Phenethyl phenylacetate

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

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

Guideline:

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 6.06 mg of test substance in 390 ml of BBM to get the final concentration of 15.56 mg/L. The sock solution was ultrasonically agitated for 30 minutes to obtain a homogenous solution for the experiment. The remaining test solutions were prepared by dilution from the above prepared stock solution under aseptic conditions. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10e4 cells/ml.

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: Chlorella vulgaris
- 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

Test temperature:

24 °C ±2°C.

pH:

6.8 ± 0.3

Nominal and measured concentrations:

2 mg/l, 3 mg/l, 4.5 mg/l, 6.75 mg/l, 10.13 mg/l and 15.19 mg/l respectively

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks
- Material, size, headspace, fill volume: Conical flasks of 100 ml size was used for the study.
- Initial cells density: 10000 cells/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
- 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
- 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 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: 2 mg/l, 3 mg/l, 4.5 mg/l, 6.75 mg/l, 10.13 mg/l and 15.19 mg/l (Nominal concentrations)
- Results used to determine the conditions for the definitive study: No data available

6.1. Test solution: The test solution was prepared in aseptic condition. The test item4-amino-5-hydroxynaphthalene-1,7-disulphonic acid was prepared by adding 500 mg of test item in 250ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring for 30minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item.

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 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 24°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.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13.89 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Reported statistics and error estimates:

To obtain a quantitative concentration-response relationship by regression analysis, a line arizing 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 experimental flasks at an equal interval of 24hrs, 48hrs and 72hrs

Experimental Flasks and test concentration	24 Hours	48 Hours	72 Hours
Control
Replicate 1	90 x 10^4	11.75 x 10^5	14.8 x 10^5
Replicate 2	74.5 x 10^4	10.5 x 10^5	15.15 x 10^5
Replicate 3	89.5 x 10^4	99 x 10^4	14 x 10^5
Test chemical
2 mg/l
Replicate 1	63.5 x 10^4	66.5 x 10^4	68 x 10^4
Replicate 2	61 x 10^4	64.5 x 10^4	65.5 x 10^4
3mg/l
Replicate 1	71.5 x 10^4	70 x 10^4	56.5 x 10^4
Replicate 2	71 x 10^4	35.5 x 10^4	53.5 x 10^4
4.5mg/l
Replicate 1	74 x 10^4	73 x 10^4	55.5 x 10^4
Replicate 2	75.5 x 10^4	61 x 10^4	49 x 10^4
6.75mg/l
Replicate 1	56.5 x 10^4	65 x 10^4	48 x 10^4
Replicate 2	53.5 x 10^4	60.5 x 10^4	48.5 x 10^4
10.13mg/l
Replicate 1	64.5 x 10^4	52.5 x 10^4	11.5 x 10^4
Replicate 2	52.5 x 10^4	41 x 10^4	12.5 x 10^4
15.19mg/l
Replicate 1	63 x 10^4	41 x 10^4	9 x 10^4
Replicate 2	54 x 10^4	42.5 x 10^4	8.5 x 10^4

 

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

	CONTROL		2mg/l		3mg/l		4.5mg/l		6.75mg/l		10.13mg/l		15.19mg/l
Average Specific Growth rate (µ )	R1	1.665	R1	1.406	R1	1.344	R1	1.338	R1	1.290	R1	0.814	R1	0.732
	R2	1.673	R2	1.394	R2	1.326	R2	1.297	R2	1.293	R2	0.841	R2	0.713
	R3	1.647
Mean of Avg. Specific growth rate	1.662		1.400		1.335		1.318		1.292		0.828		0.722
Percentage Inhibition (%I)	_		15.756		19.643		20.703		22.262		50.184		56.509

 

 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	6.7	7.2
Replicate 2	6.8	7.1
Replicate 3	6.6	7.2
Test chemical
2 mg/l
Replicate 1	6.6	7.0
Replicate 2	6.6	7.1
3 mg/l
Replicate 1	6.7	7.2
Replicate 2	6.6	7.0
4.5 mg/l
Replicate 1	6.6	7.0
Replicate 2	6.5	7.1
6.75 mg/l
Replicate 1	6.7	7.1
Replicate 2	6.6	7.2
10.13 mg/l
Replicate 1	6.8	7.2
Replicate 2	6.6	7.2
15.19 mg/l
Replicate 1	6.5	7.3
Replicate 2	6.6	7.3

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure to test chemical to various nominal test concentrations with algae Chlorella vulgaris, the EC50 value based on the growth rate inhibition of algae was determined to be 13.89 mg/L graphically and through probit analysis.

