4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Remarks:

lemna

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 January 2018 to 08 February 2018

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)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analytical samples were taken at 0 hours (initial value) from fresh test solutions, after 2 and 5 days from fresh and aged test solutions and after 7 days from aged test solutions from all test concentrations and control. For each sampling also a retain sample was taken.
All samples were stored deep frozen until they were transferred to the analytical laboratory. The samples were thawed to ambient temperature, mixed with 50 mL acetonitrile and shaken well using a flatbed shaker (15 min). If necessary, the samples were further diluted with acetonitrile/test medium (1:1, v/v) prior to analysis by HPLC-MS/MS.

Vehicle:

no

Remarks:

dissolved in test medium

Details on test solutions:

A stock solution was prepared in test medium and further diluted to achieve the required concentrations

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: duck weed
- Strain: Lemna gibba G3 (Alismatales: Araceae)
- Source: inhouse clone from primary culture by Dr. Janet Slovin, Horticulture Crops Quality Laboratory, U.S. Department of Agriculture. BARC-West, Bldg. 050 HH-4, Beltsville, MD 20705, U.S.A.
- Method of cultivation: cultured in appropriate glass beakers under continuous illumination with OSRAM light tubes (L30/32-930 and L30/W72-965), at 6500 - 10000 lux. The temperature was 24 ± 2 °C.

Before the test was started, plants were cultured under the same illumination and temperature conditions as those to be used for the toxicity test. Young, light-green plants of similar size and comprising 2 - 4 fronds were transferred onto fresh medium and cultured for 7-10 days prior to testing, with two further transfers onto fresh medium before initiating the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

no data

Test temperature:

23.5 – 23.9 °C (measured in separate vessel held under the same conditions as the test vessels)

pH:

fresh solutions: 7.41-7.61
aged solutions: 8.48-9.40 (at 0.25 and 1.00 mg/L 8.51-8.69)

Nominal and measured concentrations:

Nominal: 0, 0.00391, 0.0156, 0.0625, 0.250 and 1.00 mg/L
Measured (fresh): 89-104% of nominal
Measure (aged):

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass bottles each containing ~150 mL test medium covered with glass plate
- Aeration: no
- Renewal rate of test solution: on day 2 and 5
- No. of fronds per colony: 4 (total 12)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM:no data

TEST MEDIUM / WATER PARAMETERS: 20xAAP test medium according to the guideline

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: T8-fluorescent tubes, length 900 mm, LT30W/865 daylight; 7820 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: manual counting on day 0, 2,5 and 7 (inludes observation of appearance)
- Determination of biomass: dry weight on day 7 (for t=0 based on 6X12 fronds from the culture); dried at 60°C for about 72 hours
- Determination of frond area: no
- Other: plant development, frond size, necrosis and additional observations

RANGE-FINDING STUDY
- Test concentrations: 0, 0.010, 0.100, 1.00 and 10.0 mg/L
- Results used to determine the conditions for the definitive study: inhibition of yield and growth rate at 0.01 mg/L and above:
At 0.1 and 1.0 mg/L shortened roots, tightly assembled fronds and gibbosity were observed. At 1.00 mg/L additionally yellow, brown and partially singularized fronds were found. At t 10.0 mg/L shortened roots, partially singularised, yellow and orange fronds and small offshoots were found

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: based on yield and growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: based on yield and growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

0.084 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: based on yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

0.483 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: based on growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

0.092 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: based on yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

0.22 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: based on growth rate

Details on results:

On day 2 slightly smaller offshoots were observed at concentrations of 0.0625 to 1.00 mg/L. Additionally, at 0.250 and 1.00 mg/L roots were shortened.
On day 5 at 0.0625 mg/L shortened roots and tightly assembled and pale fronds were observed. At 0.250 mg/L, roots were shortened and gibbosity occurred, small offshoots and tightly assembled fronds were noted. At 1.00 mg/L some fronds were singularized and the fronds were brown and roots were shortened. Also gibbosity and small offshoots were found.
On day 7 small offshoots and slight gibbosity was observed at concentrations of 0.0625 to 1.00 mg/L. At 0.0625 mg/L, tightly assembled fronds were observed. Additionally at concentrations of 0.250 and 1.00 mg/L brown fronds, and small offshoots were found. At 1.00 mg/L fronds were partially singularized.

Results with reference substance (positive control):

EC50 growth rate 3,5-dichlorophenol = 6.78 mg/L (frond numbers), 4.63 mg/L (dry weight)

Frond numbers: mean values

	Mean of frond numbers				Yield based on mean frond numbers
Nominal conc. [mg/L]	Day 0	Day 2	Day 5	Day 7	7-0
Control	12	24	91	242	230
0.00391	12	23	95	262	250
0.0156	12	24	91	259	247
0.0625	12	24	79	187	175*
0.250	12	21	53	97	85*
1.00	12	21	38	47	35*

* Statistically significant different to the control

Frond numbers: inhibition of yield

Nominal conc. [mg/L]	Inhibition [%]
	7-0 days
Control	0.0
0.00391	-8.7^1)
0.0156	-7.4^2)
0.0625	23.9
0.250	63.0
1.00	84.8

