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Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 26 March 2009 and 03 April 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C18 to C50

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test material in cases where the test material is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test material with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test material and water phase. At the completion of mixing, the test material phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test material and/or leachates from the test material).

Exposures are expressed in terms of the original concentration of test material in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test material in the WAF.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

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.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Analysis of the WAF was carried out by Total Organic Carbon (TOC) analysis. Samples were taken from the uninoculated control and the 100 mg/l loading rate WAF test group at 0 and 72 hours for this analysis. Duplicate samples were taken and stored frozen (approximately -20 degree C) for
further analysis if necessary.

Vehicle:

no

Details on test solutions:

Experimental Preparation
An amount of test material 250 mg was added to the surface of 2.5 litres of culture medium to give the 100 mg/l loading rate. After the addition of the test material, 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. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test material to be present.

An aliquot (1 litre) of the WAF was inoculated with algal suspension (17.9 ml) to give the required test concentration of 100 mg/l loading rate WAF.
Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 and 72 hours (see Appendix 1- in attached section).

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test was carried out using Desmodesmus subspicatus strain CCAP 276/20. Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and
illumination at 21 ± 1 degree 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 10e3 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation
by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 degree C until the algal cell density was approximately 10e4 – 10e5 cells/ml.

The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture - The culture
medium is defined in Appendix 2 in attachment section

Test type:

static

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not Applicable

Hardness:

Not Applicable

Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test.

pH:

The pH values of each test and control flask are given in Table 2 in section any other information on results.

Dissolved oxygen:

Not Applicable

Salinity:

Not Applicable

Nominal and measured concentrations:

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.

Details on test conditions:

Exposure conditions
Six flasks each containing 100 ml of test preparation were used for the control and 100 mg/l loading rate WAF treatment group.
The control group was maintained under identical conditions but not exposed to the test material.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 2.24 x 10e5 cells per ml. Inoculation of 1 litre of test medium with 17.9 ml of this algal suspension gave an initial nominal cell density of 4 x 10e3 cells per ml and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Not stated

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Not stated

Details on results:

See Appendix 5 in attachment section for calculations for evaluating the data.

The results of the test are considered valid if the following performance criteria are met:
The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.

RESULTS
Validation of Mixing Period
Pre-study work (see Appendix 3 in attached section) indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 hours.

Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test material during the range-finding test are given in Table 1 - in section any other information on results.

The results showed no effect on growth at 10 and 100 mg/l loading rate WAF.

Based on this information a single loading rate of six replicates of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.

Definitive Test
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2 - in section any other information on results.

Daily specific growth rates for the control cultures are given in Table 3 - in section any other information on results.

Growth rates and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4 in section any other information on results.

The mean cell densities versus time for the definitive test are presented in Figure 1 - see attachment appendix 4.

Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 135 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.

Mean cell density of control at 0 hours : 4.15 x 10e3 cells per ml
Mean cell density of control at 72 hours : 5.62 x 10e5 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 27% 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 – 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Growth data
From the data given in Tables 2 and 4 (in section any other information on results), it is clear that the growth rate (r) and yield (y) of Desmodesmus
subspicatus (CCAP 276/20) were not affected by the presence of the test material over the 72-Hour exposure period.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Accordingly the following results were determined from the data:
1.3.2.1 Inhibition of growth rate
ErL*10 (0 - 72 h) : >100 mg/l loading rate WAF
ErL*20 (0 - 72 h) : >100 mg/l loading rate WAF
ErL*50 (0 - 72 h) : >100 mg/l loading rate WAF

where ErL*x 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). There were no statistically significant differences (P<0.05), between the control and 100 mg/l loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/l loading rate WAF.

Inhibition of yield
EyL*10 (0 - 72 h) : >100 mg/l loading rate WAF
EyL*20 (0 - 72 h) : >100 mg/l loading rate WAF
EyL*50 (0 - 72 h) : >100 mg/l loading rate WAF
where EyL*x is the loading rate that reduced yield by x%.

