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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:
January 23, 2017 to June 20, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
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
Specific details on test material used for the study:
Test Material Name: ETHOX 4256
Chemical Name: 2-(Bis(2-ethylhexyl)amino)ethanol
Lot/Reference/Batch Number: 26D647
Purity/Characterization (Method of Analysis and Reference): The non-GLP certificate of analysis lists the purity as 99.80% POE (1) DI 2 EH AMINE by gas chromatography (Tucker, 2016).
Test Material Stability Under Storage Conditions: The test material ETHOX 4256, Lot 26D647, was not tested for neat test material stability.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Inoculum:
The microbial inoculum consisted of activated sludge mixed liquor, collected from the oxidation ditch bioreactor at the City of Midland Wastewater Treatment Plant (Midland, Michigan) on January 24, 2017. 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 500 μm 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 1,527 mg/L. Based on this determination, an appropriate volume of the homogenized activated sludge was added to the sterilized mineral medium to yield a final MLSS concentration of 29.5 mg/L.
Duration of test (contact time):
28 d
Initial conc.:
50.2 mg/L
Based on:
ThOD
Initial conc.:
16.9 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
primary indicator of biodegradation
Parameter followed for biodegradation estimation:
CO2 evolution
Remarks:
supplemental measure of biodegradation
Parameter followed for biodegradation estimation:
DOC removal
Remarks:
supplemental measure of biodegradation
Details on study design:
Route of Administration:
The test system and route of administration was selected based on the OECD Guideline 301 (OECD, 1992), and in consideration of the physical/chemical properties of the test material. The test material occurs as a liquid that expected to have low solubility in water (1.712 mg/L, estimated) and, therefore, evaluation of biodegradability requires direct addition to the aqueous reaction mixtures on a weight or volume basis. The dispersion of such materials in the aqueous reaction mixtures is recommended for evaluating ready biodegradability, using one of several techniques described in Annex III to OECD Guideline No. 301 (OECD 1992). For this study, dispersion of the test material within the test medium was facilitated by coating the test material onto a silica gel powder. Coating of the test material onto silica gel helps to evenly disperse the test material within the test medium and maintain contact between it and the microbial inoculum. Prior to use, the silica gel (35-60 mesh) was fired in a muffle furnace at 550°C to remove any trace organic and water contaminants. The test material was mixed with silica gel at a nominal gravimetric loading of 20.2% wt. The sample manually oscillated for approximately 30 seconds, and then vortexed for 1 minute to ensure homogeneity. Weighed portions of the fired silica gel, and test material amended silica gel were analyzed for total organic carbon (by combustion) to determine the homogeneity with which the test material was incorporated onto the silica gel carrier. A weighed portion of the test material-amended silica gel was added directly to the reaction mixtures to yield the required test material concentrations.

Solubility and Stability Assessment:
A prior determination of stability in the test medium was not relevant to biodegradation studies, as the test conditions were intended to promote degradation by biodegradation, hydrolysis, and oxidation/reduction reactions.
The test material is expected to be poorly soluble in water. The extent to which the test material was dissolved and dispersed in the test medium was assessed by analyzing dissolved organic carbon (DOC) in the biodegradation reaction mixtures at test initiation.

Chemicals and Reagents:
De-ionized water used to prepare the mineral medium and test chemical/reference material stock solutions was purified through a PURELAB Ultra water treatment system (ELGA LabWater, High Wycombe, United Kingdom) producing ultrapure water. All other chemicals used were purchased from commercial sources and have appropriate documentation of identity and purity.

Mineral Medium:
A defined mineral medium was prepared as specified in OECD Guideline 301F, by dissolving appropriate volumes of concentrated mineral stock solutions in ultrapure water (Table 1). The pH of the finished mineral medium was recorded, and adjusted (if necessary) within the range of 7.2 – 7.6. The finished mineral medium was filtersterilized with a Corning 0.22 μm membrane sterilization unit prior to addition of the inoculum.

