Trisodium 8-hydroxypyrene-1,3,6-trisulphonate

Endpoint summary

Description of key information

Short term toxicity to fish:

Study was conducted to access the effect of test chemical on the growth of fish Danio rerio. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test)The test substance was soluble in water. Therefore, the test solution was prepared by dissolving 400 mg of the test substance in 1 liters of potable water (passed through reverse osmosis system) with continuous stirring. This test solution was then added to the remaining three liters of water for achieving test concentrations of 100 mg/L.This test solution was then added to the remaining three liters of water for achieving test concentrations of 100 mg/L and Zebra FishDanio reriowere exposed to these concentration for 96 hours.

Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203. After 96 hours of exposure to test item to various nominal test concentrations, LC50 was determine to be >100 mg/l . Based on the LC50, it can be consider that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Short term toxicity to aquatic invertebrate:

Aim of this study was to assess the short term toxicity of test material to aquatic invertebrates daphnia magna. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) in a static system for the total exposure period of 48 hrs.

 The stock solution 200 mg/l was prepared by dissolving yellow green powder reconstituted water. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water.10, 25 , 50 , 100 , 200 mg/lconcentrations were used in the study. Effects on immobilisation were observed for 48 hours. 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.

 The median effective concentration (EC50) for the test substance , in Daphnia magna was determined to be 500 mg/L on the basis of mobility inhibition effects in a 48 hour study. Based on the EC50 value, substance is likely to be non hazardous to aquatic invertebrate and cannot be classified as aquatic as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria:

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).The stock solution 100 mg/l was prepared by dissolving yellow- green powder in OECD growth medium . Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.

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.

The median inhibition percentage (I%) for the test substance , in algae was determined to be 7.0% at concentration 100 mg/l on the basis of growth rate inhibition effects in a 72 hour study. Based on the inhibition percentage , which indicates that the substance is likely to be non-hazardous to aquatic algae and cannot classified as per the CLP classification criteria.

Toxicity to microorganisms:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of micro-organisms of the test chemical test material.The studies are as mentioned below:

1. Toxicity value of test chemical to Vibrio fisheri was found to be EC50: 22.19+/-2.47 on the basis of inhibition of the light output after a 5 min exposure period.

2. The Microtox acute toxicity assay was performed by using a modified strain ofVibrio fischeri

Frozen samples were brought to room temperature, and centrifuged. The pH of the samples was adjusted where necessary to 6 by adding 0.5 ml 0.58 M KH2 PO4 and 70μl 1 M NaOH. Colour correction was done at 490 nm. The Microtox acute toxicity assay was performed in a Microtox 500 Analyzer on samples before and after decoloration according to the test protocols defined by the manufacturer From eight serial dilutions, the percent concentration to decrease 20% of the luminescence of amodified strain ofVibrio fischeri(EC20)after 5 min incubation was calculated with the Microtox data analysis program [Microtox Omni Software(1999) Azur Environmental, Newark, Del.]. A solution of 1 g/l ZnSO4·7H2O was used as the positive control and 1 g/l glucose as the negative control. Each EC20 reported is the average of triplicate analysis.

The concentration to decrease 50% of the bacterial luminescence in the Microtox acute assay (EC50) is normally reported. However, in most of these studies, the EC50 before or after decoloration was greater than 100% indicating that there was no toxicity or toxicity change. To better evaluate whether the decoloration process affected toxicity, the dilution required to decrease 20% of the bacterial luminescence relative to the control (EC20)was reported instead. The following rating was adapted from Coleman & Qureshi (1985) –

EC20:>100%=nontoxic;

>75–100%=slightly non-toxic;

 >50–75%=toxic;

>25–50%=moderately toxic;

<25% very toxic.

The toxicity of 100mg/l of test material determined in terms of EC20 (% dilution) was 44.6±11.6.

According to the ranking scheme for Microtox assay using EC20 values, test material can be categorized under moderately toxic category.

Thus based on the above summarised studies, test material and it’s structurally and functionally similar read across substance, it can be concluded that test material is not likely to be toxic to the micro-organisms.