Executive summary:

The study was designed to access the toxic effects of the test chemical on the green alga. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Chlorella vulgaris was used as test organism. The test substance  was prepared by adding 6.06 mg of test substance in 390 ml of BBM to get the final concentration of 15.56 mg/L. The sock solution was ultrasonically agitated for 30 minutes to obtain a homogenous solution for the experiment. The remaining test solutions were prepared by dilution from the above prepared stock solution under aseptic conditions. Six test concentrations were 2 mg/l, 3 mg/l, 4.5 mg/l, 6.75 mg/l, 10.13 mg/l and 15.19 mg/l was used and the study conducted under the static system. 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. The test is considered to be valid as met all the standard parameters. After 72 hours of exposure to test chemical to various nominal test concentrations with algae Chlorella vulgaris, the EC50 value based on the growth rate inhibition of algae was determined to be 13.89 mg/L graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was toxic and can be consider to be classified aq aquatic chronic 3 as per the CLP classification criteria.

Description of key information

The study was designed to access the toxic effects of the test chemical on the green alga. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Chlorella vulgaris was used as test organism. The test substance was prepared by adding 6.06 mg of test substance in 390 ml of BBM to get the final concentration of 15.56 mg/L. The sock solution was ultrasonically agitated for 30 minutes to obtain a homogenous solution for the experiment. The remaining test solutions were prepared by dilution from the above prepared stock solution under aseptic conditions. Six test concentrations were 2 mg/l, 3 mg/l, 4.5 mg/l, 6.75 mg/l, 10.13 mg/l and 15.19 mg/l was used and the study conducted under the static system. 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. The test is considered to be valid as met all the standard parameters. After 72 hours of exposure to test chemical to various nominal test concentrations with algae Chlorella vulgaris, the EC50 value based on the growth rate inhibition of algae was determined to be 13.89 mg/L graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was toxic and can be consider to be classified aq aquatic chronic 3 as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

13.89 mg/L

Additional information

Various experimental data of the test chemical and supporting weight of evidence studies for its structurally and functionally similar read across chemical were reviewed for the toxicity of test chemical on algae end point which are summarized as below:

 

The study was designed to access the toxic effects of the test chemical on the green alga. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Chlorella vulgaris was used as test organism. The test substance was prepared by adding 6.06 mg of test substance in 390 ml of BBM to get the final concentration of 15.56 mg/L. The sock solution was ultrasonically agitated for 30 minutes to obtain a homogenous solution for the experiment. The remaining test solutions were prepared by dilution from the above prepared stock solution under aseptic conditions. Six test concentrations were 2 mg/l, 3 mg/l, 4.5 mg/l, 6.75 mg/l, 10.13 mg/l and 15.19 mg/l was used and the study conducted under the static system. 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. The test is considered to be valid as met all the standard parameters. After 72 hours of exposure to test chemical to various nominal test concentrations with algae Chlorella vulgaris, the EC50 value based on the growth rate inhibition of algae was determined to be 13.89 mg/L graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was toxic and can be consider to be classified aq aquatic chronic 3 as per the CLP classification criteria.

 

Similar freshwater algal growth inhibition test was carried out on green algae. Test conducted in accordance with OECD Guideline 201 (Alga, Growth Inhibition Test). Desmodesmus subspicatus was used as test organism. The stock solution 100 mg/L was prepared by dissolving colourless liquid in acetone. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. 0, 6.25, 12.5, 25, 50 and 100 mg/L, respectively concentrations were used in the study. The test was performed under static conditions at a temp. of 23±2°C. Initial cell density of test organism used was 5x10e3 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. Effects on the growth rate of algae was calculated in the interval of 24, 48 and 72 hours. The median effective concentration (ErC50) for the test substance on Desmodesmus subspicatus was determined to be 34.5 mg/L on the basis of effects on growth rate in a 72 hour study. Thus based on this value, test chemical can be considered as toxic to aquatic organisms and thus can be classified in aquatic chronic category 3 as per the CLP criteria.

 

Above studies further supported by the third weight of evidence study from experimental study report. Principle of this study was to evaluate the nature of test chemical when comes in contact with the test organism. Test was conducted according to the OECD guideline 201. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as test organism. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. 0, 10,16, 26, 41, 66 mg/l, respectively concentration were used. 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 median effective concentration (ErC50) of the test substance on algae was determined to be 23.6 mg/L with 95% CI of 28.9 mg/l to 33.7 mg/l on the basis of growth rate inhibition effects in a 72 hour study. Based on the ErC50 value, the test chemical was consider likely to be hazardous to aquatic algae and can be consider to be classified in aquatic chronic 3 category.

 

Thus based on the overall studies from various sources, it was concluded that the test chemical was toxic and classified in aquatic chronic category 3 as per the CLP classification criteria.