Phenethyl benzoate

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:

key study

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 guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.25, 4 and 8 mg/L
- Sample storage conditions before analysis: Test samples were analysed immediately after sampling

Vehicle:

no

Details on test solutions:

The saturated test solution was prepared by dissolving 500mg of test chemical in 500ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 123.55 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.25, 0.5, 1, 2, 4 and 8 mg/l, respectively was from the saturated test concentrations.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Length: 8 – 14 μm
- Weight: 2 - 3 μm
- Source: Sterile, unicellular, suspension cultures of algae were obtained from the laboratory for Biological Research in Aquatic Pollution (LABRAP) at the University of Ghent in Belgium and maintained in
Laboratory.
- Method of cultivation: OECD medium

ACCLIMATION
- Culturing media and conditions (same as test or not): The medium to be used for the growth of algae was OECD medium.
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 to 24 ± 2°C

pH:

At day 0 -7.5 and day 3 - 7.28

Nominal and measured concentrations:

Test chemical concentrations used for the study were 0, 0.25, 0.5, 1, 2, 4 and 8 mg/L, respectively.

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, OECD medium was used as a test medium in the study.

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(3000-4000Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell counts were measured using microscope.
- 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: 0, 0.25, 0.5, 1, 2, 4 and 8 mg/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):

yes

Remarks:

Potassium dichromate (K2Cr2O7) was used as a reference substance.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.58 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, sickle shaped and green throughout the test duration in the control.

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50: 0.868 mg/l

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: Assessment of test concentrations

Sr. no.	Concentrations (mg/l)	Wavelength (nm)	Absorbance	Temperature (°C)
1	blank	201	0.00	25°C
2	0.06	201	0.03	25°C
3	0.12	201	0.06	25°C
4	0.25	201	0.07	25°C
5	0.5	201	0.13	25°C
6	1	201	0.27	25°C
7	2	201	0.51	25°C
8	3	201	0.76	25°C
9	4	201	1.00	25°C
10	5	201	1.22	25°C
11	6	201	1.47	25°C
12	7	201	1.70	25°C
13	8	201	1.94	25°C

 

The absorbance and concentrations were recorded at 201 nm.

Table: Concentration after analytical Determination

Sr. No	Concentrations (mg/L)	Analytical Concentrations (0 hour)	Analytical Concentrations (48 hour)
1	blank	0.00	0.00
2	4	4.55	4.34
3	8	9.85	7.34

 

Table: pH and Temperature

Test Concentration(mg/L)	Experimental Flasks	pH		Temperature °C
		0 Hours	72 hours	0 Hours	72 hours
control	R1	7.5	7.28	23	23
control	R2	7.5	7.3	23	23
control	R3	7.5	7.4	23	23
0.25	R1	7.5	7.79	23	23
0.25	R2	7.5	7.88	23	23
0.5	R1	7.6	7.70	23	23
0.5	R2	7.7	7.65	23	23
1	R1	7.7	7.64	23	23
1	R2	7.6	7.59	23	23
2	R1	7.5	7.50	23	23
2	R2	7.6	7.59	23	23
4	R1	7.7	7.51	23	23
4	R2	7.6	7.56	23	23
8	R1	7.6	7.52	23	23
8	R2	7.6	7.55	23	23

 

