Benzene-1,3,5-tricarbonyl trichloride

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05-05-2022 to 10-06-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: October 2019; signature: March 2020

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

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 (Test System) was obtained from the domestic waste sewage treatment plant Rossdorf (Germany). The plant treats predominantly domestic sewage.
- Laboratory culture: See source of inoculum/activated sludge.
- Method of cultivation: The sample of activated sewage sludge was maintained on continuous aeration upon receipt.
- Storage conditions: See pretreatment field.
- Storage length: typically < 1 week
- Preparation of inoculum for exposure: See pre-treatment field.
- Pretreatment: The sample of activated sewage sludge was maintained on continuous aeration upon receipt. The activated sludge used for this study was deposited for 15 minutes, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and again centrifuged. This procedure was repeated three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and then aerated over night until use. Determination of dry weight is made to inoculate final solution with ca. 30 mg/L dry weight activated sludge.
- Concentration of sludge: The sludge was diluted in the BOD bottles to ca. 30 mg DW/L. (actual: 28.7 mg sludge/L)
- Initial cell/biomass concentration: Not applicable.
- Water filtered: Not applicable. Culture medium was in accordance with OECD TG 301F.
- Type and size of filter used, if any: Not applicable.

Duration of test (contact time):

28 d

Initial conc.:

ca. 61.9 mg/L

Based on:

test mat.

Remarks:

Test item (test flasks 1, 2)

Initial conc.:

ca. 55.9 mg/L

Based on:

ThOD

Remarks:

corresponding to an oxygen demand of about 55.9 mg/L (ThODNH4)

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: See table 1. 500 mL of Solution A [KH2PO4: 4.25 g/L; K2HPO4: 10.88 g/L; Na2HPO4,2H2O: 16.7 g/L; NH4Cl: 0.25 g/L]; 500 mL of Solution B [MgSO4,7H2O: 11.25 g/L ]; 500 mL of Solution C [CaCl2,2H2O: 18.2 g/L]; 500 mL of Solution D [FeCl3,6H2O: 0.125 g/L] each diluted in purified water to create stock solution. In order to prepare the stock solution d) immediately before use, one drop of concentrated HCI per litre was added. 50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled to a final volume of 5000 mL with ultrapure water. The pH was 7.8 and adjusted to pH 7.5 with 1M HCl solution.
- Solubilising agent (type and concentration if used): None. Test item, reference item were directly weighed and dispersed into vessels.
- Test temperature: 22 ±1 °C
- pH: See table 1.
- pH adjusted: No (final pH was in the range of 6.8 to 6.9 for test item vessels and 7.3 to 7.4 for inoculum control, 7.6 for procedure control).
- CEC (meq/100 g): Not reported.
- Aeration of dilution water: Not reported
- Suspended solids concentration: ca. 30 mg/L dry weight (actual: 28.7 mg sludge/L)
- Continuous darkness: Yes. The test was conducted in the dark.

TEST SYSTEM
- Culturing apparatus: 500 mL glass flasks with continuous stirring (fill volume ca. 244 mL)
- Number of culture flasks/concentration: at least duplicate per concentration (test item); In duplicate (Inoculum blank control); single flasks (toxicity control, abiotic sterile control and procedure control)
- Method used to create aerobic conditions: Sealed flasks with sensor head/CO2 trap (45% KOH solution)
- Method used to create anaerobic conditions: Not applicable.
- Measuring equipment: The respirometer/manometric system used during this study is an BSB-Sensomat-System infrared-measurement system (BSB-Sensomat-System, Germany). Evolved carbon dioxide is absorbed by the CO2 trap (45% KOH solution)
- Test performed in closed vessels due to significant volatility of test substance: Not reported.
- Test performed in open system: No.
- Details of trap for CO2 and volatile organics if used: See above.

