Hydrocarbons, C18-C24, iso-alkanes <2% aromatics

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date (Date of first study specific data recording):16 August 2016; Experimental end date (Date of last study specific data recording):4 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

See test material information

Analytical monitoring:

yes

Remarks:

Analytical verification conducted to give indication of disappearance of test substance in test soil. Results based on nominal concentrations. Endpoints converted to values based on initial measured and geo. mean measured concs. for info. purposes only.

Details on sampling:

On the following test days, duplicate ca 5g samples were removed from the additional chemistry replicate at each test rate and analysed for test substance concentration:
• Day 0
• Day 1 prior to feeding
• Day 2
• Day 7 prior to feeding
• Day 14 prior to feeding
• Day 28 prior to removal of adults
• Day 28 after removal of adults
• Day 56 after the juveniles had been extracted
On sampling days, any visible food was removed prior to sampling and replaced after sampling. Two samples of 5 g were taken without removing the substrate from the jars, and avoiding the top layer of soil. Extraction solvent was added and samples were kept frozen until processed.

Vehicle:

no

Details on preparation and application of test substrate:

- Method of application to filter paper (if used): n/a
- Method of mixing into soil (if used): As the test substance was not readily soluble in water, it was applied to the test substrate using a sand carrier, and the dose expressed as mg/kg dry soil.
Sand stocks for each treatment group were prepared by adding the appropriate amount of test substance into a glass jar containing a pre-weighed volume of dry sand. The glass jars were then sealed, placed in a cement mixer, and rolled for a total of 65 minutes to homogenise the mixtures.
The test substrate was prepared in bulk, consisting of a total of 2500 g dry weight per test concentration and 5000 g dry weight for the control group. To thoroughly mix the sand stock into the test substrate, a portion of the test substrate was placed in a stainless steel Hobart mechanical mixer, and the test substance, incorporated into sand, was evenly distributed across the surface of the test substrate within the mixer. The remaining substrate was then placed on top of this, without disturbing the sand layer, prior to mixing. Each test concentration was thoroughly mixed for 5 minutes, followed by a thorough hand-turn. At this point, the appropriate amount of reverse osmosis (RO) water required to moisten the substrate to 50% MWHC was added to the mixture. This was followed by an additional 5-minute mechanical mix, another thorough hand turn, and then a final 5-minute mechanical mix. This was deemed sufficient to thoroughly incorporate the test substance into the test substrate. The control vessels were mixed with untreated sand.
The weight of the moisture control vessel was checked for moisture loss on a weekly basis and any losses due to evaporation were corrected by the addition of RO water.
Prior to weighing, the earthworms were rinsed with RO water and gently blotted dry. Worms were weighed individually and any earthworms not falling within the required range of 250 to 600 mg were excluded from the test stock. Batches of ten earthworms were then selected arbitrarily from the acclimatised, synchronised stock and weighed collectively per batch. The batches of worms were allocated randomly to each of the replicate test vessels and transferred onto the substrate surface of the appropriate replicate vessel; the vessels were re-covered with a perforated plastic film to allow gaseous exchange. The additional replicate for chemistry analysis of test soil was initiated in the same way for each test concentration and the controls. Furthermore, a moisture control satellite vessel was set up in the same way as the control test vessels but without the addition of worms.
- Controls: The control vessels were mixed with untreated sand.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Concentration of vehicle in test medium (stock solution and final test solution): n/a
- Evaporation of vehicle before use: n/a

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: Culture stocks maintained at Smithers Viscient (ESG) Limited, Harrogate, North Yorkshire.
- Age at test initiation (mean and range, SD): Earthworms were selected arbitrarily from stock cultures with a relatively homogenous age structure (they did not differ in age by more than 4 weeks). The earthworms were sexually mature (i.e. between 2 and 12 months old with clitellum).
- Weight at test initiation (mean and range, SD): Between 250 and 600 mg (wet mass)

ACCLIMATION
- Acclimation period: Prior to the start of the test, the earthworms were acclimatised for at least 24 hours in basic artificial soil substrate. Earthworms were held in a suitable container, and maintained under test conditions.
The earthworms were fed sterile, ground cow faeces during the acclimatisation period.
- Acclimation conditions (same as test or not): Yes
- Health during acclimation (any mortality observed): None reported

