test suspensions. Undissolved test substance was visible in the abiotic control, but not obvious in the test suspensions.

pH measurement

At the end of the BOD measurements the pH values were 7.1, 7.1 and 7.1 for the three test suspensions and 8.4 for the abiotic control.

Degradability based on the BOD

The BOD (theoretical value = 59.0 mg) in the test suspensions after 28-days (as corrected with the value in the inoculum blank) were -0.6, 0.0 and -0.3 mg, respectively, and the BOD in the abiotic control after 28 days was 0.4 mg.

The degradabilities based on the BOD after 28 days were calculated to be 0% (calculated values were -1, 0 and -1%) for all of the test suspensions.

Degradability based on the DOC

The DOC (theoretical value = 13.7 mg) in the test suspensions on Day 28 (as corrected with the value in the inoculum blank) were 1.8, 1.9 and 1.7 mg, respectively, and the DOC in the abiotic control on Day 28 was 0.9 mg.

Since the DOC in the abiotic control on Day 28 was less than 90% of the theoretical value, the degradability based on the DOC on Day 28 was not calculated.

Degradability based on the residual test substance amount

The residual amounts of the test substance (initial 30 mg) in the test suspensions and abiotic control on Day 28 were 28.7, 28.6, 28.7 and 29.3 mg, respectively.

The degradabilities based on the residual amount of the test substance were calculated to be 2% for all of the test suspensions.

Changes in peak shape were observed on the chromatograms of the test suspensions when compared to that of the standard test substance solution. The height of the peak shoulder at the retention time of approximately 9.3 min had risen, whereas the height of the peak top at the retention time of approximately 9.4 min had declined. No new peak was detected. Meanwhile, the chromatogram of the abiotic control showed no change in peak shape or appearance of any new peak.

Confirmation of presence or absence of transformation products

Changes in peak shape were observed on the chromatograms of the test suspensions when compared to that of the standard test substance solution. The height of the peaks at the retention time of approximately 4.2 and 4.5 minutes had declined whereas the height of the peaks at the retention time between approximately 5.0 and 6.0 minutes had risen. No new peak was detected. Meanwhile the chromatogram of the abiotic control showed no change in peak shape or appearance of any new peak.

Conclusion

From the average degradability after 28 days of 0% and 2% based on the BOD and the residual amount of test material, respectively, the test substance was found not to be readily biodegradable under the conditions of the study.

Although changes in the height of the peak shoulder and peak top were observed onthe GPC chromatograms of the test suspensions when compared to that of the standard test solutions, no shift in the time range of detected peaks had occurred. No new peak was detected, either.

Additional analyses with HPLC and LC/MS were performed to detect the presence or absence of transformation products. Although changes in the height of some peaks similar to those observed in the GPC chromatograms were also seen on the HPLC chromatograms, no shift in the time range of detected peaks had occurred. No new peak was detected either. Furthermore, mass spectra of peaks on the LC/MS total ion chromatograms (TIC) corresponding to peaks detected in the HPLC analysis were identified as being attributable to either the test substance or inoculum blank, but not to any transformation product.

Meanwhile, no change in peak shapes was observed on the GPC and HPLC chromatograms of the abiotic control when compared to those of the standard test substance solution. No new peak was detected either. Furthermore, no mass spectrum attributable to any transformation product was identified on the LC/MS TIC.

The results suggest that the changes in the peak height observed on the GPC and HPLC were due to alteration of the relative proportion of the components of the test substance, and that no new transformation product had been formed. The analysis of the standard solution of monodisperse polystyrene with known molecular weight confirmed that the retention time of the test substance peak corresponded to that of a MW < 1000.

 

Description of key information

In a ready biodegradation study performed to Japanese Guidelines the average degradability after 28 days was 0% and 2% based on the BOD and the residual amount of test material, respectively. The test substance was found not to be readily biodegradable under the conditions of the study. 
In an inherent biodegradation study the test material attained 49% degradation after 28 days based on oxygen consumption values.
As pre-exposed inoculum was used in this test, the criteria for ready biodegradability given in the OECD Guidelines do not apply, however it is considered that the test material has exhibited the potential for significant degradation to occur in the environment.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Additional information

Ready biodegradation

In a supplementary study performed in accordance with Biodegradability test of chemical substances by micro-organisms (Japanese notification, Yakusokuhatsu 0331 No. 7, Heisei 23.03.29 Seikyoku No.5, Kampokihatsu No. 110331009, March 31, 2011), the ready biodegradability of the test material was determined by exposing activated sewage sludge to a test concentration of 100 mg/L over a 28-day period. The rate of degradation was measured using biological oxygen demand (BOD) and residual test substance. The study comprised of three replicate test suspensions, an activity control using a reference substance, aniline, and an abiotic control (test material, reference substance and inoculum). Confirmation of transformation products was performed using HPLC.

Observation of contents after the end of the BOD measurement

Observations were made on growth of the activated sludge and colour of the contents in the test bottles using the inoculum blank as a reference. After the end of BOD measurement, observation on dissolution state of the test substance was also made.

