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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
no GLP
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Principles of method if other than guideline:
Trigonox 501, was administered to the flasks at 5.0 mg 3,6,9-Triethyl-3,6,9-trimethyl-1,4,7-triperoxonane /L and 5.0 mg 1,2,4,5,7,8-hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris(Ethyl and Propyl) /L, respectively. MEK and MPK were spiked at 5 mg/L to the flasks. The removal of the test substance by the formation of MEK and MPK was followed in the test flaks by measuring the MEK and MPK concentrations at time is 0, 3, and 24 hours. Primary biodegradation was calculated as the ratio of formed degradation product, after each time interval, to the theoretical maximum formed degradation product concentration. The procedure control flasks with heat killed inoculum, without inoculum and spiked with MEK and MPK were also analyzed at time is 0, 3 and 24 hours.
The pH and oxygen concentrations in the flasks were determined at the end of the test. The incubation temperature was monitored and recorded during incubation.

Analyses
The pH was measured with a pH meter (EUTECH). The temperature was measured and recorded with a Smart-Vue digital thermometer (Thermo Scientific). The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW).
The dry weight of the inoculum was determined by filtrating 30 mL of the activated sludge over a preweighed 12 µm cellulose nitrate filter. This filter and retained sludge solids were dried for minimal 1.5 hours at 104 ± 5 °C and weighed after cooling. The concentration of suspended solids (dry weight) was calculated by subtracting the mass of the weighed filter and divide by the filtrated volume.
The test units were sampled through the septa to minimize losses by volatilization using a needle and syringe. Samples were subsequently filtered using PTFE syringe filters with pores of 0.45 µm. Filtered samples were prepared immediately for analysis. The expected formation of MEK and MPK upon primary biodegradation of Trigonox 501 were measured by specific analysis.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Secondary activated sludge used as inoculum was obtained from the WWTP Nieuwgraaf in Duiven (the Netherlands). The WWTP Duiven is an activated sludge plant treating predominantly domestic wastewater. The collected activated sludge was used on the day of collection. Prior use the sludge was washed twice with tap water by separating the sludge from the supernatant through settlement. Subsequently the washed sludge was homogenized by pressing it through a needle with a syringe. Part of the washed and homogenized sludge was heated to 60°C for 15 minutes in order to kill all the biological activity. The dry weight concentration of the activated sludge in the units was 0.2 g/L.
Duration of test (contact time):
24 h
Initial conc.:
5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
See above "principle of method".
Details on results:
Test conditions
The pH of the medium in the test units ranged from 6.9 to 7.1. Temperatures in the units ranged from 21.7 to 22.2 °C. Oxygen concentrations in all units were ≥ 7.9 mg/L during the test period. These test conditions are believed to allow biodegradation by microorganisms present in the activated sludge.

Zahn-Wellens test
Alkyl hydroperoxide reductases may co-metabolic cleave the organic peroxide bonds present in Trigonox 501 resulting in the formation of a mixture of MEK and MPK, respectively. This primary biodegradation of Trigonox 501 was assessed in the modified Zahn-Wellens test by specific analyses of MEK and MPK in time. A GC-MS method was used to analyze the MEK and MPK. Validation results of the regression, accuracy, quality control, recovery, repeatability, LOQ, and system stability for the analysis are summarized in the table under "details on analytical method". The validation parameters set for both analysis methods were fulfilled.
Alkyl hydroperoxide reductase activities observed in activated sludge range between 10 to 33 μM hour-1 . g sludge dry weight-1 (Ginkel and Geerts, 2017). Assuming this range of activity Trigonox 501 should be removed within 24 hours in the Zahn Wellens test. Approximately 4 mg/L MEK + MPK will be theoretically formed upon reduction of 5 mg/L Trigonox 501. These concentrations of MEK and MPK are well detectable by the GC-MS method. Recoveries of MEK and MPK spiked at 5 mg/L to the Zahn Wellens test ranged from 86-92% and from 84 - 96%, respectively. Reliable analysis of the theoretical MEK and MPK concentrations formed in the Zahn Wellens test is therefore possible.
MEK and MPK were not formed in the tests with Trigonox 501 spiked to mineral salt medium or spiked to mineral salt medium with heat killed activated sludge. This demonstrates that in the Zahn Wellens test the peroxide bonds in Trigonox 501 are not reduced by a chemical reaction. MEK and MPK were however also not measured for Trigonox 501 in the presence of living activated sludge. Trigonox 501 is therefore not removed by alkyl hydroperoxide reductases. The active site of alkyl hydroperoxid reductase most likely cannot accommodate ketone peroxides such as Trigonox 501.

Table 2Concentrations of MEK and MPK measured in the Zahn Wellens test in time.

