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

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 1995 to 24 August 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed to a guideline and used GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition 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)

Sampling and analysis

Analytical monitoring:

not specified

Details on sampling:

NDA

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test material was prepared by a direct dispersion in water. An amount of test material was dispered in water with the aid of ultrasonic disruption and the volume adjusted to 1 L to give a 4.0 g/L stock solution. Aliquots were then removed and dispersed with activated sewage sludge, synthetic sewage and water to given the final test concentrations.
- Eluate: NDA
- Differential loading: NDA
- Controls: The control was maintained under identical conditions but not exposed to the test material.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc): N/A

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: NDA
- Method of cultivation: NDA
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of 21 °C and fed 50 mL/L of synthetic sewage sludge prior to use. The pH of the sample was 7.3 and the suspended solids equal to 3.9 g/L prior to use.
- Pretreatment: NDA
- Initial biomass concentration: Suspended solids: 3.9 g/L

Study design

Test type:

other: aerobic

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

N/A

Test conditions

Hardness:

100 mg/L as CaCO3

Test temperature:

21 °C

pH:

pH of the inoculum was 7.3

Dissolved oxygen:

NDA

Salinity:

NDA

Nominal and measured concentrations:

Nominal concentrations: 100, 180, 320, 560 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): NDA
- Material, size, headspace, fill volume: At time 0, 16 mL of synthetic sewage was diluted to 300 mL with water and 200 mL of inoculum added in a 500 mL conical flask (first control). Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the test or reference material being added. Finally a second control was prepared.
- Aeration: The mixture was aerated with compressed air via narrow bore glass tubes at a rate of approximately 0.5-1 L/min.
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- No. of organisms per vessel: N/A
- No. of vessels per concentration (replicates): 1 vessel
- No. of vessels per control (replicates): 2 controls
- No. of vessels per vehicle control (replicates): N/A
- Biomass loading rate: N/A


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory dechlorinated tap water
- Total organic carbon: NDA
- Particulate matter: NDA
- Metals: NDA
- Pesticides: NDA
- Chlorine: NDA
- Alkalinity: NDA
- Ca/mg ratio: NDA
- Conductivity: NDA
- Culture medium different from test medium: No
- Intervals of water quality measurement: NDA


OTHER TEST CONDITIONS
- Adjustment of pH: NDA
- Photoperiod: NDA
- Light intensity: Normal laboratory lighting


EFFECT PARAMETERS MEASURED: As each vessel reached 30 minutes contact time the contents of the conical flask were poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over an approximate 10 minute period (between approximately 8.6 and 1.5 mg O2/L). This procedure was repeated after 3 h contact time.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: NDA
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study
- Test concentrations: Nominal concentrations: 1.0, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Significant inhibition of respiration was observed at 1000 mg/L; however, there were no effects on respiration at 1.0, 10 and 100 mg/L. Based on this information test concentrations of 100, 180, 320, 560 and 1000 mg/L were selected for the main study.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

30 min

Dose descriptor:

IC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Effect concentrations 2

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

390 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: NDA
- Effect concentrations exceeding solubility of substance in test medium: NDA

OS 114451A EC50 (30 minutes): > 1000 mg/L*
OS 114451A EC50 (3 hours): 390 mg/L

*It was considered inappropriate to extrapolate the plot of percentage inhibition against test concentration, for the test material, to achieve an EC50 after 30 minutes on the grounds that no concentration resulted in 50 % inhibition of respiration.

Variation in respiration rates of controls 1 and 2 after 30 minutes: ± 4 %

Results with reference substance (positive control):

- Results with reference substance valid?: The validation criteria for the reference material EC50 value have been satisfied.
- Relevant effect levels: EC50 (30 minutes): 10 mg/L; EC50 (3 hours): 7.0 mg/L
- Other: NDA

Reported statistics and error estimates:

NDA

Any other information on results incl. tables

Table 1 Oxygen consumption rates and percentage inhibition values in the definitive study after 30 minutes contact time

Concentration (mg/L)	O2consumption rates (mg O2/L/min)	% inhibition
Control	-	-
R1	0.36	-
R2	0.39	-
OS 114451A	-	-
100	0.44	[17]
180	0.37	1
320	0.31	17
560	0.32	15
1000	0.43	[15]
3,5-dichlorophenol	-	-
3.2	0.36	4
10	0.16	57
32	0.07	81

[increase in respiration rate as compared to controls]

R1, R2 = Replicates

Table 2 Oxygen consumption rates and percentage inhibition values in the definitive study after 3 hours contact time

Concentration (mg/L)	O2consumption rates (mg O2/L/min)	% inhibition
Control	-	-
R1	0.40	-
R2	0.40	-
OS 114451A	-	-
100	0.44	[10]
180	0.32	20
320	0.25	38
560	0.13	68
1000	0.05	88
3,5-dichlorophenol	-	-
3.2	0.30	25
10	0.15	63
32	0.05	88

[increase in respiration rate as compared to controls]

R1, R2 = Replicates

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material, OS 114451A on the respiration rate of activated sewage sludge micro-organisms gave a 3 hour EC50 of 390 mg/L.

Executive summary:

In an assessment of the inhibitory effect on the respiration of activated sewage sludge study (525/045), the effect of the test material, OS 114451A on the respiration rate of activated sewage sludge micro-organisms gave a 3 hour EC50 of 390 mg/L, in accordance with the OECD Guidelines for Testing of Chemical (1984) No. 209 "Activated Sludge Respiration Inhibition Test".