Paraffin waxes (Fischer-Tropsch), isomerization

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 14 October 2009 and 18 November 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US Code of Federal Regulations, Title 40, Part 797, Section 1600

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Deviations:

no

Principles of method if other than guideline:

Information provided by the Sponsor gave an EL/LL50 of greater than 100 mg/l. Therefore, as toxicity was not expected, the number of test concentrations was reduced from five, as recommended in the Test Guideline, to three (10, 32 and 100 mg/l loading rate WAF). This was considered not to affect the validity of the study and reducing the number of test concentrations also reduced the number of fish used which was consistent with the Sponsors Animal Welfare policy.

Furthermore 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 and OECD 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

Vehicle:

no

Test organisms (species):

Pimephales promelas

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Duration:

33 d

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: survival and growth

Remarks on result:

other: 95 CL not reported

Duration:

33 d

Dose descriptor:

other: Lowest Observed Effect Loading Rate” (LOEL)

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: There were no significant reductions (P ≥0.05) in terms of fish length and dry weight when compared to the control at the end of the test.

Remarks on result:

other: 95% CL not reported

Duration:

33 d

Dose descriptor:

other: No Observed Effect Loading Rate (NOEL)

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: There were no significant reductions (P≥0.05) in terms of fish length and dry weight when compared to the control at the end of the test.

Remarks on result:

other: 95 % CL not reported.

RESULTS

Validation of Mixing Period

Pre-study investigational work (see attached Appendix 2) 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 the test was conducted using a 23-Hour stirring period followed by a 1-Hour settlement period.

Definitive Test

Observations

The number of dead eggs observed during the definitive test are given in the attached Table 1. 

The number of dead larvae and hatched (live) larvae observed during the definitive test are given in

the attached Tables 2 and 3 respectively.

The number of hatched larvae (non-cumulative) per day are given in the attached Table 4.

The number of dead eggs and larvae were observed to be low throughout the duration of the test with no concentration dependent effects being observed. The mean hatching success rate for the control group was 92% thereby satisfying the validation criterion of greater than 75% hatching rate. The mean hatching rate for the test loading rates was 87% to 90%.

The mean survival rate of the larvae for the control group was 95% thereby satisfying the validation criterion for post-hatch survival success rate of greater than 70%. The mean survival rates for the test loading rates ranged between 81% and 98% (see attached Table 5).

The start of egg hatching was observed to be on Day 4 of the test and completion of hatching was observed on Day 5 of the test.

There were no significant mortalities or sub-lethal effects of exposure observed in any of the test concentrations.

Sub-lethal Effects

There were no sub-lethal effects observed in the test.

Length and dry weight data.

Statistical analysis of the length data by analysis of variance (see attached Appendix 4) showed the 10, 32 and 100 mg/l loading rate WAFs not to be significantly different (P≥0.05) from the control group. 

Statistical analysis of the dry weight data showed no significant differences (P≥0.05) between the control group and 32 and 100 mg/l loading rate WAF test groups. However, the 10 mg/l loading rate WAF test group was significantly different (P<0.05) from the control group.

The observed significant differences between the control group and the 10 mg/l loading rate WAF test group were considered to be due to the presence of a small number of larger fish in the 10 mg/l loading rate WAF test group. This was considered to be due to the normal variation observed in a natural population of organisms. The larval dry weight of the 10 mg/l loading rate WAF test group was not significantly reduced compared to the control group therefore this weight difference was considered not to affect the outcome of the study.

Given this information and data assessment above it was considered that no effect on survival or growth attributable to the test material was observed. 

Lowest observed effect loading rate.

The “Lowest Observed Effect Loading Rate” (LOEL), based on nominal loading rates, was considered to be greater than 100 mg/l loading rate WAF on the basis that there were no significant reductions (P≥0.05) in terms of fish length and dry weight when compared to the control at the end of the test.

No observed effect loading rate.

The “No Observed Effect Loading Rate” (NOEL), based on nominal loading rates, was considered to be 100 mg/l loading rate WAF on the basis that there were no significant reductions (P≥0.05) in terms of fish length and dry weight when compared to the control at the end of the test.

Vortex depth measurements.

The vortex depth was recorded at the start and end of each mixing period and was observed to be a dimple at the water surface on each occasion (see attached Table 6).

Observations on test material solubility.

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

At the start and end of each mixing period, and after the 1-Hour settlement period the 10, 32 and 100 mg/l loading rates were observed to be clear, colourless water columns with an oily slick of test material at the surface. After siphoning and for the duration of the test, the 10, 32 and 100 mg/l loading rates were observed to be clear, colourless solutions. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present.

Physico-chemical measurements.

The results of the physico-chemical measurements are given in the attached Appendix 6. Temperature was maintained at 25ºC ± 2°C throughout the test, while there were no treatment related differences for oxygen concentration or pH. The temperature measurements recorded in Control Replicate R₁throughout the test by a Testo temperature logger are presented in the attached Figure 1.

The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.

The oxygen concentration for Day 12 old media control R₁, R₂and 10 mg/l LR WAF R₁were measured to be below the 60% Air Saturation Value given in the Protocol. However given that no adverse effects were observed throughout the test it was considered that this deviation had no impact on the outcome or validity of the test.

The water hardness values were observed to range from 138 to 142 mg/l as CaCO₃ at the start of the test and from 150 to 156 mg/l as CaCO₃ at termination of the test (see attached Appendix 7).

Total organic carbon analysis.

