Paraffin waxes (Fischer-Tropsch), isomerization

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 23 March 2009 and 17 April 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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. Previous experience gained from studies conducted on poorly water soluble test materials has shown that a mixing period of 24 - 48 hours is 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 results of the range-finding test a "Limit test" was conducted at a single loading rate of 100 mg/l to confirm that no immobilisation or
adverse reactions to exposure were observed.

Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control (replicates R1 - R4 pooled) and the 100 mg/l loading rate WAF test group (replicates R1 - R2 and R3 - R4 pooled) at 0 and 48 hours (see Appendix 1 in attached section).

Duplicate samples were taken and stored at approximately -20 degree C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Validation of mixing period
Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances in the WAF. A WAF of nominal loading rate of 100 mg/l was prepared, in duplicate, in deionised reverse osmosis water. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis (see Appendix 2 in attached section).

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

An amount of test material (1100 mg) was added to the surface of 11 litres of reconstituted water to give the 100 mg/l loading rate. After the
addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a
slight 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 and the WAF removed by mid-depth
siphoning (the first approximate 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic observations of the WAF were
performed and showed there to be no micro-dispersions of test material present.

The test material is a complex mixture and hence concentrations in the test preparations were not determined by analysis at the request of the
Sponsor.

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 after a 1-Hour standing period the WAF was observed to have formed a clear colourless water column with test material floating on the surface. Microscopic examination of the WAF showed there to be no micro-dispersions of test material present. After siphoning and for the duration of the test, the 100 mg/l loading rate was observed to be a clear, colourless solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20 degree C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk
transition periods. Each culture was fed daily with a suspension of algae (Chlorella sp.). Culture conditions ensured that reproduction was by
parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.

These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that
would affect the integrity or outcome of the study.


The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

The reconstituted water is defined below:

Reconstituted Water
Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l
i
Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was
approximately air-saturation value.

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test type:

static

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not Applicable

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Temperature was maintained at 21ºC throughout the test - see table of physico measurements in section in any other information for measurements.

pH:

The pH was measured at the start and termination of the test - see table of physico measurements in section in any other information for
measurements.

Dissolved oxygen:

Dissolved oxygen concentrations were recorded at the start and termination of the test - see table of physico measurements in section in any other
information for measurements

Salinity:

Not Applicable

Nominal and measured concentrations:

Based on the results of the range-finding test a "Limit test" was conducted at a single loading rate of 100 mg/l to confirm that no immobilisation or
adverse reactions to exposure were observed.

Details on test conditions:

In the definitive test 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21ºC with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.

The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

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

The test preparations were not renewed during the exposure period. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to
swim for approximately 15 seconds after gentle agitation.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Not stated

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Not stated

Details on results:

RESULTS
Validation of Mixing Period
Pre-study work (see Appendix 2 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
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table 1 -see
section any other information on results .

No immobilisation was observed at the 1.0, 10 and 100 mg/l loading rate WAFs.

Based on this information, a single loading rate of four 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 immobilisation or adverse reactions to exposure were observed.

Definitive Test
Immobilisation data
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2 see sectionany other information on results.

There was no immobilisation in 20 daphnids exposed to a 100 mg/l loading rate WAF for a period of 48 hours. Inspection of the immobilisation
data gave the following results:
Time (h) EL*50 (mg/l) 95% Confidence limits
(mg/l)
24 >100 -
48 >100 -

The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/l loading rate WAF. The No Observed Effect Loading rate is
based upon zero immobilisation at this loading rate.

Evaluation of data
An estimate of the EL*50 values was given by inspection of the immobilisation data.

Results with reference substance (positive control):

Positive Control

The positive control (Harlan Laboratories Ltd Project Number: 0039/1069) was conducted between 09 December 2008 and 11 December 2008.

Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Harlan Laboratories Ltd Project No: 0039/1069) during the positive control are given in Table 3 - see section any other information on results.

The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 1 and 2 - see attachement Appendix-3)

Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the nominal test concentrations gave the following results:

Time (h)
EC50 (mg/l) 95% Confidence limits
(mg/l)
3 > 3.2 -
24 0.82 0.71 - 0.94
48 0.71 0.61 - 0.81

The No Observed Effect Concentration after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero
immobilisation at this concentration.

The slopes and their standard errors of the response curves at 24 and 48 hours were 8.1 (SE = 1.7) and 8.6 (SE = 1.8) respectively.

