Paraffin waxes (Fischer-Tropsch), isomerization

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 16 October 2009 and 22 March 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not Applicable

Analytical monitoring:

yes

Details on sampling:

Range-finding test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
In the range-finding test Chironomus riparius larvae were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/kg (dry weight of sediment).

Definitive test
Based on the results of the range-finding test the following concentrations were assigned to the definitive test: 100, 180, 320, 560 and 1000 mg/kg (dry weight of sediment).

Vehicle:

no

Details on sediment and application:

Formulated Sediment
A defined formulated sediment was used with the following composition:
Industrial quartz sand 76% w/w
Kaolinite clay 20% w/w
Sphagnum moss peat 4% w/w

The peat was air dried and homogenised to give a particle size of less than 1 mm. The organic carbon content of the final mixture was 1.9%.


Amounts of test item (5.0, 50 and 500 mg) were each separately added to 380 g of quartz sand in a mixing vessel and mixed in a cement mixer for approximately 24 hours after which 100 g of clay and 20 g of air dried peat were added to each mixing vessel and mixed using a cement mixer for a further approximate 24 hours. An aliquot (196 ml) of deionised reverse osmosis water was added to each prepared sediment and mixed using a Kenwood Chef mixer to give the 10, 100 and 1000 mg/kg test concentrations respectively with nominal moisture content of approximately 40% of dry weight. The pH of each prepared sediment was adjusted to 7.0 ± 0.5 with calcium carbonate. The control was prepared in a similar manner, without the addition of test item.
Each prepared sediment was dispensed to a 600 ml glass beaker to give a 2 cm layer and was then covered with a 8 cm depth of reconstituted water (sediment:water ratio, 1:4). A plastic disc was placed over the sediment and the reconstituted water poured gently onto the surface of the disc in order to avoid disturbance of the sediment. The disc was removed after addition of the water. Two replicates were prepared for the control and each test concentration. This was a deviation to the study plan which stated that the range-finding test would be conducted using single replicates. This was considered not to have affected the validity of the test. The test vessels were then aerated (approximately 1 bubble/second) via narrow bore glass tubes approximately
2 – 3 cm above the sediment layer and the vessels left for seven days prior to addition of the test organisms in order to allow settlement and equilibration of test concentrations between the sediment and water phases.

After the 7-day equilibration period the aeration was stopped and 20 larvae were placed in each replicate test and control vessel and maintained in a temperature controlled room at 21ºC to 23ºC with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. Some of the room temperatures were observed to be slightly in excess of the range given in the study plan of 20 ± 2°C. This deviation was considered not to have affected the outcome or the validity of the test as no adverse effects were observed in the control group throughout the test.

The aeration was switched back on after approximately 24 hours having allowed the larvae to settle in the sediment.

The larvae were fed at a rate of 0.25 to 0.5 mg Tetramin® flake food per larva per day for the first 10 days and 0.5 to 1.0 mg Tetramin® flake food per larva per day thereafter. The Tetramin® flake food was prepared as a suspension and an appropriate volume added to the overlying water.
The measured end-point for the test was the number of live, emerged adult midges. The number of emerged adult midges was recorded daily until termination of the test after 28 days. The sex of the individual midges was also determined after emergence. The male midges were identified by the presence of plumose antennae and the female by the absence of plumose antennae.

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

Data from the control group was shared with similar concurrent studies.

Test organisms (species):

Chironomus riparius

Details on test organisms:

The test was carried out using larvae (2 – 3 days old) of Chironomus riparius derived from in-house laboratory cultures.
Larvae were maintained in glass crystalline dishes with a 10-20 mm layer of fine quartz sand covered by reconstituted water (Elendt M4) in a temperature controlled room at 21ºC to 24ºC. The lighting cycle was controlled to give a 16 hour light and 8 hour darkness cycle with 20 minute dawn and dusk transition periods. The cultures were gently aerated, so as not to disturb the substrate, through narrow bore glass tubes.

The culture vessels were housed in a sealed clear perspex cabinet (breeding box) with cotton sleeves to enable access.

The larvae were fed with Tetramin® flake food at approximately 100 to 500 mg per vessel per day given as a ground powder added to the water surface.
Any gelatinous egg masses produced by breeding adult midges were removed from the culture vessels and transferred to separate vessels and, if required, larvae used to populate new cultures at an initial density of between 100 and 200 larvae.

The diet and diluent were considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study type:

laboratory study

Test type:

not specified

Water media type:

not specified

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Remarks:

The number of emerged adult midges was recorded daily until termination of the test after 28 days.

