Leuco polysulfided 4-[(2,4-dinitrophenyl)amino]phenol

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:

2017-11-10 - 2017-11-17 (start of experiment - end of experiment)

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)

Version / remarks:

23rd March, 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 761/2009, Annex VI, C.26: „Lemna sp. Growth Inhibition Test“. Dated 24 August 2009

Version / remarks:

August 24, 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series on Testing and Assessment No. 23, Paris September 2000

Version / remarks:

September 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Expiry date: April 29, 2020

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Six replicate samples were analysed from the test solutions at the start and at the end of the renewal periods. Six replicate samples were analysed from the control as well. The samples were concentrated 5-fold before the measurement.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: As the test item is poorly soluble in deionized water as well in the test medium, preparation of test solution was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23).

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: Duckweed
- Strain: G3
- Source (laboratory, culture collection): In-house culture kept under axenic conditions; preculture was prepared at least 7 days before the tests; healthy colonies with 3-4 fronds. Originally, the culture was obtained from Friedrich Schiller Universität, Institut für Allgemeine Botanik und Pflanzenphysiologie, Jena, Germany.
- Preculture: 7 - 10 days (7 days in this study) before testing, sufficient colonies are transferred from the stock culture aseptically into fresh sterile medium and cultured under the conditions of the test prior to beginning the test.

Test type:

semi-static

Water media type:

other: 20X AAP Medium

Limit test:

yes

Total exposure duration:

7 d

Remarks on exposure duration:

At the start, frond numbers in the test vessels were recorded. The number and appearance of fronds of Lemna g. were determined in each testing vessel during the 168-hour test on the 3rd, 5th and 7th days.The test item on final biomass were also assessed.

Hardness:

Not specified

Test temperature:

The cultures were maintained at a temperature in the range of 24 +/- 2 ºC (22.2 - 24.5°C in the climate chamber and 22.9 – 23.1 °C in the test vessels.

pH:

7.72 to 8.11

Nominal and measured concentrations:

Nominal: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: Yes
- Test vessel: All-glass beakers (total capacity of 400 mL) were used and were covered by glass petri dishes, with total capacity of 400 mL were used. The volume of the test liquid in the vessels was 160 mL. Each test unit was uniquely identified with study code, test item concentration (and control) and replicate.
- Material, size, headspace, fill volume: Glass, total capacity = 400 mL, fill volume = 160 mL, - Type: Closed
- Type of cover: covered by glass petri dishes
- Aeration: No
- Agitation: No
- Renewal rate of test solution: Renewed twice during the test (on days 3 and 5).
- No. of vessels per concentration: 6
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: Yes (20 X AAP Medium)

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Illuminated continuously
- Light intensity and quality: 6500-10000 lux (with a spectral range of 400-700 nm)

EFFECT PARAMETERS MEASURED
- Determination of frond number: Manual counting
- Determination of biomass: Measured as frond counts

RANGE-FINDING STUDY (non-GLP)
In order to select appropriate test concentrations for use in the definitive test, preliminary range-finding test was conducted to determine the approximate toxicity of the test item. In the preliminary range-finding test a test item stock solution was prepared by adding 0.04 g of test item into 400 mL dilution water (20X AAP medium) to get the nominal concentration of 100 mg test substance /L. The stock solution was handled in ultrasonic bath for 10 minutes then filtrated through a membrane filter (0.22 μm) to separate the possible non-dissolved test material. The test item solution of 10 mg/L was prepared by the 10-fold dilution of the stock solution. Untreated control ran parallel in the test. The pre-test was performed with two replicates per test item treated group (containing 11 fronds in total per test vessel) for a period of 7 days. A concurrent control was run (with three replicates).
The preliminary range-finding test was not performed in compliance with the GLP-Regulations and is excluded from the Statement of Compliance.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.591 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 0.591 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.591 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

yield

Key result

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 0.591 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

yield

Results with reference substance (positive control):

The date of the last study with reference item 3,5-dichlorophenol was: 08 – 15 September 2017.
Endpoints of this study were: EyfnC50 (7 day, yield based on frond numbers): 3.980 mg/L, ErfnC50 (7 day, growth rate based on frond numbers): 6.053 mg/L, EydwC50 (7 day, yield based on dry weight): 2.906 mg/L, ErdwC50 (7 day, growth rate based on dry weight): 6.206 mg/L.

Reported statistics and error estimates:

For the determination of the LOEC and NOEC, the calculated mean growth rate and yield at the calculated test concentration were tested on significant differences to the control value using Independent Sample T-test by SPSS PC+ software program. The data were checked for homogeneity of variance by Levene’s test.

Summary of the Biological Endpoints

Endpoints for response variables (0-7 d)		Concentration(mg/L) (based on mean measured concentration)
Growth Rate (based on frond number and dry weight)	NOEC	0.591
	LOEC	> 0.591
Yield (based on frond number and dry weight)	NOEC	0.591
	LOEC	> 0.591

 

Calculation of Exposure Concentration

Nominal concentration (mg/L)	Measuredconcentration(mg/L)						Mean measuredconcentration (mg/L)
	1^strenewal period		2^ndrenewal period		3^rdrenewal period
	Start	End	Start	End	Start	End
Control	-	-	-	-	-	-	-
100 (WAF^*)	0.65	0.50	0.73	0.51	0.72	0.49	0.591

-      not detected

*   water accommodated fraction (OECD No. 23.)

