Disodium ar,ar'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[(4-chlorophenoxy)benzenesulphonate]

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:

From April 30 to July 02, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Remarks:

validation of analytical method

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

adopted on 23 March 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

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 (i.e. at 0 hour and at 7 days) in order to determine the concentration of tst item during the test.
About 10 ml of sample was collected from all test concentrations in duplicate.

Vehicle:

no

Details on test solutions:

Based on a preliminary investigation test, the test item was soluble in Swedish Standard (SIS) growth medium, hence required quantity of test item was taken in a beaker and small volume of Swedish Standard (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 Swedish Standard (SIS) growth medium and transferred again to measuring cylinder; the rinsing procedure was repeated until the complete transfer of test contents. Finally, Swedish Standard (SIS) growth medium was added up to required volume.

A homogeneous distribution of the test item within the stock solution was achieved by stirring on magnetic stirrer before preparation of the test medium.

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Source: in house maintained culture of Lemna minor, obtained from the Department of Botany, Tumkur University.
- Pre culture: pre culture (in 10 beakers containing 100 ml of culture medium) was initiated 7 days before start of the test. Pre cultures are incubated under same conditions as the test cultures.

BREEDING
- Breeding conditions: Lemna minor culture is periodically sub cultured and maintained with the illumination and temperature of 6500 to 10000 lux and 22 to 26 °C respectively.
- Pre-culture: from healthy axenic culture of Lemna minor maintained in Swedish Standard (SIS) media, pre-culture was prepared of desired colonies to provide the inoculum for test cultures.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

23.1 to 23.8 °C

pH:

6.37 to 6.54

Nominal and measured concentrations:

2.0, 4.7, 11.4, 27.3, 65.6 and 157.5 mg/l (corresponding to 1.6, 3.7, 9, 21.6, 52 and 124.7 mg a.i./l).

Details on test conditions:

TEST SYSTEM
- Test vessel: sterilized, glass beakers of 150 ml capacity were used as culture vessels to conduct the experiment.
- Fill volume: the volume of the test solution at the initiation of the test in the test vessel was 100 ml.
- Coverage: test vessels was covered to minimize accidental contamination.
- Aeration: test vessels was allowing necessary air exchange.
- No. of fronds at the start: the initial number of frond number was 9 in all treatment replicates on day 0.
- No. of vessels per concentration: 3 replicates.
- No. of vessels per control: 6 replicates

GROWTH MEDIUM
- Standard medium used: yes; Swedish Standard (SIS) growth medium.

OTHER TEST CONDITIONS
- Light intensity and quality: 6785 to 6941 lux with white fluorescent lamps.

EFFECT PARAMETERS MEASURED
- Counting of Frond Number: the frond number at the start on day 0, day 4 and on day 7 were counted in each test vessel and recorded.
- Observation of Fronds: frond numbers appearing normal or abnormal were determined at beginning of test and on day 3, day 6 and at 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) were recorded.
- Dry Weight: determined at test start from representative sample of the inoculum culture. At the end of the test, the plant material from each replicate of test concentration and control vessel was used to determine dry weight. To determine the dry weight they are blotted to remove excess water and then dried at 60 °C to a constant weight. Root fragments were included and dry weight was measured.

RANGE-FINDING STUDY
- No. of vessels per concentration: 2 replicates
- No. of vessels per control: 4 replicates
- Test concentrations: 0.013, 0.13, 1.26, 12.6, 63.13 and 126 mg/l (corresponding to 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg a.i./l).
- Results used to determine the conditions for the definitive study: the percentage of growth rate inhibition after 7 days was 5.1, 5.8, 12.2, 20.5, 43.4 and 84.9 % at 0.013, 0.13, 1.26, 12.6, 63.13 and 126 mg/l.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

84.19 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

36.49 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Counting of Frond Number
The initial number of frond number was 9 in all treatment replicates on day 0. The mean frond numbers on day 7 was 88.0 in control and 87.3, 81.7, 73.7, 56.0, 43.3 and 16.0 at the test concentrations of 2.0, 4.7, 11.4, 27.3, 65.6 and 157.5 mg/l respectively.

Observation of Fronds
All fronds appeared normal on day 0, 3, 6 and on day 7 in control and at the tested concentrations of 2.0 and 4.7 mg/L. S In some cases, chlorosis on day 6 and 7 at the tested concentrations of 11.4 mg/l.
Chlorosis on day 6 and chlorosis and colony breakup on day 7 were observed at the tested concentrations of 27.3 and 65.6 mg/l.
Chlorosis and defoliation was observed on day 6 and chlorosis, defoliation and loss of buoyancy were observed on day 7 at the tested concentration of 157.5 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. On day 0 dry weight was found to be 0.0263 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. The mean dry weight of each group was found to be 0.147 g in control and 0.145, 0.133, 0.132, 0.065, 0.049 and 0.029 g were observed in the vessels with test item concentration of 2.0, 4.7, 11.4, 27.3, 65.3 and 157.5 mg/l respectively.