Test Procedure:
The biodegradation reaction mixtures were prepared in specially designed 500-mL glass reaction vessels, each containing approximately 400 mL of the inoculated mineral medium. The reaction vessels are designed with flat glass bottoms to accommodate stirring with large PTFE-coated magnetic stir bars. The reaction vessels, stir bars, and media carboys were autoclaved one day prior to the start of the study. The various reaction mixtures which comprised the experimental design for this study are listed in Table 2 and were labeled appropriately directly on the bottle for proper identification of the vessels. Inoculum Blanks, containing the inoculated mineral medium and without added test or reference material, were prepared in duplicate. These Inoculum Blanks were used to determine mean values for cumulative O2 consumption, CO2 evolution, and DOC concentration in the absence of added test material. These mixtures also contained an amount of unamended silica gel which is approximately equal to that added in the Test Mixtures (below). Biodegradation of a reference material, aniline (101.7 mg/L), was determined in duplicate Positive Control mixtures to verify the viability of the inoculum. Biodegradation of the test material in the Test Mixtures was determined by adding the test material (as weighed portions of test material-coated silica gel) to the inoculated mineral medium (400 mL) at a concentration yielding 50.2 mg/L of theoretical oxygen demand (ThOD). A single Abiotic Control was prepared by adding mercuric chloride (309 mg/L) to inoculated mineral media containing the test material at a concentration yielding 49.9 mg/L ThOD. This Abiotic Control is used to determine the amount of O2 consumption, CO2 evolution, and changes in DOC concentrations recorded in the Test Mixtures which are attributed to abiotic reactions. A single Toxicity Control was prepared by adding test material (as weighed portions of test material-coated silica gel) at a concentration yielding 49.7 mg/L ThOD and 101.7 mg/L of aniline. This Toxicity Control was used to determine whether the concentrations of the test material used in the Test Mixtures are inhibitory to the microbial inoculum.
After addition of test material, aniline, and chemical sterilant to the appropriate vessels, the reaction mixtures were sealed and stirred for at least 30 minutes to homogenize contents prior to initiation of the test. The initial pH of each mixture was determined, and adjusted (if necessary) within the range of 7.2 – 7.6. Samples (30 mL) of the Inoculum Blanks Positive Controls, and Test Mixtures (Abiotic Control and Toxicity Control) were collected for initial DOC and nitrate/nitrite analyses. The biodegradation reaction mixtures were incubated in the darkness at a temperature in the range of 20-24°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 approximately 150 revolutions per minute (r.p.m.).

Frequency of Sampling:
Concentrations of oxygen and CO2 in the headspace of each reaction vessel were recorded at six-hour intervals over the entire 28-day test period. Upon completion of these measurements on day 28, the pH, DOC, and nitrite/nitrate concentrations in all reaction mixtures were determined.

STATISTICS AND CALCULATIONS:
Descriptive statistics (mean, standard deviation) were used where applicable.
Reference substance:
aniline
Key result
Parameter:
% degradation (O2 consumption)
Value:
-6.5
St. dev.:
14.7
Sampling time:
28 d
Remarks on result:
other:
Remarks:
Biodegradation of the material did not exceed 10% DO2, and after 28 days reached only -6.5 ± 14.7% DO2 (mean ± 1 SD, n = 2)
Details on results:
Biological Oxygen Demand (BOD):
Biological oxygen demand (BOD) is used as the primary indicator of biodegradation in the OECD 301F: Manometric Respirometry test. These measurements of BOD showed the extent of biodegradation of the test material under the conditions of this test (Figure 2). Biodegradation of the material did not exceed 10% DO2, and after 28 days reached only -6.5 ± 14.7% DO2 (mean ± 1 SD, n = 2) (Table 2).
Results with reference substance:
Biodegradation of the reference material (aniline) exceeded 60% by 6.8 days.

Test Material Coating:

Triplicate weighed portions of the test material-amended silica gel were analyzed to verify the concentration and homogeneity with which the test material was coated onto the silica gel carrier. These results showed an average carbon concentration of 209 ± 17.0 mg/g (n = 3, 1 SD), which equates to a loading of 27.6% test material by weight (based on theoretical carbon content).

Biological Oxygen Demand (BOD)

2-(Bis(2-ethylhexyl)amino)ethanol: Determination of Ready Biodegradability Using the OECD Guideline 301F - Manomentric Respirometry Test

TABLE 2. Summary of Biodegradation Based on Oxygen Consumption (DO2)

Reaction

Mixtures

Time (Days) to Achieve

DO2 (%)* at

10% DO2

60% DO2

10-d Window

Day 28

Positive

Controls

3.3

6.8

72.8 ± 10.9

92.9 ± 12.7

Test Mixtures

NA

NA

NA

-6.5 ± 14.7

Toxicity

Control

2.5

7.5

67

73.5

*Mean ± 1 SD, n = 2

CO2 Evolution:

Two of the other OECD test methods for ready biodegradability utilize measurements of CO2 evolution to indicate the extent of test material mineralization. The pass criterion for these tests is 60% of theoretical carbon dioxide evolution. While measurement of CO2 evolution is not a requirement of OECD Guideline No. 301F, these supplemental measurements of CO2 evolution confirmed the extent of test material biodegradation and ready biodegradability conclusion derived from oxygen consumption. Biodegradation of the material did not exceed 10% DCO2, and after 28 days reached -4.4 ±1.5% DCO2 (mean ± 1 SD, n = 2). Therefore, the rates and extents of biodegradation determined from CO2 evolution closely reflected those determined from BOD.

DOC Analyses:

Analyses of DOC were performed on all vessels to determine the percent degradation of the test and reference materials. It should be noted that if the test material is not fully soluble in water, measurements of DOC removal may not provide an accurate assessment of its biodegradation in this test. The analyses of the Test Mixtures at test initiation indicated a mean blank-corrected concentration of 0.0 ± 0.0 mg/L (mean ± 1 SD, n = 2). Therefore, the test material was not considered fully solubilized at the nominal concentration tested (16.9 mg/L). The mean blank-corrected DOC concentration in the Test Mixtures at day 28 was 3.8 ± 0.0 mg/L (mean ± 1 SD, n = 2). Since the test material was insoluble in water, the extent of the biodegradation could not be determined from analyses of dissolved organic carbon (DOC) removal. However, these results do suggest that some of the test material may have dissolved in the reaction mixtures.

Test Validation:

Several criteria are specified by the OECD for validating the results of its tests for ready biodegradability (OECD, 1992). These criteria are based on parameters such as inoculum viability, precision among replicate reaction mixtures, and maintenance of temperature and pH of the reaction mixtures.

The inoculum used in this test consumed an average of 41.3 mg/L oxygen over 28 days in Inoculum Blanks, where the OECD guideline indicates that this background oxygen consumption should not exceed 60 mg/L. The inoculum produced > 60% biodegradation of the reference material, aniline, within the required 10-day window prior to day 14 of the test. The 60% DO2 pass level for aniline was exceeded after 6.8 days, and biodegradation based on O2 consumption, CO2 production and DOC removal reached 92.9 ± 12.7%, 80 ± 16%, and 96.6 ± 4.1% (mean ± 1 SD, n = 2) respectively, at the end of the test, which verified the viability of the activated sludge inoculum.

For the Test Mixtures 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 (OECD, 1992). In this test, the percentage of test material biodegradation in the replicate Test Mixtures differed by 20.78% at the end of the test. The percent difference remained under 20% until Day 23.5, and never rose above 22% between Day 23.5 and the end of the test. However, the test is still considered valid because the overall conclusion does not change as the average percent degradation of the test material remained between -6.5% and -8.5% over the last 22 days of the study. In this test, the difference in percentage of aniline biodegradation in replicate Positive Control mixtures was less than 18.1% DO2 at the specified time points. The results indicate that the procedures used to prepare, incubate, and analyze the biodegradation reaction mixtures resulted in sufficient precision in the test results.

Temperature of the incubator which contained the biodegradation reaction mixtures was recorded periodically throughout the study using a calibrated min/max digital thermometer. The recorded minimum temperatures averaged 22.1 ± 0.1°C (mean ± 1 SD., n = 28) and the maximum temperatures averaged 22.3 ± 0.0°C (mean ± 1 SD., n = 28), over the entire duration of this test. Therefore, the incubation temperature fell within the required range of 20-24°C, and was maintained within the required precision of ± 1°C.

The pH of the biodegradation reaction mixtures remained within the required range of 6.0 to 8.5 over the duration of this test (Table 6). The pH of the Test Mixtures increased by no more than 0.09 pH units from their initial values over 28 days, and showed only a 0.06 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 materials evaluated in this test.

Toxicity Controls:

The Toxicity Control mixtures containing 101.7 mg/L aniline and 16.7 mg/L of the test materials showed no evidence for inhibition of the microbial inoculum by the test material. The OECD guideline specifies 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 (OECD, 1992). The onset and rate of biodegradation in the Toxicity Control mixture closely matched that in the Positive Controls. While the test material is expected to have limited solubility in water, its suspension (i.e., test material on silica gel) in the inoculated mineral medium at a concentration of 16.7 mg/L did not cause toxicity to or inhibition of the microbial inoculum.

