Potassium Lauroyl Wheat Amino Acids

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

Study period:

May 21, 2018 to May 24, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

light intensity was outside the recommended range; however in the opinion of the the study director, this deviation had no effect on the validity and integrity of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

Analysis of the samples for the verification of exposure concentrations was performed using HPLC

Details on sampling:

Duplicate samples were taken at the start and end of the 72 h exposure period. Samples were taken from remaining test media after filling test vessels for 0 h and pooled replicate flasks for 72 h.

Vehicle:

yes

Remarks:

Sterilised deionised water with added nutrients

Details on test solutions:

Preliminary solubility tests indicated that the sample was soluble after ultrasonication at >100 mg/L in water. Stock solutions were prepared for both the range-finding and definitive tests by addition of the test sample directly to nutrient growth medium in a volumetric flask of adequate volume. Calculated volumes of the stock solution were added to nutrient growth medium and made to volume to prepare the test concentrations and provide enough solution for testing and subsequent water quality measurements. The test concentrations for both the range-finding test and definitive test were prepared separately by the addition of the test sample to nutrient growth medium in volumetric flasks of adequate volume to provide enough solution for testing and subsequent water quality measurements.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata strain: CCAP 278/4 received 15 May 2018.
Source: Culture Collection of Algae and Protozoa, SAMS Ltd, Scottish Marine Institute, OBAN, Argyll PA37 1QA, Scotland, United Kingdom

Culture conditions:
Temperature: 21.2 - 21.5 deg C.
Illumination: 6270 - 7790 lux continuous white light.
Orbiting: set to 200rpm.
Culture media: Deionised water with added nutrients according to OECD 201.

The stocks of algal culture were maintained, and the tests performed, in nutrient growth medium (OECD 201) which was prepared by adding appropriate amounts of nutrient stocks to deionised water (sterilised by autoclaving at 120°C for 15 minutes) at a pH of 7.21 (adjusted to: 8.16).

Test type:

not specified

Water media type:

other: Sterilised deionised water with added nutrients

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20.7 – 21.1°C

pH:

7.55 – 8.97

Nominal and measured concentrations:

Nominal: 0 (control), 1.0, 3.2, 10, 32, and 100 mg/L
Mean measured: 0 (control), 0.392, 1.263, 4.589, 10.131 and 45.651 mg/L

Details on test conditions:

Test methods and conditions

The test was carried out according to the procedures given below, based on the guidelines produced by OECD 201: Alga Growth Inhibition Test.

Chemex SOP reference: E203 “Algal Growth Inhibition Test (Freshwater)”
Preliminary test method: A preliminary (range finding) test was conducted at concentrations of 0 (control), 0.1, 1.0, 10, 100mg/L. The duration of the preliminary study was 72 ± 2 h. There was a single replicate at each concentration.
Preliminary test results: Data from the preliminary test identified the 72-h EC50 as being >100mg/L (by growth rate) and >100mg/L (by yield).

Nominal concentration
(mg/L) Percent reduction in growth rate Percent reduction in yield
0 - 72 h 0 - 72 h
0.1 -2 -6
1.0 -7 -22
10 -42 -255
100 -17 -69

Note: Negative numbers indicate an increase in growth compared to control data. All concentrations of the test substance are reported as nominal as received.

Definitive test method:
Test period: 21 to 24 May 2018
Test duration: 72 ± 2 h
Test volume: 100 mL
Test vessel: 250 mL conical flask
Number of replicates: Six control flasks, three replicates at each test concentration.

Test concentrations:
Nominal: 0 (control), 1.0, 3.2, 10, 32, and 100mg/L
Mean measured concentrations: 0 (control), 0.392, 1.263, 4.589, 10.131 and 45.651mg/L

Composition of medium: Sterilised deionised water with added nutrients according to the OECD 201 standard.
Algal Test inoculum: From a pre-culture growing in exponential phase, under conditions described in 2.2 above. Inoculum level adjusted to give an initial cell density of 1 x 104 cells/ml.
Test conditions: Initial pH at 0 h: 8.16 (Required: 8.1±0.1). pH range in control and test concentrations throughout test: 7.55 – 8.97
Temperature range within incubator throughout test: 20.7 – 21.1°C (Required: 21-24±2°C)
Illumination range within incubator throughout test: 5660 - 10120 Lux (Required: 6x103-8x103 Lux ± 15% of recorded mean )
Orbital shaking: 200rpm (Required: 200-250rpm)

