1-acetyl-4-(3-dodecyl-2,5-dioxo-1-pyrrolidinyl)-2,2,6,6-tetramethylpiperidine

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:

1999-01-07

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed according to relevant guideline and compliant with GLP; however, the study followed the OECD 209 adopted 1984, which deviates from the updated version (2010). OECD 209 (1984) demanded fewer replicates for control and treatment groups and had no restriction on a minimum oxygen uptake rate for the control. Nonetheless, the main results of the study are considered reliable.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

adopted 1984

Deviations:

no

Remarks:

, according to OECD 209 (1984); however, the updated guideline (2010) demands a higher number of replicates (5 for the treatment groups, 6 for the control) and considers a minimum oxygen uptake rate for the control.

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

(87/302/EEC)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTION WITHOUT SOLVENT AID
For the purpose of the definitive study using a direct dispersion in water method, amounts of test material (500 mg) were dispersed in approximately 250 ml of water and subjected to ultrasonication for approximately 30 minutes. Synthetic sewage (16 ml), activated sewage sludge (200 ml) and water were added to a final volume of 500 ml to give the test concentration of 1000 mg/I.

PREPARATION OF TEST SOLUTION SOLVENT AID
Additionally the definitive study also consisted of a single test concentration prepared using a preliminary solution in auxiliary solvent method. An amount of test material (50 mg) was dissolved in acetone and the volume adjusted to 100 ml to give a 50 mg/100 ml stock solution. An aliquot (50 µl) of this stock solution was dispersed with activated sewage sludge (200 ml), synthetic sewage (16 ml) and water, to a final volume of 500 ml, to give the test concentration of 0.05 mg/I.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Solvent control: The solvent control was exposed to 100 µL/L acetone.

TEST WATER
The test water used for both the range-finding and definitive studies was laboratory tap water dechlorinated by passage through an activated carbon filter with a total hardness of approximately 100 mg/I as CaCO3.

SYNTHETIC SEWAGE
The synthetic sewage was prepared according to the guideline.

REFERENCE MATERIAL
- Reference material: 3,5-dichlorophenol, concentration tested at 3.2, 10, and 32 mg/L

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

ORIGIN OF INOCULUM
A mixed population of activated sewage sludge micro-organisms was obtained on 7 January, 1999 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic sewage.

PREPARATION OF INOCULUM
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of 21°C and was used on the day of collection. The pH of the sample was 7.5 and the suspended solids equal to 3.9 g/l prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

ca. 100 mg/I CaCO3.

Test temperature:

21°C

pH:

7.5

Nominal and measured concentrations:

Nominal concentration: 1000 mg/L, 0.050 mg/L (prepared with solvent aid)

Details on test conditions:

TEST PERFORMANCE
At the start of the test (t0), 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). The mixture was aerated with compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. Duplicate solvent control vessels were prepared by the addition of 50 µL of acetone to synthetic sewage (16 ml), activated sewage sludge (200 ml) and water to a final volume of 500 ml. Finally a second control was prepared.

TEST SYSTEM
- Test vessel: conical flask
- Fill volume: 500 mL
- Aeration: continuous
- No. of vessels for 1000 mg/L treatment group (without solvent aid): 3
- No. of vessels for 0.050 mg/L treatment group (with solvent aid): 1
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Respiration rate, after 30 min and 3 hours

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: A limit test was performed with 1000 mg/L test item prepared using direct dispersion into the test medium (excess of water solubility of the test item) as well as a 0.05 mg/L test concentration using solvent aid (referring to the limit of water solubility of the test item). In case no effect on the respiration rate for the 0.05 mg/L test item concentration was observed, the test item could be considered as nontoxic to activated sewage sludge microorganism.
- Range finding study: yes
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

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

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

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

> 0.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: test concentration prepared with solvent aid

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: test concentration prepared with solvent aid

Details on results:

RESULTS OF THE TREATMENT GROUP WITHOUT SOLVENT AID
The effect of the test material on the respiration of activated sewage sludge microorganisms gave a 3h-EC50 of >= 1000 mg/l prepared using a direct dispersion in water. Correspondingly, the NOEC was equal to 1000 mg/l.

RESULTS OF THE TREATMENT GROUP WITH SOLVENT AID
In the single test vessel prepared using a preliminary solution in auxiliary solvent to give a test concentration of 0.050 mg/l (limit of water solubility), no inhibition of the respiration rate of activated sewage sludge was observed; hence, the 3h-EC50 value was determined to be > 0.050 mg/l. The corresponding NOEC was determined equal to 0.050 mg/l. These results show ed the test item did not cause any effect on the respiration rate of activated sewage sludge micro-organisms at its identified maximum of water solubility.

VALIDATION CRITERIA
- Control and solvent control respiration: satisfied (control respiration rates are within 15% of each other)
- Reference material EC50 were: satisfied

Results with reference substance (positive control):

3h-EC50 = 7.5 mg/L (this is within the accepted range of 5-30 mg/L)

Reported statistics and error estimates:

CALCULATION RESPIRATION RATES/RESPIRATION INHIBITION
The inhibitory effect of the test and reference materials on the respiration rate was expressed as a percentage of the two control/solvent control respiration rates.

CALCULATION OF EC50 (reference material)
The percentage inhibition was plotted against the concentration and the EC50 values were derived by inspection of the graph.

