N-butyl phenyl ether

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 11, 2016 to January 3, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

The microbial inoculum consisted of secondary effluent, collected from the secondary clarifier unit at the Midland Municipal Wastewater Treatment Plant (Midland, Michigan) on July 12, 2016. This facility treats an excess of 11 million liters of wastewater per day, of which > 90% (vol.) is from domestic sources. The secondary effluent was collected one day prior to initiation of the test and was continuously aerated until upon returning to the laboratory. Prior to use, the secondary effluent was filtered through Whatman 114V filter paper. Based on preliminary results, 24 liters of mineral medium was inoculated with 7.2 mL of filtered secondary effluent. The pH of the inoculated mineral medium was measured and adjusted to 7.4 ± 0.2 using NaOH and HCl solutions. Prior to the initiation of the Closed Bottle test, the inoculated mineral medium was sparged with ambient laboratory air for at least 30 minutes, and then allowed to stand for at least 20 hours under slow stirring at the test temperature. The concentration of dissolved oxygen in the equilibrated, inoculated mineral medium was determined to be 8.34 mg/L prior to initiating the test.

Duration of test (contact time):

28 d

Initial conc.:

2.55 mg/L

Based on:

test mat.

Initial conc.:

5.04 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

The reaction mixtures were contained in 300-mL glass biological oxygen demand (BOD) bottles. The reaction mixtures were labeled appropriately directly on the bottle for proper identification of the vessels. Bottles were filled to approximately 80% capacity with inoculated mineral medium, and dosed with the test and reference materials. The bottles then were completely filled with additional inoculated mineral media and immediately sealed with ground glass stoppers and plastic covers. A sufficient number of replicate reaction mixtures were prepared to allow for duplicate analyses on several sampling days, occurring at weekly intervals and allowing for definition of the 10-day window. The sampling schedule included days 0, 5, 7, 14, 21, and 28. Inoculum Blanks, containing only the inoculated mineral medium, were used to determine mean values for dissolved oxygen consumption due to endogenous respiration of the inoculum. Portions of fired silica gel were added to these reaction mixtures, to replicate the mass of silica gel (and associated particle surface area) occurring in the biodegradation Test Mixtures (below). Positive controls, containing the reference material sodium benzoate (4.03 mg/L), were used to confirm the viability of the microbial inoculum. Test Mixtures were prepared in replicates by adding weighed portions of the test material-amended silica gel to two separate groups of Test Mixtures to attain a 2-fold difference in nominal initial concentrations of test material (e.g., 2.55 and 5.04 mg/L as test material). Two groups of Toxicity Controls, containing a combination of either 2.57 or 5.11 mg/L test material and 4.03 mg/L sodium benzoate, were prepared in replicates to determine whether the concentrations of the test material used in the test mixtures are inhibitory to the microbial inoculum. The biodegradation reaction mixtures were incubated in the darkness at an average temperature of between 20 and 24°C, and controlled within approximately ± 1°C.

Reference substance:

benzoic acid, sodium salt

Remarks:

Concentration = 4.03 mg/L

Preliminary study:

A previous test which followed the OECD Guideline 301F, using a closed-bottle respirometer system and a test material concentration of approximately 18 mg/L, showed evidence for volatilization loss and toxicity/inhibition of the microbial inoculum by the test material (Hu, 2016).

Test performance:

The results of the closed bottle test are considered as valid if dissolved oxygen depletion in the Inoculum Blanks does not exceed 1.5 mg/L after 28 days. The dissolved oxygen depletion in the Inoculum Blanks in this study was 0.82 mg/L after 28 days. In addition, the residual dissolved oxygen in the Test Mixtures did not fall below 0.5 mg/L at any time during the test.

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. In this test, the percentage of test material biodegradation in the replicate Test Mixtures differed by < 8.95% DO2 over all sample intervals of the 28-day test. The maximum difference in percentage of sodium benzoate biodegradation in replicate Positive Controls was 10.41% DO2 over the course of the study. The results indicate that the procedures used to prepare, incubate, and analyze the biodegradation reaction mixtures resulted in sufficient precisionin the test results.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

at test material concentration = 5.04 mg/L

Value:

1.4

St. dev.:

0.5

Sampling time:

28 d

Remarks on result:

other: not readily biodegradable

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

at test material concentration = 2.55 mg/L

Value:

5.4

St. dev.:

4.1

Sampling time:

28 d

Remarks on result:

other: not readily biodegradable

Details on 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 daily minimum temperatures averaged 21.8 ± 0.3°C (± 1 SD,n = 16) and the maximum temperatures averaged 22.1 ± 0.2°C (± 1 SD, n = 16), 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 7). The pH of the Test Mixtures decreased by no more than 0.14 pH units from their initial values over 28 days, and showed only an average of 0.04 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.