There were no statistically significant differences between the control and 100 mg/l loading rate WAF (P0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/l loading rate WAF.

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.

Observations on test material solubility
Observations on the test media were carried out during the mixing and testing of the WAF.

At the start of mixing the WAF was observed to have formed a clear colourless media column with a large clear oily globule of test material within the dimple. At both the end of stirring and the 1-Hour standing period the WAF was observed to have formed a clear colourless media column with globules of test material spread across the media surface.
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be pale green dispersions.

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd Project Number: 0039/1066) used potassium dichromate as the reference material.

The positive control was conducted between 18 November 2008 and 21 November 2008.

Positive Control
A positive control (Harlan Laboratories Ltd Project No: 0039/1066) used potassium dichromate as the reference material at concentrations of
0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material gave the following results:

ErC50 (0 – 72 h) : 0.52 mg/l, 95% confidence limits 0.43 – 0.62 mg/l
EyC50 (0 – 72 h) : 0.29 mg/l, 95% confidence limits 0.25 – 0.33 mg/l

No Observed Effect Concentration (NOEC) based on growth rate: 0.125 mg/l

No Observed Effect Concentration (NOEC) based on yield: 0.125 mg/l

Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.25 mg/l

Lowest Observed Effect Concentration (LOEC) based on yield: 0.25 mg/l

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

Reported statistics and error estimates:

A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/l loading rate to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Table 1: 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	3.98E+03	4.48E+05
	R2	4.27E+03	4.25E+05	-	-
	Mean	4.12E+03	4.37E+05
10	R1	4.34E+03	2.26E+05
	R2	5.07E+03	2.58E+05	15	45
	Mean	4.71E+03	2.42E+05
100	R1	4.02E+03	3.82E+05
	R2	4.27E+03	3.26E+05	5	19
	Mean	4.14E+03	3.54E+05

*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.

R₁and R₂= Replicates 1 and 2

Table 2: Cell Densities and pH Values in the DefinitiveTest

Nominal Loading Rate (mg/l)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.8	4.37E+03	3.91E+04	1.02E+05	5.33E+05	7.3
	R2	7.7	4.26E+03	2.94E+04	1.07E+05	6.33E+05	7.3
	R3	7.7	4.04E+03	2.65E+04	7.86E+04	5.02E+05	7.4
	R4	7.7	4.14E+03	2.95E+04	8.24E+04	4.91E+05	7.4
	R5	7.6	4.06E+03	2.92E+04	9.86E+04	7.21E+05	7.4
	R6	7.5	4.05E+03	2.52E+04	9.05E+04	4.91E+05	7.4
	Mean		4.15E+03	2.98E+04	9.32E+04	5.62E+05
100	R1	7.5	4.14E+03	3.21E+04	8.84E+04	5.79E+05	7.4
	R2	7.5	4.19E+03	3.03E+04	8.07E+04	5.74E+05	7.4
	R3	7.5	4.21E+03	3.04E+04	9.16E+04	4.17E+05	7.5
	R4	7.5	4.02E+03	3.26E+04	9.84E+04	5.65E+05	7.5
	R5	7.5	4.26E+03	3.30E+04	8.89E+04	5.22E+05	7.5
	R6	7.5	4.10E+03	2.61E+04	1.02E+05	4.27E+05	7.5
	Mean		4.15E+03	3.08E+04	9.17E+04	5.14E+05

*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.

