2,4-diaminobenzenesulphonic acid

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:

21.03.2017-24.03.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Experimental test result performed using standard test guideline

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

Analytical monitoring:

not specified

Vehicle:

not specified

Details on test solutions:

The stock solution 150 mg/L was prepared by dissolving light brown powder in OECD grith medium 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: Control:

Nominal and measured concentrations:

35,45,67.5,100,150 mg/L

Details on test conditions:

TEST SYSTEM- Test vessel: 150 ml Glass vessel- Type (delete if not applicable): closed (with air permeable stopper)- Sample volume: -------- ml- Initial cells density: 5x10(3) cells/ml- No. of vessels per concentration (replicates): --------GROWTH 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:

EC50

Effect conc.:

110 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI

Results with reference substance (positive control):

- Results with reference substance valid- EC50:0.75 mg/L (24 hours)

Reported statistics and error estimates:

EC50 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 the EC50 was determine to be 110 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 test solution 100 mg/l was prepared by dissolving yellow powder in OECD growth medium. Effects on the growth rate of the organism were studied (Changable). Various 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.

The median effective concentration (EC50) for the test substance 2,4-Diaminobenzenesulfonic aci

, in algae was determined to be 110 mg/L on the basis of growth rate inhibition effects in a 72 hour study. Based on the EC50 value, indicates 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 test solution 100 mg/l was prepared by dissolving yellow powder in OECD growth medium. Effects on the growth rate of the organism were studied (Changable). Various 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.

The median effective concentration (EC50) for the test substance2,4-Diaminobenzenesulfonic aci

, in algae was determined to be 110 mg/L on the basis of growth rate inhibition effects in a 72 hour study. Based on the EC50 value, indicates 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:

110 mg/L

Additional information

Various data for the target chemical 2,4-Diaminobenzenesulfonic acid (CAS No. 88-63-1) and the study of its read across substancewere reviewed to summarize the following 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 test solution 100 mg/l was prepared by dissolving yellow powder in OECD growth medium. Effects on the growth rate of the organism were studied (Changable). Various 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.

The median effective concentration (EC50) for the test substance2,4-Diaminobenzenesulfonic aci

, in algae was determined to be 110 mg/L on the basis of growth rate inhibition effects in a 72 hour study. Based on the EC50 value, indicates that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as per the CLP criteria.

Estimated 72 hrs EC50 value of test substance 2,4-Diaminobenzenesulfonic acid onPseudokirchneriella subcapitatawas determined by three different models i.e, Battery, Leadscope and SciQSAR used within Danish QSAR database (Danish QSAR database, 2016). Based on inhibition in growth rate of test organismPseudokirchneriella subcapitata, the estimated 72 hrs EC50 value was found to be 3058.404mg/l. 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.

Short term toxicity to Chlorella pyrenoidosa (green algae) study was carried out for 72 hrs (JU-CHANG HUANG and EARNEST F. GLOYNA, 1968). Emerson strain of bacteria free, experimentally reproducible cultures of Chlorella pyrenoidosa was used as a test organism. The procedure involve the use of test tubes in both the screening and final tests. These test tubes contained 15 ml of inorganic culture medium, a predetermined amount of test chemical and 5 ml of algal culture. The tubes were incubated for 72 hrs and chlorophyll content of the algal suspensions was measured every 24 hrs. For chlorophyll measurement, the chlorophyll pigment was extracted with hot methanol in two separate extractions. An algal suspension, 2.5 ml, was removed from the test tube, centrifuged, washed with distilled water, and recentrifuged in preparation of chlorophyll analysis. After discarding the supernatant, the deposited cell material was coagulated by placing the cells in a boiling water bath for about 40 sec. About 2.5 ml of methanol were used in each extraction. Finally, the chlorophyll solution was diluted to a total volume of 10 ml with an acetone-water mixture (80 per cent by volume). A Beckman Spectrophotometer, Model DB, was used to measure the chlorophyll content according to MACKIN~v (1941) and ARNON (1949). For this a wavelength of 652 m/z was used because different proportions of chlorophyll a and b least affect the results at this wavelength. Control tubes containing no test chemical was also used in the experiment. Knop's solution, including the Hutner-EDTA microelement addition, was used as the culture medium. pH of culture medium was adjusted to 7.0 using KOH before use. The test organism was maintained under steady-state conditions, provided a chlorophyll content of 38 mg/l. Environmental control was rigidly maintained. The temp. of water bath was 25 ± 1°C. The test apparatus consisted of a constant-temperature water bath, a light source containing four 200W fluorescent lamps with attached aluminum reflectors, a gas manifold to supply an air-CO2 mixture to each test tube, and a rack to hold the test tubes. A stream of 5 % CO2 in air gas mixture was supplied to culture medium in order to provide the inorganic carbon source and also to keep the algal ceils in suspension. Based on destruction of chlorophyll of test organism by test chemical Amino-1-phenol-4-sulfonic acid, the LOEC value was found to be1500 mg/l and as no toxic effect at 1000 mg/l was observed, the NOEC value was found to be 1000 mg/l. 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.

