Dipotassium dihydrogen 4-[5-[3-carboxylato-5-hydroxy-1-(4-sulphonatophenyl)-1H-pyrazol-4-yl]penta-2,4-dienylidene]-4,5-dihydro-5-oxo-1-(4-sulphonatophenyl)-1H-pyrazole-3-carboxylate

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:

10 - 19 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 761/2009, Annex, Part C, C.26: "Lemna sp. Growth Inhibition Test"

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Dipotassium dihydrogen 4-[5-[3-carboxylato-5-hydroxy-1-(4-sulphonatophenyl)-1H-pyrazol-4-yl]penta-2,4-dienylidene]-4,5-dihydro-5-oxo-1-(4-sulphonatophenyl)-1H-pyrazole-3-carboxylate

EC Number:

257-479-6

EC Name:

Dipotassium dihydrogen 4-[5-[3-carboxylato-5-hydroxy-1-(4-sulphonatophenyl)-1H-pyrazol-4-yl]penta-2,4-dienylidene]-4,5-dihydro-5-oxo-1-(4-sulphonatophenyl)-1H-pyrazole-3-carboxylate

Cas Number:

51858-17-4

Molecular formula:

C25H16N4O12S2.2K

IUPAC Name:

dipotassium dihydrogen 4-{5-[3-carboxylato-5-oxo-1-(4-sulfonatophenyl)-1,5-dihydro-4H-pyrazol-4-ylidene]penta-1,3-dien-1-yl}-5-hydroxy-1-(4-sulfonatophenyl)-1H-pyrazole-3-carboxylate

Test material form:

solid: crystalline

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test item concentrations and the control
- Sampling method: Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at test start, at each test medium renewal (day 2 and day 5). Samples from aged test solution and control were sampled at each test medium renewal and at the end of the test.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the highest test concentration of nominal 100 mg test item/L was prepared at test start and the two water renewal days by dissolving 112.2, 98.1 and 105.7 mg test item into 1122, 981 and 1057 mL test water, respectively by intense stirring for 20 to 40 min. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
- Controls: Yes (test medium only)
- Observations: The test medium showed blue coloration in all concentrations.

Test organisms

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: Duckweed
- Strain: G 3
- Source: In-house laboratory culture
- Pre-culture: For at least 7 days under test conditions with weekly media exchange.
- Method of cultivation: Under standardised conditions according to the test guidelines.

ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test.
- Any deformed or abnormal cells observed: No deformation was observed.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Test temperature:

23.4 - 24.3 °C

pH:

7.4 - 8.8 (Control)
7.5 - 8.7 (Test item concentration)

Nominal and measured concentrations:

Control, 1.0, 3.2, 10, 32 and 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: Yes, controlled environment room
Test vessel:
- Type: Open (covered with glass dishes)
- Material, size, headspace, fill volume: 250 mL glass vessels with approximately 200 mL test medium; 50 mL headspace
- Type of cover: Glass dishes
- Aeration: No aeration
- Agitation: No but the test vessels were randomized daily during the exposure period.
- Control end cells density: 255 fronds at test end (mean growth rate 0.437)
- No. of colonies per vessel: 12 fronds per vessel
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: Yes (20 x APP medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 20 x AAP medium
- Conductivity: < 5 μS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH-values were measured in all freshly prepared and aged test media; the temperature was measured daily in a test vessel filled with water and incubated under the same conditions as the test vessels.

OTHER TEST CONDITIONS
- Adjustment of pH: Adjusted with 1 M NaOH to 7.4
- Photoperiod: Continuous illumination
- Light intensity and quality: 6917 lux (mean value) with a range of 6590 to 7270 lux.

EFFECT PARAMETERS MEASURED:
- Determination of frond number: At test start frond and colony numbers were recorded. At days 2, 5 and 7 frond numbers were counted and appearance of colonies was observed.
- Determination of biomass: At test start the dry weight of a sample of fronds similar to those used to inoculate the test vessels was determined. At the end of the test the dry weight of all plants from each vessel was determined. Accordingly, the plants were collected at the end of the test.

RANGE-FINDING STUDY: non GLP pre-test
- Results used to determine the conditions for the definitive study: Based on th results of a non-GLP pre-experiment the test item concentration range of the main test were determined.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (tested at least twice a year)

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:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidential limits

Remarks:

n.d.

Effect concentrations 2

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

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:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidential limits

Remarks:

n.d.

Effect concentrations 4

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Any visual signs of phytotoxicity (abnormalities): The shape of fronds and colonies after the test period of 7 days was not different to those in the control up to and including the nominal test concentration of 10 mg test item/L. At the higher test item concentrations of 32 and 100 mg test item/L the fronds showed more gibbous growth than the control replicates after 7 days.

Any other information on results incl. tables

Table 1: Summary of Analytical Results

Sample Description [mg test item/L]	% of nominal 1)	RSD [%]	n
Control	n.a.	n.a.	12
1	98	8	12
3.2	103	3	12
10	104	1	12
32	100	2	12
100	105	2	12

1): mean value of all measured samples per treatment group RSD: relative standard deviation per treatment group n: number of analysed samples n.a.: not applicable

Table 2: Biological Results: Frond Numbers of Lemna gibba during the Test Period of 7 days

Nominal Concentration	Flask	Frond number after
[mg test item/L]	No	2 days	5 days	7days
Control	1	32	130	248
	2	29	112	259
	3	29	121	258
	M	30.0	121.0	255.0
	s	1.73	9.00	6.08
1.0	1	31	129	277
	2	29	128	282
	3	31	120	271
	m	30.3	125.7	276.7
	s	1.15	4.93	5.51
3.2	1	31	118	247
	2	32	103	245
	3	29	125	250
	m	30.7	115.3	247.3
	s	1.53	11.24	2.52
10	1	29	103	210
	2	30	93	193
	3	29	86	153
	M	29.3	94.0	185.3
	S	0.58	8.54	29.26
32	1	27	83	148
	2	27	80	141
	3	28	87	155
	M	27.3	83.3	148.0
	S	0.58	3.51	7.00
100	1	25	85	138
	2	20	60	108
	3	29	97	191
	M	24.7	80.7	145.7
	S	4.51	18.88	42.03

m: mean value; s: standard deviation starting number of fronds: 12 fronds per test vessel

Table 3: Dry Weights, Growth Rates μ (Based on Dry Weights) and Yields y (Based on Dry Weights) of Lemna gibba after 7 days of Exposure

Nominal Concentration   [mg test item/L]	Flask     No.	Dry weights (after 7 days)   [mg]	Yields y (dry weight) (after 7 days)  [mg]	Growth rates μ (dry weight) 0 - 7 days [1/day]
Control	1	31.7	29.9	0.410
	2	33.2	31.4	0.416
	3	34.9	33.1	0.424
	M	33.3	31.5	0.417
	S	1.60	1.6	0.007
1.0	1	35.1	33.3	0.424
	2	37.3	35.5	0.433
	3	33.4	31.6	0.417
	M	35.3	33.5	0.425
	S	1.96	2.0	0.008
3.2	1	32.0	30.2	0.411
	2	29.5	27.7	0.400
	3	33.5	31.7	0.418
	M	31.7	29.9	0.409
	S	2.02	2.0	0.009
10	1	25.8	24.0	0.380
	2	23.2	21.4	0.365
	3	22.3	20.5	0.360
	M	23.8	22.0	0.368
	S	1.82	1.8	0.011
32	1	20.2	18.4	0.345
	2	19.2	17.4	0.338
	3	20.9	19.1	0.350
	M	20.1	18.3	0.345
	S	0.85	0.9	0.006
100	1	18.5	16.7	0.333
	2	13.9	12.1	0.292
	3	23.8	22.0	0.369
	M	18.7	16.9	0.331
	S	4.95	5.0	0.038

m: mean value; s: standard deviation

initial dry weight at test start: 1.8 mg dry weight per test vessel

Table 4: Biological Results: Yields y (Based on Frond Numbers) and Percentage Inhibition of y (Based on Frond Numbers) during the Test Period of 7 days

Nominal Concentration	Yields y (frond number) and % inhibition of y
	0-2 days		0-5 days		0-7 days
[mg test item/L]	Y	%	Y	%	Y	%
Control	18.0	-	109.0	-	243.0	-
1.0	18.3	-1.9	113.7	-4.3	264.7	-8.9
3.2	18.7	-3.7	103.3	5.2	235.3	3.2
10	17.3	3.7	82.0	24.8*	173.3	28.7*
32	15.3	14.8	71.3	34.6*	136.0	44.0*
100	12.7	29.6*	68.7	37.0*	133.7	45.0*

- % inhibition: increase in growth relative to that of the control * mean value significantly different from the control (tested with Williams t-test, α = 0.05, one-sided)

 

Table 5: Biological Results: Growth Rates μ (Based on Frond Numbers) and Percentage Inhibition of μ (Based on Frond Numbers) during the Test Period of 7 days

Nominal Concentration	Growth rates μ (frond number) [1/day] and % inhibition of μ
[mg test item/L]	0-2 days		0-5 days		0-7 days
	µ	%	µ	%	µ	%
Control	0.458	-	0.462	-	0.437	-
1.0	0.463	-1.3	0.470	-1.7	0.448	-2.7
3.2	0.469	-2.4	0.452	2.1	0.432	1.0
10	0.447	2.4	0.411	11.0*	0.390	10.7*
32	0.412	10.1	0.387	16.1*	0.359	17.8*
100	0.355	22.5*	0.377	18.3*	0.353	19.2*

- % inhibition: increase in growth relative to that of the control * mean value significantly different from the control (tested with Williams t-test, α = 0.05, one-sided)

   

Table 6: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The doubling time of frond number in the control must be less than 2.5 days (60 h), corresponding to approximately a seven-fold increase in seven days and an average specific growth rate of 0.275 d-1.	1.6 days	Yes

 

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See Table 6 in section "Any other information on results incl tables"