Disodium 5,5'-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-amino-4-hydroxynaphthalene-2-sulphonate]

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:

Study initiation date - 08 September 2015; Experiment start date - 09 September 2015; Experiment end date - 29 October 2015; Study completion date - 28 November 2015.

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)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identification: FAT 21021/E TE
Description: Solid
Appearance: Powder
Colour: Red
Batch number: 72 (Thailand)
CAS No: 52333-30-9
Purity: >75 %
Molecular weight: 1083.06 g/mol
Expiry date: 16 February 2020
Stability of test item: Stable
Storage conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

From the stock (147 mg/L) following concentrations were prepared. All the required test concentrations were prepared for 600 ml. For 0.006, 0.06, 0.6, 6, 30 and 60 mg/600 ml, 41 µl, 408 µl, 4.08 ml, 40.82 ml, 204.08 and 408.16 ml was taken from stock and made up to 600 ml with 20X-AAP medium, respectively. For Control, 20X-AAP medium was used. For range finding experiment the concentrations selected were 0.01, 0.1, 1, 10, 50 and 100 mg/L which is equivalent to 0.006, 0.06, 0.6, 6, 30 and 60 mg /600 ml, respectively. For Limit concentration 100 mg/L which is equivalent to 130 mg/1300 ml, 884.35 ml was taken from the stock solution and made up to 1300 ml with 20X-AAP medium. For Control, 20X-AAP medium was used.

Vehicle:

yes

Details on test solutions:

SOLUBILITY ANALYSIS
100 mg of the test item was dissolved and made up to 1 L with 20X-AAP medium. Test item particles were settled down. Acetone and Dimethyl formamide was used to check the solubility but the test item was not found to be soluble. Then lesser quantity of the test item (approximately 1 mg) was made up to 1 L with 20X-AAP medium, test solution was kept on hot plate and magnetic stirrer for approximately 30 minutes, but the test item particles were settled down. The test solution was ultrasonicated approximately for 30 minutes. However, there was no impact on the solubility of the test item. Therefore, based on the sponsor communication, WAF procedure was carried out as follows: 1.0003 g of the test item was dissolved and made up to 1 L with 20X-AAP medium. The test dispersion was ultrasonicated for 15 minutes and kept on continuous stirring for 5 days (at room temperature in the dark). After stirring, the dispersion was left to settle for approximately 60 minutes and the same was filtered through a preconditioned filter paper. The undiluted filtrate was tested as WAF and it was recorded that 147 mg/Las the limit of solubility of the test item in the 20X-AAP medium.

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).

Test temperature:

Start of the test:
Control: 22.9 °C
Treatment: 22.9 °C
End of the test:
Control: 24.5-24.8 °C
Treatment: 25.1-25.4 °C

pH:

Start of the test
Control: 7.50
Treatment: 7.43
End of the test
Control: 8.70-8.73
Treatment: 8.64-8.69

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): Limit test - 6 replicates
- 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: 8440-8680 Lux

TEST CONCENTRATIONS
-For Limit Test 1.0001 g of the test item was dissolved and made up to 1L of 20X-AAP medium in a 1L standard flask and then the test dispersion was kept on the magnetic stirrer up to 5 days. The test dispersion that was kept on the magnetic stirrer was removed and allowed to settle down for approximately one hour. Then the test dispersion was filtered through the filter paper and the filtrate was used for the dose formulation, which is should be 147 mg/L concentration as per the previous analytical results.
For Limit concentration 100 mg/L which is equivalent to 130mg/1300ml, From the stock solution, 884.35 ml was taken and made up to 1300 ml with 20X-AAP medium. 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:

yield

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:

yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

RANGE FINDING EXPERIMENT
BASED ON FROND NUMBERS: The percent inhibition on the average specific growth rate on day '7' for the test concentrations of 0.01, 0.1, 1, 10, 50 and 100 mg/L was calculated as 0.02 %, 0.02 %, 0.11 %, 0.16 %, 0.16 and 0.20 %, respectively.
BASED ON FROND DRY WEIGHT:
The percent inhibition on the average growth rate on day '7' for the concentration of 0.01, 0.1, 1, 10, 50 and 100 mg/L was calculated as 0.44 %, 0.22 %, 0.44 %, 0.22 %, 0.44 and 0.44 %, respectively.

