[No public or meaningful name is available]

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:

From 08 April 2019 to 28 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

The test substance concentration in the test samples was determined by high performance liquid chromatography with mass spectrometry (HPLC-MS).

Details on sampling:

Samples of the algal populations were removed daily (24 h, 48 h and 72 h) and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Vehicle:

no

Details on test solutions:

Due to the low aqueous solubility and complex nature of the test substance, for the purposes of the study the test medium was prepared as a WAF of the test substance.

validation of Mixing Period
Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.

Range-Finding Test
The loading rate to be used in the definitive test was determined by a preliminary range finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 10 and 100 mg/L for a period of 72 hours. The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration. Nominal amounts of test substance (20 and 200 mg) were each separately added to the surface of 2 liters of culture medium to give the 10 and 100 mg/L loading rates respectively. After the addition of the test substance, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. Microscopic observations made on the 10 mg/L loading rate WAF showed there to be no micro-dispersions of test substance present and as such the aqueous phase was removed by mid-depth siphon (the first 75 to 100 mL discarded). Visual observations made on the 100 mg/L loading rate WAF indicated that a significant amount of dispersed test substance was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid depth siphoning (the first 75 to 100 mL discarded). Further filtration through two sheets of coarse filter paper was conducted to remove as much undissolved test substance as possible. Microscopic observations of the WAF were performed after filtering and showed no micro-dispersions of test substance to be present. An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (1.5 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF. The control group was maintained under identical conditions but not exposed to the test substance. At the start of the range finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours. After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter. A sample of each loading rate WAF was taken for immediate chemical analysis at 0 and 72 hours in order to determine the stability of the test substance over the test duration. Duplicate samples were taken and stored frozen for further analysis if required.

Definitive Test
Based on the result of the range finding test a "limit test" was conducted at a single loading rate of 100 mg/L to confirm that no effect on algal growth was observed.

Experimental Preparation
A nominal amount of test substance (200 mg) was added to the surface of 2 liters of culture medium to give the 100 mg/L loading rate. After the addition of the test substance, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1-Hour. Visual observations made on the WAF indicated that a significant amount of dispersed test substance was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded). Further filtration through two sheets of coarse filter paper was conducted to remove as much undissolved test substance as possible. Microscopic observations of the WAF were performed after filtering and showed no micro-dispersions of test substance to be present. An aliquot (1 liter) of the WAF was inoculated with algal suspension (3.8 mL) to give the required test concentration of 100 mg/L loading rate WAF. The concentration and stability of the test substance in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours.

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 deg C. Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E+3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 deg C until the algal cell density was approximately 10E+4 to 10E+5 cells/mL. A positive control test using potassium dichromate as the reference substance was performed twice in a 12 month period to demonstrate satisfactory conditions of the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Samples were taken at 25, 49 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Hardness:

Not specified

Test temperature:

24 ±1 °C

pH:

7.8 to 8.7

Dissolved oxygen:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal Concentration: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Initial cells density: 1000 cells/mL
- Nominal cell density of control at 0 hours: 5E+3
Mean cell density of control at 0 hours at 72 h: 1.18E+6
- No. of organisms per vessel: 5E+3
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The pH value of the control cultures was observed to increase from pH 7.9 at 0 hours to pH 8.7 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.Temperature was maintained at 24 ±1 ºC throughout the test.

- Intervals of water quality measurement: WAF was determined at initiation of the test and after 72 hours exposure

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell density determined using a Coulter® Multisizer Particle Counter

TEST CONCENTRATIONS
- Range finding study: nominal loading rates of 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: no effect on algal growth at 100 mg/L dose in range finding test.
Definative test concentration: 100 mg/L

Reference substance (positive control):

yes

Remarks:

A positive control test using potassium dichromate as the reference item was performed twice in a 12 month period to demonstrate satisfactory conditions of the test

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

NOELR

Remarks:

No Observed Effect Loading Rate

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Remarks:

No Observed Effect Loading Rate

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: 16% inhibition of yield was observed to have occurred at 100 mg/L loading rate WAF

Results with reference substance (positive control):

A positive control (Envigo Study Number YJ31TQ) used potassium dichromate as the reference substance at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. The positive control was conducted between 12 November 2018 and 03 December 2018. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference substance gave the following results:
ErC50 (0 to 72 hour) : 1.5 mg/L; 95% confidence limits 1.3 to 1.7 mg/L
EyC50 (0 to 72 hour) : 0.76 mg/L; 95% confidence limits 0.69 to 0.85 mg/L
No Observed Effect Concentration based on growth rate: 0.50 mg/L
No Observed Effect Concentration based on yield: 0.50 mg/L
Lowest Observed Effect Concentration based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration based on yield: 1.0 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference substance*.

