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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
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 (1-phenylethan-1 ol) on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).
GLP compliance:
no
Specific details on test material used for the study:
IUPAC name: 1-phenylethan-1-ol
Mol. formula: C8H10O
Molecular Weight: 122.166 g/mol
Substance form: Colourless liquid
Substance type: organic
InChI: 1S/C8H10O/c1-7(9)8-5-3-2-4-6-8/h2-7,9H,1H3
Smiles :c1(ccccc1)C(C)O
Batch No.: 2014022801R-434
Analytical monitoring:
not required
Remarks:
Stable under normal conditions
Vehicle:
not specified
Details on test solutions:
The test substance 1-phenylethanol was prepared by adding 60 µl of test substance in 300 ml of BBM to get the final concentration of 200 mg/L. 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 1 X 10e4 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)
- Iinitial cell density: 1 X 10e4 cells/ml
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:
6.9 to 7.2
Nominal and measured concentrations:
6.25 mg/l, 12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200mg/l nominal concentration were used in the study. All the six concentrations 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 having headspace of 40 ml was used for the study.
- Initial cells density: 10000 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: 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

 

24 Hours

48 Hours

72 Hours

Control

Replicate 1

72.5 x 104

11 x 105

10.25 x 105

Replicate 2

73.5 x 104

11 x 105

10.95 x 105

Replicate 3

75 x 105

10.8 x 105

10.6 x 105

CAS No. 98-85-1

6.25 mg/l

Replicate 1

81.5 x 104

92 x 105

78.5 x 104

Replicate 2

83.5 x 104

85.5 x 104

72.5 x 104

12.5 mg/l

Replicate 1

73.5 x 104

40 x 105

56.5 x 104

Replicate 2

77.5 x 104

44 x 105

57 x 105

25 mg/l

Replicate 1

58.5 x 104

61.5 x 104

58 x 105

Replicate 2

55 x 105

52.5 x 104

50 x 105

50 mg/l

Replicate 1

59.5 x 104

46.5 x 104

55.5 x 104

Replicate 2

52.5 x 104

48 x 105

53 x 105

100 mg/l

Replicate 1

40 x 105

51.5 x 104

55 x 105

Replicate 2

43 x 105

56.5 x 104

47 x 105

200 mg/l

Replicate 1

46 x 105

58.5 x 104

41.5 x 104

Replicate 2

54 x 105

55 x 105

46 x 105

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

 

CONTROL

6.25 mg/l

12.5 mg/l

25 mg/l

50 mg/l

100 mg/l

200 mg/l

Average Specific Growth rate (µ )

R1

0.775

R1

0.686

R1

0.577

R1

0.585

R1

0.571

R1

0.568

R1

0.474

 

R2

0.797

R2

0.660

R2

0.580

R2

0.536

R2

0.555

R2

0.515

R2

0.508

 

R3

0.786

 

Mean of Avg. Specific growth rate

0.786

0.673

0.578

0.561

0.563

0.542

0.491

Percentage Inhibition (%I)

_

14.392

26.453

28.673

28.372

31.109

37.530

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

7.2

7.2

Replicate 2

7.1

7.2

Replicate 3

7.2

7.1

CAS No. 98-85-1

6.25 mg/l

Replicate 1

7.1

7.2

Replicate 2

7.0

7.1

12.5 mg/l

Replicate 1

7.1

7.2

Replicate 2

7.2

7.0

25 mg/l

Replicate 1

7.2

7.1

Replicate 2

7.0

6.9

50 mg/l

Replicate 1

7.0

6.8

Replicate 2

6.9

7.0

100 mg/l

Replicate 1

7.0

6.9

Replicate 2

7.1

7.0

200 mg/l

Replicate 1

6.8

6.9

Replicate 2

7.0

7.0

Validity criteria fulfilled:
yes
Conclusions:
Based on the growth rate inhibition of green alga Chlorella vulgaris by the test chemical 1-phenylethanol (CAS No. 98-85-1), the EC50 was determine to be >200 mg/l .
Executive summary:

The study was designed to assess the toxic effects of the test compound 1-phenylethanol 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 1-phenylethan-1 ol 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 0 minutes to obtain a homogenous solution for the experiment. The test concentrations 6.25 mg/l, 12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200mg/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 1-phenylethan-1 ol 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 not toxic 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 1-phenylethanol 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 1-phenylethan-1 ol 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 0 minutes to obtain a homogenous solution for the experiment. The test concentrations 6.25 mg/l, 12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200mg/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 1-phenylethan-1 ol 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 not toxic 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

Toxicity to aquatic algae and cyanobacteria:

The toxicity to aquatic algae and cyanobacteria was summarized using two experimental studies and data from peer reviewed journal.

The study was designed to assess the toxic effects of the test compound 1-phenylethanol 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 1-phenylethan-1 ol 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 0 minutes to obtain a homogenous solution for the experiment. The test concentrations 6.25 mg/l, 12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200mg/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 1-phenylethan-1 ol 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 not toxic and can be consider to be not classified as per the CLP classification criteria.

Another experimental study was used to support the above data for the test substance. 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 of colourless liquid (purity 99.62 °/o) 100.0 mg/l was prepared by dissolving the substance in OECD growth medium .With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration inhibition percentage [%] 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 percentage [%] of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical the [%] was determine to be 1.5% at the concentration 100 mg/l. As only 1.5 % inhibition were observed, thus it was consider that the EC50 was greater than 100 mg/l. The avarage growth rate in control was 2.06d-1and the avarage growth rate for the sample was 2.03d-1on the basis of growth rate inhibition effects in a 72 hour study. Based on the [%] inhibition value, which indicates that the substance is likely to be hazardous to aquatic algae and cannot classified as per the CLP criteria.

Data from reliable peer reviewed journal was summarised to support the above experimental study data. Toxicity of test material 1 -phenylethanol was evaluated for aquatic algae and cyanobacteria. Test conducted as per the OECD guideline 201 . The EC50 value based on the growth inhibition after 72 h was observed to be >200 mg/l. Based on the above effect concentration it can be considered that test material was not toxic for aquatic algae and cyanobacteria and can not be classified as per CLP criteria.