Short term toxicityto Chlorella pyrenoidosa (green algae) study was carried out for 72 hrs (JU-CHANG HUANG and EARNEST F. GLOYNA, 1967). Emerson strain of bacteria free, experimentally reproducible cultures of Chlorella pyrenoidosa was used as a test organism. An Emerson strain of bacteria-free, experimentally reproducible cultures ofChlorella pyrenoidosawas obtained from Dr.Jack E. Myers, Professor of Zoology and Botany and Director, Laboratory of Algal Physiology ,The University of Texas. Five conc. levels of test chemical were used for the tests (100, 500, 1000, 1500 and 2000 mg/l, respectively). A test tubes was used in the screening and final tests. These test tubes contained 15 ml of inorganic culture medium, a predetermined amount of test chemical and 5 ml of algal culture. The tubes were incubated for 72 hrs and chlorophyll content of the algal suspensions was measured every 24 hrs. Control tubes containing no test chemical in the culture medium was also used in the experiment. Environmental control was rigidly maintained. The temp. of water bath was 25 ± 1°C. The special toxicity test device consisted of an aquarium complete with a light source, a gas manifold and an apparatus for holding the test tubes. In this test device, algal cultures were kept under a rigid environmental control. A stream of 5 % CO2 in air gas mixture was supplied to culture medium in order to provide the inorganic carbon source and also to keep the algal ceils in suspension. Screening tests were performed to establish the threshold toxic concentration as well as completely algicidic one. Knop's solution, including the Hutner-EDTA microelement addition, was used as the culture medium. pH of culture medium was adjusted to 7.0 using KOH before use. Kimble Product of plain culture tubes, Series 45060, was used as the test tube. These test tubes have the size of 19 x 150 mm with a volume of 25 mI. The initial algal cell density was1.0 gm/l dry weight or equivalent to 3.8 cm/ml packed cell volume and chlorophyll content was 38 mg/l. The chlorophyll pigment was extracted with hot methanol from a 2.5mlalgal suspension which was pipetted out of the test tube. The algal cells were centrifuged out of the culture medium, washed with distilled water and then centrifuged again. The supernatant was discarded and the remaining packed cell material was coagulated in a boiling water bath for a period of about 30 to 45 seconds depending on the amount of cells. Thereafter, the chlorophyll was extracted twice by using hot methanol. The extracted chlorophyll solution was then dissolved in 80 percent by volume acetone-water mixture to make a final total volume of 10 ml. The chlorophyll content was then analyzed under the spectrophotometer. Content of all extracted chlorophylls which were dissolved in 10mlof 80 percent acetone-water mixture could be determined by measuring the optical density (O.D.) at a wavelength of 652 mn. This wavelength was optimum because various proportions of chlorophylls a and b would least affect the result. A Beckman Spectrophotometer, Model DB, was used for all chlorophyll analysis. This spectrophotometer is a double beam instrument for making transmittance and absorbance measurements in the 205 to 770mn wavelength range. Based on the decrease in chlorophyll content of test organism by test chemical Amino-1- phenol-4-sulfonic acid, the LOEC value was found to be 2000 mg/l and as no toxic effect at 1000 mg/l was observed, the NOEC value was found to be 1000 mg/l. 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.

Based on the overall reported results for target and read across substance, it can be concluded that the substance 2,4-Diaminobenzenesulfonic acid can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.