Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1)

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:

01 October 2021 - 29 December 2021

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:

adopted July 22, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot number of test material: 10419
- Purity: 93 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 – 29 °C), dry and dark

Analytical monitoring:

no

Details on sampling:

- Concentrations: 5.0 mg/L, 11.50 mg/L, 26.5 mg/L, 60.8 mg/L, and 139.9 mg/L

Vehicle:

no

Details on test solutions:

The required test item was weighed to get the test item stock solution concentration as listed below.

Test Item Final Volume with Test Item Conc. (mg/L)
Weight (mg) Deionized water (mL)

1019.8 100 9484.14
5099.0 500 9484.14
268.70 250 999.56
270.20 250 1005.14
268.72 250 999.64
537.77 500 1000.25
1075.74 1000 1000.44

Test mixtures were directly pipetted into the test vessels and incubated at the test temperature under conditions of forced aeration (0.5 L/min) to keep the dissolved oxygen concentration above 60 to 70 % saturation and to maintain the sludge flocs in suspension.

Test organisms (species):

activated sludge

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected:
exit of the aeration tank of a well-operated waste water treatment plant of sewage treatment plant Honnavara road, Bheemasandara, Near Bheemasandra Lake, Tumkur Karnataka-572 107

- Method of cultivation:
activated sludge was continuously aerated before the use at the rate of 2 L/min on the day of collection

- Preparation of inoculum for exposure:
sludge was collected and coarse particles were removed by settling for a short period for 15 minutes and decanting the upper layer of finer solids. The sludge was washed off by centrifugation for a period of 10 minutes at 10,000 g to get a clear supernatant liquid. The supernatant liquid was discarded and the sludge re-suspended in chlorine-free tap water with shaking and the wash-water was removed by re-centrifuging and discarded again. The washing and centrifuging process was repeated once. The suitable suspended solids concentrations of the activated sludge were prepared in a bulk quantity.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C.

pH:

7.5 ± 0.5

Dissolved oxygen:

below 2 mg/L

Nominal and measured concentrations:

nominal concentrations: 5.0 mg/L, 11.5 mg/L, 26.5 mg/L, 60.8 mg/L, and 139.9 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 1 L glass beaker
- Type: closed
- Aeration: yes; flow rate of 0.5 L per minute
- Sludge concentration (weight of dry solids per volume): nominally 1.5 g/L of suspended solids
- Nitrification inhibitor used: N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized water from Nice (Batch No. 008288 & 109230)

OTHER TEST CONDITIONS
- Adjustment of pH: yes,m to pH 7 – 8
Reference substance: adjusted with NaOH to pH 7 – 8

Main test: djusted to 7.5 ± 0.5 using NaOH solution

- Details on termination of incubation:
The addition of 2.5 mL of ATU stock solution to an incubation mixture of final volume of 500 mL resulted in a final concentration of 11.6 mg ATU/L (10-4 mol/L) which was known to be sufficient to cause 100 % inhibition of nitrification in a nitrifying activated sludge containing 1.5 g/L suspended solids.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric factor of 2.3
- Range finding study
- Test concentrations: 10 mg/L, 100 mg/L and 1000 mg/L
- Results used to determine the conditions for the definitive study:
From the range finding results, the test item had shown that inhibitory effect on the total oxygen uptake rate of activated sludge microorganisms at 10 mg/L, 100 mg/L and 1000 mg/L after 3 hours exposure time were 1.80 %, 94.23 % and 100.06 % respectively.

Reference substance (positive control):

yes

Remarks:

3, 5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

15.99 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

47.84 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC90

Effect conc.:

143.1 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

31.52 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

71.63 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

other: EC90

Effect conc.:

162.76 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

3.89 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

17.56 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Duration:

3 h

Dose descriptor:

other: EC90

Effect conc.:

79.34 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Results with reference substance (positive control):

The EC50-value for the reference substance was found to lie between the 2 to 25 mg/L for total respiration and 5 mg/L to 40 mg/L for heterotrophic respiration and 0.1 to 10 mg/L for nitrification by Probit Finney’s Analysis.

