Paraffin waxes (Fischer-Tropsch), isomerization

Toxicity to terrestrial plants

Link to relevant study record(s)

Reference

Endpoint:

toxicity to terrestrial plants: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 22 May 2010 and 29 September 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

Following a preliminary range-finding test, five plant species; three dicotyledonous species, soybean (Glycine max), tomato (Lycopersicon
esulentum) and mustard (Sinapis alba) and two monocotyledonous species, oat (Avena sativa) and perennial ryegrass (Lolium perenne) were
exposed to concentrations of 100, 180, 320, 560 and 1000 mg/kg. The number of seedlings emerged and any mortalities and/or morphological abnormalities were determined daily for 21 or 22 days after 50% emergence in the control for each species.

Definitive test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 100, 180, 320, 560 and
1000 mg/kg using the test species soybean (Glycine max), tomato (Lycoperisicon esculentum), mustard (Sinapis alba), oat (Avena sativa) and
perennial ryegrass (Lolium perenne).

Experimental preparation
For the purpose of the definitive test the test item was added directly to the soil.

In order to reduce the organic carbon content of the soil substrate the supplied soil was mixed in a 50:50 w/w ratio with sand. A total of 17 kg (dry weight) of soil substrate was required for each test concentration. This equated to 17.6 kg (wet weight) of supplied soil therefore 8.8 kg of sand was mixed with 8.8 kg of wet soil to give the final soil substrate.

Amounts of test item (850, 1530, 2720, 4760 and 8500 mg) were each separately added to 2 kg of sand in a 5 litre plastic bottle and mixed in a cement mixer for 24 hours after which 1.5 kg (wet weight) of soil was added to each bottle and mixed using a cement mixer for a further 24 hours. A further 2.4 kg of sand and 2.4 kg (wet weight) of soil was then incorporated into each pre-mix by mixing using a Hobart A200N mixer for approximately 15 minutes to give the 100, 180, 320, 560 and 1000 mg/kg test concentrations respectively. This was prepared in duplicate.
At the request of the Sponsor, four studies were to be conducted simultaneously. Due to the method of preparation used it was not possible to prepare the test concentrations for all the studies at the same time. Therefore, the prepared test concentrations were stored at approximately 4°C prior to sowing.
Prior to sowing, the 100, 180, 320, 560 and 1000 mg/kg test concentrations were further mixed, with water added to dampen the soil, using a Hobart A200N mixer for approximately 15 minutes. Due to the amount of mixing required to conduct the four studies simultaneously, following the final mixing the soil substrate for each test concentration was transferred to pots and maintained at approximately 14°C for approximately 24 hours prior to sowing.

The control was prepared, in duplicate, in an identical manner without the addition of test item.

Vehicle:

no

Details on preparation and application of test substrate:

Not Applicable

Species:

Glycine max (G. soja)

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

The soybean seeds were obtained from Herbiseed, Twyford, UK and were supplied untreated. Upon arrival the seeds were stored under cool, dry conditions.
Details of seed variety, lot number, date received, planting depth and germination percentages are given in Table 1 in section any other
information on materials and methods.

Species:

Lycopersicon esculentum

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

The tomato seeds were obtained from Herbiseed, Twyford, UK and were supplied untreated.
Upon arrival the seeds were stored under cool, dry conditions.
Details of seed variety, lot number, date received, planting depth and germination percentages are given in Table 1 in section any other information on materials and methods.

Species:

Sinapis alba

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

The mustard seeds were obtained from Herbiseed, Twyford, UK and were supplied untreated.
Upon arrival the seeds were stored under cool, dry conditions.
Details of seed variety, lot number, date received, planting depth and germination percentages are given in Table 1 in section any other information on materials and methods.

Species:

Avena sativa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

The oat seeds were obtained from Senova Ltd., Cambridge, UK and were supplied untreated.
Upon arrival the seeds were stored under cool, dry conditions.
Details of seed variety, lot number, date received, planting depth and germination percentages are given in Table 1 in section any other
information on materials and methods.

Species:

Lolium perenne

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

The perennial ryegrass seeds were obtained from Herbiseed, Twyford, UK and were supplied untreated.
Upon arrival the seeds were stored under cool, dry conditions.
Details of seed variety, lot number, date received, planting depth and germination percentages are given in Table 1 in section any other information on materials and methods.

Test type:

other: Terrestrial plant test seedling emergence and seedling

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

22 d

Test temperature:

The temperature was maintained at 7°C to 37°C

pH:

The soil has a pH of 8

Moisture:

Not reported

Details on test conditions:

Exposure System
The exposure vessels consisted of polypropylene pots with a depth of 9.9 cm and a diameter of 13 cm. For each species, four replicates were maintained for the control and each test concentration. Each pot contained approximately 850 g (dry weight) of substrate. After planting, each pot was placed in a plant pot saucer which served as a sub-irrigation reservoir. Initial watering was via the surface to encourage germination. All subsequent watering was by sub-irrigation as required.

The study was conducted in a greenhouse equipped with artificial lighting and heating systems.

Procedure

Range-finding test
In the range-finding test three dicotyledonous species; soybean (Glycine max), tomato (Lycopersicon esculentum) and mustard (Sinapis alba) and
two monocotyledonous species; oat (Avena sativa) and perennial ryegrass (Lolium perenne) were exposed to a series of nominal test concentrations of 100 and 1000 mg/kg.

The test item was incorporated directly into the soil substrate.

In order to reduce the organic carbon content of the soil substrate the supplied soil was mixed in a 50:50 w/w ratio with sand. A total of 8.5 kg (dry weight) of soil substrate was required for each test concentration therefore 4.25 kg (dry weight) of supplied soil was required. This equated to 4.39 kg (wet weight) of supplied soil therefore 4.39 kg of sand was mixed with this to give the final soil substrate.

Amounts of test item (850 and 8500 mg) were each separately added to 2 kg of sand in a 5 litre plastic bottle and mixed in a cement mixer for 24 hours after which 1.55 kg (wet weight) of soil was added to each bottle and mixed using a cement mixer for a further 24 hours. A further 2.39 kg of sand and 2.84 kg (wet weight) of soil was then incorporated into each pre-mix by mixing using a Hobart A200N mixer for approximately 30 minutes to give the 100 and 1000 mg/kg test concentrations respectively.
At the request of the Sponsor, four studies were to be conducted simultaneously. Due to the method of preparation used it was not possible to prepare the test concentrations for all the studies at the same time. Therefore, the prepared test concentrations were stored at approximately 4°C prior to sowing.

Prior to sowing, the 100 and 1000 mg/kg test concentrations were further mixed, with water added to dampen the soil, using a Hobart A200N mixer for approximately 15 minutes.
The control was prepared in an identical manner without the addition of test item.
In the range-finding test, five seeds were placed in each test and control pot for each species. Two replicates were prepared for each concentration. Following sowing, the pots were surface irrigated to encourage germination and then placed in a randomised fashion in the greenhouse. The temperature was maintained at 9 degree C to 40 degree C with a photoperiod of approximately 16 hours light and 8 hours darkness. Some of the temperatures were observed to be outside the range given in the study plan. This deviation was considered not to affect the integrity or the outcome of the study as no morphological abnormalities were observed in the control plants.
After the initial watering the pots were watered as necessary using sub-irrigation. All the pots were monitored daily after sowing for emergence and the date when 50% emergence occurred in the control pots for each species was recorded. For 21 days following 50% emergence in the control pots the number of seedlings emerged and any mortalities and/or morphological abnormalities were determined daily in the test and control pots for each species. On Day 21 the above ground portion of each seedling was harvested and the dry weight determined by placing in pre-weighed containers and drying in an oven at approximately 60°C. For oat and perennial ryegrass the seedlings were harvested on Day 22 of the test due to Day 21 falling on a weekend. In addition, the soybean seedlings were harvested on Day 22 in error. This additional day was considered not to affect the study.
The control groups were maintained under identical conditions but not exposed to the test item. Data from the control groups were shared with similar concurrent studies.

