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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 May - 18 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
4-morpholinopropanesulphonic acid
EC Number:
214-478-5
EC Name:
4-morpholinopropanesulphonic acid
Cas Number:
1132-61-2
Molecular formula:
C7H15NO4S
IUPAC Name:
3-morpholin-4-ylpropane-1-sulfonic acid
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): 3-morpholin-4-ium-4-ylpropane-1-sulfonate
- Physical state : white powder
- Analytical purity : 99%
- Purity test date : not stated
- Lot/batch No. : 03100048/310
- Expiration date of the lot/batch : 2015-04-12
- Storage condition of test material : in a tightly closed vessel at room temperature (20 ±5 °C) under dry conditions in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
other: activated sludge, mostly domestic
Details on inoculum:
- Source of inoculum/activated sludge : The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf , Germany. Date of collection : 16 May 2014 , batch no: 20140516

- Pretreatment : The sludge was filtrated , washed with tap water twice , then washed with and re-suspended in test medium . It was then aerated for until use. The dry matter was determined as 4440 mg suspended solids/liter .
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
20 mg/L
Based on:
other: Carbon
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preparations
The medium was prepared from the stock solutions. The stock solution of the positive control was prepared and its TOC was measured. The inoculum was taken from its source, washed, aerated and the dry matter was determined. The test vessels were filled with medium and inoculum. Then, all flasks were aerated for 72 hours with purified, CO2-free, moistened air to purge the system of CO2.

Experimental Parameters
Flask volume : 1500 mL
Apparatus blanks : 2, containing mineral medium only
Controls : 2, containing mineral medium and inoculum
Positive control flasks : 2, containing positive control, mineral medium and inoculum
Test flasks : 2, containing test item, mineral medium and inoculum
Abiotic control : 1, containing test item, mineral medium and HgCl2
Toxicity control : 1, containing test item, positive control, mineral medium and inoculum
Inoculum concentration : 24.9 mg/L
Temperature : 21.4 – 24.0 °C
Duration : 28 days

The test was performed with a nominal start concentration of 20 mg organic carbon/L.
The following amounts of test item and reference item were added to the flasks: see table 1 at "any other information on materials and methods incl. tables"

Apparatus
The test vessels were aerated with purified (by activated charcoal), CO2-scrubbed, moistened air. The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1.5 m-NaOH. To control the absence of CO2, the air was then led through a flask containing a solution of Ba(OH)2 before reaching the test vessels. Magnetic stirrers were used to prevent deposition of inoculum. The emitted CO2 was trapped in 0.25-m-NaOH. Two scrubbers containing 100 ml each were connected in series to the test vessels. The initial IC value of the 0.25 m-NaOH was separately determined in each flask.

Sampling
From each front scrubber flask, ten samples were taken in order to determine the emitted CO2 (on days 0, 2, 4, 7, 9, 11, 14, 18, 23 and 29). The sample volume was 1 mL. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2. On day 28, 5 mL HCl 2-m were added to each test flask in order to drive off dissolved CO2. On day 29, samples from both scrubber flasks were taken.

CO2 determination
Analyses of the emitted CO2 were made by IC measurement using the carbon analyser TOC multi N/C 2100S, Analytik Jena. Each sample was measured at least in duplicate. The carbon analyser was calibrated with freshly prepared reference solutions containing potassium hydrogen phthalate (TC), sodium hydrogen carbonate and sodium carbonate (IC) once a week. After every start, quality control samples were measured.
Reference substance
Reference substance:
aniline
Remarks:
2.1 g/l

Results and discussion

Test performance:
All validity criteria were met.
Degradation behaviour of positive control and toxicity control was normal. Abiotic degradation was not observed. Both replicates of the test item showed very good correspondence.
As degradation in the toxicity flask was 35 % after 14 days, the test item can be stated as “not toxic towards the inoculum in a concentration of 51.7 mg/L”.
Ready degradability is defined in the guidelines as degradation surpassing 60% within 10 days after reaching a level of 10%. Therefore, the test item 3-morpholin-4-ium-4-ylpro-pane-1 -sulfonate cannot be considered as “readily biodegradable”. Degradation missed 60% within 28 days, too. Therefore, the test item is not considered as “biodegradable within 28 days”, either.
No observations were made which might cause doubts concerning the validity of the study outcome.
The result of the test can be considered valid.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
1
Sampling time:
28 d
Details on results:
The following data were determined for the test item 3-morpholin-4-ium-4-ylpropane-1 - sulfonate:
10-day-window : not detected
degradation at the end of 10-day-window : none
degradation at the end of the test : 1 %
pass level following guideline : 60% at the end of 10-day-window

BOD5 / COD results

Results with reference substance:
Degradation of the reference item (Aniline) was 65 % after nine days.