* In-house data from the last 5 years shows a mean ErC50 value of 1.4 mg/L (standard deviation = 0.26) and a mean EyC50 value of 0.66 mg/L (standard deviation = 0.14).

Results

Validation of Mixing Period

Preliminary investigational work indicated that there was no significant increase in the amount of dissolved test substance present when the preparation period was extended for longer than 24 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test substance during the range finding test are given in Table 1. The results showed no effect on growth rate at 10 and 100 mg/L loading rate WAF. Based on this information a single loading rate of six replicates of 100 mg/L was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed. Chemical analysis of the 10 and 100 mg/L loading rate WAF test preparations at 0 hours showed measured test concentrations of less than the limit of quantification (LOQ) and 0.097 mg/L respectively were obtained. Measured test concentrations of less than the LOQ and 0.031 mg/L were obtained at 72 hours for the 10 and 100 mg/L loading rate WAFs respectively. The decline in measured test concentration in the 100 mg/L loading rate WAF was considered to be due to either instability of the test substance under the conditions of the test and/or adsorption of the test substance to the algal cells present. It was therefore considered appropriate to provide samples for analysis from the definitive test at 0, 24, 48 and 72 hours in order to better define the rate in decline. Analysis of an additional sample prepared with the omission of algal cells at 72 hours would determine if the decline in measured concentrations was attributable to adsorption to the algal cells present.

Table1: Cell Densities and Percentage Inhibition of Growth from the Range‑finding Test

Nominal Loading Rate (mg/L)		Cell Densities*(cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	6.92E+03	1.71E+06	-	-
	R2	7.22E+03	1.25E+06
	Mean	7.07E+03	1.48E+06
10	R1	6.28E+03	1.50E+06	[1]	11
	R2	5.75E+03	1.13E+06
	Mean	6.01E+03	1.32E+06
100	R1	6.75E+03	1.27E+06	3	21
	R2	5.95E+03	1.08E+06
	Mean	6.35E+03	1.17E+06

* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks

R = Replicate

- = Not applicable

[ ] = Increase in growth as compared to controls

Definitive Test

Chemical Analysis of Test Loading Rates

Analysis of the 100 mg/L loading rate WAF at 0 hours showed a measured test concentration of 0.055 mg/L was obtained. A decline in measured test concentration was observed after each 24-Hour period to 0.018, 0.0038 and 0.0012 mg/L at 24, 48 and 72 hours, respectively. A sample of the 100 mg/L loading rate WAF which contained no algal cells was incubated alongside the test samples to provide a sample for uninoculated analysis at 72 hours. A decline in measured test concentration to 0.0098 mg/L was observed. These results indicated that not only was the test substance unstable under the conditions of the test, it was also adsorbing to the algal cells present. However, given that toxicity cannot be attributed to a single component or mixture of components but to the test substance as a whole, the results were based on nominal loading rates only.

Growth Data

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3.  Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4. The mean cell densities versus time for the definitive test are presented in Figure 1. From the data given in Table 2 and Table 4, it is clear that whilst the growth rate (r) of Pseudokirchneriella subcapitata (CCAP 278/4) was not affected by the presence of the test substance over the 72 Hour exposure period, a slight effect on yield (y) was observed. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Table 2: Cell Densities and pH Values in the Definitive Test

Nominal Loading Rate (mg/L)		pH	Cell Densities*(cells per mL)			pH
		0 Hour	25 Hour	49 Hours	72 Hours	72 Hour
Control	R1	7.9	3.46E+04	1.89E+05	1.12E+06	8.7
	R2		3.58E+04	1.99E+05	1.14E+06
	R3		3.61E+04	1.98E+05	1.17E+06
	R4		3.19E+04	1.87E+05	1.20E+06
	R5		3.35E+04	1.95E+05	1.16E+06
	R6		3.47E+04	2.02E+05	1.28E+06
	Mean		3.44E+04	1.95E+05	1.18E+06
100	R1	7.8	2.94E+04	1.52E+05	1.00E+06	8.7
	R2		2.75E+04	1.41E+05	8.69E+05
	R3		2.95E+04	1.52E+05	9.15E+05
	R4		2.92E+04	1.68E+05	1.04E+06
	R5		3.06E+04	1.64E+05	1.06E+06
	R6		2.87E+04	1.64E+05	1.06E+06
	Mean		2.92E+04	1.57E+05	9.91E+05

* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from3counts for each of the replicate flasks

R=Replicate

Table 3:Daily Specific Growth Rates for the Control Cultures in the Definitive Test

Treatment		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 to 1	Day 1 to 2	Day 2 to 3
Control	R1	0.077	0.071	0.077
	R2	0.079	0.071	0.076
	R3	0.079	0.071	0.077
	R4	0.074	0.074	0.081
	R5	0.076	0.073	0.078
	R6	0.077	0.073	0.080
	Mean	0.077	0.072	0.078

R=Replicate

Table 4: Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.075	-	1.11E+06	-
	R2	0.075		1.13E+06
	R3	0.076		1.16E+06
	R4	0.076		1.20E+06
	R5	0.076		1.16E+06
	R6	0.077		1.27E+06
	Mean	0.076		1.17E+06
	SD	0.001		5.56E+04
100	R1	0.074	3	9.95E+05
	R2	0.072	5	8.64E+05
	R3	0.072	5	9.10E+05
	R4	0.074	3	1.03E+06
	R5	0.074	3	1.05E+06
	R6	0.074	3	1.06E+06
	Mean	0.073	4	9.86E+05	16
	SD	0.001		8.13E+04

* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from3counts for each of the replicate flasks

R=Replicate

- =Not applicable

SD   = Standard deviation

Accordingly the following results were determined from the data:

Inhibition of Growth Rate

ErL10 (0 to 72 hour) : >100 mg/L loading rate WAF

ErL20 (0 to 72 hour) : >100 mg/L loading rate WAF

ErL50 (0 to 72 hour) : >100 mg/L loading rate WAF

Where ErLx is the loading rate that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100 mg/L loading rate WAF test group using a Student’s t test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). Whilst a statistically significant difference (P<0.05), was observed between the control and 100 mg/L loading rate WAF test group, inspection of the data showed that inhibition values ranged from 3% to 5% which was not considered to be of biological significance.  As such the No Observed Effect Loading Rate (NOEL) based on growth rate was considered to be 100 mg/L loading rate WAF.

Inhibition of Yield

EyL10 (0 to 72 hour) : <100 mg/L loading rate WAF

EyL20 (0 to 72 hour) : >100 mg/L loading rate WAF

EyL50 (0 to 72 hour) : >100 mg/L loading rate WAF

Where EyLx is the loading rate that reduced yield by x%.

It was not possible to determine an exact EyL10 value as 16% inhibition of yield was observed to have occurred at 100 mg/L loading rate WAF. Statistical analysis of the yield data was carried out. A statistically significant difference (P<0.05), was observed between the control and 100 mg/L loading rate WAF and therefore the No Observed Effect Loading Rate (NOEL) based on yield was less than 100 mg/L loading rate WAF.

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 235 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Nominal cell density of control at 0 hours : 5.00 x 10E+3 cells per mL

Mean cell density of control at 72 hours : 1.18 x 10E+6 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 5% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%. The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Water Quality Criteria

The pH values of the control and each test concentration are given in Table 2. The pH value of the control cultures was observed to increase from pH 7.9 at 0 hours to pH 8.7 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines. Temperature was maintained at 24 ±1 ºC throughout the test.

Vortex Depth Measurements

The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a slight dimple at the media surface.

Observations on Test Substance Solubility

Observations on the test media were carried out during the mixing and testing of the WAF. At the start of the mixing period the 100 mg/L loading rate WAF was observed to have formed a clear colorless media column with white chunks of test substance floating at the media surface. At the end of stirring a slightly hazy media column with fine particulates of test substance floating throughout the media column and at the media surface was observed. Following a 1-Hour standing period the 100 mg/L loading rate WAF was observed to have formed a slightly hazy media column with fine particulates of test substance suspended within the media column and floating at the media surface. As visual inspection of the WAF at the end of standing showed there to be undissolved test substance present, the WAF was removed by filtration through a glass wool plug. Further filtration through two sheets of coarse filter paper was employed to remove as much undissolved test substance as possible. Microscopic examination of the WAF after filtration showed there to be no micro-dispersions of test substance present. At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

Conclusion

The effect of the test substance on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values based on both inhibition of growth rate and yield of greater than 100 mg/L loading rate WAF.  The No Observed Effect Loading Rate (NOEL) based on growth rate was 100 mg/L loading rate WAF.  A statistically significant difference in terms of inhibition of yield was observed between the control and 100 mg/L loading rate WAF test group and as such the NOEL based on yield was considered to be less than 100 mg/L loading rate WAF.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72 h EL50, NOEL of the test substance to aquatic algae, based on growth rate and yield, were determined to be >100, 100, >100 and <100 mg/L (nominal loading rate), respectively.

