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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 301F, EPA OPPTS 835.3110, EU Methid C.4-D and in accordance with the Principles of Good Laboratory Practice (GLP).
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Remarks:
The identity and purity of the reference substance was characterized by laboratory methods which did not comply with GLP. Stability of the test and reference substances was not characterized.
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
Remarks:
same as above
Qualifier:
according to
Guideline:
EPA OPPTS 835.3100 (Aerobic Aquatic Biodegradation)
Deviations:
no
Remarks:
same as above
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: The microbial inoculum consisted of activated sludge mixed liquor, collected from the oxidation ditch bioreactor at the Midland Municipal Wastewater Treatment Plant (Midland, Michigan). This facility treats an excess of 11 million liters of wastewater per day, of which > 90% is from domestic sources. The activated sludge was collected one day prior to initiation of the test, and was continuously aerated until used. Prior to use, the activated sludge was screened through 500μm nylon mesh, and briefly homogenized in a Waring blender (Waring Products Inc., Torrington, Connecticut). The mixed liquor suspended solids (MLSS) content of the homogenized sludge was determined gravimetrically to be 1,355 mg/l. Based on this determination, 355 ml of the homogenized activated sludge was added to 18 liters of the sterilized mineral medium to yield a final MLSS concentration of 27 mg/l.
Duration of test (contact time):
28 d
Initial conc.:
88.6 mg/L
Based on:
ThOD/L
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The biodegradation reaction mixtures were prepared in specially designed 1-liter glass reaction vessels, each containing a 500 ml portion of the inoculated mineral medium. The reaction vessels are designed with flat glass bottoms to accommodate stirring with large PTFE-coated magnetic stir bars. These vessels are also fitted with 20 x 105 mm glass side baffles to facilitate complete mixing/aeration of the stirred reaction mixtures. Inoculum Blanks, containing only the mineral medium and the sludge inoculum, were prepared in duplicate. These Inoculum Blanks were used to determine mean values for cumulative O2 consumption and CO2 evolution in the absence of added test substance. Biodegradation of a reference substance, aniline (101.3 mg/l), was determined in duplicate Positive Control mixtures to verify the viability of the inoculum. The Test Suspensions were prepared by adding the test material to the inoculated mineral medium (500 ml) at a concentration yielding 88.6 mg/l of theoretical oxygen demand (ThOD). A single Toxicity Control reaction mixture was similarly prepared by combining the test substance and 101.3 mg/l aniline in the inoculated mineral medium. The rate and extent of aniline degradation in the Toxicity Control reaction mixture was used to assess whether the test substance was inhibitory to the microbial inoculum. A single Abiotic Control mixture was prepared by adding mercuric chloride (239.8 mg/l) to inoculated mineral medium containing the test substance. This Abiotic Control was used to determine the amount of O2 consumption or CO2 evolution recorded in the Test Suspensions which is attributed to abiotic reactions. Volatility Control mixtures were prepared in duplicate by adding the test substance at a concentration of 36.3 mg/l in sterile (autoclaved) mineral medium. The volatility control mixtures were prepared to examine any potential loss of the test material over the course of the study due to volatilization.