Sample

Concentration (mg/L)

MEK

MPK

5 mg/L Tx-301

(t=0 , 3 and 24 hours)

Mineral salts medium

<LOQ

n.a.

Mineral salts medium and heat killed inoculum

<LOQ

n.a.

Mineral salts medium and inoculum

<LOQ

n.a.

5 mg/L Tx-501

(t=0 , 3 and 24 hours)

Mineral salts medium

<LOQ

<LOQ

Mineral salts medium and heat killed inoculum

<LOQ

<LOQ

Mineral salts medium and inoculum

<LOQ

<LOQ

5 mg/L MEK t=0 hours

Mineral salts medium and inoculum

4.6

n.a.

5 mg/L MEK t=3 hours

Mineral salts medium and inoculum

4.3

n.a.

5 mg/L MEK t=24 hours

Mineral salts medium and inoculum

4.3

n.a.

5 mg/L MPK t=0 hours

Mineral salts medium and inoculum

n.a.

4.4

5 mg/L MPK t=3 hours

Mineral salts medium and inoculum

n.a.

4.8

5 mg/L MPK t=24 hours

Mineral salts medium and inoculum

n.a.

4.2

n.a. =not applicable

Interpretation of results:
not inherently biodegradable
Conclusions:
MEK and MPK were not formed in the tests with Trigonox 501 spiked to mineral salt medium or spiked to mineral salt medium with heat killed activated sludge. This demonstrates that in the Zahn Wellens test the peroxide bonds in Trigonox 501 are not reduced by a chemical reaction. MEK and MPK were however also not measured for Trigonox 501 in the presence of living activated sludge. Trigonox 501 are therefore not removed by alkyl hydroperoxide reductases. The active site of alkyl hydroperoxid reductase most likely cannot accommodate ketone peroxides such as Trigonox 501.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov 2014 - Jan 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD GLP study well described
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Secondary activated sludge (06-11-2014) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge plant treating predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the BOD bottles (van Ginkel and Stroo, 1992).
Duration of test (contact time):
60 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

Nutrients, stocks and administration
The nutrient medium of the Closed Bottle test contained per liter of deionized water; 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. Accurate administration of the test material, and mineral oil were accomplished with solid stocks of 3.0 mg test material/g silica gel and 3.0 mg mineral oil/g silica gel, respectively. Subsequently 0.12 and 0.2 g of silica gel dosed with mineral oil and test substance, respectively were added to the respective bottles. Next the bottles were filled with nutrient medium with inoculum and closed. The control bottles contained 0.2 g silica gel. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L in water.
Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
7
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
7
Sampling time:
60 d

Theoretical oxygen demand (ThOD)

The calculated theoretical oxygen demand (ThOD) of active substance (reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl-3,6,9-trimethyl-9-npropyl- 1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl-1,2,4,5,7,8- hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl-1,2,4,5,7,8-hexoxonane), and mineral oil are 1.9 and 3.5 mg/mg, respectively. The ThOD of test material containing active ingredient, and mineral oil is 2.9 mg/mg. The ThOD of sodium acetate is 0.8 mg/mg.

Toxicity

Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of the test substance to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected (Table I). Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

Test conditions

The pH of the media was 7.3 at the start of the test. The pH of the medium at day 28 was 7.3 (control with silica gel) and 7.2 (control, test, and mineral oil). Temperatures were within the prescribed temperature range of 22 to 24°C.

Validity of the test

The validity of the test is demonstrated by an endogenous respiration of 0.8 mg/L at day 28 (Table I). Furthermore, the differences of the replicate values at day 28 were less than 20%.The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 87. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Biodegradability

The mineral oil was biodegraded by 64% at day 28 in the Closed Bottle test (Figure and Tables). The reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6- diethyl-3,6,9-trimethyl-9-n-propyl-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9- di-n-propyl-1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl-1,2,4,5,7,8- hexoxonane (active ingredient) was not biodegraded in the Closed Bottle test (Figure and Tables). The 7% achieved at days 7, 14, 28, 42, and 60 is considered as not significant due to the low concentration the active ingredient and the high concentration of the biodegradable mineral oil. The active ingredient should therefore not be classified as readily biodegradable. Lack of biodegradation does not mean that the test substance is recalcitrant in nature. The stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable, not fulfilling specific criteria
Conclusions:
The reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl- 3,6,9-trimethyl-9-n-propyl-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-npropyl- 1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl-1,2,4,5,7,8- hexoxonane was biodegraded 7% at day 28 and 60.
Executive summary:

ABSTRACT

In order to assess the biotic degradation, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

