Reaction mass of Amines, C10-14-branched and linear alkyl, [2,4-dihydro-4-[(2-hydroxy- 4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-); Amines, C10-14-branched and linearalkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-) (1:1) and Amines, C10-14-branchedand linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Amines, C10-14-branched and linear alkyl, [2,4-dihydro-4-[(2-hydroxy- 4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-); Amines, C10-14-branched and linearalkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-) (1:1) and Amines, C10-14-branchedand linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-)

EC Number:

943-144-8

IUPAC Name:

Reaction mass of Amines, C10-14-branched and linear alkyl, [2,4-dihydro-4-[(2-hydroxy- 4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-); Amines, C10-14-branched and linearalkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-) (1:1) and Amines, C10-14-branchedand linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-)

Test material form:

solid

Details on test material:

- Physical state / Appearance: Solid / red

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test substance is a poorly water soluble mixture; therefore a water accommodated fraction (WAF) was prepared following general guidance provided in OECD 23. The test concentrations 1-32 mg/L were prepared by directly adding test substance to test medium. The mixtures were stirred for about 2 days. Undissolved test substance was removed by centrifugation (approximately 20 min at about 17700 G). After centrifugation, undissolved test substance was observed in the test concentrations 1-32 mg/L and the Tyndall effect was positive. The centrifuged test solutions appeared slightly pink up to red. Then filtration with a membrane filter (Nalgene, pore width 0.2 µm) was done. The first 50-100 mL of filtered solution was discarded (used to condition the filter). After filtration, the Tyndall effect was negative. The highest test solution appeared slightly pink and clear. The lower concentrations were colorless and clear. The aqueous fraction of the test solution, after separation of the undissolved material, is considered the water saturated fraction (WSF) in test media. Due to the very low loading rates required, it was not possible to prepare each test solution separately. Therefore, the lowest test concentration was prepared by diluting the water saturated fraction (WSF) into test medium according to guidance in OECD 23.
Fresh test solutions were prepared for renewal on day 3 to maximize the exposure to all soluble components of the test substance.

Test organisms

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: Gibbous duckweed
- Strain: Clone G3
- Source (laboratory, culture collection): The stock culture was obtained from BASF SE, Crop Protection Ecology and Environmental Analytics, Speyerer Strasse 2, 67117 Limburgerhof, Germany.
- Age of inoculum (at test initiation): An inoculum culture of Lemna gibba (7-10 days old at 24 °C 2°) is used to initiate the test (study day 0).
- Method of cultivation: A culture is maintained continuously at the test facility. Before the exposure an inoculum culture is prepared with sufficient colonies of Lemna transferred aseptically into fresh sterile 20X AAP media. The inoculum culture is incubated under test conditions for 7 days prior to test initiation.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Test temperature:

23.5-24.6 °C ± 2

Nominal and measured concentrations:

0 (control), 0.32, 1, 3.2, 10 and 32 mg/L as loading rate based on test substance mass without correction for purity or composition

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes (Infors HT Multitron Pro controlled climate cabinet)
- Test vessel: Glass petri dishes covered with glass plates (nominal volume 300 mL) (colorless glass with a minimum water depth of 20 mm)
- Test volume: 160 mL
- Renewal rate of test solution (frequency/flow rate): In order to insure constant exposure conditions this study was conducted as a static-renewal exposure. The renewal was done at day 3.
- Initial colonies: 11 fronds/ test vessel at the start of exposure
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
The test medium (20X AAP medium) was prepared as described in OECD Guideline for Testing of Chemicals, No. 221 Lemna sp. Growth inhibition Test, Mar 2006, Annex 4.
Growth medium intended for testing was prepared at least 2 days before use to allow the pH to stabilize. The test medium (20X AAP medium) was prepared as described in OECD Guideline for Testing of Chemicals, No. 221 Lemna sp. Growth inhibition Test, Mar 2006, Annex 4.
Growth medium intended for testing was prepared at least 2 days before use to allow the pH to stabilize. The pH of growth medium was checked prior to use and readjusted if necessary by the addition of 0.1 or 1 M NaOH or HCl.
The test media was sterilized after preparation and prior to inoculation.

