Tetrabutylammonium bromide

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed according to the guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Principles of method if other than guideline:

This study was designed to access the toxic effects of the test chemical on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The test substance was prepared by adding 50 mg of test substance in 100 ml of BBM to get the final concentration of 500 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. The test solution was prepared in aseptic condition. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/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
- Source (laboratory, culture collection): National Environmental Engineering Research Institute (NEERI), Nagpur (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 and 72 hrs

Test temperature:

24°C ±2°C.

pH:

6.7 - 7.3

Nominal and measured concentrations:

3.125 mg/l, 6.25 mg/l, 12.5 mg/l, 25 mg/l, 50 mg/l and 100 mg/l nominal concentration were used. All the six concentration were in geometric series spaced by a factor of 2.

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: 10000cells/ml
- 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

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: continuous, uniform fluorescent illumination(1500Lux)

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: 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: 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.

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

204.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: calculated from equation through probit analysis

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.

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

Experimental Flasks and test concentration	24 Hours	48 Hours	72 Hours
Control
Replicate 1	87.5 x 10^4	10 x 10^5	13.25 x 10^5
Replicate 2	91.5 x 10^4	11.55 x 10^5	14.65 x 10^5
Replicate 3	86 x 10^4	11.3 x 10^5	12.65 x 10^5
Test chemical
3.125 mg/l
Replicate 1	74 x 10^4	63 x 10^4	73 x 10^4
Replicate 2	72.5 x 10^4	70 x 10^4	70.5 x 10^4
6.25 mg/l
Replicate 1	67.5 x 10^4	63 x 10^4	63 x 10^4
Replicate 2	66 x 10^4	35.5 x 10^4	60 x 10^4
12.5 mg/l
Replicate 1	66.5 x 10^4	65 x 10^4	61 x 10^4
Replicate 2	62.5 x 10^4	67 x 10^4	61.5 x 10^4
25 mg/l
Replicate 1	60.5 x 10^4	60.5 x 10^4	49 x 10^4
Replicate 2	57.5 x 10^4	59.5 x 10^4	52 x 10^4
50 mg/l
Replicate 1	55 x 10^4	57.5 x 10^4	19 x 10^4
Replicate 2	55.5 x 10^4	58.5 x 10^4	25 x 10^4
100 mg/l
Replicate 1	47 x 10^4	60 x 10^4	12 x 10^4
Replicate 2	46.5 x 10^4	55.5 x 10^4	17 x 10^4

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

	CONTROL		3.125 mg/l		6.25 mg/l		12.5 mg/l		25 mg/l		50 mg/l		100 mg/l
Average Specific Growth rate (µ )	R1	1.628	R1	1.430	R1	1.381	R1	1.370	R1	1.297	R1	0.981	R1	0.828
	R2	1.662	R2	1.418	R2	1.364	R2	1.373	R2	1.317	R2	1.072	R2	0.944
	R3	1.613
Mean of Avg. Specific growth rate	1.634		1.424		1.372		1.371		1.307		1.027		0.886
Percentage Inhibition (%I)	_		12.877		16.023		16.100		20.044		37.168		45.784

Table 3: Depicting pH values at test initiation (0 Hours) and test termination (72 Hours)

Experimental Flasks and test concentration	0 Hours	72 Hours
CONTROL
Replicate 1	7.0	7.2
Replicate 2	7.1	7.3
Replicate 3	7.0	7.2
Test chemical
3.125 mg/l
Replicate 1	7.2	7.3
Replicate 2	7.1	7.2
6.25 mg/l
Replicate 1	7.3	7.2
Replicate 2	7.2	7.1
12.5 mg/l
Replicate 1	7.2	7.2
Replicate 2	7.1	7.2
25 mg/l
Replicate 1	7.1	7.1
Replicate 2	7.1	7.0
50 mg/l
Replicate 1	7.0	7.2
Replicate 2	7.1	7.2
100 mg/l
Replicate 1	6.9	6.8
Replicate 2	7.0	6.7

Validity criteria fulfilled:

yes

Conclusions:

Based on the growth rate inhibition of green alga Chlorella vulgaris by the test chemical, the EC50 was determine to be 204.70 mg/l.

Executive summary:

The study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). The test substance was prepared by adding 50 mg of test substance in 100 ml of BBM to get the final concentration of 500 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. The test solution was prepared in aseptic condition. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. 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 chemical to various nominal test concentrations, EC50 was determine to be 204.70 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

Description of key information

The study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). The test substance was prepared by adding 50 mg of test substance in 100 ml of BBM to get the final concentration of 500 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. The test solution was prepared in aseptic condition. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. 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 chemical to various nominal test concentrations, EC50 was determine to be 204.70 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

204.7 mg/L

Additional information

Based on the various experimental data for the target chemical study have been reviewed to determine the nature of test chemical on the growth of aquatic algae and cyanobacteria. The studies are as mentioned below:  

The study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). The test substance was prepared by adding 50 mg of test substance in 100 ml of BBM to get the final concentration of 500 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. The test solution was prepared in aseptic condition. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. 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 chemical to various nominal test concentrations, EC50 was determine to be 204.70 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

 

First study was supported by the second experimental study for target chemical from peer reviewed journal. Freshwater algal growth inhibition test was carried out for 96 hr on Pseudokirchneriella subcapitata with the test chemical according to OECD Guideline 201 (Alga, Growth Inhibition Test) and EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II), respectively. The study was based on the effects of the test compound on Pseudokirchneriella subcapitata in a flow through fresh water system. The green microalga used as the model algal strain in this study, Selenastrum capricornutum ATCC-22662, was obtained from the National Institute Environmental Research, Korea. The stock alga was cultivated in 250 ml Erlenmeyer flasks, containing 200 ml sterilized nitrate-enriched BBM medium prepared in triple distilled water, to avoid nitrogen limitation during the high-density culture. Experiments were performed in 250 ml Erlenmeyer flasks, containing 55ml of sterilized culture medium, inoculated with 5ml samples of 7-day cultured algae. Solutions of ionic liquids and organic solvents were subsequently added to the test flasks. The flasks then were placed on a shaker incubator at 170 rpm and 25 deg C, with 24 h light supplied via warm-white fluorescent tubes, with an average illumination of 3075 mEm2 s1. At each determined exposure date, the optical density of the algal biomass was estimated at 438nm using a spectrophotometer (UV mini-1240, Shimadzu, Kyoto, Japan) and average specific growth rate determined. The dry cell weight, corresponding to the optical density, was determined from the linear relationship; dry cell weight (g/l) = 0.1329 optical density. Based upon the effect on the growth rate of the test organism Pseudokirchneriella subcapitata, the 48, 72 and 96hr EC50 value was determined to be 300.84, 1542.94 and 721.689 mg/L, respectively. 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.

 

Thus based on the above studies, it was conclude that the test chemical was nontoxic to the growth of algae and cyanobacteria and can be consider to be not classified as per the CLP classification criteria.