SAMPLING
- Sampling frequency: Daily.
- Sampling method: The respirometer/manometric system used during this study is an BSB-Sensomat-System infrared-measurement system (Germany).
- Sterility check if applicable: Not reported.
- Sample storage before analysis: Not applicable.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes. See table 1.
- Abiotic sterile control: Yes.
- Toxicity control: Yes.
- Other: Positive reference control (Sodium Benzoate)

Reference substance:

benzoic acid, sodium salt

Remarks:

103.3 mg/L

Test performance:

1. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test item at the end of 10-day window. Therefore, the test is considered valid. The difference of duplicate values at day 23 (end of the 10-day window) was 16%.
2. The BOD of the inoculated blank control was 12 mgO2/L and < 60 mgO2/L after 28 days
3. The pH at day 28 was in the range of 6.0 to 8.5 (actual pH: 6.8 to 6.9 for test item vessels and 7.3 to 7.4 in inoculum control and 7.6 in procedure control)
4. The toxicity test attained 67% degradation at 14 days and 84% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment microorganisms used in the study.
5. Sodium Benzoate attained 95% degradation at 14 days and 100% at 28 days thereby confirming the suitability of the inoculum and test conditions.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

mean (n=2) / test concentration ca. 61.9 mg/L nominal test item or 55.9 mg/L ThOD

Value:

90.8

Sampling time:

28 d

Remarks on result:

other: test flasks 1 and 2 ; 10-day window met on day 23 ; mean biodegradation day 23 = 81.9% ; < 20% repeatability criteria was met at the end of the 10-day window on day 23 (actual: 16%); mean biodegradation day 28 = 90.8%

Details on results:

(i) In the abiotic control the Oxygen Demand was 0 mg O2/L.
(ii) In the inoculum blank controls, the oxygen demand was 12 mg O2/L and therefore less than 60 mgO2/L satisfying the requirements of the guideline.
(iii) In the procedure control: sodium Benzoate attained 95% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.
(iv) In the two toxicity controls, the degradation was 67% after 14 days and 84% after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was >25% within 14 days.
(v) In all the test, reference item and controls the pH was in the range of 6.8 to 7.6 (thereby was in the range of 6.0 to 8.5 required by the guideline)
(vi) The difference of duplicate values for the degradation of the test item at the plateau, at the end of the test OR at the end of the 10-day window should be less than 20%. The validity criterion was met for the end of the 10-day window and was actual: 16%. [The repeatability was not met for the end of the test, on day 28. However, since the validity criteria was met for the end of the 10-day window on day 23 the test is valid according to OECD TG 301, para. 24 'Validity of Tests', page: 6 - 7].
(vii) The mean test item biodegradation conducted in duplicate, was 90.8% at day 28. The 10-day window criteria was met. The test item is readily biodegradable.

Results with reference substance:

Sodium Benzoate attained 95% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The mean biodegradation was 90.8% at day 28 conducted in duplicate. The 10-day window criteria was met. The test item is readily biodegradable.

Executive summary:

The ready biodegradability test was carried out according to OECD TG 301F guideline under GLP. The test item, at a concentration of ca. 61.9 mg/L nominal (or 55.9 mg/L ThODNH14) was exposed to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment plant which treats predominantly domestic sewage (Rossdorf, Germany) with culture medium in sealed culture vessels in the dark at 22°C ± 1°C for 28 days. The sludge was diluted in the BOD bottles to ca. 30 mg DW/L. The degradation of the test item was assessed by the measurement of daily oxygen consumption on days 0 and 28 using a respirometer/manometric system which utilised a BSB-Sensomat-System : infrared measurement system (BSB-Sensomat-System, Germany). Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control and/or an abiotic control were used for validation purposes. In the test inoculum blank the oxygen uptake was ca. 12 mg O2/L. The pH value at the end of the test period 28 days was 6.8 to 6.9 in the test item systems and 7.6 in the reference item system. In the toxicity control, the degradation was 67% after 14 days and 84% after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was >25% within 14 days. The test system met the validation criteria of the guideline. Sodium Benzoate attained 95% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions. In the test vessels, a degradation of 102% and 79% after 28 days was found for the two replicates, respectively. The mean biodegradation percentage at the end of the 28-day exposure period was 90.8% (ThODNH4). The 10-day windows began on day 13 after application, the mean value was calculated to be 21.4% biodegradation (ThODNH4). Therefore, the end of the 10-day window was day 23. The mean biodegradation percentage at the end of the 10-day window was 81.9% (ThODNH4); the criterion of the 10-day window was passed. The difference of duplicate values for the degradation of the test item at the plateau, at the end of the test or at the end of the 10-day window should be less than 20%. The validity criterion was met for the end of the 10-day window and was 16%. Therefore the test is considered valid. Under the conditions of this study, the mean biodegradation was 90.8% at day 28 conducted in duplicate. The 10-day window criteria was met. The test item is readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