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

19.5 - 19.9 ºC

pH:

5.69 - 6.40

Moisture:

43.3 - 48.84

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Glass test vessel, size not stated.
- Amount of soil or substrate: 500 g dry weight of artificial soil substrate maintained at nominally 50% maximum water holding capacity (MWHC).
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per vehicle control: n/a

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: The artificial soil substrate used in this study was OECD 10% Peat soil, as recommended in OECD Guidelines, and was prepared in-house, at Smithers Viscient (ESG) Limited, Harrogate, North Yorkshire, by mixing a batch using the constituents and proportions detailed below:
• 10% Sphagnum peat. The peat was received 2mm sieved from LBS worldwide Ltd. The wet dry ratio was measured in-house prior to mixing.
• 20% Kaolin clay. The clay was received dry from Potclays Ltd., and had a kaolinite content above 30%.
• 70% Quartz (silica) sand (predominately fine). The sand was received dry, from Minerals Marketing Ltd.
Calcium carbonate was added to adjust the substrate to pH 6.03. The moisture content of the soil was determined prior to testing, and also the maximum water holding capacity (MWHC) was determined.

- Storage (condition, duration): The substrate was mixed and stored in a sealed container prior to use in the test.
- Stability and homogeneity of test material in the medium: Not reported

OTHER TEST CONDITIONS
- Photoperiod: 16:8 light:dark cycle
- Light intensity: 536 - 553 Lux
The moisture content and pH were determined at the start and end of the study.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The monitored endpoints were adult mortality and sub-lethal effects (including abnormal behaviour or appearance compared to the control earthworms and changes in weight) four weeks after the experiment start and the number of surviving juveniles eight weeks after experiment start.

VEHICLE CONTROL PERFORMED: yes/no: n/a

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approximately 2.99
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study: none

Nominal and measured concentrations:

Nominal: Control (0), 25, 74, 222, 667, 2000 mg/kg dry soil.
Initial measured concentrations: 20, 54, 159, 458 and 1253 mg/kg dry soil.
Geometric mean measured concentrations: 21, 36, 97, 264 and 799 mg/kg dry soil.

Reference substance (positive control):

yes

Remarks:

Carbendazim

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

74 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

780 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 2 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

2 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 2 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

weight change

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

2 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

weight change

Details on results:

- Mortality at end of exposure period: None
- Total mass of adults at beginning of test: Not reported
- Changes in body weight of live adults (% of initial weight) at end of exposure period: Mean per treatment:
Control: -16%;
25 mg/kg dry soil concentration: -16%;
74 mg/kg dry soil concentration: -5%;
222 mg/kg dry soil concentration: -7%;
667 mg/kg dry soil concentration: -2%;
2000 mg/kg dry soil concentration: 1%;
- No. of offspring produced: mean per replicate:
Control: 146;
25 mg/kg dry soil concentration: 127;
74 mg/kg dry soil concentration: 122;
222 mg/kg dry soil concentration: 90;
667 mg/kg dry soil concentration: 82;
2000 mg/kg dry soil concentration: 21;
- No. of unhatched cocoons: Not reported
- Morphological abnormalities: None reported
- Behavioural abnormalities: None reported
- Other biological observations: None reported

Results with reference substance (positive control):

- Results with reference substance valid?
- Relevant effect levels: EC50: 2.09 mg/kg dry soil

Reported statistics and error estimates:

Statistical analysis of the adult mortality data and reproduction data, was undertaken using CETIS version 1.8.6.8, based on the nominal test concentrations, initial measured concentrations and geometric mean measured concentrations. The following methods were used;
• The NOEC for adult mortality was determined using a Wilcoxon/Bonferroni Adj Test, and the EC50 was determined using Linear Interpolation (ICPIN).
• The NOEC for number of juveniles was determined using a Bonferroni Adj t Test, and the EC50 was determined using Linear Interpolation (ICPIN).
In deviation to the protocol, and because adult weight change data was similar across all treatment levels, the endpoints for weight change data were determined empirically. In addition to this, because there were no adult mortalities at Day 28, the LC50 for adult mortality was also determined empirically.

