Reaction products of Hydrogen amino-4-hydroxynaphthalene-2-sulfonate, Potassium substituted-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate and Sodium substituted-({4-[(2-chloroethyl)sulfonyl]butanoyl}amino)benzenesulfonate

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item: FAT 40875/A TE
Physical appearance: Dark red powder
Purity: 86.4% all organic components
Batch No: BOP 05-17 (BS-ROE 1550 NIM 25+26+27)
Manufactured date: 20 December 2017
Expiry date: 11 December 2022
pH : 4.3 (aq. soln. 2 %w/w at room-temperature)
Recommended storage condition: Refrigeration (+2 to +8 °C) and protection from light

Analytical monitoring:

yes

Details on sampling:

The test concentration along with the control was analyzed for the test item concentration at the beginning and at the end of each renewal during the test. For analysis, single composite sample was drawn from prepared test concentration and from the control.

Vehicle:

yes

Details on test solutions:

To prepare test concentration of 100 mg/L, 100 mg of test item (after grounding with mortar and pestle) was mixed with test medium along with sonication followed by making up to 1 L in volumetric flask using test medium. For control (G1), 1 L of test medium was used.
The test was performed on a black, non-reflecting surface due to coloured nature of test medium as per the OECD Guidance Document No. 23.

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: Lemna
- Source (laboratory, culture collection): Stock culture maintained at: Eurofins Agroscience Services Ecotox GmbH, Aquatic Toxicology, Eutinger Strasse 24 D-75223 Niefern Öschelbronn, Germany.

ACCLIMATION
- Acclimation period: At least seven days before testing, sufficient colonies of Lemna weretransferred aseptically into fresh sterile medium and cultured for 7 - 10 days under the conditions of the test.
- Culturing media and conditions (same as test or not): Same as test.
- Any deformed or abnormal cells/plants observed: No

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Test temperature:

Range finding test: 24.0 to 24.6 °C
Definitive test: 23.7 to 24.9 °C

pH:

Range finding test: 6.86 to 7.76
Definitive test: 6.68 to 7.17

Nominal and measured concentrations:

Definitive Test: 0 (control), 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes
- Test vessel: Beaker
- Type: closed covered with petri plates.
- Material, size: Glass, 250 mL.
- Aeration: yes
- Agitation: no
- No. of colonies per vessel: Three
- No. of fronds per colony: Four
- No. of vessels per concentration (replicates): six
- No. of vessels per control (replicates): six

GROWTH MEDIUM
- Standard medium used: yes - Swedish Standard (SIS) Medium Stock Solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water.
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous illumination
- Light intensity and quality: 6500 to 10000 Lux

EFFECT PARAMETERS MEASURED :
- Determination of frond number: Yes
- Determination of biomass: Yes, Dry weight
- Shoot length: No

RANGE-FINDING STUDY
- Test concentrations: 0 (control), 1, 10, 50 and 100 mg/L.
- Results used to determine the conditions for the definitive study: Based on the results of the range finding test, the definitive test was carried out using the limit test concentration of 100 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

> 66.81 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

> 66.81 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

> 66.81 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

> 66.81 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

yield

Details on results:

Fronds were found to be normal in appearance in control and test item treated group (except permissible level of frond mortalities in two replicates of treated group) at the end of the test. Test medium was clear and red in colour in treatment group. Test medium was similar in appearance on days 3, 5 and 7 as compared at the start of the exposure.

Results with reference substance (positive control):

The inhibition of growth rate for frond numbers was 0,0.8, 25.3, 36.7 and 100% and yield was 0, 2.1, 52.0, 66.1 and 100% for dry weight was 0, 0.5, 29.5. 43.7 and 100% and yield was 0, 1.1, 58.0, 73.5 and 100% at the tested coocentralions of 0.3, 1, 3, 9 and 27 rng/L, respectively as compared with the control.

Reported statistics and error estimates:

The statistical analysis of the growth rate and yield data was evaluated using licensed copies of SYSTAT Statistical package version 12.0. The growth rate and yield for frond number and dry weight was tested for normality (Shapiro-Wilk test) and homogeneity of variance before performing further analysis. The growth rate and yield for frond number and dry weight data were found to be normal and homogeneous and performed two sample t-test (for NOEC evaluation).

Test Item Solubility: Test item was tested for its solubility in the test medium and formed red coloured solution (along with sonication) with the test medium at 10 mg/mL (performed as a non-GLP test).

Analysis of Test Concentrations

The test item was recoverable using the analytical method at nominal concentrations of 2.05 and 205 mg/L in the test medium. The stability test results concluded that the test item was found to be unstable in the test medium at the test conditions up to 24 h at nominal concentrations of 2.05 and 205 mg/L.

