Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

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

Study period:

2017-04-28 to 2017-08-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006-07-11

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

EC Number:

304-519-6

EC Name:

Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Cas Number:

94276-33-2

Molecular formula:

C16 H12 Cr N4 O9 S . C11 H25 N O . H

IUPAC Name:

hydrogen 3-[(2-ethylhexyl)oxy]propan-1-amine 11-methyl-4-nitro-12-(phenylcarbamoyl)-6-sulfonato-8λ³-oxa-10λ³-oxa-1λ⁴,13-diaza-9-chromatricyclo[7.4.0.0²,⁷]trideca-1(13),2(7),3,5,10-pentaene-9,9,9-tris(ylium)-8,12-diid-9-olate

Test material form:

solid

Details on test material:

Physical state / Appearance: Solid / orange to brown

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, without exposure to light in a tightly sealed container.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: standard solution 500 µg/mL of the test substance in a mixture of acetone and deionized water (1:1, v/v). Working solutions containing 50, 25, 10, 5, 2.5, 1 and 0.5 µg/mL in deionized water.
- Sampling method: Each sample in a volume of 10 mL (i.e. control sample, test sample, sample fortified with the standard) was analyzed. If necessary the sample was diluted using de-ionized water.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
The test item is a complex mixture which in incompletely soluble in water. The composition of the soluble fraction changes with the loading. Thus the definitive test was performed using test item concentrations: filtrate of a loading of 100 mg/L, filtrate of a loading of 57 mg/L, filtrate of a loading of 32 mg/L, filtrate of a loading of 18 mg/L, filtrate of a loading of 10 mg/L, plus the control. The four highest test item concentrations were prepared by weighing appropriate amounts of the test item in glass flasks with 20X AAP and mixing with the 20X AAP medium [SOP/W/7]. The mixtures and the control were mechanically shaken for 48 hours (90 revolutions per minute, darkness, room temperature) and filtered through a conditioned nitrocellulose membrane filter (0.45 µm, Millipore) [SOP/W/37]. The preparation of the lowest test item concentration for exposure was not technically feasible in form of separately weighed loading rates. Therefore it was prepared in the following way. After one hour of mechanically shaking, a part of an additionally prepared dispersion of 2.2 mg/L (568 mL) was collected with a metrical cylinder and mixed with 432 mL of 20X AAP medium to obtain a required concentration of 1.25 mg/L. Care was taken, that the dispersion was as homogeneous as possible when the aliquot was removed. It was taken during mechanical shaking. The diluted dispersion was mechanically shaken for 48 hours (90 revolutions per minute, darkness, room temperature) and filtered through a conditioned nitrocellulose membrane filter (0.45 µm, Millipore), [SOP/W/37]. The conditioning was performed by pre-filtering a small volume of each test item concentration or the control to saturate the filter (and disposing it) before filtering the volumes for exposure. Each filtrate was visually homogeneous, transparent and colored directly after preparation and throughout exposure period between renewals.

- Controls: reference material, 3,5-dichlorophenol

Test organisms

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: swollen duckweed
- Strain: CPCC 310
- Source: cultivated in a standard laboratory culture at the Institute of Industrial Organic Chemistry, Branch Pszczyna, Department of Ecotoxicology, Laboratory of Aquatic Toxicology, according to the OECD Guideline No. 221 (2006) recommendations. The culture of Lemna gibba CPCC 310 on agar bevels was received from Canadian Phycological Culture Centre (CPCC), Department of Biology, University of Waterloo, Ontario, Canada.
- Method of cultivation: Duckweed Lemna gibba was transferred from agar bevels to the fresh 20X AAP medium in glass beakers with a capacity of 600 mL with transparent lids and incubated at room temperature with constant illumination (pre-culturing). The duckweed culture was inoculated to fresh medium once a week [SOP/W/66]. A pre-culture was started eight days before exposure. Only organisms in good physiological condition without any discoloration were used for the inoculation of the test item concentrations and the control.

