A mixture of isomers of branched tetracosane

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 January 1995 and 3 April 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

As stated below

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity for poorly water soluble complex mixtures, the Sponsor requested modification of the standard methods for the preparation of aqueous media. Methods involving the continuous stirring of complex mixtures, like oil products have been used, particularly for exposing robust organisms like fish. Such approaches are unsuitable for small organisms like Daphnia and algae, which will be damaged by the turbulence and physically fouled by undissolved material (recent evidence has suggested that for some products, physical fouling of fish may also lead to inconsistent results. As an alternative, the use of solvents or surfactants to solubilise or disperse poorly soluble products has been widely advocated in the past. This approach results in the production of aqueous media in which the test material is in a different form from that encountered following accidental release unto the environment. In addition, there may be interaction between the surfactant/solvent and the product. The approach recommended by CONCAWE for oil products (1) and subsequently endorsed by several important regulatory authorities in the EC (2,3) and elsewhere, is to expose organisms to the “Water Accommodated Fraction” (WAF) of the oil product. Using this approach, aqueous medium is prepared by mixing the product with water for a prolonged period (usually 24 hours), with continuous stirring to ensure equilibration between the product and water phases. At the completion of mixing, the product phase is separated and organisms exposed to the aqueous phase (which will contain dissolved material, along with any part of the product that may be present in emulsified or dispersed form). Exposures are expressed in terms of the original concentration of product in water at the preparation of WAF (the loading rate), irrespective of the actual concentration of product dissolved in the water.

GLP compliance:

yes (incl. QA statement)

Remarks:

The work described was performed in compliance with the UK Principles of Good Laboratory Practice (The United Kingdom Compliance Programme, Department of Health 1989).

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of isomers of branched tetracosane

EC Number:

417-060-2

EC Name:

A mixture of isomers of branched tetracosane

Cas Number:

151006-61-0

Molecular formula:

C24 H50

IUPAC Name:

A mixture of isomers of branched tetracosane

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Standard solutions were prepared in hexane at nominal concentrations of 10, 100, 1000 and 10000 mg/L.

- Sampling method:
An aliquot (500 ml) of each test sample was extracted with three portions (3 x 50 mL) of dichloromethane, each extract being filtered through a bed of anhydrous sodium sulphate. The combined extracts were evapourated to dryness and the residue dissolved in hexane (2.0 mL).

- Sample storage conditions before analysis:
Samples used immediately.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
An amount of test material (4.0 g) was dispersed onto the surface of 2 litres of culture medium to give a loading rate of 2000 mg/L and then stirred for 24 hours prior to the study start; care was taken to avoid vortex formation or gross mixing. Stirring was stopped after 24 hours and the mixture allowed to stand for 1 hour prior to removal of the aqueous phase or Water Accommodated Fraction.

- Eluate:
Not applicable

- Differential loading:
Not recorded

- Controls:
Control vessel contained 200mL of the test media without being exposed to the test material

- Chemical name of vehicle:
Not applicable

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material:
Observations on test material solubility were carried out during the mixing and testing of the Water Accommodated Fraction. At 24 hours prior to the study start and at the start of the mixing period, the test material was observed to be contained within the vortex and floating on the surface of the test medium. However after 20 hours of stirring and 4 hours of standing the test material was observed to be floating on the surface of the test medium only.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name:
Green Algae

- Strain:
CCAP 278/4.

- Source (laboratory, culture collection):
Culture Centre for Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria.

- Age of inoculum (at test initiation):
Not recorded

- Method of cultivation:
Cultures were maintained in the laboratory by the transfer of algal cells to fresh culture medium approximately once per week. The culture was maintained in the laboratory at a temperature of 21 +/- 1 deg C under continous illumination (intensity approximately 7000 lux) and constant aeration.

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
Same as test

Cultures were maintained in the laboratory by the transfer of algal cells to fresh culture medium approximately once per week. The culture was maintained in the laboratory at a temperature of 21 +/- 1 deg C under continous illumination (intensity approximately 7000 lux) and constant aeration.

- Any deformed or abnormal cells observed:
No abnormal or deformed cells observed.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not recorded.

Test temperature:

The temperature within the incubator was recorded every 3.2 minutes using a Tinytalk temperature logger.

Temperature was maintained at 24 +/- Deg C throughout the study (See Attachment 1).

pH:

The pH of each control and test flask was determined at initiation of the study and after 96 hours exposure.