Water quality measurements:
The temperature (to 0.1 deg C) and light intensity (lux) within the incubator was recorded at the beginning of the study, after 24, 48 h and at the end of the 72 h test period. The pH (to 0.01) and temperature (to 0.1 deg C) were recorded for each test and control solution at the beginning of the test and on the pooled replicates at the end of the 72-h test period.

Observations/frequency:
Cell densities were measured microscopically by direct cell counts on each test and control replicate, in sextuplicate at 24 h for the control and triplicate thereafter, and in triplicate at 24, 48 and 72 h (±2h) for the test concentrations. The cell counts were made using a haemocytometer and microscope.

Analysis of test substance:
Duplicate samples were taken at the start and end of the 72 h exposure period. Samples were taken from remaining test media after filling test vessels for 0 hours and pooled replicate flasks for 72 h. Analysis of these samples for the verification of exposure concentrations was performed using HPLC.

Calculation of results:
Growth curves for each test concentration were plotted as logarithm of the mean cell density against time. The inhibition of growth was calculated using both the biomass (increase in cell yield) and the growth rate method. Where possible, the EC50 values were estimated graphically and 95% confidence limits calculated according to the method of ToxCalc™ Version 5.0 “Comprehensive Toxicity Data Analysis and Database Software”, copyright 1994-1996(2).

Reference Substance:
A separate reference study (ENV 11396) was run from 16 to 19 January 2017 using potassium dichromate (K2Cr2O7) as a means of checking the test procedure and sensitivity of the test species. ISO 8692:2012 quotes the 72-h ErC50 as 1.19mg/L (SD = 0.27), the result obtained in the reference test should be in the range 0.65 and 1.73mg/L (mean value ± 2 x standard deviation). The ErC50 obtained was 1.06mg/L and all validity criteria were met.

Study plan deviation
Definitive test: states that the light should be at an “intensity in the range 6000 - 8000 lux at the level of the test flasks and will not vary by more than ± 15% from the mean value across all test vessels.”. A minimum lux value of 5660 and maximum of 10120 Lux was recorded, 340 lux below and 2120 above the recommenced range. The range was also outside the ± 15% recommended range. The cell density in the control cultures increased by the required amount (a factor of at least 16) within the 72-h test period. Therefore, we can attribute any changes in cell density in the test concentration to be due to the test substance. Also, there were no cell morphological abnormalities within the control units. All necessary validity criteria was met and in the opinion of the study director, this deviation had no effect on the validity and integrity of the study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

ca. 90.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 67.2-145

Remarks:

ErC50 = equivalent to 63.2 mg a.i./L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass and growth rate

Remarks on result:

other: Determined by Bonferroni t Test

Remarks:

NOEC = equivalent to 6.98 mg a.i./L

Key result

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

ca. 31.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits: 24.9-39.2

Remarks:

EyC50 = equivalent to 21.99 mg a.i./L

Key result

Duration:

72 h

Dose descriptor:

other: ErC10

Effect conc.:

ca. 22.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 4.61-37

Remarks:

ErC10 = equivalent to 15.77 mg a.i./L

Key result

Duration:

72 h

Dose descriptor:

other: EyC10

Effect conc.:

ca. 13.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits: 2.95-19.8

Remarks:

EyC10 = equivalent to 9.7 mg a.i./L

Results with reference substance (positive control):

A separate reference study (ENV 11396) was run from 16 to 19 January 2017 using potassium dichromate (K2Cr2O7) as a means of checking the test procedure and sensitivity of the test species. ISO 8692:2012 quotes the 72-h ErC50 as 1.19mg/L (SD = 0.27), the result obtained in the reference test should be in the range 0.65 and 1.73mg/L (mean value ± 2 x standard deviation). The ErC50 obtained was 1.06mg/L and all validity criteria were met.