Hardness:

The water hardness values for the control were determined to be 330 and 350 mg/l as CaCO3 on the Days 0 and 28 respectively, and to range from 184 to 372 and 344 to 364 mg/l as CaCO3 for the test groups on the Days 0 and 28 respectively. The theoretical hardness of the Elendt M4 medium is 250 mg/l as CaCO3. The increase in hardness was considered to be due to the addition of calcium carbonate to the sediment during preparation to adjust the pH to 7.0 ± 0.5.

Test temperature:

Water temperature was maintained at 20ºC to 23ºC throughout the test.

pH:

See Appendix 4 in attached section - Physico-Chemical Measurements

Dissolved oxygen:

See Appendix 4 in attached section - Physico-Chemical Measurements

Salinity:

Not Applicable

Ammonia:

The ammonia concentrations were determined to be 0.443 and 0.060 mg/l as NH4 for the control on the Days 0 and 28 respectively and to range from 0.427 to 0.573 and 0.052 to 1.48 mg/l as NH4 for the test groups on the Days 0 and 28 respectively.

Nominal and measured concentrations:

Based on the results of the range-finding test the following concentrations were assigned to the definitive test: 100, 180, 320, 560 and 1000 mg/kg (dry weight of sediment).

Details on test conditions:

Experimental Preparation
Approximately seven days prior to the start of the test, the test item was prepared by direct addition to the sediment. Homogeneity of mixing trials indicated that extraction of the test item from the prepared ‘wet’ sediment was problematic. Analysis of the prepared ‘dry’ sediment prior to the addition of the water to adjust the moisture content, showed results within 80% to 120% of nominal value. It was considered possible that the addition of water to the prepared sediment may have affected the homogeneity of the prepared sediment. As the prepared ‘dry’ sediment analysis showed near nominal values, the method of preparation was therefore revised in that the prepared ‘dry’ sediment was added directly to the test vessels. In this way it could be assumed that the correct concentration was added to each test vessel. Water was then added to each individual test vessel to adjust the moisture content of the sediment. Analysis could then be performed using the entire sediment in the test vessel without sub-sampling. Analysis of the ‘wet’ sediment prepared in this way also showed low results indicating a possible interaction between the water, calcium carbonate and solvents during extraction.

In order to maintain consistency with the homogeneity trials, and to ensure the correct concentrations were added to each test vessel by sub-sampling the ‘dry’ sediment only, the revised method of preparation was used for the definitive test.

Amounts of test item (100, 180, 320, 560 and 1000 mg) were each separately added to 760 g of quartz sand in a mixing vessel and mixed in a cement mixer for 24 hours after which 200 g of clay and 40 g of air dried peat were added to each vessel and mixed using a cement mixer for a further 24 hours. Each prepared sediment was then further mixed using a Kenwood Chef mixer for approximately 15 minutes to ensure homogeneity. The pH of each prepared sediment was adjusted to 7.0 ± 0.5 with calcium carbonate. The control was prepared in a similar manner, without the addition of test item.

An aliquot (50 g) of the control and each prepared sediment was separately dispensed to 250 ml glass jars. The moisture content of the sediment was adjusted to approximately 40% by adding an aliquot (20 ml) of deionised reverse osmosis water to each jar and carefully mixing to give a 1.5 cm layer of sediment. This was then covered with a 6 cm depth of reconstituted water (sediment:water ratio, 1:4). A plastic disc was placed over the sediment and the reconstituted water poured gently onto the surface of the disc in order to avoid disturbance of the sediment. The disc was removed after addition of the water. The test vessels were then aerated (approximately 1 bubble/second) via narrow bore glass tubes approximately 2 – 3 cm above the sediment layer and the vessels left for seven days prior to addition of the test organisms in order to allow settlement and equilibration of test concentrations between the sediment and water phases.

Four replicates were prepared for each of the control and 100, 180, 320, 560 and 1000 mg/kg test concentrations, plus an additional two replicates of each for sacrificing on Day 10 of the exposure period. A further four replicates were prepared for the control and each test concentration, without the addition of larvae, for sacrificing on Days -7 and 0 for chemical analysis.
Analysis for the concentration of the test item in the ‘dry’ and ‘wet’ sediment was performed on Day -7 (the day of sediment preparation) to confirm correct dosing of the test system (see Appendix 2 - in attached section).
The concentration of the test item in the whole ‘wet’ sediment, interstitial (pore) water and overlying water were verified by chemical analysis on Days 0 and 28 (see Appendix 2 - in attached section).

Analysis was also conducted on the ‘wet’ sediment on Days -7, 0 and 28 after pre-drying the sediment at approximately 60°C (see Appendix 2 - in attached section).

Exposure conditions
In the definitive test 250 ml glass jars were used. After the 7-day equilibration period the aeration was stopped and 20 larvae were placed in each test and control vessel and maintained in a temperature controlled room at 19ºC to 24ºC with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The aeration was switched back on after approximately 24 hours having allowed the larvae to settle in the sediment.

The larvae were fed at a rate of 0.25 to 0.5 Tetramin® flake food per larva per day for the first 10 days and 0.5 to 1.0 mg Tetramin® flake food per larva per day thereafter. The Tetramin® flake food was prepared as a suspension in water and an appropriate volume added to the overlying water.
On Day 10 of the exposure period, two of the extra replicates prepared for the control and each test concentration were sacrificed for the determination of larval survival and weight. The sediment was sieved and live and dead larvae counted. The dry weight of the surviving larvae per test vessel was determined and the mean individual dry weight per vessel calculated.

The measured end-point for the test was the number of live, emerged adult midges. The number of emerged adult midges was recorded daily until termination of the test after 28 days. The sex of the individual midges was also determined after emergence. The male midges were identified by the presence of plumose antennae and the female by the absence of plumose antennae. Any egg masses produced prior to termination were also recorded and removed from the test vessels to prevent re-introduction of larvae into the sediment. The number of visible pupae that failed to emerge were counted separately. Any abnormal behaviour was also recorded.

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

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Remarks on result:

other: Not stated

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

560 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Remarks on result:

other: Not stated

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

development rate

Remarks on result:

other: Not stated

Details on results:

See any other information on results incl. tables section.

RESULTS

Range-findingTest

Emergence data (number of adult midges emerging from the test vessels) from the exposure of Chironomus riparius larvae to the test item during the range finding test are given in Table 1 - in attached section

Inspection of the data indicated that whilst the 10 and 100 mg/kg test concentrations showed similar emergence to the control, fewer midges emerged in the 1000 mg/kg test concentration compared to the control.

Based on this information test concentrations of 100, 180, 320, 560 and 1000 mg/kg were selected for the definitive test.

DefinitiveTest

Day 10 larval survival and growth

The Day 10 larval survival and growth data during the definitive test are given in Tables 2 and 3 - in attached section

Inspection of the data showed no differences in larval survival between the control and each test group. No significant differences (P≥0.05) were observed in larval growth, in terms of mean larval dry weight, between the control and each test group.

Mortality data

Emergence data (number of adult midges emerging from the test vessels) from the exposure of Chironomus riparius larvae to the test item during the definitive test are given in Table 4 - see in attached section.

Inspection of the emergence data showed similar numbers of emerged adult midges in the control and 100, 180, 320 and 560 mg/kg test concentrations. However, fewer adult midges were observed to have emerged from the 1000 mg/kg test concentration compared to the control. Following normalisation against the control the observed effect at 1000 mg/kg was determined to be less than 50% therefore an EC₅₀value could not be calculated.

Inspection of the emergence data gave the following results based on nominal test concentrations of spiked sediment:

Time (days)	EC50(emergence) mg/kg
28	>1000

The No Observed Effect Concentration after 28 days was 560 mg/kg. The No Observed Effect Concentration was based on no significant reduction in emergence.

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

The sediment was sieved at the end of the study and the number of visible pupae that failed to emerge were counted (see Table 5 in attached section).

Emergence ratio data

Statistical analysis of the emergence ratio data, transformed by the square root arcsin function in order to obtain an approximate normal distribution and to equalise the variance, using Dunnett’s multiple comparison procedure (Dunnett 1955) (see Appendix 3 in attached section) showed no significant differences (P³0.05) between the control and 100, 180, 320 and 560 mg/kg test groups. However, significant differences were observed between the control and 1000 mg/kg test group.

Development site

The mean development rate per vessel was calculated for each test concentration (see Table 6 in attached section). The overall mean development rates for the control and each test concentration were calculated and an EC₅₀(development rate) estimated by inspection of the data.

The EC₅₀(development) rate based on nominal concentration was estimated to be greater than 1000 mg/kg.

No observed effect concentration

The No Observed Effect Concentration was 560 mg/kg on the basis that no biologically significant reduction in emergence was observed after 28 days and additionally no sub-lethal effects were observed at 560 mg/kg.