The presence of the test material did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. The reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl- 3,6,9-trimethyl-9-n-propyl-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-npropyl- 1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6,9-tri-n-propyl-1,2,4,5,7,8- hexoxonane was biodegraded 7% at day 28 and 60. The test material was therefore not biodegraded in the Closed Bottle test (28 days and prolonged) and should therefore not be classified as readily biodegradable. The lack of biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

The test is valid as shown by an endogenous respiration of 0.9 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 81% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
no GLP
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Principles of method if other than guideline:
Trigonox 301, was administered to the flasks at 5.0 mg 3,6,9-Triethyl-3,6,9-trimethyl-1,4,7-triperoxonane /L and 5.0 mg 1,2,4,5,7,8-hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris(Ethyl and Propyl) /L, respectively. MEK and MPK were spiked at 5 mg/L to the flasks. The removal of the test substance by the formation of MEK and MPK was followed in the test flaks by measuring the MEK and MPK concentrations at time is 0, 3, and 24 hours. Primary biodegradation was calculated as the ratio of formed degradation product, after each time interval, to the theoretical maximum formed degradation product concentration. The procedure control flasks with heat killed inoculum, without inoculum and spiked with MEK and MPK were also analyzed at time is 0, 3 and 24 hours.
The pH and oxygen concentrations in the flasks were determined at the end of the test. The incubation temperature was monitored and recorded during incubation.

Analyses
The pH was measured with a pH meter (EUTECH). The temperature was measured and recorded with a Smart-Vue digital thermometer (Thermo Scientific). The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW).
The dry weight of the inoculum was determined by filtrating 30 mL of the activated sludge over a preweighed 12 µm cellulose nitrate filter. This filter and retained sludge solids were dried for minimal 1.5 hours at 104 ± 5 °C and weighed after cooling. The concentration of suspended solids (dry weight) was calculated by subtracting the mass of the weighed filter and divide by the filtrated volume.
The test units were sampled through the septa to minimize losses by volatilization using a needle and syringe. Samples were subsequently filtered using PTFE syringe filters with pores of 0.45 µm. Filtered samples were prepared immediately for analysis. The expected formation of MEK and MPK upon primary biodegradation of Trigonox 501 were measured by specific analysis.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Secondary activated sludge used as inoculum was obtained from the WWTP Nieuwgraaf in Duiven (the Netherlands). The WWTP Duiven is an activated sludge plant treating predominantly domestic wastewater. The collected activated sludge was used on the day of collection. Prior use the sludge was washed twice with tap water by separating the sludge from the supernatant through settlement. Subsequently the washed sludge was homogenized by pressing it through a needle with a syringe. Part of the washed and homogenized sludge was heated to 60°C for 15 minutes in order to kill all the biological activity. The dry weight concentration of the activated sludge in the units was 0.2 g/L.
Duration of test (contact time):
24 h
Initial conc.:
5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
See above "principle of method".
Details on results:
Test conditions
The pH of the medium in the test units ranged from 6.9 to 7.1. Temperatures in the units ranged from 21.7 to 22.2 °C. Oxygen concentrations in all units were ≥ 7.9 mg/L during the test period. These test conditions are believed to allow biodegradation by microorganisms present in the activated sludge.

Zahn-Wellens test
Alkyl hydroperoxide reductases may co-metabolic cleave the organic peroxide bonds present in Trigonox 301 resulting in the formation of MEK. This primary biodegradation of Trigonox 301 was assessed in the modified Zahn-Wellens test by specific analyses of MEK in time. A GC-MS method was used to analyze the MEK. Validation results of the regression, accuracy, quality control, recovery, repeatability, LOQ, and system stability for the analysis are summarized in the table under "details on analytical method". The validation parameters set for both analysis methods were fulfilled.
Alkyl hydroperoxide reductase activities observed in activated sludge range between 10 to 33 μM hour-1 . g sludge dry weight-1 (Ginkel and Geerts, 2017). Assuming this range of activity Trigonox 301 should be removed within 24 hours in the Zahn Wellens test. Approximately 4 mg/L MEK will be theoretically formed upon reduction of 5 mg/L Trigonox 301. These concentrations of MEK is well detectable by the GC-MS method. Recoveries of MEK spiked at 5 mg/L to the Zahn Wellens test ranged from 86-92%. Reliable analysis of the theoretical MEK concentrations formed in the Zahn Wellens test is therefore possible.
MEK was not formed in the tests with Trigonox 301 spiked to mineral salt medium or spiked to mineral salt medium with heat killed activated sludge. This demonstrates that in the Zahn Wellens test the peroxide bonds in Trigonox 301 are not reduced by a chemical reaction. MEK was however also not measured for Trigonox 301 in the presence of living activated sludge. Trigonox 301 is therefore not removed by alkyl hydroperoxide reductases. The active site of alkyl hydroperoxid reductase most likely cannot accommodate ketone peroxides such as Trigonox 301.