Reported statistics and error estimates:

Statistical methods used in ToxCalc v5.0: Maximum Likelihood-Logit

Results

Analytical results:

Concentration of test substance (mg/L) determined by HPLC UV analysis 

Nominal Concentration (mg/l)	Concentrations of test substance (mg/L) calculated from measured active ingredient concentrations at the following sampling times
	0hrs	72hrs	Mean measured
1	0.880	0.686	0.392
3.2	2.926	2.128	1.263
10	10.295	8.062	4.589
32	30.584	9.940	10.131
100	102.130	80.473	45.651

 

 

Average recoveries of test substance (mg/L), percentage recovery compared to nominal concentrations and aged solutions as a percentage of fresh solutions.

  

Nominal Concentration (mg/l)	Concentrations of test substance (mg/L) calculated from measured active ingredient concentrations at the following sampling times
	0hrs	72hrs	Mean
1	87.973	68.629	39.150
3.2	91.433	66.500	39.483
10	102.954	80.621	45.894
32	95.576	31.062	31.659
100	102.130	80.473	45.651

Analytical measurements of the freshly prepared exposure solutions indicated that the initial measured concentrations were within 20% of nominal concentrations. However analytical measurement of the aged exposure solutions showed that all of the concentrations were not within 20% of the initial measured concentrations and therefore were also not within the ± 20% of nominal concentrations. The mean measured concentrations were determined to be 0 (control), 0.392, 1.263, 4.589, 10.131 and 45.651 mg/L. Due to poor recovery, all effect concentrations were reported as nominal concentrations of the test substance.

Test results:

Cell densities were measured microscopically by direct cell counts on each test and control replicate, in sextuplicate at 24 h for the control and triplicate thereafter, and in triplicate at 24, 48 and 72 h (±2 h) for the test concentrations. All results in this study are calculated from the measured cell densities.

 

Cell density measurements

Mean initial cell density: Approximately 1 x 10⁴cells/mL based upon inoculation volume, not counted microscopically.

Nominal concentration (mg/L)	Replicate	Cell density measurements (cells/mL x 10^4)
		24 h	48 h	72 h
0 (Control)	1	4.8	23.7	78.7
	2	3.5	16.7	44.7
	3	4.3	23.0	66.3
	4	3.7	17.0	83.3
	5	4.2	13.0	82.0
	6	5.3	15.3	86.3
	Mean	4.3	18.1	73.6
1.0	1	3.0	16.0	74.3
	2	3.0	17.7	64.7
	3	4.7	24.0	62.7
	Mean	3.6	19.2	67.2
3.2	1	3.3	21.7	79.3
	2	5.7	21.7	80.3
	3	6.3	23.7	80.7
	Mean	5.1	22.4	80.1
10	1	1.7	17.7	106.0
	2	1.7	15.3	93.3
	3	3.3	24.7	59.3
	Mean	2.2	19.2	86.2
32	1	1.7	8.3	23.3
	2	3.3	11.7	37.3
	3	2.7	15.7	45.7
	Mean	2.6	11.9	35.4
100	1	1.3	4.0	7.0
	2	2.3	5.3	9.7
	3	4.3	5.7	5.7
	Mean	2.6	5.0	7.5

Growth curves of logarithm of cell density against time are given in Graph 1. For graphs, kindly refer to the attached background material section of the IUCLID.

 

Test observations

The algal cells were examined microscopically during the determination of the cell density, all cells within the control and all test concentrations appeared normal, no morphological abnormalities were observed. When observed visually, the control conical flasks appeared clear and colourless after 24 h and a green colour after 48 and 72 h. All test concentrations appeared clear and colourless after 24 h. 1, 3.2 and 10mg/L test concentrations appeared a green colour after 48 and 72 h, 32mg/L appeared slightly green after 48 and 72 h and 100mg/L remained clear and colourless throughout the test.

 

Percent inhibition

Nominal concentration (mg/L)	Percent reduction in growth rate		Percent reduction in yield
	0 - 48 h	0 - 72 h	0 - 48 h	0 - 72 h
1.0	-2	2	-7	9
3.2	-8	-3	-25	-9
10	-2	-4	-7	-17
32	15	17	36	53
100	44	54	77	91

Note: Negative numbers indicate an increase in growth compared to the unexposed controls.