R₁- R₆= Replicates 1 to 6

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

		Daily Specific Growth Rate (cells/ml/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.095	0.040	0.069
	R2	0.083	0.054	0.074
	R3	0.079	0.045	0.077
	R4	0.083	0.043	0.074
	R5	0.083	0.051	0.083
	R6	0.077	0.053	0.070
	Mean	0.083	0.048	0.075

R₁- R₆= Replicates 1 to 6

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 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.068		5.29E+05
	R2	0.070		6.29E+05
	R3	0.067		4.98E+05
	R4	0.067	-	4.87E+05	-
	R5	0.072		7.17E+05
	R6	0.067		4.87E+05
	Mean	0.069		5.58E+05
	SD	0.002		9.48E+04
100	R1	0.069	0	5.75E+05
	R2	0.069	0	5.70E+05
	R3	0.065	6	4.13E+05
	R4	0.069	0	5.61E+05
	R5	0.068	1	5.18E+05
	R6	0.065	6	4.23E+05
	Mean	0.068	2	5.10E+05	9
	SD	0.002		7.42E+04

*In accordance with the OECD test guideline only the mean value for yield is calculated

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

Table 5: Vortex Depth Measurements at the Start and End of the Mixing Period

	Nominal Loading Rate (mg/l)
	Control		100
	*	+	*	+
Height of Media Column (cm)	15	15	15	15
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present

*= Start of mixing period

+= End of mixing period

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated and gave EL50 values of greater than 100 mg/l
loading rate WAF. Correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

Executive summary:

Introduction.

A study was performed to assess the effect of the test material 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’ on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 440/2008.

Methods.

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a Water Accommodated Fraction (WAF) of the test material, 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 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Results.

Exposure of Desmodesmus subspicatus to the test material gave EL*₅₀values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours. The results obtained showed that measured concentrations of less than the limit of quantitation (LOQ) were obtained at both 0 and 72 hours.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, and the dissolved test material was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

*EL = Effective Loading Rate

Reference 2

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:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C8 to C26, without GLP and analytics but acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

not specified

Principles of method if other than guideline:

This test was performed according to the scaled down version of the U.S. EPA algal bottle test (U.S. EPA, 1998) that involves using microliter volumes of test solutions contained in 96-well microplates as opposed to milliliter volumes (Environment Canada, Biological Test method: EPS 1/RM/25).

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
Water accommodated fractions (WAF’s) were prepared in sealed vessels with a small headspace according to the CONCAWE methodology for the ecotoxicological testing of petroleum products (CONCAWE report, 1993). WAF’s were prepared using 1L reverse osmosis permeate (ROp) water from a Milli-Q system. The required amount of test substance was weighed on microscope glass cover slips and added to ROp. The vessels were sealed and vigorously shaken (200 rpm’s) for 24 hours on an orbital shaker. The mixtures were poured into separation funnels and left for two hours to separate. The water fractions were transferred to other vessels and used in the test.

Test organisms (species):

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

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Study result do not specify different endpoints for biomass and growth rate.

Table 1: Acute toxicity to Selenastrum capricornutum   

Diesel Fuel	NOEL (mg/L)
Test material	>1000
FCC simulated crude derived refinery	>1000
Test material + FCC	>1000
DHC simulated crude derived refinery	>1000
Test material+ DHC	>1000

Validity criteria fulfilled:

not specified

Conclusions:

The test substance 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' is non-toxic to S. capricornutum [NOEL > 1000 mg/L, nominal, water accommodated fraction].

Executive summary:

The algal growth inhibition test was used to assess the impact of the diesels to freshwater algae using Selenastrum capricornutum. This test was performed according to the scaled down version of the U.S. EPA algal bottle test (U.S. EPA, 1998) that involves using microliter volumes of test solutions contained in 96-well microplates as opposed to milliliter volumes (Environment Canada, Biological Test method: EPS 1/RM/25). In this test the growth of unicellular green algae is used to determine the effect of a test substance. The study demonstrated that 'Distillates (Fischer-Tropsch), C8-26 - branched and linear' was non-toxic (NOEL >1000 mg/L) to Selenastrum capricornutum.

Reference 3

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:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C8 to C26, without GLP and analytics but acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable, since neither a surrogate nor an analogue material was used in the test.