Table 2Concentrations of MEK and MPK measured in the Zahn Wellens test in time.

Sample

Concentration (mg/L)

MEK

MPK

5 mg/L Tx-301

(t=0 , 3 and 24 hours)

Mineral salts medium

<LOQ

n.a.

Mineral salts medium and heat killed inoculum

<LOQ

n.a.

Mineral salts medium and inoculum

<LOQ

n.a.

5 mg/L Tx-501

(t=0 , 3 and 24 hours)

Mineral salts medium

<LOQ

<LOQ

Mineral salts medium and heat killed inoculum

<LOQ

<LOQ

Mineral salts medium and inoculum

<LOQ

<LOQ

5 mg/L MEK t=0 hours

Mineral salts medium and inoculum

4.6

n.a.

5 mg/L MEK t=3 hours

Mineral salts medium and inoculum

4.3

n.a.

5 mg/L MEK t=24 hours

Mineral salts medium and inoculum

4.3

n.a.

5 mg/L MPK t=0 hours

Mineral salts medium and inoculum

n.a.

4.4

5 mg/L MPK t=3 hours

Mineral salts medium and inoculum

n.a.

4.8

5 mg/L MPK t=24 hours

Mineral salts medium and inoculum

n.a.

4.2

n.a. =not applicable

Interpretation of results:
not inherently biodegradable
Conclusions:
MEK was not formed in the tests with Trigonox 301 spiked to mineral salt medium or spiked to mineral salt medium with heat killed activated sludge. This demonstrates that in the Zahn Wellens test the peroxide bonds in Trigonox 301 are not reduced by a chemical reaction. MEK was however also not measured for Trigonox 301 in the presence of living activated sludge. Trigonox 301 are therefore not removed by alkyl hydroperoxide reductases. The active site of alkyl hydroperoxid reductase most likely cannot accommodate ketone peroxides such as Trigonox 301.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: complete and under GLP
Qualifier:
according to
Guideline:
other: Closed Bottle Test, OECD method 301 and EU test method C.6.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Duration of test (contact time):
28 d
Reference substance:
other: sodium acetate
Parameter:
% degradation (O2 consumption)
Value:
3
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
65
Sampling time:
140 d
Details on results:
Points of degradation plot (test substance):
0 % degradation after 7 d
0 % degradation after 14 d
3 % degradation after 28 d
13 % degradation after 56 d
39 % degradation after 84 d
59 % degradation after 120 d
65 % degradation after 140 d
Results with reference substance:
Points of degradation plot (reference substance):
59 % degradation after 7 d
75 % degradation after 14 d
Validity criteria fulfilled:
yes
Interpretation of results:
other: not readily but inherently biodegradable
Conclusions:
Under the conditions of the test, methylethylketone peroxide trimer is not readily biodegradable, but is inherently biodegradable from the results of the extended biodegradation test.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
09-1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: under GLP and complete
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
EU method C.7 of 92/69/EEC
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Duration of test (contact time):
28 d
Parameter:
% degradation (CO2 evolution)
Value:
9
Validity criteria fulfilled:
yes
Interpretation of results:
not inherently biodegradable
Conclusions:
test is valid

Description of key information

The result of an OECD 301D tests indicate that the 1,2,4,5,7,8-Hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris(Ethyl and Propyl) derivatives was biodegraded 7% at day 28 and 60. An OECD 302B study did not show biodegradation either.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

In order to assess the biotic degradation, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

The presence of the test material did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. 1,2,4,5,7,8-Hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris(Ethyl and Propyl) derivatives was biodegraded 7% at day 28 and 60. The test material was therefore not biodegraded in the Closed Bottle test (28 days and prolonged) and should therefore not be classified as readily biodegradable. The lack of biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

No biodegradation was found in the OECD 302B study. MEK and MPK were not formed in the tests with Trigonox 501 spiked to mineral salt medium or spiked to mineral salt medium with heat killed activated sludge. This demonstrates that in the Zahn Wellens test the peroxide bonds in Trigonox 501 are not reduced by a chemical reaction. MEK and MPK were however also not measured for Trigonox 501 in the presence of living activated sludge. Trigonox 501 are therefore not removed by alkyl hydroperoxide reductases. The active site of alkyl hydroperoxid reductase most likely cannot accommodate ketone peroxides such as Trigonox 501.

Studies with the main constituent (CAS 24748 -23 -0) showed similar results. The substance was not found readily biodegradable and neither inherently biodegradable.