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Environmental fate & pathways

Biodegradation in water and sediment: simulation tests

Administrative data

Endpoint:
biodegradation in water: sewage treatment simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units)
Principles of method if other than guideline:
-The primary settled sewage was collected weekly and stored in the refrigerator until required instead of a daily collection of wastewater.
-The units consisted of aeration vessels capable of holding only 0.35 L from which the liquor was then passed continuously to settler of 0.30 L capacities.
These minor deviations do not have an effect on the outcome of the test.

-The CAS test unit was modified, after finalizing the standard test, to determine the removal by evaporation. A gas-scrubbing system was set-up and validated.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
EC name 1,1,3,3-Tetramethylbutyl hydroperoxide
Batch/Lot no. 1504510829
Purity 92.5%
Appearance Clear colourless liquid
Chemical stability Stable under test conditions
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 01-06-2018
Radiolabelling:
no

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on source and properties of surface water:
N/A
Details on source and properties of sediment:
N/A
Details on inoculum:
Secondary activated sludge to inoculate the test at the start was collected on 27-09-2017 from the wastewater treatment plant (WWTP) Nieuwgraaf in Duiven, The Netherlands. The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic sewage. 0.35 liter of secondary activated sludge containing approximately 3 g/L dry weight was used as an inoculum for each CAS unit.
Secondary activated sludge was collected weekly throughout the test period. Primary settled sewage was collected from the same plant weekly and stored frozen until required. Before use 10 mL/L of a NaHCO3 (10 g/L) solution was added.
Duration of test (contact time):
42 d
Initial test substance concentration
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
The CAS test was operated for a period of 8 weeks and was performed according to ISO (1995), EC (1988) and OECD (1981) test guidelines. The test and control unit were not coupled. The units were started with activated sludge. The aeration was achieved by operating an air-lift. The aeration rate was regulated (8-9 L/hour) so that the activated sludge was kept in suspension and the dissolved oxygen concentration was at least 2 mg/L. This oxygen concentration in the aeration vessel was measured at least two times a week. The domestic sewage was supplied at a rate of approximately 1.4 L/day to give a hydraulic retention time of 6 hours. The flow was checked by measuring the total volume of effluent
over a 24-hour period. After brushing, 35 mL of sludge was daily removed from the aeration tank to maintain a sludge retention time of 10 days. To maintain an activated sludge concentration between 2 and 3 g/L dry weight in the CAS unit unacclimated secondary activated sludge containing approximately 3 g/L dry weight was added daily (Table I). Effluent samples (50 mL) were taken from the settler.
1,1,3,3-Tetramethylbutyl hydroperoxide was directly added to the test unit using a syringe pump. To prevent precipitation of the stock suspension in the syringe the suspension was stirred by using a small magnetic stirrer bar inside the syringe. The flow rate of the syringe pump was 9.6 mL/day giving a nominal test substance influent concentration of 30 mg/L at a sewage supply rate of 1.4 L/day.
The NPOC values were primarily used to assess the performance of biological treatment system fed with 1,1,3,3-tetramethylbutyl hydroperoxide containing wastewater and to preliminary follow the removal of the test substance during the test period. NPOC values of the last period of the test were used to calculate the mean removal percentage. The daily removal percentages were calculated by the following equation: 100 x (CT-(Ct-Cc)) / CT. Where CT is the carbon content of the test compound added to the settled sewage, Ct is the carbon found as NPOC in the effluent of the CAS unit spiked with the test substance and Cc is the carbon found as NPOC in the effluent of the control CAS unit.
The NPOC analysis values in the test and control unit were treated as paired observations. Outliers of the mean difference (Xd) series were eliminated according to the Dixon test at a 95% probability level.
From the set of 'n' paired observations the mean difference (Xd) and the standard deviation (Sd) were calculated. The Sd is calculated with the following formula Sd = Öå(`x -X)/n-1. The statistical significance of the observed difference was then assessed from the t-statistics given by the following equation: t-value = |Xd| x Ön/Sd. The critical value of t at the required confidence level was obtained from statistical tables for a one tailed test with n-1 degrees of freedom. A significant difference is obtained when the calculated t-value is higher than the critical t-value.
The percentage biodegradation/removal was given by; (SL-Xd)/SL x 100 where Xd the mean difference and SL is the spiking level, both values being expressed in mg/L carbon. The 95% confidence interval of the mean difference was calculated as follows: tn x Sd /Ön where tn is the t statistic for a two-tailed test, n-1 degrees of freedom, P = 0.05.
Specific analyses of 1,1,3,3-tetramethylbutyl hydroperoxide were used to determine the primary removal of the test substance. The removal percentage of 1,1,3,3-tetramethylbutyl hydroperoxide was determined with the following equation; (Is-Es)/Is x 100, where Is is the nominal test substance concentration in the influent and Es is the mean of the measured test substance concentrations in the effluent.
The concentration of the test substance on the activated sludge (Csludge) and the theoretical maximum concentration on sludge are used to assess the removal of the test substance by adsorption. Provided either biodegradation nor evaporation of the test substance occurs in the system, the theoretical maximum concentration of 1,1,3,3-tetramethylbutyl hydroperoxide adsorbed onto the sludge is; Cmax adsorption = Is x SRT/HRT, where SRT is the sludge retention time and HRT is the hydraulic retention time (both expressed in days). The % removal of 1,1,3,3-tetramethylbutyl hydroperoxide by adsorption is 100 x Csludge/Cmax adsorption.

