N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)Glycine

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-11-15 to 2022-02-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 1992

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Purity, including information on contaminants, isomers, etc.: 100.0% (HPLC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Recommended storage temperature: room temperature. Store in a dry place, keep away from moisture. Protect from heat and direct sunlight.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: No decomposition if used as directed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Due to its low solubility, the test item was weighed onto weighing paper and added directly into the test vessels. 54.8 – 54.9 mg of the test item were added into the test vessels with a liquid volume of 1.5 L, corresponding to a concentration of 19.9 – 20.0 mg/L organic carbon.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from the municipal wastewater treatment plant AZV Breisgauer Bucht was used as inoculum with a concentration corresponding to 30 mg dry solids per litre. The treatment plant clarifies predominantly domestic wastewater and has a capacity of 600,000 inhabitant equivalents. Sampling date of activated sludge was 30 November 2021. The dry solid content of the activated sludge was 4.7 g/L. It was determined by weight measurements after drying at 105°C for 4 hours (mean of triplicate measurements). The activated sludge was washed twice with tap water by settling the sludge, decanting the supernatant and re-suspending the sludge.

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

inorg. C analysis

Parameter followed for biodegradation estimation:

CO2 evolution

Parameter followed for biodegradation estimation:

other: ThCo2 (Theoretical CO2 ecolution)

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium and CO2-absorption medium (0.2 M NaOH)
- Test temperature: 19.9 – 21.9°C
- pH: ranged between 7.0 and 7.8 at test end
- Aeration: yes, 30-100 ml/min
- Continuous darkness: no, diffuse light for 28 days

TEST SYSTEM
- Culturing apparatus: Gas wash bottles (2000 ml volume)
- Number of culture flasks/concentration: One test concentration and three replicates
- Method used to create aerobic conditions: The test vessels are aerated by the passage of carbon dioxide-free air.
- Measuring equipment: Total carbon analyser (TOC-L), Non-dispersive infrared gas analyser (NDIR)
- Details of trap for CO2 and volatile organics if used: The CO2 produced in the reactors was absorbed in two 250 mL gas wash bottles in series each filled with 200 mL 0.2 M NaOH. Sampling was performed through the lateral connecting pieces through the butyl rubber septum using 10 mL PE syringes.

SAMPLING
- Sampling frequency: IC-measurements were performed on day 0, 4, 7, 11, 14, 21 and 28. CO2 dissolved in water was measured on day 28. pH was measured at day 12 and day 40.
- Sampling method: 8 mL NaOH were sampled from the first of the two CO2 absorber flasks

CONTROL AND BLANK SYSTEM
- Inoculum blank: containing only inoculum
- Abiotic control: without inoculum containing test item and sodium azide
- Toxicity control: containing inoculum and reference compound sodium benzoate

Reference substance:

other: sodium benzoate

Test performance:

The temperature was 19.9 – 21.9°C throughout the whole study. It was therefore just below the required range of 22 ± 2°C. However, the deviation from the target temperature occurred on only one day and was with 0.1°C very small. An effect on the test result can therefore be excluded.

The IC-concentration of the NaOH in the second CO2-absorber flasks in line, used as protective flasks, was ≤ 9 ppm in all but one flask and was not considered in the data processing, because CO2 absorption from room air during the filling process was its source. In absorber flask no. 19 belonging to the abiotic control vessel), an unusually high IC concentration of >70 mg/L was measured.
Presumably, the sealing film on the sample vial had come loose and the sample absorbed CO2 from the room air. Since the IC value in the first absorber was also inconspicuous, the increased IC concentration did not affect the test result.

Key result

Parameter:

other: % of ThCO2

Value:

26.3

Sampling time:

28 d

Remarks on result:

other: with considering the IC in the liquid phase

Details on results:

The degradation of the test item at the end of the test was 26.3% of ThCO2 (28 days with considering the IC in the liquid phase, mean of three replicates).
The test item did not reach the criteria for ready biodegradability (60% of ThCO2 within 28 days and within a 10-d window). The degradation in the abiotic control at the end of the test was 12.5%.

Results with reference substance:

The degradation of the reference control after 4 days was >60%.

Table 1: Ultimate biodegradation after x days [% of ThCO2]

Reactor	Day	0	4	7	11	14	21	28	28 with IC reactor
15	Test flasks	0	-1.6	7.6	15.7	20.1	24.4	26.4	26.4
16		0	1.3	4.3	8.7	13.2	22.4	24.5	24.4
17		0	1.7	2.9	5.7	10.3	23.6	28.3	28.1
4	Reference flasks	0	69.7	76.2	81.6	86.3	89.8	91.5	91.7
5		0	72.1	78.0	85.7	87.4	89.7	91.1	91.2
6		0	68.6	82.5	87.4	89.0	90.1	92.9	93.0
18	Toxicity control	0	37.3	40.6	48.8	52.2	54.8	56.7	56.7
19	Abiotic control	0	9.0	10.5	10.0	3.0	10.2	11.3	12.5

 

The degradation extent in the toxicity control within 14 days was 52.2%. According to the guideline, the test substance had no inhibitory effect on the inoculum.

In the abiotic control, a degradation of 12.5% was determined at the end of the test (see table 1). Whether the CO2 evolution measured in the abiotic control was caused by an abiotic decomposition of the test item or by an unintended entry of CO2 into the test system (e.g. bacterial growth in the aeration tubes) cannot be as certained since no abiotic blank was included in the test design.

The biodegradation of the reference compound reached the pass level of 60% ThCO2 by day 4.

The IC content of the test item reactor at the beginning of the test was 1.3% of the TC and therefore less than 5%.

The CO2 evolution in the inoculum blank at the end of the test was 22.3 mg/L and therefore below 40 mg/L.

The difference of extremes of replicate values of the test item at the end of the test was less than 20%.

Validity criteria fulfilled:

yes

Remarks:

The test is valid according to OECD 301 B (July 1992).

Interpretation of results:

not readily biodegradable

Conclusions:

The degradation of the test item at the end of the test was 26.3% of ThCO2 (28 days with considering the IC in the liquid phase, mean of three replicates).
The test item did not reach the criteria for ready biodegradability (60% of ThCO2 within 28 days and within a 10 days window).

Executive summary:

A solution of N-[2-[bis(carboxymethyl)amino]ethy|]-N-(1-oxononyl)G|ycine in a mineral medium was inoculated and incubated for 28 days under aerobic conditions. During this period. degradation was followed by IC and CO2 determinations. N-[2-[bis(carboxymethyl)amino]ethy|]-N-(1-oxononyl)G|ycine showed 26.3% degradation of ThCO2 after 28 days.

N-[2-[bis(carboxymethyl)amino]ethy|]-N-(1-oxononyl)G|ycine is considered to be "Not Readily Biodegradable".

Description of key information

The test item was determined to be not readily biodegradable in an OECD 301 test.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater