2-[3-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)prop-1-enyl]-1,3,3-trimethyl-3H-indolium acetate

Toxicity to aquatic plants other than algae

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-[3-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)prop-1-enyl]-1,3,3-trimethyl-3H-indolium acetate

EC Number:

265-479-2

EC Name:

2-[3-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)prop-1-enyl]-1,3,3-trimethyl-3H-indolium acetate

Cas Number:

65122-08-9

Molecular formula:

C25H29N2.C2H3O2

IUPAC Name:

2-[3-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)prop-1-enyl]-1,3,3-trimethyl-3H-indolium acetate

Test material form:

liquid

Sampling and analysis

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 item concentrations and control.
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 on a horizontal flatbed shaker for 15 min. If necessary, the samples were further diluted with acetonitrile/test medium (1:1, v/v) prior to analysis by HPLC-MS/MS.

Test solutions

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

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.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

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: 7.40-7.61
aged solutions: 8.51-9.49 ( 100 ug/L 8.55-8.96)

Nominal and measured concentrations:

Nominal: 0, 0.160, 0.800, 4.00, 20.0 and 100 ug/L
Measured (fresh): 74-133% of nominal
Measure (aged): geometric mean concentrations: 0, 0.0496, 0.192, 1.04, 7.20 and 52 ug/L

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: 2-4 (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; 780 0 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 based on frond number at 0.01 mg/L and above
On day 3 shortened roots and yellow fronds were observed from 0.100 to 10.0 mg/L. Additionally at 0.100 mg/L fronds were tightly assembled and gibbosity was observed. At 1.00 and 10.0 mg/L fronds were partially singularized. Also, at 1.00 mg/L, the edge of the fronds was coloured red.
On day 5 at 0.0100 mg/L shortened roots and partially small offshoots were found. Small offshoots were also observed at 0.100 mg/L. Shortened roots, as well as yellow and partially singularized fronds were observed at test item concentrations 0.100 and 10.0 mg/L. At 1.00 mg/L shortened roots, and singualised, yellow fronds with red coloured edge were found.
On day 7 at 0.0100 mg/L shortened roots, small offshoots, tightly assembled fronds and gibbosity were found. Small offshoots and gibbosity were also observed at 0.100 mg/L. Shortened roots, as well as yellow and partially singularized fronds were observed at test item concentrations 0.100 and 10.0 mg/L. At 1.00 mg/L shortened roots, and singualised, yellow fronds were found.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.192 µg/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

Effect concentrations 2

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1.04 µg/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

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

5.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: based on yield

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

33.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: based on growth rate

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

14.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: based on yield

Effect concentrations 6

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

27.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: based on growth rate

Details on results:

On day 2 shortened roots and tightly assembled fronds were observed at 100 µg/L.
On day 5 shortened roots and gibbosity were observed at concentrations of 4.00 to 100 µg/L. Additionally at concentrations of 20.0 and 100 µg/L small offshoots and tightly assembled fronds were found. At 100 µg/L additionally, fronds were brown.
On day 7 shortened roots and gibbosity were observed at concentrations of 4.00 to 100 µg/L. Additionally at concentrations of 20.0 and 100 µg/L small offshoots and tightly assembled fronds were found. At 100 µg/L additionally, fronds were brown and partially singularized.

Results with reference substance (positive control):

EC50 growth rate 3,5-dichlorophenol = 6.69 mg/L (frond numbers), 11.0 mg/L (dry weight)

Any other information on results incl. tables

Frond numbers:yield

	Mean of frond numbers				Yield based on mean frond numbers	Inhibition (%)
	Days
Nominal conc. [µg/L]	0	2	5	7	7-0	7-0 days
Control	12	24	91	242	230	0.0
0.160	12	24	91	258	246	-7.0^1)
0.800	12	25	93	256	244	-6.1^2)
4.00	12	23	79	191	179*	22.2
20.0	12	23	60	108	96*	58.3
100	12	19	35	42	30*	87.0

* Statistically significant different to the control (Dunnetts-t-test, left-sided)

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

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

 

Frond numbers: growth rates

	Days			Days
Nominal conc. [µg/L]	2	5	7	2	5	7
	Mean growth rate µ [d^-1]			Inhibition of growth rat (%)
Control	0.3387	0.4039	0.4290	0.0	0.0	0.0
0.160	0.3389	0.4051	0.4383	-0.1	-0.3	-2.2^1)
0.800	0.3602	0.4102	0.4371	-6.3	-1.6	-1.9^2)
4.00	0.3230	0.3772	0.3952*	4.6	6.6	7.9
20.0	0.3240	0.3229	0.3129*	4.3	20.1	27.1
100	0.2198	0.2155	0.1798*	35.1	46.6	58.1

* Statistically significant different to the control (Dunnetts-t-test, left-sided)

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. [µg/L]	2	5	7	7
	Td[days]			Td[hours]
Control	2.046	1.716	1.616	38.8
0.160	2.045	1.711	1.581	37.9
0.800	1.924	1.690	1.586	38.1
4.00	2.146	1.838	1.754	42.1
20.0	2.139	2.147	2.215	53.2
100	3.154	3.216	3.855	92.5

Dry weight:

	Mean dry weight [g]		Yield based on mean dry weight [g]		Inhibition [%]
Nominal conc. [µg/L]	Days
	0	7		0.0	7-0
Control	0.0106	0.0435		-11.6^1)	0.0329
0.160		0.0473		-10.0^2)	0.0367
0.800		0.0468		10.0	0.0362
4.00		0.0402		38.9	0.0296
20.0		0.0307		81.2	0.0201*
100		0.0168			0.0062*

* Statistically significant different to the control (Bonferroni-Holms corrected Welch test, left-sided)

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

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

 

 

 

 

 

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The EC50 based on growth rate is 27.1 ug/L based on biomass, The NOEC based on growth rate is 0.192 ug/L based on biomass

Executive summary:

In a study with a semi-static design (renewal on day 2 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, 0.160, 0.800, 4.00, 20.0 and 100 ug/L (mean measured concentrations 0, 0.0496, 0.192, 1.04,  7.20 and 52 ug/L).

Significant inhibitory effects were determined for yield and growth rate of frond numbers and biomass at test item concentrations of 1.04 ug/L (measured) and above. The EC50-value for yield of frond numbers was determined to be 5.50 ug/L (measured), the EC50-value for growth rate of frond numbers was determined to be 33.3 ug/L (measured). The EC50-value for yield of dry weight was determined to be 14.6 ug/L (measured), the EC50-value for growth rate of dry weight was determined to be 27.1 ug/L (measured).