After 8 weeks the CAS test unit was modified to determine the removal by evaporation.
Modification of the CAS unit included closing of the top and inlet of the aeration vessel, closing of the top and outlet of the settler and removing the effluent with a pump from the settler. The pressure in the gas phases of aeration vessel and settler was kept equal through an open connection. Operation of the CAS test remained unchanged and during a period of seventeen hours air from the aeration vessel was directed through three scrubbers in series containing a known amount of acetonitrile as scrubber liquid.
Subsequently the acetonitrile from the scrubbers was sampled and analyzed for the 1,1,3,3- tetramethylbutyl hydroperoxide and 2-hydroxy-2,4,4-trimethylpentane content. Evaporation of the test substance was calculated by the following equation: 100 x Cs / Cn Where Cn is the nominal mass of the test compound added to the settled sewage over the seventeen hour period and Cs is the total mass of test compound found in the three scrubbers over the same period.
To validate the gas-scrubbing system, the degree of scrubbing of the test substance and its primary biodegradation product from the gas-phase was determined. A CAS unit containing 480 mL demineralized water supplemented with 1,1,3,3-tetramethylbutyl hydroperoxide (99.7 mg/L) and 2- hydroxy-2,4,4-trimethylpentane (105.3 mg/L) was places in series with three scrubbers containing a known amount of acetonitrile as scrubber liquid. Subsequently, the same aeration gas-flow as used in the test CAS units (8-9 L/hour) was directed during seventeen hours from the aeration vessel through the three scrubbers in series. The concentration of 1,1,3,3-tetramethylbutyl hydroperoxide and 2- hydroxy-2,4,4-trimethylpentane was determined in the scrubber liquids.

