5,5'-{ethane-1,2-diylbis[thio-1,3,4-thiadiazole-5,2-diyldiazene-2,1-diyl(5-amino-3-tert-butyl-1H-pyrazole-4,1-diyl)]}diisophthalic acid

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental study was run 9th to 16th November 2011. The final report was issued 12th Jan 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Report on substance meets the criteria for classification as reliable without restriction according to Klimisch et al (1997).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tetra potassium 5,5'-[ethane-1,2-diylbis[thio-1,3,4-thiadiazole-5,2-diyldiazene-2,1-diyl(5-amino-3-tert-buyl-1H-pyrazole-4,1-diyl)]}diisophthalate

EC Number:

700-616-6

Cas Number:

849608-59-9

Molecular formula:

C36H32K4N14O8S4

IUPAC Name:

Tetra potassium 5,5'-[ethane-1,2-diylbis[thio-1,3,4-thiadiazole-5,2-diyldiazene-2,1-diyl(5-amino-3-tert-buyl-1H-pyrazole-4,1-diyl)]}diisophthalate

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The concentrations of test material in the test solutions was measured on exposure Day 0 and Day 7 using HPLC. Samples for analysis were taken on Day 0 from from excess test solutions and Day 7 from a single test replicate solution of each concentration. All samples on exposure Day 0 and Day 7 were analysed both pre and post centrifugation.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Test solutions: Nominal concentrations of the test material of 0.36, 1.1, 3.7, 12, 38 and 120 mg/L and a control.
- Method: A primary stock of the test substance with a nominal concentration of 120 mg/L was prepared by the addition of 0.240g of test substance to 2000ml of dilution water. The stock solution was stirred for 20 minutes. The resultant solution was observed to be a yellow to orange coloured solution that was slightly cloudy. The remaining test solutions were prepared by dilution of appropriate volumes of the primary stock to final volumes of 1000mL with culture medium. All test solutions were stirred for 5 minutes. The resultant solutions were increasingly dark, yellow or yellow to orange coloured clear solutions.

Test organisms

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: Duckweed
- Strain: ST strain
- Source (laboratory, culture collection): laboratory cultures maintained in house at test lab
- Age of inoculum (at test initiation): 9 days

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Test temperature:

24 +/- 2 degrees centigrade

pH:

pH of test concentrations ranged from 6.8 to 7.0 at the start of the test amd 8.1 to 8.6 at the end of the test. This exceeds the maximum expected increase of 1.5 units quoted in the OECD guideline. It is also stated however that a pH increase of more than 1.5 units in the control does not invalidate a test as long as the validity criteria are met.

Nominal and measured concentrations:

The measured concentrations in the test solutions ranged from 24 to 70% of the nominal concentrations at the start of the test and 13 to 59% of the nominal concentrations at the end of the test. The percentage loss over the course of the study ranged from 0 to 68% (with the exception of the 1.1mg/L nominal test concentration where the measured concentration increased from 0.47 to 0.52 mg/L). The geometric mean of the measured concentration ranged from 20 to 61% of the nominal values. The geometric mean measured concentration of test substance was used for the calculation and reporting of results.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass dishes of 300mL nominal capacity fitted with loose fitting lids containing 160mL of test solution.
- No. of fronds per test vessel: 12 (from 3-5 plants)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used as recommended by OECD, Swedish Standard (SIS)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Culture medium

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination of 9600 lux.

EFFECT PARAMETERS MEASURED
- Determination of biomass: The increase in dry weight (yield) was calculated by subtraction of the estimated initial weight from the measured dry weight at day 7.
- Determination of frond number: The increase in number of fronds (yield) over the 7 day test peiod was calculated for each test vessel by subtracting 12 (the number of fronds inocluated on day 0) from the day 7 counts.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

> 28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 7

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 9

Key result

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

> 28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 10

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

It was noted during Day 5 and Day 7 observations that some small particulates were present on the bottom of the test vessels in the 120 mg/L nominal test concentrations. It was also noted on Day 7 that a green coloured tinge, thought to be algal growth, was observed on the test vessels edges in all replicates. Neither of these observations was considered to have had an effect on the integrity of the study.

Reported statistics and error estimates:

The data for increase in frond number, dry weight and their respective growth rates were examined by one-way analysis of variance and Dunnett's procedure was used to identify significant differences (p<0.05) from the control.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

During this study, there were no significant differences in the increase in the number of fronds (yield) between the control and any of the test concentrations. Based on geometric mean measured values the NOEC was therefore determined to be 28 mg/L and the LOEC >28mg/L. As there was no observable reduction in the frond number increase the EC10, EC20 and EC50 were all calculated to be >28mg/L.

As was observed for frond number, there were no significant differences in the increase in the plant dry weight (yield) between the control and any of the test concentrations in this study. Based on geometric mean measured concentrations the NOEC was determined to be 28mg/L and the LOEC >28 mg/L. As there was no observable reduction in plant dry weight increase the EC10, EC20 and EC50 were all calculated to be >28mg/L.

As for the yield data, there were no significant differences in the average specific growth rates between the control and any of the test concentrations in this study. Based on geometric mean measured values the NOEC for growth rate based on both frond number or dry weight was therefore determined to be 28 mg/L and the LOEC >28mg/L. As there was no observable reduction in the frond number increase the EC10, EC20 and EC50 were all calculated to be >28mg/L.

Executive summary:

Introduction.

A study was performed to assess the effect of the test material on the growth of the freshwater plant Lemna minor. The method followed that described in OECD Guidelines for Testing of Chemicals. Test Guideline 221, Lemna sp. Growth Inhibition Test. Adopted 23 March 2006.

Methods.

A primary stock of test substance with a nominal concentration of 120 mg/L was prepared. Control and nominal concentrations of 0.36, 1.1, 3.7, 12, 38 and 120 mg/L. The measured concentration of the test substance was not maintained within ±20% of the nominal concentrations during the study. Therefore the calculation and reporting of results is based on geometric mean measured concentrations.

Results and Conclusion.

Based on frond number, the results for both yield and average specific growth rate were: NOEC = 28 mg/L and LOEC, EC10, EC20 & EC50 >28 mg/L.

Based on dry weight, the results for both yield and average specific growth rate were: NOEC = 28 mg/L and LOEC, EC10, EC20 & EC50 >28 mg/L.

The test substance is considered to be non-toxic to duckweed (Lemna minor) based on average specific growth rate.