Table: Cell count and percent inhibition

		DAY 0				DAY 1				DAY 2				DAY 3
Experimental Sets	TEST CONC.s (mg/L)	Count 1	Count 2	Average	Ln(Av)	Count 1	Count 2	Average	Ln(Av)	Count 1	Count 2	Average	Ln(Av)	Count 1	Count 2	Average	Ln(Av)	Daily Growth rate 0-1 days	Daily Growth rate 1-2 days	Daily Growth rate 2-3 days	Average Growth rate 0-3 days	Standard deviation	Mean	Coefficient of Variation	Inhibition	Percent inhibition
Control 1	0.0	10000	10000	10000	9.21	40000	40000	40000	10.60	70000	80000	75000	11.23	210000	220000	215000	12.28	1.39	0.63	1.05	1.02	0.004	1.03	0.004
Control 2	0.0	10000	10000	10000	9.21	30000	40000	35000	10.46	80000	80000	80000	11.29	220000	220000	220000	12.30	1.25	0.83	1.01	1.03
Control 3	0.0	10000	10000	10000	9.21	50000	40000	45000	10.71	70000	80000	75000	11.23	210000	230000	220000	12.30	1.50	0.51	1.08	1.03
Conc. 1REP 1	0.25	10000	10000	10000	9.21	40000	40000	40000	10.60	50000	50000	50000	10.82	140000	120000	130000	11.78	1.39	0.22	0.96	0.85	0.007	0.85	0.008	0.17
Conc. 1REP 2	0.25	10000	10000	10000	9.21	40000	30000	35000	10.46	50000	60000	55000	10.92	120000	130000	125000	11.74	1.25	0.45	0.82	0.84					17.48
Conc. 1REP 3	0.25	10000	10000	10000	9.21	35000	40000	37500	10.53	55000	50000	52500	10.87	125000	130000	127500	11.76	1.32	0.34	0.89	0.85
Conc. 2REP 1	0.5	10000	10000	10000	9.21	30000	30000	30000	10.31	40000	40000	40000	10.60	120000	120000	120000	11.70	1.10	0.29	1.10	0.83	0.081	0.77	0.105	0.25
Conc. 2REP 2	0.5	10000	10000	10000	9.21	30000	30000	30000	10.31	50000	40000	45000	10.71	90000	80000	85000	11.35	1.10	0.41	0.64	0.71					25.24
Conc. 2REP 3	0.5	10000	10000	10000	9.21	30000	30000	30000	10.31	45000	40000	42500	10.66	130000	75000	102500	11.54	1.10	0.35	0.88	0.78
Conc. 3REP 1	1	10000	10000	10000	9.21	30000	30000	30000	10.31	30000	40000	35000	10.46	100000	90000	95000	11.46	1.10	0.15	1.00	0.75	0.009	0.74	0.012	0.28
Conc. 3REP 2	1	10000	10000	10000	9.21	25000	30000	27500	10.22	30000	30000	30000	10.31	90000	90000	90000	11.41	1.01	0.09	1.10	0.73					28.16
Conc. 3REP 3	1	10000	10000	10000	9.21	20000	30000	25000	10.13	30000	35000	32500	10.39	95000	90000	92500	11.43	0.92	0.26	1.05	0.74
Conc. 4REP 1	2	10000	10000	10000	9.21	20000	30000	25000	10.13	30000	20000	25000	10.13	80000	70000	75000	11.23	0.92	0.00	1.10	0.67	0.000	0.67	0.000	0.35
Conc. 4REP 2	2	10000	10000	10000	9.21	30000	30000	30000	10.31	30000	20000	25000	10.13	70000	80000	75000	11.23	1.10	-0.18	1.10	0.67					34.95
Conc. 4REP 3	2	10000	10000	10000	9.21	27500	27500	27500	10.22	20000	30000	25000	10.13	70000	80000	75000	11.23	1.01	-0.10	1.10	0.67
Conc. 5REP 1	4	10000	10000	10000	9.21	10000	20000	15000	9.62	20000	10000	15000	9.62	60000	70000	65000	11.08	0.41	0.00	1.47	0.62	0.087	0.56	0.154	0.45
Conc. 5REP 2	4	10000	10000	10000	9.21	20000	20000	20000	9.90	10000	20000	15000	9.62	50000	40000	45000	10.71	0.69	-0.29	1.10	0.50					45.63
Conc. 5REP 3	4	10000	10000	10000	9.21	15000	15000	15000	9.62	15000	15000	15000	9.62	60000	50000	55000	10.92	0.41	0.00	1.30	0.57
Conc. 6REP 1	8	10000	10000	10000	9.21	20000	20000	20000	9.90	20000	10000	15000	9.62	40000	30000	35000	10.46	0.69	-0.29	0.85	0.42	0.026	0.39	0.066	0.62
Conc. 6REP 2	8	10000	10000	10000	9.21	10000	10000	10000	9.21	10000	10000	10000	9.21	30000	30000	30000	10.31	0.00	0.00	1.10	0.37					62.14
Conc. 6REP 2	8	10000	10000	10000	9.21	20000	10000	15000	9.62	15000	10000	12500	9.43	40000	25000	32500	10.38	0.40	-0.18	0.95	0.39

 

Validity criteria:

