Trisodium bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]chromate(3-)

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:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

November 22, 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

100.04 mg of the test item was dissolved and made up to 100 ml with test medium (20X-AAP) in standard flask to make a stock concentration of 1 mg/ml. From the stock following concentrations were prepared. All the required test concentrations were prepared for 600 ml. For, 0.06, 0.6, 6 and 60 mg/600ml, 60 µl, 600 µl, 6 ml and 60 ml was taken from stock and made up to 600 ml with 20X-AAP medium respectively. For 15mg/600ml, 15 mg of test item was directly added to the 600ml 20X-AAP medium. For Control, 20X-AAP medium was used.

For Limit concentration ,100 mg/L which is equivalent to 130mg/1300ml. 130.04 mg of the test item was dissolved and made up to 1300 ml of 20X-AAP medium. From this 1200 ml of stock was directly used. For Control, 20X-AAP medium was used.

Vehicle:

yes

Details on test solutions:

For Limit concentration ,100 mg/L which is equivalent to 130mg/1300ml. 130.04 mg of the test item was dissolved and made up to 1300ml of 20X-AAP medium. From this 1200 ml of stock was directly used. For Control, 20X-AAP medium was used.

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Strain: Lemna gibba (Duckweed)
- Source (laboratory, culture collection): In-house culture, RCC Laboratories India Private Limited (Originally procured by Department of Plant Biology & Plant Biotechnology Madras Christian College, Chennai)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Post exposure observation period:

Frond number in each replicate was counted on days ‘0’, ‘3’, ‘5’ and ‘7’.
A representative sample (approximately 12 fronds) of pre-culture was checked for its average dry weight on day ‘0’ of exposure and dry weight of plant material from each test and control vessel on day ‘7’ of exposure.
Observation was performed on day ‘0’, ‘3’, ‘5’ and ‘7’ for the abnormal appearance or any developmental changes due to the effect of the test item (if any).

Hardness:

None

Test temperature:

Temperature in test vessel (°C) Start of the test
Control : 23.5
Treatment : 23.5
End of the test
Control : 24.8-25.1
Treatment : 25.1-25.3

pH:

Medium pH range Start of the test
Control : 7.54
Treatment : 7.48
End of the test
Control : 8.69-8.72
Treatment : 8.72-8.75

Nominal and measured concentrations:

Range finding experiment: 0.1, 1, 10, 25, 50 and 100 mg/l.
Main experiment: 100 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 ml Glass Beaker
- No. of organisms per vessel: 12 fronds (3 plants)
- No. of vessels per concentration (replicates): Main Experiment - 3 replicates for each concentration
- No. of vessels per control (replicates): Control - 6 Replicates.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Start of the test


- Photoperiod: Continuous Light
- Light intensity and quality: 8460-8650 Lux

TEST CONCENTRATIONS
-For range finding experiment the concentrations selected were 0.1, 1, 10, 25, 50 and 100 mg/L.
For Limit concentration ,100 mg/L which is equivalent to 130mg/1300ml. 130.04 mg of the test item was dissolved and made up to 1300 ml of 20X-AAP medium. From this 1200 ml of stock was directly used. For Control, 20X-AAP medium was used.

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

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:

other: Frond Dry Weight; Yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Results with reference substance (positive control):

ErC50 of 3,5-Dichlorophenol based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 4.30 mg/L with a lower confidence limit of 4.15 mg/L and the upper confidence limit of 4.46 mg/L.
EyC50 of 3,5-Dichlorophenol based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was calculated as 2.38 mg/L with a lower confidence limit of 2.29 mg/L and the upper confidence limit of 2.48 mg/L.
ErC50 of 3,5-Dichlorophenol based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was calculated as 3.02 mg/L with a lower confidence limit of 2.89 mg/L and the upper confidence limit of 3.16 mg/L.
EyC50 of 3,5-Dichlorophenol based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was calculated as 1.53 mg/L with a lower confidence limit of 1.46 mg/L and the upper confidence limit of 1.59 mg/L.

Reported statistics and error estimates:

Mean coefficient of variation of average specific growth rate in replicate control and treated cultures were calculated. No analysis was done as the study is concluded with the Limit Test.

Validity criteria fulfilled:

yes

Conclusions:

ErC50 (7 days) > 100 mg/l

Executive summary:

Method

In a GLP compliant OECD guideline 221 followed study, Lemna gibba (Duckweed) were exposed to the test item at test concentration 100 mg/L. Initially on day ‘0’, 12 fronds were inoculated in beaker containing 200 ml of test medium (20X-AAP medium). Three replicates for each test concentration and six replicates for control were maintained. The test item was formulated in 20X-AAP medium. Prior to exposure,Lemna gibba was pre-cultured in the 20X-AAP medium for period of ‘8’ days and the pre-culture was found healthy.

After exposure,Lemna gibba was observed for frond count on days ‘3’, ‘5’ and ‘7’. Dry weight of the fronds were recorded at start (pre-culture representative samples) and at the end of the test.

Fronds were observed for their appearance on days ‘3’, ‘5’ and ‘7’. All fronds were found normal and healthy in all replicates of control and test concentration.

The doubling time (Td) of frond number in the control group was calculated as 1.608 day-1.

Results

Frond Numbers:

From the average specific growth rate recorded with the test concentration 100 mg/l the test concentration that inhibited 50 % growth (ErC50) was calculated as > 100 mg/l.

The percent inhibition on the average specific growth rate on day ‘7’ for the test concentration of 100 mg/l was calculated as 0.00 %.

From the yield recorded with the test concentration,100 mg/l, the concentration that inhibited 50 % yield (EyC50) was calculated as >100 mg/l.

The percent inhibition on the yield on day ‘7’ for the test concentration of 100 mg/l was calculated as 0.00 %.

Frond Dry Weight:

From the average specific growth rate recorded with the test concentration, 100 mg/l, the concentration that inhibited 50 % growth (ErC50) was calculated as > 100 mg/l.

The percent inhibition on the average growth rate on day ‘7’ for the concentration of 100 mg/l was calculated as 0.21 %.

From the yield recorded with the test concentration,100 mg/l the concentration that inhibited 50% yield (EyC50) was calculated as > 100 mg/l.

The percent inhibition on the yield on day ‘7’ for the test concentration of 100 mg/L was calculated as 0.71 %.

Conclusion

ErC50 based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was found to be > 100 mg/L.

EyC50 based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was found to be > 100 mg/L

ErC50 based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was found to be > 100 mg/L

EyC50 based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was found to be >100 mg/L.

Description of key information

ErC50 > 100mg/l

EyC50 >100mg/l

Additional information

No studies on the "Toxicity to aquatic plants other than algae" are available for the substance in itself nevertheless a study have been conducted with an analogue molecule (Similar Substance 01). Further information are reported in the Read Across justification attached to section 13.

In a GLP compliant OECD guideline 221 followed study, Lemna gibba (Duckweed) were exposed to the test item at test concentration 100 mg/l. Three replicates for each concentration and six replicates as a control were maintained. Observations and frond number counts were made on days ‘0’, ‘3’, ‘5’ and ‘7’. The following results were observed: ErC50 based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was found to be > 100 mg/l; EyC50 based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was found to be > 100 mg/l; ErC50 based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was found to be > 100 mg/l; EyC50 based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was found to be >100 mg/l.  All fronds were found normal and healthy in all replicates of control and test concentration after 7 days leading to the end point being determined as ErC50 > 100mg/l and EyC50 > 100mg/l.