Disodium 2-(11-oxido-3-oxo-3H-dibenzo[c,h]xanthen-7-yl)benzoate

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:

15 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)

Version / remarks:

23 March 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 0.14, 0.41, 1.23, 3.7, 11.11, 33.33 and 100.0 mg/L
- Sampling method: Concentrations were determined at the beginning and at the end of the test period. During the range finding study and main study, analytical samples (plus retain samples) were taken from a randomly chosen replicate per treatment group at beginning and at the end of the test (at 0 hour and 7 days) in order to determine the concentration of the test item during the test. About 10 mL of sample was collected from all test concentrations in duplicates.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solution was freshly prepared in the Swedish Standard (SIS) growth medium. Required quantity of test item was taken in a beaker, to this small volume of SIS growth medium was added and stirred well using glass rod. After complete solubility, test formulation was transferred to measuring cylinder, beaker was rinsed with SIS growth medium and transferred again to measuring cylinder. The rinsing procedure was repeated until the complete transfer of test contents. Finally the volume made up to required volume using SIS growth medium. A homogeneous distribution of the test item within the stock solution was achieved by stirring on magnetic stirrer before preparation of the test medium. The test concentrations were prepared by taking appropriate amount of stock solutions and adding up SIS growth medium to the required volume.
- Chemical name of vehicle: Based on the in house solubility/suspendibitity test, the test item was soluble in both Steinberg Medium and Swedish Standard (SIS) growth medium. Hence, SIS growth medium was selected as the vehicle for preparation of test item formulation.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: common duckweed
- Source: Department of Botany, Tumkur University
- Age of inoculum (at test initiation): The pre culture was initiated 7 days before the start of the test.
- Method of cultivation: Lemna minor culture is periodically sub cultured at least once in 3 months and maintained with the illumination and temperature of 6500 to 10000 lux and 22 to 26 °C respectively. Lemna minor was maintained separately on Steinberg medium and Swedish Standard (SIS media). From healthy axenic culture of Lemna minor, pre-culture was prepared of desired colonies to provide the inoculum for test cultures.

ACCLIMATION
- Acclimation period: 7 days
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

no data.

Test temperature:

24.7 to 25.4 °C

pH:

6.37 to 6.77

Dissolved oxygen:

no data.

Salinity:

no data.

Conductivity:

no data.

Nominal and measured concentrations:

Nominal: 0, 0.14, 0.41, 1.23, 3.7, 11.11, 33.33 and 100.0 mg/L
Measured: 0, 0.13, 0.39, 1.24, 3.49, 11.17, 31.80 and 101.65 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Type: open
- Material, size, headspace, fill volume: glass, 150 mL, 50 mL, 100 mL
- Aeration: No
- No. of fronds per colony: 9
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Light intensity and quality: 9425 - 9935 lux

EFFECT PARAMETERS MEASURED
- The frond number at the start on day 0, day 4 and on day 7 was counted in each test vessel and recorded. Frond numbers appearing normal or abnormal were determined at beginning of test on day 3, day 6 and at the end of the test. Changes in plant development (e.g. frond size, appearance, indication of necrosis, chlorosis or gibbosity, colony break-up or loss of buoyancy and in root length and appearance) was recorded.

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L

Reference substance (positive control):

yes

Remarks:

3,5 - Dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

30.982 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL: 22.942 - 44.366 mg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

9.411 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL: 7.311 - 12.437 mg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

47.687 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL: 33.920 - 71.828 mg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

108.866 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL: 80.554 - 153.387 mg/L

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1.323 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL: 0.652 - 1.957 mg/L

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.446 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL: 0.233 - 0.712 mg/L

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1.69 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL: 0.866 - 2.753 mg/L

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

5.182 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL: 3.120 - 7.7731 mg/L

Reported statistics and error estimates:

The no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) were determined by Hypothesis testing (ToxRat Pro version 3.2.1) using individual replicates values of the specific growth rate (µ).

Couting of Found Number

During main study, the initial number of frond number in all treatment replicates on day 0 was 12. Mean frond numbers per group was 66.0, 68.3, 61.0, 54.0, 46.7, 40.3, 26.0 and 14.3 in control and in the tested concentrations of 0.14, 0.41, 1.23, 3.7, 11.11, 33.33 and 100 mg/L on day 7.

Table 1: Frond number observation during exposure (main study)

Test Conc.  (mg/L)	R	Frond Number on Day
		0	4	7
0	1	9	21	72
	2	9	19	75
	3	9	23	58
	4	9	24	63
	5	9	18	58
	6	9	24	70
	Mean	9	21.5	66.0
	±SD	0	2.6	7.3
	CV	0	12.0	11.1
0.14	1	9	21	72
	2	9	23	69
	3	9	20	64
	Mean	9	21.3	68.3
	±SD	0	1.5	4.0
	CV	0	7.2	5.9
0.41	1	9	19	64
	2	9	21	62
	3	9	17	57
	Mean	9	19.0	61.0
	±SD	0	2.0	3.6
	CV	0	10.5	5.9
1.23	1	9	19	54
	2	9	20	50
	3	9	14	58
	Mean	9	17.7	54.0
	±SD	0	3.2	4.0
	CV	0	18.2	7.4
3.7	1	9	16	43
	2	9	12	45
	3	9	18	52
	Mean	9	15.3	46.7
	±SD	0	3.1	4.7
	CV	0	19.9	10.1
11.11	1	9	16	31
	2	9	21	42
	3	9	18	48
	Mean	9	18.3	40.3
	±SD	0	2.5	8.6
	CV	0	13.7	21.4
33.33	1	9	13	29
	2	9	16	21
	3	9	12	28
	Mean	9	13.7	26.0
	±SD	0	2.1	4.4
	CV	0	15.2	16.8
100	1	9	10	12
	2	9	12	15
	3	9	13	16
	Mean	9	11.7	14.3
	±SD	0	1.5	2.1
	CV	0	13.1	14.5

R: Replicate; SD: Standard Deviation; CV: Coefficient of Variation.

Observation of fronds
During main study, all fronds appeared normal on day 0, 3, 6 and on day 7 in control and in tested concentrations of 0.14, 0.41, 1.23 and 3.7 mg/L. Some frond appeared normal and some fronds showed chlorosis on day 6 and 7 in the tested concentrations of 11.11 and 33.33 mg/L. Fronds showed chlorosis on day 6 and 7 in the tested concentration of 100 mg/L.

Dry Weight
Dry weight was determined at test start from representative sample of the inoculum culture. Consistent with the approach taken in the test. Day 0 dry weight was found to be 0.0306 g.
At the end of the test the plant material from each replicate of test concentration and control vessel were used to determine dry weight. Mean dry weight of each group was found to be 0.121 g in control and 0.126, 0.117, 0.108, 0.105, 0.101, 0.082 and 0.038 g were observed in the tested concentration of 0.14, 0.41, 1.23, 3.7, 11.11, 33.33 and 100.0 mg/L.

Douling Time

Doubling time (Td) of frond number in control group was found to be 2.442 days with and average specific growth rate of 0.284 d-1 during main study.

Average Specific Growth Rate
The average specific growth rate from day 0 to day 7, for frond numbers was found to be 0.284, 0.289, 0.273, 0.256, 0.235, 0.212, 0.150 and 0.065 in control and in the tested concentrations of 0.14, 0.41, 1.23, 3.7, 11.11, 33.33 and 100 mg/L.
The average specific growth rate from day 0 to day 7, for biomass (dry weight) was found to be 0.197, 0.203, 0.191, 0.180, 0.176, 0.170, 0.138 and 0.031 in control and in the tested concentration of 0.14, 0.41, 1.23, 3.7, 11.11, 33.33 and 100 mg/L.

Percent Inhibition in Growth Rate
Percent inhibitions in growth rate for frond number was found to be -2.0, 3.8, 9.9, 17.4, 25.3, 47.1 and 77.0 % were observed in the tested concentration of 0.14, 0.41, 1.23, 3.7, 11.11, 33.33  and 100 mg/L.
Percent inhibition in growth rate in biomass (Dry weight) was found to -2.9, 3.0, 8.5, 10.5, 13.5, 29.8 and 84.1 % were observed in the tested concentrations of 0.14, 0.41, 1.23, 3.7, 11.11, 33.33 and 100 mg/L.

Percent Inhibition in Yield
Percent inhibition in yield in frond number was found to be -4.0, 8.8, 21.1, 33.9, 45.1,  70.2 and 90.7 % were observed in the tested concentrations 0.14, 0.41, 1.23, 3.7, 11.11, 33.33 and 100 mg/L.
Percent inhibition in yield in biomass (Dry weight) was found to -5.5, 5.4, 14.7, 18.0, 22.7, 43.9 and 91.7 % were observed in the tested concentration of 0.14, 0.41, 1.23, 3.7, 11.11, 33.33 and 100 mg/L.