 

EC_x, NOEC values by yield (E_yC_x) and growth rate (E_rC_x)

Exposure Period (h)	ErCxvalue mg/L (95% confidence limits)			EyCxvalue mg/L (95% confidence limits)
	ErC10	ErC20	ErC50	EyC10	EyC20	EyC50
0 to 48	25.2 (2.73 - 42.4)	44.2 (13.0 - 63.0)	116 (83.8 - 280)	13.7 (5.41 - 20.8)	21.7 (11.5 - 29.8)	47.6 (36.0 - 64.2)
0 to 72	22.6 (4.61 - 37.0)	37.7 (14.2 - 53.5)	90.6 (67.2 - 145)	13.9 (2.95 - 19.8)	18.8 (6.83 - 24.1)	31.5 (24.9 - 39.2)
NOEC (0-72h)	10mg/L (Determined by Bonferroni t Test)^$*			10mg/L (Determined by Bonferroni t Test)^$^
Statistical methods used in ToxCalc v5.0: Maximum Likelihood-Logit ^$Following Shapiro-Wilk’s Test for normality of distribution * Bartlett’s Test which indicated equal variances. ^ Equality of variances could not be confirmed. All concentrations of the test substance are reported as nominal as received.

Test validity criteria

The control cell density should increase by a factor of more than 16 in 72 h, corresponding to a specific growth rate of 0.92 d^-1. The measured control cell density increase was recorded as 73.6, corresponding to a specific growth rate of 1.425 d^-1. The variation coefficient of the control specific growth rate should not exceed 7% for the 72h period and 35% for each time period. The variation coefficient for the 72h period was 5.78% and for each time period 19.96%. These fulfil the validity criteria of the study and it is therefore compliant with the OECD Guideline for Testing of Chemicals reference 201 Alga, Growth Inhibition Test 2011.

 

Control growth rate data coefficient of variation calculations

Control replicate no.	Cell density (1.0 x 10^4cells/ml)				0-72 hr average specific growth rates
	0 hr	24 hr	48 hr	72 hr
1	1.0	4.8	23.7	78.7	1.46
2	1.0	3.5	16.7	44.7	1.27
3	1.0	4.3	23.0	66.3	1.40
4	1.0	3.7	17.0	83.3	1.47
5	1.0	4.2	13.0	82.0	1.47
6	1.0	5.3	15.3	86.3	1.49
			Mean:		1.43
			Standard deviation:		0.0826
			Coefficient of variation:		5.78

Note: 0-h cell density based upon inoculation volume and not counted microscopically.

 

Control replicate no.	Day-by-day specific growth rates			Coefficient of variation
	0-24 hr	24-48 hr	48-72 hr
1	1.57	1.60	1.20	15.29
2	1.25	1.56	0.98	22.97
3	1.46	1.68	1.06	22.45
4	1.31	1.52	1.59	9.89
5	1.44	1.13	1.84	24.21
6	1.67	1.06	1.73	24.94
Mean coefficient of variation:				19.96

 Note:0-h cell density based upon inoculation volume, not counted microscopically.

 

Discussion

The definitive test conducted from 21 to 24 May 2018 was performed according to the OECD 201 (2011) guideline.The growth curves illustrated in Graph 1 demonstrate that the algae in the control were in logarithmic growth for the duration of the study.The 48 h EC(r)₅₀ and EC(y)₅₀ of test substance to Pseudokirchneriella subcapitata were 116 mg/L and 47.6 mg/L respectively (both determined by Maximum Likelihood-Logit). Graphical representations of the 0 -48 h EC(r)_x and EC(y)_x values are given in Graphs 2 and 3, respectively. The 72 h EC(r)₅₀ and EC(y)₅₀ of test substance to Pseudokirchneriella subcapitata were 90.6mg/L and 31.5mg/L respectively (both determined by Maximum Likelihood-Logit). Graphical representations of the 0 -72 h EC(r)_x and EC(y)_x values are given in Graphs 4 and 6 respectively. The 0 to 72 -h NOEC(r) was 10 mg/L and the 0 -72 h LOEC was 32 mg/L (both determined by Bonferroni t Test). The algal cells were examined microscopically during the determination of cell concentration. All cells within the control and test concentrations appeared normal, no abnormalities were observed. All validity criteria for the definitive test were met.

