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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:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
A few modifications from the test guideline were introduced:
• Test substance and initial biodegradation product concentrations were measured by specific analysis to demonstrate primary biodegradation of the test substance.
• Analysis of dissolved organic carbon and chemical oxygen were not performed.
• Test substance and activated sludge dry weight concentration were approximately 5 mg/L and 0.2 g/L, respectively.
• The initial step in the biodegradation of the test substance is expected to be a cometabolic step. The quality of the sludge was therefore not checked with a reference substance which is degraded through growth-linked biodegradation.
• The test was operated for 2 days.
• The test was performed in triplicate with four different procedure controls.

The modified Zahn-Wellens test was performed according to the study plan, developed from the OECD Test Guideline 302 B This guideline is equivalent to EEC Guideline C.9. (1988) and ISO Test Guideline 9888 (1999).
Use was made of 3 test flasks containing mineral salt medium, test substance, and inoculum; 1 flaks containing mineral salt medium and inoculum (procedure control 1); 1 flaks containing mineral salt medium and test substance (procedure control 2); 1 flaks containing mineral salt medium test substance and acidified inoculum (procedure control 3); 1 flaks containing mineral salt medium, inoculum and 2-hydroxy-2,4,4,-trimethylpentane (procedure control 4). The test substance and 2-hydroxy-2,4,4-trimethylpentane were administered to the flasks at 4.1 mg 1,1,3,3-tetramethylbutyl hydroperoxide/L and 5.0 mg 2-hydroxy-2,4,4-trimethylpentane /L, respectively. The removal of the test substance and formation of 2-hydroxy-2,4,4-trimethylpentane was followed in the test flaks by measuring the test substance and its initial degradation product at time is 0, 3, 6, 24, 30, and 48 hours. Procedure controls were analysed on their 1,1,3,3-tetramethylbutyl hydroperoxide and 2-hydroxy-2,4,4-trimethylpentane content at the start and end of the test (time 0 and 48 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.
The primary biodegradation was calculated as the ratio of the test substance removed to the test substance concentration added at the start of the test. The measured 2-hydroxy-2,4,4-trimethylpentane concentration was compared with the theoretical alcohol concentration formed upon complete primary degradation of the test substance.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch/Lot no.: 1504510829
Purity: 92.5% Trigonox TMBH-L, 82.8% 1,1,3,3-Tetramethylbutyl hydroperoxide (see certificate of analysis)
Appearance: Clear colourless liquid
Chemical stability: Stable under test conditions
Solubility: 277 – 908 mg/L (EPISuite 2012)
Vapor pressure: 431 Pa (25 °C) (EPISuite 2012)
Log Koc: KOW method = 2.4; MCI method = 2.8 (calculated values EPISuite v4.10)
Toxicity microorganisms: EC10 = 33 mg/L and EC50 = 138 mg/L (Geerts, 2012)
Storage: In the dark between -5ºC and 25 ºC
Stability under storage conditions: Stable
Expiry date: 06-01-2019 (month/day/year)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Secondary activated sludge used as inoculum was obtained (10-09-2018) 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 acidification (pH 1.9) using HCl (12M) to kill all the biological activity. The acidified sludge was used in a procedure control and for the recovery validation of the 1,1,3,3-tetramethylbutyl hydroperoxide and 2-hydroxy-2,4,4,-trimethylpentane analysis. The dry weight concentration of the activated sludge in the units was 0.2 g/L.
Duration of test (contact time):
48 h
Initial conc.:
4.1 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Parameter followed for biodegradation estimation:
other: degradation product analysis
Details on study design:
Mineral salt medium and stock solutions
The nutrient medium of the Zahn-Wellens test contained per liter of deionized water: 85.0 mg KH2PO4, 217.5 mg K2HPO4, 266 mg Na2HPO4, 5 mg NH4Cl, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O. The test and reference substance were administered using stock solutions of 0.2 g/L in deionized water.