Average Specific Growth Rate
The average specific growth rate from day 0 to day 7, for frond numbers, was found to be 0.326 in the control and 0.325, 0.315, 0.300, 0.260, 0.224 and 0.081 in the vessels with test item at the concentrations of 2.0, 4.7, 11.4, 27.3, 65.3 and 157.5 mg/l respectively. The average specific growth rate from day 0 to day 7, for dry weight, was found to be 0.246 in the control and 0.244, 0.231, 0.230, 0.127, 0.084 and 0.013 in the vessels with test item at the concentration of 2.0, 4.7, 11.4, 27.3, 65.3 and 157.5 mg/l respectively.

Percent Inhibition in Growth Rate
Percent inhibitions in growth rate for frond number was found to be 0.3, 3.2, 7.8, 20.2, 31.2 and 75 % in the vessels with test item at the concentration of 2.0, 4.7, 11.4, 27.3, 65.6 and 157.5 mg/l, respectively. Percent inhibition in growth rate in dry weight was found to 0.7, 6.0, 6.5, 48.2, 65.9 and 94.7 % were observed in the vessels with test item at the concentrations of 2.0, 4.7, 11.4, 27.3, 65.6 and 157.5 mg/l, respectively.

Percent Inhibition in Yield
Percent inhibition in yield in frond number was found to be 0.9, 8.0, 18.1, 40.5, 56.6 and 91.1 % in the vessels with test item at the concentrations 2.0, 4.7, 11.4, 27.3, 65.6 and 157.5 mg/l respectively. Percent inhibition in yield in dry weight was found to 1.6, 12.2, 12.9, 67.6, 81.2 and 97.6 % in the vessels with test item at the concentration of 2.0, 4.7, 11.4, 27.3, 65.6 and 157.5 mg/l respectively.

RANGE-FINDING STUDY
Counting of Frond Number
The initial frond number was 9 in all treatment replicates on day 0. The mean frond numbers on day 7 was 79.0 in control and 71.0, 69.5, 60.5, 50.5, 31.0 and 12.5 at the test concentrations of 0.013, 0.13, 1.26, 12.6, 63.13 and 126 mg/l respectively.

Observation of Fronds
All fronds appeared normal on day 0, 3, 6 and on day 7 in control and in the vessels with test item concentrations of 0.013, 0.13, 1.26 and 12.6 mg/l.
In some cases defoliation and chlorosis were observed on day 6; chlorosis, defoliation and loss of buoyancy were observed on day 7 at the tested concentration of 63.13 mg/l. Chlorosis, defoliation and loss of buoyancy was observed on days 6 and 7 at the tested concentration of 126 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. On day 0 dry weight was found to be 0.0247 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. The mean dry weight of each group was found to be 0.210 g in control and 0.213, 0.205, 0.200, 0.190, 0.135 and 0.059 g were observed in the vessels with test item concentration of 0.013, 0.13, 1.26, 63.13, and 126 mg/l respectively.

Average Specific Growth Rate
The average specific growth rate from day 0 to day 7, for frond numbers was found to be 0.310 in the control and 0.294, 0.292, 0.272, 0.246, 0.175 and 0.047 in the vessels with test item at the concentrations of 0.013, 0.13, 1.26, 12.6, 63.13 and 126 mg/l respectively.
The average specific growth rate from day 0 to day 7, for dry weight, was found to be 0.306 in control and 0.308, 0.303, 0.299, 0.291, 0.243 and 0.123 in the vessels with test item at the concentrations of 0.013, 0.13, 1.26, 12.6, 63.13 and 126 mg/l respectively.

Percent Inhibition in Growth Rate
Percent inhibition in growth rate, for frond number, was found to 5.1, 5.8, 12.2, 20.5, 43.4 and 84.9 % in the vessels with test item at the concentrations of 0.013, 0.13, 1.26, 12.6, 63.13 and 126 mg/l, respectively. Percent inhibition in growth rate in dry weight was found to -0.7, 1.0, 2.2, 4.7, 20.6 and 59.9 % in the vessels with test item at the concentrations of 0.013, 0.13, 1.26, 12.6, 63.13 and 126 mg/l, respectively.