 

Analytical measurements of the freshly prepared exposure solutions indicated that the initial measured concentrations were within 20% of nominal concentrations. However analytical measurement of the aged exposure solutions showed that all of the concentrations were not within 20% of the initial measured concentrations and therefore were also not within the ± 20% of nominal concentrations. All effect concentrations are reported as nominal concentrations of the test substance.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72 h ErC50, ErC10, EyC50, EyC10, and NOEC values for the test substance with freshwater green algae, were determined to be 90.6, 22.6, 31.5, 13.9 and 10 mg/L (nominal) (i.e., equivalent to 63.2, 15.77, 21.99, 9.7 and 6.98 mg a.i./L) respectively

Executive summary:

A study was conducted to determine the acute toxicity study of the test substance,'potassium lauroyl wheat amino acids' (active: 69.8%), to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. A preliminary (range finding) test was conducted at concentrations of 0 (control), 0.1, 1.0, 10, 100mg/L. Data from the preliminary test identified the 72-h EC50 to be >100mg/l (for growth rate as well as for yield). Therefore, in the main study, six control and three replicate algal cultures were exposed to test substance at nominal concentrations of 0 (control; sixuplicates), 1.0, 3.2, 10, 32, and 100 mg/L in sterilised deionised water (with added nutrients) for 72 h. Six replicates of the culture medium was employed. Inoculum level was adjusted to give an initial cell density of 1 × 104 cells/mL. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0 (control), 1.0, 3.2, 10, 32, and 100 mg/L in sterilised deionised water (with added nutrients) for 72 h. Analytical measurements of the test concentrations were carried out by taking duplicate samples at the start and end of the 72 h exposure period using HPLC. Samples were taken from remaining test media after filling test vessels for 0 h and pooled replicate flasks for 72 h.Analytical measurements at 0 h indicated that the initial measured concentrations were within 20% of nominal concentrations. However analytical measurement at 72 h showed that all of the concentrations were not within 20% of the initial measured concentrations and therefore were also not within the ± 20% of nominal concentrations. The meanmeasured concentration of the test substances were determined to be 0 (control), 0.392, 1.263, 4.589, 10.131 and 45.651 mg/L respectively. Therefore, due to the poor recovery, all effect concentrations were reported as nominal concentrations of the test substance.Cell densities were measured microscopically by direct cell counts on each control replicate at 24 h and on each test replicate at 24, 48 and 72 h (±2 h) using haemocytometer and microscope. The inhibition of growth was calculated using both the biomass (increase in cell yield) and the growth rate method. Where possible, the EC50 values were estimated graphically and 95% confidence limits calculated according to the method of ToxCalc™ Version 5.0. Following microscopical observations of algal cells within the control and all test concentrations appeared normal, no morphological abnormalities were observed. When observed visually, the control conical flasks appeared clear and colourless after 24 h and a green colour after 48 and 72 h. All test concentrations appeared clear and colourless after 24 h and 1, 3.2 and 10 mg/L test concentrations appeared a green colour after 48 and 72 h, 32 mg/L appeared slightly green after 48 and 72 h and 100mg/L remained clear and colourless throughout the test. The measured control cell density increase was recorded as 73.6, corresponding to a specific growth rate of 1.425 per day. The variation coefficient for the 72h period was 5.78% and for each time period 19.96%. Therefore, all validity crtieria were considered to be fulfilled according to the guideline. The ErC50 and ErC10 values (for growth rate) and EyC50 and EyC10 values (for cell particle density) were calculated to be 90.6 (95% confidence interval: 67.2-145), 22.6 (95% confidence interval: 4.61-37), 31.5 (95% confidence interval: 24.9-39.2), 13.9 (95% confidence interval: 2.95-19.8) mg/L (nominal), respectively. The NOEC value was determined to be 10 mg/L (nominal), for both growth rate and cell particle densities. Under the study conditions, 72 h ErC50, ErC10, EyC50, EyC10, and NOEC values for the test substance with freshwater green algae, were determined to be 90.6, 22.6, 31.5, 13.9 and 10 mg/L (nominal) (i.e., equivalent to 63.2, 15.77, 21.99, 9.7 and 6.98 mg a.i./L) respectively (Chemex, 2018).

