Trisodium 2-[[4-[[4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulphonatonaphthyl]azo]-2,5-dimethylphenyl]azo]benzene-1,4-disulphonate

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Remarks:

Lemna

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

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:

Name: FAT 40075/D
Batch no: BS-1113243
EAS Test item code: 2014-000184
Appearance / colour: solid / orange
CAS number: 70210-13-8
Expiry date: 14 Mar 2017
Date of analysis: 03 Dec 2013
Storage conditions: cool (+1 to +10 °C), dark, dry

Analytical monitoring:

yes

Details on sampling:

Samples were taken at 0 hours (initial value) from fresh test solution, after 2 and 5 days from fresh and aged test solution and after 7 days from aged test solution from control and all test item concentrations. Nominal test item concentrations of 6.25, 12.5, 25.0, 50.0, 100 mg/L and control were analysed at 0, 2, 5 and 7 days. After sampling, the test medium samples (0.5 mL) were mixed with 0.5 mL methanol and stored deep-frozen (below -18 °C) until analysis.

Vehicle:

yes

Details on test solutions:

A stock solution (11470 mg/L) of the test item was prepared in N,N-dimethylformamide and a dilution (100 mg/L) in methanol/water (1:1, v/v). The stock solution and the dilution were used for fortification of recovery samples.

Test organisms (species):

Lemna gibba

Details on test organisms:

The test organism was the aquatic plant duckweed, Lemna gibba G3 (Alismatales: Araceae). The plants used in this study were taken as aseptic clones from the cultivation of the testing facility. They were primarily cultured by Dr. Janet Slovin, Horticulture Crops Quality Laboratory, U.S. Department of Agriculture. BARC-West, Bldg. 050 HH-4, Beltsville, MD 20705, U.S.A.

The plants were cultured in appropriate glass beakers under continuous illumination with OSRAM light tubes (L30/32-930 and L30/W72-965), at 6500 - 7000 lux. The temperature was 24 ± 2 °C. Before the test was started, plants were cultured under the same illumination and temperature conditions as those to be used for the toxicity test. Young, light-green plants of similar size and comprising 2 - 5 fronds were transferred onto fresh medium and cultured for ≥ 3 weeks prior to testing, with two further transfers onto fresh medium before initiating the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

23.0 – 24.6 °C

pH:

7.45 – 7.64

Nominal and measured concentrations:

0, 6.25, 12.5, 25, 50 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes
- Test vessel: 250 mL glass beakers filled with 150 mL
- No. of colonies per vessel: 3-4
- No. of fronds per colony: 12
- No. of vessels per concentration (replicates): 1

GROWTH MEDIUM
- Standard medium used: yes (20xAAP Medium)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 7 days
- Light intensity and quality: continuous illumination with OSRAM light tubes (L30/32-930 and L30/W72-965), at 6500 - 7000 lux.

Study period:
Study initiation date: 11 Jun 2014
Start of the experimental phase: 25 Jun 2014
End of the experimental phase (biological part): 04 Jul 2014
End of the experimental phase (analytical part): 05 Jul 2014
Draft Report (biological part): 14 Jul 2014
Draft Report (analytical part): 13 Aug 2014
Final Report: 26 Sep 2014

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

55.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

On day 5 and 7 the fronds looked slightly pale and roots were coloured orange at 25 and 50 mg/L. At 100 mg/L the fronds were pale with short roots and discolouration, offshoots were smaller.

Reported statistics and error estimates:

The statistical evaluation for day 7 was performed for yield of frond numbers, growth rate of frond numbers, growth rate dry weight and yield of dry weight. A test for normality of the data was performed by calculating the Shapiro-Wilk’s statistic, a test for homogeneity of the data was performed according to Levene. The NOEC and LOEC were determined by using a multiple comparison method (Dunnett`s t-test and Jonkheere-Terpstra). The EC10 and EC20 values for yield of frond numbers and dry weight and growth rate of dry weight were determined by probit analysis following Gompertz procedure. The EC50 value for yield of dry weight was determined by probit analysis following Gompertz procedure. The calculation of EC50 values for yield and growth rate of frond numbers and growth rate of dry weight was not indicated since inhibition was below 50 %. The calculation of EC20 value for growth rate of frond numbers was not indicated since inhibition was below 20 %. The evaluation of data was performed by SAS® (2002-2010).