The pH values of each test and control flask are as follows:
0 h = 7.5
96 h = 9.7 +/- 0.2

Dissolved oxygen:

Not recorded

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal concentration
Range-finding study: 1000 mg/l
Definitive study: 1000 mg/l

Quantitation of the test and control samples by GC-MS shows that the solubility of the many components of the test material in aqueous media is lower than the limit of quantifitation. Therefore the test concentrations for the Acute Toxicity to Seleneastrum capricornutum could not be verified by chemical analysis.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250ml glass conical flask, each containing 100mL of Water Accommodated Fraction.
- Aeration: Not recorded

- Type of flow-through (e.g. peristaltic or proportional diluter):
Not applicable

- Renewal rate of test solution (frequency/flow rate):
Not recorded

- Initial cells density:
At initiation of the study the culture contained a nominal cell density of 10E4 cells per mL.

- Control end cells density:
Mean cell density of control @ 96 h : 1.51E6 cells/ml

- No. of organisms per vessel:
Not applicable

- No. of vessels per concentration:
6

- No. of vessels per control :
3

- No. of vessels per vehicle control:
Not applicable

GROWTH MEDIUM
- Standard medium used: yes

- Detailed composition if non-standard medium was used:
Not applicable


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Not recorded

- Total organic carbon:
The concentration of Total Organic Carbon (TOC) in the test samples from the Water Accommodated Fraction WAF) of the test material was determined by Total Organic Carbon analysis.

Approximately 20 mL of the 1000 mg/L loading rate Water Accommodated Fraction was taken for analysis at 0 and 96 hours.

Standard solutions of potassium hydrogen phthalate were prepared in deionised reverse osmosis water at a nominal concentration of 50 mg carbon/litre and used for calibration of the carbon analyser.

The standard and sample solutions were analysed for TOC iusing a Dohrmann DC-190 high temperature Total Organic Carbon Analyser using the following conditions:

Total Carbon Channel
Temperature : 900 deg C
Carrier gas : Zero grade oxygen
Carrier gas flow rate : 200 ml/min regulated at 50 psi
Catalyst : Platinum
Injection volume : 250 micoL

Inorganic Carbon Channel
Temperature : 20 deg C
Carrier gas : Zero grade oxygen
Carrier gas flow rate : 200 ml/min regulated at 50 psi
Catalyst : 20% phosphoric acid
Injection volume : 250 microL

The results of the Total Organic Carbon analysis generated for the test samples taken throughout the study have been detailed in Table A.

- Particulate matter:
None recorded

- Metals:
MgCl2.6H2O : 12 mg/L
MgSO4.7H2O : 15 mg/L
FeCl3.6H2O : 0.08 mg/L
ZnCl2 : 3E-3 mg/L
CuCl2.2H2O : 1E-5 mg/L

- Pesticides:
None recorded

- Chlorine:
NH4Cl : 15 mg/L
MgCl2.6H2O : 12 mg/L
CaCl2.2H2O : 18 mg/L
FeCl2.6H2O : 0.08 mg/L
MnCl2.4H2O : 0.415 mg/L
ZnCl2 : 3E-3 mg/L
CoCl2.6H2O : 1.5E-3 mg/L
CuCl2.2H2O : 1E-5 mg/L

- Alkalinity:
The pH of this medium after equilibration with air is approximately 7.5.

- Ca/mg ratio:
CaCl2.2H2O - 18 mg/L : MgCl2.6H2O - 12 mg/L MgSO4.7H2O - 15 mg/L

- Conductivity:
Not recorded

- Culture medium different from test medium:
No

- Intervals of water quality measurement:
Not recorded


OTHER TEST CONDITIONS
- Sterile test conditions:
Not recorded

- Adjustment of pH:
No

- Photoperiod:
Continuous illumination

- Light intensity and quality:
Illumination approximately 7000 lux)

- Salinity (for marine algae):
Not applicable


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:
Samples were taken at 0, 24, 48, 72 and 96 hours and the absorbance measured at 665 nm using a Jenway 6100 Spectrophotometer. The cell densities of the control cultures at 0, 24, 48, 72 and 96 hours, were determined by direct counting with the aid of a haemocytometer to confirm that the absorbance values were sufficiently well correlated with cell density values to be used to monitor the growth of the test cultures.

- Chlorophyll measurement:
Not recorded

- Other:
Comparison of areas under the growth curves.
The area under the curve is taken to be an index of growth and was calculated using the following equation:

A = (N1 - N0 / 2) x t1 + (N1 + N2 - 2N0 / 2) x (t2 - t1) + (Nn-1 + Nn -2N0 / 2) x (tn - tn-1)

where:
A = area
N0 = absorbance at t0
N1 = absorbance at t1
Nn = absorbance at tn
t1 = time of first measurement (hours from start)
tn = time of nth measurement (hours from start)

Percentage inhibition of growth at each test concentration (IA) was calculated by comparing the area under the test curve (At) with that under the control curve (Ac) using the following equation:

IA = (Ac - At / Ac) x100

IA values are given in Table 5. The ELR50 value with respect to biomass, EbLR50 (72 and 96 h), was determined by inspection of the area under the growth curve data after 72 and 96 hours.

Comparison of growth rates
The average maximum growth rate (µ) for each culture was also calculated, from the straight section of the growth curve (Attachment 1, Figure 2) using the following equation:

µ = ln Nn -ln N1 / tn - t1

Percentage reductions in growth rate were calculated as previous and the results given in Table 5. The ELR50 value with respect to growth rate, ErLR50 (24 - 48 h), was determined by inspection of the growth rates for the period 24 - 48 hours.


TEST CONCENTRATIONS
- Spacing factor for test concentrations:
No spacing factors for test concentrations

- Justification for using less concentrations than requested by guideline:
Not applicable

- Range finding study
For the purpose of the range-finding study, an amount of test material (4.0 g) was dispersed onto the surface of 2 litres of culture medium to give a loading rate of 2000 mg/L and then stirred for 24 hours prior to the study start; care was taken to avoid vortex formation or gross mixing. Stirring was stopped after 24 hours and the mixture allowed to stand for 1 hour prior to removal of the aqueous phase or Water Accommodated Fraction. An aliquot (100 ml) of this 2000 mg/L loading rate Water Accommodated Fraction was diluted 50:50 with algal suspension to give the required test concentration of 1000 mg/l loading rate Water Accommodated Fraction.
The Water Accommodated Fractions were not prepared by stirring the culture medium to give a vortex of 20-25% of the water column height due to the range-finding study being carried out prior to the request of the sponsor to carry out mixing with the production of a vortex.


- Test concentrations:
The test concentration to be used in the definitive study was determined by a preliminary range-finding study. The range-finding study was conducted by exposing Selenastrum capricornutum cells to a 1000 mg/L loading rate Water Accommodated Fraction for a period of 96 hours.

- Results used to determine the conditions for the definitive study:
Based on the result of the range-finding study a "limit test" was conducted for the definitive study at a test concentration of 1000 mg/L loading rate Water Accommodated Fraction to confirm that at the maximum test concentration defined by the Sponsor, no effect on algal growth was observed.

Reference substance (positive control):

no

Remarks:

No reference substance used

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

other: ELR50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

The results are based on an initial loading rate of 2000 mg/L which was diluted by the addition of the algal suspension to give an equivalent loading rate of 1000 mg/L.

Basis for effect:

growth rate

Remarks on result:

other: CL not determined

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CL not determined

Details on results:

- Exponential growth in the control (for algal test):
yes

- Observation of abnormalities (for algal test):
All test and control cultures were inspected microscopically at 96 hours. There were no abnormalities detected in any of the control or test cultures at 1000 mg/L loading rate Water Accommodated Fraction.

- Any stimulation of growth found in any treatment:
Growth was not affected by the presence of 1000 mg/L loading rate Water Accommodated Fraction of the test material, Alkane 2, over the 96 hour exposure period.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
GC-MS analysis of the test and control samples suggested that the solubility of the many components of the test material in aqueous media was lower than the threshold needed to overcome the inherent background hydrocarbon concentration.
Consequently, the concentration if the test material in the test sample was no higher than the background levels of hydrocarbon based products exhibited in the control samples, even after extensive precautions were undertaken in both the production and subsequent analysis of the WAFs to eradicate the interference.
Therefore, the test concentrations for the Algal Inhibition study could not be verified by GC analysis.

- Effect concentrations exceeding solubility of substance in test medium:
Observations on test material solubility were carried out during the mixing and testing of the Water Accommodated Fraction. At 24 hours prior to the study start and at the start of the mixing period, the test material was observed to be contained within the vortex and floating on the surface of the test medium. However after 20 hours of stirring and 4 hours of standing the test material was observed to be floating on the surface of the test medium only.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

A Students t-test was carried out on the area under the growth curve data at 96 hours for the control and 1000 mg/L loading rate Water Accomodated test concentration to determine any statistically significant differences between the test and control groups.

Statistical analysis of the area under the growth curve data was carried out for the control and 1000 mg/L loading rate Water Accommodated Fraction test group using a Students t-test. There were no statistically significant differences (P>=0.05), between the control and 1000 mg/L loading rate Water Accommodated Fraction test group and therefore the "No Observed Effect Concentration" (NOEC) is given as >= 1000 mg/L loading rate Water Accommodated Fraction.

