2-[2-[4-(diethylamino)phenyl]vinyl]-1,3,3-trimethyl-3H-indolium acetate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Data is for read across chemicals

Justification for type of information:

Data is from handbook or collection of data

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE was prepared from different studies conducted on two algal species.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

WoE 2: The test chemical was prepared by adding 0.278 mg of test item in 250 ml of BBM to get the final concentration of 1.112 mg/L. This stock solution was kept for stirring for 04 minutes 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 cell density of the culture was kept 5 X 1000 cells/ml.

WoE 3: The stock solution (10 mg/L) was prepared by dissolving yellow powder in OECD growth medium. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.

Test organisms (species):

other: Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum), Desmodesmus subspicatus

Details on test organisms:

TEST ORGANISM
WoE 2:
- Common name: green alga
- Source (laboratory, culture collection): Sterile, unicellular, suspension cultures of algae were obtained from the laboratory for Biological Research in Aquatic Pollution (LABRAP) at the university of Ghent in Belgium
- 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

WoE 3:
TEST ORGANISM
- Common name: Green algae
- 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 available

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

24, 48, 72 hrs

Test temperature:

22 °C±2°C to 23±2°C

pH:

Sample at concentration 0.64 mg/L: pH= 7.9 changed to pH= 7.8 during the test
Control: pH= 8.0 changed to pH= 7.4 during the test

Nominal and measured concentrations:

2: Six test concentration were 0.3 mg/l, 0.39 mg/l, 0.507 mg/l, 0.659 mg/l, 0.856 mg/l and 1.112 mg/l
3: 0, 0.02, 0.04, 0.08, 0.16, 0.32 and 0.64 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks
- Material, size, headspace, fill volume: 100 ml conical flasks filled with 60 ml was used for the study.
- Initial cells density: 5000 cells/ml
- No. of organisms per vessel: 5000 cells/ml
- No. of vessels per concentration (replicates): Two replicates for each test concentration
- No. of vessels per control (replicates): Three replicates for Control

GROWTH MEDIUM
- Standard medium used: yes
- 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: Yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: White fluorescent light illumination 3000-4000Lux

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
- Other: 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.
- Test concentrations: Six test concentration were: 0.3mg/l,0.39mg/l,0.507mg/l,0.659mg/l,0.856mg/l,1.112mg/l (Nominal concentrations)
- Results used to determine the conditions for the definitive study: Mortality of test organisms


Other:
Incubation :
1. 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.
2. The test vessels were incubated with a continuous, uniform fluorescent illumination (1500Lux).
3. 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.
4. 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.
5. Study duration : The experimental phase of the study was lasted for a period of 72 hours.


WoE 2:
Details on test conditions
TEST SYSTEM
- Test vessel: 50 ml Glass vessel
- Type (delete if not applicable): closed (with air permeable stopper)
- Sample volume: 15 ml
- Initial cells density: 5x10(3) cells/ml
- No. of vessels per concentration (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 6000-8000 lx

EFFECT 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:

3. Potassium dichromate (K2Cr2O7)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.086 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: WoE - 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: WoE - 3

Details on results:

The microscopic observations were also noted in each of the experimental flasks. All the cells appeared healthy, round and green throughout the test duration in the control and in the experimental flask also no significant changes were observed.

Results with reference substance (positive control):

Results with reference substance (positive control)
Results with reference substance valid
- EC50: 0.75 mg/L

Reported statistics and error estimates:

WoE 2: 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.

WoE 3: EC50 was calculated using nonlinear regression by the software Prism 4.0.

Validity criteria fulfilled:

yes

Conclusions:

WoE 2: Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the EC50 was determine to be 1.086 mg/l. Based on the EC50, it can be concluded that the chemical was toxic to the growth of algae.
WoE 3: Based on the growth rate inhibition on Desmodesmus subspicatus after the exposure of test chemical for 72 hours, the EC50 was determined to be 0.13 mg/L.
Under the test condition it is observed that the chemical consider to be toxic as cause toxicity on Pseudokirchneriella subcapitata and Desmodesmus subspicatus at concentrations ranges from 1.086 to 0.13 mg/l.