09-03-2022 to 07-04-2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met. The source and target substances are related by virtue of the source substance being a ‘common breakdown product’ of the target substance. The source and target demonstrate ‘chemical similarity’ : since the source is the structurally-similar degradation product (i.e. similarity through (bio) transformation) from the target. The target substance hydrolytically degrades in water in a matter of seconds/minutes at all relevant pH 4, 7 and 9 forming the source substance. See related endpoint information in IUCLID: section 5.1.2: hydrolysis.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in attachment to IUCLID section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is based on the hypothesis that the source and target substances are related by virtue of the source substance being a ‘common breakdown product’ of the target substance. The source and target demonstrate ‘chemical similarity’ : since the source is the structurally-similar degradation product (i.e. similarity through (bio) transformation) from the target. The target substance hydrolytically degrades in water in a matter of seconds/minutes at all relevant pH 4, 7 and 9 forming the source substance. See related endpoint information in IUCLID: section 5.1.2: hydrolysis.
Further information is included in attachment to IUCLID section 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source and target chemicals have comparable chemical similarity. Further information is included in attachment to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
The source substance is a the ‘common breakdown product’ of the target substance. The source and target demonstrate ‘chemical similarity’ : since the source is the structurally-similar degradation product (i.e. similarity through (bio) transformation) from the target. The target substance hydrolytically degrades in water in a matter of seconds/minutes at all relevant pH 4, 7 and 9 forming the source substance. As a consequence they should be considered chemically similar substances due to routes of common (a)biotic degradation and/or metabolism and common or similar degradants. See related endpoint information in IUCLID: section 5.1.2: hydrolysis. Further information is included in attachment to IUCLID section 13

4. DATA MATRIX
Further information is included in attachment to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Remarks:

Reporting format for the analogue approach

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: October 2019; signature: March 2020

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

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 (Test System) was obtained from the domestic waste sewage treatment plant Bensheim (Germany). The plant treats predominantly domestic sewage.
- Laboratory culture: See source of inoculum/activated sludge.
- Method of cultivation: The sample of activated sewage sludge was maintained on continuous aeration upon receipt.
- Storage conditions: See pretreatment field.
- Storage length: typically < 1 week
- Preparation of inoculum for exposure: See pre-treatment field.
- Pretreatment: The sample of activated sewage sludge was maintained on continuous aeration upon receipt. The activated sludge used for this study was deposited for 15 minutes, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and again centrifuged. This procedure was repeated three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and then aerated over night until use. Determination of dry weight is made to inoculate final solution with ca. 30 mg/L dry weight activated sludge.
- Concentration of sludge: The sludge was diluted in the BOD bottles to ca. 30 mg DW/L. (actual: 28.7 mg sludge/L)
- Initial cell/biomass concentration: Not applicable.
- Water filtered: Not applicable. Culture medium was in accordance with OECD TG 301F.
- Type and size of filter used, if any: Not applicable.

Duration of test (contact time):

28 d

Initial conc.:

ca. 103.1 mg/L

Based on:

test mat.