Analytical results

During main study, samples from the test concentration was analyzed on day 0 and on day 7 and the results reported that test samples of Mordant Red 27 are found to be with in the acceptable range ± 20 % recovery to the nominal concentration.

 

Table 2: Analytical results during the main study

Day 0			Day 7
Nominal Conc. (mg/L)	Conc. Obtained (mg/L)	% Recovery	Nominal Conc. (mg/L)	Conc. Obtained (mg/L)	% Recovery
0.0	-	-	0.0	-	-
0.14	0.14	100	0.14	0.13	92.86
0.41	0.38	94.46	0.41	0.39	93.90
1.23	1.29	104.48	1.23	1.24	100.41
3.70	3.57	96.49	3.70	3.49	94.32
11.11	11.41	102.70	11.11	11.17	100.50
33.33	32.39	97.18	33.33	31.80	95.40
100.0	103.66	103.66	100.0	101.65	101.65

 

Validity criteria of the test
The Lemna growth inhibition test fulfills the validity criteria of the OECD guideline 221:
The doubling time of frond number in the control was 2.469 days (59.2 hours) and 2.442 days (58.6 hours), corresponding to average specific growth rate of 0.281 and 0.284  d-1 during range finding study and main study respectively, (validity criterion: growth rate ≥0.275 d-1, doubling time <2.5 d).

Validity criteria fulfilled:

yes

Conclusions:

In a growth inhibition test with Lemna minor exposed to Mordant Red 27 over 7 days exposure, the ErC50 and EyC50 values for growth rate and yield for frond number was found to be 30.982 (95% CL: 22.942 and 44.366 mg/L) and 9.411 mg/L (95% CL: 7.311 and 12.437 mg/L), respectively. The ErC50 and EyC50 values for growth rate and yield for biomass (dry weight) was found to be 108.866 (80.554 and 153.387 mg/L) and 47.687 mg/L (33.920 and 71.828 mg/L), respectively. The ErC10 and EyC10 values for growth rate and yield for frond number was found to be 1.232 (95% CL: 0.652 – 1.957) and 0.446 mg/L (95% CL: 0.233 - 0.712), respectively. The ErC10 and EyC10 values for growth rate and yield for biomass (dry weight) was found to be 5.182 (95% CL: 3.120 – 7.731) and 1.690 mg/L (95% CL: 0.866 – 2.753), respectively. The NOEC (No observed effect concentration) and LOEC (Lowest observed effect concentration) for frond number was found to be 1.230 and 0.410 mg/L. The NOEC (No observed effect concentration) and LOEC (Lowest observed effect concentration) for dry weight (Biomass) was found to be 1.230 and 0.410 mg/L.

Executive summary:

The Lemna sp. Growth Inhibition Test was performed with the test substance according to the OECD Guideline 221. This test was performed to determine ErC50 for % Inhibition of Growth, % Inhibition of yield EyC50 along with determination of Lowest Observed Effect Concentration (LOEC) and No Observed Effect Concentration (NOEC) for frond number and measurement variable (dry weight).

Lemna minor was exposed to the test item by aqueous exposure under static condition. Swedish Standard (SIS) growth medium was used to prepare test medium for exposure. Based on the results of range finding study, the main study was performed at the test concentration of 0.14, 0.41, 1.23, 3.7, 11.11, 33.33 and 100.0 mg/L. The ErC50 for % Inhibition of Growth, % Inhibition of yield EyC50 along with determination of LOEC and NOEC for frond number and measurement variable (dry weight) was determined. Lemna minor were observed for frond number on day 0, 4 and on day 7. Observation for fronds was made at beginning of the test and on day 3, 6 and at end of the test day 7. During main study, all fronds appeared normal on day 0, 3, 6 and on day 7 in control and in tested concentrations of 0.14, 0.41, 1.23 and 3.7 mg/L. Some fronds appeared normal and some fronds showed chlorosis on day 6 and 7 in the tested concentrations of 11.11 and 33.33 mg/L. Fronds showed chlorosis on day 6 and 7 in the tested concentration of 100 mg/L.

