Reaction mass of Chromate(1-), [N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthalenyl]acetamidato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-, hydrogen, compd. with N-cyclohexylcyclohexanamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1) and hydrogen bis[N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthyl]acetamidato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1)

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:

24 Mar 2017 to 05 Apr 2017

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:

yes

Remarks:

see "Principles of method if other than guideline"

Principles of method if other than guideline:

In the experimental part of the study, a deviation from the OECD Test Guideline No. 221 (2006) ‘Lemna sp., growth inhibition test’, the study plan and the SOP/W/47 concerning the temperature occurred. The temperature recorded during exposure in the definitive test was in the range of 24.6 – 26.4 °C and hence the temperature fluctuations was 1.8°C. However, for the last 3 hours of exposure the temperature was higher than 26°C by 0.4°C, what did not impact the generated results.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

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

Analytical monitoring:

yes

Details on sampling:

At exposure initiation, three abiotic samples (i.e. samples taken from separate test vessels which did not contain any fish) of each treatment were collected. One of them was transferred for chemical analyses at exposure initiation, whereas the second one was stored under test conditions for 24 h (time between renewals) and the third one was stored under test conditions till exposure termination and transferred for chemical analyses.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The separate portions of the test item were weighed directly into test medium in a glass flask. Every portion was quantitatively transferred into the respective glass vessel by multiple washing with test medium and filling up to the respective total volume. The resulting mixtures and the control (i.e. test medium without test item) was incubated at temperature approx. 30 °C for 48 h with continuous mechanical shaking at 90 rpm in darkness. Next, every mixture and the control was kept at room temperature for another 24 h without shaking and with continuous aeration. Every mixture and the control were separately filtered through a pre-conditioned cellulose filter and next through a preconditioned nitrocellulose filter of 0.22 µm pores (Millipore 0.22 µm GSTF, Merck). The filtrates were collected and used for exposure. Each filtrate was visually homogeneous and without any visibly non-dissolved particles. Glass test vessels were crystallisers pre-conditioned by filling up with the respective filtrate, discarding the contents and refilling to be used for exposure. Each filtrate was visually homogeneous and without any visibly non-dissolved particles.

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: Duckweed
- Strain: CPCC 310
- Source: Canadian Phycological Culture Centre (CPCC), Department of Biology, University of Waterloo, Ontario, Canada.
- Method of cultivation: 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.

CULTURING
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. A pre-culture was started nine days before exposure. Only organisms in good physiological condition without any discolouration were used for the inoculation of the test item concentrations and the control.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

304.1 mg/L (as CaCO3)

Test temperature:

24.6 - 26.4 °C. However, for the last 3 hours of exposure the temperature was higher than 26°C by 0.4°C (see 'Deviations' under 'Any other information on materials and methods incl. tables').

pH:

- pH measured in fresh samples: 7.40 - 7.93
- pH measured in spent samples: 8.43 - 8.96

Nominal and measured concentrations:

- Nominal loading rates: 0 (control), 1.76, 3.88, 8.54, 18.8, 41.3, 90.9, 200 mg/L.

Details on test conditions:

TEST SYSTEM
- Incubation chamber: Yes / thermostatic chamber (incubator, ILW400STD Pol-Eko-Aparatura, Poland)
- Test vessel: Glass crystallisers with a depth of 4 cm and a diameter of 7 cm were used.
- Type: Closed; transparent lids were used to minimise evaporation and accidental contamination, allowing necessary air exchange.
- Pre-conditioning: test vessels were pre-conditioned by filling up with the respective filtrate, discarding the contents and refilling to be used for exposure.
- Fill volume: 150 mL
- Renewal rate of test solution: Daily
- No. of colonies per vessel: 3
- No. of fronds per colony: 3
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: 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.

TEST MEDIUM / WATER PARAMETERS
- Alkalinity: 304.1 mg/L as CaCO3 in the culture medium
- Culture medium different from test medium: No
- Intervals of water quality measurement: Daily, before and after renewals

OTHER TEST CONDITIONS
- Photoperiod: Constant illumination
- Light intensity and quality: 7210 - 8010 lux. The light intensity was measured at exposure initiation, twice during exposure and at exposure termination with a 2π receptor lux-meter (Sonopan L-100, Poland).
- Light type: Fluorescent

EFFECT PARAMETERS MEASURED: frond number, plant development (frond size, shape and appearance) and growth rate.
In order to quantify the test item related effects on vegetative growth over a period of 7 days, the number of fronds in each replicate was counted. In the definitive test the total number of fronds in each test vessel was counted on days 2, 4 and at exposure termination. Only visibly distinct fronds were counted. At the same time, observations of plant development were performed: frond size, shape and appearance (necrosis, chlorosis, gibbosity or bending of fronds), colony breakup or loss of buoyancy, root length and appearance. Growth of plant cultures in the test item loading rates was compared with that of the control. The dry weight of the representative sample of the duckweed culture used as the inoculum was measured after exposure initiation. The dry weight of all plants from each test vessel was measured after exposure termination. All colonies (with roots) were transferred onto previously weighed microscopic slides and dried at approximately 60°C in a laboratory oven until constant weight.

