Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, aluminium salt

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:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Experimental result using standard method of analysis

Justification for type of information:

Data is from the Experimental reports.

Qualifier:

according 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 Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt ; CAS No. 129-17-9) 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:

-CAS No: 129-17-9
-Chemical Name: Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt
-Nature of chemical: Organic
-Batch no: I/13-14/1002
-Physical state: Solid Dark violet powder
-Date of manufacturing: January, 2014
-Date of expiry: December, 2022
-Purity: 93.48 % (dye content by GC)
-Stability: Stable in recommended storage conditions
-Storage conditions: Ambient Temp (23 to 27 °C)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The test solution was prepared in aseptic condition. The test item Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt was prepared by adding 70 mg of test item in 350 ml of BBM to get the final concentration of 200 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 104 cells/ml. Care was taken to have a homogeneous solution for the experiment.

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

No data available

Test temperature:

24 °C ±2°C

pH:

6.8 ±0.3

Dissolved oxygen:

No data available

Salinity:

No data available

Nominal and measured concentrations:

6.25 mg/L, 12.5 mg/L, 25 mg/L, 50mg/L, 100 mg/L and 200mg/L (Nominal)

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: 54x105 cells/mL
- Control end cells density: No data available
- No. of organisms per vessel: 10000cells/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 and 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 Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt was prepared by adding 70 mg of test item in 350 ml of BBM to get the final concentration of 200 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 104 cells/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 24°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.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

123.386 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: EC50 was found graphically through probit analysis.

Details on results:

After 72 hours of exposure to Hydrogen [4-[4-(diethylamino)-2', 4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt (CAS No. 129-17-9) to various nominal test concentration, EC50 was found to be 123.386 mg/L through probit analysis.

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	41.5 x 10^4	70 x 10^4	89 x 10^4
Replicate 2	49 x 10^4	65 x 10^4	86 x 10^4
Replicate 3	44.5 x 10^4	71.5 x 10^4	91.x 10^4
CAS No. 129-17-9
6.25 mg/l
Replicate 1	39.5 x 10^4	53 x 10^4	35.5 x 10^4
Replicate 2	37 x 10^4	49.5 x 10^4	35 x 10^4
12.5 mg/l
Replicate 1	39 x 10^4	48 x 10^4	36 x 10^4
Replicate 2	36 x 10^4	35.5 x 10^4	39.5 x 10^4
25 mg/l
Replicate 1	31 x 10^4	53 x 10^4	33 x 10^4
Replicate 2	39 x 10^4	50.5 x 10^4	29.5 x 10^4
50 mg/l
Replicate 1	26.5 x 10^4	39 x 10^4	26.5 x 10^4
Replicate 2	27.5 x 10^4	33.5 x 10^4	25 x 10^4
100 mg/l
Replicate 1	20 x 10^4	17.5 x 10^4	13 x 10^4
Replicate 2	18.5 x 10^4	13 x 10^4	11.5 x 10^4
200 mg/l
Replicate 1	11 x 10^4	75 x 10^4	70 x 10^4
Replicate 2	14.5 x 10^4	90 x 10^4	35 x 10^4

 

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

	CONTROL		6.25 mg/l		12.5 mg/l		25 mg/l		50 mg/l		100 mg/l		200 mg/l
Average Specific Growth rate (µ )	R1	1.496	R1	1.180	R1	1.194	R1	1.165	R1	1.092	R1	0.855	R1	0.649
	R2	1.485	R2	1.185	R2	1.225	R2	1.128	R2	1.073	R2	0.814	R2	0.417
	R3	1.505
Mean of Avg. Specific growth rate	1.495		1.187		1.209		1.146		1.082		0.834		0.533
Percentage Inhibition (%I)	_		20.595		19.091		23.314		27.60		44.195		64.352

 

 

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	6.5	7.5
Replicate 2	6.6	7.5
Replicate 3	6.6	7.4
CAS No. 129-17-9
6.25 mg/l
Replicate 1	6.5	6.7
Replicate 2	6.5	6.8
12.5 mg/l6.7
Replicate 1	6.6	6.8
Replicate 2	6.5	6.8
25 mg/l
Replicate 1	6.5	6.7
Replicate 2	6.5	6.7
50 mg/l
Replicate 1	6.5	6.7
Replicate 2	6.5	6.7
100 mg/l
Replicate 1	6.6	6.7
Replicate 2	6.5	6.7
200 mg/l
Replicate 1	6.5	6.6
Replicate 2	6.5	6.6

 

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure to Hydrogen [4-[4-(diethylamino)-2', 4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt (CAS No. 129-17-9) to various nominal test concentration, EC50 was found to be 123.386 mg/L through probit analysis.