Results and discussion

Test performance:
Test conditions:
The temperature of MLSS in both CAS units ranged from 20.0 to 22.7°C and the pH of the effluents varied from 7.0 to 7.4. The oxygen concentrations measured in both units were always ≥2.5 mg/L. These test conditions are believed to allow biodegradation by microorganisms present in activated sludge.
The CAS test was started with a high concentration of aerobic microorganisms (2.5 - 3.0 g/L dry weight) maintained by the daily addition of primary settled sewage and sludge from a full-scale treatment plant.
The daily removal of 35 mL of activated sludge from the aeration vessel resulted in a sludge retention time of 10 days. The dry weight of the MLSS in the CAS units ranged from 2.3 to 3.1 g/L.
The performance of the control unit was checked by measuring the COD removal (day 14 and day 49) and the concentrations of ammonium and nitrite in the influent and effluent (day 14). At day 14 the COD contents in the influent and effluent were 315 and 27 mg/L, respectively. At day 42, the COD levels in the influent and effluent were 368 and 29 mg/L, respectively. COD removal percentages were 91 (day 14) and 92 (day 49). The nitrite-nitrogen concentrations in the influent and effluent at day 14 were < 0.6 mg/L. The ammonium-nitrogen concentrations at day 14 were 59 mg/L (influent) and < 1 mg/L (effluent). These results demonstrate that the criteria as prescribed in the OECD 303A guideline are met and the test is therefore valid.
Mean total recovery
Compartment:
other: water / sediment, material (mass) balance
Remarks on result:
other: See below
% Degradationopen allclose all
Key result
% Degr.:
> 99.2
Parameter:
test mat. analysis
Remarks on result:
other: mean removal from effluent
Key result
% Degr.:
< 0.1
Parameter:
test mat. analysis
Remarks on result:
other: removal by sludge adsorption
Key result
% Degr.:
< 0.4
Parameter:
test mat. analysis
Remarks on result:
other: removal by volatilization
Key result
% Degr.:
> 99.5
Parameter:
test mat. analysis
Remarks on result:
other: removal by biodegradation
Half-life of parent compound / 50% disappearance time (DT50)
Compartment:
other:
Type:
other:
Temp.:
20 °C
Remarks on result:
other: Not derivable from this test
Transformation products:
no
Details on transformation products:
The test substance dosed as an emulsion to the CAS unit most likely adsorbs to the activated sludge. Nevertheless, the NPOC results demonstrate that the continuous activated sludge system treating domestic wastewater spiked with di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide removes 100% of the organic carbon of the test substance and formation of water-soluble substances during the biodegradation of di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide can be excluded
Evaporation of parent compound:
no
Volatile metabolites:
no
Residues:
no

Any other information on results incl. tables

Assessment of removal by non-purgeable organic carbon (NPOC) content:

Both units were preconditioned for one week. After this week sludge wastage was started and the test substance was introduced to one of the units with a syringe pump. The test substance was added by spiking the influent with a suspension of the test substance to a predetermined concentration of 30 mg/L. The calculated carbon content of 30 mg/L test substance in the influent of the reactor was 16.5 mg/L. This carbon content was calculated using the 1,1,3,3 - tetramethylbutylhydroxide concentration in the test substance. The measured concentration of the

NPOC in an aqueous solution with 30 mg/L test substance was 19.1 mg/L. After filtration through an 8 μm cellulose nitrate filter the NPOC was 18.9 mg/L. No losses of the test substance are therefore expected by the filtration step. The measured carbon content of the test substance is slightly higher compared to the calculated concentration, most likely as a result of impurities present in the test substance. The lower theoretical carbon content of 16.5 mg/L (CT) was used to calculate the carbon removal percentages. The calculated organic carbon removal in the CAS test is therefore a conservative (worst-case) calculation of the removal.

In the first week after the introduction of the test substance a NPOC removal of ~ 80% was achieved.

The NPOC removal from day 9-16 was lower again and ranged from 47-64%. This increase of the NPOC content in the effluent of the CAS test unit was probably the result of the formation of 2-hydroxy-2,4,4-trimethylpentane. After day 16 the NPOC removal increased to removals ≥100% suggesting biodegradation of the formed 2-hydroxy-2,4,4-trimethylpentane. NPOC removal percentages reached a plateau starting at about day 23. From day 23 until the end of the test the organic carbon concentrations in the effluent of the CAS unit fed with 1,1,3,3-tetramethylbutyl hydroperoxide were generally (12 out of 14 sampling dates) lower than in the effluent of the control CAS unit. From day 22 to 42 (end of test), 15 samples were taken to assess a mean NPOC removal percentage. One outlier (day 22) was identified using the Dixon test. Subsequently, the remaining data were used in a statistical test. The mean difference between the NPOC in the effluents of control and test unit was -2.5 ± 1.2 mg/L (95 percent confidence interval).

