Reaction mass of Cobaltate(2-), [2-[[[4-hydroxy-3-[[2-oxo-1-[(phenylamino)carbonyl]propyl]azo]phenyl]sulfonyl]amino]benzoato(3-)][2-[[2-hydroxy-5-[(phenylamino)sulfonyl]phenyl]azo]-3-oxo-N-phenylbutanamidato(2-)]-, disodium and Cobaltate(3-), bis[2-[[[4-hydroxy-3-[[2-oxo-1-[(phenylamino)carbonyl]propyl]azo]phenyl]sulfonyl]amino]benzoato(3-)]-, trisodium and sodium bis[2-[[2-hydroxy-5-[(phenylamino)sulphonyl]phenyl]azo]-3-oxo-N-phenylbutyramidato(2-)]cobaltate(1-)

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:

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

Adequacy of study:

key study

Study period:

From September the 09th to October the 28th, 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

Details for read across approach are included into the IUCLID section 13.2.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

Adopted on 26 March 2006

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analytical verification of the test item in the exposure medium samples were carried out in the test item concentrations of low (10 mg/l), intermediate (33.75 mg/l) and high (113.91 mg/l) on days day 0 and day 7.

Details on test solutions:

100 mg of the test item was added to 1l of 20X- AAP medium in a 1l standard flask, ultrasonicated for 15 minutes 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 should be 203 mg/l concentration as per the previous analytical results.
20X- AAP medium was selected as vehicle based on the solubility check of the test item.
From the stock solution, each required test concentration was prepared as follows: 31.03 ml, 44.33 ml, 75.37 ml, 103.89 ml, 149.66 ml, 224.43 ml and 350.69 ml were taken and made up to 600 ml except 6.3, 21.09 and 71.19 mg/625 ml with 20X-AAP medium. For Control, 20X-AAP medium was used.

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Strain: Lemna gibba (Duckweed)
- Source: in-house culture, testing facility.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

Test vessels: 22.9 - 25.5 °C
Control: 22.9 - 24.9 °C

pH:

Test vessels: 7.38-8.77
Control: 7.5 - 8.79

Nominal and measured concentrations:

Nominal 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 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: 3 replicates
- No. of vessels per control: 6 Replicates.

GROWTH MEDIUM
- Standard medium used: 20X-AAP medium.

OTHER TEST CONDITIONS
- Photoperiod: Continuous Light
- Light intensity and quality: 8520 - 8640 Lux

EFFECT PARAMETERS MEASURED
Frond number in each replicate was counted on days 0, 3, 5 and 7.
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.

RANGE FINDING TEST
- No. of vessels per concentration: 3 replicates
- No. of vessels per control: 6 Replicates.
- Concentrations: 0.01, 0.1, 1, 10, 50 and 100 mg/l.

VALIDITY CRITERIA
For the test to be valid, the doubling time of frond number in the control has to be less than 2.5 days (60 h), corresponding to approximately a seven-fold increase in seven days and an average specific growth rate of 0.275 d-1.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

86.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

87.38 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

51.84 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

51.08 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

ErC50 (7d): 86.90 mg/l (nominal), based on frond numbers
EyC50 (7d): 51.84 mg/l (nominal), based on frond numbers

From the average specific growth rate recorded, the test concentration that inhibited 50 % growth (ErC50), based on frond number, was calculated as 86.90 mg/l; the percent inhibition on the average specific growth rate on day 7 for the test concentrations of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 was calculated as 0.00 %, 0.23 %, 4.59 %, 7.34 %, 20.92 %, 41.38 % and 66.13 % respectively.  
From the yield recorded, the concentration that inhibited 50 % yield (EyC50), based on frond number, was calculated as 51.84 mg/l. The percent inhibition on the yield on day 7 for the test concentrations of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/l was calculated as 0.00 %, 0.72 %, 13.59 % 20.89 %, 49.21 %, 74.96 % and 90.84 % respectively.  
The no observed effect concentration (NOEC) was calculated as 15 mg/l and the low observed effect concentration (LOEC) was calculated as 25.5 mg/l.

From the average specific growth rate recorded, the concentration that inhibited 50 % growth (ErC50), based on dry weight, was calculated as 87.38 mg/l. The percent inhibition on the average growth rate on day 7 for the concentration of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/l was calculated as 0.12 %, 0.31 %, 4.13 %, 6.15 %, 17.23 %, 53.41 % and 59.12 % respectively.  
From the yield recorded, the concentration that inhibited 50 % yield (EyC50), on the basis of dry weight, was calculated as 51.08 mg/l. The percent inhibition on the yield on day 7 for the test concentrations of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/l was calculated as 0.44 %, 1.06 %, 13.16 %, 18.99 %, 44.86 %, 85.87 % and 88.96 % respectively.
The no observed effect concentration (NOEC) was calculated as 15 mg/l and the low observed effect concentration (LOEC) was calculated as 25.5 mg/l.

MEASURED CONCENTRATIONS
Day 0 mean recovery results: 97.95, 97.37 and 97.60 % for 10, 33.75 and 113.91 mg/l, respectively.
Day 7 mean recovery results: 97.27, 96.58 and 96.75 % for 10, 33.75 and 113.91 mg/l, 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.