Executive summary:

The effect of test item Hydrogen [4-[4-(diethylamino)-2',4'- disulphonatobenzhydrylidene] cyclohexa- 2,5-dien-1-ylidene] diethylammonium, sodium salt, (CAS No.129-17-9) 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 and 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 123.386 mg/L. Thus, based on this value, it can be concluded that the test chemical can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.

Description of key information

The effect of test item Hydrogen [4-[4-(diethylamino)-2',4'- disulphonatobenzhydrylidene] cyclohexa- 2,5-dien-1-ylidene] diethylammonium, sodium salt, (CAS No.129-17-9) was studied on the growth of fresh water green algaChlorella 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 and 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 123.386 mg/L. Thus, based on this value, it can be concluded that the test chemical can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

123.386 mg/L

Additional information

Following studies include the experimental studies for the structurally similar read across to conclude the toxicity extent of Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, aluminium salt (CAS: 15876-40-1) towards aquatic algae is summarized as follows:

Expermental study from ABITEC for test item Hydrogen [4-[4-(diethylamino)-2',4'- disulphonatobenzhydrylidene] cyclohexa- 2,5-dien-1-ylidene] diethylammonium, sodium salt, (CAS No.129-17-9) was studied on the growth of fresh water green algaChlorella 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 and 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 123.386 mg/L. Thus, based on this value, it can be concluded that the test chemical can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.

Similar Experimental study from ABITEC, s. r. o for structurally similar read across 2,7-Naphthalenedisulfonic acid, 3-hydroxy-4-[(4-sulfo-1-naphthalenyl), sodium salt; Amaranth dye; trisodium 3-hydroxy-4-[(4-sulfonato-1-naphthyl)diazenyl]naphthalene-2,7-disulfonate (CAS: 915 -67 -3) suggests that

Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance 2,7-Naphthalenedi- sulfonic acid, 3-hydroxy-4-[(4-sulfo- 1-naphthal - enyl), sodium salt (Amaranth dye) according to OECD Guideline 201. The test substance was dissolved in OECD growth medium and tested at the concentrations 0, 12.5, 25, 50, 100, 200 mg/L. Effects on the growth rate of the organism were studied. The median effective concentration (ErC50) for the test substance, 2,7-Naphthalenedisulfonic acid, 3-hydroxy-4-[(4-sulfo-1- -naphthalenyl), sodium salt, in Desmodesmus subspicatus was determined to be 356.2 mg/L. This value indicates that the substance is likely to be non-hazardous to aquatic algae and can not be classified as toxic as per the CLP criteria.

Further is also assissted by the Experimental study from ABITEC, s. r. o forstructurally similar read across

disodium 3-[(2,4-dimethyl-5-sulfonatophenyl)diazenyl]-4-hydroxynaphthalene-1-sulfonate (CAS: 4548-53-2) Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance Disodium 3-[(2,4-dimethyl-5-sulphonatophenyl) azo]-4-hydroxynaphthalene-1-sulphonate according to OECD Guideline 201.The test substance was dissolved in OECD growth medium and tested at the concentrations 0, 0, 12.5, 25, 50, 100 and 200 mg/L. Effects on the growth rate of the organism were studied. The median effective concentration (ErC50) for the test substance, Disodium 3-[(2,4-dimethyl-5-sulphonatophenyl)azo]-4-hydroxynaphthalene-1-sulphonate, in Desmodesmus subspicatus was determined to be 276.1 mg/L. Based on this E_rC50 value and after comparing with CLP criteria for aquatic classification of the substance it is concluded that the substance Disodium 3-[(2,4-dimethyl-5-sulphonatophenyl)azo]-4-hydroxynaphthalene-1-sulphonate does not exhibit toxicity to aquatic algae (Desmodesmus subspicatus).

Thus based on the effect concentrations which is in range of 123.386 mg/l to 356.2 mg/L gives the conclusion that test substance Hydrogen [4-[4-(diethylamino)-2',4' disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, aluminium salt (CAS: 15876-40-1) was likely to be not toxic to algae at environmentally relevant concentrations and applying weight of evidence approach it can be considered to be“not classified”as per the CLP classification criteria for aquatic environment.