Remarks:

Test item (test flasks 1, 2)

Initial conc.:

ca. 169.3 mg/L

Based on:

ThOD

Remarks:

corresponding to an oxygen demand of about 169.3 mg/L (ThODNH4)

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: See table 1. 500 mL of Solution A [KH2PO4: 4.25 g/L; K2HPO4: 10.88 g/L; Na2HPO4,2H2O: 16.7 g/L; NH4Cl: 0.25 g/L]; 500 mL of Solution B [MgSO4,7H2O: 11.25 g/L ]; 500 mL of Solution C [CaCl2,2H2O: 18.2 g/L]; 500 mL of Solution D [FeCl3,6H2O: 0.125 g/L] each diluted in purified water to create stock solution. In order to prepare the stock solution d) immediately before use, one drop of concentrated HCI per litre was added. 50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled to a final volume of 5000 mL with ultrapure water. The pH was 7.7 and adjusted to pH 7.5 with 1M HCl solution.
- Solubilising agent (type and concentration if used): None. Test item, reference item were directly weighed and dispersed into vessels.
- Test temperature: 22 ±1 °C
- pH: See table 1.
- pH adjusted: No (final pH was in the range of 8.0 to 8.1 for test item vessels and 7.2 for inoculum control, 7.7 for procedure control).
- CEC (meq/100 g): Not reported.
- Aeration of dilution water: Not reported
- Suspended solids concentration: ca. 30 mg/L dry weight (actual: 28.7 mg sludge/L)
- Continuous darkness: Yes. The test was conducted in the dark.

TEST SYSTEM
- Culturing apparatus: 500 mL glass flasks with continuous stirring (fill volume ca. 244 mL)
- Number of culture flasks/concentration: at least duplicate per concentration (test item); In duplicate (Inoculum blank control); single flasks (toxicity control, abiotic sterile control and procedure control)
- Method used to create aerobic conditions: Sealed flasks with sensor head/CO2 trap (45% KOH solution)
- Method used to create anaerobic conditions: Not applicable.
- Measuring equipment: The respirometer/manometric system used during this study is an BSB/BOD Sensor System (BSB-Sensomat-System, Germany). Evolved carbon dioxide is absorbed by the CO2 trap (45% KOH solution)
- Test performed in closed vessels due to significant volatility of test substance: Not reported.
- Test performed in open system: No.
- Details of trap for CO2 and volatile organics if used: See above.

SAMPLING
- Sampling frequency: Daily.
- Sampling method: The respirometer/manometric system used during this study is an BSB/BOD Sensomat-System (Germany).
- Sterility check if applicable: Not reported.
- Sample storage before analysis: Not applicable.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes. See table 1.
- Abiotic sterile control: Yes.
- Toxicity control: Yes.
- Other: Positive reference control (Sodium Benzoate)

Reference substance:

benzoic acid, sodium salt

Remarks:

101.6 mg/L

Test performance:

1. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test item at end of 10-day window (as applicable) and on day 28. Therefore, the test is considered valid. The difference of duplicate values at days 17 and 28 was 13% and 1%, respectively.
2. The BOD of the inoculated blank control was 25 mgO2/L and < 60 mgO2/L after 28 days
3. The pH at day 28 was in the range of 6.0 to 8.5 (actual pH: 8.0 to 8.1 for test item vessels and 7.2 in inoculum control and 7.7 in procedure control)
4. The toxicity test attained 77% degradation at 14 days and 86% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment microorganisms used in the study.
5. Sodium Benzoate attained 86% degradation at 14 days thereby confirming the suitability of the inoculum and test conditions.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

mean (n=2) / test concentration ca. 103.1 mg/L nominal test item or 169.3 mg/L ThOD

Value:

93.5

Sampling time:

28 d

Remarks on result:

other: test flasks 1 and 2 ; 10-day window met ; mean biodegradation day 28 = 93.5%

Details on results:

(i) In the abiotic control the Oxygen Demand was 0 mg O2/L.
(ii) In the inoculum blank controls the oxygen demand was 25 mg O2/L and therefore less than 60 mgO2/L satisfying the requirements of the guideline.
(iii) In the procedure control: sodium Benzoate attained 86% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.
(iv) In the two toxicity controls, the degradation was 77% after 14 days and 86% after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was >25% within 14 days.
(v) In all the test, reference item and controls the pH was in the range of 7.2 to 8.1 (thereby was in the range of 6.0 to 8.5 required by the guideline)
(vi) The difference of duplicate values for the degradation of the test item at the plateau, at the end of the test and at the end of the 10-day window should be less than 20 %. The validity criterion was met for the period of plateau phase.
(vii) The mean test item biodegradation conducted in duplicate, was 93.5% at day 28. The 10-day window criteria was met. The test item is readily biodegradable.