Executive summary:

A study was conducted to determine the acute toxicity of the test substance to the unicellular green alga Pseudokirchneriella subcapitata according to OECD 201 Guideline and EU Method C.3, in compliance with GLP. Due to the low aqueous solubility and complex nature of the test substance for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test substance. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a WAF of the test substance, at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 deg C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Particle Counter. Analysis of the 100 mg/L loading rate WAF at 0 hours showed a measured test concentration of 0.055 mg/L. A decline in measured test concentration was observed after each 24-hour period to 0.018, 0.0038 and 0.0012 mg/L at 24, 48 and 72 hours, respectively. A sample of the 100 mg/L loading rate WAF which contained no algal cells was incubated alongside the test samples to provide a sample for uninoculated analysis at 72 hours. A decline in measured test concentration to 0.0098 mg/L was observed. These results indicated that not only was the test substance unstable under the study conditions, it was also adsorbing to the algal cells present. However, given that the toxicity cannot be attributed to a single component or a mixture of components but to the test substance as a whole, the results were based on nominal loading rates only. Exposure of Pseudokirchneriella subcapitata to the test substance gave EL50 values based on both inhibition of growth rate and yield of greater than 100 mg/L loading rate WAF.  The No Observed Effect Loading Rate (NOEL) based on growth rate was 100 mg/L loading rate WAF. A statistically significant difference in terms of inhibition of yield was observed between the control and 100 mg/L loading rate WAF test group and as such the NOEL based on yield was considered to be less than 100 mg/L loading rate WAF. It was considered unnecessary to test at loading rates in excess of 100 mg/L loading rate WAF. The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%. Under the study conditions, the 72 h EL50, NOEL of the test substance to aquatic algae, based on growth rate and yield, were determined to be >100, 100, >100 and <100 mg/L (nominal loading rate), respectively (Covance, 2019).

Description of key information

Based on the study results, the 72 h EL50, NOEL of the test substance to aquatic algae, based on growth rate and yield, were determined to be >100, 100, >100 and <100 mg/L (nominal loading rate), respectively (Covance, 2019).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

A study was conducted to determine the acute toxicity of the test substance, ‘Reaction products of hexadecyl dihydrogen phosphate, dihexadecyl hydrogen phosphate, hexadecan-1-ol, stearic acid, esters of C18 (branched and linear) fatty acids with C18 (branched and linear) alcohols, and potassium hydroxide’ to the unicellular green alga Pseudokirchneriella subcapitata according to OECD 201 Guideline and EU Method C.3, in compliance with GLP. Due to the low aqueous solubility and complex nature of the test substance for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test substance. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a WAF of the test substance, at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 deg C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Particle Counter. Analysis of the 100 mg/L loading rate WAF at 0 hours showed a measured test concentration of 0.055 mg/L. A decline in measured test concentration was observed after each 24-hour period to 0.018, 0.0038 and 0.0012 mg/L at 24, 48 and 72 hours, respectively. A sample of the 100 mg/L loading rate WAF which contained no algal cells was incubated alongside the test samples to provide a sample for uninoculated analysis at 72 hours. A decline in measured test concentration to 0.0098 mg/L was observed. These results indicated that not only was the test substance unstable under the study conditions, it was also adsorbing to the algal cells present. However, given that the toxicity cannot be attributed to a single component or a mixture of components but to the test substance as a whole, the results were based on nominal loading rates only. Exposure of Pseudokirchneriella subcapitata to the test substance gave EL50 values based on both inhibition of growth rate and yield of greater than 100 mg/L loading rate WAF.  The No Observed Effect Loading Rate (NOEL) based on growth rate was 100 mg/L loading rate WAF. A statistically significant difference in terms of inhibition of yield was observed between the control and 100 mg/L loading rate WAF test group and as such the NOEL based on yield was considered to be less than 100 mg/L loading rate WAF. It was considered unnecessary to test at loading rates in excess of 100 mg/L loading rate WAF. The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%. Under the study conditions, the 72 h EL50, NOEL of the test substance to aquatic algae, based on growth rate and yield, were determined to be >100, 100, >100 and <100 mg/L (nominal loading rate), respectively (Covance, 2019).