Hydrocarbons, C15-C19, n-alkanes, isoalkanes,

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:

25 April 2016 – 20 June 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

Details on sampling:

- Concentrations: All test concentrations.
- Sampling method: At Day 0, an additional chemistry replicate was initiated for each test rate. This vessel was treated in the exact same way as the test replicates, with addition of adults at Day 0, removal of adults on Day 28, and removal of juveniles on Day 56, in addition to weekly feeding/moisture adjustment as necessary.
On day 0, 1 prior to feeding, 2, 7 prior to feeding, 14 prior to feeding, 28 prior to removal of adults, 28 after removal of adults, and day 56 after the juveniles had been extracted, duplicate ca 5g samples were removed from the additional chemistry replicate at each test rate and analysed for test substance concentration.
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.
- Sample storage conditions before analysis:
Extraction solvent was immediately added and samples were kept frozen until processed.

Vehicle:

no

Details on preparation and application of test substrate:

A definitive test was undertaken using test substance concentrations of Control (0), 25, 74, 222, 667 and 2000 mg/kg dry soil.
Four replicate vessels were prepared for each test rate and eight replicate vessels were prepared for the control(s).
For the purpose of analysis of test soil, an additional replicate was initiated for each test rate.

Test Substrate
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. The substrate was mixed and stored in a sealed container prior to use in the test.
- 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 5.93. The moisture content of the soil was determined prior to testing, and also the maximum water holding capacity (MWHC) was determined following procedures described in Annex 2 of OECD Guideline 222.

- Method of mixing into soil (if used): Each replicate consisted of one glass test vessel containing ten earthworms in the equivalent of approximately 500 g dry weight of artificial soil substrate maintained at
nominally 50% maximum water holding capacity (MWHC).
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 70 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. 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.

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, and originated from Bias Labs Ltd, Fife.
- Age at test initiation (mean and range, SD): 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:

19.4 to 20.6 ºC

pH:

5.48 to 6.26

Moisture:

29.5 to 33.1%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Glass vessel. Size not reported.
- Amount of soil or substrate: 500 g dry weight
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
For the purpose of analysis of test soil, an additional replicate was initiated for each test rate.
- No. of replicates per control: 8
- No. of replicates per vehicle control: N/a

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate):
- 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 5.93.
- Organic carbon (%): Not reported
- Maximum water holding capacity (in % dry weight): determined but not reported
- CEC: Not reported
- Pretreatment of soil: None reported
- Storage (condition, duration): Not reported
- Stability and homogeneity of test material in the medium: Degradation of test substance expected. Test material mixed well to ensure homogeneity.

OTHER TEST CONDITIONS
- Photoperiod: 16:8 light:dark cycle
- Light intensity: 536 to 698 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The light intensity was measured at the start and at the end of the study.
Test vessels were maintained at 20 ± 2°C. Temperature was recorded throughout the test using a min/max thermometer.
The moisture content and pH were determined at the start and end of the study.

At Day 28, the substrate was spread out onto disposable plastic film and examined. Earthworms were classified as dead if they did not respond to gentle mechanical stimulus to the anterior end. Due to rapid decomposition under test conditions, missing earthworms were also considered dead. Any observed behavioural (e.g. inability to burrow into the soil), morphological (e.g. open wounds), or pathological symptoms were also recorded at this time.
Following observations on day 28 the test substrate was carefully replaced in the test vessels and the adult worms were discarded.
On Day 56, the vessels were placed in a water bath set to between 40–50ºC, leaving approximately the top 2 cm of substrate above the water level. The vessels were then left for approximately 20 to 30 minutes to allow the juvenile worms to move to the surface of the substrate and away from the heat.
The juvenile worms were then removed from the surface of the substrate and counted; once all visible worms were removed, the soil was spread out over a tray covered with a disposable plastic film. Any juvenile worms remaining in the substrate were removed and counted, to determine the total number of juvenile worms per vessel.