Growth rates (µ) and Percentage Inhibition ofµbased on Frond Number

Concentration (mg/L)		Growth rate (µ) and % inhibition ofµ
Nominal	Measured	0–168 h(based on frond number)
		µ	% Iµ
Control	-	0.3534	-
100 (WAF)	0.591	0.3535	-0.02^*

*: negative value indicates increase in comparison to the control.

Growth rates (µ) and Percentage Inhibition ofµbased on Dry Weight

Concentration (mg/L)		Growth rate (µ) and % inhibition ofµ
Nominal	Measured	0–168 h(based on dry weight)
		µ	% Iµ
Control	-	0.39742	-
100 (WAF)	0.591	0.38439	3.28

Yield (y) and Percentage Inhibition of Yield based on Frond Number

Concentration (mg/L)		Yield (y) and % inhibition of yield
Nominal	Measured	0–168 h(based on frond number)
		y	% Iy
Control	-	119.83	-
100 (WAF)	0.591	119.83	0.00

 Yield (y) and Percentage Inhibition of Yield based on Dry Weight

Concentration (mg/L)		Yield (y) and % inhibition of yield
Nominal	Mean measured	0–168 h(based on dry weight)
		y	% Iy
Control	-	0.00838	-
100 (WAF)	0.591	0.00761	9.24

Symptoms, Changes of Lemna gibba Plants Observed during the Test

Concentration (mg/L)		3^rdday of the experiment		5^thday of the experiment		7^thday of the experiment
Nominal	Mean measured	Symptoms	Degree of change	Symptoms	Degree of change	Symptoms	Degree of change
Control	-	–	–	–	–	–	–
100 (WAF)	0.591	–	–	–	–	–	–

"–":the plants were healthy, there was not any symptom observed

Validity criteria fulfilled:

yes

Conclusions:

In a Growth Inhibition Test with Lemna gibba according to OECD 221 and Commission Regulation (EC) No 761/2009, Annex VI, C.26 the 7-d LOEC of the test item for growth rate and biomass were determined to be > 0.591 mg/L (geom. mean measured). The 7-d NOEC of the test item both for growth rate and biomass was determined to be 0.591 mg/L (geom.mean measured).

Executive summary:

The toxicity of the test item to Lemna gibba was assessed in a semi-static study according to OECD Guideline 221, Commission Regulation (EC) No 761/2009, Annex VI, C.26 and GLP principles. As the test item is poorly soluble in deionized water as well in the test medium, preparation of test solutions was performed using the WAF method (in accordance with OECD Series on Testing and Assessment No. 23). Based on the results of the preliminary experiment, the nominal concentration of 100 mg/L was investigated in the main study (limit test). The measured concentrations deviated more than 20 % from the nominal at the start and at the end of the renewal periods, therefore the geometric mean of the measured concentrations were calculated to determine exposure concentration. The corresponding calculated concentration was 0.591 mg/L (based on total product). The test included six replicates and the control group with six replicates.400 mL Petri dishes-covered glass beakers were filled with 160 mL testing solutions and two Lemna gibba colonies with four, and one colony with three fronds were added to each vessel. Growth and morphological changes were assessed on days 3, 5 and 7. For determination of the test item concentrations, samples were taken from the test solution and control at the start and at the end of each renewal period. Test item concentration was determined using UV/VIS spectrophotometric method. For determination of the NOEC and LOEC values Independent Sample T-test was used (2-tailed, α = 0.05). All validity criteria were met and therefore the study can be considered as valid. The test item had no toxic effect at aquatic saturation (i.e. limit test concentration) on Lemna gibba; the overall LOEC is higher than the nominal concentration of 100 mg/L (solubility level) of the test item in the test medium, which corresponds to the mean measured concentration of 0.591 mg/L, based on water accommodated fraction of the total product.

Description of key information

The test item had no toxic effect at aquatic saturation (i.e. limit test concentration) on Lemna gibba; the overall LOEC is higher than the nominal concentration of 100 mg/L of the test item in the test medium, which corresponds to the mean measured concentration of 0.591 mg/L, based on water accommodated fraction of the total product.

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:

0.591 mg/L

Additional information

The toxicity of the test item toLemna gibbawas assessed in a semi-static study according to OECD Guideline 221, Commission Regulation (EC) No 761/2009, Annex VI, C.26 and GLP principles. As the test item is poorly soluble in deionized water as well in the test medium, preparation of test solutions was performed using the WAF method (in accordance with OECD Series on Testing and Assessment No. 23). Based on the results of the preliminary experiment, the nominal concentration of 100 mg/L was investigated in the main study (limit test). The measured concentrations deviated more than 20 % from the nominal at the start and at the end of the renewal periods, therefore the geometric mean of the measured concentrations were calculated to determine exposure concentration. The corresponding calculated concentration was 0.591 mg/L (based on total product). The test included six replicates and the control group with six replicates. 400 mL Petri dishes-covered glass beakers were filled with 160 mL testing solutions and twoLemna gibbacolonies with four, and one colony with three fronds were added to each vessel. Growth and morphological changes were assessed on days 3, 5 and 7. For determination of the test item concentrations, samples were taken from the test solution and control at the start and at the end of each renewal period. Test item concentration was determined using UV/VIS spectrophotometric method. For determination of the NOEC and LOEC values Independent Sample T-test was used (2-tailed, α = 0.05). All validity criteria were met and therefore the study can be considered as valid. The test item had no toxic effect at aquatic saturation (i.e. limit test concentration) onLemna gibba; the overall LOEC is higher than the nominal concentration of 100 mg/L (solubility level) of the test item in the test medium, which corresponds to the mean measured concentration of 0.591 mg/L, based on water accommodated fraction of the total product.