RANGE-FINDING STUDY
- Test item loading rates: 0 (control), 5, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: At exposure termination, the growth rate inhibition based on the frond number was 13.2% in the filtrate of a loading of 5 mg/L, 9.6% in the filtrate of a loading of 10 mg/L and 20.1% in the filtrate of a loading of 100 mg/L in comparison to the control. See 'Any other information on materials and methods incl. tables' for an overview of the results of the range-finding study.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

EL10

Effect conc.:

45.2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 37.3 - 55.1 mg/L

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

188.5 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 159.0 - 240.2 mg/L

Duration:

7 d

Dose descriptor:

EL10

Effect conc.:

45.7 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 28.3 - 60.3 mg/L

Details on results:

At exposure termination, in the test item loading rates no distinctive changes from the development of plants in the control were observed. In the filtrates with a loading of up to 41.3 mg/L, no inhibition of growth rate based on frond number was observed. At the filtrate of a loading of 90.9 mg/L, inhibition of growth rate based on frond number was 15.5%, which increased up to 24.6% inhibition at a loading of 200 mg/L. In 'Any other information on results incl. tables' the results are tabulated and additional effect values are presented.

Results with reference substance (positive control):

The 7-d ErC50 and 7-d ErC10 based on frond number of the reference substance were determined to be 13.91 and 10.61 mg/L, respectively. The 7-d EyC50 and 7-d EyC10 based on frond number of the reference substance were determined to be 13.05 and 9.51 mg/L, respectively.

Reported statistics and error estimates:

Probit method calculations and analysis by Shapiro-Wilk’s Test on Normal Distribution, Levene’s Test on Variance Homogeneity (with Residuals), Williams multiple sequential t-test procedure.

Table: Inhibition of growth rate and yield at exposure termination (day 7) - definitive test

Treatment	Based on frond number		Based on dry weight
	% Inhibition of growth rate	% Inhibition of yield	% Inhibition of growth rate	% Inhibition of yield
Filtrate of the control	0	0	0	0
Filtrate of a loading of 1.76 mg/L	-2.9	-0.8	-4.4	-14.9
Filtrate of a loading of 3.88 mg/L	-4.3	-11.8	-5.9	-21.1
Filtrate of a loading of 8.54 mg/L	-1.5	-4.2	-5.2	-17.4
Filtrate of a loading of 18.8 mg/L	-3.5	-9.5	-5.5	-19.1
Filtrate of a loading of 41.3 mg/L	-1.6	-4.2	-1.8	-6
Filtrate of a loading of 90.9 mg/L	15.5	33.8	3.2	9.8
Filtrate of a loading of 200 mg/L	24.6	48.7	11.2	30.3

Table: Frond number and dry weight

Filtrate of a loading of	Frond number			Dry weight [mg]
	day 2	day 4	day 7	day 7
Control mean	25.0	54.2	96.7	35.6
standard deviation	1.10	3.66	3.78	3.75
1.76 mg/L mean	21.7	50.7	103.7	40.6
standard deviation	1.15	2.52	8.50	4.07
3.88 mg/L mean	23.3	51.0	107.0	42.7
standard deviation	1.15	4.36	4.36	6.60
8.54 mg/L mean	22.0	49.0	100.3	41.5
standard deviation	0.00	2.00	6.81	1.17
18.8 mg/L mean	23.3	51.0	105.0	42.0
standard deviation	0.58	4.36	6.24	4.04
41.1 mg/L mean	22.7	49.0	100.3	37.6
standard deviation	1.15	5.29	4.73	4.52
90.9 mg/L mean	22.0	43.3	67.0	32.2
standard deviation	1.00	1.53	4.36	3.71
200 mg/L mean	21.3	43.7	54.0	25.3
standard deviation	0.58	1.53	4.00	2.98

 

