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Diss Factsheets

Environmental fate & pathways

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:
08 Feb - 12 Apr 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom, London, UK

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(piperidin-4-yl)morpholine
EC Number:
627-580-3
Cas Number:
53617-35-9
Molecular formula:
C9H18N2O
IUPAC Name:
4-(piperidin-4-yl)morpholine

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): Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK
- Laboratory culture: no
- Storage length: used on the day of collection
- Preparation of inoculum for exposure: washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC); The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C.
- Suspended solids concentration: 3.0 g/L (determined by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.)
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
15.8 mg/L
Based on:
test mat.
Initial conc.:
10 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: according to guideline
- Test temperature: 21 - 24 °C
- pH: 7.4 ± 0.2
- pH adjusted: if necessary using diluted hydrochloric acid or sodium hydroxide solution
- Aeration of dilution water: The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer. The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
- Continuous darkness: yes

TEST SYSTEM
- Number of culture flasks/concentration: 2
- Method to create aerobic conditions: Approximately 24 h prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 30 mL of inoculum and aerated overnight.
- Measuring equipment: The samples were analyzed for IC using either a Shimadzu TOC-VCSH TOC analyzer or a Shimadzu TOC-LCSH TOC analyzer. The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter. The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter.
- Test performed in closed vessels due to significant volatility of test substance: test vessels were sealed
- Details of trap for CO2 and volatile organics if used: The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.
- Suspended solids concentration: 30 mg suspended solids/L
- IC analysis: All samples were analyzed for IC immediately. The remainder of all samples with the exception of the Day 0 samples were frozen for further analysis if required. On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.

SAMPLING
- Sampling frequency: The first absorber vessel was sampled on days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29.
- Sampling method: Samples (2 mL) were taken from the first CO2 absorber vessels.
- Sample storage before analysis: analysed immediately

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 2 bottles
- Abiotic sterile control: no
- Toxicity control: yes, 1 bottle
- Procedure control: yes, 2 bottles

STATISTICAL ANALYSIS
- Statistical analysis of the Day 29 IC values for the inoculum control and test item vessels was carried out using a Student’s t-test to determine any statistically significant differences between the test and control groups.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
The results obtained from the samples taken for DOC analysis from the preliminary investigational indicated that the test item did not adsorb to filter matrices or to activated sewage sludge. Therefore, for the purpose of the study, the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test item.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
22
Sampling time:
28 d
Details on results:
Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 81% biodegradation after 14 days and 83% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

The toxicity control attained 30% biodegradation after 14 days and 47% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

Table 1: Percentage biodegradation

Day

Biodegradation [%]

Procedure control

Test item

Toxicity control

0

0

0

0

2

46

0

29

6

54

0

32

8

66

0

37

10

68

2

35

14

81

1

30

21

84

0

34

28

82

19

45

29*

83

22

47

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable