1-Propanaminium, N,N,N-trimethyl-3-(octadecyloxy)-, chloride (1:1)

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 July 2007 to 30 July 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EEC Commission Directive 87/302/EEC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 30 August 2005 Date of Signature: 21 November 2005

Test material

Test material information

Constituent 1

Reference substance name:

trimethyl[3-(octadecyloxy)propyl]azanium chloride

EC Number:

700-414-8

Cas Number:

23328-71-4

Molecular formula:

C24H52ClNO

IUPAC Name:

trimethyl[3-(octadecyloxy)propyl]azanium chloride

Details on test material:

- Name of test material (as cited in study report): 3-Octadecyloxypropyl-N,N,N-trimethylammonium chloride
- Molecular formula (if other than submission substance): Not applicable
- Molecular weight (if other than submission substance): Not applicable
- Smiles notation (if other than submission substance): Not applicable
- InChl (if other than submission substance): Not applicable
- Structural formula attached as image file (if other than submission substance): Not applicable
- Substance type: white solid
- Physical state: solid
- Analytical purity: Not reported
- Impurities (identity and concentrations): Not reported
- Composition of test material, percentage of components: Not reported
- Isomers composition: Not reported
- Purity test date: Not reported
- Lot/batch No.: Exp. I-070518
- Expiration date of the lot/batch: Not reported
- Radiochemical purity (if radiolabelling): Not applicable
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable
- Stability under test conditions: Analysis of stability of the test material in the test preparations was not appropriate to the Test Guideline.
- Storage condition of test material: room temperature in the dark
- Other: Not reported.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not reported.

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Not applicable.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: For the purpose of the definitive test, the test material was dissolved directly in water.
An amount of test material (2000 mg) was dissolved in water with the aid of ultrasonication for approximately 45 minutes and the volume adjusted to 1 litre to give a 2000 mg/l stock solution from which serial dilutions were made to give 200 and 20 mg/l stock solutions. An aliquot (250 ml) of the 20 mg/l stock solution was dispersed with synthetic sewage (16 ml), activated sewage sludge (200 ml) and water, to a final volume of 500 ml, to give the required concentration of 10 mg/l. Similarly, aliquots (80 ml and 250 ml of the 200 mg/l stock solution and 80 ml and 250 ml of the 2000 mg/l stock solution) were used to prepare the test concentrations of 32, 100, 320 and 1000 mg/l. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls:
Negative control: Maintained under identical conditions but not exposed to the test material.
Positive control: See reference substance section.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not reported.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was used on the day of collection. The pH of the sample was 7.5 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 ml of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 4.0 g/l prior to use.

A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
16 g Peptone
11 g Meat extract
3 g Urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
dissolved in 1 litre of water with the aid of ultrasonication.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not applicable.

Test conditions

Hardness:

A total hardness of approximately 140 mg/l as CaCO3.

Test temperature:

21±1deg C.

pH:

pH 7.6 - 7.9 at the start of the exposure.
pH 8.1 - 8.4 at the end of the exposure.

Dissolved oxygen:

(After 30 minutes of contact time)
3.8 - 7.9 mg O2/l of initial O2 reading.
1.9 - 7.2 mg/O2l of final O2 reading.
(After 3 hours of contact time)
4.7 - 7.9 mg O2/l of initial O2 reading.
1.6 - 7.5 mg/O2l of final O2 reading.

Salinity:

Not reported.

Nominal and measured concentrations:

Range-finding test: 1.0, 10, 100 and 1000 mg/l (nominal).
Definitive test: 10, 32, 100, 320, and 1000 mg/l (nominal).