Test units
The test was performed in 1 L conical flasks closed gas tight with PTFE coated silicone rubber seals. Aeration in the flasks was achieved by agitating at 100 rpm in a shaking incubator at ~ 23 °C. The working volume in the flasks was 0.4 L.
Reference substance:
other: 2-Hydroxy-2,4,4,-trimethylpentane
Key result
Parameter:
% degradation (test mat. analysis)
Value:
> 40
Sampling time:
3 h
Key result
Parameter:
% degradation (test mat. analysis)
Value:
>= 90
Sampling time:
24 h
Key result
Parameter:
other: formation of 2-hydroxy-2,4,4-trimethylpentane
Value:
> 84 - < 89
Sampling time:
24 h
Details on results:
Results from procedure controls showed that a) 1,1,3,3-Tetramethylbutyl hydroperoxide and 2-hydroxy-2,4,4-trimethylpentane were not present in the mineral salt medium with activated sludge used at the start of the test, b) 1,1,3,3-Tetramethylbutyl is stable over 48 hours in the Zahn-Wellens test medium without activated sludge, c) 2-hydroxy-2,4,4-trimethylpentane was not degraded by activated sludge during the 48 hours.
The recovery of the test substance in the presence of acidified activated sludge was however unexpected low. 1,1,3,3-Tetramethylbutyl hydroperoxide was recovered 79% and <10% after incubation periods of 0 and 48 hours, respectively. The biological activity was apparently not inhibited adequately through acidification. This is also indicated by the formation of 2-hydroxy-2,4,4-trimethylpentane (59% of the test item on molar basis) after 48 hours. Activity still being present after acidification does not allow drawing a final conclusion about the possible removal mechanism (chemical or biochemically) of 1,1,3,3-tetramethylbutyl hydroperoxide. The unexpected result of this procedure control does not influence the outcome of the test because it is obvious that 1,1,3,3-tetramethylbutyl hydroperoxide is rapidly removed by either a chemical or biological reaction catalysed by activated sludge in the modified Zahn-Wellens test.
In the test units >40% of 1,1,3,3-tetramethylbutyl hydroperoxide is removed with 3 hours. No lag period was therefore detected. Stoichiometric formation of 2-hydroxy-2,4,4-trimethylpentane after 3 hours demonstrates that the organic peroxide bond of the test substance is destroyed and that removal of the test item is not the result of adsorption. A complete (≥ 90%) removal of 1,1,3,3-tetramethylbutyl hydroperoxide was already achieved after 24 hours. Complete removal of the test substance is also demonstrated by the stoichiometric formation of 2-hydroxy-2,4,4-trimethylpentane (84 – 89% recovery). The concentration of 2-hydroxy-2,4,4-trimethylpentane did not decrease between 24 and 48 hours, demonstrating that 2-hydroxy-2,4,4-trimethylpentane was not susceptible to biodegradation during 2-day exposure period.

Test conditions
The pH of the medium in the test units ranged from 7.0 to 7.1 (Table I). The pH of the procedure controls ranged from 7.0 to 7.5 with the exception of the procedure control containing acidified activated sludge (Table I). The pH in this procedure control was 5.9, which is lower than prescribed in the guideline (pH 6.5-8.0). This lower pH however does not affect the result (no effect on the biology) because it was intended to have no biological activity in this procedure control. Temperatures in the units ranged from 21.8 to 22.1 °C. Oxygen concentrations in all units were ≥ 7.9 mg/L during the test period.
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
In conclusion, the modified Zahn-Wellens test demonstrates that 1,1,3,3-tetramethyl-butyl hydroperoxide is rapidly converted into 2-hydroxy-2,4,4-trimethylpentane in the presence of activated sludge. These results allows classification of 1,1,3,3-tetramethylbutyl hydroperoxide (parent) as inherently biodegradable.
Executive summary:

The biodegradability of 1,1,3,3-tetramethylbutyl hydroperoxide was assessed by using the Zahn-Wellens test guidelines (OECD Test Guideline 302 B (1992), ISO Test Guideline 9888 (1999) and EEC Guideline C.9.) with some modifications. The modified Zahn-Wellens test is designed to determine primary biodegradation of a test substance. 1,1,3,3-Tetramethylbutyl hydroperoxide was added to an aqueous solution containing mineral salts and relatively high numbers of unadapted microorganisms. 1,1,3,3-Tetramethylbutyl hydroperoxide and 2-hydroxy-2,4,4-trimethylpentane concentrations were monitored in time using HPLC and GC analysis, respectively. Both analytical methods were satisfactory with regard to the linearity, repeatability of the injections, limit of quantification (LOQ), recovery, and specificity.In the modified Zahn-Wellens test a complete (≥ 90%) removal of 1,1,3,3-tetramethylbutyl hydroperoxide was achieved within 24 hours without a detectable lag phase. In the presence of activated sludge 1,1,3,3-tetramethylbutyl hydroperoxide was rapidly transformed into 2-hydroxy-2,4,4-trimethylpentane. This conversion was stoichiometrically as demonstrated by the 84 – 89% recovery of the alcohol. 2-Hydroxy-2,4,4-trimethylpentane was not removed during the 48-hour incubation period in the modified Zahn-Wellens test. 1,1,3,3-Tetramethylbutyl hydroperoxide (parent) should therefore be classified as inherently biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 23 2011 - August 23 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 301D study carried out under GLP
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
ammonium chloride was not added to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound).
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
n/a
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge (17-06-2011) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This activated sludge plant treats 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
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:
Nutrients and stock solutions
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. Sodium acetate was added to the bottles using aqueous stock solution of 1.0 g/L. Accurate administration of water-insoluble 1,1,3,3-tetramethylbutyl hydroperoxide was accomplished by dosing the test substance on silica gel (3.0 mg/g).