Abiotic Controls:

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 material. The mixture contained 16.8 mg/L of test material in the inoculated mineral medium, which was chemically sterilized by addition of 309 mg/L HgCl2. The Abiotic Control mixture exhibited no O2 consumption or CO2 production over the duration of the 28-day test (data not shown). Therefore, the O2 consumption and CO2 production in the Test Mixtures was solely attributed to biodegradation of the test material.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Biodegradation of 2-(bis(2-ethylhexyl)amino)ethanol failed to exceed the 60% pass criterion for demonstrating “ready biodegradability” in the manometric respirometry test. The results of this test therefore demonstrate that 2-(bis(2-ethylhexyl)amino)ethanol cannot be classified as “readily biodegradable”, according to the OECD 301F: Manometric Respirometry Test (OECD, 1992).
Executive summary:

The ready biodegradability of 2-(bis(2-ethylhexyl)amino)ethanol was determined using the OECD Guideline No. 301F: Manometric Respirometry Test. This study employed a series of

biodegradation reaction mixtures containing activated sludge inoculum collected from the City of Midland Wastewater Treatment Plant (Midland, Michigan), which was suspended in a defined

mineral medium at a concentration of 29.5 mg/L (dry solids). Biodegradation of the test material was evaluated in Test Mixtures at a concentration of 16.9 mg/L, which was equivalent to 50.2 mg/L theoretical oxygen demand (ThOD). Reaction mixtures were incubated in the dark at a constant temperature between 20 to 24°C, and maintained within ± 1°C. Oxygen consumption in the

biodegradation reaction mixtures was continuously recorded at 6 hr intervals, using an automated respirometer system. Biodegradation of 2-(bis(2-ethylhexyl)amino)ethanol failed to exceed the

pass level of 60% ThOD consumption. At the end of the 28 day test, the oxygen consumption in the Test Mixtures was less than that observed in the Inoculum Blanks, indicating no (0%) biodegradation occurred. A Toxicity Control mixture, containing both aniline (101.7 mg/L) and the test material (16.7 mg/L), showed no evidence for inhibition of the microbial inoculum. Oxygen

consumption observed in the reaction mixtures could be attributed solely to biological activity, as no net O2 consumption was measured in an Abiotic Control mixture containing the test material and a chemical sterilant (HgCl2). Thus, 2-(bis(2-ethylhexyl)amino)ethanol is not inhibitory of the inoculum and can not be classified as “readily biodegradable.”

Other results of this test met or exceeded each of the OECD-specified criteria for validation of the ready biodegradability test. These include parameters such as viability of the inoculum, control of

pH and temperature, and precision in percentage biodegradation recorded among replicate test mixtures containing a biodegradable reference material. Biodegradation of the reference material

(aniline) exceeded 60% by 6.8 days, verifying the viability of the activated sludge inoculum. Therefore, the results of this study are considered fully valid.

Description of key information

In the key study, the ready biodegradability of 2-(bis(2-ethylhexyl)amino)ethanol was determined using the OECD Guideline No. 301F: Manometric Respirometry Test. This study employed a series of biodegradation reaction mixtures containing activated sludge inoculum collected from the City of Midland Wastewater Treatment Plant (Midland, Michigan), which was suspended in a defined mineral medium at a concentration of 29.5 mg/L (dry solids). Biodegradation of the test material was evaluated in Test Mixtures at a concentration of 16.9 mg/L, which was equivalent to 50.2 mg/L theoretical oxygen demand (ThOD). Reaction mixtures were incubated in the dark at a constant temperature between 20 to 24°C, and maintained within ± 1°C. Oxygen consumption in the biodegradation reaction mixtures was continuously recorded at 6 hr intervals, using an automated respirometer system. Biodegradation of 2-(bis(2-ethylhexyl)amino)ethanol failed to exceed the pass level of 60% ThOD consumption. At the end of the 28 day test, the oxygen consumption in the Test Mixtures was less than that observed in the Inoculum Blanks, indicating no (0%) biodegradation occurred. A Toxicity Control mixture, containing both aniline (101.7 mg/L) and the test material (16.7 mg/L), showed no evidence for inhibition of the microbial inoculum. Oxygen consumption observed in the reaction mixtures could be attributed solely to biological activity, as no net O2 consumption was measured in an Abiotic Control mixture containing the test material and a chemical sterilant (HgCl2). Thus, 2-(bis(2-ethylhexyl)amino)ethanol is not inhibitory of the inoculum and cannot be classified as “readily biodegradable.”

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information