Additional information

Short term toxicity to fish:

Short term toxicity to fish was evaluate based on the experimental report and other peer reviewed journal.

Study was conducted to access the effect of test chemical on the growth of fish Danio rerio. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test)The test substance was soluble in water. Therefore, the test solution was prepared by dissolving 400 mg of the test substance in 1 liters of potable water (passed through reverse osmosis system) with continuous stirring. This test solution was then added to the remaining three liters of water for achieving test concentrations of 100 mg/L.This test solution was then added to the remaining three liters of water for achieving test concentrations of 100 mg/L and Zebra FishDanio reriowere exposed to these concentration for 96 hours.

Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203. After 96 hours of exposure to test item to various nominal test concentrations, LC50 was determine to be >100 mg/l . Based on the LC50, it can be consider that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

In a data from peer reviewed journal,to study the effects of test material on aquatic invertebrate acute toxicity tofish test was carried out for 96.0 hr under static condition. 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 fish in 96 hours exposure to the test chemical (LC50) was found to be greater than 500mg/l. Since the LC50 for the test chemical is greater than 100 mg/l , it falls in the LOW concern category in the aquatic toxicity ratings. It can be concluded from the value that the test material is not toxic to the aquatic invertebrate and can be considered as “not classified” as per the classification criteria for aquatic environment.

In another peer reviewed journal , ecotoxicological assessments were based on the determination of acute toxicity to daphniae according to DIN 38412, Part 11 (Deutsches Institut für Normung e.V. 1982), and 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.Test materialis classified as safe for use in water tracing.It can be concluded that the test substance is not toxic to the aquatic invertebrateand can be considered as “not classified” as per the classification criteria for aquatic environment.

Based on the LC50, it can be consider that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Short term toxicity to aquatic invertebrate:

The short term toxicity to aquatic invertebrate was evalauted based on experimental report and peer reviewed journal.

Aim of this study was to assess the short term toxicity of test material to aquatic invertebrates daphnia magna. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) in a static system for the total exposure period of 48 hrs.

 The stock solution 200 mg/l was prepared by dissolving yellow green powder reconstituted water. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water.10, 25 , 50 , 100 , 200 mg/lconcentrations were used in the study. Effects on immobilisation were observed for 48 hours. 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.

 The median effective concentration (EC50) for the test substance , in Daphnia magna was determined to be 500 mg/L on the basis of mobility inhibition effects in a 48 hour study. Based on the EC50 value, substance is likely to be non hazardous to aquatic invertebrate and cannot be classified as aquatic as per the CLP classification criteria.

In a peer reviewed journal,to study the efffects of test material on aquatic invertebrate test was carried for 48 hr.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 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 greater than 100mg/l.

Since the LC50 for the test materialis greater than 100 mg/l, it falls in the LOW concern category in the aquatic toxicity ratings. Based on the result, thetest substancewas considered as "not classified" according to the CLP regulations.

In another peer reviewed journal,toxicity tests on the dyes were carried out using standard bioassay using the copepod acute toxicity test (Tisbe battagliai 48 hr LC50).Stock solutions of test dyes were prepared using filtered aerated seawater (0.2 μm). Serial dilutions were then prepared from the main stock to produce the test concentration range. Initial tests, or range finders, were carried out on bothTisbebattagliai. The range finder tests covered a concentration range of 0.001 to 1000 mg/L, where needed this was increased to 4000mg/L for tracer dye test material . The observations allowed for a narrower range to be achieved for more focused definitive studies.The T.battagliai bioassay followed the guidelines in ISO (1999) with no further modifications. These bioassays were carried out in 12 well polystyrene plates, each well contained 5mls of test solution and 5T.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. Water quality checks for temperature, salinity, dissolved oxygen and pH were carried out at the start and the end of each test to ensure that physical parameters remained within the acceptable range, as set out in the test protocols, for each bioassay. All parameters were within acceptable limits, e.g. temperature (21°C ± 3°C), salinity (32 ± 2 %), dissolved oxygen (³80%) and pH (7.8 - 8.2) forT.battagliai bioassay.

