3-morpholinopropylamine

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-31 to 2018-01-22

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-morpholinopropylamine

EC Number:

204-590-2

EC Name:

3-morpholinopropylamine

Cas Number:

123-00-2

Molecular formula:

C7H16N2O

IUPAC Name:

3-morpholin-4-ylpropan-1-amine

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 6H706
- Expiration date of the lot/batch: 2018-08-11
- Purity: 99.7%
- Purity test date: 2017-10-10 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, at 20 ± 5 °C. Keep container tightly closed in a dry and well-ventilated place.
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): aerobic activated sewage sludge from the aeration stage of the local wastewater treatment plant, ARA Birs (Birsfelden / Switzerland), which treats predominantly domestic sewage.
- Storage conditions: During the holding period of one day prior to use, the sludge was aerated with CO2-free air at room temperature.
- Preparation of inoculum for exposure: The aerobic activated sewage sludge was washed three times by centrifugation and resuspension. The supernatant (liquid phase) was decanted and the solid material resuspended in tap water and after the final wash in mineral medium. Aliquots of the homogenized final sludge suspension were weighed, thereafter dried and the dry weight of the suspended solids was determined.
- Pretreatment: no
- Concentration of sludge: final concentration of 30 mg dry material per liter
- Water filtered: yes, the purified water used in the test was ultrapure water obtained from an ELGA PURELAB Option-Q water purification system.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

26 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

15.2 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral medium was prepared according to the testing guidelines and is considered not to contain any contaminant that would affect the integrity or outcome of the study. The purified water used in the test was ultrapure water obtained from an ELGA PURELAB Option-Q water purification system. Analytical grade salts were dissolved in ultrapure water to obtain the following stock solutions:
* stock solution 1) KH2PO4: 8.50 g/L
K2HPO4: 21.75 g/L
Na2HPO4 × 2H2O: 33.40 g/L
NH4Cl: 0.50 g/L
The pH of this solution was 7.4.
* stock solution 2) CaCl2 × 2H2O: 36.40 g/L
* stock solution 3) MgSO4 × 7H2O: 22.50 g/L
* stock solution 4) FeCl3 × 6H2O: 0.25 g/L, stabilized with one drop of concentrated HCl per liter.
* To obtain the final mineral medium, 10 mL of stock solution No. 1 and 1 mL each of stock solutions Nos. 2, 3 and 4 were combined and made up to 1000 mL with ultrapure water.
- Additional substrate: no
- Test temperature: 22-24°C
- pH: 7.4 - 7.5, measured prior to test start (Day 0), in each test vessel after the addition of test and/or reference item and at the end of incubation (Exposure Day 28).
- pH adjusted: yes. The pH was adjusted from 7.8 to 7.4 with a diluted hydrochloric acid solution.
- Aeration of dilution water: bottles fitted with an aeration glass tube reaching nearly the bottom of the vessel. the test suspension was continuously stirred with the help of magnetic stirrers.
- Continuous darkness: yes (darkroom)

TEST SYSTEM
- Culturing apparatus: 5-liter amber glass bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: CO2-free air was bubbled through the solution in each test vessel at a rate of 30-100 mL/min. CO2-free air was produced by passing compressed air through a bottle, containing about 4000 mL of a 2 M NaOH solution to trap CO2 and further through glass column containing self-indicating soda lime granules (Carbosorb® from VWR International GmbH, Darmstadt, Germany).
- Measuring equipment: IC analyses were performed using a TOC infrared gas analyzer equipped with an automatic sampler (i.e. vario TOC cube from Elementar Analysensysteme GmbH, Langenselbold, Germany).
- Details of trap for CO2 and volatile organics if used: The CO2 evolved from the test vessels were collected in 2 x 500 mL Drechsel bottles (gas-absorbtion flasks) containing 300 and 200 mL of 0.05 M NaOH. The air leeding connections between the test vessel and the absorption flasks were done by glass tubes.

SAMPLING
- Sampling frequency: For analysis of the evolved CO2, samples were taken on Exposure Day 2, 5, 7, 9, 12, 14, 19, 21, 28 (before acidification), and 29 (after driving off residual CO2).
- Sampling method: On each sampling day, an aliquot of 10.0 mL was withdrawn from the absorber flask nearest to the test vessel for analysis of inorganic carbon (IC). Additional samples for analysis of IC were withdrawn from the second absorber flask of all test vessels on Exposure Day 14 and at the end of the exposure period on Exposure Day 28 in order to correct for any carry over of CO2.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 2 replicates with only inoculum
- Toxicity control: yes, 1 replicate
- Procedure control: yes, 2 replicates

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Test performance:

- The percentage degradation of the reference item reached with 75 % by Exposure Day 5 the pass level for ready biodegradability (criterion: at least 60 % in a 10-day window by Day 14).
- The total CO2 evolution in the inoculum controls at the end of the test was 20 and 22 mg CO2/L (corresponding to 17 and 18 mg C per vessel, respectively; criterion: less than 40 mg CO2/L, corresponding to 33 mg C per vessel).
- The IC content in the test item vessels was less than 5 % of the TC, i.e. according to the validity criterion.

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 6.2

Sampling time:

28 d

Remarks on result:

other: mean of 2 replicates

Details on results:

The criterion for ready biodegradability (at least 60% biodegradation within 10 days of biodegradation exceeding 10%) was not met

BOD5 / COD results

Results with reference substance:

The percentage degradation of the reference item reached with 75 % by Exposure Day 5 the pass level for ready biodegradability (criterion: at least 60 % in a 10-day window by Day 14), confirming suitability of the activated sludge.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

A 28-d ready biodegradability test (OECD 301B, modified Sturm test) using unadapted activated sludge from a predominantly domestic waste water treatment plant indicated that the test item N-morpholinopropylamine was not readily biodegradable under the conditions of the test (initial concentration 26 mg/L). The test substance showed only 6.2% biodegradation (based on % ThCO2). The test substance did not inhibit microbial activity at the concentration used in the test. The results of the test can be considered reliable without restriction.