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

Nominal concentration (mg/l)	Initial O2reading (mg O2/l)	Measurement period (minutes)	Final O2reading (mg O2/l)	O2Consumption rates (mg O2/l)	% Inhibition
Control                                   R1                                             R2	7.5 6.4	10 10	3.0 1.9	0.45 0.45	- -
Treatment group             1000     R1 without solvent aid          1000     R2                                  1000     R3	6.7 7.2 6.1	10 10 10	2.3 3.1 1.7	0.44 0.41 0.44	2 9 2
3.5-dichlorophenol           3.2 10 32	7.5 8.2 8.5	10 10 10	3.9 6.9 7.8	0.36 0.13 0.07	20 71 84
Solvent control                           R1                                              R2	7.2 7.4	10 10	2.9 3.2	0.43 0.42	- -
Treatment group             0.050 with solvent aid	6.7	10	2.3	0.44	[4]

[increase in respiration rate as compared to controls or solvent controls]

R₁– R₃= Replicates

Validity criteria fulfilled:

yes

Remarks:

according to OECD 209 (1984); however, the study does not fulfill the validity criteria of the updated version (2010) w.r.t. the minimum oxygen uptake rate of the control.

Conclusions:

The derived effect concentrations (nominal) on the respiration of microorganisms were as following: 3h-EC50 > 1000 mg/L and 3h-NOEC value >= 1000 mg/L.

Executive summary:

A study was performed to assess the effect of the test material on the respiration rate of activated sewage sludge microorganisms. The method followed the OECD guideline 209 adopted 1984, the EEC Commission Directive 87/302/EEC and US EPA Ecological Effects Test Guidelines OPPTS 850.6800. The study was conducted in compliance with GLP.

In the presence of activated sewage sludge and with the addition of a synthetic sewage the test material was aerated for a period of 3 hours at 21°C. The rate of respiration was determined after 30 minutes and 3 hours contact time and compared to data of a control and reference group (reference substance 3,5-dichlorophenol).

In order to satisfy the requirements of all followed guidelines, the definitive study was conducted using two methods of dispersing the test material in the test system: i.e., one using a single test concentration of nominal 0.050 mg/L using auxiliary solvent (acetone), the other using a test concentration of nominal 1000 mg/L prepared by direct dispersion in the test water with the aid of ultrasonication (corresponding to a limit test).

Results of the study showed that in the single test vessel of 0.050 mg/L test item, no inhibition of the respiration rate of activated sewage sludge microorganisms was observed; hence, the 3h-EC50 value was determined to be > 0.050 mg/L. Correspondingly, the 3h-NOEC was greater than or equal to 0.050 mg/L. These results showed that at the limit of water solubility (<0.05 mg/L) the test material had no effect on the respiration rate of activated sewage sludge. Furthermore, the test material did not show any negative effect on the respiration rate of the microorganisms at test item concentrations in excess of the water solubility level (1000 mg/L test item). Hence, the 3h-EC50 value for the limit test was determined to be > 1000 mg/L. The corresponding 3h-NOEC was determined to be greater or equal to 1000 mg/L. Summarising, the results of the study showed that the test item had no negative effect on the respiration rate of activated sludge microorganism at neither its limit of water solubility nor at concentrations in excess of its water solubility level.

The study fulfilled the validity criteria of OECD 209 (1984). However, the OECD 209 adopted 1984 deviates from the update version (2010) in such that it demanded fewer replicates for the control and treatment groups and had no restriction on a minimum oxygen uptake rate for the control. With respect to the number of replicates, in this study three replicates were used for the limit test item concentration (1000 mg/L) and two for the controls (where in the updated version five replicates for the treatment group and six for the control are required). Despite these deficiencies, as the test substance did not cause any negative effects towards the respiration rate of the activated sludge microorganisms and correspondingly the respiration rate of the treatment group and control replicates (total of five replicates) did not differ more than 10% from each other, the results of the study were considered reliable.

Description of key information

3h-EC50 (microorganisms) > 1000 mg/L (nominal); 3h-NOEC (microorganisms) >= 1000 mg/L (nominal)

Additional information

The reliable study was performed to assess the effect of the test material on the respiration rate of activated sewage sludge microorganisms. The method followed the OECD guideline 209 adopted 1984, the EEC Commission Directive 87/302/EEC and US EPA Ecological Effects Test Guidelines OPPTS 850.6800. The study was conducted in compliance with GLP.

With regard to the low water solubility of the test item (<0.05 mg/L), a limit test was performed using a nominal test concentration of 1000 mg/L test item. Additional treatment groups consisted of a nominal test concentration group of 0.050 mg/L test item using the auxiliary solvent acetone, a reference substance group (3,5-dichlorophenol) and controls.

The test was performed under aerobic conditions with activated sewage sludge and synthetic sewage over the period of 3 hours. The test item did not cause any negative effect on the respiration rate of microorganism – neither at its limit of water solubility nor at a concentration in excess of the test item water solubility. Hence, the 3h-EC50 value for the limit test was determined to be > 1000 mg/L; the corresponding 3h-NOEC was determined to be >= 1000 mg/L.

The study fulfilled the validity criteria of OECD 209 (1984); however, the OECD 209 (1984) deviates from the update version (adopted 2010). Despite related deficiencies, the results of the present study were considered reliable (RL2).