Analytical monitoring:

no

Details on sampling:

- Concentrations:
Loading rates (mg/L):
I. 100% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear': 1000
II. 76% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear', 24% ULSD: 3, 10, 30, 100, 300, 1000
II. 76% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear', 24% ULSD: 3, 10, 30, 100, 300, 1000
III. 50% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear', 50% ULSD: 3, 10, 30, 100, 300, 1000
IV. 24% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear', 76% ULSD: 100, 220, 460, 1000
V. 100% ULSD: 1, 2.2, 4.6, 10, 22, 46, 100
VI. Anullex BX: 1000
- Sampling method: Cell counts were made, using a Coulter Multisizer, on samples taken from each flask at the start of the test and then at approximately 24 h intervals
- Sample storage conditions before analysis: Not applicable

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test samples are mixtures containing a number of moderately volatile components with a range of relatively low water solubilities.
Therefore water accommodated fraction (WAF) methodology was used for production of test media. Vessels containing the medium and test sample
were sealed, leaving only a small headspace, and the contents stirred at 150 rpm for approximately 72 hours (+/- 2 hours). After stirring, the
contents of the vessels were left to settle (1-2 hours) to allow the undissolved material to separate out.
- Eluate: Not applicable (static test)
- Differential loading: Not applicable
- Controls: Control media were subject to the same regime but did not contain the test sample.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not observed.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Strain: Nygaard
- Age of inoculum (at test initiation): No information available
- Method of cultivation: Axenic stock cultures of R. subcapitata (formerly Selenastrum capricornutum) are maintained on agar plates. These are used
to inoculate liquid cultures which, while in exponential phase growth, are in turn used to inoculate test solutions.

ACCLIMATION
- Acclimation period: No information available
- Culturing media and conditions (same as test or not): Maintenance of cultures: Agar plates are prepared by adding 3% mass/volume agar to liquid
medium (Appendix B) prior to autoclaving. On cooling the agarified medium is poured into 9 cm diameter sterile plastic petri dishes and allowed to
set. The algal cultures are streaked onto these plates and maintained under continuous illumination at nominally 21-25 °C in a Gallenkamp vertical
incubator. The cultures are renewed approximately every week. Cultures in liquid medium (Appendix B) are initiated with cells transferred on a sterile
loop from an agar plate. The cultures are grown as 100 mL batch cultures in 250 mL Erlenmeyer flasks for up to 4 days. The cultures are maintained at nominally 21-25 °C in a Gallenkamp orbital incubator under constant illumination.
- Any deformed or abnormal cells observed: No observations reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

total hardness as mg/L CaCO3: 170 mg/L

Test temperature:

Test with 'Distillates (Fischer-Tropsch), C8-26 - branched and linear':
22.9-24.1 °C (reported at hourly intervals)
II. Test with ULSD:
22.4-23.6 °C (reported at hourly intervals),
III. Test with Annulex BX:
23.3-23.7 °C (reported at hourly intervals),
IV. Test with 24% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear' and 76% ULSD:
22.6-23.6 °C (reported at hourly intervals),
V. Test with 50% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear' and 50% ULSD:
22.9-23.6 °C (reported at hourly intervals),
VI. Test with 76% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear' and 24% ULSD:
21.4-23.4 °C (reported at hourly intervals)

pH:

I. Test with 'Distillates (Fischer-Tropsch), C8-26 - branched and linear':
pH at test initiation (T = 0 h): 7.4-7.7,
pH at test finalisation (T = 48 h): 8.7-9.0
II. Test with ULSD:
pH at test initiation (T = 0 h): 7.5-8.2,
pH at test finalisation (T = 48 h): 8.1-9.3
III. Test with Annulex BX:
pH at test initiation (T = 0 h): 8.4-8.6,
pH at test finalisation (T = 48 h): 9.5
IV. Test with 24% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear' and 76% ULSD:
pH at test initiation (T = 0 h): 7.5-8.3,
pH at test finalisation (T = 48 h): 7.4-9.7
V. Test with 50% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear' and 50% ULSD:
pH at test initiation (T = 0 h): 7.6-8.0,
pH at test finalisation (T = 48 h): 8.1-9.8
VI. Test with 76% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear' and 24% ULSD:
pH at test initiation (T = 0 h): 7.9-8.3,
pH at test finalisation (T = 48 h): 9.4-9.5.

