bis(2,2,6,6-tetramethyl-1-(phenylthio)piperidin-4-yl)carbonate

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:

2019-07-09 - 2019-09-27

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))

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: (EPA 712-C-014): OCSPP 850.3300

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYÉI (April 21, 2016)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Test item: At the start of the test defined amounts of the test item (5 x 1 mg; 5 x 3 mg; 5 x 9.6 mg; 5 x 30 mg; 5 x 96 mg and 5 x 300 mg) were directly weighed (administered) into each test flask and the subsequent calculations refer to the initial weighed nominal concentrations.
Reference Item:
A stock solution of 3,5-Dichlorophenol is prepared according to the following guideline procedure: 1 g of 3,5-Dichlorophenol is dissolved in 1000 mL of water (warm water and/or short ultrasonication (< 1 min) can be used to accelerate the dissolution). The pH of the solution is checked and adjusted, if necessary, with 1N NaOH or H2SO4 to pH 7-8.
In the present study 0.1 g 3,5-Dichlorophenol was dissolved in warm purified, deionized water. The pH of the stock solution (after cooling) was adjusted with 1N NaOH to 7.42 to fall within the range of 7 – 8. After pH setting the volume of this solution was completed ad 100 mL, in a volumetric flask.
ATU: According to the OECD Guideline No. 209 the N-allylthiourea solution was prepared by dissolving 0.232 g of N-allylthiourea in 100 mL of purified, deionized water.

- Controls:
Blank Control (CB):
In the definite test eight controls (according to the number of the available O2 electrodes) four at the start and four at the end of the test series were investigated.
Abiotic Control (CA):
The abiotic control was not repeated because no oxygen uptake was observed in the abiotic control group in the preliminary test.
Reference Control (R):
The reference item 3,5-Dichlorophenol was tested at three concentrations with three parallels (at the nominal test concentrations of 2, 7 and 24.5 mg/L) under otherwise identical test conditions; as the test mixtures.
Nitrification Control (CN):
The nitrification respiration was not significant in the preliminary test and it was assumed that the heterotrophic oxygen uptake equals the total uptake and no significant nitrification was occurring. The definite test was performed in one set of test vessels, without ATU addition, but for informative reason a nitrification control group (containing N-allylthiourea) was investigated with three parallels.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 10 July 2019 (one day before the test).
- Preparation of inoculum for exposure: The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice.
An aliquot of the final sludge suspension was weighed (5.482 g wet weight), dried and the ratio of wet sludge to dry weight (0.4358 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (30 g dry weight that was equivalent to 377.4 g wet sludge) was suspended in isotonic saline solution (ad. 10 L) to yield a concentration equivalent to about 3 g per litre (on dry weight basis).
(In the test containers (300 mL final volume) the final concentration of suspended solids, containing 150 mL inoculum was 1.5 g per litre on dry weight basis.)
The above concentration calculation accounts for the dilution resulting from feeding with synthetic sewage. The activated sludge was not used on the day of the collection, but continuously aerated (2 L/minute) at the test temperature (20 ± 2 °C) for about 24 hours (one day) and fed with 50 mL synthetic sewage/L activated sludge.
The pH of the activated sludge inoculum was checked after preparation (pH: 7.52). pH adjustment of the inoculum was considered not necessary.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

The oxygen concentration was measured with O2 electrode (working based on LDO method) under stirred conditions and was recorded for about 6.5-10 minutes.

Test temperature:

19.8 °C in average (minimum: 18.0 °C, maximum: 22.0 °C)

pH:

7 - 8

Nominal and measured concentrations:

3.2; 10; 32; 100; 320 and 1000 mg/L nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer glass bottles of approximately 300 mL volume.
- Material, size, headspace, fill volume: glass, 300 mL volume
- Aeration: with compressed air (0.5 L/minute)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 8 replicates blank control , 1 replicate with 3 parallels nitrification control, 3 replicates reference control at 3 concentrations (2, 7 and 24.5 mg/L nominal))
- No. of vessels per abiotic control (replicates): The abiotic control was not repeated because no oxygen uptake was observed in the abiotic control group in the preliminary test.
- Sludge concentration (weight of dry solids per volume): 3 g/L
- Nutrients provided for bacteria: Synthetic Sewage (fed daily with 50 mL synthetic sewage/L activated sludge)
- Nitrification inhibitor used (delete if not applicable): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test media were prepared freshly before the test (for composition see Table 1)

