Naphtha (Fischer-Tropsch), light, C4-10-branched and linear

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 10 February 2011 and 11 March 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

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 items, 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 item in cases where the test item 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 item 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 item and water phase. At the completion of mixing, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Purity: 100%
Batch number: not provided
Storage conditions: room temperature in the dark

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 100 mg/l loading rate WAF at 0 and 72 hours for this analysis (background material). Duplicate samples were taken and stored at approximately 4°C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Define Test
Experimental Preparation
An amount of test item (250 mg) was added to the surface of 2.5 litres of culture medium in a stirring 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 litres) of the WAF was inoculated with algal suspension (28.3 ml) to give the required test concentration of 100 mg/l loading rate WAF.
Additional samples of the control and 100 mg/l loading rate WAF were prepared with the omission of algal cells at 0 hours and incubated alongside the test from which samples were taken for Total Organic Carbon (TOC) analysis at 0 and 72 hours (see Appendix 4, background material)).

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.

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), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Appendix 2, background material). 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.

The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture with the addition of 500 mg/l of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system (Herman et al 1990). Culture Medium, see Appendix 2, background material.

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not Applicable

Hardness:

Not Applicable

Test temperature:

Temperature was maintained at 24 ± 1°C throughout the test. The temperature within the incubator was recorded daily.

pH:

The pH of the control and 100 mg/l loading rate WAF test concentration was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.

Dissolved oxygen:

Not Applicable

Salinity:

Not Applicable

Conductivity:

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
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 characterised by a cell density of 3.53 x 105 cells per ml. Inoculation of 2 litres of test medium with 28.3 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.

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

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture with the addition of 500 mg/l of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system (Herman et al 1990), see Apendix 2, background material.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 other: mg/l loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

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, background material) indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 hours. Therefore for the purposes of the test the WAF was prepared using a 23-Hour stirring period followed by a 1-Hour settlement period prior to removal of the aqueous phase for testing.

Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item 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. 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, in section any other information on results.
The mean cell densities versus time for the definitive test are presented in Figure 1, see background material.
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.

Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 87 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 : 5.07 x 10e3 cells per ml
Mean cell density of control at 72 hours : 4.42 x 10e5 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 13% 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, 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.
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:

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), however all other loading rates were significantly different (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 WAFs.
At both the start and end of the mixing period and following the 1-Hour settlement period the 100 mg/l loading rate WAF was observed to have formed a clear colourless media column with a slick of test item at the media surface. Microscopic observations made on the WAF prior to siphoning indicated that there were no globules or micro-dispersions of test item present.
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 green dispersions.

Results with reference substance (positive control):

Positive Control
A positive control (Harlan Laboratories Ltd Project No: 41100094) 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) : 1.2 mg/l, 95% confidence limits 0.98 – 1.4 mg/l
EyC50 (0 – 72 h) : 0.48 mg/l, 95% confidence limits 0.43 – 0.54 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: 0.5 mg/l

Lowest Observed Effect Concentration (LOEC) based on yield: 0.5 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

NominalLoading Rate (mg/l)		Cell density (cells per ml)		Inhibition Values (%)
		0Hours	72Hours	Growth Rate	Yield
Control	R1	5.26E+03	5.27E+05	-	-
	R2	4.82E+03	4.13E+05
	Mean	5.04E+03	4.70E+05
10	R1	5.16E+03	4.87E+05	0	[2]
	R2	5.10E+03	4.74E+05
	Mean	5.13E+03	4.80E+05
100	R1	5.05E+03	4.37E+05	2	4
	R2	5.05E+03	4.67E+05
	Mean	5.05E+03	4.52E+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 and R2 = Replicates 1 and 2

[Increase in growth compared to controls]