Definitive test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 100, 180, 320, 560 and 1000 mg/kg using the test species soybean (Glycine max), tomato (Lycoperisicon esculentum), mustard (Sinapis alba), oat (Avena sativa) and perennial ryegrass (Lolium perenne).



Exposure conditions
At the start of the test five seeds were placed in each test and control pot at random. Four replicate pots were prepared for the control and each test concentration for each species. Following sowing, the pots were surface irrigated to encourage germination and then placed in a randomised fashion in a greenhouse. The temperature was maintained at 7 degree C to 37 degree C with a photoperiod of approximately 16 hours light and 8 hours darkness. After the initial watering the pots were watered as necessary using sub-irrigation.

The control groups were maintained under identical conditions but not exposed to the test item. Data from the control groups were shared with similar concurrent studies.

All the pots were monitored daily after sowing for emergence and the date when 50% emergence occurred in the control pots for each species was recorded. For 21 days following 50% emergence in the control pots, the number of seedlings emerged and any mortalities and/or morphological abnormalities were determined daily in the test and control pots for each species. On Day 21 the above ground portion of each seedling (except soybean) was harvested and the dry weight determined by placing in pre-weighed containers and drying in an oven at approximately 60°C. The soybean seedlings were harvested on Day 22 of the test due to Day 21 falling on a weekend. This additional day was considered not to affect the study.

Nominal and measured concentrations:

Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 100, 180, 320, 560 and 1000 mg/kg using the test species soybean (Glycine max), tomato (Lycoperisicon esculentum), mustard (Sinapis alba), oat (Avena sativa) and perennial ryegrass (Lolium perenne).

Reference substance (positive control):

no

Species:

Glycine max (G. soja)

Duration:

22 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks:

and growth based on final dry weight

Remarks on result:

other: 95 % CL Not stated

Species:

Glycine max (G. soja)

Duration:

22 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks:

and growth based on final dry weight

Remarks on result:

other: 95 % CL Not stated

Species:

Lycopersicon esculentum

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks:

and growth based on final dry weight

Remarks on result:

other: 95 % CL Not stated

Species:

Lycopersicon esculentum

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks:

and growth based on final dry weight

Remarks on result:

other: 95 % CL Not stated

Species:

Sinapis alba

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Remarks:

and growth based on final dry weight

Remarks on result:

other: 95 % CL Not stated

Species:

Sinapis alba

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks:

and growth based on final dry weight

Remarks on result:

other: 95 % CL Not stated

Species:

Avena sativa

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks:

and growth based on final dry weight

Remarks on result:

other: 95 % CL Not stated

Species:

Avena sativa

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks:

and growth based on final dry weight

Remarks on result:

other: 95 % CL Not stated

Species:

Lolium perenne

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks:

and growth based on final dry weight

Remarks on result:

other: 95 % CL Not stated

Species:

Lolium perenne

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

seedling emergence

Remarks:

and growth based on final dry weight

Remarks on result:

other: 95 % CL Not stated

Details on results:

RESULTS
Range-finding Test
A summary of the percentage emergence and mortality for each species during the range-finding test is given in Table 2 - see in attached section.
No morphological effects were observed throughout the duration of the test.

Statistical analysis using Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the emergence and shoot dry weight data for each species.

There were no significant differences (P<0.05) between the control and all the test groups in terms of emergence for each species.
There were no significant decreases in shoot dry weight (P<0.05) between the control and all the test groups at test termination for oat, perennial ryegrass, tomato and mustard.

