Aluminium, 2-(1,3-dihydro-3-oxo-5-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic acid complex

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-09-2016 to 05-09-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed using standard test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Principles of method if other than guideline:

Aim of this study was to evaluate the nature of chemical when comes in contact with the test organism. Test was conducted according to the OECD guideline 201.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): C.I. Food Blue 1- Molecular formula (if other than submission substance): C16H8N2S2O8Na2- Molecular weight (if other than submission substance): 449.375 g /mol- Substance type: Organic- Physical state: Dark-blue powder or granules- Smiles notation (if other than submission substance): C1(=C2\C(c3cc(S(O)(=O)=O)ccc3N2)=O)\C(c2cc(S(O)(=O)=O)ccc2N1)=O.[Al+3]

Analytical monitoring:

not specified

Vehicle:

not specified

Details on test solutions:

The stock solution 100 mg/l was prepared by dissolving blue powder in OECD growth medium. The stock solution was kept in ultrasonic bath for 5 min. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM- Common name: - Strain: 86.81 SAG- Source (laboratory, culture collection): Institute of botany of the ASCR- Initial biomass concentration: 5x10(3) cells /ml - Method of cultivation: No data availableACCLIMATION - No data available- Acclimation period:- Culturing media and conditions (same as test or not):- Any deformed or abnormal cells observed:

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

23±2°C

pH:

Test at highest concentration: 7.5 did not changeControl: 8.2 changes to 7.6 during tet

Nominal and measured concentrations:

0, 12, 20, 35, 60 and 100 mg/l concentration were used

Details on test conditions:

TEST SYSTEM- Test vessel: 50 ml Glass vessel- Type (delete if not applicable): closed (with air permeable stopper)- Sample volume: 7.5 ml- Initial cells density: 5x10(3) cells/ml- No. of vessels per concentration (replicates): 3GROWTH MEDIUM- Standard medium used: yesOTHER TEST CONDITIONS- Adjustment of pH: No- Photoperiod: Continuous- Light intensity and quality: 6000-8000 lxEFFECT PARAMETERS MEASURED (with observation intervals if applicable) :- Determination of cell concentrations: microscope with counting chamber Cyrus I or electronic particle counter.- Other: ErC50 was calculated using non-linear regression by the software Prism 4.0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

187.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI was 108-326.7 mg/l

Results with reference substance (positive control):

- Results with reference substance valid- EC50: 0.69 mg/L (24 hours)

Reported statistics and error estimates:

ErC50 was calculated using non linear regression by the software Prism 4.0

Validity criteria fulfilled:

yes

Conclusions:

Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical C.I. Food Blue 1 the ErC50 was determine to be 187.8 mg/l.

Executive summary:

Aim of this study was to evaluate the nature of chemical test chemical when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201. The stock solution 100 mg/l was prepared by dissolving blue powder in OECD growth medium. The stock solution was kept in ultrasonic bath for 5 min. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. 0, 12, 20, 35, 60 and 100 mg/l concentration were used. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determine after an exposure period of 72 hrs. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical  C.I. Food Blue 1 the ErC50 was determine to be 187.8 mg/l. Based on the ErC50 value, it was concluded that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as per the CLP criteria.

Description of key information

Aim of this study was to evaluate the nature of chemical test chemical when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201. The stock solution 100 mg/l was prepared by dissolving blue powder in OECD growth medium. The stock solution was kept in ultrasonic bath for 5 min. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.0, 12, 20, 35, 60 and 100 mg/l concentration were used. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determine after an exposure period of 72 hrs. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical  C.I. Food Blue 1 the ErC50 was determine to be 187.8 mg/l. Based on the ErC50 value, it was concluded that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as per the CLP criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

187.8 mg/L

Additional information

Summarized result for the toxicity of test chemical (Cas no. 16521 -38 -3) on the growth and other biological activity of aquatic algae and cyanobacteria are as follows:

Aim of 1st experimental study 2018, was to evaluate the nature of chemical test chemical when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201. The stock solution 100 mg/l was prepared by dissolving blue powder in OECD growth medium. The stock solution was kept in ultrasonic bath for 5 min. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.0, 12, 20, 35, 60 and 100 mg/l concentration were used. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determine after an exposure period of 72 hrs. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical  C.I. Food Blue 1 the ErC50 was determine to be 187.8 mg/l. Based on the ErC50 value, it was concluded that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as per the CLP criteria.

First study was supported by second experimental report 2016. The study was designed to assess the toxic effects of the test compound aluminium, 2-(1,3-dihydro-3-oxo-5-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3- oxo-1H-indole-5- sulfonic acid complex on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution was prepared in aseptic condition. The test item aluminium, 2-(1,3-dihydro-3-oxo-5-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic acid complex was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/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 concentrations chosen were set up to the water solubility limit. 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 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. 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) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate. As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201. Based on the growth inhibition of green alga Chlorella vulgaris by the test chemical aluminium, 2-(1,3-dihydro-3-oxo-5-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic acid complex, the EC50 was determine to be > 200 mg/l.. Based on the EC50, it can be concluded that the test chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

Similar Short term toxicity study were carried out for 72hr to check the toxicity of test chemical on algae Pseudokirchneriella subcapitata. Algae was exposed the concentration of 30mg/L under static condition. Test chemical strongly stimulate the algal growth indicating the nutritional effects . Pseudokirchneriella subcapitata when exposed for 72 hr, 164% growth was observed at the test concentration of 30mg/L .Therefore the  NOEC for test chemical can be considered as 30mg/L. Based on the NOEC value the substance may not be classified.

Thus based on the overall studies it was concluded that the chemical was nontoxic and not classified as per the CLP classification criteria.