OTHER TEST CONDITIONS
- Temperature: 20.2 - 20.9 °C
- Adjustment of pH: not necessary

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The oxygen consumption rates (R), the specific respiration rates (Rs), the mean values, standard deviations, coefficients of variances and percentage inhibitions (IT) were calculated for each control (blank, reference, nitrification, abiotic) and for each treatment using Excel for Windows software.
3-hour EC10, EC50, EC80

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study
- Test concentrations: 10; 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: The 3-hour EC10 value of the test item is < 1000 mg/L; and the 3-hour EC50 and EC80 values are higher than 1000 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

53.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

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

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

Details on results:

Validity of the Study
The specific respiration rate of the blank controls (without the test substance or reference substance) was 27.71 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (higher than 20 mg/gh) with a coefficient of variation of 3.00 %. The 3-hour EC50 of the reference item 3,5-Dichlorophenol (for the used activated sludge batch) was 17.9 mg/L. This is within the range of 5 mg/L to 25 mg/L, as required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was considered as not necessary).

Oxygen Consumption Rates in the Test Item Concentrations
Based on the results of the preliminary experiments neither inclusion of abiotic controls nor differentiation between heterotrophic respiration and nitrification was considered as necessary.
Inhibitory effect of the test item was not observed at the lowest examined concentration of 3.2 mg/L. The obtained average inhibition of 2.94 % noticed at 10 mg/L, remained within the biological variability range of the applied test system. The calculated oxygen consumption rates and corresponding inhibitory tendencies showed a slight dose-related tendency up to the concentration of 100 mg/L (where 20.45 % inhibition was noticed), and remained nearly the same (~20-22 %) in the concentration range of 100-1000 mg/L.

Nitrification Controls
An additional nitrification control was examined in the test with three parallels to check the possible nitrification potential of the applied activated sludge batch. By applying the nitrification control the differentiation between the total, heterotrophic and nitrification respiration was possible. The total respiration (RT) was 41.56 mg/Lh and the heterotrophic respiration (RH) was 41.23 mg/Lh. The nitrification respiration (RN) of 0.33 mg/Lh was calculated according to the following equation: RN= RT-RH.
The obtained nitrification respiration of 0.33 mg/Lh was considered to be within the biological variability range of the applied test system, since it is lower than 5 % of the average RT in the blank controls (2.08 mg/Lh).
According to the above calculations it was assumed that the heterotrophic oxygen uptake equals the total uptake.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: The following concentrations of the positive reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 2, 7 and 24.5 mg/L. In comparison to the blank controls the oxygen consumption rate of the activated sludge was inhibited by 14 % at the lowest concentration of 2 mg/L and at the nominal concentrations of 7 and 24.5 mg/L, the oxygen consumption rate was inhibited by 23 % and 60 %, respectively.
The 3-hour EC50 of 3,5-Dichlorophenol was calculated (Probit analysis, IBM® SPSS® Statistics software) to be 17.9 mg/L, (95 % confidence limits: 13.2-27.6 mg/L).

Reported statistics and error estimates:

The examined concentrations did not cover the appropriate inhibition range for exact EC50 calculation. However, the obtained values allowed the performance of the statistical (Probit) analysis and calculation of EC10 value.

Specific Respiration Rates in the Test Item Concentrations
The specific respiration rates were compared with the blank control values using Dunnett’s t-test (2-sided, α = 0.05). The specific respiration rates did not differ statistically significantly from the blank control at the concentrations of 3.2 and 10 mg/L (Dunnett’s t-test, α = 0.05). The specific respiration rates differ statistically significantly from the blank control at the concentration range of 32-1000 mg/L (Dunnett’s t-test, α = 0.05). Consequently, the NOEC can be statistically and biologically determined as 10 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the earlier performed preliminary test (statistically significant inhibition of oxygen consumption by the test substance at the concentration of 1000 mg/L) a definite test was performed.
In the present definite test, based on measured oxygen consumption values and calculated specific respiration rates the 3-hour EC10 value of the test item was calculated as 53.4 mg/L (within the range of 10 mg/L to 100 mg/L), the 3-hour EC50 and EC80 values of the test item are higher than 1000 mg/L.
Based on the statistical and biological evaluation in this test the NOEC was determined as 10 mg/L.

Executive summary:

The definite test on the activated sludge respiration inhibition of the test item was conducted following OECD 209 and EU Method C.11, under GLP compliance. The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions.

The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours.