Description of key information

Based on the study results, the 72 h ErC50, ErC10, EyC50, EyC10, and NOEC values for the test substance with freshwater green algae, were determined to be 90.6, 22.6, 31.5, 13.9 and 10 mg/L (nominal) (i.e., equivalent to 63.2, 15.77, 21.99, 9.7 and 6.98 mg a.i./L) respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

63.2 mg/L

EC10 or NOEC for freshwater algae:

15.77 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the test substance,'potassium lauroyl wheat amino acids' (active: 69.8%), to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. A preliminary (range finding) test was conducted at concentrations of 0 (control), 0.1, 1.0, 10, 100mg/L. Data from the preliminary test identified the 72-h EC50 to be >100mg/l (for growth rate as well as for yield). Therefore, in the main study, six control and three replicate algal cultures were exposed to test substance at nominal concentrations of 0 (control; sixuplicates), 1.0, 3.2, 10, 32, and 100 mg/L in sterilised deionised water (with added nutrients) for 72 h. Six replicates of the culture medium was employed. Inoculum level was adjusted to give an initial cell density of 1 × 104 cells/mL. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0 (control), 1.0, 3.2, 10, 32, and 100 mg/L in sterilised deionised water (with added nutrients) for 72 h. Analytical measurements of the test concentrations were carried out by taking duplicate samples at the start and end of the 72 h exposure period using HPLC. Samples were taken from remaining test media after filling test vessels for 0 h and pooled replicate flasks for 72 h.Analytical measurements at 0 h indicated that the initial measured concentrations were within 20% of nominal concentrations. However analytical measurement at 72 h showed that all of the concentrations were not within 20% of the initial measured concentrations and therefore were also not within the ± 20% of nominal concentrations. The meanmeasured concentration of the test substances were determined to be 0 (control), 0.392, 1.263, 4.589, 10.131 and 45.651 mg/L respectively. Therefore, due to the poor recovery, all effect concentrations were reported as nominal concentrations of the test substance.Cell densities were measured microscopically by direct cell counts on each control replicate at 24 h and on each test replicate at 24, 48 and 72 h (±2 h) using haemocytometer and microscope. The inhibition of growth was calculated using both the biomass (increase in cell yield) and the growth rate method. Where possible, the EC50 values were estimated graphically and 95% confidence limits calculated according to the method of ToxCalc™ Version 5.0.Following microscopical observations of algal cells within the control and all test concentrations appeared normal, no morphological abnormalities were observed. When observed visually, the control conical flasks appeared clear and colourless after 24 h and a green colour after 48 and 72 h. All test concentrations appeared clear and colourless after 24 h and 1, 3.2 and 10 mg/L test concentrations appeared a green colour after 48 and 72 h, 32 mg/L appeared slightly green after 48 and 72 h and 100mg/L remained clear and colourless throughout the test. The measured control cell density increase was recorded as 73.6, corresponding to a specific growth rate of 1.425 per day. The variation coefficient for the 72h period was 5.78% and for each time period 19.96%. Therefore, all validity crtieria were considered to be fulfilled according to the guideline. The ErC50 and ErC10 values (for growth rate) and EyC50 and EyC10 values (for cell particle density) were calculated to be 90.6 (95% confidence interval: 67.2-145), 22.6 (95% confidence interval: 4.61-37), 31.5 (95% confidence interval: 24.9-39.2), 13.9 (95% confidence interval: 2.95-19.8) mg/L (nominal), respectively. The NOEC value was determined to be 10 mg/L (nominal), for both growth rate and cell particle densities. Under the study conditions, 72 h ErC50, ErC10, EyC50, EyC10, and NOEC values for the test substance with freshwater green algae, were determined to be 90.6, 22.6, 31.5, 13.9 and 10 mg/L (nominal) (i.e., equivalent to 63.2, 15.77, 21.99, 9.7 and 6.98 mg a.i./L) respectively (Chemex, 2018).