The mean removal percentage calculated with this mean difference was 115 ± 7 (95% confidence). The t-statistics (n = 14) did exceed the critical value and the mean difference is therefore statistically significant. The statistically significant better removal of the organic carbon in the test unit cannot be explained. Nevertheless, the results demonstrate that the continuous activated sludge system treating domestic wastewater spiked with 1,1,3,3-tetramethylbutyl hydroperoxide removes 100% of the organic carbon of the test substance and formation of water-soluble substances during the biodegradation of 1,1,3,3-tetramethylbutyl hydroperoxide can be excluded.

Specific analysis of 1,1,3,3-tetramethylbutyl hydroperoxide and 2-hydroxy-2,4,4 -trimethylpentane:

The removal of 1,1,3,3-tetramethylbutyl hydroperoxide and the formation of 2-hydroxy-2,4,4 -trimethylpentane in the CAS test was assessed by using specific HPLC and GC analyses, respectively.

HPLC analysis confirmed the nominal concentration (101% recovery) of the stock suspension dosed to the CAS unit. The stock suspension dosed to the CAS unit was found stable during four days storage at room temperature (maximum time of stock emulsion in syringe) as shown by the 99 % recovery of the test substance. Influent, effluent and sludge samples were spiked with 1,1,3,3-tetramethylbutyl hydroperoxide to determine the recovery and stability of the test substance during storage in refrigerator and freezer. Acceptable recoveries between 70 and 110% were obtained for effluent and sludge samples spiked at 3.1, 29.8 mg/L and 12.7, 144 mg/L, respectively. Recoveries of influent spiked with 3.1 and 29.8 mg/L 1,1,3,3-tetramethylbutyl hydroperoxide ranged from 82-165% and from 78 - 92%. Analysis of 1,1,3,3-tetramethylbutyl hydroperoxide in the influent is therefore only reliable at concentrations of ≥ 29.8 mg/L.

To determine the recovery and stability of 2-hydroxy-2,4,4-trimethylpentane during storage in refrigerator and freezer influent, effluent and sludge samples were spiked. Acceptable recoveries

between 70 and 110% were obtained for 2-hydroxy-2,4,4-trimethylpentane spiked at 24 mg/L in effluent and influent. 2-hydroxy-2,4,4-trimethylpentane spiked to sludge (24 mg/L) was mainly recovered in the supernatant (106-107%). Concentrations of 2-hydroxy-2,4,4-trimethylpentane in the sludge extract were below the LOQ. Adding the <LOQ concentration to the concentration measured in the supernatant resulted in recoveries from the sludge ranging from ≥ 106 to <111 %. The upper recovery limit of ≤ 110% is slightly exceeded for one of the sludge samples.

Analysis of 2-hydroxy-2,4,4-trimethylpentane analysis in sludge is however still regarded valid because a worst case recovery calculation was used.

Assessment of 1,1,3,3-tetramethylbutyl hydroperoxide degradation using specific analysis:

The concentration of 1,1,3,3-tetramethylbutyl hydroperoxide in the effluent of the test unit from day 38 to 42 were below 0.2 mg/L which was the determined LOQ. This concentration corresponds with >99.2% removal of the test substance from the influent.

The concentrations of 1,1,3,3-tetramethylbutyl hydroperoxide in the mixed liquid suspended solids (activated sludge) of the reactor monitored on days 41, 42, 44 and 45 were <2.2, <0.4, <0.4 and <0.4 mg/L, respectively. Based on these concentrations the removal of 1,1,3,3-tetramethylbutyl hydroperoxide by adsorption to activated sludge ranged from <0.04 - < 0.22%.

Alkyl hydroperoxide reductases present in activated sludge bacteria are expected to transform 1,1,3,3 -tetramethylbutyl hydroperoxide rapidly to 2-hydroxy-2,4,4-trimethylpentane. 2-Hydroxy-2,4,4 -trimethylpentane is predicted to be non-readily biodegradable (Episuite, 2012). Measurement of the formation of 2-hydroxy-2,4,4-trimethylpentane was therefore expected possible in the CAS test. However all measured concentrations of 2-hydroxy-2,4,4-trimethylpentane in samples from the effluent (day 38, 39, 40, 41, 42) and mixed liquid suspended solids (day 41, 42, 44, 45) were below the determined LOQ. Not being able to detect 2-hydroxy-2,4,4-trimethylpentane in the effluent is in agreement with the high NPOC removal determined.

From day 49 to 50 (steady state conditions in the CAS test) the removal of the test substance and 2 -hydroxy-2,4,4-trimethylpentane through evaporation was determined during a 17-hour period of operation. The gas-scrubbing system was validated using a known amount of test substance and alcohol spiked to the CAS unit. After seventeen hours 78% and 49% of the spiked test substance and 2-hydroxy-2,4,4-trimethylpentane was still present in the liquid phase of the CAS test, respectively. It was also demonstrated that the test substance and alcohol were retained by the

three gas scrubbers (no test substance detected in the last scrubber). The mass balance over the validation gas-scrubbing system after the seventeen hours aeration period showed that 97.1% and 103.4% of the nominal concentration was recovered by analysis for the test substance and 2-hydroxy-2,4,4-trimethylpentane, respectively. During the seventeen hour period 24.8 mg of test substance was dosed to the test CAS unit, which corresponds with a maximum formation of 22.1 mg 2-hydroxy-2,4,4-trimethylpentane. The concentrations of 1,1,3,3 -tetramethylbutyl hydroperoxide and 2-hydroxy-2,4,4-trimethylpentane found after seventeen hours in the liquid of the gas scrubbers of the test CAS unit were all < LOQ. The LOQ concentrations were used

to calculate the removal by evaporation as a worst case assumption for the removal by evaporation.

The total amount of 1,1,3,3-tetramethylbutyl hydroperoxide in the three gas scrubbers was calculated to be < 93 μg which corresponds with < 0.4 % removal by evaporation. The total amount of 2-hydroxy-2,4,4-trimethylpentane in the three gas scrubbers was calculated to be < 460 μg which corresponds with < 2.1 % removal by evaporation. 2-Hydroxy-2,4,4-trimethylpentane, if formed in the CAS test, is therefore not removed by evaporation or adsorption but most likely by biodegradation.

With an average removal of 1,1,3,3-tetramethylbutyl hydroperoxide from the influent of >99.2%, an average removal by adsorption of <0.1%, a removal by evaporation of <0.4%, and no formation of water soluble organic substances it may be concluded that in properly operating conventional biological wastewater treatment plants the removal of 1,1,3,3-tetramethylbutyl hydroperoxide will be primarily achieved through biodegradation.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See quality criteria in report (COD removal, ammonium and nitrite concentrations )
Conclusions:
The mean removal percentage of 1,1,3,3-tetramethylbutyl hydroperoxide in the test unit as quantified with the specific analysis in the effluent from day 38 to 42 was >99.2%. 1,1,3,3-Tetramethylbutyl hydroperoxide concentrations in the sludge of the reactor sampled on days 41, 42, 44 and 45 were <2.2, <0.4, <0.4 and <0.4 mg/L, respectively. The mean removal percentage of 1,1,3,3-tetramethylbutyl hydroperoxide from the influent through adsorption onto sludge assessed is therefore <0.1%. The removal by volatilization, determined by analysing the test substance derived from the air of the aeration vessel through three gas-scrubbers in series, was less than 0.4%. The majority of the removal (>99.5%) for 1,1,3,3-tetramethylbutyl hydroperoxide in the CAS unit is therefore ascribed to
biodegradation. Concentrations of the expected degradation product 2-hydroxy-2,4,4-trimthylpentane measured in effluent, MLSS, and the gas scrubbing liquids from the CAS test were all below 1 mg/L (< limit of quantification) suggesting biodegradation of the alcohol upon formation.