Reported statistics and error estimates:

ECx-values including their associated lower and upper 95 % confidence limits for the parameter were calculated using statistical methods by Probit Finney’s Analysis.
The statistical analysis were used to determine the NOEC, per-vessel statistics (individual vessels were considered as replicates) were necessary. Adverse effects of the test item compared to the control were investigated using one-tailed (smaller) hypothesis testing at p ≤ 0.05 using Annova, Dunnett t-test.

The mean inhibition of total respiration for the test item with five replicates at the tested concentrations of 5.0 mg/L, 11.5 mg/L, 26.5 mg/L, 60.8 mg/L, and 139.9 mg/L were 0.38 %, 6.86 %, 21.22 %, 49.47 % and 93.08 % respectively.

The mean inhibition of heterotrophic respiration for the test item with added ATU inhibitor at the tested concentrations of 5.0 mg/L, 11.5 mg/L, 26.5 mg/L, 60.8 mg/L, and 139.9 mg/L were -15.01%, -6.29 %, 7.65 %, 31.04 % and 87.82 % respectively.

The mean inhibition of nitrification for the test item at the tested concentrations of 5.0 mg/L, 11.5 mg/L, 26.5 mg/L, 60.8 mg/L, and 139.9 mg/L were 25.76 %, 28.31 %, 46.34 %, 81.82 % and 102.21 % respectively.

In order to check the activity of the test system and the test conditions, 3, 5-dichlorophenol was chosen as reference substance and the test was carried out and the reference toxicity was determined. The EC50-value for the reference substance was found to lie between the 2 to 25 mg/L for total respiration and 5 mg/L to 40 mg/L for heterotrophic respiration and 0.1 to 10 mg/L for
nitrification by Probit Finney’s Analysis.

 

Validity criteria fulfilled:

yes

Conclusions:

The Assessment of Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1) is non- toxic at the concentration of 5 mg/L to activated sludge of a sewage treatment plant.
The EC10, EC50 and EC90 of the test item for total respiration are 15.99 mg/L, 47.84 mg/L and 143.10 mg/L respectively.
The EC10, EC50 and EC90 of the test item for heterotrophic respiration are 31.52 mg/L, 71.63 mg/L and 162.76 mg/L respectively.
The EC10, EC50 and EC90 of the test item for nitrification are 3.89 mg/L, 17.56 mg/L and 79.34 mg/L respectively.

Executive summary:

Activated Sludge Respiration Inhibition Test was conducted according to OECD Guideline No. 209 carried out with the test item Assessment of Diethyl sulphate,

compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1). The main test was carried out under the test conditions with the test item concentrations 5.0 mg/L, 11.50 mg/L, 26.5 mg/L, 60.8 mg/L, and 139.9 mg/L.

The EC10, EC50 and EC90 of the test item without the inhibitor ATU are 15.99 mg/L, 47.84 mg/L and 143.10 mg/L respectively.

The EC10, EC50 and EC90 of the test item for heterotrophic respiration are 31.52 mg/L, 71.63 mg/L and 162.76 mg/L respectively.

The EC10, EC50 and EC90 of the test item for nitrification are 3.89 mg/L, 17.56 mg/L and 79.34 mg/L respectively.

Description of key information

Assessment of Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1Himidazole-1-ethanol (1:1) is non- toxic at the  concentration of 5 mg/L to the activated sludge of a sewage treatment plant. The mean inhibition of total respiration for the test item with five replicates at the tested concentrations of 5.0 mg/L, 11.5 mg/L, 26.5 mg/L, 60.8 mg/L, and 139.9 mg/L were 0.38 %, 6.86 %, 21.22 %, 49.47 % and 93.08 % respectively.
The EC10, EC50 and EC90 of the test item without the inhibitor ATU are 15.99 mg/L, 47.84 mg/L and 143.10 mg/L respectively.
The EC10, EC50 and EC90 of the test item for heterotrophic respiration are 31.52 mg/L, 71.63 mg/L and 162.76 mg/L respectively.
The EC10, EC50 and EC90 of the test item for nitrification are 3.89 mg/L, 17.56 mg/L and 79.34 mg/L respectively.

Key value for chemical safety assessment

EC50 for microorganisms:

47.84 mg/L

EC10 or NOEC for microorganisms:

5 mg/L