Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

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 using standard OECD test guidelines
Qualifier:
equivalent or similar to
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 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
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
The test solution was prepared in aseptic condition. The test item Tetrylammonium bromide was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring for 24 hours 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 of the culture was kept 1 X 104cells/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
- Strain:Chlorella vulgaris
- Source (laboratory, culture collection): 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.
- 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
Total exposure duration:
72 h
Test temperature:
22 °C ±2°C
pH:
6.8 ±0.3
Nominal and measured concentrations:
Six test concentration were: 6.25mg/l,12.5mg/l,25mg/l,50mg/l,100mg/l,200mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flasks
- Type (delete if not applicable): No data available
- 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: 29.16 x104 cells/mL
- Control end cells density: No data available
- No. of organisms per vessel: 10000cells/m
l- 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 availableGROWTH 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 availableEFFECT 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,200mg/l (Nominal concentrations)- Results used to determine the conditions for the definitive study: No data available6.1. Test solution: The test solution was prepared in aseptic condition. The test item Tetrylammonium bromide was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring for 24 hours 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 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.
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
Details on results:
Control vessels: The microscopic observations were noted down in each of the control vessel. All the cells appeared healthy, round and green throughout the study duration in the control. Also, the drift in pH in the control vessels did not increase by >1.5 units when observed on 72 hours as compared to 0 hours. The average pH drift observed in the control vessels was 0.11 units.Test vessels: The effect of the test item on the green algae Chlorella vulgaris culture was observed at nominal test concentration of >6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L, 100 mg/L 200 mg/L. All the six concentrations were in geometric series spaced by a factor of 2. The microscopic observations were also noted in each of the test vessel. All the cells appeared healthy, round and green throughout the study duration and no significant changes were observed up to the concentration of 200 mg/l. EC50 was found to be >200 mg/l graphically through probitanalysis.
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 test concentration

24 Hours

48 Hours

72 Hours

Control

Replicate 1

20000

36400

46400

Replicate 2

20800

37200

47200

Replicate 3

22800

38800

48000

CAS No. 71 -91 -0

6.25 mg/l

Replicate 1

18000

34000

35600

Replicate 2

18800

30000

37600

12.5 mg/l

Replicate 1

16800

28400

35200

Replicate 2

18000

26800

34000

25 mg/l

Replicate 1

16800

27200

33200

Replicate 2

22800

29600

32400

50 mg/l

Replicate 1

20400

26400

30800

Replicate 2

18800

26800

29200

100 mg/l

Replicate 1

19200

24000

28800

Replicate 2

19600

22800

26800

200 mg/l

Replicate 1

20400

19600

26000

Replicate 2

21200

20400

25200

 

 

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

0.511

R1

0.423

R1

0.419

R1

0.399

R1

0.374

R1

0.352

R1

0.318

 

R2

0.517

R2

0.441

R2

0.407

R2

0.391

R2

0.357

R2

0.328

R2

0.308

 

R3

0.522

 

Mean of Avg. Specific growth rate

0.517

0.432

0.413

0.395

0.366

0.340

0.313

Percentage Inhibition (%I)

_

16.409

20.016

23.454

29.222

34.149

39.429

 

 

 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

6.60

6.68

Replicate 2

6.61

6.74

Replicate 3

6.62

6.75

Average

6.61

6.72

CAS No. 71 -91 -0

6.25 mg/l

Replicate 1

6.75

6.34

Replicate 2

6.76

6.38

12.5 mg/l

Replicate 1

6.65

6.44

Replicate 2

6.64

6.49

25 mg/l

Replicate 1

6.65

6.54

Replicate 2

6.66

6.63

50 mg/l

Replicate 1

6.63

6.53

Replicate 2

6.64

6.52

100 mg/l

Replicate 1

6.63

6.52

Replicate 2

6.62

6.52

200 mg/l

Replicate 1

6.63

6.53

Replicate 2

6.64

6.53

 

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
After 72 hours of exposure to test item to various nominal test concentrations, EC50 was found to be >200 mg/l graphically and through probit analysis.
Executive summary:

The effect of test item 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, 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 >200 mg/L. Thus based on this value, it can be concluded that the substance can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP classification criteria.

Description of key information

The effect of test item 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, 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 >200 mg/L. Thus based on this value, it can be concluded that the substance can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:
200 mg/L

Additional information

Summarized result of the toxicity of test chemical on the growth and mobility of test chemical on the growth of aquatic algae and cyanobacteria. As mention below:

 

In an experimental key study the effect of test item 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, 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 >200 mg/L. Thus based on this value, it can be concluded that the substance can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP classification criteria.

 

Similarly in the second study from experimental study 2017, 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 solution 100 mg/l was prepared by dissolving white powder in OECD growth medium. Limit test at 100 mg/l was performed. 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. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical N, N, N-triethylethanaminium bromide, only 2.6% inhibition were observed at 100 mg/l. As the less effect were observed i.e only 2.6% inhibition were obtain at 100 mg/l, so on that basis chemical was consider as nontoxic and can be consider to be not classified as per the CLP classification criteria.

 

 

Similarly Short term toxicity to Pseudokirchneriella subcapitata (green algae) study was carried out for 48 hrs. The study was performed according to OECD Guideline 201 and ISO 8692 method. The study was based on the effects of the test compound on Pseudokirchneriella subcapitata (green algae) in a static fresh water system. The stock solutions were prepared with the growth media. All stock solutions were adjusted to the pH of the control media before use. The mini-scale algal growth inhibition tests were conducted with 4 ml of medium and 17 ml of CO2 -enriched headspace (1% CO2). The vials were closed with a Teflon covered membrane, and CO2 was added with a syringe. The diameter of the glass vial was 1.87 cm, which gave a glass surface area in contact with water of 11.3 cm2. The growth rates (exponential growth) of the cultures were used as the test parameter. Six control replicates were used together with 9 to 13 test concentrations with only one replicate to optimize the dose–response modelling and for cost-effectiveness. In combination with the CO2-enriched headspace, the resulting test pH was 7.0±0.2. The control growth rates were 1.7 to 1.9/d. The test vials were incubated and vigorously shaken on a microplate shaker (100 oscillations/min) in continuous white-fluorescent light (30W/33; Philips, Amsterdam, The Netherlands) with an intensity of 80µE/m2/s and a temperature of 20±1ᵒC. Cell density in the inoculation culture was counted on a particle counter (Coulter Counter Z2; Beckman Coulter, Hialeah, FL, USA). Algal biomasses were determined at the start, after 24 h, and at the end (48 h) from acetone pigment extractions. The growth rate in each vial was calculated directly from the log-transformed fluorescence measurements. EC50s and EC10s with 95% confidence limits using probit or Weibull models are calculated by nonlinear regression on the whole dataset using a dose–response regression program with variance weighting and proper inverse estimation. Based on effect on growth rate of the test organism Pseudokirchneriella subcapitata(green algae), the 48 hr EC50, EC10 value (by probit model) was determined to be 345 and 164 mg/l, respectively and on the basis effect on growth rate, the 48 hr EC50, EC10 value (by Weibull model), was determined to be 357 and 138 mg/l, respectively. Thus, based on the EC50 value, it can be concluded that the substance can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP classification criteria.

Based on the overall studies chemical was consider as nontoxic and not classified as per the CLP classification criteria.