Percent Inhibition in Yield
Percent inhibition in yield in frond number was found to 11.4, 13.6, 26.4, 40.7, 68.6 and 95.0 % in the vessels with test item at the concentrations of 0.013, 0.13, 1.26, 12.6, 63.13 and 126 mg/l respectively. Percent inhibition in yield in dry weight was found to -1.8, 2.5, 5.2, 10.8, 40.3 and 81.7 % in the vessels with test item at the concentration of 0.013, 0.13, 1.26, 12.6, 63.13 and 126 mg/l respectively.

APPEARANCE OF TEST MEDIUM
The test medium was clear in all the test concentration during range finding and main study on day 0 except at high test concentration deposition of test item in the bottom of test vessels was observed at highest test concentration 126.3 and 157.5 mg/l was observed on day 7 during range finding and main study respectively.

MEASURED CONCENTRATIONS
During the range finding and the main study, the test concentrations on day 0 (0 hour) and on day 7 were found to be within in the acceptable range ± 20 % recovery to the nominal concentration.

Results with reference substance (positive control):

ErC50 (7d): 3.70 mg/l, based on frond number
ErC50 (7d): 3.29 mg/l, based on dry weight
The ErC50 value for the frond number of 3.70 is in good agreement with the results of an international ring test between 2.7 and 3.4 mg/l (n=52).
Hence, this test with toxic standard establishes the acceptability of test system response and test procedures followed.

SUMMARY OF IMMOBILIZATION DURING THE MAIN STUDY

Test Conc. (mg/l)	Frond Number		Measurement variable (Dry weight)
	% Inhibition in	% Inhibition in	% Inhibition in	% Inhibition in
	Growth Rate	Yield	Growth Rate	Yield
0	-	-	-	-
2	0.3	0.9	0.7	1.6
4.7	3.2	8	6	12.2
11.4	7.8	18.1	6.5	12.9
27.3	20.2	40.5	48.2	67.6
65.6	31.2	56.6	65.9	81.2
157.5	75	91.1	94.7	97.6

TEST CONCENTRATIONS DURING THE MAIN STUDY

Day 0			Day 7
Nominal Conc. (mg/l)	Measured Conc. (mg/l)	% Recovery	Nominal Conc. (mg/l)	Measured Conc. (mg/l)	% Recovery
0	-	-	0	-	-
2	1.84	92.00	2	1.82	91.00
4.7	4.18	88.94	4.7	4.24	90.21
11.4	10.42	91.40	11.4	10.4	91.18
27.3	22.99	84.20	27.3	22.91	83.92
65.6	57.47	87.61	65.6	56.44	86.03
157.5	137.66	87.41	157.5	137.01	88.26

SUMMARY OF IMMOBILIZATION DURING THE RANGE-FINDING STUDY

Test Conc. (mg/l)	Frond Number		Measurement variable (Dry weight)
	% Inhibition in	% Inhibition in	% Inhibition in	% Inhibition in
	Growth Rate	Yield	Growth Rate	Yield
0	-	-	-	-
0.013	5.1	11.4	-0.7	-1.8
0.13	5.8	13.6	1.0	2.5
1.26	12.2	26.4	2.2	5.2
12.6	20.5	40.7	4.7	10.8
63.13	43.4	68.6	20.6	40.3
126	84.9	95.0	59.9	81.7

TEST CONCENTRATIONS DURING THE RANGE-FINDING STUDY

Day 0			Day 7
Nominal Conc. (mg/l)	Measured Conc. (mg/l)	% Recovery	Nominal Conc. (mg/l)	Measured Conc. (mg/l)	% Recovery
0	-	-	0	-	-
0.013	-	-	0.013	-	-
0.13	0.11	84.62	0.13	-	-
1.26	1.09	86.51	1.26	1.02	80.95
12.63	10.60	83.89	12.63	10.024	81.08
63.13	53.19	84.25	63.13	51.69	81.87
126.3	108.64	86.22	126.3	103.16	81.68

Validity criteria fulfilled:

yes

Remarks:

The doubling time of frond number in the control was 2.236 days (53.66 hours) and 2.129 days (51.09 hours), corresponding to average specific growth rate of 0.310 and 0.326 d-1 during range finding study and the main study, respectively

Conclusions:

ErC50 (7d): 84.19 mg/l (nominal), based on frond number
EyC50 (7d): 36.49 mg/l (nominal), based on frond number

Executive summary:

The test item was assessed for Lemna sp., Growth Inhibition Test as per the OECD Guidelines for Testing of Chemicals (Section 2), Effects on Biotic Systems, Guideline No. 221, “Lemna sp., Growth Inhibition Test” adopted by the Council on 23 March 2006.