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test item GTL base oil ['Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'] to the sediment-dwelling larvae of Chironomus riparius has been investigated and gave a 28-Day EC50 (emergence) of greater than 1000 mg/kg. The No Observed Effect Concentration was 560 mg/kg. The EC50 (development rate) based on nominal test concentrations was greater than 1000 mg/kg.

Executive summary:

Introduction

A study was performed to assess the toxicity of the test item GTL base oil ['Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'] to the sediment-dwelling larvae of Chironomus riparius. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 218 (April 2004) “Sediment-Water Chironomid Toxicity Test using Spiked Sediment”.

Methods

Following a preliminary range-finding test 80 larvae of Chironomus riparius were exposed in groups of twenty (four replicates of 20 larvae per concentration) to formulated sediment spiked with test item over a range of concentrations of 100, 180, 320, 560 and 1000 mg/kg for a period of 28 days. The numbers of emerged adult midges were recorded daily.

A further 40 larvae (two replicates of 20 larvae) of each test group were prepared and sacrificed on Day 10 of the exposure period to determine the 10-Day larval survival and growth data.

Results

The 28-Day EC₅₀ (reduction in emergence) based on nominal test concentrations was greater than 1000 mg/kg. 

The No Observed Effect Concentration was 560 mg/kg.

The EC₅₀ (development rate) based on nominal test concentrations was greater than 1000 mg/kg.

Analytical work indicated that extraction of the test item from the prepared ‘wet’ sediment was problematic, possibly due to an interaction between the water, calcium carbonate and solvents during extraction. Analysis was therefore also conducted on the ‘dry’ sediment following mixing with the test item but prior to the addition of the water.

Analysis of the ‘dry’ sediment on the day of preparation (Day -7) showed measured concentrations to range from 92% to 111% of nominal with the exception of the 180 mg/kg test concentration which showed a measured concentration of 73% of nominal value. Analysis of the corresponding duplicate sample stored frozen prior to analysis, showed a measured concentration of 90% of nominal value indicating that the initial analysis was erroneous. The results from the ‘dry’ sediment analysis indicated that the test item had been correctly prepared.

Analysis of the ‘wet’ sediment on Days -7, 0 and 28 showed measured concentrations to range from less than the limit of quantitation to 48% of nominal value. Analysis was also conducted on the ‘wet’ sediment after pre-drying the sediment at approximately 60°C. Analysis of these pre-dried samples on Days -7, 0 and 28 showed measured concentrations to range from 63% to 89% of nominal value.

Analysis of the overlying and interstitial ‘pore’ water on Days 0 and 28 showed measured concentrations of less than the limit of quantitation of the analytical method.

Description of key information

- (28d) EC50 and (28d) NOEC for Chironomus riparius (OECD 218): >1000 mg/kg sediment dw (nominal, based on: development rate) and ≥1000 mg/kg sediment dw (nominal, based on: development rate), respectively [test mat. GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0)];

- since hydrocarbons with carbon numbers >C50 are considered too hydrophobic, it is expected that the study results of this read-across substance (covering the entire low molecular weight fraction of the registration substance) are also applicable for the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' containing higher molecular weight components (C25-150, about 30-55 % >C50);

- due to the density of the registration substance (ca. 845 kg/m³), the substance floats on the surface of water and thus exposure to sediment is not anticipated.

Additional information

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

Due to the density of the registration substance (ca. 845 kg/m³), the substance floats on the surface of water and thus exposure to sediment is not anticipated; although, if the substance does come into contact with sediment or particulate matter, it may sorb to it due to high log Koc values of its constituents.

Supporting information of the closely related substances GTL base oil (CAS 848301-69-9, EC 482-220-0)  - consisting of linear and branched alkanes (paraffins) with a small amount of cycloalkanes in the carbon range from C18 to C50 - indicate that (saturated) hydrocarbons in the carbon range C18-C50 are not toxic to sediment-dwelling organisms at high dose levels. In this OECD 218 guideline study with Chironomus riparius, the EC₅₀ was >1000 mg/kg dw and NOEC ≥1000 mg/kg dw for development; the EC₅₀ was >1000 mg/kg dw and NOEC = 560 mg/kg dw for mortality and emergence rate.

Since hydrocarbons with carbon numbers >C50 are considered too hydrophobic, it is expected that the results are also applicable for the registration substance containing higher molecular weight components (C25-150, about 30-55 % >C50).