Reaction mass of sodium 3-[[4-[(4-ethoxy-m-tolyl)azo]-1-naphthyl]azo]benzenesulphonate and sodium 3-[[4-[(4-ethoxyphenyl)azo]-1-naphthyl]azo]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 February 10 to March 17, 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:

validated analytical method

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

adopted on 23 March 2006

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The 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 7 days) in order to determine the concentration of test item during the test.
About 10 ml of sample was collected from all test concentration in duplicates.

Vehicle:

no

Details on test solutions:

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 suspension, 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 was used as the test system for the study.
- Pre-culture: from healthy axenic culture of Lemna minor, pre-culture was prepared of desired colonies to provide the inoculum for test cultures.

BREEDING CONDITIONS
- Subculturing: Lemna minor culture is periodically subcultured.
- Illumination: 6500 to 10000 lux
- Temperature: 22 to 26 °C.
- Medium: Lemna minor was maintained separately on Steinberg medium and Swedish Standard (SIS) media.

PRECULTURE
- Test vessels: 10 beakers containing 100 ml of culture medium.
- Duration: preculture was initiated 7 days before start of the test.
- Incubation conditions: pre cultures are incubated under same conditions as the test cultures.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Test temperature:

Test vessels: 24.7 - 25.3 °C
Control: 24.4 - 25.3 °C

pH:

Test vessels: 6.82 - 6.94
Control: 6.37 - 6.53

Nominal and measured concentrations:

Nominal 100.0 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: sterilized, glass beakers of 150 ml capacity.
- Type of cover: the test vessels were covered with perforated transparent sheet to minimize accidental contamination, while allowing necessary air exchange.
- No. of fronds per colony: the initial Lemna minor colonies comprising 3 fronds; each test replicates contain 9 fronds.
- No. of vessels per concentration: 6 replicates.
- No. of vessels per control: 6 replicates.

GROWTH MEDIUM
- Standard medium used: yes; Swedish Standard (SIS). Stock solution was prepared freshly during every renewal period in the Swedish Standard (SIS) growth medium.

OTHER TEST CONDITIONS
- Light intensity and quality: 6671 to 6981 lux during pre-culture and dose-range finding test and 9641 to 9864 lux during the main test, 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 were recorded.
- Dry Weight: dry weight was determined at test start from representative sample of the inoculum culture. Consistent with the approach taken in the test. 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. Any root fragments were included and dry weight was measured.

ENVIRONMENTAL PARAMETERS
The temperature (culture medium) and light intensity (at points the same distance from the light source as the Lemna fronds) was monitored once in a day during pre-culture and exposure during range finding study and limit test. The pH of the test concentrations (fresh and spent) were measured on day 0 and on day 7 at the termination of the test during range finding and limit test.

RANGE-FINDING STUDY
- Test concentrations: 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/l.
- Replicates: 2 replicates in each concentration and 4 replicates in control.
- Results used to determine the conditions for the definitive study: range finding study indicates that the test item has no toxic effects at concentrations up to 100 mg/l, hence limit test was undertaken as the main study.

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 2.410 days (57.8 hours) and 2.442 days (58.6 hours), corresponding to average specific growth rate of 0.288 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).

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

MAIN STUDY
During the main test, the initial number of frond number in all treatment replicates on day 0 was 9. The mean frond numbers on Day 7 was 65.8 and 64.5, in the control and in the treated group, reapectively.
All fronds appeared normal on day 0, 3, 6 and on day 7 in control and in all the vessels with test item (i.e. at the concentration of 100 mg/l).
Dry weight was determined at test start from representative sample of the inoculum culture. On day 0 dry weight was found to be 0.0269 g; at the end of the test the mean dry weight was found to be 0.170 g in control and 0.160 g were observed in the vessels with test item (at the concentration of 100.0 mg/l).

The average specific growth rate from day 0 to day 7, for frond numbers, was found to be 0.284 in control and 0.281 in the vessels with test substance (i,e, at the concentration of 100.0 mg/l).
The average specific growth rate from day 0 to day 7, for dry weight, was found to be 0.259 in control and 0.254 in the vessels with test substance (i,e, at the concentration of 100.0 mg/l).

The NOEC (No observed effect concentration) for all parameters related to frond number and dry weight was found to be equal/higher than 100.0 mg/l.

MEASURED CONCENTRATIONS
During the range finding and limit test, samples from the test concentration was analyzed on day 0 and on day 7 and the results reported that test samples of test item are found to be within the acceptable range ± 20 % recovery to the nominal concentration.

DOSE RANGE FINDING STUDY
The mean frond numbers on day 7 was 67.5 in the control and 63.0, 63.0, 63.0, 61.0, 61.0 and 59.0 in the vessels with test item concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100 mg/l.
All fronds appeared normal on day 0, 3, 6 and on day 7 in control and in all the vessels with test item (i.e. at the concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100 mg/l).
Dry weight was determined at test start from representative sample of the inoculum culture. On day 0 dry weight was found to be 0.0224 g; at the end of the test the mean dry weight was found to be 0.368 g in control and 0.364, 0.349, 0.352, 0.347, 0.349 and 0.347 in the vessels with test item at the concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/l.