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 of FAT 21021/E based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was found to be > 100 mg/L.
EyC50 of FAT 21021/E based upon the frond numbers observed for a period of ‘7’ days for Lemna gibba was found to be >100 mg/L.
ErC50 of FAT 21021/E based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was found to be >100 mg/L.
EyC50 of FAT 21021/E based upon the frond dry weight observed for a period of ‘7’ days for Lemna gibba was found to be >100 mg/L.
The no observed effect concentration (NOEC) was calculated as >100 mg/L.

Executive summary:

An GLP-compliant OECD guideline 221 study was performed to determine the effects of the test material FAT 21021/E. The exponentially growing culture of Lemna gibba (Duckweed) with an observation period of ‘7’ days. The effect of test item on the growth of the Lemna gibba culture compared with control was determined and the concentration that inhibited growth rate by 50 % (ErC50) and the concentration that inhibited yield by 50 % (EyC50) each of which in turn based upon frond number and frond dry weight were calculated. Based on the solubility of test item, 20X- AAP medium was selected as vehicle for dose formulation. For Limit concentration, Lemna gibba (Duckweed) were exposed to the test item (FAT 21021/E) at test concentration of 100 mg/L. Water Accommodated Fraction (WAF) was prepared and it was analytically recorded that 147 mg/L as the limit of solubility of the test item in the 20X-AAP medium. The WAF filtrate (147 mg/L) was considered as the stock from which 100 mg/L was prepared. 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.

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 0.02 %. From the yield recorded with the different 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 0.07 %. The no observed effect concentration (NOEC) was calculated as >100 mg/L.

 

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 0.53 %. The no observed effect concentration (NOEC) was calculated as >100 mg/L.

Description of key information

The average specific growth rate (frond number) had an EC50 of >100 mg/L. The NOEC was 32 mg/L and the LOEC was 100 mg/L.
The average specific growth rate (dry weight) had an EC50 of >100 mg/L. The NOEC was 100 mg/L.
The yield (frond number) had an EC50 of >100 mg/L. The NOEC was 32 mg/L and the LOEC was 100 mg/L.
The yield (dry weight) had an EC50 of >100 mg/L. The NOEC was 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:

100 mg/L

EC10 or NOEC for freshwater plants:

100 mg/L

Additional information

A GLP-compliant OECD guideline 221 study was performed to determine the effects of the test material FAT 21021/E. The exponentially growing culture of Lemna gibba (Duckweed) with an observation period of ‘7’ days. The effect of test item on the growth of the Lemna gibba culture compared with control was determined and the concentration that inhibited growth rate by 50 % (ErC50) and the concentration that inhibited yield by 50 % (EyC50) each of which in turn based upon frond number and frond dry weight were calculated. Based on the solubility of test item, 20X- AAP medium was selected as vehicle for dose formulation. For Limit concentration, Lemna gibba (Duckweed) were exposed to the test item (FAT 21021/E) at test concentration of 100 mg/L. Water Accommodated Fraction (WAF) was prepared and it was analytically recorded that 147 mg/L as the limit of solubility of the test item in the 20X-AAP medium. The WAF filtrate (147 mg/L) was considered as the stock from which 100 mg/L was prepared. After exposure, Lemna gibba was observed for frond count on days ‘3’, ‘5’ and ‘7’. Dry weight of the fronds was 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.

 

Frond Numbers:

From the average specific growth rate recorded with the test concentration 100mg/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 0.02 %. From the yield recorded with the different test concentration,100 mg/L, the concentration that inhibited 50% yield (EyC50) was calculated as >100mg/L. The percent inhibition on the yield on day ‘7’ for the test concentration of 100 mg/L was calculated as 0.07 %. The no observed effect concentration (NOEC) was calculated as >100 mg/L.

 

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 0.53 %. The no observed effect concentration (NOEC) was calculated as >100 mg/L.