Validity criteria fulfilled:

yes

Remarks:

doubling time of frond number in the control was 1.608/d, corresponding to around 20.42 fold increase and average growth rate was 0.431/d over the test period

Conclusions:

ErC50 (7d): 86.90 mg/l (nominal), based on frond numbers
EyC50 (7d): 51.84 mg/l (nominal), based on frond numbers

Executive summary:

Following OECD guideline 221, Lemna gibba (Duckweed) were exposed to the test item at different test concentrations of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 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. 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 concentrations except 113.91 mg/l where necrosis and root filaments were turned brown colour on day 7.  

From the average specific growth rate recorded, the test concentration that inhibited 50 % growth (ErC50), based on frond number, was calculated as 86.90 mg/l; the percent inhibition on the average specific growth rate on day 7 for the test concentrations of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 was calculated as 0.00 %, 0.23 %, 4.59 %, 7.34 %, 20.92 %, 41.38 % and 66.13 % respectively.  

From the yield recorded, the concentration that inhibited 50 % yield (EyC50), based on frond number, was calculated as 51.84 mg/l. The percent inhibition on the yield on day 7 for the test concentrations of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/l was calculated as 0.00 %, 0.72 %, 13.59 % 20.89 %, 49.21 %, 74.96 % and 90.84 % respectively.  

The no observed effect concentration (NOEC) was calculated as 15 mg/l and the low observed effect concentration (LOEC) was calculated as 25.5 mg/l.  

From the average specific growth rate recorded, the concentration that inhibited 50 % growth (ErC50), based on dry weight, was calculated as 87.38 mg/l. The percent inhibition on the average growth rate on day 7 for the concentration of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/l was calculated as 0.12 %, 0.31 %, 4.13 %, 6.15 %, 17.23 %, 53.41 % and 59.12 % respectively.  

From the yield recorded, the concentration that inhibited 50 % yield (EyC50), on the basis of dry weight, was calculated as 51.08 mg/l. The percent inhibition on the yield on day 7 for the test concentrations of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/l was calculated as 0.44 %, 1.06 %, 13.16 %, 18.99 %, 44.86 %, 85.87 % and 88.96 % respectively.

The no observed effect concentration (NOEC) was calculated as 15 mg/l and the low observed effect concentration (LOEC) was calculated as 25.5 mg/l.  

Analytical verification of the test item in the exposure medium samples were carried out in the test item concentrations of low (10 mg/l), intermediate (33.75 mg/l) and high (113.91 mg/l) on days day 0 and day 7 and the measured concentrations resulted to be in the range of ± 20 % of the nominal ones.

Conclusion

ErC50 (7d): 86.90 mg/l (nominal), based on frond numbers

EyC50 (7d): 51.84 mg/l (nominal), based on frond numbers

Description of key information

ErC50 (7d): 86.90 mg/l (nominal), based on frond numbers

EyC50 (7d): 51.84 mg/l (nominal), based on frond numbers

Key value for chemical safety assessment

EC50 for freshwater plants:

86.9 mg/L

EC10 or NOEC for freshwater plants:

15 mg/L

Additional information

There is no information about the potential toxicity to aquatic plants of Acid Yellow 235, thus the available data on structural analogous Similar Substance 01 has been taken into account. The read across approach can be considered as reliable and adequate for the purpose; details and explanations are detailed in the report attached to the IUCLID section 13.2.

Following OECD guideline 221, Lemna gibba were exposed to the Similar Substance 01, at different test concentrations of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/l. From the average specific growth rate recorded, the test concentration that inhibited 50 % growth (ErC50), based on frond number, was calculated as 86.90 mg/l; the percent inhibition on the average specific growth rate on day 7 for the test concentrations of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 was calculated as 0.00 %, 0.23 %, 4.59 %, 7.34 %, 20.92 %, 41.38 % and 66.13 % respectively.  

From the yield recorded, the concentration that inhibited 50 % yield (EyC50), based on frond number, was calculated as 51.84 mg/l. The percent inhibition on the yield on day 7 for the test concentrations of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/l was calculated as 0.00 %, 0.72 %, 13.59 % 20.89 %, 49.21 %, 74.96 % and 90.84 % respectively.  

The no observed effect concentration (NOEC) was calculated as 15 mg/l and the low observed effect concentration (LOEC) was calculated as 25.5 mg/l.  

From the average specific growth rate recorded, the concentration that inhibited 50 % growth (ErC50), based on dry weight, was calculated as 87.38 mg/l. The percent inhibition on the average growth rate on day 7 for the concentration of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/l was calculated as 0.12 %, 0.31 %, 4.13 %, 6.15 %, 17.23 %, 53.41 % and 59.12 % respectively.  

From the yield recorded, the concentration that inhibited 50 % yield (EyC50), on the basis of dry weight, was calculated as 51.08 mg/l. The percent inhibition on the yield on day 7 for the test concentrations of 10, 15, 25.5, 33.75, 50.63, 75.94 and 113.91 mg/l was calculated as 0.44 %, 1.06 %, 13.16 %, 18.99 %, 44.86 %, 85.87 % and 88.96 % respectively.

The no observed effect concentration (NOEC) was calculated as 15 mg/l and the low observed effect concentration (LOEC) was calculated as 25.5 mg/l.