Table: Section-by-section growth rate and growth rate, based on frond number

Filtrate of a loading of	Section-by-section growth rate*			Growth rate
	0-2 d	2-4 d	4-7 d	0-7 d
Control mean	0.510	0.386	0.193	0.339
standard deviation	0.022	0.025	0.018	0.006
1.76 mg/L mean	0.439	0.425	0.238	0.349
standard deviation	0.026	0.050	0.041	0.012
3.88 mg/L mean	0.476	0.390	0.248	0.354
standard deviation	0.025	0.040	0.024	0.006
8.54 mg/L mean	0.447	0.400	0.239	0.344
standard deviation	0.000	0.020	0.036	0.010
18.8 mg/L mean	0.476	0.390	0.241	0.351
standard deviation	0.012	0.030	0.023	0.009
41.3 mg/L mean	0.461	0.384	0.240	0.344
standard deviation	0.025	0.029	0.051	0.007
90.9 mg/L mean	0.447	0.339	0.145	0.287
standard deviation	0.023	0.023	0.014	0.010
200 mg/L mean	0.431	0.358	0.070	0.256
standard deviation	0.013	0.012	0.013	0.011

* growth rate = [ln (frond number on day 2) - ln (frond number on day 0)]/2 days.

EFFECT VALUES

7-d ErL20 based on frond number: 206.4 mg/L (95% CL: 157.3 - 287.4 mg/L)

7-d LOErLR based on frond number: 90.9 mg/L

7-d NOErLR based on frond number: 41.3 mg/L

7-d EyL20 based on frond number: 74.3 mg/L (95% CL: 55.0 - 90.1 mg/L)

7-d LOEyLR based on frond number: 90.9 mg/L

7-d NOEyLR based on frond number: 41.3 mg/L

7-d ErL50 based on dry weight: > 200 mg/L

7-d ErL20 based on dry weight: > 200 mg/L

7-d ErL10 based on dry weight: > 200 mg/L

7-d LOErLR based on dry weight: 200 mg/L

7-d NOErLR based on dry weight: 90.9 mg/L

7-d EyL50 based on dry weight: > 200 mg/L

7-d EyL20 based on dry weight: 160.9 mg/L

7-d EyL10 based on dry weight: 40.3 mg/L (95% CI: 15.2 – 120.3 mg/L)

7-d LOEyLR based on dry weight: 200 mg/L

7-d NOEyLR based on dry weight: 90.9 mg/L

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on materials and methods incl. tables'

Conclusions:

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

Description of key information

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

Additional information

The toxicity to aquatic plants other than algae was determined in a study according to OECD TG 221 and in compliance with GLP criteria (IPO, 2017). In this study 3 replicates of 3 uniform, healthy-looking duckweed (L. gibba), with 3 fronds each, were introduced in test vessels containing filtrates of the test substance with the following loading rates: 0 (control), 1.76, 3.88, 8.54, 18.8, 41.3, 90.9 and 200 mg/L. As plants were exposed to a nominal loading rate of the test substance, effect concentrations are expressed as nominal. Plants were exposed for 7 days under semi-static conditions. Analytical quantification of the test substance was performed by measuring the total organic carbon (TOC) using a validated method. In order to quantify the test item related effects on vegetative growth over a period of 7 days, the number of fronds in each replicate was counted. In the definitive test the total number of fronds in each test vessel was counted on days 2, 4 and at exposure termination. At the same time, observations of plant development were performed: frond size, shape and appearance (necrosis, chlorosis, gibbosity or bending of fronds), colony breakup or loss of buoyancy, root length and appearance. Growth of plant cultures in the test item loading rates was compared with that of the control. At exposure termination, the inhibition of growth rate and the inhibition of yield in comparison to the control based on frond number and based on dry weight are estimated. The inhibition of growth rate based on frond number was between -4.3% and up to 24.6%. The inhibition of yield based on frond number was between -11.8% and up to 48.7%. The inhibition of growth rate based on dry weight was between -5.9% and up to 11.2%. The inhibition of yield based on dry weight was between -21.1% and up to 30.3% in comparison to the control. At exposure termination, in the test item loading rates no distinctive changes from the development of plants in the control were observed. The 7-d ErL50-values for inhibition of fronds and dry weight were both > 200 mg/L. The 7-d ErL10-values for inhibition of fronds and dry weight were 45.2 mg/L (95% CL: 37.3 - 55.1 mg/L) and > 200 mg/L, respectively. The 7-d EyL50-values for inhibition of fronds and dry weight were 188.5 mg/L (95% CL: 159 - 240.2 mg/L) and > 200 mg/L, respectively. The 7-d EyL10-values for inhibition of fronds and dry weight were 45.7 mg/L (95% CL: 28.3 - 60.3 mg/L) and 40.3 mg/L (95% CL: 15.2 - 120.3 mg/L), respectively.