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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 November 2017 to 31 January 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
17 July 1992
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
Details on test material:
CAS 1629579-82-3
Molecular formula C18H32N2O11
EC 818-033-1

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was determined to be 4.3 g/L in the concentrated sludge. Before use, the sludge was allowed to settle (41 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.

Reason for selection
The test has been accepted internationally for determining the 'ready' biodegradability of test items under aerobic conditions.
Duration of test (contact time):
ca. 28 d
Details on study design:
Readily biodegradable are those test items giving a result of at least 60% biodegradation within 28 days. This pass level must be reached within the 10 days immediately following the attainment of 10% biodegradation (10-day window).
Theoretical carbon dioxide (ThCO2) is the quantity of carbon dioxide calculated (mg) to be produced from the known or measured carbon content of the test item when fully mineralized; also expressed as mg carbon dioxide evolved per mg test item.
Total Organic Carbon (TOC) of a sample is the sum of the organic carbon in solution and in suspension.

Test concentration and preparation of test solutions

Bis-Aminopropyl Diglycol Dimaleate was a clear colourless to pale yellow liquid with a water content of 74%. The test item was tested in duplicate at a concentration of 96.5 mg/L, corresponding to 12 mg TOC/L. The organic carbon content was based on the molecular formula.
Since Bis-Aminopropyl Diglycol Dimaleate was easily soluble in water, the test media were prepared using a stock solution of 1 g/L in Milli- RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). A weighed amount of 999.4 mg of Bis- Aminopropyl Diglycol Dimaleate was dissolved in Milli- RO water and made up to 1000 mL. After stirring for 20 minutes, the final stock solution was clear and colourless. Aliquots of 193 mL of the stock solution were added to the test item bottles A and B and to the toxicity control. These test bottles contained medium with microbial organisms (final volume: 2 litres). The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms.
Any residual volumes were discarded.

Test procedure and conditions

Test duration 28 days for the inoculum blank and test item (last CO2 measurement on day 29).
14 days for the positive and toxicity control (last CO2 measurement on day 15).
During the test period, the test media were aerated and stirred continuously.

Test vessels 2 litre brown coloured glass bottles.
Milli- RO water Tap-water purified by reverse osmosis (Milli- RO) and subsequently passed over activated carbon.

Stock solutions of mineral components
A) 8.50 g KH2PO4
21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in Milli- RO water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli- RO water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli- RO water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli- RO water and made up to 1 litre.

Mineral medium
1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli- RO water.

Barium hydroxide
0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.

Synthetic air (CO2 < 1 ppm)
A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).

Illumination The test media were excluded from light.

Preparation of Bottles
Pre-incubation medium
The day before the start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

Type and number of bottles

Test suspension: containing test item and inoculum (2 bottles). Inoculum blank: containing only inoculum (2 bottles) Positive control: containing reference item and inoculum (1 bottle). Toxicity control: containing test item, reference item and inoculum (1 bottle).

At the start of the test (day 0), test and reference item were added to the bottles containing the microbial organisms and mineral components. The volumes of suspensions were made up to 2 litres with Milli- RO water, resulting in the mineral medium described before. Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

Determination of CO2
Experimental CO2 production
The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).

Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test item. Titrations for the positive and toxicity control were made over a period of at least 14 days.
Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers were moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.
On the penultimate day, the pH of respective test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 15 (positive and toxicity control) and on day 29 (remaining vessels).
Theoretical CO2 production
The theoretical CO2 production was calculated from the molecular formula.

Measurements and Recordings

At the start of the test (day 0) and on the penultimate day (day 14 for the positive and toxicity control and day 28 for the inoculum blanks and test item), before addition of concentrated HCl.

Temperature of medium
Continuously in a vessel with Milli- RO water in the same room.

Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradationopen allclose all
Key result
% degradation (CO2 evolution)
ca. 50
Sampling time:
28 d
Key result
% degradation (CO2 evolution)
ca. 51
Sampling time:
28 d
Details on results:
Acceptability of the Test

1.    The positive control item was biodegraded by at least 60% (73%) within 14 days.
2.    The difference of duplicate values for %-degradation of the test item was always less than 20 (≤ 3%).
3.    The total CO2release in the blank at the end of the test did not exceed 40 mg/L (38.6 mg CO2per 2 litres of medium, corresponding to 19.3 mg CO2/L).
4.    The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli- RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).

Since all criteria for acceptability of the test were met, this study was considered to be valid.

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

BOD5 / COD results

Results with reference substance:
Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve.

Any other information on results incl. tables

Theoretical CO2Production

The ThCO2of Bis-Aminopropyl Diglycol Dimaleate was calculated to be 0.45 mg CO2/mg.

The ThCO2of sodium acetate was calculated to be 1.07 mg CO2/mg.

If applicable the ThCO2per test bottle are given in the subscript of the tables (see Appendix 1).


All data are presented in Appendix 1. The results of CO2production and biodegradation in blank bottles, background bottles and each test bottle are listed in Table2 to 8.

Table 9 contains the comparison of biodegradation of Bis-Aminopropyl Diglycol Dimaleate in bottles A and B.

Figure 1 (attached) shows the curves for biodegradation of the two bottles with Bis-Aminopropyl Diglycol Dimaleate, the positive control and the toxicity control.

The relative biodegradation values calculated from the measurements performed during the test period revealed 50% and 51% biodegradation of Bis-Aminopropyl Diglycol Dimaleate (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control, more than 25% biodegradation occurred within 14 days (58%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.

Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve (see also paragraph 5.1).

Monitoring of temperature and pH

The temperature recorded in a vessel with water in the same room varied between 22 and 23°C. The pH values of the different test media are presented in Table 1.

Table 1          
pH Values of Different Test Media

Test medium:

At the start of the test:

On day 14:

On day 28:

Blank control (A)

7.9 → 7.61



Blank control (B)

7.9 → 7.61



Positive control

7.9 → 7.61



Bis-Aminopropyl Diglycol Dimaleate (A)

7.8 → 7.51



Bis-Aminopropyl Diglycol Dimaleate (B)

7.7 → 7.61



Toxicity control

7.7 → 7.61



¹: Adjusted using 1 M HCl (Merck, Darmstadt, Germany)

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
In conclusion, Bis-Aminopropyl Diglycol Dimaleate was not readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

The objectiveofthe studywas to evaluate the test item Bis-Aminopropyl Diglycol Dimaleate for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with the supernatant of activated sludge; Carbon dioxide (CO2) evolution test (modified Sturm test). The study procedures described in this report were in compliance with the OECD guideline No. 301 B, 1992.

In conclusion, Bis-Aminopropyl Diglycol Dimaleate was not readily biodegradable under the conditions of the modified Sturm test presently performed.