In conclusion, the CAS test demonstrates that 1,1,3,3-tetramethylbutyl hydroperoxide is almost completely removed from the wastewater in conventional biological wastewater treatment plants.
1,1,3,3-Tetramethylbutyl hydroperoxide is most likely removed primarily by biodegradation.
Executive summary:

The continuous activated sludge (CAS) test was performed according to ISO Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. Microorganisms were exposed to

1,1,3,3-tetramethylbutyl hydroperoxide in the CAS test. To this end, 1,1,3,3-tetramethylbutyl hydroperoxide was spiked as an emulsion to domestic wastewater at a nominal test substance influent

concentration of 30 mg/L (16.5 mg/L carbon; calculated) for a period of 42 days and included a control fed with domestic wastewater only.

The mean organic carbon removal percentage of 1,1,3,3-tetramethylbutyl hydroperoxide calculated over 14 measurements obtained from day 23 to 42 of the test was 115±7% (95% confidence interval).

The difference in organic carbon concentrations of the control and test measured is statistically significant. The statistically significant higher NPOC removal in the CAS unit dosed with 1,1,3,3-

tetramethylbutyl hydroperoxide cannot be explained. Nevertheless the NPOC results do indicate that the CAS system treating domestic wastewater spiked with 1,1,3,3-tetramethylbutyl hydroperoxide

removes 100% of the organic carbon of the test substance and do exclude formation of water-soluble substances during the biodegradation of 1,1,3,3-tetramethylbutyl hydroperoxide.

An accurate assessment of the removal of 1,1,3,3-tetramethylbutyl hydroperoxide and the formation of the degradation product 2-hydroxy-2,4,4-trimethylpentane was established by specific analyses. The HPLC method for the determination of 1,1,3,3-tetramethylbutyl hydroperoxide and the GC method for the analysis of 2-hydroxy-2,4,4-trimethylpentane were satisfactory with regard to the regression, accuracy of the calibration standards (including the quality control standards), reproducibility, limit of quantification (LOQ), system stability, and recoveries of spiked influent, effluent and mixed liquid suspended solids (MLSS) samples.

The mean removal percentage of 1,1,3,3-tetramethylbutyl hydroperoxide in the test unit as quantified with the specific analysis in the effluent from day 38 to 42 was >99.2%. 1,1,3,3-Tetramethylbutyl

hydroperoxide concentrations in the sludge of the reactor sampled on days 41, 42, 44 and 45 were <2.2, <0.4, <0.4 and <0.4 mg/L, respectively. The mean removal percentage of 1,1,3,3-tetramethylbutyl hydroperoxide from the influent through adsorption onto sludge assessed is therefore <0.1%. The removal by volatilization, determined by analysing the test substance derived from the air of the aeration vessel through three gas-scrubbers in series, was less than 0.4%. The majority of the removal (>99.5%) for 1,1,3,3-tetramethylbutyl hydroperoxide in the CAS unit is therefore ascribed to biodegradation. Concentrations of the expected degradation product 2-hydroxy-2,4,4-trimthylpentane measured in effluent, MLSS, and the gas scrubbing liquids from the CAS test were all below 1 mg/L (< limit of quantification) suggesting biodegradation of the alcohol upon formation.

In conclusion, the CAS test demonstrates that 1,1,3,3-tetramethylbutyl hydroperoxide is almost completely removed from the wastewater in conventional biological wastewater treatment plants.

1,1,3,3-Tetramethylbutyl hydroperoxide is most likely removed primarily by biodegradation.