4-Piperidinone, 2,6-diethyl-2,3,6-trimethyl-1-(1-phenylethoxy)-

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Performed according to OECD-Guideline under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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

Details on test material:

Identity: TKA 40270 (CGPS 345)
Batch no.: Lot 02243FC7, Op. 1/07
Purity: Confidential Information (see Analytical Certificate No: 38291-1)
Expiration date: 01-Oct-2012
Solubility in water: <0.21 mg/L
Aggregate state / physical form at room temperature: Liquid
Color: Colorless to yellow
Storage conditions: Storage at under 18 °C may lead to partial solidification. To reliquify, heat contents to 25-35 °C.Protect from light. (At RCC: At room temperature at about 20 °C, away from direct sunlight.)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal concentration test 100 mg/L (Limit Test)
- Sampling after 3 hours incubation time; then O2 was continuously measuared

Test solutions

Vehicle:

no

Details on test solutions:

The final nominal concentration tested was 100 mg/L. No stock solution could be prepared due to the low water solubility of the test item.
The test item was handled in the dark due to its light sensitivity. Therefore, 50.07 mg of the test item was first weighed onto a glass slide and then
transferred to the designated test vessel. Then, 284 ml, tap water was added to each vessel.
The test item was mixed into the tap water by ultrasonic treatment for fifteen minutes and intense stirring for 24 hours at room temperature in the
dark to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. No emulsifiers or solvents were used.
After stirring, the appearance of the test medium was evaluated, and the test concentration was clearly above the water solubility limit of TKA 40270
(CGPS 345) under the test conditions. Then, 16 mL synthetic wastewater and 200 mL activated sludge inoculum were added (see table on next page).

Test organisms

Test organisms (species):

activated sludge

Details on inoculum:

The study was performed with aerobic activated sludge from a wastewater treatment plant treating predominantly domestic wastewater. The sludge
was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During
the holding period of three days prior to use, the sludge was fed daily with 50 mL synthetic wastewater per liter and was kept at room temperature
under continuous aeration until use, Before use, the dry weight of the activated sludge was measured again in the inoculum used for the test, The pH of the activated sludge inoculum was 7.4.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

at least 10 minutes

Test conditions

Hardness:

Synthetic wastewater was used

Test temperature:

20°C

pH:

The pH was 7.5 - 8.1 for the test solution

Dissolved oxygen:

8.7 mg/L

Nominal and measured concentrations:

Nominal: 100 mg/L

Details on test conditions:

At the start of the test (after the stirring period of 24 hours), synthetic wastewater and activated sludge inoculum were added. The inoculum had a
sludge concentration of 3.4 g/L dry weight (corresponding to about 1.4 g dry material per liter test medium). The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) first to a control, secondly to the test solutions of the reference item, thirdly to the test suspension of the test item, and finally to the second control.
During the incubation period of 3 hours the single test medium and the controls were continuously aerated by intense stirring on magnetic stirrers
to avoid possible foaming and/or stripping of the test item. The concentration of dissolved oxygen did not drop below 2.5 mg/L during the
incubation period. Just before measurement of the respiration rates the dissolved oxygen concentrations were at least 8.7 mg/L. The temperature in the test media, measured in one control, was 20 °C at the start and at the end of the incubation period. Then the dissolved oxygen concentration was measured with an oxygen electrode, and was continuously recorded. During measurement, the samples were continuously stirred on a magnetie
stirrer. The oxygen consumption rate (in mg 02 /L minute) was determined from the linear part of the respiration curve in the range 6.5 - 2.5 mg
02/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the
respiration curve. In case of low oxygen consumption the rate was determined over a period of at least ten minutes.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The test item TKA 40270 (CGPS 345) had no significant inhibitory effect (=15%) on the respiration rate of activated sludge after the incubation period of 3 hours at the limit test concentration of 100 mg/L. Based on the physical appearance of the test suspension, the saturation concentration (water solubility limit of TKA 40270 (CGPS 345) under the test conditions) was <100 mg/L and resulted in less than 15% inhibition after three hours contact time.
Thus, the 3-hour NOEC (EC15) of TKA 40270 (CGPS 345) to activated sludge microorganisms was at least 100 mg/L. This value might even be higher but concentrations above 100 mg/L were not tested. The 3-hour EC20, EC50, and EC80 could not be calculated but were clearly higher than
100 mg/L.

Results with reference substance (positive control):

The reference item 3,5-dichlorophenol was tested in parallel under identical test conditions, and functioned as a positive control. The nominal
concentrations tested were 5, 16 and 50 mg/L. A stock solution of 3,5-dichlorophenol was prepared according to the test guidelines:
0.5 g 3,5-dichlorophenol was dissolved in 10 mL 1 M NaOH and diluted to about 30 mL with purified water. Excess NaOH was neutralized with
0.5 mol/L H2SO4 to the point of incipient precipitation.
The 3-hour EC5O of the reference item 3,5-dichlorophenol was calculated to be 15 mg/L (the 95% confidence limits could not be calculated). The
3-hour EC5O is within the guideline-recommended range of 5—30 mg/L, confirming suitability of the activated sludge used.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 3-hour NOEC (EC15) of the test item to activated sludge microorganisms was at least 100 mg/L. This value might even be higher but concentrations above 100 mg/L were not tested. The 3-hour EC20, EC50, and EC80 could not be calculated but were clearly higher than 100 mg/L.

Executive summary:

A limit test was performed in accordance with the EU Commission Directive 92/69/EEC (C.1-C.3, [1]) to demonstrate that the test item has no toxic effect on activated sludge up to the nominal test concentration of 100 mg/L. The respiration rates (oxygen consumption) of activated sludge were measured. The respiration rate in the presence of the test item was compared to the mean respiration rate of two controls.

At the test item concentration of 100 mg/L, the test item was not fully dissolved in the test medium. Thus, the test concentration was clearly above the water solubility limit of TKA 40270 (CGPS 345) under the test conditions. The test item TKA 40270 (CGPS 345) had no significant inhibitory effect (=15%) on the respiration rate of activated sludge after the incubation period of 3 hours at the limit test concentration of 100 mg/L. Based on the physical appearance of the test suspension, the saturation concentration (water solubility limit of TKA 40270 (CGPS 345) under the test conditions) was reached and no inhibitory effect was determined after three hours contact time.