9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthylium, salt with 4,5-dihydro-5-oxo-1-(4-sulphophenyl)-4-[(4-sulphophenyl)azo]-1H-pyrazole-3-carboxylic acid (3:1)

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 test result performed using standard OECD test guidelines

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 itemSodium fluorescein; CAS No. 518-47-8) 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:

yes

Specific details on test material used for the study:

Name of test material (as cited in study report): Sodium fluorescein
Molecular formula (if other than submission substance): C20H10O5.2Na
Substance type: Organic
Physical state: Solid
Lot/batch No.: L19201Z1605
Expiration date of the lot/batch: April, 2021
Storage condition of test material:
1) Ambient Temp (23 to 27 °C) (Store in cool place. Keep containers tightly closed in a dry and well- ventilated place)
2) In Refrigerator (4 °C)
3) In Freezer (-10 To 0 °C)
Purity: 98.56%
Other: Stable in recommended storage conditions
Solubility in water: 552.4 g/l

Analytical monitoring:

yes

Details on sampling:

No data available

Vehicle:

no

Details on test solutions:

No data available

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

TEST ORGANISM
- Common name:green alga
- Strain:Chlorella vulgaris
- Source (laboratory, culture collection):laboratory
- 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

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

24, 48, 72 hrs.

Hardness:

No data available

Test temperature:

22 °C ±2°C

pH:

6.8 ±0.3

Dissolved oxygen:

No data available

Salinity:

No data available

Nominal and measured concentrations:

Six test concentration were: 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l, 100mg/l and 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: 38.787x105 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 itemDisodium 2-(3-oxo-6-oxidoxanthen-9-yl) benzoate was prepared by adding 50 mg of test item in 250ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring for 1 hr to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. 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 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.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

209.24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Reported statistics and error estimates:

To obtain a quantitative concentration-response relationship by regression analysis, a line arizing 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	3.72 × 10^4	5.32 × 10^4	6.92 × 10^4
Replicate 2	4.08 × 10^4	5.68 × 10^4	7.28 × 10^4
Replicate 3	3.96 × 10^4	5.56 × 10^4	7.96 × 10^4
CAS No. 518-47-8
6.25 mg/l
Replicate 1	3.08 × 10^4	3.32 × 10^4	4.12 × 10^4
Replicate 2	3.08 × 10^4	3.52 × 10^4	4 × 10^4
12.5 mg/l
Replicate 1	2.92 × 10^4	2.68 × 10^4	3.88 × 10^4
Replicate 2	3 × 10^4	2.8 × 10^4	3.72 × 10^4
25 mg/l
Replicate 1	2.28 × 10^4	2.76 × 10^4	3.68 × 10^4
Replicate 2	2.12 × 10^4	2.8 × 10^4	3.64 × 10^4
50 mg/l
Replicate 1	2.2 × 10^4	3 × 10^4	3.4 × 10^4
Replicate 2	2.36 × 10^4	2.76 × 10^4	3.32 × 10^4
100 mg/l
Replicate 1	2.16 × 10^4	2.32 × 10^4	3 ×10^4
Replicate 2	2.36 × 10^4	2.28 × 10^4	2.84 × 10^4
200 mg/l
Replicate 1	2.32 × 10^4	2.2 × 10^4	2.68 × 10^4
Replicate 2	2.28 × 10^4	2.04 × 10^4	2.68 × 10^4

 

 

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	0.645	R1	0.472	R1	0.452	R1	0.434	R1	0.408	R1	0.366	R1	0.329
	R2	0.662	R2	0.462	R2	0.438	R2	0.431	R2	0.400	R2	0.348	R2	0.329
	R3	0.691
Mean of Avg. Specific growth rate	0.666		0.467		0.445		0.432		0.404		0.357		0.329
Percentage Inhibition (%I)	_		29.876		33.194		35.062		39.346		46.386		50.660

 

 

 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	6.15	6.30
Replicate 2	6.25	6.34
Replicate 3	6.30	6.40
Average	6.23	6.35
CAS No. 518-47-8
6.25 mg/l
Replicate 1	6.28	6.25
Replicate 2	6.27	6.24
12.5 mg/l
Replicate 1	6.25	6.31
Replicate 2	6.27	6.49
25 mg/l
Replicate 1	6.28	6.54
Replicate 2	6.27	6.50
50 mg/l
Replicate 1	6.32	6.52
Replicate 2	6.30	6.52
100 mg/l
Replicate 1	6.51	6.50
Replicate 2	6.50	6.51
200 mg/l
Replicate 1	6.56	6.51
Replicate 2	6.55	6.50

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure to test item Disodium 2-(3-oxo-6-oxidoxanthen-9-yl)benzoate to various nominal test concentrations,EC50 was 209.24 mg/l graphically and through probit analysis.

Executive summary:

The effect of test item Disodium 2-(3-oxo-6-oxidoxanthen-9-yl) benzoate, 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 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l, 100mg/l and 200mg/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 209.24 mg/l.

Thus, based on the EC50 value, test substance 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

Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, toxicity on Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was predicted for 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis (ethylamino)-2,7-dimethyl - xanthylium (65138-66-1). The EC50 value was estimated to be 186.836 mg/l when 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthylium exposed to Desmodesmus subspicatus for 72hrs.

Key value for chemical safety assessment

EC50 for freshwater algae:

186.836 mg/L

Additional information

Study was conducted for evaluatin the toxic nature of chemical 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthylium (65138-66-1). Summarized results are as follows:

In the first predicted report for target chemical from QSAR toolbox 2017 study was conducted on the toxicity of chemical on the growth of algae. Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, toxicity on Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was predicted for 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthylium (65138-66-1). The EC50 value was estimated to be 186.836 mg/l when 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthylium exposed to Desmodesmus subspicatus for 72hrs.

 

In the second weight of evidence study for (518-47-8), UERL report. The effect of test item Disodium 2-(3-oxo-6-oxidoxanthen-9-yl) benzoate, 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 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l, 100mg/l and 200mg/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 209.24 mg/l. Thus, based on the EC50 value, test substance can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.

 

Similarly in the third weight of evidence study for RA chemical (504-44-3). Short term toxicity of Caprolactone on the growth of algae Scenedesmus subspicatus was studied by the exposure of chemical with the daphnia for 48 hrs. The test was performed according the OECD guideline 201. Test conducted at the different nominal concentrations 0, 102, 256, 640, 1600, 4000 mg/l. Positive control i.e. potassium dichromate was used which shows biomass inhibition at the concentration of 0.47 mg/l. After the 72 hrs. Of exposure the NOEC was 256, LOEC observed at 640 mg/l, EC10 was 484 mg/l, EC50 1217 mg/l, EC90 was observed at > 4000 mg/l. Based on the inhibition of biomass of Scenedesmus subspicatus (Algae) due to the exposure of chemical Caprolactone, the EC50 was 1217 mg/l. Thus the chemical was consider as non-toxic and can be consider to be not classified as toxic as per the CLP classification criteria.

 

Based on the various studies it was concluded that the chemical 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthylium (65138-66-1) was consider as non-toxic and can be consider to be not classified as toxic as per the CLP classification criteria.