TEST INITIATION, MAINTAINANCE AND TOC MEASUREMENT
Colonies consisting of 2 to 4 visible fronds were transferred from the inoculum culture and randomly assigned to the test vessels under aseptic conditions. Each test vessel should contain a total of 9 to 12 fronds. The number of fronds and colonies were the same in each test vessel. The test vessels were impartially distributed in an incubator and continuous illumination. Vessel positions were altered after each assessment. The temperature was monitored within the incubator and in a separate vessel filled with deionized water. At the end of the test, the pH was measured in all replicates of the control and treatment groups; the pH at test initiation was measured in the respective bulk solutions.
The TOC values of all the test solutions were measured at the start and end of one renewal interval as an additional water quality parameter.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: The pH of growth medium was checked prior to use and readjusted if necessary by the addition of 0.1 or 1 M NaOH or HCl.
- Photoperiod: Continuous
- Light intensity and quality: 8559 lux (±15%) at a wave length of 400 - 700 nm.
- Illumination: Artificial light, type universal white (OSRAM L 25), permanent illumination. To minimize the potential effect of slight variations in illumination.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Frond number
The frond number in the test vessels were counted at the start of the test, after day 3, day 5 and after end of exposure the total number of fronds was counted in each replicate. Every frond visibly projecting beyond the edge of the parent frond was counted. Observations on the appearance of the fronds included necrosis, chlorosis, changes in plant size or shape and root growth were documented. The TOC values of all the test solutions were measured at the start and end of one renewal interval as an additional water quality parameter.
Dry weight
The biomass based on dry weight was determined at the start of exposure from a sample of the inoculum culture representative of what was used to begin the test, and at the end of the test with the plant material from each treatment and control vessel. All colonies were collected from each of the test vessels and rinsed with deionized water. They were blotted to remove excess water and then dried at 60°C for 5 days to a constant weight. Any root fragments were included.

RANGE-FINDING STUDY
According to the test guidelines, at least 5 concentrations in a geometric series with a separation factor of ≤3.2 should be used.
The test concentrations were selected on the basis of range finding tests (experimental conduct in accordance with GLP but without a GLP Status). The results of the 7 day range finding tests can be found below (as nominal concentrations based on frond number).
The raw data of the range finding test are archived together with the raw data of this study.

Reference substance (positive control):

yes

Remarks:

3,5 Dichlorphenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

EL10

Effect conc.:

1.42 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Effect concentrations 2

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

> 32 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Remarks on result:

other: EL50 (determined mathematically) = 43.9 mg/L

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EL10

Effect conc.:

1.94 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

> 32 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Remarks on result:

other: EL50 (determined mathematically) = 49.4 mg/L

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EL10

Effect conc.:

0.236 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

biomass

Effect concentrations 6

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

4.97 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

biomass

Effect concentrations 7

Duration:

7 d

Dose descriptor:

EL10

Effect conc.:

0.362 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

biomass

Effect concentrations 8

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

5.07 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

biomass

Details on results:

The duckweed population in the control vessels showed exponential growth, increasing from 11 fronds per vessel to an average of 430.0 fronds per vessel in the control after 7 days (corresponding to an average specific growth rate of 0.523 d-1). The dry weight increased from 1.8 mg (sample of the inoculum culture at start of exposure) to an average of 42.6 mg per vessel in the control at test termination.

VALIDITY CRITERIA
This test was fully compliant with all the following validity criteria required by the corresponding test guidelines and is considered valid.
• The doubling time of frond number in the control was 1.3 days (<2.5 days).

Results with reference substance (positive control):

In order to verify that the Lemna cultures are responding normally to toxic stress, tests with a reference substance (3,5 Dichlorphenol) are conducted. Reference substance tests are conducted according to OECD 221 guidelines and in accordance with GLP, but without a GLP status. The results from the reference substance test are compared to 3,5 Dichlorphenol EC50 values published in OECD Guideline, which represent the typical response range for the Lemna species tested.

