N,N,4-trimethylpiperazine-1-ethylamine

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:

2017-06-28 to 2017-06-28

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)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PFW160141
- Expiration date of the lot/batch: 2018-12-30
- Purity test date: 2016-07-19 (certificate of analysis release date)
- Purity: 99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data

Analytical monitoring:

no

Remarks:

As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and is reflected in the GLP compliance statement.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. 2500 mg of test item was dissolved in water and the volume adjusted to 1L to give a 2500 mg/L stock solution from which dilutions were made to give 250 and 25 mg/L stock solutions. An aliquot (200 mL) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (200 mL) of the 250 mg/L and 2500 mg/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg/L. The 1000 mg/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity. The pH of the test item stock solutions was adjusted to 7.0 - 8.0 if necessary.
- Controls: yes, the control group was maintained under identical conditions but not exposed to the test item.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no data

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 27 June 2017 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage
- Method of cultivation: 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.
- Pretreatment: On the day of collection the activated sewage sludge (10 liters) was fed synthetic sewage (500 mL). The pH of the sample on the day of the test was 7.2 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 3 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 one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.
- Initial biomass concentration: 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

Post exposure observation period:

After 3 h of exposure, oxygen consumption was recorded for apprimately 10 minutes.

Hardness:

Not reported

Test temperature:

20”C

pH:

7.9 - 10.7 prior to adjustment and 7.9 after adjustment

Dissolved oxygen:

5.62 - 8.14 mg O2/L or 63 - 91% of Dissolved Oxygen Saturation Level of 8.9 mg O2/L

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Range-Finding Test: 10, 100 and 1000 mg/L
Based on these results the main test was not performed

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL conical flask
- Aeration: The activated sewage sludge sample was maintained on continuous aeration
- No. of vessels per concentration (replicates): 1 and 3 for the highest test item concentration
- No. of vessels per control (replicates): 4
- Sludge concentration (weight of dry solids per volume): The suspended solids concentration was equal to 3.0 g/L prior to use.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test item stock solutions were measured using a Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 to pH 8.0 if necessary.
- Photoperiod and Light intensity: The test was conducted under normal laboratory lighting.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs 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 the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x10
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

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 data
- Blank controls oxygen uptake rate: 26.73 mg O2/L.h
- Coefficient of variation of oxygen uptake rate in control replicates: 1.2%

Results with reference substance (positive control):

- The reference item gave a 3-Hour EC50value of 6.5 mg/L, 95% confidence limits 5.0 to 8.5 mg/L which suggested the suitability of the test conditions and the inoculum

Reported statistics and error estimates:

- ECx: The percentage inhibition values were plotted against concentration for the reference item only, 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. The EC10, EC20, EC50 and EC80 values for the test item were determined by inspection of the inhibition of respiration rate data. 95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).
- NOEC: In order to determine te NOEC, one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O2/L)	Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 8.9 mg O2/L
Control	R1	6.09	68
	R2	5.81	65
	R3	6.09	68
	R4	6.09	68
Test Item	10	6.00	67
	100	6.37	72
	1000 R1	6.10	69
	1000 R2	6.19	70
	1000 R3	5.62	63
3,5-dichlorophenol	3.2	6.86	77
	10	7.02	79
	32	8.14	91

R₁– R₄= Replicates 1 to 4

Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2Reading (mg O2/L)	O2Consumption Rates (mg O2/L/hour)	% Inhibition
Control	R1	6.1	6	2.1	40.00	-
	R2	5.8	6	1.8	40.00	-
	R3	5.4	5	2.1	39.60	-
	R4	5.5	5	2.1	40.80	-
Test Item	10	5.2	5	1.9	39.60	1
	100	5.2	5	2.0	38.40	4
	1000 R1	5.4	5	2.1	39.60	1
	1000 R2	5.7	5	2.4	39.60	1
	1000 R3	5.1	5	1.8	39.60	1
3,5-dichlorophenol	3.2	6.9	10	2.5	26.40	34
	10	7.2	10	4.4	16.80	58
	32	8.2	10	7.5	4.20	90

R₁– R₄= Replicates 1 to 4

 

Validity criteria fulfilled:

yes

Conclusions:

An activated sludge respiration inhibition test (according to OECD guideline 209) with activated sludge from a predominantly domestic sewage treatment plant resulted in a 3-h EC50 > 1000 mg Jeffcat TAP/L. In conclusion, Jeffcat TAP was not toxic to activated sludge at or below a loading rate of 1000 mg/L. The results of the study can be considered reliable without restriction.

Executive summary:

A study was performed to assess the effect of the test item 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)".

Methods

Activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures 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

Results

The effect of the test item on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

The reference item gave a 3-Hour EC₅₀value of 6.5 mg/L, 95% confidence limits 5.0 to 8.5 mg/L

Description of key information

The study of Best (2017), investigating the toxicity of the test substance to microorganisms according to OECD guideline 209, was considered as the key study for endpoint coverage. The 3-h EC50 was >1000 mg/L. The 3-h NOEC was 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L