A significant decrease in shoot dry weight (P<0.05) was observed between the control and the 100 mg/kg test group at test termination for soybean. A review of the data indicated that this result was probably due to a single seedling which showed a greatly reduced weight compared to the remainder of the seedlings. It was therefore considered that this single value may have skewed the analysis. Given that no significant decrease in shoot dry weight was observed between the control and 1000 mg/kg test concentration, this was considered not to affect the outcome of the study.
For perennial ryegrass and mustard a significant increase in shoot dry weight was observed between the control and the 100 and 1000 mg/kg test concentrations respectively. This was considered not to affect the outcome of the test as the effect observed was not a reduction in weight.
Based on this information, test concentration of 100, 180, 320, 560 and 1000 mg/kg were selected for each species for the definitive test.

Definitive Test
Emergence Data
Soybean (Glycine max), tomato (Lycoperisicon esculentum), mustard (Sinapis alba), oat (Avena sativa) and perennial ryegrass (Lolium perenne) reached 50% emergence in the control on Days 9, 10, 4, 5 and 7 after sowing respectively.
A summary of the percentage emergence for each species are given in Tables 3 to 7 - see in attached section

Statistical analysis using Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the emergence data for each species.

There were no significant differences (P<0.05) between the control and all the test groups in terms of emergence for each species.
Inspection of the emergence data based on the nominal test concentrations gave the following results:

Species EC50 (emergence) (mg/kg)
Soybean >1000
Tomato >1000
Mustard >1000
Oat >1000
Perennial Ryegrass >1000

The No Observed Effect Concentration for each species was 1000 mg/kg. The No Observed Effect Concentration is based upon no significant differences (P<0.05) between the test concentrations and the control.

Morphological Effects
A summary of the percentage mortality, for each species is given in Table 8 - see in attached section

No morphological abnormalities were observed for each species throughout the test.

Shoot Dry Weight Data
Shoot mean dry weight values for each species are given in Tables 9 to 13 - see in attached section.

Individual values for shoot dry weights are given in Appendix 1 - see in attached section

Statistical analysis using Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the shoot dry weight data for each species.
There was no significant decrease in shoot dry weight (P<0.05) between the control and all the test groups in terms of shoot dry weights at test termination for all test species.

A significant increase in shoot dry weight was observed at test concentrations of 180 and 320 mg/kg for perennial ryegrass and 560 and 1000 mg/kg for mustard. Given that the effect observed was not a detrimental effect, this was considered not to affect the outcome of the study.
Inspection of the shoot dry weight data based on the nominal test concentrations gave the following results:

Species EC50 (growth) (mg/kg)
Soybean >1000
Tomato >1000
Mustard >1000
Oat >1000
Perennial Ryegrass >1000

The No Observed Effect Concentration for each species was 1000 mg/kg. The No Observed Effect Concentration is based upon no significant differences (P<0.05) between the test concentrations and the control.

Physico-chemical measurements
The temperature was maintained at 7 degree C to 37 degree C and the relative humidity at 20% to 89% with a photoperiod of approximately 16 hours light and 8 hours darkness.

Some of the temperatures were observed to be outside the range given in the study plan. This deviation was considered not to affect the integrity or the outcome of the study as no morphological abnormalities were observed in the control plants.

CONCLUSION
The EC50 (emergence) and EC50 (growth based on final dry weight) for the test item based on nominal test concentrations for the five species tested were as follows:
Species EC50 (emergence) (mg/kg) No Observed Effect Concentration (mg/kg) EC50
(growth) (mg/kg) No Observed Effect Concentration (mg/kg)
Soybean >1000 1000 >1000 1000
Tomato >1000 1000 >1000 1000
Mustard >1000 1000 >1000 1000
Oat >1000 1000 >1000 1000
Perennial Ryegrass >1000 1000 >1000 1000

Reported statistics and error estimates:

Statistical analysis of the emergence and dry weight data for each species was performed using Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Due to the nature and quantity of the tables, all tables have been attached (see "Attached background material").