The ErC50 and EyC50 values for growth rate and yield for frond number was found to be 30.982 (95% CL: 22.942 and 44.366 mg/L) and 9.411 mg/L (95% CL: 7.311 and 12.437 mg/L), respectively. The ErC50 and EyC50 values for growth rate and yield for biomass (dry weight) was found to be 108.866 (80.554 and 153.387 mg/L) and 47.687 mg/L (33.920 and 71.828 mg/L), respectively. The ErC10 and Ey10 values for growth rate and yield for frond number was found to be 1.232 (95% CL: 0.652 – 1.957) and 0.446 mg/L (95% CL: 0.233 - 0.712), respectively. The ErC10 and EyC10 values for growth rate and yield for biomass (dry weight) was found to be 5.182 (95% CL: 3.120 – 7.731) and 1.690 mg/L (95% CL: 0.866 – 2.753), respectively. The NOEC (No observed effect concentration) and LOEC (Lowest observed effect concentration) for frond number was found to be 1.230 and 0.410 mg/L. The NOEC (No observed effect concentration) and LOEC (Lowest observed effect concentration) for dry weight (Biomass) was found to be 1.230 and 0.410 mg/L.

Description of key information

In a growth inhibition test with Lemna minor exposed to Mordant Red 27 over 7 days exposure, the ErC50 and EyC50 values for growth rate and yield for frond number was found to be 30.982 (95% CL: 22.942 and 44.366 mg/L) and 9.411 mg/L (95% CL: 7.311 and 12.437 mg/L), respectively. The ErC50 and EyC50 values for growth rate and yield for biomass (dry weight) was found to be 108.866 (80.554 and 153.387 mg/L) and 47.687 mg/L (33.920 and 71.828 mg/L), respectively. The ErC10 and Ey10 values for growth rate and yield for frond number was found to be 1.232 (95% CL: 0.652 – 1.957) and 0.446 mg/L (95% CL: 0.233 - 0.712), respectively. The ErC10 and EyC10 values for growth rate and yield for biomass (dry weight) was found to be 5.182 (95% CL: 3.120 – 7.731) and 1.690 mg/L (95% CL: 0.866 – 2.753), respectively. The NOEC (No observed effect concentration) and LOEC (Lowest observed effect concentration) for frond number was found to be 1.230 and 0.410 mg/L. The NOEC (No observed effect concentration) and LOEC (Lowest observed effect concentration) for dry weight (Biomass) was found to be 1.230 and 0.410 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:

30.982 mg/L

EC10 or NOEC for freshwater plants:

1.232 mg/L

Additional information

The Lemna sp. Growth Inhibition Test was performed with the test substance according to the OECD Guideline 221. This test was performed to determine ErC50 for % Inhibition of Growth, % Inhibition of yield EyC50 along with determination of Lowest Observed Effect Concentration (LOEC) and No Observed Effect Concentration (NOEC) for frond number and measurement variable (dry weight).

Lemna minor was exposed to the test item by aqueous exposure under static condition. Swedish Standard (SIS) growth medium was used to prepare test medium for exposure. Based on the results of range finding study, the main study was performed at the test concentration of 0.14, 0.41, 1.23, 3.7, 11.11, 33.33 and 100.0 mg/L. The ErC50 for % Inhibition of Growth, % Inhibition of yield EyC50 along with determination of LOEC and NOEC for frond number and measurement variable (dry weight) was determined. Lemna minor were observed for frond number on day 0, 4 and on day 7. Observation for fronds was made at beginning of the test and on day 3, 6 and at end of the test day 7. During main study, all fronds appeared normal on day 0, 3, 6 and on day 7 in control and in tested concentrations of 0.14, 0.41, 1.23 and 3.7 mg/L. Some fronds appeared normal and some fronds showed chlorosis on day 6 and 7 in the tested concentrations of 11.11 and 33.33 mg/L. Fronds showed chlorosis on day 6 and 7 in the tested concentration of 100 mg/L. The ErC50 and EyC50 values for growth rate and yield for frond number was found to be 30.982 and 9.411 mg/L respectively. With 95 % confidence limits for growth rate of 22.942 and 44.366 mg/L and  95 % confidence limits for yield 7.311 and 12.437 mg/L respectively. The ErC50 and EyC50 values for growth rate and yield for biomass (dry weight) was found to be 108.866 and 47.687 mg/L respectively. The NOEC and LOEC for frond number was found to be 1.230 and 0.410 mg/L. The NOEC and LOEC for dry weight (Biomass) was found to be 1.230 and 0.410 mg/L.