4-[3-(1-naphthylamino)propyl]morpholine

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Lot/Reference/Batch Number: ZA01212016
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 94.4% by liquid chromatography with identification by nuclear magnetic resonance spectroscopy and liquid chromatography mass spectrometry.
Test Material Stability Under Storage Conditions: The test material was determined to be stable for 2 weeks at 54°C which is equivalent to 24 months under ambient storage conditions as tested under USEPA OPPTS Guideline 830.6313.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Considering the OECD guideline recommendation to avoid (where possible) the use of organic carrier solvents, the (neat) test material was added directly to the reaction mixtures by direct weight addition while the reaction mixtures were vigorously stirred and/or aerated. This mixing action was expected to have dispersed and dissolved the test material in a manner similar to what would be expected to occur in an actual wastewater treatment scenario.
A stock solution of the reference material, 3,5-dichlorophenol, was prepared in ultrapure water at a concentration of 496 mg/L. The pH of the solution was adjusted to 7.63 with 1N NaOH and H2SO4.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge was obtained from the Midland Municipal Wastewater Treatment Plant (Midland, Michigan). This facility treats an excess of 11 million liters of wastewater daily, of which > 90% is from domestic sources. Upon return to the laboratory, the activated sludge was washed by allowing the sludge to settle for approximately 5 minutes, decanting off the supernatant and adding the appropriate amount of ultrapure water to bring to volume. The washing procedure was repeated twice. The mixed liquor suspended solids (MLSS) was determined to be 6,440 mg/L by standard procedures and then was adjusted to approximately 3,100 mg/L.
Since the sludge was not used on the day of collection, 50 mL of synthetic sewage was added to each liter of the activated sludge prepared as described above. The sludge was aerated at a temperature falling in the range of 19.4°C to 20.0°C until used the following day. Before use, the pH was checked and adjusted to pH 6.45 using 1N NaOH. The MLSS was re-assayed and determined to be 2,720 mg/L. The MLSS content was adjusted once more by allowing the sludge to settle for 10 minutes, and then removing 370 mL of supernatant. Upon re-analysis, the MLSS content was 2,870 mg/L. This MLSS concentration yielded a final concentration of 1,435 mg/L in the final reaction mixtures.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Remarks on exposure duration:

The incubation was considered to begin with the initial contact of the activated sludge inoculum with the other constituents of the final mixture. Each incubation was started in turn, at conveniently timed intervals of 15 min.

Test temperature:

19.4 degrees C

pH:

7.5 ± 0.5.

Nominal and measured concentrations:

Nominal Concentrations: 0 (Control), 10, 100, and 1000 mg/L

Details on test conditions:

TThe following experimental procedure was followed to evaluate respiration inhibition by the test and reference materials at a 3-hour contact period at approximately 20 ± 2°C. The exact temperature was not recorded. However, temperature of the activated sludge was taken prior to the dosing of the reaction mixtures. This recorded temperature was 19.4ºC, indicating that the temperature in the room at the start of the incubation period was within the required range. This test evaluated a range of three test material concentrations (i.e., 10, 100 and 1000 mg/L) to estimate the range of concentrations suitable for a definitive test. The absence of inhibition of oxygen consumption by the test material in a preliminary test can be used to demonstrate that a definitive test is unnecessary, but triplicates of the highest tested concentration in the preliminary test are needed to make this determination. Since no statistically significant inhibition of oxygen consumption was observed at the high test concentration (i.e., 1000 mg/L) in the range-finding test, the range-finding test was considered to be the definitive test, and no further testing at higher or lower concentrations was necessary.

All reaction mixtures were labeled with their appropriate designation directly on the reaction containers throughout the duration of the study.The various mixtures needed for the test in synthetic sewage were prepared but the inoculum was withheld, and the requisite portions of sludge were added to each vessel of the series. The incubation was considered to begin with the initial contact of the activated sludge inoculum with the other constituents of the final mixture. Each incubation was started in turn, at conveniently timed intervals of 15 min. Reaction mixtures were prepared in 1-liter graduated cylinders and aerated by bubbling purified compressed air through the reaction mixture at 0.57-0.85 L/min.
At time “0,” 16 mL of the synthetic feed, 234 mL water, and 250 mL of microbial inoculum were mixed (final volume 500 mL) in a 1-liter graduated cylinder. This mixture, designated as control FB1, was aerated by bubbling purified compressed air through the reaction mixture at 0.57-0.85 L/min.
At time “15 minutes” (15 minutes is an arbitrary, but convenient time interval), the above procedure was repeated with the reference material, 3,5-dichlorophenol. Three reference material reaction mixtures (reference mixtures FR1-3) were prepared in sequence at 15-minute intervals.
Similarly, test reaction mixtures were prepared by adding the neat test material to the reaction mixtures. Five test reaction mixtures (FT1-5) were prepared as specified in the guideline in sequence at 15-minute intervals to evaluate respiratory inhibition by the test material. Finally, a second control mixture (FB2) was prepared and evaluated, as described for control FB1.
After 3 hours of contact time, the contents of the first cylinder (FB1) were poured into a 300-mL BOD bottle, the dissolved oxygen electrode was inserted, and the dissolved oxygen concentration of the reaction mixture was recorded over a period of up to 10 minutes. This oxygen consumption determination was repeated for the contents of each additional graduated cylinder at 15-minute intervals, in such a way that the contact time for each reaction mixtures is 3 hours.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Respiration of each test mixtures containing 10, 100, and 1,000 mg/L of the test material were measured after three hours contact time with activated sludge. Nominal 4-(3-(1-naphthylamino)propyl)morpholine concentrations as high as 1,000 mg/L did not inhibit the respiration rate of the activated sludge by ≥ 50% compared to control mixtures (no test chemical added). Therefore, an exact EC50 value for 4-(3-(1-naphthylamino)propyl)morpholine could not be determined, and three hour EC50 value was determined empirically as EC50 > 1,000 mg/L.