VEHICLE CONTROL PERFORMED: N/a

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approximately 3
- Justification for using less concentrations than requested by guideline: N/a
- Range finding study
No rangefinding study reported
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Nominal and measured concentrations:

Nominal: Control (0), 25, 74, 222, 667 and 2000 mg/kg dry soil.
Initial Measured: 23, 61, 133, 429 and 1337 mg/kg dry soil.
Geometric mean measured: 21, 47, 81, 224 and 779 mg/kg dry soil.
Due to the complex nature of the test substance, all endpoints are presented as nominal concentrations of test substance. These values have been converted to initial measured concentrations and geometric mean measured concentrations for information purposes only.

Reference substance (positive control):

yes

Remarks:

Carbendazim

Key result

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

777 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Confidence limit 323-1155

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

222 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

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

Key result

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

Key result

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:

other: adult weight change

Key result

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:

other: adult weight change

Details on results:

- Mortality at end of exposure period: 0
- Total mass of adults at beginning of test: See table
- Changes in body weight of live adults (% of initial weight) at end of exposure period: See table
- No. of offspring produced: See table
- 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? Yes
- Relevant effect levels: EC50 2.09 mg/kg dry soil
- Other:

Reported statistics and error estimates:

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
- 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: Mean Treatment Mortality for Adult E. fetida

	Mortality	Mortality	Mortality
Nominal Concentration (mg/kg dry soil)	Number of E. fetida Exposed	Day 28 Number of Mortalities	Total (%)
Control (0)	80	0	0
25	40	0	0
74	40	0	0
222	40	0	0
667	40	0	0
2000	40	0	0

Table: Mean Live Weight of Individual E. fetida per Treatment and Percentage Weight Change from Day 0 to Day 28

	Live weight (mg)
Nominal Concentration (mg/kg dry soil)	Day 0	Day 28	Change (%)
Control (0)	297	370	25
25	320	381	19
74	304	365	20
222	311	384	24
667	292	386	32
2000	310	359	16

Table: Mean Number of Juvenile Worms per replicate at Day 56

Nominal Concentration (mg/kg)	Mean Number of Juveniles	% Difference Compared to the Control
0	57	n/a
25	40	29
74	48	16
222	44	23
667	31	46
2000	13	77

Coefficient of Variance for controls = 14.56%

Table: Results per endpoint

	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 %	25	19	20	24	32	16
Mean Number of Juveniles	57	40	48	44	31	13
Difference in Juveniles when Compared to the Control %	n/a	29	16	23	46	77

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on survival, growth and reproduction of earthworms (Eisenia fetida) has been investigated. The results are as follows:
28-day NOEC Adult Survival 2000 mg/kg dry soil
28-day LC50 Adult Survival >2000 mg/kg dry soil
28-day NOEC Adult Weight Change 2000 mg/kg dry soil
28-day EC50 Adult Weight Change >2000 mg/kg dry soil
56-day NOEC Reproduction 222 mg/kg dry soil
56-day EC50 Reproduction 777 mg/kg dry soil

Executive summary:

Introduction

A study was performed to assess the chronic toxicity of the test item to 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 the disappearance of the test substance in test soil. A DT₅₀ for the disappearance of the test substance was calculated to be 22.5 days.

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 = 222 mg/kg dry soil

EC₅₀ value based on reproduction = 777 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 test substance on survival and growth of Eisenia fetida.

A 56-day EC₅₀ value of 777 mg/kg dry soil and NOEC value of 222 mg/kg dry soil (nominal concentrations) have been determined for the effects of the test substance on reproduction of Eisenia fetida.

Additional information

Measured toxicity data are available for Shell GTL Solvent GS270 (Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics) with the soil macroorganism Eisenia fetida (Shell, 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).

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 222 mg/kg dry soil and the EC₅₀ value for reproduction is 777 mg/kg dry soil, based on nominal concentrations.