Dissolved oxygen:

Data not reported

Salinity:

according to standard nutrient addition

Nominal and measured concentrations:

Nominal concentrations:
Loading rates (mg/L):
I. 100% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear': 1000
II. 76% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear', 24% ULSD: 3, 10, 30, 100, 300, 1000
II. 76% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear', 24% ULSD: 3, 10, 30, 100, 300, 1000
III. 50% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear', 50% ULSD: 3, 10, 30, 100, 300, 1000
IV. 24% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear', 76% ULSD: 100, 220, 460, 1000
V. 100% ULSD: 1, 2.2, 4.6, 10, 22, 46, 100
VI. Anullex BX: 1000.

Nominal concentrations were not confirmed by analytical measurements.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 287 mL full volume Erlenmeyer flask, glass
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): None
- Initial cells density: The culture inoculum volume introduced at the start of the test is sufficient to give an initial concentration of 5000 cells/mL in
the vessels. The cell concentration of an exponentially growing starter culture is measured using a Coulter Counter or Coulter Multisizer and the
required inoculum volume calculated. All flasks used for a particular test are inoculated from the same starter culture.
- Control end cells density: Three out of each set of four flasks containing test sample and six of each set of control flasks were inoculated with
sufficient R. subcapitata to give an initial concentration of approximately 5000 cells/mL. The remaining flasks were not inoculated but used to
determine background particle counts in the absence of R. subcapitata.
- No. of organisms per vessel: approximately 5000 cells per mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 7
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: Not applicable

TEST MEDIUM / WATER PARAMETERS
information not available

OTHER TEST CONDITIONS
- Sterile test conditions: no data
- Adjustment of pH: no
- Photoperiod: 16 h light 8 h dark cycle
- Light intensity and quality: artificial light (intensity unknown)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
Results from the R. subcapitata test were evaluated using two growth measures: (a) Area under the growth curve (A).
(b) Average specific growth rate (μ).
- Chlorophyll measurement: No
- Other: None

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: 10, 100 , 1000 mg/L
- Results used to determine the conditions for the definitive study:
Obtained range-finding EC50 values were:
for 'Distillates (Fischer-Tropsch), C8-26 - branched and linear': > 1000 mg/L
for AGO-ULSD: 10-100 mg/L,
for Annulex BX: > 1000 mg/L

Reference substance (positive control):

yes

Remarks:

AGO - Ultra Low Sulphur Diesel (ULSD)

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

average specific growth rate

Remarks on result:

other: Water-accommodated fraction

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: area under the growth curve

Remarks on result:

other: Water-accommodated fraction

Duration:

72 h

Dose descriptor:

EC0

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: area under the growth curve

Remarks on result:

other: Water-accommodated fraction

Duration:

72 h

Dose descriptor:

EC0

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

average specific growth rate

Remarks on result:

other: Water-accommodated fraction

Details on results:

Acute toxicity to R. subcapitata was determined in sealed tests, the duration of which were 72 hours. As the test substance was a mixture of poorly
soluble, volatile components they were tested as water accommodated fractions (WAFs) prepared in sealed vessels. Test results were expressed as
the loading rates (mg/L) which had no effect (NOEL) or which caused 50% inhibition when compared with unamended controls (EL50).

