3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic 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:

06.09.2016 – 09.09.2016

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. / Objective of this study was to observe the action of test chemical when it exposed with the Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) for 72 hrs. Test conducted in accordance with OECD Guideline 201 (Alga, Growth Inhibition Test).

GLP compliance:

no

Analytical monitoring:

not specified

Vehicle:

not specified

Details on test solutions:

The stock solution 100 mg/L was prepared by dissolving red 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.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Institute of botany of the ASCR
- Age of inoculum (at test initiation): 5x103 cells/ml
- Method of cultivation: No data

ACCLIMATION
- Acclimation period: No data
- Culturing media and conditions (same as test or not): No data
- Any deformed or abnormal cells observed: No data

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

72 hour ±1 hour

Post exposure observation period:

No data

Hardness:

No data

Test temperature:

23°C ± 2°C

pH:

The sample at concentration 10.0 mg/L : pH = 7.9 changed to pH = 7.5 during the test,
Control : pH = 8.0 changed to pH = 7.4 during the test

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

0, 0.6, 1.2, 2.5, 5.0 and 10.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50ml glass vessel
- Type : closed (with air-permeable stopper)
- Material, size, headspace, fill volume: 7.5 ml per replicate in 50 ml glass vessel
- Aeration: No data
- Type of flow-through (e.g. peristaltic or proportional diluter): No data
- Renewal rate of test solution (frequency/flow rate): No data
- Initial cells density: 5x103 cells/ml
- No. of vessels per concentration (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: Without adjustment
- Photoperiod: Continuous
- Light intensity and quality: 6000 lx - 8000 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Microscope with counting chamber Cyrus I or electronic particle counters.
- Chlorophyll measurement: No
- Other: ErC50 was calculated using non-linear regression by the software Prism 4.0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 6.9 to 8.1 mg/L

Results with reference substance (positive control):

- Results with reference substance valid
- ErC50: 0.69 mg/L K2Cr2O7

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 7.5 mg/l.

Executive summary:

Aim of this study was to evaluate the nature of chemical test chemical 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6- hydroxy-3-oxoxanthen-9-yl)benzoic acid 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 red 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.Various concentrationes 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 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6- hydroxy-3-oxoxanthen-9-yl)benzoic acid, in algae was determined to be 7.5 mg/L on the basis of growth rate inhibition effects in a 72 hour study. Based on the EC50 value, which indicates that the substance is likely to be hazardous to aquatic algae and can be classified as aquatic chronic 2 category as per the CLP classification criteria.

Description of key information

The median effective concentration (ErC50) for the test substance, 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid, in a freshwater algae Desmodesmus subspicatus was determined to be 7.5 mg/L with 95% confidence limit of 6.9 -8.1 mg/L on the basis of effects on growth rate in a 72 hour study.

Key value for chemical safety assessment

EC50 for freshwater algae:

7.5 mg/L

Additional information

Various experimental studies following OECD guideline for toxicity to aquatic algae and cyanobacteria with the target substance 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (CAS No. 18472-87-2), were summarized as follows:

 

Aim of this study was to evaluate the nature of chemical test chemical 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6- hydroxy-3-oxoxanthen-9-yl)benzoic acid 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 red 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.Various concentrationes 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 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6- hydroxy-3-oxoxanthen-9-yl)benzoic acid, in algae was determined to be 7.5 mg/L on the basis of growth rate inhibition effects in a 72 hour study. Based on the EC50 value, which indicates that the substance is likely to be hazardous to aquatic algae and can be classified as aquatic chronic 2 category as per the CLP classification criteria.

Moreover,The study was designed to assess the toxic effects of the test compound 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3 -oxoxanthen- 9-yl)benzoic acid (CAS No. 18472-87-2) 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 in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test substance (3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3 -oxoxanthen- 9-yl)benzoic acid (CAS No. 18472-87-2) 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/ sonication for 0 minutes to obtain a homogenous solution for the experiment. The test concentrations 0.5mg/l, 1.6mg/l, 5.12mg/l, 16.384mg/l, 52.429mg/l and 167.772mg/l 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.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.After 72 hours of exposure to test item 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3 -oxoxanthen- 9-yl)benzoic acid (CAS No. 18472-87-2) to various nominal test concentrations, EC50 was determine to be 166.06 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was non hazardous and can be consider to be not classified as per the CLP classification criteria.

 

So, based on the above experimental studies for target substance 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (CAS No. 18472-87-2), the median effective concentration (EC50) was found to be in the range of 7.5–166.06 mg/L on the basis of growth rate effect in a 72 hours of exposure. Although data from UERL suggests that the substance is not classified but as the ABITEC (2016) data has K1 reliability and the test was performed using OECD guidelines which has the value of EC50 as 7.5 mg/L in a 72 hours of study and also the substance was found to be not rapidly degradable, indicating that 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3 -oxoxanthen -9-yl)benzoic acid (CAS No. 18472-87-2) exhibits toxicity to aquatic aquatic algae in the chronic 2 classification category, criteria of CLP. The same has been considered for the purpose of classification and chemical safety assessment.