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Open.
- Material, size, headspace, fill volume: 250 ml darkened glass Biological Oxygen Demand (BOD) bottle as a measuring vessel.
- Aeration: No.
- Type of flow-through : Not applicable.
- Renewal rate of test solution: Not applicable.
- No. of organisms per vessel: Not applicable.
- No. of vessels per concentration: 1
- No. of vessels per control: 2
- No. of vessels per vehicle control: Not applicable.
- Biomass loading rate: Not reported.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The test water used for both the range-finding and definitive tests was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Total organic carbon: (Average) 1.429 mg/l
- Particulate matter: Not reported.
- Metals: (Average)
Al <10.596 ug/l
Pb <1.113 µg/l
Fe <28.038 µg/l
Cd <0.213 µg/l
Cr <0.551 ug/l
Cu <0.021 mg/l
Mn <1.723 ug/l
Ni <2.325 µg/l
Hg <0.013 µg/l
- Pesticides: (Average) 0.019 µg/l
- Chlorine: (Average) 0.274 mg/l
- Alkalinity: (Average) pH 7.655
- Ca/mg ratio: Not reported.
- Conductivity: 403.577 µS/cm at 20°C
- Culture medium different from test medium: No
- Intervals of water quality measurement: Not reported.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod and light intensity: Under normal laboratory lighting.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Observations were made on the test preparations throughout the test period, and the pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Spacing of 3.125 - 3.2 with concentrations of 10, 32, 100, 320, and 1000 mg/l.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study
- Test concentrations: A series of nominal test concentrations of 1.0, 10, 100 and 1000 mg/l.
- Results used to determine the conditions for the definitive study: See table 1. Significant inhibition of respiration was observed at 100 and 1000 mg/l, however, there were no significant effects on respiration at 1.0 and 10 mg/l. Based on this information test concentrations of 10, 32, 100, 320 and 1000 mg/l were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

440 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% confidence limits of 330 - 590 mg/l

Effect concentrations 2

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

See the table 2 for the results. The data generated from the test concentrations employed in the test showed that no concentration resulted in greater than 80% inhibition after 30 minutes contact time. This test concentration range was chosen in order that a No Observed Effect Concentration (NOEC) value and 3-Hour EC50 value was attained. It was not possible to obtain 95% confidence limits for the test material after 30 minutes contact time as the data generated did not fit the models available for the calculation of these limits.

In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O2/l and 2.5 mg O2/l respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
Observations made throughout the test period showed that at all test concentrations employed no undissolved test material was visible.

Results with reference substance (positive control):

- Results with reference substance valid?
yes - See Table 3. Variation in respiration rates of controls 1 and 2 after 30 minutes was ± 3%, and ± 0% after 3 hours contact time.
The validation criteria for the control respiration rates and reference material EC50 values were therefore satisfied.

Reported statistics and error estimates:

Not reported.

Any other information on results incl. tables

Table 2 Results of definitive test with the test material

	ECx(30 Minutes) (mg/l)	95% Confidence Limits (mg/l)	ECx(3 Hours) (mg/l)	95% Confidence Limits (mg/l)
EC20	20	-	60	-
EC50	750	-	440	330 - 590
EC80	>1000	-	940	-
NOEC	10	-	10	-

Table 3 Results of definitive test with a reference substance

	ECx(30 Minutes) (mg/l)	95% Confidence Limits (mg/l)	ECx(3 Hours) (mg/l)	95% Confidence Limits (mg/l)
EC20	4.1	-	3.1	-
EC50	11	8.8 - 14	9.3	7.6 - 11
EC80	30	-	27	-

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of 440 mg/l, 95% confidence limits 330 - 590 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 10 mg/l.

Executive summary:

Introduction.

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", EEC Commission Directive 87/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Methods.

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous solution of the test material at concentrations of 10, 32, 100, 320 and 1000 mg/l for a period of 3 hours at a temperature of approximately 21°C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 30 minutes and 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

Results.

The effect of the test material on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀of 440 mg/l, 95% confidence limits 330 - 590 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was10mg/l.

The reference material gave a 3-Hour EC₅₀value of 9.3 mg/l, 95% confidence limits 7.6 - 11 mg/l.