A preliminary test was carried out with the test item under the study code 911-209-4242 (Final Report: April 15, 2019). In the preliminary test clear dose-related effect of the test item was established at the examined concentrations of 10, 100 and 1000 mg/L and the results demonstrate a statistically significant inhibition of oxygen consumption by the test item at the highest examined concentration of 1000 mg/L.

Based on the above preliminary information about the test item caused effect on the activated sludge inoculum, in the present definite test the test item was investigated at the nominal concentrations of 3.2, 10, 32, 100, 320 and 1000 mg/L. Defined amounts of the test item were added directly into the test vessels.

In parallel with the test item treatments 3,5-Dichlorophenol as positive reference control in concentrations of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control and nitrification controls were investigated.

The definite test was performed without abiotic controls, based on the results of the preliminary test where abiotic controls were tested at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed.

All validity criteria of the study were met.

Inhibitory effect of the test item was not observed at the lowest examined concentration of 3.2 mg/L. The obtained average inhibition of 2.94 % noticed at 10 mg/L, remained within the biological variability range of the applied test system. The observed-calculated oxygen consumption rates and corresponding inhibitory tendencies showed a slight dose-related tendency up to the concentration of 100 mg/L (where 20.45 % inhibition was noticed), and remained nearly the same (~20-22 %) in the concentration range of 100-1000 mg/L.

The specific respiration rates did not differ statistically significantly from the blank control at the concentrations of 3.2 and 10 mg/L (Dunnett’s t-test, α = 0.05). Statistically significant differences (from the blank control values) were established at the concentration range of 32-1000 mg/L (Dunnett’s t-test, α = 0.05).

In the present definite test, based on measured oxygen consumption values and calculated specific respiration rates the 3-hour EC₁₀ value of the test item was calculated as 53.4 mg/L (within the range of 10 mg/L to 100 mg/L), the 3-hour EC₅₀ and EC₈₀ values of the test item are higher than 1000 mg/L.

Based on the statistical and biological evaluation in this test the NOEC was determined as 10 mg/L.

Description of key information

Definite test, OECD 209, GLP, static, results: EC₁₀ (3h) 53.4 mg/L (nominal) and EC50 (3h) > 1000 mg/L (nominal)

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

53.4 mg/L

Additional information

The definite test on the activated sludge respiration inhibition of the test item was conducted following OECD 209 and EU Method C.11, under GLP compliance. The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions.

The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours.

A preliminary test was carried out with the test item under the study code 911-209-4242 (Final Report: April 15, 2019). In the preliminary test clear dose-related effect of the test item was established at the examined concentrations of 10, 100 and 1000 mg/L and the results demonstrate a statistically significant inhibition of oxygen consumption by the test item at the highest examined concentration of 1000 mg/L.

Based on the above preliminary information about the test item caused effect on the activated sludge inoculum, in the present definite test the test item was investigated at the nominal concentrations of 3.2, 10, 32, 100, 320 and 1000 mg/L. Defined amounts of the test item were added directly into the test vessels.

In parallel with the test item treatments 3,5-Dichlorophenol as positive reference control in concentrations of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control and nitrification controls were investigated.

The definite test was performed without abiotic controls, based on the results of the preliminary test where abiotic controls were tested at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed.

All validity criteria of the study were met.

Inhibitory effect of the test item was not observed at the lowest examined concentration of 3.2 mg/L. The obtained average inhibition of 2.94 % noticed at 10 mg/L, remained within the biological variability range of the applied test system. The observed-calculated oxygen consumption rates and corresponding inhibitory tendencies showed a slight dose-related tendency up to the concentration of 100 mg/L (where 20.45 % inhibition was noticed), and remained nearly the same (~20-22 %) in the concentration range of 100-1000 mg/L.

The specific respiration rates did not differ statistically significantly from the blank control at the concentrations of 3.2 and 10 mg/L (Dunnett’s t-test, α = 0.05). Statistically significant differences (from the blank control values) were established at the concentration range of 32-1000 mg/L (Dunnett’s t-test, α = 0.05).

In the present definite test, based on measured oxygen consumption values and calculated specific respiration rates the 3-hour EC₁₀ value of the test item was calculated as 53.4 mg/L (within the range of 10 mg/L to 100 mg/L), the 3-hour EC₅₀ and EC₈₀ values of the test item are higher than 1000 mg/L.

Based on the statistical and biological evaluation in this test the NOEC was determined as 10 mg/L.