ACCLIMATION
- Culturing media and conditions (same as test): The 20X AAP medium recommended by the OECD Guideline No. 221 (2006) was the culturing medium for test organism and the diluent/solvent for the test item. The 20X AAP medium was the source of nutrients for plant growth. The 20X AAP medium was prepared on the basis of deionised water [SOP/W/71] by adding stock solutions of reagent-grade chemicals [SOP/W/18, SOP/W/47]. The medium was sterilised by autoclaving [SOP/W/56] before addition of the stock solution of sodium hydrogen carbonate – the buffering ingredient. After 24 h of aeration the pH value of test medium was measured and adjusted with 0.1 M HCl, if necessary. The stock solutions were renewed on regular basis for Lemna gibba culturing and stored in a refrigerator. The alkalinity determined for 20X AAP medium was 289 mg/L as NaHCO3, the hardness was 284 mg/L as CaCO3 [SOP/W/48, SOP/W/82, SOP/W/92].

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Test temperature:

24.2 - 24.8 ºC

pH:

Beginn: 7.28 – 7.77
Termination: 7.85 – 8.82

Conductivity:

1616 µS/cm

Nominal and measured concentrations:

Nominal: 1.25, 2.5, 5.0, 10.0, 20.0 mg/L (loading)

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: no
- Test vessel: crystallizers
- Type: open
- Material, size, headspace, fill volume: glass, depth of 4 cm and a diameter of 7 cm containing 150 mL.
- Type of cover: Transparent lids
- Agitation: no
- Renewal rate of test solution (frequency): 24 h
- No. of colonies per vessel: 3
- No. of fronds per colony: 3
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: constant illumination
- Light intensity and quality: cool white light with a mean light intensity in the range of 8043 - 8200 lux.

EFFECT PARAMETERS MEASURED:
- Determination of frond number: manual counting on day 2, 4 and 7
- Determination of biomass: dry weight. All colonies (with roots) were transferred onto previously weighed microscopic slides and dried at approximately 60 ºC in a laboratory oven until constant weight.

- Other: frond size, shape and appearance (necrosis, chlorosis, gibbosity or bending of fronds), colony break-up or loss of buoyancy, root length and appearance.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

5.09 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

7 d

Dose descriptor:

EL20

Effect conc.:

2.14 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

7 d

Dose descriptor:

EL10

Effect conc.:

1.36 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Details on results:

- Any visual signs of phytotoxicity (abnormalities):
The morphology of plants was observed in each test item concentration and the control on days 2, 4 and at exposure termination. The morphological effects on treated plants were compared with the appearance of colonies in the control.
In the 1.25 mg/L dose group, no distinctive changes from the development of plants in the control were observed during the study period.
Day 2:
In the 2.5 mg/L dose group separating of root was observed. In the 5.0 mg/L dose group colony break up, separating of roots and spots of chlorosis were observed. In the 10 mg/L dose group, colony break up, separating of rootdose groups and chlorosis were observed. In the 20 mg/L dose group, colony break up, separating of roots and necrosis were observed.
Day 4:
In the 2.5 mg/L dose group, separating of roots and chlorosis were observed. In the 5.0 mg/L dose group, colony break up, separating of roots and chlorosis were observed. In the 10 mg/L and t20 mg/L dose group, colony break up, separating of roots and necrosis were observed.
Termination:
In the 2.5 mg/l and 5.0 mg/L dose group, colony break up, separating of roots and chlorosis were observed. In the 10 mg/L and 20 mg/L dose group, colony break up, separating of roots and necrosis were observed.

Reported statistics and error estimates:

The ELx values were calculated with a probit method. For the calculations and statistical analyses, the ToxRat Professional commercial software was used.
The statistical tests performed with data on growth rate based on frond number were: Shapiro-Wilk’s Test on Normal Distribution confirmed normal distribution of data, Levene’s Test on Variance Homogeneity showed that variances are homogeneous, Williams Multiple Sequential t-test Procedure showed a significant difference between all test item loading rates compared with the control.

Any other information on results incl. tables

In the definitive test, the following validity criteria specified in OECD Guideline No. 221 (2006) were met:

- The doubling time of frond number in the control was 1.9 days, criterion: less than 2.5 days (the factor of frond number in the control between 0 and 7 day was 12.9.

- The average specific growth rate in the control between day 0 and day 7 was 0.364 d-1 (minimum requirement: higher than 0.275 d-1).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test substance is acutely toxic for aquatic plants. However, based on the long-term (chronic) value, the test substance is very likely not chronicly harmful to aquatic organisms.