Executive summary:

Data available for the test chemical has been reviewed to determine the toxicity of test chemical on the growth of aquatic algae. The studies are as mentioned below:

 

The study was designed to access the toxic effects of the test chemical on the green alga Pseudokirchneriella subcapitata. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Six test concentrations were 0.3mg/l, 0.39mg/l, 0.507mg/l, 0.659mg/l, 0.856mg/l, 1.112mg/l used and the study conducted under the static system. 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. Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the EC50 was determine to be 1.086 mg/l. Based on the EC50, it can be concluded that the chemical was toxic to the growth of algae.

 

The above study was further supported by the second study from experimental report. Freshwater algal growth inhibition test was carried out on green algae Desmodesmus subspicatus with the substance. Test conducted under the static system for 72 hours and according to the OECD Guideline 201 (Alga, Growth Inhibition Test). The test substance was prepared in OECD growth medium and tested at the concentrations 0, 3, 6, 12, 24 and 50 mg/L. Effects on the growth rate of the organism were studied. 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) of the test substance on algaewas determined to be 0.13 mg/L on the basis of growth rate inhibition effects in a 72 hour study. Basedon the EC50 value, it is concluded that the substance is likely to be hazardous to aquatic algae and canbe consider to be classified as aquatic acute/ chronic 1 category as per the CLP classification criteria.

Thus based on the experimental data from various sources conducted on two different species of algae, concluded that the test chemical is toxic and can be consider to be classified in aquatic acute category 1/ chronic 1 as per the CLP classification criteria.

Description of key information

WoE 2: Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the EC50 was determine to be 1.086 mg/l. Based on the EC50, it can be concluded that the chemical was toxic to the growth of algae.

WoE 3: Based on the growth rate inhibition on Desmodesmus subspicatus after the exposure of test chemical for 72 hours, the EC50 was determined to be 0.13 mg/L.

Under the test condition it is observed that the chemical consider to be toxic as cause toxicity on Pseudokirchneriella subcapitata and Desmodesmus subspicatus at concentrations ranges from 1.086 to 0.13 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.13 mg/L

Additional information

Data available for the test chemical and structurally and functionally similar read across chemicals extracted using mechanistic approach has been reviewed to determine the toxicity of test chemical on the growth of aquatic algae. The studies are as mentioned below:

 

The study was designed to access the toxic effects of the test chemical on the green alga Pseudokirchneriella subcapitata. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Six test concentrations were 0.3mg/l, 0.39mg/l, 0.507mg/l, 0.659mg/l, 0.856mg/l, 1.112mg/l used and the study conducted under the static system. 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. Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the EC50 was determine to be 1.086 mg/l. Based on the EC50, it can be concluded that the chemical was toxic to the growth of algae.

 

The above study was further supported by the second study from experimental report. Freshwater algal growth inhibition test was carried out on green algae Desmodesmus subspicatus with the substance. Test conducted under the static system for 72 hours and according to the OECD Guideline 201 (Alga, Growth Inhibition Test). The test substance was prepared in OECD growth medium and tested at the concentrations 0, 3, 6, 12, 24 and 50 mg/L. Effects on the growth rate of the organism were studied. 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) of the test substance on algaewas determined to be 0.13 mg/L on the basis of growth rate inhibition effects in a 72 hour study. Basedon the EC50 value, it is concluded that the substance is likely to be hazardous to aquatic algae and canbe consider to be classified as aquatic acute/ chronic 1 category as per the CLP classification criteria.

Thus based on the experimental data from various sources conducted on two different species of algae, concluded that the test chemical is toxic and can be consider to be classified in aquatic acute category 1/ chronic 1 as per the CLP classification criteria.