The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/l (sd = 0.21).

Table1: Cumulative Immobilisation Data in the Range-finding Test

Nominal Loading Rate (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
1.0	0	0
10	0	0
100	0	0

Table 2: Cumulative Immobilisation Data in the DefinitiveTest

Nominal Loading Rate (mg/l)		Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)
		24 Hours			48 Hours
		No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control	R1	0	0	0	0	0	0
Control	R2	0			0
Control	R3	0			0
Control	R4	0			0
100	R1	0	0	0	0	0	0
100	R2	0			0
100	R3	0			0
100	R4	0			0

R₁– R₄= Replicates 1 to 4

Table 3: Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	3 Hours				24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0	0	0	0	0
0.56	0	0	0	0	1	1	2	10	2	2	4	20
1.0	0	0	0	0	7	8	15	75	9	9	18	90
1.8	0	0	0	0	10	10	20	100	10	10	20	100
3.2	0	0	0	0	10	10	20	100	10	10	20	100

R₁– R₂= Replicates 1 and 2

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 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 acute toxicity of the test material ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’ to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/l for 48 hours at a temperature of 21ºC under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.

The 48-Hour EL^[1]₅₀ for the test material to Daphnia magna based on nominal loading rates was 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. The test material is a complex mixture and hence compound specific analysis of the WAFs was not performed. However samples of the control and 100 mg/l loading rate WAF were taken at 0 (fresh media) and 48 hours (old media) for Total Organic Carbon (TOC) analysis. Given 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 organic carbon concentrations were below the limit of quantitation at 0 and 48 hours, the results were based on nominal loading rates only.

The 48-Hour EC₅₀ for the reference material to Daphnia magna based on nominal concentrations was 0.71 mg/l with 95% confidence limits of 0.61 – 0.81 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

^{[1] EL = Effective Loading rate}

Description of key information

- (48h) EL50 for Daphnia: >100 mg/l  [OECD 202; test mat. GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0); (WAFs) (nominal) based on: mobility];

- as hydrocarbons with carbon numbers > C50 are considered too hydrophobic and immobile to cause significant aquatic toxicity, the study results of this read-across substance (covering the entire low molecular weight fraction of the registration substance) are expected to represent the maximum achievable toxicity of the registration substance containing higher molecular weight constituents (C25-150, about 30-55 % >C50);

- acute toxicity of the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' to aquatic invertebrates is considered to be no effects at the water solubility limit (saturation). 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

Measured acute (short-term) toxicity data are not available for the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization'.

However, reliable data of the closely related substance GTL base oil (CAS 848301-69-9, EC 482-220-0) can be used to address the endpoint, which is entirely appropriate to draw conclusions on the short-term toxicity of the registration substance to aquatic invertebrates.
The test item GTL base oil - consisting of linear and branched alkanes (paraffins) with a small amount of cycloalkanes in the carbon range from C18 to C50 - covers the complete low molecular weight fraction of the registration substance. Since hydrocarbons with carbon numbers > C50 are considered too hydrophobic and immobile to cause significant aquatic toxicity, the study results of this read-across substance are expected to represent the maximum achievable toxicity of the registration substance containing higher molecular weight constituents (C25-150, about 30-55 % >C50).

 

The short-term toxicity of the closely related substance GTL base oil to freshwater invertebrate Daphnia magna (water flea) has been determined in a GLP study conducted in accordance with OECD Test Guideline 202 (Priestly, 2009). 

WAFs of the sample were prepared by stirring for approximately 23 hours and left to stand for 1 hour before drawing off the aqueous phase – the WAF – for testing. Static exposures were then carried out according to GLP.

Total Organic Carbon (TOC) analysis of the freshly prepared test preparations showed amount of carbon present within the control and the 100 mg/l loading rate WAF test vessels to be less than the limit of quantitation (LOQ) for the method within fresh and old media. Therefore the results from the TOC analysis did not provide definitive evidence of the stability of the test preparations.

The test results, expressed as the EL50value, showed that the sample was not hazardous to Daphnia magna at a loading rate of 100 mg/l.

 

Based on these results,

• the (48h) EL50 (freshwater invertebrates) of the registration substance is considered to be >100 mg/l and


• the acute toxicity to aquatic invertebrates is considered to be no effects at the water solubility limit (saturation).