Any other information on results incl. tables

Table 1 : Absorbance Values from the Range-Finding Study

Nominal Loading Rate (mg/l)		Absorbance Value
		0 hours	96 hours
Control	R1	0.03	1.125
	R2	0.03	1.218
	Mean	0.03	1.172
1000	R1	0.03	1.119
	R2	0.03	1.122
	Mean	0.03	1.121

Table 2 : Mean Cell Density and Absorbance Values of the Control Culture in the Definitive Study

		0h	24h	48h	72h	96h
Control	Mean cell density* (cells/mL)	1.22E+04	6.47E+04	2.13E+05	5.69E+05	1.51E+06
	Mean Absorbance Value	0.024	0.066	0.226	0.614	1.098

* Mean cell density values represent the mean number of cells per ml calculated from the mean of the cell counts from 3 fields of view for each of the replicate flasks.

Table 3: Absorbance and pH Values in the Definitive Study

Nominal Loading Rate (mg/L)		pH	Absorbance values					pH
		0h	0h	24h	48h	72h	96h	96h
Control	R1	7.5	0.024	0.064	0.216	0.612	1.104	9.6
	R2	7.5	0.024	0.066	0.240	0.600	1.090	9.7
	R3	7.5	0.024	0.068	0.223	0.631	1.099	9.9
	Mean		0.024	0.066	0.226	0.614	1.098
	R1	7.5	0.024	0.071	0.184	0.600	1.109	9.7
	R2	7.5	0.024	0.070	0.211	0.601	1.111	9.7
	R3	7.5	0.024	0.062	0.214	0.614	1.114	9.6
	R4	7.5	0.024	0.063	0.230	0.620	1.120	9.7
	R5	7.5	0.024	0.070	0.239	0.605	1.100	9.8
	R6	7.5	0.024	0.071	0.228	0.607	1.094	9.8
	Mean		0.024	0.068	0.218	0.608	1.108

Table 4: Inhibition of Growth

Nominal Loading Rate (mg/L)	Area under curve @72 h	% Inhibition	Area under curve @ 96h	% Inhibition	Growth rate (24-48 h)	% Inhibition
Control	12.936	-	32.924	-	0.051	-
1000	12.72	2	32.72	1	0.049	4

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of Alkane 2 on the growth of Selenastrum capricornutum has been investigated and gave ELR50* values of greater than 1000 mg/L loading rate Water Accommodated Fraction. Correspondingly the No Observed Effect Concentration was greater than or equal to 100 mg/L loading rate Water Accommodated Fraction.

These results are based on an initial loading rate of 2000 mg/L which was diluted by the addition of the algal suspension to give an equivalent loading rate of 1000 mg/L.

* ELR = Effective Loading Rate

Executive summary:

1. Methods

A study was performed to assess the effect of the test material, Alkane 2, on the growth of Selenastrum capricornutum. The method (Safepharm Standard Method 11.ECO) followed that described in the OECD Guidelines for Testing of Chemicals (1984) No. 201, "Algal Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC) and US EPA Code of Federal Regulations, Title 40; Part 797 Section 1050.

2. Procedure

Following a preliminary range-finding study, Selenastrum capricornutum was exposed to a Water Accommodated Fraction of the test material at a loading rate of 1000 mg/L (six replicate flasks) for 96 hours, under constant illumination and shaking at a temperature of 24 deg C.

Samples of the algal populations were removed daily and absorbance values determined for each control and treatment group.

3. Results

Exposure of Selenastrum capricornutum to the test material gave ELR50* values of greater than 1000 mg/L loading rate Water Accommodated Fraction and correspondingly the No Observed Effect Concentration was greater than or equal to 1000 mg/L loading rate Water Accommodated Fraction.

It was considered unnecessary and unrealistic to test at concentration in excess of 1000 mg/L loading rate Water Accommodated Fraction.

The above results are based on an initial loading rate of 2000 mg/L which was diluted by the addition of the algal suspension to give an equivalent loading rate of 1000 mg/L.

Analysis of the Water Accommodated Fraction was carried out by Total Organic Carbon (TOC) analysis on test samples of fresh media at 0 hours and old media at 96 hours.

The results of the TOC analysis showed the concentrations of carbon in the test vessels to be around the limit of detection of the analytical method. Given this and the low water solubility of the test material it is considered that these results do not provide definite evidence of stability of the test preparations.