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Toxicity to aquatic algae and cyanobacteria

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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:
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 compound Tetraethylammonium chloride (CAS No. 56-34-8) 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 Tetraethylammonium chloride(CAS No. 56-34-8) was prepared by adding 54.675 mg of test substance in 250 ml of BBM to get the final concentration of 218.7 mg/l. The remaining test solutions were prepared by dilution from the above stock solution. All the final test solutions were prepared under 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, 72 hrs
Test temperature:
22 °C±2°C
Nominal and measured concentrations:
0.9mg/l,2.7mg/l,8.1mg/l,24.3mg/l,72.9mg/l,218.7mg/l All the six concentration were in geometric series spaced by a factor of 3.
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.:
> 200 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

Test vessels and

0 Hours

24 Hours

48 Hours

72 Hours

test concentration

 

 

 

 

Control

 

 

 

 

Replicate1

 

13600

34000

41600

Replicate2

 

13600

22000

44800

Replicate3

 

12800

21600

36800

CAS No. 56-34-8

 

 

 

 

0.9mg/l

 

 

 

 

Replicate1

 

18800

30400

35200

Replicate2

 

17200

29600

34400

2.7mg/l

 

 

 

 

Replicate1

 

17200

28800

32400

Replicate2

 

16400

28000

31600

8.1mg/l

 

 

 

 

Replicate1

 

14800

26000

30000

Replicate2

 

14400

24800

28400

24.3mg/l

 

 

 

 

Replicate1

 

14000

23200

26800

Replicate2

 

12800

22000

25200

72.9mg/l

 

 

 

 

Replicate1

 

11600

20400

25200

Replicate2

 

10800

19600

24800

218.7mg/l

 

 

 

 

Replicate1

 

12000

19200

23200

Replicate2

 

11200

18000

21200

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

 

CONTROL

0.9mg/l

2.7mg/l

8.1mg/l

24.3mg/l

72.9mg/l

218.7mg/l

Average

R1

0.475

R1

0.419

R1

0.391

R1

0.366

R1

0.328

R1

0.308

R1

0.280

Specific

R2

0.499

R2

0.411

R2

0.383

R2

0.347

R2

0.308

R2

0.302

R2

0.250

Growth rate (µR3

0.434

 

 

 

 

 

 

 

 

 

 

 

 

)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean of Avg.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Specific

 

0.469

 

0.415

 

0.387

 

0.357

 

0.318

 

0.305

 

0.265

growth rate

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Percentage

 

_

 

11.521

17.474

23.992

32.235

34.987

 

43.485

Inhibition (%I)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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

Test vessels and

0 Hours

72 Hours

test concentration

 

 

CONTROL

 

 

Replicate1

6.80

6.97

Replicate2

6.81

6.97

Replicate3

6.80

6.95

Average

6.8

6.96

CAS No.56-34-8

 

 

0.9mg/l

 

 

Replicate1

6.64

6.86

Replicate2

6.66

7.01

2.7mg/l

 

 

Replicate1

6.63

6.90

Replicate2

6.70

6.89

8.1mg/l

 

 

Replicate1

6.69

6.90

Replicate2

6.73

7.01

24.3mg/l

 

 

Replicate1

6.68

6.95

Replicate2

6.76

6.96

72.9mg/l

 

 

Replicate1

6.75

6.87

Replicate2

6.71

6.91

218.7mg/l

 

 

Replicate1

6.8

6.92

Replicate2

6.67

7.02

Validity criteria fulfilled:
yes
Conclusions:
Based on the growth inhibition of green alga Chlorella vulgaris by the test chemical , the EC50 was determine to be >200 mg/l
Executive summary:

The study was designed to assess the toxic effects of the test compound Tetraethylammonium chloride(CAS No. 56-34-8) on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

Test was 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 60 ml so that a sufficient amount of head space was left. The test substance was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring/ sonication for 10 minutes to obtain a homogenous solution for the experiment. The test concentrations 0.9mg/l, 2.7mg/l, 8.1mg/l, 24.3mg/l,72.9mg/l, 218.7mg/l 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.

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.

As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item Tetraethylammonium chloride to various nominal test concentrations, EC50 was determine to be >200 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was non hazardous and can be consider to be not classified as per the CLP classification criteria.

Description of key information

Based on the growth inhibition of green alga Chlorella vulgaris by the test chemical , the EC50 was determine to be >200 mg/l

Key value for chemical safety assessment

EC50 for marine water algae:
200 mg/L

Additional information

Following three studies of target chemical to conclude the aquatic algae toxicity of Tetraethylammonium chloride (CAS: 56-34-8) is summarized below:

The study was designed to assess the toxic effects of the test compound Tetraethylammonium chloride (CAS No. 56-34-8) on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Test was 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 60 ml so that a sufficient amount of head space was left. The test substance was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring/ sonication for 10 minutes to obtain a homogenous solution for the experiment. The test concentrations 0.9mg/l, 2.7mg/l, 8.1mg/l, 24.3mg/l,72.9mg/l, 218.7mg/l 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. 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 was 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) was 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. As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201 After 72 hours of exposure to test item Tetraethylammonium chloride to various nominal test concentrations, EC50 was determine to be >200 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was non hazardous and can be consider to be not classified as per the CLP classification criteria.

In a similar study, Aim of this study was to evaluate the nature of chemical test chemical when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201. The stock solution 100 mg/l was prepared by dissolving white powder in OECD growth medium. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. 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 determined after an exposure period of 72 hrs. The % inhibition for the test substance Tetraethylammonium chloride (CAS 56 -34 -8), in algae was determined to be 0.5% on the basis of growth rate inhibition effects in a 72 hour study. Based on the % inhibition, which indicates that the substance is likely to be non-hazardous to aquatic algae and can be classified as Not classified as per the CLP classification criteria.

Thus based on the effect concentrations which is >200 mg/l give the conclusion that test substance Tetraethylammonium chloride (CAS: 56-34-8) is likely to be not toxic to aquatic algae at environmentally relevant concentrations and can be considered to be“Not Calssified”as per the CLP classification criteria.