Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

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:

18th October 1995 to 15 November 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.4-F (Determination of the "Ready" Biodegradability - MITI Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

EC Number:

421-660-1

EC Name:

Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

Molecular formula:

Hill formula: C50 H96 N2 O10 CAS formula: C42 H74 O8. 2(C4 H11 N O)

IUPAC Name:

bis((2-hydroxyethyl)dimethylazanium) (4E)-3-{[2-({3-carboxylato-3-[(2E)-hexadec-2-en-2-yl]propanoyl}oxy)ethoxy]carbonyl}-4-methyloctadec-4-enoate

Constituent 2

Reference substance name:

BIS(DIMETHYL-(2-HYDROXYETHYL)AMMONIUM) 1,2-ETHANEDIYL-BIS (2-HEXADECENYLSUCCINATE)

IUPAC Name:

BIS(DIMETHYL-(2-HYDROXYETHYL)AMMONIUM) 1,2-ETHANEDIYL-BIS (2-HEXADECENYLSUCCINATE)

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms were obtained from 10 different sampling sites around the UK; 3 city domestic sewage plants (Liverpool, Belper and Bristol), 1 industrial sewage plant (Derby), 3 river samples (Mersey, Trent and Severn), 1 lake water sample (Allestree Lake) and 2 sea water samples (Skegness and Southport). The sample types and volumes were as follows:
City sewage: 1 litre of return sewage at each sewage disposal point
Rivers, lakes and sea: 1 litre of surface water and 1 litre of surface soil on the bank/beach which is in contact with the atmosphere
- Laboratory culture: NDA
- Method of cultivation: NDA
- Storage conditions: NDA
- Storage length: NDA
- Preparation of inoculum for exposure: The sample mixutres were allowed to settle adn the floating foreign matter was removed and the supernatant filtered. The filtrate was then mixed with ~2 L of supernatant removed from a previously established culture and transfered to a cluture vessel. The pH was adjusted and the solution aerated. The culture was then allowed to settle and ~1/3 of the volume of the supernatant removed. An equal volume of 0.1 % synthetic sewage was added and the aeration re-started. Synthetic sewage was prepared by dissolving glucose, peptone and monopotassium phosphate in deionised water at a concentration of 0.1 % (w/v).
- Pretreatment: NDA
- Concentration of sludge: NDA
- Initial cell/biomass concentration: NDA - the sludge was found to have an active micorflora including a variety of protozoa, including ciliates, flagellates and a large population of motile bacteria.
- Water filtered: yes
- Type and size of filter used, if any: reverse osmosis (Elgastat Spectrum R01)

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST CONDITIONS
- Composition of medium: To 1 L of water was added 3 mL each of the following solutions:
Solution 1:
K2HPO4 = 21.75 g/L
KH2PO4 = 8.5 g/L
Na2HPO4.12H2O = 44.60 g/L
NH4Cl = 1.70 g/L
Solution 2: MgSO4.7H2O = 22.50 g/L
Solution 3: CaCl2 = 27.5 g/L
Solution 4: FeCl3.6H2O = 0.25 g/L
- Additional substrate: Not used
- Solubilising agent (type and concentration if used): Not used
- Test temperature: 25 ± 1 °C. The temperature of the water bath was recorded daily and the pH of each control, test and standard material vessel were recorded on days 0 and 28.
- pH: 7.0 ± 1.0
- pH adjusted: yes - sodium hydroxide or phosphoric acid
- CEC (meq/100 g): NDA
- Aeration of dilution water: The culture mixutre was constantly aerated.
- Suspended solids concentration of the test samples: 30 mg/L
- Continuous darkness: yes
- Other: None


TEST SYSTEM
- Culturing apparatus: All control, test and standard material vessels were placed in the CES Multi-Channel Aerobic Respirometer.
- Number of culture flasks/concentration: Three replicate bottles were used at a test material concentration of 100 mg/L.
- Method used to create aerobic conditions: The samples were aerated.
- Method used to create anaerobic conditions: N/A
- Measuring equipment: The DOC analyses were carried out using an Ionics 1555B and a Dohrmann DC 190 Carbon Analyser. The carbon analyses were by the method of high temperature catalytic conversion.
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system: NDA
- Details of trap for CO2 and volatile organics if used: The test system consists of a sample flask sealed by a manometric cell/ CO2 trap immersed in a temperature controlled water bath.
- Other: The samples were stirred for the duration of the study.