Experimental Sets	TEST CONC.s (mg/L)	Count 1	Count 2	Average	Ln(Av)	Count 1	Count 2	Average	Ln(Av)	Count 1	Count 2	Average	Ln(Av)	Count 1	Count 2	Average	Ln(Av)	Daily Growth rate 0-1 days	Daily Growth rate 1-2 days	Daily Growth rate 2-3 days	Average Growth rate 0-3 days	Standard deviation	Mean	Coefficient of Variation	% CV
Control 1	0.0	10000	10000	10000	9.21	40000	40000	40000	10.60	70000	80000	75000	11.23	210000	220000	215000	12.28	1.39	0.63	1.05	1.02	0.004	1.028	0.0043	0.43
Control 2	0.0	10000	10000	10000	9.21	30000	40000	35000	10.46	80000	80000	80000	11.29	220000	220000	220000	12.30	1.25	0.83	1.01	1.03
Control 3	0.0	10000	10000	10000	9.21	50000	40000	45000	10.71	70000	80000	75000	11.23	210000	230000	220000	12.30	1.50	0.51	1.08	1.03
																	Mean	1.38104	0.65537	1.04696
																	SD	0.12574	0.15962	0.03271
																	CV	0.0910	0.2436	0.0312
																	% of CV	9.1	24.4	3.1
														Criteria No. 2 ) ≤ 35%			Mean of % CV of Average specific / section by section growth rate			12.19

Validity criteria fulfilled:

yes

Remarks:

* The average increase in cell count in control group is >16 fold. * The coefficent of varion between replictes was <7 % * coefficent of variation in the in control f % CV of Average specific <35%.

Conclusions:

Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the 72 hr median effect concentration (EC50) was determined to be 4.58 mg/l (calculated from equation through probit analysis).

Executive summary:

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The saturated test solution was prepared by dissolving 500mg of test chemical in 500ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 123.55 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.25, 0.5, 1, 2, 4 and 8 mg/l, respectively was from the saturated test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 23 °C and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. the initial pH in the control vessels were 7.3 in all the replicates at day 0 and followed by between 7.28 to 7.4. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 4.58 mg/l (calculated from equation through probit analysis). On the basis of this value, chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.

Description of key information

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata (Experimental study report, 2020). The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The saturated test solution was prepared by dissolving 500mg of test chemical in 500ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 123.55 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.25, 0.5, 1, 2, 4 and 8 mg/l, respectively was from the saturated test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 4.58 mg/l (calculated from equation through probit analysis). On the basis of this value, chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

4.58 mg/L

Additional information

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

 

In an experimental study from study report (2020),a freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The saturated test solution was prepared by dissolving 500mg of test chemical in 500ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 123.55 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.25, 0.5, 1, 2, 4 and 8 mg/l, respectively was from the saturated test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 4.58 mg/l (calculated from equation through probit analysis).

 

Another toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae (Experimental study report, 2017). The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test) in a static system. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5000 cells /ml was used as a test organism. The stock solution 50.0 g/l was prepared by dissolving test chemical 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. Test chemical concentrations were not verified analytically. Nominal test chemical conc. used for the study were 0, 0.7, 1.2, 2.0, 3.5, 5.8 and 10 mg/l, respectively. Desmodesmus subspicatus were exposed to test chemical in 50 ml glass vessel in a volume of 15 ml of liquid solution containing both the chemical and media. Control solution vessel containing OECD medium without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. In the control test vessel containing OECD growth medium without test chemical, the coefficient of variation of average growth rate in replicates during the whole test period was 2.0% i.e, not exceeded 7%, the biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation for section by section specific growth rate in the control cultures was not exceeded 35%, indicating that the validity criteria has been fulfilled. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr median effect concentration (ErC50) value was determined to be 1.6 mg/l (95 % CI 1.3 - 2.1 mg/l) (nominal concentration). Thus, based on the EC50 value, test chemical can be considered to be toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.

 

For the test chemical, toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae (2017). The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test). Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5000 cells /ml was used as a test organism. The stock solution 50 g/l was prepared by dissolving test chemical in acetone. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium. Test solutions were kept in ultrasonic bath for 20 min and then were inoculated by algae. Test chemical concentrations were not verified analytically. Nominal test chemical conc. used for the study were 0, 0, 2.5, 3.8, 5.6, 8.4 and 12.7 mg/l, respectively. Study was performed in a static fresh water system for 72 hrs. Desmodesmus subspicatus were exposed to test chemical in 50 ml glass vessel in a volume of 15 ml of liquid solution containing both the chemical and media. Control solution vessel containing OECD medium without the test chemical and other control vessel containing OECD medium plus acetone without test chemical were also setup during the study. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. In the control test vessel containing OECD growth medium without test chemical, the coefficient of variation of average growth rate in replicates during the whole test period was 0.7% in control and 1.1% in control plus acetone i.e, not exceeded 7%, the biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation for section by section specific growth rate in the control cultures was not exceeded 35%, indicating that the validity criteria has been fulfilled. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr median effect concentration (ErC50) value was determined to be 5.5 mg/l (95% C. I. = 4.6 to 6.8 mg/l) (nominal concentration).

 

On the basis of the above results, it can be concluded that the test chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.