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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
Study period:
14.03.2017 to 17.03.2017
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 compound Benzimidazole on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material (as cited in study report): Benzimidazole
- Molecular formula :C7H6N2
- Molecular weight: 118.1384 g/mol
- Substance type:Organic
- Physical state:Solid
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test solution was prepared in aseptic condition. The test item Benzimidazole 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 30 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 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:
6.25mg/l,12.5mg/l, 25mg/l, 50mg/l,100mg/l, 200mg/l 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.:
> 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

24 Hours

48 Hours

72 Hours

test concentration

 

 

 

Control

 

 

 

Replicate1

16800

32800

50000

Replicate2

17200

35200

52400

Replicate3

18000

36800

55600

Test chemical

 

 

 

6.25mg/l

 

 

 

Replicate1

12000

21600

33200

Replicate2

13200

20800

34800

12.5mg/l

 

 

 

Replicate1

13600

18800

33600

Replicate2

16400

19600

31600

25mg/l

 

 

 

Replicate1

16000

20400

31200

Replicate2

15200

20400

30800

50mg/l

 

 

 

Replicate1

15600

22000

29200

Replicate2

15600

20400

28400

100mg/l

 

 

 

Replicate1

14000

19600

26800

Replicate2

12400

16800

25200

200mg/l

 

 

 

Replicate1

12800

17600

23600

Replicate2

12800

14800

25200

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

6.58

6.70

Replicate2

6.59

6.68

Replicate3

6.62

6.68

Average

6.6

6.69

Test chemical

 

 

 

6.25mg/l

 

 

Replicate1

6.65

6.71

Replicate2

6.66

6.67

12.5mg/l

 

 

Replicate1

6.68

6.66

Replicate2

6.65

6.68

25mg/l

 

 

Replicate1

6.66

6.69

Replicate2

6.69

6.74

50mg/l

 

 

Replicate1

6.66

6.74

Replicate2

6.69

6.75

100mg/l

 

 

Replicate1

6.68

6.74

Replicate2

6.67

6.75

200mg/l

 

 

Replicate1

6.92

6.81

Replicate2

6.92

6.80


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

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

The test solution was prepared in aseptic condition. The test item Benzimidazole 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 30 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 of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. 6.25mg/l,12.5mg/l, 25mg/l, 50mg/l,100mg/l, 200mg/l concentration were used in the study. All the six concentrations were in geometric series spaced by a factor of 2.

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 Benzimidazole 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 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 Benzimidazole on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). The test solution was prepared in aseptic condition. The test item Benzimidazole 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 30 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 of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. 6.25mg/l,12.5mg/l, 25mg/l, 50mg/l,100mg/l, 200mg/l concentration were used in the study. All the six concentrations were in geometric series spaced by a factor of 2. 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 Benzimidazole 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 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:
200 mg/L

Additional information

Summarized result for the toxicity of test chemical Benzimidazole (CAS No. 51 -17 -2) on the growth of aquatic algae and cyanobacteria are as follows:

In the first experimental report, study was designed to assess the toxic effects of the test compound Benzimidazole on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). The test solution was prepared in aseptic condition. The test item Benzimidazole 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 30 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 of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. 6.25mg/l,12.5mg/l, 25mg/l, 50mg/l,100mg/l, 200mg/l concentration were used in the study. All the six concentrations were in geometric series spaced by a factor of 2. 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 Benzimidazole 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 nontoxic and can be consider to be not classified as per the CLP classification criteria.

Similarly in the second study freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance Benzimidazole to OECD Guideline 201. The stock solution (120 mg/L) was prepared by dissolving whiite powder in OECD growth medium. The solution was kept in ultrasonic bath for 20 mins. Test solutions  of required concentrations  were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Conc. of test chemical used for the study was 0, 7.5, 15, 30, 60 and 120 mg/L, respectively. The test was performed under static conditions in a static fresh water system at a temp. of 23±2°C. Initial cell density of test organism used was 5x10(3) cells/ml. Determination of cell counting involve the use of microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. The median effective concentration (ErC50) for the test substance Benzimidazole in Desmodesmus subspicatus was determined to be 26.8 mg/L on the basis of effects on growth rate in a 72 hour study. Based on the ErC50 value, chemical consider to be toxic to the growth of aquatic algae and cyanobacteria and classified as aquatic chronic 3 as per the CLP classification criteria. But as the chemical was readily biodegradable in water, thus it not exist for longer period of time. Thus on that basis chemical consider to be nontoxic and not classified as per the CLP classification criteria.

Thus based on the above both studies, chemical Benzimidazole (CAS No. 51 -17 -2) was consider to be nontoxic and not classified as per the CLP classification criteria.