Lemna minor was exposed under static condition. Swedish Standard (SIS) was chosen as growth medium. Based on the results of range finding study, the main study was performed at the test concentration of 2.0, 4.7, 11.4, 27.3, 65.6 and 157.5 mg/l (corresponding to 1.6, 3.7, 9, 21.6, 52 and 124.7 mg a.i./l).

During main study, all fronds appeared normal on day 0, 3, 6 and on day 7 in control and at the tested concentrations of 2.0 and 4.7 mg/l. Some fronds appeared normal and clinical sign of chlorosis on day 6 and 7 at the tested concentrations of 11.4 mg/l.

Some fronds appeared normal and clinical sign of chlorosis on day 6 and some fronds appeared normal and clinical sign of chlorosis and colony breakup on day 7 at the tested concentrations of 27.3 and 65.6 mg/l.

Clinical signs of chlorosis and defoliation was observed on day 6 and clinical signs of chlorosis, defoliation and loss of buoyancy was observed on day 7 in the tested concentration of 157.5 mg/l.

The test concentration was analyzed on day 0 and day 7 and they resulted to be within the acceptable range ± 20 % of recovery to the nominal concentration.

The following results were obtained:

ErC50 (7d): 84.19 mg/l (nominal), based on fron number (95 % confidence limits of 68.10 to 104.09 mg/l)

EyC50 (7d): 36.49 mg/l (nominal), based on frond number (95 % confidence limits of 30.72 to 43.34 mg/l)

ErC50 (7d): 33.90 mg/l (nominal), based on dry weight (95 % confidence limits of 26.24 to 43.79 mg/l)

EyC50 (7d): 21.57 mg/l (nominal), based on dry weight (95 % confidence limits of 15.92 to 29.21 mg/l)

Conclusion

ErC50 (7d): 84.19 mg/l (nominal), based on frond number

EyC50 (7d): 36.49 mg/l (nominal), based on frond number

Description of key information

ErC50 (7d): 84.19 mg/l (nominal), based on frond number

EyC50 (7d): 36.49 mg/l (nominal), based on frond number

Key value for chemical safety assessment

EC50 for freshwater plants:

84.19 mg/L

Additional information

The test item was assessed for Lemna sp., Growth Inhibition Test as per the OECD Guidelines 221.

Lemna minor was exposed under static condition. Swedish Standard (SIS) was chosen as growth medium. Based on the results of range finding study, the main study was performed at the test concentration of 2.0, 4.7, 11.4, 27.3, 65.6 and 157.5 mg/l (corresponding to 1.6, 3.7, 9, 21.6, 52 and 124.7 mg a.i./l).

During main study, all fronds appeared normal on day 0, 3, 6 and on day 7 in control and at the tested concentrations of 2.0 and 4.7 mg/l. Some fronds appeared normal and clinical sign of chlorosis on day 6 and 7 at the tested concentrations of 11.4 mg/l.

Some fronds appeared normal and clinical sign of chlorosis on day 6 and some fronds appeared normal and clinical sign of chlorosis and colony breakup on day 7 at the tested concentrations of 27.3 and 65.6 mg/l.

Clinical signs of chlorosis and defoliation was observed on day 6 and clinical signs of chlorosis, defoliation and loss of buoyancy was observed on day 7 in the tested concentration of 157.5 mg/l.

The test concentration was analyzed on day 0 and day 7 and they resulted to be within the acceptable range ± 20 % of recovery to the nominal concentration.

The percent inhibitions in growth rate for frond number was found to be 0.3, 3.2, 7.8, 20.2, 31.2 and 75 % in the vessels with test item at the concentration of 2.0, 4.7, 11.4, 27.3, 65.6 and 157.5 mg/l, respectively. Percent inhibition in growth rate in dry weight was found to 0.7, 6.0, 6.5, 48.2, 65.9 and 94.7 % were observed in the vessels with test item at the concentrations of 2.0, 4.7, 11.4, 27.3, 65.6 and 157.5 mg/l, respectively.

The percent inhibition in yield in frond number was found to be 0.9, 8.0, 18.1, 40.5, 56.6 and 91.1 % in the vessels with test item at the concentrations 2.0, 4.7, 11.4, 27.3, 65.6 and 157.5 mg/l respectively. Percent inhibition in yield in dry weight was found to 1.6, 12.2, 12.9, 67.6, 81.2 and 97.6 % in the vessels with test item at the concentration of 2.0, 4.7, 11.4, 27.3, 65.6 and 157.5 mg/l respectively.