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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental results of read across chemicals
Justification for type of information:
Data for the target chemical is summarized based on the structurally similar read across chemicals.
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: As mention below
Principles of method if other than guideline:
WoE report is prepared based on short term toxicity to aquatic algae study:
1 and 2
GLP compliance:
not specified
Test material information:
Composition 1
Specific details on test material used for the study:
- Name of test material : Aluminum, 2-(1,3-dihydro-1,3-dioxo-2H-inden-2-ylidene)-1,2-dihydro-6,7-quinolinedisulfonate complexes
- Molecular formula : C18H11NO8S2.Al
- Molecular weight : 460.39 g/mol
- Smiles notation : O=C1c2ccccc2C(=O)C1=C1C=Cc2cc3c4cc2N{-}1.[Al]{3+}(.O{-}S3(=O)=O).O{-}S4(=O)=O
- Substance type : Organic
- Physical state : Solid
Analytical monitoring:
yes
Remarks:
In 1st study chemical was analytically monitorized
Vehicle:
no
Test organisms (species):
other: Chlorella vulgaris and Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
1. TEST ORGANISM
- Common name:green alga
- Strain:Chlorella vulgaris
- Source (laboratory, culture collection):laboratory
- Method of cultivation:BBM

ACCLIMATION
- Culturing media and conditions (same as test or not):The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrients, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM
- Any deformed or abnormal cells observed: no

2. - Common name: Raphidocelis subcapitata
- Method of cultivation: Algae were routinely cultured in Cyanobacteria BG-11 Freshwater Solution
Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Test temperature:
1. 24 °C ±2°C
2. 25±1 °C
pH:
1. 6.8 ±0.3
Nominal and measured concentrations:
1. Six test concentration were:
6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l, 100mg/l and 200mg/l
2.100 mg/L.
Details on test conditions:
1. - No. of vessels per concentration (replicates):Two replicates for each test concentration
- No. of vessels per control (replicates):Three replicates for Control
-Test solution: The test solution was prepared in aseptic condition. The test item was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring for 24 hours to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 104cells/ml. Care was taken to have a homogeneous solution for the experiment.
6.2. Test vessels: All the tests were carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60ml so that a sufficient amount of head space was left.
6.3. Replicates: For the assessment of algal growth, the study was conducted in replicates. The control vessel was maintained in triplicates as recommended in the OECD guideline and the test concentrations were selected in geometric series which were maintained in duplicates.
Incubation:
i) The temperature of the orbital shaking incubator was kept constant throughout the period of exposure of the experiment. The temperature was maintained at 24 ° C±2°C.
ii) The test vessels were incubated with a continuous, uniform (1500Lux).
iii) The pH of the control cultures needs to be noted during the study and the pH of the control medium should not increase by more than 1.5 units during the test.
iv) The orbital shaking incubator was set at a speed of 120 revolutions per minute throughout the study period. This is to provide constant shaking to the algal cells to keep them in suspension and to ensure that they do not settle down on the bottom of the test vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod:16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality:White Fluorescent Light

2. -Algae were exposed to the treatment in sterile 24-well plates with 2 mL of treatment solution to the microplate well, for three days.

- Algal stock solution containing 1.01E6 cells/mL was added (20 μL) to each well to obtain a starting algal density of 1E4 cells/mL.
Plates were incubated for 72 h at 25±1 °C.
on an orbital shaker (90 rpm) in a thermostatic chamber.
- Light intensity and quality: provided by a 2 W LED Unit for each plate.
Reference substance (positive control):
yes
Remarks:
In 2nd study potassium dichromate were used
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.079 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: For 1st study Other details not known
Key result
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
1.12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: For 2 study chemical was toxic
Results with reference substance (positive control):
2 .potassium dichromate 0.10– 1.8 mg/L
Reported statistics and error estimates:
1. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) was determined.

1.

Table 1: Showing the average cell count using Haemocytometer of the test vessels at an equal interval of 24hrs, 48hrs and 72hrs

Test vessels and test concentration

24 Hours

48 Hours

72 Hours

Control

Replicate 1

1.6 ×104

3.6 ×104

4.84 ×104

Replicate 2

1.6×104

4 ×104

5.52 ×104

Replicate 3

1.52 ×104

4 ×104

5.08 ×104

Test chemical

6.25mg/l

Replicate 1

3.24 ×104

2.28 ×104

1.96 ×104

Replicate 2

3.32 ×104

2.36 ×104

1.84 ×104

12.5mg/l

Replicate 1

3.08 ×104

2.32 ×104

1.72 ×104

Replicate 2

3.16×104

2.28 ×104

1.8 ×104

25mg/l

Replicate 1

3.12 ×104

2.95 ×104

1.68 ×104

Replicate 2

3.16 ×104

2.32 ×104

1.68 ×104

50mg/l

Replicate 1

3.08 ×104

2.28 ×104

1.72 ×104

Replicate 2

3 ×104

2.16 ×104

1.4 ×104

100mg/l

Replicate 1

2.92 ×104

1.92 ×104

1.48 ×104

Replicate 2

2.84 ×104

1.8 ×104

1.4 ×104

200mg/l

Replicate 1

2.84 ×104

1.68 ×104

1.36 ×104

Replicate 2

2.75 ×104

1.72 ×104

1.24 ×104

 

 

Table 2: Showing the values of average specific growth rate and percentage inhibition after an interval of 72 hours

 

CONTROL

 6.25mg/l

12.5mg/l

25mg/l

50mg/l

100mg/l

200mg/l

Average Specific Growth rate (µ )

R1

0.526

R1

0.224

R1

0.181

R1

0.173

R1

0.181

R1

0.131

R1

0.102

 

R2

0.569

R2

0.203

R2

0.196

R2

0.173

R2

0.112

R2

0.112

R2

0.071

 

R3

0.542

 

Mean of Avg. Specific growth rate

0.056

0.214

0.188

0.173

0.146

0.121

0.087

Percentage Inhibition (%I)

_

60.818

65.479

68.306

73.156

77.747

84.037

 

 

 

 

 

Table 3: Depicting pH values at 0 Hours and after 72 Hours of test item exposure to algae

 

Test vessels and test concentration

0 Hours

72 Hours

CONTROL

Replicate 1

6.70

6.78

Replicate 2

6.70

6.75

Replicate 3

6.70

6.84

Average

6.7

6.79

Test chemical

6.25mg/l

Replicate 1

6.74

6.95

Replicate 2

6.74

7.07

12.5mg/l

Replicate 1

6.75

6.88

Replicate 2

6.76

6.94

25mg/l

Replicate 1

6.82

7.50

Replicate 2

6.80

7.14

50mg/l

Replicate 1

6.82

6.93

Replicate 2

6.83

6.13

100mg/l

Replicate 1

6.84

6.95

Replicate 2

6.86

6.88

200mg/l

Replicate 1

6.90

7.05

Replicate 2

6.90

6.90

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
1. After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be 2.079 mg/l graphically and through probit analysis.
2. The effect concentration of test material on green algae on Raphidocelis subcapitata for growth rate after 72 h was observed to be 1.20 mg/l
Based on the above study chemical was consider as toxic and classified as aquatic chronic 2 as per the CLP classification criteria.
Executive summary:

Short term toxicity of Aluminum, 2-(1,3-dihydro-1,3-dioxo-2H-inden-2-ylidene)-1,2-dihydro-6,7-quinolinedisulfonate complexes (94891 -32 -4) on the growth and other biological activity of aquatic algae is predicted on the basis of it structurally and functionally similar read across chemicals. The studies are as mentioned below:

 

Short term toxicity study was performed to determine the toxic effect of the structurally and functionally similar read across chemicals on the aquatic algae and cyanobacteria. The effect of test item was studied on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l, 100mg/l and 200mg/l. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be 2.079 mg/l. Based on the EC50, chemical was consider as toxic and classified as aquatic chronic 2 as per the CLP classification criteria.

 

Similar study was performed for another structurally and functionally similar read across chemicals. The 72 h algal growth inhibition test with Raphidocelis subcapitata was done according to OECD 201 (2011) for the test chemical. Algae were routinely cultured at 25±1 °C with light in Cyanobacteria BG-11 Freshwater Solution. The culture was split twice a week, adding fresh medium. Algae were exposed to the treatment in sterile 24-well plates with 2 mL of treatment solution to the respective microplate well, for three days. The effect concentration of test material on green algae on Raphidocelis subcapitata for growth rate after 72 h was observed to be 1.20 mg/l.

 

Thus based on the overall studies for the structurally and functionally similar read across chemical for Aluminum, 2-(1,3-dihydro-1,3-dioxo-2H-inden-2-ylidene)- 1,2-dihydro-6,7- quinolinedisulfonate complexes (94891-32-4), it can be concluded that the test chemical was toxic and classifies as aquatic chronic 2 as per the CLP classification criteria.

Description of key information

1. After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be 2.079 mg/l graphically and through probit analysis.

2. The effect concentration of test material on green algae on Raphidocelis subcapitata for growth rate after 72 h  was observed to be 1.20 mg/l

Based on the above study chemical was consider as toxic and classified as aquatic chronic 2 as per the CLP classification criteria.

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
2.079 mg/L

Additional information

Short term toxicity of Aluminum, 2-(1,3-dihydro-1,3-dioxo-2H-inden-2-ylidene)-1,2-dihydro-6,7-quinolinedisulfonate complexes (94891 -32 -4) on the growth and other biological activity of aquatic algae is predicted on the basis of it structurally and functionally similar read across chemicals. The studies are as mentioned below:

 

Short term toxicity study was performed to determine the toxic effect of the structurally and functionally similar read across chemicals on the aquatic algae and cyanobacteria from study report. The effect of test item was studied on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l, 100mg/l and 200mg/l. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be 2.079 mg/l. Based on the EC50, chemical was consider as toxic and classified as aquatic chronic 2 as per the CLP classification criteria.

 

Similar study was performed for another structurally and functionally similar read across chemicals from peer reviewed journal. The 72 h algal growth inhibition test with Raphidocelis subcapitata was done according to OECD 201 (2011) for the test chemical. Algae were routinely cultured at 25±1 °C with light in Cyanobacteria BG-11 Freshwater Solution. The culture was split twice a week, adding fresh medium. Algae were exposed to the treatment in sterile 24-well plates with 2 mL of treatment solution to the respective microplate well, for three days. The effect concentration of test material on green algae on Raphidocelis subcapitata for growth rate after 72 h was observed to be 1.20 mg/l.

 

Thus based on the overall studies for the structurally and functionally similar read across chemical for Aluminum, 2-(1,3-dihydro-1,3-dioxo-2H-inden-2-ylidene)- 1,2-dihydro-6,7- quinolinedisulfonate complexes (94891-32-4), it can be concluded that the test chemical was toxic and classifies as aquatic chronic 2 as per the CLP classification criteria.