Dialuminium tris[2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate]

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:

2016-11-08 to 2016-11-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed using standard OECD test guidelines.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Principles of method if other than guideline:

This study was designed to assess the effect of the test item Di aluminium tris [2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate] (CAS No. 15876-39-8) on the growth of green alga Chlorella vulgaris. The study was conducted in accordance with “OECD guideline for testing of chemicals No. 201 – Alga, growth inhibition test”.

GLP compliance:

no

Specific details on test material used for the study:

Name of test material: Di aluminium tris [2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate]
- Molecular formula: C20H8Br4O5.2/3Al
- Molecular weight: 1991.5992 g/mol
- Physical state: Solid

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The test solution was prepared in aseptic condition. The test item dialuminium tris[2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate] was prepared by adding 60 mg of test item in 300 ml of BBM to get the final concentration of 300 mg/L. This stock solution was kept for stirring for 48 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 of the culture was kept 1 X 104cells/ml. Care was taken to have a homogeneous solution for the experiment.

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

TEST ORGANISM
- Common name:green alga
- Strain:Chlorella vulgaris
- Source (laboratory, culture collection): The fresh water green alga Chlorella vulgaris, was used as the test system (organism). Sterile, unicellular, suspension cultures of algae were obtained from National Environmental Engineering Research Institute (NEERI), Nagpur and maintained at Unique Ecotox Research Laboratory, Nagpur. The culture was examined under the microscope to confirm that it was unicellular, healthy and not contaminated.
- Method of cultivation: Bold’s Basal Medium(BBM)

ACCLIMATION
- Culturing media and conditions (same as test or not):The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrients, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM.
- Any deformed or abnormal cells observed:no

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

22 °C ±2°C

pH:

6.8 ±0.3

Nominal and measured concentrations:

Six test concentration were: 6.25mg/l,12.5mg/l,25mg/l,50mg/l,100mg/l,200mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks
- Type (delete if not applicable): No data available
- Material, size, headspace, fill volume: Conical flasks of 100 ml size was used for the study.
- Aeration: No data available
- Type of flow-through (e.g. peristaltic or proportional diluter): No data available
- Renewal rate of test solution (frequency/flow rate): No data available
- Initial cells density: 16.45 x104 cells/mL
- Control end cells density: No data available
- No. of organisms per vessel: 10000 cells/ml
- No. of vessels per concentration (replicates): Two replicates for each test concentration
- No. of vessels per control (replicates): Three replicates for Control
- No. of vessels per vehicle control (replicates): No data available

GROWTH MEDIUM
- Standard medium used: No data available
- Detailed composition if non-standard medium was used: The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrients, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM.


OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No data available
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(1500Lux)
- Salinity (for marine algae): No data available

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: 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.
- Chlorophyll measurement: No data available
- Other: 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: All the six concentrations were in geometric series spaced by a factor of 2.
- Justification for using less concentrations than requested by guideline: No data available
- Range finding study: No data available
- Test concentrations: Six test concentration were: 6.25mg/l,12.5mg/l,25mg/l,50mg/l,100mg/l,200mg/l (Nominal concentrations)
- Results used to determine the conditions for the definitive study: No data available

6.1. Test solution: The test solution was prepared in aseptic condition. The test item dialuminium tris[2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate] was prepared by adding 60 mg of test item in 300 ml of BBM to get the final concentration of 300 mg/L. This stock solution was kept for stirring for 48 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 of the culture was kept 1 X 104cells/ml. Care was taken to have a homogeneous solution for the experiment.

6.2. Test vessels: All the tests were 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 60ml so that a sufficient amount of head space was left.

6.3. Replicates: For the assessment of algal growth, the study was conducted in replicates. The control vessel was maintained in triplicates as recommended in the OECD guideline and the test concentrations were selected in geometric series which were maintained in duplicates.

6.4. Incubation:
i) The temperature of the orbital shaking incubator was kept constant throughout the period of exposure of the experiment. The temperature was maintained at 22°C ± 2°C.
ii) The test vessels were incubated with a continuous, uniform fluorescent illumination(1500Lux).
iii) The pH of the control cultures needs to be noted during the study and the pH of the control medium should not increase by more than 1.5 units during the test.
iv) The orbital shaking incubator was set at a speed of 120 revolutions per minute throughout the study period. This is to provide constant shaking to the algal cells to keep them in suspension and to ensure that they do not settle down on the bottom of the test vessel.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

164.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Control vessels: The microscopic observations were noted down in each of the control vessel. All the cells appeared healthy, round and green throughout the study duration in the control. Also, the drift in pH in the control vessels did not increase by >1.5 units when observed on 72 hours as compared to 0 hours. The average pH drift observed in the control vessels was 0.28 units.

Test vessels: The effect of the test item on the green algae Chlorella vulgaris culture was observed at nominal test concentration of >6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,200 mg/L. All the six concentrations were in geometric series spaced by a factor of 2. The microscopic observations were also noted in each of the test vessel. All the cells appeared healthy, round and green throughout the study duration and no significant changes were observed up to the concentration of 200 mg/l whereas, black dotted rings were observed inside the algal cells in concentrations of 50 mg/l, 100 mg/l and 200 mg/l. EC50 was found to be 164.10 mg/l graphically through probit analysis.

Reported statistics and error estimates:

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) was determined.

Table 1: Showing the average cell count using Haemocytometer of the test vessels at an equal interval of 24hrs, 48hrs and 72hrs

Test vessels and test concentration	24 Hours	48 Hours	72 Hours
Control
Replicate 1	24400	29200	46000
Replicate 2	22800	28000	47600
Replicate 3	19600	26000	45600
CAS No. 15876 -39 -8
6.25 mg/l
Replicate 1	24400	21200	24400
Replicate 2	21200	20800	24800
12.5 mg/l
Replicate 1	21200	22800	23600
Replicate 2	20400	23200	24800
25 mg/l
Replicate 1	24400	21200	24400
Replicate 2	21200	20800	22800
50 mg/l
Replicate 1	28400	21600	20800
Replicate 2	26800	22400	24800
100 mg/l
Replicate 1	26400	22000	21200
Replicate 2	23600	22800	23600
200 mg/l
Replicate 1	21200	22800	22000
Replicate 2	20400	22000	20000

 

 

Table 2: Showing the values of average specific growth rate and percentage inhibition after an interval of 72 hours

	CONTROL		6.25mg/l		12.5mg/l		25mg/l		50mg/l		100mg/l		200mg/l
Average Specific Growth rate (µ )	R1	0.509	R1	0.297	R1	0.286	R1	0.297	R1	0.244	R1	0.250	R1	0.263
	R2	0.520	R2	0.303	R2	0.308	R2	0.275	R2	0.303	R2	0.286	R2	0.231
	R3	0.506
Mean of Avg. Specific growth rate	0.512		0.300		0.294		0.286		0.273		0.268		0.247
Percentage Inhibition (%I)	_		41.342		42.428		44.082		46.543		47.539		51.725

 

 

 Table 3: Depicting pH values at 0 Hours and after 72 Hours of test item exposure to algae

Test vessels and test concentration	0 Hours	72 Hours
CONTROL
Replicate 1	6.98	7.05
Replicate 2	6.99	7.30
Replicate 3	6.97	7.42
Average	6.98	7.26
CAS No. 15876 -39 -8
6.25 mg/l
Replicate 1	7.26	7.13
Replicate 2	7.27	7.14
12.5 mg/l
Replicate 1	7.26	7.20
Replicate 2	7.26	7.18
25 mg/l
Replicate 1	7.25	7.34
Replicate 2	7.24	7.30
50 mg/l
Replicate 1	7.23	7.52
Replicate 2	7.23	7.41
100 mg/l
Replicate 1	7.29	7.41
Replicate 2	7.28	7.42
200 mg/l
Replicate 1	7.32	7.43
Replicate 2	7.31	7.46

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure to test chemical dialuminium tris[2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate] (CAS No. 15876-39-8) to various nominal test concentrations, EC50 was found to be 164.10 mg/l graphically and through probit analysis. It ca be concluded form the valiue that the dialuminium tris[2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate] (CAS No. 15876-39-8) is not toxic to the aquatic environment.

Executive summary:

The effect of test item dialuminium tris[2 -(2,4,5,7 -tetrabromo-6 -oxido-3 -oxoxanthen-9 -yl)benzoate], CAS No. 15876-39-8 was studied on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,200 mg/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 164.10 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 classification criteria.

Description of key information

The effect of test item dialuminium tris[2 -(2,4,5,7 -tetrabromo-6 -oxido-3 -oxoxanthen-9 -yl)benzoate], CAS No. 15876-39-8 was studied on the growth of fresh water green alga Chlorella vulgaris (UERL Study report, Sustainability Support Services (Europe) AB, 2016). The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,200 mg/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 164.10 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 classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

164.1 mg/L

Additional information

Following studies from experimental reports following standard testing procedures by UERL and ABITEC 2016, to conclude the toxicity extent of

Di aluminium tris [2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate] (CAS: 15876-39-8) towards aquatic invertebrate is summarized as follows:

Study from UERL 2016,indicates that The effect of test item dialuminium tris[2 -(2,4,5,7 -tetrabromo-6 -oxido-3 -oxoxanthen-9 -yl)benzoate], CAS No. 15876-39-8 was studied on the growth of fresh water green alga Chlorella vulgaris (UERL Study report, Sustainability Support Services (Europe) AB, 2016). The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,200 mg/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 164.10 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 classification criteria.

Similar study from experimental reports following standard testing procedures by ABITEC 2016, suggests that the, A freshwater algal growth inhibition exposure assessment was used to test how a range of concentrations of CAS 15876-39-8 exerts different degrees of toxic effects on the growth of Desmodesmus subspicatusunder otherwise identical test conditions. The test was performed in close resemblance to OECD guideline 201 by ABITEC in Prague, Crez Republic. The standardized protocol used to obtain the herein described results was by the testing laboratory referred to as “CSN EN ISO 8692”. The 72 hours EC50 was by the testing laboratory defined as the concentration estimated to inhibit 50 per cent of the algae cell growth compared to unexposed cells after 27 hours of exposure. The nominal concentrations used for the exposure assessment were as follows: 5, 10, 20, 40 and 80 mg/L. The positive control/reference substance used in the tested showed an expected result and gave a EC50 that corresponded to previous exposures with this chemical in Desmodesmus subspicatus.In conclusion, the EC50 for CAS 15876-39-8 was 72.6 mg/L (95% 61.6-85.5 mg/L) after 72 hours of exposure.

Thus theDialuminium tris[2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate]is not toxic to the aquatic environment can be considerd as "not classiified." as per CLP regulation.

Thus based on the effect concentrations which is in the range 72.6 mg/L mg/l to 164.10 mg/l give the conclusion that test substance Di aluminium tris [2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate] (CAS: 15876-39-8) was likely to be not toxic to algae at environmentally relevant concentrations and can be considered to be“not classified”as per the CLP classification criteria.