Docosane

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:

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

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)

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

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

Remarks on result:

other: Not stated

Key result

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

Description of key information

The 72 -hour E_rL₅₀ for GTL Base Oil Distillates was determined to be >100 mg/L WAFs (Desmodesmus subspicatus).

This data is used for read across to Docosane.

Additional information

The toxicity of a sample of GTL Base Oil Distillates has been determined in a test with the unicellular alga Desmodesmus subspicatus. The tests were 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 EL₅₀ and 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.