Table 1: Results

	Nominal concentration (mg/kg dry soil)
Endpoints	Control (0)	25	74	222	667	2000
28-day Mean Mortality %	0	0	0	0	0	0
Worm Live Weight Change %	-16	-16	-5	-7	-2	1
Mean Number of Juveniles	146	127	122	90	82	21
Difference in Juveniles when Compared to the Control %	n/a	13	17	38	44	86

Validity criteria fulfilled:

yes

Conclusions:

Measured toxicity data are available for Shell GTL Solvent GS310 (Hydrocarbons, C18-C24, n-alkanes, isoalkanes, <2% aromatics) with the soil microorganism Eisenia fetida (Smithers Viscient, 2017). The test was conducted in accordance with OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)), using artificial soil substrate.
As the test substance was not readily soluble in water, it was applied to the test substrate using a sand carrier, and the dose expressed as mg/kg dry soil. Five test substance concentrations of Control (0), 25, 74, 222, 667 and 2000 mg/kg dry soil were tested. Analysis of test concentrations was carried out using gas chromatography with flame ionisation detection (GC-FID). However, due to the complex nature of the test substance, results are reported in terms of the nominal concentrations.
After 28 days, no effects on survival and growth of adult earthworms was observed. For both endpoints, the E(L)C50 and NOEC values are reported as >2000 mg/kg dry soil and 2000 mg/kg dry soil, respectively, based on nominal concentrations. Effects on reproduction were observed. The 56 day NOEC value for reproduction is 74 mg/kg dry soil and the EC50 value for reproduction is 780 mg/kg dry soil, based on nominal concentrations.

Executive summary:

Introduction

A study was performed to assess the potential effects of the test substance on mortality, sub-lethal effects and reproduction of Eisenia fetida. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 222, "Earthworm Reproduction Test".

 

Methods

As the test substance was not readily soluble in water, it was applied to the test substrate using a sand carrier, and the dose expressed as mg/kg dry soil.

A definitive test was undertaken using test substance concentrations of Control (0), 25, 74, 222, 667 and 2000 mg/kg dry soil, in which mature Eisenia fetida were exposed to the test substance for a period of 28 days, after which the adult worms were removed and cocoons allowed to hatch and juveniles to mature for a further 28 days.

Four replicate vessels were prepared for each test concentration and eight replicate vessels were prepared for the control(s), each replicate consisting of one glass test vessel containing ten earthworms.

The monitored endpoints were adult mortality and sub-lethal effects (including abnormal behaviour or appearance compared to the control earthworms and changes in weight) four weeks after the experiment start and the number of surviving juveniles eight weeks after experiment start. From these, EC₅₀ and NOEC values for reproduction were obtained, where possible.

 

Results

Analytical verification of test substance concentrations is not a guideline requirement of OECD Guideline 222. However, this was conducted to give an indication of the disappearance of Shell GTL Solvent GS 310 in test soil. Due to the complex nature of the test substance, all results are based on nominal concentrations only. Endpoints have been converted to values based on initial measured concentrations and geometric mean measured concentrations for information purposes only.

Based on nominal concentrations of the test substance, the results are as follows:

28-day NOEC value for adult E. fetida survival = 2000 mg/kg dry soil

28-day LC₅₀ value for adult E. fetida survival = >2000 mg/kg dry soil

NOEC value based on adult weight change = 2000 mg/kg dry soil

EC₅₀ value based on adult weight change = >2000 mg/kg dry soil

NOEC value based on reproduction = 74 mg/kg dry soil

EC₅₀ value based on reproduction = 780 mg/kg dry soil

The validity criteria were met, and therefore these data are considered to be valid.

Description of key information

A 28-day E(L)C₅₀ value of >2000 mg/kg dry soil and NOEC value of 2000 mg/kg dry soil (nominal concentrations) (highest concentration tested) have been determined for effects of the registered substance on survival and growth of Eisenia foetida. These data are from the Eisenia foetida reproduction study.