Test procedures
The Closed Bottle test was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Administration was accomplished by dosing 0.03 g of test substance on 10 g of silica gel in a 50-mL serum flask. Only part of the top layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw top with alumiminium foil and the content was mixed vigorously. Subsequently 0.2 g of silica gel dosed with test substance was added to the test bottles. The resulting concentration of test substance in the bottles was 2.0 mg/L. Use was made of 10 bottles containing only inoculum (not agitated), 6 bottles containing inoculum and sodium acetate (not agitated), 10 bottles containing inoculum and test substance (agitated), and 10 bottles with 0.2 g silica gel containing inoculum (agitated). The contents of bottles were agitated with magnetic stirrers at 600 rpm. The concentrations of the test substance and sodium acetate in the bottles were 2.0 and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were com-pletely filled without air bubbles. The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28. One extension from the protocol of the Closed Bottle test was intro-duced. The Closed Bottle test was prolonged by measuring the course of the oxygen decrease in the bottles of day 28 using a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle
Reference substance:
acetic acid, sodium salt
Remarks:
>99% pure
Test performance:
The validity of the test is demonstrated by an endogenous respiration of 0.8 mg/L at day 28. 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 85. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
7 d
Remarks on result:
other: test extended to 60 days
Details on results:
Theoretical oxygen demand (ThOD)
The calculated theoretical oxygen demand (ThOD) of 1,1,3,3-tetramethylbutyl hydroperoxide is 2.5 mg/mg. It is assumed that the ThOD of the impurities (~10%) is also 2.5 mg/mg. g. ThOD of sodium acetate is 0.78 mg/mg.

Test conditions
The pH of the media was 7.2 at the start of the test. The pH of the medium at day 28 was 7.2 (controls) and 7.1 (test). Temperatures were within the prescribed temperature range of 22 to 24°C.
Results with reference substance:
see Table below

Oxygen consumption (mg/L) and the percentages biodegradation of the test substance, 1,1,3,3-tetramethylbutyl hydroperoxide (BOD/ThOD) and sodium acetate (BOD/ThOD) in the Closed Bottle test.

Time (days)

Oxygen consumption (mg/L)

Biodegradation (%)

Test substance

Acetate

Test substance

Acetate

 

 

 

 

 

0

0.0

0.0

0

0

7

0.0

4.2

0

78

14

-0.1

4.6

0

85

21

-0.1

 

0

 

28

0.0

 

0

 

42

-0.1

 

0

 

60

-0.1

 

0

 

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
1,1,3,3-Tetramethylbutyl hydroperoxide is not biodegraded in the Closed Bottle test and should therefore not be classified as readily biodegradable. Lack of biodegradation does not mean that 1,1,3,3-tetramethylbutyl hydroperoxide is recalcitrant in nature. The stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.
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 (OECD TG 301), EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

1,1,3,3-Tetramethylbutyl hydroperoxide did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. 1,1,3,3-Tetramethylbutyl hydroperoxide was 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 1,1,3,3-tetramethylbutyl hydroperoxide 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 0.8 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 85% 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.

Description of key information

One OECD 301D GLP test is available where 1,1,3,3-Tetramethylbutyl hydroperoxide is not biodegraded in the Closed Bottle test and should therefore not be classified as readily biodegradable.

In a modified Zahn-Wellens test (OECD 302B) performed under GLP complete removal (90%) of 1,1,3,3 -Tetramethylbutyl hydroperoxide was achieved within 24h, the substance can therefore be classified as inherently biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information