Results with reference substance:

Sodium Benzoate attained 86% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

For the target substance: the mean biodegradation is considered to be 93.5% at day 28 conducted in duplicate and the 10-day window criteria met. The target substance: is considered readily biodegradable.

Executive summary:

The ready biodegradability test was carried out on a source substance according to OECD TG 301F guideline under GLP. The test item, at a concentration of ca. 103.1 mg/L nominal (or 169.3 mg/L ThODNH14) was exposed to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment plant which treats predominantly domestic sewage (Bensheim, Germany) with culture medium in sealed culture vessels in the dark at 22°C ± 1°C for 28 days. The sludge was diluted in the BOD bottles to ca. 30 mg DW/L. The degradation of the test item was assessed by the measurement of daily oxygen consumption on days 0 and 28 using a respirometer/manometric system which utilised a BSB/BOD Sensor (BSB-Sensomat-System, Germany). Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control and/or an abiotic control were used for validation purposes. In the test inoculum blank the oxygen uptake was ca. 25 mg O2/L. The pH value at the end of the test period 28 days did not exceed 8.1 in the test item systems and 7.7 in the reference item system. In the toxicity control, the degradation was 77% after 14 days and 86% after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was >25% within 14 days. The test system met the validation criteria of the guideline. Sodium Benzoate attained 86% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions. In the test vessels, a degradation of 93% and 94% after 28 days was found for the two replicates, respectively. The 10-day windows began on day 7 after application, the mean value was calculated to be 14% biodegradation (ThODNH4). Therefore, the end of the 10-day window was day 17. The mean biodegradation percentage at the end of the 10-day window was 74.5% (ThODNH4); the criterion of the 10 day window was passed. The mean biodegradation percentage at the end of the 28-day exposure period was 93.5% (ThODNH4). Under the conditions of this study, the mean biodegradation was 93.5% at day 28 conducted in duplicate. The 10-day window criteria was met. The test item is readily biodegradable.
For the target substance: the mean biodegradation is considered to be 93.5% at day 28 conducted in duplicate and the 10-day window criteria met. The target substance: is considered readily biodegradable.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-03-2022 to 07-04-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Reason / purpose for cross-reference:

other: Read-Across Target Endpoint Study Record (ESR)

Reason / purpose for cross-reference:

read-across: supporting information

Remarks:

Reporting format for the analogue approach

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: October 2019; signature: March 2020

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

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 (Test System) was obtained from the domestic waste sewage treatment plant Bensheim (Germany). The plant treats predominantly domestic sewage.
- Laboratory culture: See source of inoculum/activated sludge.
- Method of cultivation: The sample of activated sewage sludge was maintained on continuous aeration upon receipt.
- Storage conditions: See pretreatment field.
- Storage length: typically < 1 week
- Preparation of inoculum for exposure: See pre-treatment field.
- Pretreatment: The sample of activated sewage sludge was maintained on continuous aeration upon receipt. The activated sludge used for this study was deposited for 15 minutes, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and again centrifuged. This procedure was repeated three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and then aerated over night until use. Determination of dry weight is made to inoculate final solution with ca. 30mg/L dry weight activated sludge.
- Concentration of sludge: The sludge was diluted in the BOD bottles to ca. 30 mg DW/L. (actual: 28.7 mg sludge/L)
- Initial cell/biomass concentration: Not applicable.
- Water filtered: Not applicable. Culture medium was in accordance with OECD TG 301F.
- Type and size of filter used, if any: Not applicable.

Duration of test (contact time):

28 d

Initial conc.:

ca. 103.1 mg/L

Based on:

test mat.

Remarks:

Test item (test flasks 1, 2)

Initial conc.:

ca. 169.3 mg/L

Based on:

ThOD

Remarks:

corresponding to an oxygen demand of about 169.3 mg/L (ThODNH4)

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: See table 1. 500 mL of Solution A [KH2PO4: 4.25 g/L; K2HPO4: 10.88 g/L; Na2HPO4,2H2O: 16.7 g/L; NH4Cl: 0.25 g/L]; 500 mL of Solution B [MgSO4,7H2O: 11.25 g/L ]; 500 mL of Solution C [CaCl2,2H2O: 18.2 g/L]; 500 mL of Solution D [FeCl3,6H2O: 0.125 g/L] each diluted in purified water to create stock solution. In order to prepare the stock solution d) immediately before use, one drop of concentrated HCI per litre was added. 50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled to a final volume of 5000 mL with ultrapure water. The pH was 7.7 and adjusted to pH 7.5 with 1M HCl solution.
- Solubilising agent (type and concentration if used): None. Test item, reference item were directly weighed and dispersed into vessels.
- Test temperature: 22 ±1 °C
- pH: See table 1.
- pH adjusted: No (final pH was in the range of 8.0 to 8.1 for test item vessels and 7.2 for inoculum control, 7.7 for procedure control).
- CEC (meq/100 g): Not reported.
- Aeration of dilution water: Not reported
- Suspended solids concentration: ca. 30 mg/L dry weight (actual: 28.7 mg sludge/L)
- Continuous darkness: Yes. The test was conducted in the dark.

TEST SYSTEM
- Culturing apparatus: 500 mL glass flasks with continuous stirring (fill volume ca. 244 mL)
- Number of culture flasks/concentration: at least duplicate per concentration (test item); In duplicate (Inoculum blank control); single flasks (toxicity control, abiotic sterile control and procedure control)
- Method used to create aerobic conditions: Sealed flasks with sensor head/CO2 trap (45% KOH solution)
- Method used to create anaerobic conditions: Not applicable.
- Measuring equipment: The respirometer/manometric system used during this study is an BSB/BOD Sensor System (BSB-Sensomat-System, Germany). Evolved carbon dioxide is absorbed by the CO2 trap (45% KOH solution)
- Test performed in closed vessels due to significant volatility of test substance: Not reported.
- Test performed in open system: No.
- Details of trap for CO2 and volatile organics if used: See above.

SAMPLING
- Sampling frequency: Daily.
- Sampling method: The respirometer/manometric system used during this study is an BSB/BOD Sensomat-System (Germany).
- Sterility check if applicable: Not reported.
- Sample storage before analysis: Not applicable.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes. See table 1.
- Abiotic sterile control: Yes.
- Toxicity control: Yes.
- Other: Positive reference control (Sodium Benzoate)

Reference substance:

benzoic acid, sodium salt

Remarks:

101.6 mg/L

Test performance:

1. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test item at end of 10-day window (as applicable) and on day 28. Therefore, the test is considered valid. The difference of duplicate values at days 17 and 28 was 13% and 1%, respectively.
2. The BOD of the inoculated blank control was 25 mgO2/L and < 60 mgO2/L after 28 days
3. The pH at day 28 was in the range of 6.0 to 8.5 (actual pH: 8.0 to 8.1 for test item vessels and 7.2 in inoculum control and 7.7 in procedure control)
4. The toxicity test attained 77% degradation at 14 days and 86% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment microorganisms used in the study.
5. Sodium Benzoate attained 86% degradation at 14 days thereby confirming the suitability of the inoculum and test conditions.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

mean (n=2) / test concentration ca. 103.1 mg/L nominal test item or 169.3 mg/L ThOD

Value:

93.5

Sampling time:

28 d

Remarks on result:

other: test flasks 1 and 2 ; 10-day window met ; mean biodegradation day 28 = 93.5%

Details on results:

(i) In the abiotic control the Oxygen Demand was 0 mg O2/L.
(ii) In the inoculum blank controls the oxygen demand was 25 mg O2/L and therefore less than 60 mgO2/L satisfying the requirements of the guideline.
(iii) In the procedure control: sodium Benzoate attained 86% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.
(iv) In the two toxicity controls, the degradation was 77% after 14 days and 86% after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was >25% within 14 days.
(v) In all the test, reference item and controls the pH was in the range of 7.2 to 8.1 (thereby was in the range of 6.0 to 8.5 required by the guideline)
(vi) The difference of duplicate values for the degradation of the test item at the plateau, at the end of the test and at the end of the 10-day window should be less than 20 %. The validity criterion was met for the period of plateau phase.
(vii) The mean test item biodegradation conducted in duplicate, was 93.5% at day 28. The 10-day window criteria was met. The test item is readily biodegradable.

Results with reference substance:

Sodium Benzoate attained 86% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The mean biodegradation was 93.5% at day 28 conducted in duplicate. The 10-day window criteria was met. The test item is readily biodegradable.

Executive summary:

The ready biodegradability test was carried out according to OECD TG 301F guideline under GLP. The test item, at a concentration of ca. 103.1 mg/L nominal (or 169.3 mg/L ThODNH14) was exposed to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment plant which treats predominantly domestic sewage (Bensheim, Germany) with culture medium in sealed culture vessels in the dark at 22°C ± 1°C for 28 days. The sludge was diluted in the BOD bottles to ca. 30 mg DW/L. The degradation of the test item was assessed by the measurement of daily oxygen consumption on days 0 and 28 using a respirometer/manometric system which utilised a BSB/BOD Sensor (BSB-Sensomat-System, Germany). Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control and/or an abiotic control were used for validation purposes. In the test inoculum blank the oxygen uptake was ca. 25 mg O2/L. The pH value at the end of the test period 28 days did not exceed 8.1 in the test item systems and 7.7 in the reference item system. In the toxicity control, the degradation was 77% after 14 days and 86% after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was >25% within 14 days. The test system met the validation criteria of the guideline. Sodium Benzoate attained 86% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions. In the test vessels, a degradation of 93% and 94% after 28 days was found for the two replicates, respectively. The 10-day windows began on day 7 after application, the mean value was calculated to be 14% biodegradation (ThODNH4). Therefore, the end of the 10-day window was day 17. The mean biodegradation percentage at the end of the 10-day window was 74.5% (ThODNH4); the criterion of the 10 day window was passed. The mean biodegradation percentage at the end of the 28-day exposure period was 93.5% (ThODNH4). Under the conditions of this study, the mean biodegradation was 93.5% at day 28 conducted in duplicate. The 10-day window criteria was met. The test item is readily biodegradable.

Description of key information

Biodegradation: benzene-1,3,5-tricarbonyl trichloride : ‘readily biodegradable’ ; mean biodegradation 90.8% (28-days; 10-day window met), OECD TG 301F, 2022

Supporting information:

Biodegradation: target : benzene-1,3,5-tricarbonyl trichloride : ‘readily biodegradable’ ; mean biodegradation 93.5% (28-days; 10-day window met), OECD TG 301F, 2022
Read-Across: source data : benzene-1,3,5-tricarboxylic acid : OECD TG 301F, 2022

Note: The source and target substances are related by virtue of the source substance being a ‘common breakdown product’ of the target substance. The target substance hydrolytically degrades in water in a matter of seconds/minutes at all relevant pH 4, 7 and 9 forming the source substance. The read-across hypothesis has been demonstrated experimentally.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