Results with reference substance (positive control):

The EC50 value determined for the reference material, 3,5-dichlorophenol was 4.98 mg/L. This concentration falls within the expected EC50 concentration range specified in the guideline of 2 to 25 mg/L.

Reported statistics and error estimates:

A statistical comparison of respiration rates resulted in determination of a no observed effect concentration (NOEC) of 1,000 mg/L. Observation of the oxygen uptake rate data indicated that the impact of increased test material concentrations on measured respiration rates did not follow a monotonic response. The statistical significance of the difference between controls and the 1,000 mg/L treated group was determined by a t-test, with the null hypothesis being no difference. First a folded F-test was used to test for equality of variances. When the F-test was significant, the Satterthwaite p-value from the t-test was used to determine significance. Otherwise, the pooled p-value from the t-test was used to determine significance. The folded F-test p-value was 0.0505 and was not significant at alpha=0.05, therefore the pooled p-value from the t-test was used to determine significance. The pooled t-test p-value was 0.0811, therefore the treated group was not statistically different from the control group at alpha=0.05. Consequently, the statistically-derived no-observed-effect concentration was determined to be 1,000 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The potential for 4-(3-(1-naphthylamino)propyl)morpholine to inhibit the respiration of municipal activated sludge was evaluated using the OECD 209 “Activated Sludge, Respiration Inhibition Test.” This study assessed the potential effect of a range of test chemical concentrations on measured activated sludge respiration rates following s a three hour contact period. The 4-(3-(1-naphthylamino)propyl)morpholine material, at nominal concentrations of 10, 100, and 1,000 mg/L did not show ≥ 50% inhibition of the activated sludge respiration rate after three hours. Therefore, the EC50 value was determined empirically to be EC50 > 1,000 mg/L. A statistical comparison of respiration rates resulted in determination of a no observed effect concentration (NOEC) of 1,000 mg/L. 4-(3-(1-naphthylamino)propyl)morpholine material is expected to have very low potential for adversely affecting biological wastewater treatment operations.

Executive summary:

The potential for4-(3-(1-naphthylamino)propyl)morpholineto inhibit the respiration ofmunicipal activated sludge was evaluated using the OECD Guideline 209 “Activated Sludge, Respiration Inhibition Test.” This test method assesses the potential effect of a range of test chemical concentrations on measured activated sludge respiration rates following a three-hour contact period. The4-(3-(1-naphthylamino)propyl)morpholinematerial, at nominal concentrations of 10, 100, and 1000 mg/L, did not show > 50% inhibition of the respiration rate of the activated sludge after three hours. Therefore, the EC₅₀value was determined empirically to be EC₅₀> 1,000 mg/L. A statistical comparison of respiration rates resulted in determination of a no observed effect concentration (NOEC) of 1,000 mg/L. Further testing of potential impact of the test material on nitrifying activity of activated sludge was deemed to be unnecessary, because respiration of nitrifying bacteria represents a relatively small proportion of total activated sludge respiration, and very high concentrations of test material were necessary to impact total respiration. 4-(3-(1-naphthylamino)propyl)morpholinematerial is expected to have very low potential for adversely affecting biological wastewater treatment operations.

Description of key information

Thepotential for 4-(3-(1-naphthylamino)propyl)morpholineto inhibit the respiration ofmunicipal activated sludge was evaluated using the OECD Guideline 209 “Activated Sludge, Respiration Inhibition Test.” This test method assesses the potential effect of a range of test chemical concentrations on measured activated sludge respiration rates following a three-hour contact period. The 4-(3-(1-naphthylamino)propyl)morpholine material, at nominal concentrations of 10, 100, and 1000 mg/L, did not show > 50% inhibition of the respiration rate of the activated sludge after three hours. Therefore, the EC₅₀value was determined empirically to be EC₅₀> 1,000 mg/L. A statistical comparison of respiration rates resulted in determination of a no observed effect concentration (NOEC) of 1,000 mg/L. Further testing of potential impact of the test material on nitrifying activity of activated sludge was deemed to be unnecessary, because respiration of nitrifying bacteria represents a relatively small proportion of total activated sludge respiration, and very high concentrations of test material were necessary to impact total respiration.  4-(3-(1-naphthylamino)propyl)morpholinematerial is expected to have very low potential for adversely affecting biological wastewater treatment operations.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L