SAMPLING
- Sampling frequency: The appearance of the test material dispersions were recorded on days 0 and 28. Samples of control, test and standard material solutions were removed on days 0 and 28 for measurements of oxygen consumption, DOC analysis and compound specific analyses (HPLC). IR was also performed at the start and termination of the study.
- Sampling method: NDA
- Sterility check if applicable: NDA
- Sample storage before analysis: NDA
- Other: None


CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: Yes
- Toxicity control: No
- Other: None


STATISTICAL METHODS: Not used

Reference substance

Reference substance:

aniline

Results and discussion

Preliminary study:

N/A

Test performance:

Comparison of the IR spectrum of the test material at initiation, with that at termination of the study showed the test material to be stable over the duration of the study.
The results of the study confirm that no hydrolysis of the test material occurred over the study period and that loss of parent test material in the inoculated test vessels was due to microbial degradation.

% Degradation

% Degradation 1

Parameter:

% degradation (O2 consumption)

Value:

38

Sampling time:

28 d

Remarks on result:

other: Mean value of three replicates

% Degradation 2

Parameter:

% degradation (DOC removal)

Value:

42

Sampling time:

28 d

Remarks on result:

other: Mean value of three replicates

% Degradation 3

Parameter:

% degradation (test mat. analysis)

Value:

100

Sampling time:

28 d

Remarks on result:

other: Mean value of three replicates

Details on results:

Points of degradation plot (test substance):
0 % degradation after 1 d
7.7 % degradation after 3 d
21 % degradation after 5 d
24.7 % degradation after 7 d
26.7 % degradation after 9 d
27.7 % degradation after 11 d
28 % degradation after 13 d
29.3 % degradation after 16 d
31.7 % degradation after 19 d
35 % degradation after 22 d
37 % degradation after 25 d
38.3 % degradation after 28 d

OS 114451A attained 44, 32 and 39 % degradation with a mean of 38 % degradation calculated from the oxygen consumption values after 28 days. The degradation rates calculated from the results of the DOC analyses were 38, 37 and 50 % with a mean of 42 % degradation. The results of the compound specific analyses showed that the test amterial attained a mean of 100 % degradation after 28 days.

BOD5 / COD results

Results with reference substance:

Points of degradation plot (reference substance):
0 % degradation after 1 d
0 % degradation after 3 d
16 % degradation after 5 d
51 % degradation after 7 d
59 % degradation after 9 d
61 % degradation after 11 d
62 % degradation after 13 d
66 % degradation after 16 d
67 % degradation after 19 d
71 % degradation after 22 d
76 % degradation after 25 d
79 % degradation after 28 d

Aniline attained 100 % degradation calculated from the results of the DOC analyses.

Any other information on results incl. tables

Table 1 Summary of results on days 7, 14, 21 and 28 calculated from oxygen consumption values, DOC analyses and the residual test material analyses

Identification (test material (100 mg/L) plus inoculums	Degradation (%)
	Oxygen consumption				Day 28
	Day 7	Day 14	Day 21	Day 28	DOC analyses	Compound specific analyses
R1	27	32	39	44	38	100
R2	23	27	29	32	37	100
R3	24	27	34	39	50	100
Mean	25	29	34	38	42	100

R1, R2, R3 = Replicates 1, 2 and 3

Applicant's summary and conclusion

Interpretation of results:

other: not readily biodegradable

Conclusions:

OS 114451A is not readily biodegradable according to OECD Guideline 301C given that the degradation rates calcluated from oxygen consumption values were less than 60 %.

Executive summary:

In an assessment of ready biodegradability study, modified MITI test (525/044), the test material (OS 114451A) was determined to not be readily biodegradable according to OECD Guideline 301C given that the degradation rates calcluated from oxygen consumption values were less than 60 %.