Validity criteria fulfilled:

yes

Conclusions:

The EC50 (emergence) and EC50 (growth based on final dry weight) for the test item GTL base oil ['Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'] based on nominal test concentrations for the five species tested were as follows:

Species EC50 (emergence)(mg/kg) NOEC (mg/kg) EC50 (growth) mg/kg) NOEC (mg/kg)

Soybean >1000 1000 >1000 1000
Tomato >1000 1000 >1000 1000
Mustard >1000 1000 >1000 1000
Oat >1000 1000 >1000 1000
Perennial Ryegrass >1000 1000 >1000 1000

Executive summary:

Introduction.

A study was performed to assess the effects of the test item GTL base oil ['Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'] on the emergence and growth of five plant species. The method followed that described in the OECD Guidelines for the Testing of Chemicals: Guideline 208: Terrestrial (Non-target) Plant Test: Seedling Emergence and Seedling Growth Test (July 2006).

 

Methods.

Following a preliminary range-finding test, five plant species; three dicotyledonous species, soybean (Glycine max), tomato (Lycopersicon esulentum) and mustard (Sinapis alba) and two monocotyledonous species, oat (Avena sativa) and perennial ryegrass (Lolium perenne) were exposed to concentrations of 100, 180, 320, 560 and 1000 mg/kg. The number of seedlings emerged and any mortalities and/or morphological abnormalities were determined daily for 21 or 22 days after 50% emergence in the control for each species.

 

Results.

The EC₅₀ (emergence) and EC₅₀ (growth based on final dry weight) for the test item based on nominal test concentrations for the five species tested were as follows:

Species	EC50(emergence) (mg/kg)	No Observed Effect Concentration (mg/kg)	EC50 (growth) (mg/kg)	No Observed Effect Concentration (mg/kg)[1]
Soybean	>1000	1000	>1000	1000
Tomato	>1000	1000	>1000	1000
Mustard	>1000	1000	>1000	1000
Oat	>1000	1000	>1000	1000
Perennial Ryegrass	>1000	1000	>1000	1000

^{[1] No Observed Effect Concentration (growth) based on the concentration where no significant decrease was observed for dry weight compared to the control and no morphological abnormalities were observed}

Description of key information

(21 or 22 d) EC₅₀ of ≥1000 mg/kg soil dw; G. max, L. esulentum, S. alba, A. sativa, L. perenne [OECD 208; GLP; test mat. GTL base oil (CAS 848301-69-9, EC 482-220-0)]

Key value for chemical safety assessment

Short-term EC50 or LC50 for terrestrial plants:

1 000 mg/kg soil dw

Long-term EC10, LC10 or NOEC for terrestrial plants:

1 000 mg/kg soil dw

Additional information

No terrestrial plants growth tests are available for the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' itself.

Five plant species, three dicotyledonous species, soybean (Glyicine max), tomato (Lycopersicon esulentum) and mustard (Sinapis alba) and two monocotyledonous species, oat (Avena sativa) and perennial ryegrass (Lolium perenne) were exposed to the closely related substance GTL Base Oil (covering the carbon range from C18 to C50) at concentrations of 100, 180, 320, 560 and 1000 mg/kg dw in a seedling emergence and growth test. The study was conducted according to OECD 208 and to GLP, however no analytical monitoring was carried out (Goodband, 2011d).

The number of seedlings which emerged, mortalities and morphological abnormalities were recorded daily for 21 or 22 days after 50% emergence in the controls for each species. It was not possible to establish an EC₅₀ with these species and NOEC values have been reported to be at least 1000 mg/kg soil dw for all the organisms tested and across all endpoints.

It is concluded that the results are also applicable for the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' containing higher molecular weight constituents (C25-C150, about 30-55 % >C50) since hydrocarbons >C50 are expected to be less bioavailable (due to the low water solubility and enhanced size).