Any other information on results incl. tables

Table 1 : Emitted Carbon (in mg/L)

Day  Control 1  Control 2  Positive Control 1 Positive Control 2  Test 1  Test 2  Abiotic Control   Toxicity Control
0,27 0,62 0,28 0,39  0,45 0,70 -0,01 0,50 
0,79 1,04  3,60 5,63 1,31 1,17  0,01 4,37 
1,19 1,62 9,97 11,22 2,10 1,77 0,10 10,39 
1,74 2,44 15,50 15,12 2,73 2,33 0,06 13,93 
11  2,03 2,73 16,25 16,14 3,10 2,73 0,19 14,75 
14  2,06 2,73 16,20 16,31 2,81 2,93 0,15 16,53 
18  2,40 3,36 18,56 18,84 3,54 3,45 0,21 18,94 
23  2,41 3,37 18,11 17,35 3,31 3,35 0,19 18,45 
29  2,86 4,35 19,73 18,79 3,86 3,71 0,12 19,68 

Table 2 : Degradation-Values in %

Day  Positive Control 1  Positive Control 2  Positive Control Mean Test 1  Test 2  Test Mean  Abiotic Control   Toxicity Control
-0,8 -0,2 -0,5 0,1 1,3 0,7 0,0 0,1 
13,2 23,2  18,2 2,0 1,3 1,6  0,1 8,6 
42,1 48,2 45,1 3,4 1,8 2,6 0,5 22,2 
65,9 64,0 65,0 3,2 1,2 2,2 0,3 29,3 
11  68,1 67,6 67,9 3,6 1,7 2,7 1,0 30,6 
14  67,9 68,4 68,1 2,1 2,7 2,4 0,7 35,0 
18  77,1 78,5 77,8 3,3 2,9 3,1 1,0 39,8 
23  74,8 71,0 72,9 2,1 2,3 2,2 0,9 38,5 
29  79,2 74,6 76,9 1,3 0,5 0,9 0,6 39,8 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
All validity criteria were met
Interpretation of results:
other: not readily biodegradable
Conclusions:
The study was performed according to OECD 301B/EU C.4-C without deviations and therefore considered to be of the highest quality (reliability Klimisch 1).
The test item 3-morpholin-4-ium-4-ylpropane-1-sulfonate was tested using a concentration of nominally 20 mg organic carbon/L (corresponding to 51.5 mg 3-morpholin-4-ium-4- ylpropane-1-sulfonate/L) in test medium.
Aniline was chosen as positive control.
Activated sludge was used as inoculum (concentration in the test 24.9 mg dry matter/L). The test was left running for 28 days.
All validity criteria were met. Degradation of the positive control was 65 % after nine days.
The following data were determined for the test item 3-morpholin-4-ium-4-ylpropane-1 - sulfonate:
10-day-window : not detected
degradation at the end of 10-day-window : none
degradation at the end of the test : 1 %
pass level following guideline : 60% at the end of 10-day-window
Therefore, regardless of the 10-day-window, 3-morpholin-4-ium-4-ylpropane-1-sulfonate is not biodegradable following OECD 301 B/EU C.4-C.
Executive summary:

The biodegradability of the substance 3-morpholin-4-ium-4-ylpropane-1-sulfonate was investigated according to OECD guideline 301B and EU method C.4 –C. The study was conducted in compliance with the Principles of Good Laboratory Practice (GLP). Activated sludge collected from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, NW-Lachen-Speyerdorf, Germany, which treats predominantly domestic sewage was used as inoculum. The test item was tested using a concentration of 20 mg organic carbon/L (corresponding to 51.5 mg 3-morpholin-4-ium-4- ylpropane-1-sulfonate/L) in test medium following OECD 301B/EU-Guideline C.4-C. Aniline was chosen as positive control. The test was left running for 28 days. Analyses of the emitted CO2 were made by IC measurement using the carbon analyzer TOC multi N/C 2100S, Analytik Jena. All validity criteria were met. Degradation of the positive control was 65% after nine days.

The following data could be determined for the test item 3-morpholin-4-ium-4-ylpropane-1-sulfonate :

10-day-window : not detected

degradation at the end of 10-day-window : none

degradation at the end of the test : 1 %

pass level following guideline : 60% at the end of 10-day-window

 

Therefore, regardless of the 10-day-window, 3-morpholin-4-ium-4-ylpropane-1-sulfonate is not biodegradable following OECD 301 B/EU C.4-C.