The active ingredient concentration analysis in test concentration showed that the percent agreement with claimed concentration was 62.34 to 106.54 % (considering all the sample intervals from fresh samples) at the start of exposure and 15.32 to 69.91 % (considering all the sample intervals from aged samples) at the end of renewal periods (24 h test medium renewal cycle). Since the analysis results of samples drawn at the start of exposure (except on days 0, 1, 3, 4, 5 and 6 during the start of exposure) and end of renewal period was less than the acceptable limit (80 to 120 % of the claimed concentration with an RSD of ≤ 20%). Hence the nominal concentration of test item group was recalculated and presented as mean measured concentration. No measurable concentration of test item was recorded in the control samples. The calculated mean measured concentration was 66.81 mg/L against the nominal concentration of 100 mg/L.

VALIDITY OF THE TEST

The Lemna growth inhibition test fulfills the validity criteria of the OECD guideline 221: The factor of frond number increase, measured in the control between day 0 and day 7, was 13.8 corresponding to an average specific growth rate for frond number in the control of 0.375 per day and to a doubling time of 1.850 days (validity criterion: growth rate ≥ 0.275 per day, doubling time < 2.5 days).

Validity criteria fulfilled:

yes

Conclusions:

The NOEC for growth rate and yield (for frond number and dry weight) on the basis of the mean measured concentration of the test item was higher than 66.81 mg test item/L (nominal concentration of test item was 100 mg/L).

Executive summary:

The effect of FAT 40875/A TE was tested on the growth of freshwater vascular aquatic plant, Lemna minor. This test was conducted in accordance with OECD test guideline 221, in a GLP certified laboratory.

A range finding test was conducted at concentration of 0 (control), 1, 10, 50 and 100 mg/L. No toxicity was observed at maximum concentration.

Based on the results of the range finding test, the definitive test was carried out using the limit test concentration of 100 mg/L; along with a control (each group had six replicates). Lemna was exposed in semi-static mode with renewal of test medium at every 24 h interval. Frond observations were made on days 3, 5 and 7. Dry weight was determined at the start (day 0) and at the end of the test (day 7).

3,5-Dicholorphenol was used as a positive control. The test medium served as vehicle control. Analysis of test item concentration in the test samples was carried out using HPLC.

 

Fronds were found to be normal in appearance in control and test item treated group (except permissible level of frond mortalities in two replicates of treated group) at the end of the test. Test medium was clear and red in colour in treatment group. Test medium was similar in appearance on days 3, 5 and 7 as compared at the start of the exposure.

 

The test item was recoverable using the analytical method at nominal concentrations of 2.05 and 205 mg/L in the test medium. The stability test results concluded that the test item was found to be unstable in the test medium. The analysis results of samples drawn at the start of exposure and end of renewal period was less than the acceptable limit, thus the nominal concentration of test item group was recalculated and presented as mean measured concentration. No measurable concentration of test item was recorded in the control samples. The calculated mean measured concentration was 66.81 mg/L against the nominal concentration of 100 mg/L.

 

Based on the findings of the study, the NOEC for growth rate and yield (for frond number and dry weight) on the basis of the mean measured concentration of the test item was higher than 66.81 mg test item/L (nominal concentration of test item was 100 mg/L).

 

The NOECr (growth rate of frond numbers) was found to be 1 mg/L in Positive control (3,5- Dicholorophenol).

Description of key information

The NOEC for growth rate and yield (for frond number and dry weight) on the basis of the mean measured concentration of the test item was higher than 66.81 mg test item/L (nominal concentration of test item was 100 mg/L).

Key value for chemical safety assessment

EC50 for freshwater plants:

66.81 mg/L

EC10 or NOEC for freshwater plants:

66.81 mg/L

Additional information

The effect of FAT 40875/A TE was tested on the growth of freshwater vascular aquatic plant, Lemna minor. This test was conducted in accordance with OECD test guideline 221, in a GLP certified laboratory.

A range finding test was conducted at concentration of 0 (control), 1, 10, 50 and 100 mg/L. No toxicity was observed at maximum concentration.

Based on the results of the range finding test, the definitive test was carried out using the limit test concentration of 100 mg/L; along with a control (each group had six replicates).Lemna was exposed in semi-static mode with renewal of test medium at every 24 h interval. Frond observations were made on days 3, 5

 

Fronds were found to be normal in appearance in control and test item treated group (except permissible level of frond mortalities in two replicates of treated group) at the end of the test. Test medium was clear and red in colour in treatment group. Test medium was similar inappearance on days 3, 5 and 7 as compared at the start of the exposure.

 

Based on the findings of the study, the NOEC for growth rate and yield (for frond number and dry weight) on the basis of the mean measured concentration of the test item was higher than 66.81 mg test item/L (nominal concentration of test item was 100 mg/L).