Reaction mass of Amines, C10-14-branched and linear alkyl, 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-branched and 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

Study period:

14 Feb 2017 to 27 Feb 2017

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)

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, 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-branched and 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:

943-145-3

IUPAC Name:

Reaction mass of Amines, C10-14-branched and linear alkyl, 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-branched and 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

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: The sample of the test item was stored at ambient temperature without exposure to light in a tightly sealed container.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

From each filtrate and the control, two samples were collected for chemical determinations (100 mL each). One was transferred to chemical analyses at exposure initiation, the others were stored under test conditions.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test item is a complex mixture. Every constituent contributes to a different degree to overall solubility, depending on its own individual solubility and its mass fraction in the test item. For this reason, the loading rates were prepared by using the water accommodated fraction (WAF) approach followed by filtration. Appropriate amounts of the test item were weighed separately in glass flasks with 20X AAP medium and mixed with appropriate volume of the 20X AAP medium. The mixtures of the loadings were visually heterogeneous (with undissolved particles) and colored. The mixtures and the control (20X AAP medium) were left for mechanical shaking for 48 hours (30ºC, 90 rpm, darkness). After 48 hours of mechanical shaking, the mixtures (visually heterogeneous) and the control were left at room temperature for a settling phase (no shaking). After 24 hours of settling, the mixtures were still visually heterogeneous. All mixtures and the control were filtrated, first through a conditioned paper filter (qualitative cellulose filter) and next through a conditioned nitrocellulose membrane filter (HAWG filter, 0.22 µm, Millipore). Each filtrate was visually homogeneous (without any visible undissolved particles.

Test organisms

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: Duckweed
- Strain: CPCC 310
- Source: 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: 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.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Hardness:

304.1 mg/L as CaCO3

Test temperature:

24.2 - 25.2 °C

pH:

- Fresh samples: 7.24 - 7.74
- Spent samples: 7.99 - 8.78

Nominal and measured concentrations:

- Nominal loading rates: 0 (control), 2.56, 6.4, 16, 40 and 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Incubation chamber: yes / thermostatic chamber (incubator with shaker, 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.
- 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: 304.9 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: 7713 - 7863 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: number of fronds, plant development (frond size, shape and apperance) and growth rate.
The total number of fronds in each test vessel was counted on days 3, 5 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. 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.

PRELIMINARY NON-GLP TEST
- Test item loading rates: 0 (control), 0.1 (diluted), 1 (diluted), 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: 42.1% and 53.6% growth rate inhibition at 10 and 100 mg/L, respectively. See 'Any other information on materials and methods incl. tables'.

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.:

74.77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 68.62 - 82.04 mg/L

Effect concentrations 2

Key result

Duration:

7 d

Dose descriptor:

EL10

Effect conc.:

2.23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 68.62 - 82.04 mg/L

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

8.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 7.91 - 8.75 mg/L

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EL20

Remarks:

EL10 not determined

Effect conc.:

1.73 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 1.56 - 1.90 mg/L

Details on results:

At exposure termination, in the filtrates of a loading of 2.56 mg/L, no distinctive changes from the development of plants in the control were observed. In the filtrate of a loading of 6.4 mg/L, shortened roots were observed. In the filtrates of a loading of 100, 40 and 16 mg/L, separated roots were observed. Inhibition of growth rate was 9.4% at 2.56 mg/L and increased in a dose-dependent manner up to 43% inhibition at 100 mg/L. In 'Any other information on results incl. tables' the results are tabulated and additional effect values are presented.

VALIDITY CRITERIA
In the definitive test, the following validity criteria specified in the OECD Guideline No. 221 (2006) were met:
- The doubling time of frond number in the control was 1.5 days, criterion: less than 2.5 days (the factor of frond number in the control between 0 and 7 day was 28.1).
- The average specific growth rate in the control between day 0 and day 7 was 0.476 d-1 (minimum requirement: higher than 0.275 d-1).

Results with reference substance (positive control):

EFFECT LEVELS BASED ON FROND NUMBER:
- 7-d ErC50: 9.94 mg/L (95% CL: 8.14 - 12.12 mg/L)
- 7-d ErC10: 4.71 mg/L (95% CL: 1.10 - 6.48 mg/L)
- 7-d EyC50: 6.60 mg/L (95% CL: 3.77 - 9.77 mg/L)
- 7-d EyC10: 3.05 mg/L (95% CL: 0.48 - 4.77 mg/L)
- Results with reference substance valid? Yes

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), Step-down Jonckheere -Terpstra test procedure, Williams multiple sequential and t-test procedure.