A 56-day EC₅₀ value of 780 mg/kg dry soil and NOEC value of 74 mg/kg dry soil (nominal concentrations) have been determined for the effects of the registered substance on reproduction of Eisenia foetida.

Additional information

Measured data are available with the registered substance. An earthworm reproduction test has been carried out with Shell GTL Solvent GS310 (Hydrocarbons, C18-C24, isoalkanes, <2% aromatics) with the soil macroorganism Eisenia foetida (Clarke, 2017). The test was conducted in accordance with OECD Guideline 222 (Earthworm Reproduction Test (Eisenia foetida/Eisenia andrei)), using artificial soil substrate.

As the test substance was not readily soluble in water, it was applied to the test substrate using a sand carrier, and the dose expressed as mg/kg dry soil. Five test substance concentrations of Control (0), 25, 74, 222, 667 and 2000 mg/kg dry soil were tested. Analysis of test concentrations was carried out using gas chromatography with flame ionisation detection (GC-FID). However, due to the complex nature of the test substance, results are reported in terms of the nominal concentrations.

After 28 days, no effects on survival and growth of adult earthworms were observed. For both endpoints, the E(L)C₅₀ and NOEC values are reported as >2000 mg/kg dry soil and 2000 mg/kg dry soil, respectively, based on nominal concentrations. 

Effects on reproduction were observed. According to the study report, the 56-day NOEC value for reproduction is 74 mg/kg dry soil and the EC₅₀ value for reproduction is 780 mg/kg dry soil, based on nominal concentrations.

Supporting studies are read-across from two GTL-derived substances in the relevant carbon number range for Hydrocarbons, C18-C24, isoalkanes, <2% aromatics:

An acute earthworm toxicity study is available for GTL Gasoil (Priestly, 2010b). The test was conducted in accordance with OECD 207 and GLP, however no analytical monitoring of the test substance in the exposure medium was carried out. Earthworms (Eisenia foetida) were exposed to a single concentration of GTL Gasoil at a nominal loading rate of 1000 mg/kg of soil (dry weight) for a period of 14 days. There were no significant mortalities or sub-lethal effects observed during the 14-day exposure period.

The 14-day acute LC₅₀ for earthworms was therefore determined to be greater than 1000 mg/kg dw.

A short-term earthworm toxicity study is available for GTL Base Oil Distillates (Priestly, 2010b). The test was conducted in accordance with OECD 207 and GLP. Earthworms (Eisenia foetida) were exposed to a single concentration of GTL Base Oil Distillates at a nominal loading rate of 1000 mg/kg of soil (dry weight) for a period of 14 days in artificial soil. There were no significant mortalities or sub-lethal effects during the 14-day exposure period. The 14-d acute LC₅₀ for earthworms was therefore determined to be greater than 1000 mg/kg dw.

In addition, an earthworm reproduction study, conducted in accordance with OECD 222 and GLP has also been performed with GTL Gasoil (Schmidt, 2011a). No analytical monitoring was carried out. Adult earthworms were exposed in treated soils for a period of four weeks, at test concentrations of 10, 32, 100, 320, and 1000 mg per kg dry soil. Additionally, a control and a reference item treatment were tested in parallel. After this period, the adults were removed from the test vessels and the survival and growth rate were determined. The cocoons and juvenile earthworms remained in the vessels for additional four weeks. The reproduction rate was determined by counting the number of offspring hatched from the cocoons after this additional test period of four weeks. The NOEC of this study based on survival, growth and reproduction parameters was at least 1000 mg/kg dw. A LOEC could not be established due to the absence of adverse effects at the highest dose level.

A long-term toxicity study with E. foetida has been conducted with Base Oil Distillates (Schmidt, 2011b) in accordance with GLP and OECD 222. Adult test organisms were exposed to 10, 32, 100, 320 and 1000 mg/kg dw soil for 4 weeks and then removed, while the juvenile earthworms remained in the vessels for an additional 4 weeks. Adult mortality and mean change in body weight were recorded, as well as mean reproduction rate of juveniles. Based on these parameters an LC₅₀ and a LOEC could not be determined and a NOEC of at least 1000 mg/kg dw has been determined.