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: For detailed results refer to attached table with summary results for reference substance ULSD
- Other: NOEC (for detailed results refer to attached table with summary results for reference substance ULSD)

Reported statistics and error estimates:

I. Calculation of EbL50 values by comparison of areas under the growth curve (A):
The percentage inhibition of culture growth at each loading rate based on the areas under the growth curves (IA) was calculated as the difference
between the mean area under the control curve (Ac) and the mean of area under the growth curve at each loading rate (As)

II. Calculation of ErL50 values by comparison of average specific growth rates (μ):
The percentage reduction in average specific growth rate at each loading rate of test substance compared to the control value was determined after
24, 48 and 72 h exposure. These percentage reduction values were used to estimate 0-24, 0-48 and 0-72 h ErL50 values (loading rate used to
prepare WAF, calculated to cause 50% reduction in culture growth based on average specific growth rate).

III. Calculation of no observed effect loading rate (NOEL):
Williams' test (Williams 1971, 1972) was used to compare differences in areas under the growth curves and average specific growth rates between
control and treated cultures. The highest concentration at which there was no significant difference (p <5%) between treated and control cultures was identified as the NOEL.

Table 1: Summary table: Results with Raphidocelis subcapitata:

	A (area under growth curve)		µ (average specific growth rate)
Test Item	48 hour EL50 (mg/L)	NOEL (mg/L)	48 hour EL50 (mg/L)	NOEL (mg/L)
100% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear'	>1000	1000	>1000	1000
76% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear' : 24% ULSD	>1000	300	>1000	300
50% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear' : 50% ULSD	100-300	10	>1000	10
24% 'Distillates (Fischer-Tropsch), C8-26 - branched and linear' : 76% ULSD	30-100	<3	100-300	<3
100% ULSD	10 -22	<1	22-46	<1
Anullex BX	>1000	<1000	>1000	1000

Validity criteria fulfilled:

yes

Conclusions:

The test substance 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' is non-toxic to R. subcapitata [EL50 > 1000 mg/L, nominal, water accommodated fraction].

Executive summary:

The acute toxicity of 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' was determined according to OECD guideline 201. Acute toxicity to Raphidocelis subcapitata was determined in sealed tests, the duration of which were 72 hours. The study demonstrated that the substance was non-toxic (NOEL >1000 mg/L) to Raphidocelis subcapitata. Using the European Union Criteria for classifying ‘dangerous substances’ (EU, 1993), 'Distillates (Fischer-Tropsch), C8-26 - branched and linear' would be classified as ‘not harmful’ to aquatic organisms.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 7th 2014 to 19 August 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C15 to C19

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Test Organism Observations
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Vehicle:

no

Details on test solutions:

 Experintal Preparation
A nominal amount of test item (230 mg) was added to the surface of 2.3 liters of culture medium in a sealed vessel with minimal headspace to give the 100 mg/L loading rate. After the addition of the test item, 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. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
 
An aliquot (2 liters) of the 100 mg/L loading rate WAF was inoculated with algal suspension (17 mL) to give the required test concentration of 100 mg/L loading rate WAF.
 
Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 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 in the laboratory under constant aeration and illumination at 21 ± 1°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 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 104 – 105 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Not stated in report

Test temperature:

24 +/- 1°C recorded daily

pH:

Measured using a Hach HQ30d Flexi handheld meter.

Dissolved oxygen:

Not stated in report

Salinity:

Not applicable as a freshwater study

Nominal and measured concentrations:

Range finding test: 10 and 100 mg/l loading rate
Definitive test: 100 mg/l loading rate

Details on test conditions:

As in the range-finding test 250 mL glass conical flasks were used. Six flasks each completely filled with test preparation were used for the control and 100 mg/L loading rate WAF treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 5.85 x 105 cells per mL. Inoculation of 2 liters of test medium with 17 mL of this algal suspension gave an initial nominal cell density of 5 x 103 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

growth rate

Details on results:

From the data given in Tables 2 and 4 (please refer to the attached documents), it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item over the 72-Hour exposure period.

