(1R)-2-[5-(2-halo-3-methoxyphenyl)-3-[2-halo-6-(trihalomethyl)benzyl]- 4-alkyl-2,6-dioxoheterocyclyl]-1-phenylethanaminium salicylate

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24th August 2018 to 14th November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: MEP, P.R. China, the Guidelines for the Testing of Chemicals, Effects on Biotic Systems (the Second Edition), No. 209 "Activated Sludge, Respiration Inhibition Test". (2013.8).

GLP compliance:

yes

Remarks:

SEPA, P.R. China, the Guidelines of Chemical Testing Good Laboratory Practices (HJ/T 155-2004).

Test material

Test material information

Sampling and analysis

Details on sampling:

DOC for total respiration was measured in 10 minutes intervals after 3 hours contact time until 6 h.

Test solutions

Details on test solutions:

To prepare test item mixtures, the test item was directly weighed into the test vessel and distilled water was added.

Test organisms

Details on inoculum:

The activated sludge was derived from a treatment plant receiving predominantly domestic sewage. A fresh sample of activated sludge was collected from the aeration tank of Shenyang North Sewage Treatment Plant 1 day before the test was started and the sludge was fed daily with synthetic sewage feed.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

20.6 - 21.3 °C

Nominal and measured concentrations:

The definitive test was conducted with 4 concentrations of 10, 100, 300 and 1000 mg/L. 10, 100, 300 mg/L were tested with 1 replicate, 1000 mg/L was tested with triplicate and an abiotic control at concentration of 1000 mg/L was performed.

Details on test conditions:

Test water: distilled water
Air for aeration: clean oil-free air, flow rate 0.5 ~ 1 L/min

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Results and discussion

Effect concentrations

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 4 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Based on the results of test item test, the EC50 mg/L of test item for total respiration resulted in a total of >4000. Validity criteria were met throughout test.

Results with reference substance (positive control):

3,5-dichlorophenol was used as a reference item in order to check the sensitivity of the activated sludge used as microbial inoculum on the day of exposure. The reference item was tested at 0.5, 1.3, 3.5, 9.4 and 25 mg/L with 1 replicate. Based on the results of the reference item test, the EC50 ( 3 hours) of the reference substance for the definitive test was:
EC50 (mg/L)=2.7 mg/L with a 95% confidence limit of 1.9 ~ 3.9 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of test item test, the EC50 (3 hours) of test item for total respiration resulted in a total of >4000 mg/L, indicating that the substance would not be of a concern, in terms of toxicity, to the sludge microorganisms which are responsible for the biodegradation of organic material in waste water treatment plant (as the EC50 is >100 mg/L).

Executive summary:

The study was conducted to determine the inhibition effects of E Stage 3 intermediate on micro-organisms from activated sludge (largely bacteria) by measuring their respiration rate. The contact time for exposure was 3 hours. 

The range-finding test was conducted with four concentrations of 10, 100, 300 and 1000 mg/L. 10, 100, 300 mg/L were tested with 1 replicate. 1000mg/L was tested in triplicate and an abiotic control at a concentration of 1000mg/L was performed. Inoculums blank controls were performed with 6 replicates. The rate of respiration was determined after 3 hours contact time. 

According to the results of the range-finding test, the definitive test was carried out at 6 concentration of 4, 16, 63, 250, 1000 and 4000mg/L with five replicates. Inoculums blank controls were performed with 6 replicates. A reference item test with 3,5-dichlorophenol (3,5-DCP) was conducted the same as the range-finding test.  

3,5-DCP was used as a reference item in order to check the sensitivity of the activated sludge used as a microbial inoculum on the day of exposure. The reference item was tested at 0.5, 1.3, 3.5, 9.4 and 25 mg/L with one replicate. 

The specific oxygen uptake rate of the blank controls (without the test item or reference item) was 21.0 mg/(h·g SS) oxygen both in the range-finding and definitive test. The coefficient of variation of oxygen uptake rate in control replicates was 3.2% and 1.1% in the range-finding and definitive tests. The validity criteria have therefore been satisfied.

Based on the results of the reference test item, the EC50 of 3,5-DCP for total respiration is as follows: 

Test	EC50 (mg/L)
Range Finding Test	4.6
Definitive	2.7

Based on the results of the test item in the definitive test, the EC₅₀ (3 hours) of test item for respiration is as follows: 

Inhibition of respiration	EC50 (mg/L)
Total	>4000