Biodegradation in the Toxicity Controls (sodium benzoate + test substance) reached approximately 27% and 41% DO2 after 28 days in the high and low concentrations of test material, respectively. The toxicity control for the high concentration of test material (22.5% degraded at 14 days) did not pass guideline recommendation (>25%degradation at 14 days). However, the test material is assumed to not be inhibitory to the microbial inoculum under the test conditions because the oxygen depletion value was greater than that expected based on the sum of the oxygen depletion values from the Test Mixtures and Positive Controls, thereby confirming that the test material was not toxic to the micro-organisms used in the test.

Results with reference substance:

The inoculum used in this test produced > 60% biodegradation of the reference material, sodium benzoate prior to day 14 of the test, as required by the OECD 301D guideline.

Summary of Biodegradation Based on Oxygen Consumption (DO2) - %Degradation of ~2.5 mg/L Test Material

Incubation Time (Days)	Test Chemical	Positive Controls	Toxicity Controls
0	0.0 ± 0.0*	0.1 ± 0.1	0.0 ± 0.1
5	-0.8 ± 0.3	-	-
7	-1.1 ± 0.1	68.3 ± 7.4	31.2 ± 1.3
14	-0.8 ± 0.4	77.0 ± 0.5	32.7 ± 0.2
21	2.4 ± 0.3	78.5 ± 2.3	36.6 ± 0.5
28	5.4 ± 4.1	77.5 ± 0.2	41.2 ± 0.9

*Mean ± 1SD

Summary of Biodegradation Based on Oxygen Consumption (DO2) - %Degradation of ~5.0 mg/L Test Material

Incubation Time (Days)	Test Chemical	Positive Controls	Toxicity Controls
0	0.0 ± 0.0*	0.1 ± 0.1	0.0 ± 0.1
5	-0.3 ± 0.1	-	-
7	-0.8 ± 0.2	68.3 ± 7.4	20.1 ± 0.3
14	0.3 ± 0.5	77.0 ± 0.5	22.5 ± 0.9
21	0.6 ± 0.1	78.5 ± 2.3	24.8 ± 0.2
28	1.4 ± 0.5	77.5 ± 0.2	26.8 ± 2.4

*Mean ± 1SD

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The butyl phenyl ether test material did not achieve 60% biodegradation within 28 days of the start of the test detailed in OECD 301D guideline. The results of this test demonstrated that butyl phenyl ether cannot be classified as “readily biodegradable”, according to the OECD 301D: Closed Bottle Test.

Executive summary:

The ready biodegradability of butyl phenyl ether was evaluated using the OECD Guideline No. 301D: Closed Bottle Test. This study employed a series of biodegradation reaction mixtures containing secondary effluent inoculum collected from the City of Midland Wastewater Treatment Plant (Midland, Michigan), which was suspended in a defined mineral medium. Since the test material is moderately volatile and poorly soluble, the test material was coated onto silica gel to facilitate its dispersion in the biodegradation reaction mixtures. Biodegradation of the test material was evaluated in reaction mixtures at two concentrations containing 2.6 mg/L and 5.0 mg/L of the test material. Consumption of dissolved oxygen in the biodegradation reaction mixtures was determined after 5, 7, 14, 21, and 28 days. At the end of the 28 day test, the extent of biodegradation based on dissolved oxygen consumption reached 5.4 ± 4.1% and 1.4 ± 0.5% (mean ± 1 SD) for the 2.6 and 5.0 mg/L treatments, respectively. Thus, butyl phenyl ether cannot be classified as “readily biodegradable” according to the OECD Guideline 301D: Closed Bottle Test. The 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 mixtures containing a biodegradable reference material. Biodegradation of the reference material (sodium benzoate) exceeded 60% prior to day 14, verifying the viability of the activated sludge inoculum. Therefore, the results of this study are considered fully valid.

Description of key information

Biodegradation of the test material was evaluated in reaction mixtures at two concentrations containing 2.6 mg/L and 5.0 mg/L of the test material. At the end of the 28 day test, the extent of biodegradation based on dissolved oxygen consumption reached 5.4 ± 4.1% and 1.4 ± 0.5% (mean ± 1 SD) for the 2.6 and 5.0 mg/L treatments, respectively. Thus, butyl phenyl ether cannot be classified as “readily biodegradable” according to the OECD Guideline 301D: Closed Bottle Test.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed