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Short term toxicity to fish:

Various studies from peer reviewed journals for the target chemical Fluorescein sodium (Cas no. 518-47-8) were reviewed to summarize the following information:

1.The Progressive Fish – Culturist, Vol. 31 (3), Pg. no. 139-142, 1969:

Short term toxicity study to 3 different species of fish was carried out for 96 hrs.The test organism include-

1.      Rainbow trout (Salmo gairdneri)

2.      Channel catfish (Ictalurus punctatus) and

3.      Bluegill (Lepomis macrochirus)

The dye fluorescein sodium (9-(0-carboxyphenyl)-6-hydroxy-3-isoxanthenone) was acquired from Matheson, Coleman, and Bell in a dry powder in a grade.The dye for the toxicity study was weighed separately for each vessel.Stock solutions of the water-soluble dye was prepared for concentrations of 5 and 100 p.p.m. in the antimycin-dye mixtures. Because antimycin is relatively insoluble in water, it was first dissolved in acetone and then diluted with water.The conc. of test chemical used for the study was 5 and 100 mg/l (5 and 100 ppm), respectively.

The toxicity study was performed at temperature of 21°C. LC50 value was determined at 24, 48 and 96 hrs.Based on mortality of test organism Rainbow trout, Channel catfish and Bluegill, the 24, 48 and 96 hrs LC50 values was given-

Rainbow trout:LC50 values at

24 hrs - 4198 mg/l

48 hrs - 3420 mg/l

96 hrs –1372 mg/l

 Channel catfish:LC50 values at

24 hrs -3828 mg/l

48 hrs -2826 mg/l

96 hrs –2267 mg/l

Bluegill:LC50 values at

24 hrs -5000 mg/l

48 hrs -4898 mg/l

96 hrs –3433 mg/l

2. The Journal of Toxicological Sciences, Vol. 3, Pg. no. 205-214, 1978:

Short term toxicity study toOryzias latipeswas carried out for 48 hrs and survival rate (in %) was determined. As no mortality of test animal was observed after 48 hrs, the LC0 value was found to be 3,000 mg/l. Thus, the survival rate of fish after 48 hrs was found to be 100%.

3. The Journal of Toxicological Sciences, Vol. 3, Pg. no. 205-214, 1978 and 1979:

Short term toxicity study toOryzias latipeswas carried out for 48 hrs and TLm (median tolerance limit) value was determined. The TLm value was found to be 3,000 mg/l at 48 hrs.

TLm (median tolerance limit) value consider to be median lethal concentration (LC50) thus based on mortality of test fishes, the 48 hr LC50 value was found to be 3,000 mg/l.

4. Environmental Monitoring and Assessment, Volume 38, Issue 1, pp 75-96; 1995:

Ecological assessments for both acute and chronic effects for the 3 life forms of fish was conduct.During experimentThe Median Lethal Concentration which is estimated to be lethal to 50% of fish in 96 hours exposure to the test chemical (LC50) was found to be 2200mg/l.

5. Hydrogeology Journal (2001) 9:321–325:

Ecotoxicological assessments were based on the determination of acute toxicity to fish according to DIN 38412, Part 31 (Deutsches Institut für Normung e.V. 1989). For the fish test, zebra fish were used as a modification of the guideline.

The Median Lethal Concentration which is found to be lethal to 0% of zebrafish in a 96 hours exposure to the test chemical (LC0) was found to be greater than 10mg/l

The fluorescent dyes that showed no effect upon either the genotoxicity or the ecotoxicity tests were classified by the Working Group as SAFE for use in water tracing thus Uranine is classified as safe for use in water tracing.

Thus on the basis of the above all experimental results from peer reviewed journals concluded that the target chemical Fluorescein sodium (Cas no.518-47-8) can be non- hazardous to fish.

Short term toxicity to aquatic invertebrates:

Various studies for the target chemical fluorescein sodium (518-47-8)along with its read across substance were reviewed to summarize the following information: 

The toxicity of dye, fluorescein sodium salt, to Daphnia pulex was assessed using acute level toxicological test (W.K. Walthall, J.D. Stark). The test organisms were obtained from cultures at or beyond the third filial (F3) generation. The final concentrations used in the generation of the acute mortality regression are 0, 150, 250, 400, 600 and 800 mg/liter. No control mortality occurred in the acute toxicity experiments. For each concentration tested, 25 ml of test solution was transferred into a 30-ml plastic cup and five neonates were transferred into the test container using a disposable glass pipette. Each concentration was replicated a minimum of four times. The testing regime was 48 h static, nonrenewal. The LC50for Fluorescein sodium was found to be 337 (278±403) mg/liter for Daphnia pulex neonates on the basis of the results obtained from mortality and the response predicted by regression analysis.

 

Short term toxicity study was conducted (Kelly A. Stockton, Christine M. Moffitt) to determine the aquatic toxicity of sodium fluorescein.The dose concentration used are0.2, 20.0, 50.0 71.0 100.0 141.0 and 200.0mg/L. The snail behavior was observed every 15 minutes during the first hour of exposure, and then every hour for the next 4 h. At the end of a 24h exposure, we poured the test system into small sieve to recover all snails, and then rinsed the snail three times with clean, aged laboratory water. The snails are then placed s into small cups with aged water, assessed mortality immediately, then again at 24 and 48 h. Each beaker was inspected with the aid of a dissecting microscope and watched for snail movement, and touched individual snails that were not active with a probe to elicit movement or tactile response. Each trial was replicated three times over two weeks for a total of 9 beakers tested at each concentration. Probit model and normal distribution were used for estimating the probability of mortality. Based on the experiment the median lethal concentration was determined to be 377 mg/L for snails.

Ecological assessments (Malcolm S. Field, Ronald G. Wilhelm, James F. Quinlan, and Thomas J. Aley) included both acute and chronic effects for the 3 life forms: fish, Cladocera daphnia, and algae. The concern levels identified by use of the SAR technique are expressed as high, moderate and low. These values are toxicity values, not exposure values. SAR results are generally regarded by EPA as being reliable.

Dyes with aquatic toxicity values below 1mg/l received a high concern rating, those with toxicity values ranging from 1-100mg/l received a moderate concern rating and those with toxicity values greater than 100mg/l received a low concern rating.

The Median Lethal Concentration which is estimated to be lethal to 50% of Cladocera (water flea) in 48 hours exposure to the test chemical (LC50) was found to be 165mg/l.

 

Toxicity tests (A marine toxicity assessment of selected tracer dyes using standard bioassays )on the tracer dyes (Fluorescein)

were carried out using standard bioassay using the Copepod acute toxicity test .The test were conducted according to ISO (1999) Guidelines - ISO 14669 Water Quality - Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea). These bioassays were carried out in 12 well polystyrene plates, each well contained 5mls of test solution and 5 T.battagliaicopepodites (approximately 4-6 days old). There were 4 replicates per concentration and the test was carried out over a period of 48 hours. The Tisbe were observed using a binocular microscope and mortality recorded. All parameters were within acceptable limits. A 48 h Zinc reference study was carried for quality assurance purposes alongside the tracer dyes to test the sensitivity of the species population for validity of the study. The results showed that EC50 for Fluorescein was determined to be 160.7 mg/l and the chemical was considered as not classified according to the CLP regulations.

Based on the studies on fluorescein sodium and read across, the test chemical was considered as a not classified to short term toxicity to aquatic invertebrates and also according to the CLP regulations.

Toxicity to aquatic algae and cyanobacteria

Various study and predicted data for the target chemical Sodium fluorescein (CAS no 518-47-8) were reviewed to summarize the following information:

The effect of test itemDisodium 2-(3-oxo-6-oxidoxanthen-9-yl) benzoate,CAS No. 518-47-8 was studied on the growth of fresh water green algaChlorella vulgaris[UERL Study Report, Sustainability Support Services (Europe) AB (Report no. 518-47-8/01/2016/AT), 2016].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 209.24 mg/l. Thus, based on the EC50 value, test substance can be considered asnon-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.