After addition of test substance, aniline, and chemical sterilant to the appropriate vessels; the pH of the reaction mixtures were measured and adjusted as necessary to 7.4 ± 0.2, then stirred for one hour to homogenize their contents prior to initiation of the test. At the study initiation (day 0) aliquots of the reaction mixtures were collected for dissolved organic carbon (DOC) analysis. Prior to measurement of initial oxygen and CO2 concentrations, the headspace volume of each individual reaction vessel was determined by the automated respirometer system. A temperature reference vessel, consisting of a biodegradation reaction vessel containing 500 ml water, was incubated and stirred alongside the biodegradation reaction mixtures in this test. The water temperature in this vessel was recorded by a submerged NIST-traceable thermocouple whenever a respirometric measurement was recorded for the Test Suspensions (i.e. at six-hour intervals for each mixture). The biodegradation reaction mixtures were incubated in the darkness at a temperature of approximately 22 ± 2°C, and maintained within ± 1°C of the average incubation temperature. The reaction mixtures were continuously stirred by a PTFE-coated magnetic stir bar rotating at a setting of 150 rpm.
Reference substance:
aniline
Remarks:
Lot #00914PH, 99.5%, A.C.S. Reagent Grade (Sigma Aldrich, St. Louis, Missouri)
Preliminary study:
not applicable
Test performance:
not applicable
Parameter:
% degradation (O2 consumption)
Value:
74
St. dev.:
4.1
Sampling time:
10 d
Parameter:
% degradation (O2 consumption)
Value:
90.1
St. dev.:
6.5
Sampling time:
28 d
Parameter:
% degradation (CO2 evolution)
Value:
53.7
St. dev.:
4.1
Sampling time:
10 d
Parameter:
% degradation (CO2 evolution)
Value:
69.3
St. dev.:
5.5
Sampling time:
28 d
Parameter:
% degradation (DOC removal)
Value:
98.3
St. dev.:
0.1
Sampling time:
28 d
Details on results:
By the end of the 28-day test, biodegradation of the test substance reached 90.1 ± 6.5% DO2 (mean ± 1SD). The time required for biodegradation to exceed 10% DO2 (i.e., the lag period) was 0.6 days and the 60% DO2 level was exceeded after 5.9 days.

Biodegradation of the substance exceeded 10% DCO2 after 1.6 days and after 28 days reached 69.3 ± 5.5 % DCO2 (mean ± 1SD). Therefore, the rates and extents of biodegradation determined from CO2 evolution closely reflected those determined from BOD

DOC analyses were performed on the Test Suspensions at the beginning and the end of the test. The Day 0 analyses indicated that the added test substance was fully dissolved in the Test Suspensions, Abiotic Control, and Volatility Controls. Measured DOC ranged from 20.7 – 24.1 mg/l, which accounts for between 86 and 100% of the added test substance (36.3 mg/l at 66.6% carbon). The extent of DOC removal determined for the Test Suspensions averaged 98.3 ± 0.1 % after 28 days. The day 28 DOC values for the Abiotic Controls and the Volatility Controls were near 0 mg/l.These results indicate complete loss of test material from the reaction mixtures through volatilization over the 28 day test period. This loss occurs incrementally over the course of the test, when the “dead volume” of the closed-loop respirometer system is flushed with fresh air after each 6-hr. measurement interval. Although the test substance showed sufficient degradation to meet/exceed the criteria for ready biodegradability, the extent of ultimate biodegradation recorded during this test may have been suppressed somewhat due to this volatilization loss.
Results with reference substance:
The Toxicity Control mixture containing 101.3 mg/l aniline and 36.3 mg/l of the test substance passed the OECD guideline specifying that toxicity or inhibition of the inoculum is indicated when net oxygen consumption remains less than 25% of the total applied ThOD over the first 14 days of the test. The biodegradation in the Toxicity Control mixture closely matched that in the Positive Controls.

The inoculum used in this test produced > 60% biodegradation of the reference substance, aniline, within the required 10-day window prior to day 14 of the test. The 60% DO2 pass level was exceeded after 7.7 days, and biodegradation based on O2 consumption, CO2 production and DOC removal reached 27.2%, 92.1% and 99.1%, respectively, at the end of the test. Although the extent of aniline degradation determined from oxygen consumption was corrected for measured nitrification of ammonia, it is recognized that this correction can be imprecise. This fact, combined with additional cellular respiration and decay from new cells produced during growth on the high concentration of aniline, can result in apparent extents of degradation which exceed 100% of ThOD. Such results are by no means considered to indicate an invalid test condition or measurement method. 

For the Test Suspensions and Positive Controls, the extent of biodegradation recorded for replicate reaction mixtures must not differ by more than 20% DO2 at the end of the 10-day window, plateau of degradation, or the end of the test. In this test, the percentage of test substance biodegradation in the replicate Test Suspensions differed by < 9.2% DO2 over all sample intervals of the 28-day test. During the course of the study one of the replicate positive control samples developed a leak. The loss of associated data for this vessel negated any comparison of replicate reaction mixtures for the Positive Control samples. 

