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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5th November 2018 to 9th November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: MEP, P.R. China, the Guidelines for the Testing of Chemicals, No. 201 "Algal Growth Inhibition Test". (2013.8)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EC, DG Health and Consumer Protection: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data reqs for Annex II (part A sec 4) & Annex III (Part A sec 5) of Dir 91/414 SANCO/3029/99 rev 4 11/07/00

GLP compliance:

yes

Test material

Test material information

Sampling and analysis

Analytical monitoring:

yes

Remarks:

HPLC/MS/MS

Details on sampling:

Analysis frequency: in 0-hour fresh solution and 72-hour old solution

Test solutions

Details on test solutions:

A saturated solution (SS) at nominal concentration of 100 mg/L was prepared. A weight of 0.2005g of test item was dissolved into a 2000ml volumetric flask with algal medium using a stirring rate of 420 rpm for 24 hours. Solutions were filtrated with 0.45 μm membrane.

250, 125, 62.5 and 31.3mL saturated solutions were pipetted and diluted respectively with 250, 375, 437.5 and 468.7mL test water to get 50% 25%, 12.5% and 6.25 % saturated concentration

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Source: Institute of Hydrobiology, Chinese Academy of Sciences and cultured continuously in the facility.
Batch No.: 20181102
Initial Biomass Concentration: 5 x 10 3 cell/mL

Study design

Test type:

static

Water media type:

not specified

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

0, 24, 48 and 72 hours

Test conditions

Test temperature:

22.9 - 24 °C

Nominal and measured concentrations:

Measured concentrations: the mean concentrations in 0 h media were 1.71, 3.33, 7.33, 13.4 and 30.4 mg/L. The mean concentrations in 72 h media were 1.37, 2.54, 6.28, 10.6 and
24.4.0mg/L. The overall mean concentrations were 1.54, 2.94, 6.31, 12.0 and 27.4mg/L.

Details on test conditions:

A saturated solution (SS) at nominal concentration of 100 mg/L was prepared. A weight of 0.2005g of test item was dissolved into a 2000ml volumetric flask with algal medium using a stirring rate of 420 rpm for 24 hours. Solutions were filtrated with 0.45 μm membrane.

250, 125, 62.5 and 31.3mL saturated solutions were pipetted and diluted respectively with 250, 375, 437.5 and 468.7mL test water to get 50% 25%, 12.5% and 6.25 % saturated concentration

Each solution was divided into 3 replicates and the control was 100ml Algal Growth Medium for each replicate.

The biomass of the pre-cultured algae solution was counted 4 times. The mean concentration was 3.5 x 10 6 cell/mL. 0.140ml of pre-cultured algal solution was added to each replicate vessel, mixed well, resulting in an initial test biomass of 5 x 10 3 cell/mL. All test flasks were capped with air-permeable membranes.
The algal biomass was measured daily (24, 48 and 72 hours) during the test period. The biomass was measured by manual cell counting using a microscope four times. Any abnormal symptoms were recorded.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.82 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.37 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 72 h median effect concentration of average specific growth rate (Er,C10, Er, C50) and yield (Ey, C10, Ey, C50) and 95% confidence limits of E Stage 3 intermediate to Desmodesmus subspicatus are as follows:

ErC10: 3.39 mg/L 95% Confidence Limit 1.68 - 6.82 mg/L

EyC10: 2.37 mg/L 95% Confidence Limit 1.39 - 4.06 mg/L

ErC50: 5.13 mg/L 95% Confidence Limit 2.58 - 10.22 mg/L

EyC50: 3.82 mg/L 95% Confidence Limit 2.15 - 6.78 mg/L

Executive summary:

An algal growth inhibition study of E Stage 3 intermediate was conducted for a period of 72 hours.

Based on the results of the preliminary test, the definitive test was conducted at the concentration of saturated concentration 100mg/L, 50% 25%, 12.5% and 6.25 % saturated concentration. The control group was treated in parallel. There were 3 replications for the test concentrations and control and 100ml for each replication. The algal species used was Desmodesmus subspicatus and the initial cell count was 5 x 10³ cell/mL. 

Algal growth medium recommended by the OECD guideline was used. 250ml glass conical flasks were used. During the 72 h test period, the cell concentration in each flask was determined by manual cell counting by microscope at 24, 48 and 72 hours after the start of the test. At the end of the test, the biomass was 2.39 x 10⁵ cell/mL in control cultures which had increased exponentially by a factor of 47.8 over the 72 h test period. During the test period, the mean co-efficient of variation of section-by-section specific growth rate was 26.8% in the control replicates. The co-efficient of variance of average specific growth rates during the whole test period was 1.34% in the control replicates. During the 72 hours test period, there was no abnormal appearance of algae observed in the control or in 100%, 50%, 25%, 12.5% and 6.25 % saturated solution replicates. After 72 hours exposure, there were apparent inhibition effects on growth at the test concentrations compared to the control. The inhibition rate increased with increasing concentrations.

The concentration of test item was determined in samples of test media by HPLC-MS/MS method. For 6.25, 12.5, 25, 50 and 100 %saturated concentrations the mean concentrations were as follows: 

in 0 h media were 1.71, 3.33, 7.33, 13.4 and 30.4 mg/L, the mean concentrations in 72 h media were 1.37, 2.54, 6.28, 10.6 and 24.4.0mg/L. The overall mean concentrations were 1.54, 2.94, 6.31, 12.0 and 27.4mg/L.

 

The 72 h median effect concentration of average specific growth rate (Er,C10, Er, C50) and yield (Ey, C10, Ey, C50) and 95% confidence limits of E Stage 3 intermediate to Desmodesmus subspicatus are as follows:

E_rC₁₀: 3.39 mg/L 95% Confidence Limit 1.68 - 6.82 mg/L

E_yC₁₀: 2.37 mg/L 95% Confidence Limit 1.39 - 4.06 mg/L

E_rC₅₀: 5.13 mg/L 95% Confidence Limit 2.58 - 10.22 mg/L

E_yC₅₀: 3.82 mg/L 95% Confidence Limit 2.15 - 6.78 mg/L