Total Organic Carbon (TOC) analysis (see attached Appendix 3) of the test preparations showed no significant differences in the amount of carbon present within the 10, 32 and 100 mg/l loading rate WAF test vessels when compared to the control vessels. This was expected due to the low aqueous solubility of the test material. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that the majority of the results were around the limit of quantitation of the analytical method.

The dissolved test material may have been one or several components of the test material. Given that toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole, the results were based on nominal loading rates only.

Validity criteria fulfilled:

yes

Conclusions:

The application of the test material to newly laid eggs of fathead minnows was considered to have no effect on the survival or growth of the larvae.
The No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

Executive summary:

Introduction.

A study was performed to assess the effects of the test material ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’ on freshly hatched larvae of the fathead minnow (Pimephales promelas). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 210, "Fish, Early-Life Stage Toxicity Test”, US Code of Federal Regulations, Title 40, Part 797, Section 1600 and the US EPA Draft Ecological Effects Test Guideline OPPTS 850.1400.

Methods.

Based on data supplied by the Sponsor, newly laid eggs were exposed toWater Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 10, 32 and 100 mg/lfor a period of 33 days at a temperature of 25 ºC ± 2°C under semi-static test conditions. 

The number of mortalities or any sub-lethal effects of exposure in each test and control vessel were recorded daily until termination of the test (28 days post-hatch). At test termination the length and dry weight of the surviving fish were measured.

Results. 

Over the duration of the test there were no significant mortalities or sub-lethal effects of exposure resulting from the exposure of fathead minnow (Pimephales promelas) larvae to nominal loading rates of 10, 32 and 100 mg/l loading rate WAF.

The mean hatching rate ranged from 87% to 92% and the mean survival rate ranged from 81% to 98%. The fish length and dry weight data obtained at termination of the test are summarised as follows:

	Nominal Loading Rate (mg/l)
	Control	10	32	100
Body Length (mean ± standard deviation, mm)	15.05 ± 1.16	15.37 ± 1.75	15.41 ± 1.06	15.34 ± 0.99
Dry Weight (mean ± standard deviation, mg)	11.7 ± 2.8	13.6 ± 4.7	12.4 ± 2.8	12.2 ± 2.8

Statistical analysis of these data showed there were no significant reductions (P≥0.05) between the control and all the test groups in terms of fish length or dry weight.

Conclusion.

Given the above results and information it was considered that the test material had no effect on the survival or growth of newly laid eggs of fathead minnows.

The “Lowest Observed Effect Loading Rate” (LOEL), based on nominal loading rates, was considered to be greater than 100 mg/l loading rate WAF on the basis that there were no significant reductions (P≥0.05) in terms of fish length and dry weight when compared to the control at the end of the test.

The “No Observed Effect Loading Rate” (NOEL), based on nominal loading rates, was considered to be 100 mg/l loading rate WAF on the basis that there were no significant reductions (P≥0.05) in terms of fish length and dry weight when compared to the control at the end of the test.

Total Organic Carbon (TOC) analysis of the test preparations showed no significant differences in the amount of carbon present within the 10, 32 and 100 mg/l loading rate WAF test vessels when compared to the control vessels. This was expected due to the low aqueous solubility of the test material. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that the majority of the results were around the limit of quantitation of the analytical method.

The dissolved test material may have been one or several components of the test material. Given that toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole, the results were based on nominal loading rates only.

Description of key information

- NOELR (33 d) for freshwater fish (Pimephales promelas): ≥100 mg/l [OECD 210; test mat. GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0); (WAFs) (nominal) based on: survival and reproduction];

- 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);

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

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

Measured long-term toxicity data are not available for the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization'.
However, for the endpoint, reliable data of the closely related substance GTL base oil (CAS 848301-69-9, EC 482-220-0) can be used, which is entirely appropriate to draw conclusions on the long-term toxicity of the registration substance to fish.
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).

 

A Fish Early Life Stage study has been conducted with GTL base oil using the freshwater species Pimephales promelas (fathead minnow) (Priestly, 2010a). The test was conducted in accordance with OECD 201 and was compliant with GLP. TOC content was analysed in the test media for stability and exposure concentration.

Newly laid eggs were exposed to water accommodated fractions (WAFs) of the test material over a range of nominal loading rates of 10, 32 and 100 mg/l for a period of 33 days at a temperature of 25°C ± 2°C under semi-static test conditions. 

There were no significant mortalities or sub-lethal effects at any loading rate. The mean hatching rate ranged from 87 to 92% and the mean survival rate ranged from 81 to 98%. There were no statistically significant reductions in mean fish length or dry weight between the control group and the 32 and 100 mg/l WAFs loading rate. However the observations at the 10 mg/l loading rate WAF were significantly different from the control. This was considered by the authors to be due to normal biological variation. The report concluded that there were no effects on survival or growth of newly laid eggs of fathead minnows when exposure to loading rates up to 100 mg/l GTL Distillates. The No Observed Effect Loading Rate (NOELR) was therefore determined to be at least 100 mg/l.

Total Organic Carbon (TOC) analysis of the freshly prepared test preparations showed amount of carbon present within the 10, 32 and 100 mg/l loading rate WAF test vessels to range from less than the limit of quantitation (LOQ) for the method (1.0 mg C/l) to 11.64 mg C/l. TOC analysis of the old test media showed amount of carbon present to range from less than the LOQ to 2.75 mg C/l There was no significant difference in TOC content between control and exposure vessels. This was expected due to the low water solubility of the test substance. 

 

Based on these results,

• the NOELR (freshwater fish) of the registration substance is considered to be >100 mg/l and


• the long-term toxicity of the registration substance to fish is considered to be no effects at the water solubility limit (saturation).