The recorded values from the temperature reference vessel averaged 22.6 ± 0.7 °C (± 1 SD, n=896), and the minimum and maximum recorded temperatures were 22.0 and 24.6 °C, respectively, over the entire duration of this test. An initial rise in temperature to 24.6°C was observed through day 3 of the study. The incubator room temperature was lowered and the temperature of the reference vessel returned to normal. The temporary rise in temperature did not impact the results of the study.

 

The pH of the biodegradation reaction mixtures remained within the required range of 6.0 to 8.5 over the duration of this test. The pH of the Test Suspensions decreased by no more than 0.19 pH units from their initial values over 28 days, and showed only a 0.03 pH unit (maximum) difference relative to the Inoculum Blanks at the end of the test. This minimal variation in pH indicates that the mineral medium contained adequate buffering capacity for the inoculum and test substances evaluated in this test.

 

A single Abiotic Control mixture was included in the experimental design to determine the extent to which abiotic processes may result in degradation of the test substance. The mixture contained 36.3 mg/l of test substance in the inoculated mineral medium, which was chemically sterilized by addition of 239.8 mg/l HgCl2. The Abiotic Control mixtures exhibited no O2consumption or CO2production over the duration of the 28-day test (data not shown).

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Biodegradation began soon after addition to the test mixtures, and after 28 days reached 90.1 ± 6.5% (mean ± 1 SD) based on biological oxygen demand (BOD). The pentyl propionate test substance exceeded 60% biodegradation within 5.9 days of the start of the test and within the 10-day window detailed in OECD 301F. The results of this test demonstrated that Pentyl Propionate can be classified as “readily biodegradable”, according to the OECD 301F: Manometric Respirometry Test.
Executive summary:

The ready biodegradability of pentyl propionate was evaluated using the OECD Guideline No.301F: Manometric Respirometry Test. Given the compound’s aqueous solubility (< 0.5% @ 20ºC), pentyl propionate was added directly to the biodegradation reaction mixtures (36.3 mg/L).

 

Biodegradation began soon after addition to the test mixtures, and after 28 days reached 90.1 ± 6.5% (mean ± 1 SD) based on biological oxygen demand (BOD). The pentyl propionate test substance exceeded 60% biodegradation within 5.9 days of the start of the test and within the 10-day window detailed in OECD 301F. Pentyl propionate can be classified as readily biodegradable.

 

Biodegradation of a reference substance (aniline) exceeded 60% by 7.7 days verifying the viability of the activated sludge inoculum. A Toxicity Control mixture, containing both aniline (101.3 mg/L) and the test substance (36.3 mg/L), showed no evidence for inhibition of the microbial inoculum. Oxygen consumption and CO2 evolution observed in the reaction mixtures could be attributed solely to biological activity, as no net O2 consumption or CO2 evolution was measured in an Abiotic Control mixture containing the test substance and a chemical sterilant (HgCl2). The results of this test met or exceeded each of the OECD-specified criteria for validation of the ready biodegradability tests. These include parameters such as viability of the inoculum, control of pH, temperature, and precision in percentage biodegradation recorded among replicate test mixtures.

Description of key information

Based on the biodegradation study (OECD TG 301F-Manometric Respirometry Test), Pentyl propionate is considered to be readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

In a GLP study conducted according to OECD TG 301F (Manometric Respirometry Test), biodegradation of pentyl propionate began soon after addition to the test mixtures, and after 28 days reached 90.1 ± 6.5% (mean ± 1 SD) based on biological oxygen demand (BOD) and exceeded 60% biodegradation within 5.9 days of the start of the test and within the 10-day window detailed in OECD 301F. The results of this test demonstrated that Pentyl Propionate can be classified as “readily biodegradable”, according to the OECD 301F: Manometric Respirometry Test..