 

72 hrs aquatic toxicity study was predicted (SSS QSAR Prediction Model, 2016) to assess toxic effects of the test compound Sodium fluorescein

(CAS No. 518 -47 -8) and the results were predicted. The study was based on the effects of the test compound on Pseudokirchneriella subcapitata in a static fresh water system. The predicted data suggests theeffective concentration (EC50) for the test compound Sodium fluorescein (CAS No. 518 -47 -8) was estimated to be 107.5449 mg/l on the basis of population.Thus, based on this value. it can be concluded that the substance can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.

 

 

Ecotoxicological assessment of chemical Fluorescein was estimated (Malcolm S. Field et. al; 1995) to evaluate the toxicity of chemical to green algae. The concern levels identified by use of the SAR technique are expressed ad HIGH, MODERATE, LOW. These values are toxicity values, not exposure values. SAR results are generally regarded by EPA as being reliable.Dyes with aquatic toxicity values below 1mg/l received a HIGH concern rating; those with toxicity values ranging from 1-100mg/l received a MODERATE concern rating and those with toxicity values greater than 100mg/l received a LOW concern rating. The Median Effective Concentration which is estimated to be sublethal to 50% of Green Algae in 96 hours exposure to the test chemical (EC50) was determined to be <10 mg/l. As the EC50 for the test chemical lies between 1 – 100mg/l, it falls in moderate category in the aquatic toxicity ratings.Based on the EC50 value, it can be concluded that the substance can be classified as aquatic chronic category 2. Since the partition coefficient of the test chemical is 0.34, Fluorescein can be considered asnon-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.

 

Based on the overall reported results of study and predicted data for target substance, it can be concluded that the substance can be considered as non-toxic to aquatic organisms and thus can be classified as not hazardous as per the CLP criteria.

Toxicity to micro-organisms:

Toxicity to micro-organisms study of test chemical Fluorescein sodium was conducted onStaphylococcus aureusof enterotoxigenic strain ATCC 13565.

 

For the preparation of dye solution, a test portion (approximately 10 mg) was placed in a 10-ml volumetric flask, which was wrapped in aluminum foil, and dissolved by adding aqueous sodium hydroxide (approx. 0.4% NaOH) (1 ml) and water (9 ml).

 

Inoculum was prepared inbrain heart infusion broth (BHI). Cultures were incubated shaking (200 rpm) at 37°C. All the experiments were conducted under conditions of standard room illumination (fluorescent ceiling light, Sylvania Octron 4100K) in tubes containing 3 ml of medium. Growth was analyzed by measuring turbidity (absorbance at a wavelength of 660 to 680 nm) with a spectrophotometer (Spectronic 21D). Experiments were initiated with ̴0.2 ml of inoculum from a fresh culture grown aerobically at an optical density (OD) of ̴1. Colony counts were measured on BHI plates.

 

For the study,a 300-µl aliquot of dye solution (1 mg/ml i.e 100 mg/l) was mixed with 0.7 ml of BHI and then added to a tube of diluted culture. The tubes were aerated vigorously, and the OD was measured every 45 min to check culture growth. The survival rate was measured by plating serial dilutions of a sample taken during the experiment on BHI plates.

 

During the study period of 250 mins, OD was measured every 45 mins to check culture growth.

 

Experiment was performed in triplicate on separate days to ascertain reproducibility.

 

As the test substance do not have any activity againstS. aureusi.e, no difference was observed between the treated test tubes and the control tubes, the NOEC value was found to be100 mg/l.

Other supporting study of target chemical(Nepalese Journal of Ophthalmologyl, Vol. 4(7), Pg. no. 80-83, 2012.) indicate thatinhibition in growth of test organismsS. aureus,Coagulase negativeStaphylococcus, E. coliandP. aeruginosa,the MIC value was found to be 5000 mg/l.

Thus overall both experimental studies indicate that the test substance Fluorescein sodium is non toxic to micro-organisms.