Results with reference substance (positive control):

A positive control (Harlan Study Number 41303826) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

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 item gave the following results:

ErC50 (0 – 72 h) : 1.1 mg/L; 95% confidence limits 0.91 – 1.2 mg/L
EyC50 (0 – 72 h) : 0.51 mg/L; 95% confidence limits 0.45 – 0.59 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

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

Reported statistics and error estimates:

Inhibition of growth rate
ErL10 (0 - 72 h) : >100 mg/L loading rate WAF
ErL20 (0 - 72 h) : >100 mg/L loading rate WAF
ErL50 (0 - 72 h) : >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 a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant decreases in growth rate (P0.05), between the control and 100 mg/L loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/L loading rate WAF.


Inhibition of Yield
EyL10 (0 - 72 h) : >100 mg/L loading rate WAF
EyL20 (0 - 72 h) : >100 mg/L loading rate WAF
EyL50 (0 - 72 h) : >100 mg/L loading rate WAF

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

Statistical analysis of the yield data was carried out as per the above. There were no statistically significant decreases in yield between the control and 100 mg/L loading rate WAF (P0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 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 58 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.

 

Mean cell density of control at 0 hours         :          7.34 x 10³cells per mL

Mean cell density of control at 72 hours       :          4.26 x 10⁵cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 20% 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 – 72 h) was 3% 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

Temperature was maintained at 24 ± 1 ºC throughout the test.

 

The pH value of the control cultures (see Table 2) was observed to increase from pH 8.7 at 0 hours to pH 9.6 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH unites after 72 hours and was therefore within the limits given in the Test Guidelines.

 

Vortex Depth Measurements

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

  

Observations on Test Item Solubility

Observations on the test media were carried out during the mixing and testing of the WAF.

 

At both the start and end of the mixing period, and following a 1-Hour standing period, the 100 mg/l loading rate WAF was observed to have formed a clear colorless media column with an oily globule of test item floating at the media surface. Microscopic examination of the WAF showed there to be no globules or micro-dispersions of test item present.

 

At the start of the test all control and 100 mg/L loading rate WAF 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.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

 

 Methods…

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

 

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to a Water Accommodated Fraction (WAF) of the test item, 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 ± 1 °C.

 

Due to the potentially volatile nature of the test item, testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. Following the recommendations of published data (Hermanet al1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.

 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

 

Results…….

Total Organic Carbon (TOC) analysis of the 100 mg/L loading rate WAF test preparation at 0 hours showed a measured concentration of 4.93 mg C/L was obtained. A decline in measured carbon concentration was observed at 72 hours to 0.79 mg C/L.

 

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

 

Exposure ofPseudokirchneriella subcapitatato the test item gave EL₅₀values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

 

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.

Description of key information

- (72h) ErL50 for algae (Desmodesmus subspicatus): >100 mg/l  [OECD 201; test mat. Distillates (Fischer–Tropsch), heavy, C18–C50 branched, cyclic and linear (WAFs) (nominal) based on: growth rate];

- (72h) ErL50 for algae (Pseudokirchneriella subcapitata): >1000 mg/l [OECD 201; test mat. Distillates (Fischer–Tropsch), C8–C26 branched and linear (WAFs) (nominal) based on: growth rate];

- (72h) ErL50 for algae (Pseudokirchneriella subcapitata): >1000 mg/l [US EPA Algal Bottle Test; Distillates (Fischer–Tropsch), C8–C26 branched and linear (WAFs) (nominal) based on: growth rate];

- (72h) NOELR for algae (Pseudokirchneriella subcapitata): >100 mg/l [OECD 201; test mat. Hydrocarbons, C15-C19, n-alkanes, isoalkanes < 2% aromatics (WAFs) (nominal) based on: growth rate].