 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. This test uses several concentrations of Zinc sulphate 0 (control), 0.1, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L forT.battagliai bioassay. Atest is considered valid if the mortality ranges fall with the expected limits, i.e no more than 10% mortality forT.battagliai.

 The results showed lower levels of toxicity for test material for the test species, however, it is difficult to rule out whether these are considered to be safe for the marine environment.The LC50 for test material was 2703.1 mg/l. Test material was the least toxic tracer dye among the tracers tested. Based on the result, the chemical was considered as not classified for short term toxicity to aquatic invertebrates according to the CLP regulation.

Based on the EC50 value, substance is likely to be non hazardous to aquatic invertebrate and cannot be classified as aquatic as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria:

Toxicity to aquatic algae and cyanobacteria was evlauted based on experimental data and data from peer reviewed journal,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).The stock solution 100 mg/l was prepared by dissolving yellow- green powder in OECD growth medium . Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.

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.

The median inhibition percentage (I%) for the test substance , in algae was determined to be 7.0% at concentration 100 mg/l on the basis of growth rate inhibition effects in a 72 hour study. Based on the inhibition percentage , which indicates that the substance is likely to be non-hazardous to aquatic algae and cannot classified as per the CLP classification criteria.

.

According to peer revieved journal,to study the efffects of test material on aquatic algae test was carried for 96 hr.The study was performed under static conditions in a fresh water system. The concern levels identified by use of the SAR technique are expressed ad HIGH, MODERATE, LOW.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 (EC50) value for test materialon Green algae in a 96 hour study was determined to be 20 mg/L.Since the EC50 for the test chemical lies between 1 – 100mg/l, it falls in MODERATE category in the aquatic toxicity ratings.Thus based on the EC50 value, it can be concluded that the substance can be classified as aquatic chronic category 3.

As the relibility of study report is high and the test has been carried out according to OECD guideline , test substance is not considered to have toxic effect on aquatic algae , and hence test substance is not classified as per CLP criteria.

Toxicity to microorganisms:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of micro-organisms of the test chemical test material.The studies are as mentioned below:

1. Toxicity value of test chemical to Vibrio fisheri was found to be EC50: 22.19+/-2.47 on the basis of inhibition of the light output after a 5 min exposure period.

2. The Microtox acute toxicity assay was performed by using a modified strain ofVibrio fischeri

Frozen samples were brought to room temperature, and centrifuged. The pH of the samples was adjusted where necessary to 6 by adding 0.5 ml 0.58 M KH2 PO4 and 70μl 1 M NaOH. Colour correction was done at 490 nm. The Microtox acute toxicity assay was performed in a Microtox 500 Analyzer on samples before and after decoloration according to the test protocols defined by the manufacturer From eight serial dilutions, the percent concentration to decrease 20% of the luminescence of amodified strain ofVibrio fischeri(EC20)after 5 min incubation was calculated with the Microtox data analysis program [Microtox Omni Software(1999) Azur Environmental, Newark, Del.]. A solution of 1 g/l ZnSO4·7H2O was used as the positive control and 1 g/l glucose as the negative control. Each EC20 reported is the average of triplicate analysis.

The concentration to decrease 50% of the bacterial luminescence in the Microtox acute assay (EC50) is normally reported. However, in most of these studies, the EC50 before or after decoloration was greater than 100% indicating that there was no toxicity or toxicity change. To better evaluate whether the decoloration process affected toxicity, the dilution required to decrease 20% of the bacterial luminescence relative to the control (EC20)was reported instead. The following rating was adapted from Coleman & Qureshi (1985) –

EC20:>100%=nontoxic;

>75–100%=slightly non-toxic;

 >50–75%=toxic;

>25–50%=moderately toxic;

<25% very toxic.

The toxicity of 100mg/l of test material determined in terms of EC20 (% dilution) was 44.6±11.6.

According to the ranking scheme for Microtox assay using EC20 values, test material can be categorized under moderately toxic category.

Thus based on the above summarised studies, test material and it’s structurally and functionally similar read across substance, it can be concluded that test material is not likely to be toxic to the micro-organisms.