The EC50(7d) of the reference substance 3,5 Dichlorphenol was 10.3 mg/L (experiment date: 03 Apr 2017, project number: 63E0308/02E014).
This result is within the range of 5.9 – 13.3 mg/L and indicates that the culture of Lemna gibbqa used in this study is responding normally to toxic stress.

Any other information on results incl. tables

Table 3: Lemna morphology and visual signs of phytotoxicity

Following signs of phytotoxicity were observed in the treatment groups on day 3, 5 and 7.

 

Loading rate [mg/L]	Vessel No.	Morphological changes, signs of phytotoxicity
		Day 3		Day 5	Day 7
0 (control)	1 - 6	0		0	0
0.32	1 - 3	0		0	0
1	1 - 3	0		3 (+)	3 (+)
3.2	1 - 3	4, 6		4, 6, 9 (a)	4, 6, 9(a)
10	1 - 3	4, 6		4, 6, 9 (a, b)	4, 6, 9(a, b)
32	1 - 3	5, 6, 8		3, 5, 6, 8, 9 (a, b)	1 (+++), 3, 5, 6, 8, 9 (a, b)
Explanation of abbreviations: 0:            No findings 1:            frond or root yellowing (chlorosis) 2:            fronds humped or swollen (gibbosity) 3:            small fronds 4:            shortened roots 5:            no roots			6:            plants fragmented (fronds and/or roots) 7:            dead white plant tissue (necrosis) 8:            plants colored by test substanz (fronds and/or roots) 9:            others (a: inelastic; b: without structure) (+):          slight effects (++):       medium effects (+++):     strong effects

Table 4: Physical and chemical characteristics of the test medium used.

 

Date of preparation	pH value	pH value after adjustment	Date of measurement
28 Aug 2017	8.3	7.5	28 Aug 2017
01 Sep 2017	8.3	7.4	01 Sep 2017

 

Table 5: pH values

pH values in the test solutions after 0 and 3 days – 1st Interval

 

Loading rate [mg/L]	pH value after 0 days^a	pH value after 3 days
		Replicate 1	Replicate 2	Replicate 3	Replicate 4	Replicate 5	Replicate 6
0 (control)	7.7	8.7	8.7	8.7	8.6	8.7	8.7
0.32	7.8	8.6	8.6	8.6
1	7.9	8.7	8.7	8.6
3.2	7.9	8.6	8.6	8.6
10	7.9	8.6	8.7	8.6
32	7.9	8.6	8.6	8.6

^awithout Lemna

 

Table 6: pH values in the test solutions after 3 and 7 days – 2nd Interval

 

Loading rate [mg/L]	pH value after 3 days^a	pH value after 7 days
		Replicate 1	Replicate 2	Replicate 3	Replicate 4	Replicate 5	Replicate 6
0 (control)	7.7	9.2	9.0	9.0	9.0	9.0	9.0
0.32	7.8	9.1	9.0	9.1
1	7.9	9.2	9.0	8.9
3.2	7.9	8.7	8.8	8.8
10	7.9	8.6	8.6	8.7
32	7.9	8.6	8.6	8.6

^awithout Lemna

 

Table 7: TOC values

TOC values were measured over one renewal interval as an additional water quality measure.

 

Nominal concentration / Loading rate [mg/L]	day 0 (fresh solution)	day 3 (aged solution)
0 (control)	2.2	2.4
0.32	2.2	2.3
1	2.2	2.5
3.2	2.1	2.4
10	2.3	2.3
32	2.3	2.4

 

Table 8: Dissolution behavior

The following observations were recorded for the dissolution behavior of the test substance in the test water:

 

Nominal concentration [mg/L]	day 0	day 3		day 5	day 7
	Fresh test solution	Old test solution	Fresh test solution	Old test solution	Old test solution
0 (control)	0	0	0	0	0
0.1	0	0	0	0	0
0.32	0	0	0	0	0
1	0	0	0	0	0
3.2	0	0	0	0	0
10	1	1, 2	1	1, 2	1, 2
Explanation of abbreviations: 0:          no remarkable observations, clear test medium 1:          coloration caused by the test substance (slightly pink) 2:           precipitation from the test medium at the bottom of the test vessel (crumbs)

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test substance is acutely harmful for aquatic plants.