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Measured toxicity data are not available for 'Paraffin waxes (Fischer-Tropsch), full-range, C15-50 - branched and linear':

The toxicity of the closely related substance GTL Base Oil Distillates (covering the carbon range from C18 to C50) has been determined by Harlan (Vryenhoef, 2009) in a test with the unicellular alga Desmodesmus subspicatus. The test was conducted in accordance with OECD Test Guideline 201.

Desmodesmus subspicatus were exposed to water accommodated fractions (WAFs) of the test material over a single nominal loading rates of 100 mg/l for a period of 72 hours. The WAFs were prepared by stirring for 23 hour and left to stand for 1 hour before drawing off the aqueous phase – the WAF – for testing. Static exposures were then carried. The study was carried out according to GLP and analytical monitoring, TOC analysis, also took place. 

The test results, expressed as the EL₅₀and NOELR values, showed that the sample was not toxic to algae at a loading rate of 100 mg/l.

Total Organic Carbon (TOC) analysis of the freshly prepared test preparations showed amount of carbon present within the 100 mg/l loading rate WAF test vessels to be less than the limit of quantitation (LOQ) for the method (1.0 mg C/l) in fresh and old test media samples.

The acute toxicity of the supporting substance GTL Gasoil (covering the carbon range from C8 to C26) has been determined by Palmer (2001) in a test with the unicellular alga Raphidocelis subcapitata. The test was conducted in accordance with OECD Test Guideline 201.

WAFs of the sample were prepared in sealed vessels with minimum headspace by stirring for approximately 72 hours. The contents of the vessels were left to stand for 1-2 hours before drawing off the aqueous phase – the WAF – for testing. Static exposures were then carried out in completely full, sealed vessels. The test was not subject to GLP and the test media were not analysed for stability or for exposure concentration of the test substance. However in other respects it was considered to fulfil the requirements of current best practice.

The test results, expressed as the EL₅₀and NOEL values, showed that the sample was not acutely toxic to algae at a loading rate of 1000 mg/l.

Albertus and Phillips (2005) have also reported results for an acute toxicity test carried out on the supporting substance GTL Gasoil with the freshwater alga Selenastrum capricornutum.

The test was carried out using a scaled-down version of the US EPA algal bottle test method. In all cases the test media were water-accommodated fractions of the test sample that were prepared in sealed vessels with a small headspace according to the CONCAWE methodology for the ecotoxicological testing of petroleum products (CONCAWE report, 1993).

The test results, expressed as the EL₅₀and NOEL values, showed that the sample was not acutely toxic to algae at a loading rate of 1000 mg/l.

Finally, measured toxicity data are available for 'Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics' to the freshwater green alga Pseudokirchneriella subcapitata (Vryenhoef, 2014c). The test was conducted under static (no renewal of the test media) conditions in accordance with OECD Test Guideline 201 and method C3 of EC Regulation No. EC 761/2009. Appropriate modifications to the test and media preparation procedures were made to take account of the test substance containing multiple constituents, having low solubility in water and being potentially volatile. No effects on growth (expressed in terms of yield (y) and growth rate (r)) of P. subcapitata were observed after 72 hours exposure to the test medium prepared as a water-accommodated fraction (WAF) at a loading rate of 100 mg/l; 72-hour E_yL₅₀ and E_rL₅₀ values were >100 mg/l and NOELR values were ≥100 mg/l. Total Organic Carbon (TOC) analysis of the 100 mg/l loading rate WAF test preparation at 0 hours showed a measured concentration of 4.93 mg C/l. A decline in measured carbon concentration was observed at 72 hours to 0.79 mg C/l. The results of the test are considered to be reliable.

Conclusion:

Algal growth inhibition tests are available for three GTL-derived substances in the relevant carbon number range for the registered substance. In the absence of adverse effects in these studies it can be concluded that, based on weight of evidence, the ELR50(algae) for 'Paraffin waxes (Fischer-Tropsch), full-range, C15-50 - branched and linear' is >100 mg/l and the NOELR is ≥100 mg/l.