The average specific growth rate from day 0 to day 7, for frond numbers, was found to be 0.288 in control and 0.278, 0.278, 0.277, 0.273, 0.273 and 0.268 in the vessels with test item at the concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/l.
The average specific growth rate from day 0 to day 7, for dry weight, was found to be 0.400 in control and 0.398, 0.392, 0.394, 0.392, 0.392 and 0.391 in the vessels with test item at the concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/l.

Results with reference substance (positive control):

The ErC50 values for growth rate of frond number and dry weight were calculated to be 3.66 mg/l and 3.26 mg/l, respectively.
The ErC50 value for the frond number of 3.66 is in good agreement with the results of an international ring test between 2.7 and 3.4 mg/l (n=52).

INHIBITION IN GROWTH RATE AND YIELD DURING MAIN STUDY

Test conc. (mg/l)	% Inhibition Growth Rate		% Inhibition Yield
	Frond number	Dry weight	Frond number	Dry weight
0	0	0	0	0
100	1.1	1.9	2.3	6.9

INHIBITION IN GROWTH RATE AND YIELD DURING RANGE FINDING STUDY

Test conc. (mg/l)	% Inhibition Growth Rate		% Inhibition Yield
	Frond number	Dry weight	Frond number	Dry weight
0	0	0	0	0
0.01	3.4	0.4	7.7	1.6
0.1	3.5	1.9	7.7	5.8
1	3.6	1.5	7.7	5.0
10	5.0	2.1	11.1	6.4
50	5.0	1.8	11.1	5.8
100	6.9	2.1	14.5	6.5

MEASURED CONCENTRATIONS

MAIN STUDY

Nominal Concentration (mg/l)	Acutal concentration (mg/l)	% Recovery	Actual concentration (mg/l)	% Recovery
	Day 0		Day 7
0	-	-	-	-
100	102.5	102.5	99.43	99.43

-: Not applicable

RANGE FINDING STUDY

Nominal Concentration (mg/l)	Actual concentration (mg/l)	% Recovery	Actual concentration (mg/l)	% Recovery
	Day 0		Day 7
0	-	-	-	-
0.01	-	-	-	-
0.1	-	-	-	-
1	0.98	98.0	0.94	93.5
10	9.61	96.1	9.78	97.8
50	48.05	96.1	48.14	96.28
100	96.33	96.33	95.94	95.94

-: Not applicable

Validity criteria fulfilled:

yes

Remarks:

doubling time of frond number in the control was 2.410 days and 2.442 days, corresponding to average specific growth rate of 0.288 and 0.284 d-1 during range finding study and main study, respectively

Conclusions:

ErC50 (7d) > 100 mg/l, based on frond number and dry weight
EyC50 (7d) > 100 mg/l, based on frond number and dry weight

Executive summary:

The test item was assessed for Lemna sp., Growth Inhibition Test as per the OECD Guidelines for Testing of Chemicals 221, “Lemna sp., Growth Inhibition Test” adopted on 2006. The Lemna minor was selected as test organism and exposed to the substance under static condition.

Based on the results of range finding study, a limit test was performed as main experiment at the test concentration of 100.0 mg/l. Lemna minor were observed for frond number on day 0, 4 and on day 7. Observation for fronds was made at beginning of test and on day 3, 6 and at end of the test.

All fronds appeared normal during 7 days of exposure in control and at the test concentration of 100 mg/l during limit test.

The NOEC (No observed effect concentration) for all parameters related to frond number and dry weight was found to be equal/higher than 100.0 mg/l. Therefore, the ErC50 for the test endpoint growth rate of the primary measurement variable frond number and dry weight was found to be higher than 100.0 mg/l, as well as the EyC50 for the test endpoint yield of the primary measurement variable frond number and dry weight.

Conclusion

ErC50 (7d) > 100 mg/l, based on frond number and dry weight

EyC50 (7d) > 100 mg/l, based on frond number and dry weight

Description of key information

ErC50 (7d) > 100 mg/l, based on frond number and dry weight

EyC50 (7d) > 100 mg/l, based on frond number and dry weight

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:

100 mg/L

Additional information

The test item was assessed for Lemna sp., Growth Inhibition Test as per the OECD Guidelines for Testing of Chemicals 221, “Lemna sp., Growth Inhibition Test” adopted on 2006. The Lemna minor was selected as test organism and exposed to the substance under static condition.

Based on the results of range finding study, a limit test was performed as main experiment at the test concentration of 100.0 mg/l. Lemna minor were observed for frond number on day 0, 4 and on day 7. Observation for fronds was made at beginning of test and on day 3, 6 and at end of the test.

All fronds appeared normal during 7 days of exposure in control and at the test concentration of 100 mg/l during limit test.

The NOEC (No observed effect concentration) for all parameters related to frond number and dry weight was found to be equal/higher than 100.0 mg/l. Therefore, the ErC50 for the test endpoint growth rate of the primary measurement variable frond number and dry weight was found to be higher than 100.0 mg/l, as well as the EyC50 for the test endpoint yield of the primary measurement variable frond number and dry weight.