Table 2: Cell Densities and pH Values in the DefinitiveTest

Nominal Loading Rate (mg/l)		pH	Cell Densities* (cells per ml)				pH
		0 h	0h	24 h	48 h	72 h	72 h
Control	R1		5.12E+03	2.71 E+04	1.30E+05	4.38E+05
	R2		5.07E+03	2.42E+04	1.23E+05	4.28E+05
	R3		5.10E+03	2.35E+04	1.04E+05	3.65E+05
	R4	8.3	5.03E+03	2.33E+04	1.17E+05	4.35E+05	9.7
	R5		5.06E+03	2.26E+04	1.28E+05	4.96E+05
	R6		5.02E+03	2.29E+04	1.23E+05	4.89E+05
	Mean		5.07E+03	2.39E+04	1.21E+05	4.42E+05
100	R1		5.03E+03	2.11 E+04	8.42E+04	4.24E+05
	R2		4.94E+03	2.85E+04	1.07E+05	4.26E+05
	R3		5.09E+03	2.14E+04	1.08E+05	4.02E+05
	R4	8.3	5.06E+03	3.15E+04	1.39E+05	4.52E+05	9.6
	R5		4.40E+03	2.02E+04	8.67E+04	4.27E+05
	R6		5.12E+03	2.01 E+04	9.57E+04	3.76E+05
	Mean		4.94E+03	2.38E+04	1.03E+05	4.18E+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)
	Day0 -1	Day1 - 2	Day2 - 3
Control R1	0.070	0.065	0.051
R2	0.066	0.068	0.052
R3	0.065	0.062	0.052
R4	0.064	0.067	0.055
R5	0.063	0.072	0.056
R6	0.063	0.070	0.057
Mean	0.065	0.067	0.054

R₁- R₆= Replicates 1 to 6

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

	GrowthRate (cells/ml/hour)		Yield (cells/ml)
	0-72h	% Inhibition	0-72h	%Inhibition*
Control R1	0.062	-	4.33E+05	-
R2	0.062		4.23E+05
R3	0.060		3.60E+05
R4	0.062		4.30E+05
R5	0.064		4.90E+05
R6	0.064		4.84E+05
Mean	0.062		4.37E+05
SD	0.002		4.75E+04
Control R1	0.062	0	4.19E+05
R2	0.062	0	4.21 E+05
R3	0.061	2	3.97E+05
R4	0.063	[2]	4.47E+05
R5	0.062	0	4.22E+05
R6	0.060	3	3.71 E+05
Mean	0.062	1	4.13E+05	5
SD	0.001		2.57E+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

[Increase in growth as compared to controls]

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:

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 (Effective Loading rate) (72 h) values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate (72 h) 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 alga Pseudokirchneriella 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) 440/2008.

Methods.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was 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. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter. The test item was known to be volatile and hence testing was conducted in completely filled, stoppered test vessels in order to minimise possible losses due to volatilisation. Following the recommendations of published data (Herman et al 1990) 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.

Results.

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 (72 h) values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate (72 h) 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 prepared with the omission of algal cells was performed at 0 and 72 hours. Analysis at 0 hours showed a measured test concentration of 8.04 mg C/l was obtained whilst a decline to less than the limit of quantitation (LOQ) which was considered to be 1.0 mg C/l was observed at 72 hours. This decline was considered to be due to a combination of possible instability, biodegradation and volatility despite every precaution having been taken during exposure to minimise such losses. 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.

*EL = Effective Loading Rate

Description of key information

-(72 h) NOEL rate for Pseudokirchneriella subcapitata: 100 mg/l (WAF, nominal, based on: growth rate) [OECD 201; test mat. C4-C10 branched and linear hydrocarbons (light) – Naphtha]

-(72 h) EL50 for Pseudokirchneriella subcapitata: > 100 mg/l (WAF, nominal, based on: growth rate) [OECD 201; test mat. C4-C10 branched and linear hydrocarbons (light) – Naphtha]

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Toxicity to algae was investigated according to OECD Guideline 201. Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 (Effective Loading rate) (72 h) values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate (72 h) was 100 mg/l loading rate WAF.