Ethyltriphenylphosphonium acetate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 February 2018 - 15 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test item was dispersed directly in water.
Nominal amounts of test item (9, 16, 28, 50 and 90 mg (three replicates of each)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 18, 32, 56, 100 and 180 mg/L (three replicates of each).

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected:
A mixed population of activated sewage sludge micro-organisms was obtained on 28 February 2018 for the range-finding test and on 14 March 2018 for the definitive test from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the day of collection the activated sewage sludge (9.5 liters) was fed synthetic sewage (475 mL). The pH of the sample on the day of the test was 7.3 measured using a Hach HQ40d Flexi handheld 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 three times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC 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 3.0 g/L prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20°C

pH:

prior to adjustment: 4.7 - 5.1; after adjustment: 7.0 - 8.0

Dissolved oxygen:

5.58 - 8.11 mg O2/L

Nominal and measured concentrations:

18, 32, 56, 100 and 180 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL conical flask, 500 mL fill volume
- Type (delete if not applicable): closed
- Aeration: clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Nutrients provided for bacteria:
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
Each constituent was dissolved in a final volume of 1 liter of water with the aid of ultrasonication.
The suspended solids concentration was equal to 3.0 g/L prior to use.

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Statistically significant toxic effects were shown at all the test concentrations employed.
Based on these results and after discussion with the Sponsor, it was considered necessary to perform a definitive test in order to obtain an EC50 value for the test item. Test concentrations of 18, 32, 56, 100 and 180 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

47 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% confidence limits

Remarks:

32 - 69 mg/L

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

0.54 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no undissolved test item visible during observation time
- Blank controls oxygen uptake rate: mean 43.7 mg O2/L/hour
- Coefficient of variation of oxygen uptake rate in control replicates: 6.2%

Results with reference substance (positive control):

- Results with reference substance valid?
- Relevant effect levels: 3 h EC50 = 7.4 mg/L, 95% confidence limits 5.6 to 9.8 mg/L

Reported statistics and error estimates:

The percentage inhibition values were plotted against concentration, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line.
95% confidence limits were calculated for the EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).

Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Definitive Test

Test Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2 Reading (mg O2/L)	O2 Consumption Rates (mg O2/L/hour)	Inhibition (%)
Control	R1	3.8	2	2.2	48.00	-
	R2	4.1	3	2.1	40.00	-
	R3	3.9	2	2.4	45.00	-
	R4	4.4	3	2.3	42.00	-
	R5	5.0	4	2.1	43.50	-
	R6	4.9	4	2.0	43.50	-
Test Item	18 R1	6.1	8	2.4	27.75	36
	18 R2	6.1	9	2.0	27.33	37
	18 R3	6.2	10	1.9	25.80	41
	32 R1	6.3	10	2.4	23.40	46
	32 R2	6.7	10	2.8	23.40	46
	32 R3	6.5	10	2.5	24.00	45
	56 R1	6.5	10	3.0	21.00	52
	56 R2	6.9	10	3.5	20.40	53
	56 R3	7.0	10	3.5	21.00	52
	100 R1	6.8	10	3.8	18.00	59
	100 R2	7.1	10	4.2	17.40	60
	100 R3	6.9	10	4.1	16.80	62
	180 R1	7.2	10	5.0	13.20	70
	180 R2	7.5	10	5.2	13.80	68
	180 R3	7.3	10	5.1	13.20	70
3,5-Dichlorophenol	3.2	6.2	8	2.1	30.75	30
	10	6.9	10	3.8	18.60	57
	32	8.0	10	7.0	6.00	86

R = Replicate

- = Not applicable

Validity Criteria

• The EC50 value (3-Hour contact time) for the reference item, 3,5-Dichlorophenol, was 7.4 mg/L.

• The specific respiration rate of the controls was 29.11 mg oxygen per gram dry weight of sludge per hour.

• The coefficient of variation of oxygen uptake in the control vessels was 6.2%.

All validation criteria for the study were therefore satisfied.

Validity criteria fulfilled:

yes

Conclusions:

The 3-Hour EC50 of ETPPAAc to activated sewage sludge was 47 mg/L (95% confidence limits 32 to 69 mg/L).

Executive summary:

This study was performed to assess the effect of ETPPAAc on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 18, 32, 56, 100 and 180 mg/L (three replicates) for a period of 3 hours at a measured temperature of approximately 20 °C with the addition of a synthetic sewage as a respiratory substrate.

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

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of 47 mg/L, 95% confidence limits 32 to 69 mg/L. The 3 h EC10 was 0.54 mg/L.

The reference item gave a 3-Hour EC50 value of 7.4 mg/L, 95% confidence limits 5.6 to 9.8 mg/L.

Description of key information

3 h EC50 = 47 mg/L, 95% confidence limits 32 to 69 mg/L, 3 h EC10 = 0.54 mg/L (OECD TG 209)

Key value for chemical safety assessment

EC50 for microorganisms:

47 mg/L

EC10 or NOEC for microorganisms:

0.54 mg/L

Additional information

This study was performed to assess the effect of ETPPAAc on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 18, 32, 56, 100 and 180 mg/L (three replicates) for a period of 3 hours at a measured temperature of approximately 20 °C with the addition of a synthetic sewage as a respiratory substrate.

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

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of 47 mg/L, 95% confidence limits 32 to 69 mg/L. The 3 h EC10 was 0.54 mg/L.

The reference item gave a 3-Hour EC50 value of 7.4 mg/L, 95% confidence limits 5.6 to 9.8 mg/L.