¹⁾Value was omitted for EC_{10, 20, 50}-calculation

²⁾Value was set to zero for EC_{10, 20, 50}-calculation

 

Frond numbers: mean growth rates

	Days
Nominal conc. [mg/L]	2	5	7
	Mean growth rate µ [d^-1]
Control	0.3387	0.4039	0.4290
0.00391	0.3250	0.4145	0.4403
0.0156	0.3389	0.4052	0.4386
0.0625	0.3392	0.3769	0.3925*
0.250	0.2872	0.2979	0.2989*
1.00	0.2717	0.2322	0.1938*

* Statistically significant different to the control

Frond numbers: inhibition of growth rates

	Days
Nominal conc. [mg/L]	2	5	7
	Inhibition growth rate [%]
Control	0.0	0.0	0.0
0.00391	4.0	-2.6	-2.6^1)
0.0156	-0.1	-0.3	-2.2^2)
0.0625	-0.1	6.7	8.5
0.250	15.2	26.2	30.3
1.00	19.8	42.5	54.8

¹⁾Value was omitted for EC_{10, 20, 50}-calculation

²⁾Value was set to zero for EC_{10, 20, 50}-calculation

Frond numbers: mean doubling times (T_d)

	Days
Nominal conc. [mg/L]	2	5	7	7
	Td[days]			Td[hours]
Control	2.046	1.716	1.616	38.8
0.00391	2.133	1.672	1.574	37.8
0.0156	2.045	1.711	1.580	37.9
0.0625	2.043	1.839	1.766	42.4
0.250	2.413	2.327	2.319	55.7
1.00	2.551	2.985	3.577	85.8

 

Dry weight: mean values

	Mean dry weight [g]		Yield based on mean dry weight [g]
Nominal conc. [mg/L]	Days
	0	7		7-0
Control	0.0106	0.0435		0.0329
0.00391		0.0514		0.0408
0.0156		0.0502		0.0396
0.0625		0.0375		0.0269*
0.250		0.0236		0.0130*
1.00		0.0145		0.0039*

* Statistically significant different to the control

Dry weight: inhibition of yield

Nominal conc. [mg/L]	Inhibition [%]
	7-0 days
Control	0.0
0.00391	-24.0^1)
0.0156	-20.4^2)
0.0625	18.2
0.250	60.5
1.00	88.1

¹⁾Value was omitted for EC_{10, 20, 50}-calculation

²⁾Value was set to zero for EC_{10, 20, 50}-calculation

Dry weight: mean growth rate and inhibition of growth rate

	Day 7
Nominal conc. [mg/L]	Mean growth rate µ [d^-1]	Inhibition Growth rate [%]^4)
Control	0.2014	0.0
0.00391	0.2254	-11.9^1)
0.0156	0.2222	-10.3^2)
0.0625	0.1806*	10.3
0.250	0.1141*	43.3
1.00	0.0451*	77.6

¹⁾Value was omitted for EC_{10, 20, 50}-calculation

²⁾Value was set to zero for EC_{10, 20, 50}-calculation

* Statistically significant different to the control

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

The EC50 based on growth rate is 0.22 mg/L based on biomass, The NOEC based on growth rate is 0.00156 mg/L based on biomass

Executive summary:

In a study with a semi-static design (renewal on day 3 and 5) the vegative growth of Lemna gibba G3 was observed for a test period of 7 days by measuring the increase of frond numbers and dry weight. Lemna was exposed at nominal concentrations of 0, 0.00391, 0.0156, 0.0625, 0.250 and 1.00 mg/L (mean measured concentrations 0, 0.000665, 0.00156, 0.0194, 0.138 and 0.660 mg/L).

Significant inhibitory effects were determined for yield and growth rate of frond numbers and biomass at test item concentrations of 0.0194 mg/L (measured) and above. The EC50-value for yield of frond numbers was determined to be 0.00385 mg/L (measured), the EC50-value for growth rate of frond numbers was determined to be 0.483 mg/L (measured). The EC50-value for yield of dry weight was determined to be 0.0916 mg/L (measured), the EC50-value for growth rate of dry weight was determined to be 0.220 mg/L (measured).

Description of key information

In a study with a semi-static design (renewal on day 3 and 5) the vegative growth of Lemna gibba G3 was observed for a test period of 7 days by measuring the increase of frond numbers and dry weight. Lemna was exposed at nominal concentrations of 0, 0.00391, 0.0156, 0.0625, 0.250 and 1.00 mg/L (mean measured concentrations 0, 0.000665, 0.00156, 0.0194, 0.138 and 0.660 mg/L)

Significant inhibitory effects were determined for yield and growth rate of frond numbers and biomass at test item concentrations of 0.0194 mg/L (measured) and above. The EC50-value for yield of frond numbers was determined to be 0.00385 mg/L (measured), the EC50-value for growth rate of frond numbers was determined to be 0.483 mg/L (measured). The EC50-value for yield of dry weight was determined to be 0.0916 mg/L (measured), the EC50-value for growth rate of dry weight was determined to be 0.220 mg/L (measured).

Key value for chemical safety assessment

EC50 for freshwater plants:

0.22 mg/L

EC10 or NOEC for freshwater plants:

0.002 mg/L