Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-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 sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

EC Number:

915-758-6

Molecular formula:

C32H18CrN6O8.Na / C32H22CrN10O8.Na / C32H20CrN8O8.Na

IUPAC Name:

Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

Test material form:

solid

Details on test material:

Appearance:
- physical state: solid
- color: dark brown

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test substance was stored at ambient temperature.

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Concentration control analysis was not performed because a sufficiently sensitive method for analyses in the required concentration range was not available. However all reasonable efforts were taken to produce a saturated solution of all soluble components of the test substance in test media. Since the test substance is a multi-constituent material, the test solution is considered a water accommodated fraction (WAF). The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration. According to OECD 23, for tests with chemicals that can not be quantified by analytical methods at the concentrations causing effects, the effect concentration can be expressed based loading rate (for mixtures).

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test substance is poorly water soluble and a complex mixture. Therefore, the test solutions were prepared separately for each test concentration following general guidance provided in OECD 23. Each test solution was prepared by directly adding the required mass of test substance to test medium and stirring for approximately 2 days.
After centrifugation, undissolved test substance was still observed and the Tyndall effect was positive. Then filtration with a membrane filter (pore width 0.2 µm) was done. The first 50-100 mL of filtered solution was discarded (used to condition the filter). The aqueous fraction of the test solution, after separation of the undissolved material, is considered the water saturated fraction (WSF) in test media. In the filtered solution the Tyndall effect was negative.
The control was visibly colorless and clear. The test solution 1-32 mg/L were visibly colorless up to beige, proportional to the test concentrations. The Tyndall effect in this test solutions were negative.
The highest test solution appeared yellow-brown and the Tyndall effect was positive despite previous filtration.

The exposure was started after separation of the undissolved material. According to OECD 23, test solutions of poorly soluble substances separated from the remaining undissolved fraction were considered equivalent to the nominal or loading concentration used to prepare them.
Fresh test solutions were prepared on day 0 for renewal on day 2.
The control solutions were treated in the same way.

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.
- 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 9 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:

24 ±2°C

pH:

7.7 - 9.2

Nominal and measured concentrations:

0 (control), 1, 3.2, 10, 32, 100 mg/L as loading rate

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes (Vötsch Industrietechnik GmbH Bioline (VB1014) controlled climate cabinet. 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)
- Aeration:
- Agitation: yes/no
- 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 2.
- No. of colonies per vessel: 11 fronds/ test vessel at the start of exposure
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 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 the three highest test solutions were measured at the start and end of one renewal interval as an additional water quality parameter.


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic fresh water (20X AAP media)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: 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 HC
- Photoperiod: Continuous
- Light intensity and quality: 8284 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 2, 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.
- 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 6 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 a range finding test (experimental conduct in accordance with GLP but without a GLP Status). The results of the 7 days range finding test were (as nominal concentrations):
ErC50 = between 10 and 100 mg/L
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 (as nominal concentrations based on frond number) can be found in table 2

Reference substance (positive control):

yes

Remarks:

3,5 Dichlorphenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Effect concentrations 2

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

12.4 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

27.8 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

biomass

Effect concentrations 6

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

3.59 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

biomass

Effect concentrations 7

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

38.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

biomass

Effect concentrations 8

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

2.97 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

biomass

Details on results:

No additional adverse effects or abnormal behavior were observed in any of the test groups.
The duckweed population in the control vessels showed exponential growth, increasing from 11 fronds per vessel to an average of 331.0 fronds per vessel in the control after 7 days (corresponding to an average specific growth rate of 0.486 d-1). The dry weight increased from 1.5 mg (sample of the inoculum culture at start of exposure) to an average of 36.8 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 1.4 days (<2.5 days).

Any other information on results incl. tables

Table 3: Lemna morphology and visual signs of phytotoxicity

Loading rate [mg/L]	Vessel No.	Morphological changes, signs of phytotoxicity
		Day 3		Day 5	Day 7
0 (control)	1 - 6	0		0	0
1	7 - 9	0		0	0
3.2	10 - 12	0		0	3
10	13 - 15	0		8	8
32	16 - 18	8		3, 8	3, 8
100	19 - 21	8		3, 8	3, 8
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 (+):          slight effects (++):       medium effects (+++):     strong effects

Table 4: TOCvalues

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

 

Loading rate [mg/L]		day 0 (fresh solution)		day 3 (aged solution)
0 (control)		2.4		2.3
10		2.5		2.3
32		3.1		2.7
100		4.8		4.2

 

The TOC in the treatment groups were higher than in the control test medium. The higher TOC is likely due to soluble organic components from the test substance. The TOC value confirms qualitatively that the daphnids in the WAF were exposed to soluble organic components from the test substance.

 

Table 5: Dissolution behavior

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

Loading rate [mg/L]	day 0	day 2		day 5	day 7
		Old test solution	Fresh test solution
0 (control)	0	0	0	0	0
1	0	0	0	0	0
3.2	0	0	0	0	0
10	1	1	1	1	1
32	1	1	1	1	1, 2
100	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 (yellow-brown) 2:           dark precipitation, undissolved test substance at the bottom of the test vessel

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

the test substance is with high probability not acutely harmful to aquatic plants.