The contents in the activity control, the test suspensions and abiotic control bottles were white. Growth of the sludge was observed in the activity control in contrast with the inoculum blank, whereas no growth of the sludge was observed in the test suspensions. Undissolved test substance was visible in the abiotic control, but not obvious in the test suspensions.

pH measurement

At the end of the BOD measurements the pH values were 7.1, 7.1 and 7.1 for the three test suspensions and 8.4 for the abiotic control.

Degradability based on the BOD

The BOD (theoretical value = 59.0 mg) in the test suspensions after 28-days (as corrected with the value in the inoculum blank) were -0.6, 0.0 and -0.3 mg, respectively, and the BOD in the abiotic control after 28 days was 0.4 mg.

The degradabilities based on the BOD after 28 days were calculated to be 0% (calculated values were -1, 0 and -1%) for all of the test suspensions.

Degradability based on the DOC

The DOC (theoretical value = 13.7 mg) in the test suspensions on Day 28 (as corrected with the value in the inoculum blank) were 1.8, 1.9 and 1.7 mg, respectively, and the DOC in the abiotic control on Day 28 was 0.9 mg.

Since the DOC in the abiotic control on Day 28 was less than 90% of the theoretical value, the degradability based on the DOC on Day 28 was not calculated.

Degradability based on the residual test substance amount

The residual amounts of the test substance (initial 30 mg) in the test suspensions and abiotic control on Day 28 were 28.7, 28.6, 28.7 and 29.3 mg, respectively.

The degradabilities based on the residual amount of the test substance were calculated to be 2% for all of the test suspensions.

Changes in peak shape were observed on the chromatograms of the test suspensions when compared to that of the standard test substance solution. The height of the peak shoulder at the retention time of approximately 9.3 min had risen, whereas the height of the peak top at the retention time of approximately 9.4 min had declined. No new peak was detected. Meanwhile, the chromatogram of the abiotic control showed no change in peak shape or appearance of any new peak.

Confirmation of presence or absence of transformation products

Changes in peak shape were observed on the chromatograms of the test suspensions when compared to that of the standard test substance solution. The height of the peaks at the retention time of approximately 4.2 and 4.5 minutes had declined whereas the height of the peaks at the retention time between approximately 5.0 and 6.0 minutes had risen. No new peak was detected. Meanwhile the chromatogram of the abiotic control showed no change in peak shape or appearance of any new peak.

From the average degradability after 28 days of 0% and 2% based on the BOD and the residual amount of test material, respectively, the test substance was found not to be readily biodegradable under the conditions of the study.

Although changes in the height of the peak shoulder and peak top were observed onthe GPC chromatograms of the test suspensions when compared to that of the standard test solutions, no shift in the time range of detected peaks had occurred. No new peak was detected, either.

Additional analyses with HPLC and LC/MS were performed to detect the presence or absence of transformation products. Although changes in the height of some peaks similar to those observed in the GPC chromatograms were also seen on the HPLC chromatograms, no shift in the time range of detected peaks had occurred. No new peak was detected either. Furthermore, mass spectra of peaks on the LC/MS total ion chromatograms (TIC) corresponding to peaks detected in the HPLC analysis were identified as being attributable to either the test substance or inoculum blank, but not to any transformation product.

Meanwhile, no change in peak shapes was observed on the GPC and HPLC chromatograms of the abiotic control when compared to those of the standard test substance solution. No new peak was detected either. Furthermore, no mass spectrum attributable to any transformation product was identified on the LC/MS TIC.

The results suggest that the changes in the peak height observed on the GPC and HPLC were due to alteration of the relative proportion of the components of the test substance, and that no new transformation product had been formed. The analysis of the standard solution of monodisperse polystyrene with known molecular weight confirmed that the retention time of the test substance peak corresponded to that of a MW < 1000.

Inherent biodegradation

A key study was performed to assess the biodegradability of the test material in an aerobic aqueous media. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301F, “Ready Biodegradability; Manometric Respirometry Test” referenced as method C.4-D of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC) and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 Paragraph (q), however the inoculum used in the test was pre-exposed to the test material in an attempt to produce an acclimated population of bacteria that was capable of degrading the test material.

The test material at a concentration of 50 mg/L was exposed to pre-exposed sewage sludge with culture medium in sealed culture vessels in the dark at 21 5 0.8°C for 28 days. The degradation of the test material was assessed by the measurement of daily oxygen consumption values. Control solutions with inoculum and the standard material, aniline, together with a toxicity control were used for validation purposes.

Pre-exposure of the inoculum to the test material was performed by the addition of test material at concentrations of 4, 8 and 8 mg carbon/L to the inoculum on Days 0, 7 and 11 of the pre-exposure period.

The test material attained 49% degradation after 28 days based on oxygen consumption values.

As pre-exposed inoculum was used in this test, the criteria for ready biodegradability given in the OECD Guidelines do not apply, however it is considered that the test material has exhibited the potential for significant degradation to occur in the environment.