Summary;

	Test item [mg/L] nominal
Endpoint	EC10	EC20	EC50	NOEC	LOEC
Yield of frond numbers	19.6	40.2	>100	25	50
Growth rate of frond numbers	55.5	>100	>100	25	50
Yield of dry weight	45.1	61.7	98.9	50	100
Growth rate of dry weight	66.8	89.7	>100	50	100

Validity criteria fulfilled:

yes

Conclusions:

The nominal EC50-value for yield of frond numbers, growth rate of frond numbers and growth rate of dry weight was determined to be >100 mg/L.

Executive summary:

The aim of the study was to assess the effect of the test item FAT 40075/D on the growth of Lemna gibba according to OECD Guideline 221.

Test item: FAT 40075/D, content of a.i. (nominal): 65 % (w/w);

Test species: Lemna gibba G3 (Duckweed).

A semi-static main test with nominal concentrations of 0 (control), 6.25, 12.5, 25.0, 50.0 and 100 mg/L was performed. Vegetative growth was observed for a test period of 7 days by measuring the increase of frond numbers and dry weight. The following endpoints (NOEC/LOEC, EC10, 20, 50) were determined:

- frond numbers and dry weight

- growth rates of frond numbers and dry weight

- yield of frond numbers and dry weight

- doubling times of frond numbers

The initial mean measured concentration of FAT 40075/D in the test item solutions was 106 % of nominal. The measured concentration of FAT 40075/D in the test item solutions ranged from 98 to 117 % of nominal with an overall mean concentration of fresh and aged samples of 108 % of nominal. Significant inhibitory effects were determined for yield of frond numbers and for growth rate of frond numbers at 50.0 and 100 mg/L (nominal). Significant inhibitory effects were determined for yield dry weight and for growth rate of dry weight at 100 mg/L. The overall LOEC was therefore determined to be 50.0 mg/L, the corresponding NOEC at 25.0 mg/L. The nominal EC10-value for yield of frond numbers was determined to be 19.6 mg/L, the nominal EC10-value for yield of dry weight was determined to be 45.1 mg/L. The nominal EC10 - value for growth rate of dry weight was determined to be 66.8 mg/L. The nominal EC10 -value for growth rate of frond numbers was determined to be 55.5 mg/L. The nominal EC20-value for yield of frond numbers was determined to be 40.2 mg/L, the nominal EC20-value for yield of dry weight was determined to be 61.7 mg/L. The nominal EC20 - value for growth rate of dry weight was determined to be 89.7 mg/L. The nominal EC20 -value for growth rate of frond numbers was determined to be >100 mg/L. The nominal EC50-value for yield of frond numbers, growth rate of frond numbers and growth rate of dry weight was determined to be >100 mg/L. The nominal EC50-value for yield of dry weight was determined to be 98.9 mg/L. The test is valid as the doubling time of control frond numbers was calculated to be 37.9 h, corresponding to 1.581 days at day 7. This is less than the maximum allowable doubling time set in OECD guideline 221 (60 h, 2.5 days).

Description of key information

The aim of the study was to assess the effect of the test item FAT 40075/D on the growth of Lemna gibba according to OECD Guideline 221. A semi-static main test with nominal concentrations of 0 (control), 6.25, 12.5, 25.0, 50.0 and 100 mg/L was performed. Significant inhibitory effects were determined for yield of frond numbers and for growth rate of frond numbers at 50.0 and 100 mg/L (nominal). Significant inhibitory effects were determined for yield dry weight and for growth rate of dry weight at 100 mg/L. The overall LOEC was therefore determined to be 50.0 mg/L, the corresponding NOEC at 25.0 mg/L. The nominal EC10-value for yield of frond numbers was determined to be 19.6 mg/L, the nominal EC10-value for yield of dry weight was determined to be 45.1 mg/L. The nominal EC10 - value for growth rate of dry weight was determined to be 66.8 mg/L. The nominal EC10 -value for growth rate of frond numbers was determined to be 55.5 mg/L. The nominal EC20-value for yield of frond numbers was determined to be 40.2 mg/L, the nominal EC20-value for yield of dry weight was determined to be 61.7 mg/L. The nominal EC20 - value for growth rate of dry weight was determined to be 89.7 mg/L. The nominal EC20 -value for growth rate of frond numbers was determined to be >100 mg/L. The nominal EC50-value for yield of frond numbers, growth rate of frond numbers and growth rate of dry weight was determined to be >100 mg/L. The nominal EC50-value for yield of dry weight was determined to be 98.9 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:

100 mg/L

EC10 or NOEC for freshwater plants:

25 mg/L