Any other information on results incl. tables

Table: Inhibition of growth rate and yield, definitive test

Filtrate of a loading of	Based on frond number		Based on dry weight
	% Inhibition at exposure termination (day 7) (growth rate)	% Inhibition at exposure termination (day 7) (yield)	% Inhibition at exposure termination (day 7) (growth rate)	% Inhibition at exposure termination (day 7) (yield)
Control	0	0	0	0
2.56 mg/L	7.1	22.2	9.4	31
6.4 mg/L	17.3	45.6	17.4	49.6
16 mg/L	33.2	69.4	28.8	67.7
40 mg/L	42.9	79	33.7	73.9
100 mg/L	51.1	84.9	43	82.3

Table: Frond number and dry weight

Filtrate of a loading of	Frond number			Dry weight [mg]
	day 3	day 5	day 7	day 7
Control mean	38.8	103.3	253.3	44.4
standard deviation	2.04	5.65	27.78	5.62
2.56 mg/L mean	31	79	199	30.9
standard deviation	3	3.61	9.17	0.97
6.4 mg/L mean	33.3	71	142	22.9
standard deviation	3.51	4.36	10.82	2.06
16 mg/L mean	29	54.3	83.7	15
standard deviation	3.61	5.69	6.66	2.7
40 mg/L mean	26.7	46	60.3	12.3
standard deviation	1.53	1.73	3.21	0.71
100 mg/L mean	25.7	39	46	8.7
standard deviation	0.58	2	2.65	0.47

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
	0-3 d	3-5 d	5-7 d	0-7 d
Control mean	0.487	0.489	0.446	0.476
standard deviation	0.0181	0.0166	0.0319	0.0166
2.56 mg/L mean	0.411	0.469	0.462	0.442
standard deviation	0.0324	0.0357	0.0098	0.0065
6.4 mg/L mean	0.435	0.379	0.346	0.394
standard deviation	0.035	0.0283	0.0092	0.0111
16 mg/L mean	0.388	0.315	0.217	0.318
standard deviation	0.0407	0.0607	0.0153	0.0116
40 mg/L mean	0.362	0.273	0.135	0.272
standard deviation	0.0193	0.0311	0.0407	0.0075
100 mg/L mean	0.349	0.209	0.082	0.233
standard deviation	0.0075	0.0167	0.0096	0.0083

EFFECT VALUES

7-d ErL20 based on frond number: 7.45 mg/L (95% CL: 6.69 - 8.23 mg/L)

7-d LOErLR based on frond number: < 2.56 mg/L

7-d NOErLR based on frond number: < 2.56 mg/L

7-d EyL20 based on frond number: 1.73 mg/L (95% CL: 1.56 - 1.90 mg/L)

7-d LOEyLR based on frond number: < 2.56 mg/L

7-d NOEyLR based on frond number: < 2.56 mg/L

7-d ErL50 based on dry weight: 169.56 mg/L (95% CL: 136.46 - 220.29 mg/L)

7-d ErL20 based on dry weight: 8.68 mg/L (95% CL: 7.12 – 10.27 mg/L)

7-d ErL10 based on dry weight: 1.83 mg/L (95% CL: 1.28 - 2.47 mg/L)

7-d LOErLR based on dry weight: < 2.56 mg/L

7-d NOErLR based on dry weight: < 2.56 mg/L

7-d EyL50 based on dry weight: 7.08 mg/L (95% CL: 6.53 - 7.65 mg/L)

7-d EyL20 based on dry weight: n.d.

7-d EyL10 based on dry weight: n.d.

7-d LOEyLR based on dry weight: < 2.56 mg/L

7-d NOEyLR based on dry weight: < 2.56 mg/L

n.d. - not determined

Table: Concentration and stability of TOC, definitive test

Day	Mean TOC concentration in filtrate [mg/L]
	Control	2.56 mg/L	6.4 mg/L	16 mg/L	40 mg/L	100 mg/L
Day 0 fresh	1.36	2.49	2.74	2.97	3.49	6
Day 1 old	1.02	2.41	2.92	3.07	3.51	4.96
Day 1 fresh	1.06	2.65	--	--	--	4.36
Day 2 old	< LoQ	3.06	--	--	--	4.85
Day 2 fresh	< LoQ	3.1	--	--	--	5.02
Day 3 old	1.22	2.21	--	--	--	4.46
Day 3 fresh	< LoQ	2.51	--	--	--	4.77
Day 4 old	< LoQ	2.83	--	--	--	6.95
Day 4 fresh	< LoQ	2.57	--	--	--	6.96
Day 5 old	< LoQ	2.76	--	--	--	4.92
Day 5 fresh	< LoQ	2.99	--	--	--	5.47
Day 6 old	< LoQ	2.82	--	--	--	6.57
Day 6 fresh	< LoQ	2.98	--	--	--	6.79
Day 7 old	1.25	2.61	--	--	--	6.17

LoQ = 1.0 mg C/L

The results were corrected by the value determined for the control sample in a series and presented as the concentrations of total

organic carbon (TOC)

TOC - total organic carbon

-- no value/not determined

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

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