Key study : benzene-1,3,5-tricarbonyl trichloride, OECD TG 301F, 2022 : The ready biodegradability test was carried out according to OECD TG 301F guideline under GLP. The test item, at a concentration of ca. 61.9 mg/L nominal (or 55.9 mg/L ThODNH14) was exposed to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment plant which treats predominantly domestic sewage (Rossdorf, Germany) with culture medium in sealed culture vessels in the dark at 22°C ± 1°C for 28 days. The sludge was diluted in the BOD bottles to ca. 30 mg DW/L. The degradation of the test item was assessed by the measurement of daily oxygen consumption on days 0 and 28 using a respirometer/manometric system which utilised a BSB-Sensomat-System : infrared measurement system (BSB-Sensomat-System, Germany). Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control and/or an abiotic control were used for validation purposes. In the test inoculum blank the oxygen uptake was ca. 12 mg O2/L. The pH value at the end of the test period 28 days was 6.8 to 6.9 in the test item systems and 7.6 in the reference item system. In the toxicity control, the degradation was 67% after 14 days and 84% after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was >25% within 14 days. The test system met the validation criteria of the guideline. Sodium Benzoate attained 95% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions. In the test vessels, a degradation of 102% and 79% after 28 days was found for the two replicates, respectively. The mean biodegradation percentage at the end of the 28-day exposure period was 90.8% (ThODNH4). The 10-day windows began on day 13 after application, the mean value was calculated to be 21.4% biodegradation (ThODNH4). Therefore, the end of the 10-day window was day 23. The mean biodegradation percentage at the end of the 10-day window was 81.9% (ThODNH4); the criterion of the 10-day window was passed. The difference of duplicate values for the degradation of the test item at the plateau, at the end of the test or at the end of the 10-day window should be less than 20%. The validity criterion was met for the end of the 10-day window and was 16%. Therefore the test is considered valid. Under the conditions of this study, the mean biodegradation was 90.8% at day 28 conducted in duplicate. The 10-day window criteria was met. The test item is readily biodegradable.

 

Key study : Read-Across: TARGET (benzene-1,3,5-tricarbonyl trichloride) from SOURCE (benzene-1,3,5-tricarboxylic acid) : OECD TG 301F, 2022 : The ready biodegradability test was carried out on a source substance according to OECD TG 301F guideline under GLP. The test item, at a concentration of ca. 103.1 mg/L nominal (or 169.3 mg/L ThODNH14) was exposed to activated sewage sludge micro-organisms obtained from the secondary treatment stage of the sewage treatment plant which treats predominantly domestic sewage (Bensheim, Germany) with culture medium in sealed culture vessels in the dark at 22°C ± 1°C for 28 days. The sludge was diluted in the BOD bottles to ca. 30 mg DW/L. The degradation of the test item was assessed by the measurement of daily oxygen consumption on days 0 and 28 using a respirometer/manometric system which utilised a BSB/BOD Sensor (BSB-Sensomat-System, Germany). Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control and/or an abiotic control were used for validation purposes. In the test inoculum blank the oxygen uptake was ca. 25 mg O2/L. The pH value at the end of the test period 28 days did not exceed 8.1 in the test item systems and 7.7 in the reference item system. In the toxicity control, the degradation was 77% after 14 days and 86% after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was >25% within 14 days. The test system met the validation criteria of the guideline. Sodium Benzoate attained 86% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions. In the test vessels, a degradation of 93% and 94% after 28 days was found for the two replicates, respectively. The 10-day windows began on day 7 after application, the mean value was calculated to be 14% biodegradation (ThODNH4). Therefore, the end of the 10-day window was day 17. The mean biodegradation percentage at the end of the 10-day window was 74.5% (ThODNH4); the criterion of the 10 day window was passed. The mean biodegradation percentage at the end of the 28-day exposure period was 93.5% (ThODNH4). Under the conditions of this study, the mean biodegradation was 93.5% at day 28 conducted in duplicate. The 10-day window criteria was met. The test item is readily biodegradable.
For the target substance: the mean biodegradation is considered to be 93.5% at day 28 conducted in duplicate and the 10-day window criteria met. The target substance: is considered readily biodegradable.

Note: The source and target substances are related by virtue of the source substance being a ‘common breakdown product’ of the target substance. The target substance hydrolytically degrades in water in a matter of seconds/minutes at all relevant pH 4, 7 and 9 forming the source substance. The read-across hypothesis has been demonstrated experimentally. Refer to study OECD TG 301F (2022) conducted on benzene-1,3,5-tricarbonyl trichloride for further information.