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

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to terrestrial arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 – 21 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 213 (Honeybees, Acute Oral Toxicity Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 214 (Honeybees, Acute Contact Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Application method:

other: oral and contact in two separate tests

Specific details on test material used for the study:

See test material information

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Tween 80

Details on preparation and application of test substrate:

PREPARATION AND APPLICATION OF TEST MATERIAL
- Method:
Procedures for the Oral Application Bioassay
Oral dosing was conducted by providing each vessel of ten honeybees with 200 μL of test dose into the feeder. It is assumed that due to the honeybees’ trophallaxis (behaviour of sharing food) that each honeybee consumes 20 μL of dose since the entire dose was consumed.
Due to the insolubility of Shell GTL Solvent GS 270 in RO water, a dispersing agent, Tween-80, was used to absorb the test substance; this was then topped up using 50% (w/v) sucrose solution and placed in an ultrasonic bath for approximately 10 minutes until a homogenous dispersion was reached. To show that the Tween-80 was not toxic to the honeybees, a solvent control test rate was set-up within the test using the same amount of the dispersing agent. See Table 1 in "Any other information on materials and methods including tables" for dosing preparation.
Approximately 1 hour prior to administration of the oral dose, the sucrose feed was removed in order to starve the honeybees. The 200 μL oral dose was placed into the feeding reservoir present within the test vessel. Control honeybees were provided with 50% sucrose solution only. All vessels were weighed prior to filling and when filled. As the entire dose was consumed this data is not reported.
Oral doses were observed at hourly intervals until the honeybees had consumed the entire dose. After complete consumption, the feeding tubes were replenished with untreated 50% sucrose solution for the remainder of the study. All orally dosed test vessels consumed the full dose after 4 hours from dose addition time.

Procedures for the Contact Application Bioassay
Contact test solutions were prepared in RO water at concentrations giving the required test dose in 1 μL of solution.
As with the oral dose, due to the insolubility of Shell GTL Solvent GS 270 in RO water, the dispersing agent, Tween-80, was used to absorb the test substance; this was then topped up using RO water and placed in an ultrasonic bath for approximately 10 minutes until a homogenous dispersion was reached. A solvent control test rate was also set-up within the test using the same amount of the dispersing agent. See Table 2 in "Any other information on materials and methods including tables" for dosing preparation.
To prepare the honeybees for treatment, they were re-anaesthetised in the test vessels using CO2 gas. Aliquots (1 μL) of each appropriate test solution were placed on the dorsal thorax of each bee using a calibrated pipette. The control treatments were dosed with 1 μL RO water/bee, the solvent control were dosed with 1 μL RO water/Tween-80 mixture/bee. All honeybees were provided with access to a feeder containing 50% sucrose solution only.

- Spray deposition (if applicable): n/a
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Tween-80
- Concentration of vehicle in test medium (stock solution and final test solution): See Table 1 and 2 in "Any other information on materials and methods including tables".
- Volume of test solution applied: See Table 1 and 2 in "Any other information on materials and methods including tables".

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: Honeybee
- Source: Collected by apiarist, details of whom are maintained in the study raw data.
- Taxonomic confirmation [yes / no]: Not reported
- By whom? Not reported
- Age at test initiation (mean and range, SD): Not reported
- Stage at test initiation: Not reported
- Weight at test initiation (mean and range, SD): Not reported
- Date of collection: 19 October 2015
- Cultural background (if honeybees): Healthy active worker honeybees, that showed no discernible signs of, or were treated for, Nosema infection or infestation with Varroa mites, were collected without the use of smoke from a queenright hive on the day of test initiation. All honeybees for each part of the testing were obtained from the same supplier hive.
- Disease free: Yes
- Kept according to standard practices: yes

ACCLIMATION
- Acclimation period: none
- Acclimation conditions (same as test or not): n/a
- Feeding: n/a
- Health during acclimation (any mortality observed): n/a

RATIONALE FOR SELECTION OF SPECIES (if other than requested by test guideline): n/a

Study type:

laboratory study

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

23.5 to 24.8

pH (if soil or dung study):

n/a

Humidity:

50 to 65

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 90 mm petri dishes with a smaller 35 mm petri dish inside the larger dish.
- Amount of soil or dung or substrate: A layer of absorbent paper (e.g. filter paper) was placed in the base of the vessel
- Depth of dung (cm): n/a
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 3
- No. of replicates per control: 2
- No. of replicates per vehicle control: 2

SOURCE AND PROPERTIES OF SUBSTRATE (if soil or dung)
n/a

OTHER TEST CONDITIONS
- Photoperiod: not reported
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
All vessels of honeybees were examined at approximately 4, 24 and 48 hours after treatment.
Details of all toxic effects were recorded. Lethal and sub-lethal effects were documented as follows:
 Category U (unaffected): Honeybees act normally compared with controls
 Category A (slight effect): Honeybees may be hyperactive compared with controls or may appear disorientated and movement may be slower than controls
 Category B (moderate effect): Honeybees exhibit partial paralysis and poor co-ordination of movement. Honeybees may find it difficult to right themselves if they fall on their backs. Honeybees thus affected may get worse (Category C) or may recover (particularly with pyrethroid insecticides)
 Category C (severe effect): Honeybees exhibit almost complete paralysis. Honeybees appear moribund (almost dead), cannot walk and show only very feeble movements of legs and antennae. Honeybees may revert to Category B, but often die
 Category D (dead): No response at all to stimulation when touched or blown upon, legs often curl inwards

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study
- Test concentrations: An oral and contact range-finder limit test was conducted at control (0), solvent control, 0.01, 0.1, 1.0, 10 and 100 μg product/bee.
- Results used to determine the conditions for the definitive study: Only range-finder limit test was conducted

Nominal and measured concentrations:

Nominal: control (0), solvent control, 0.01, 0.1, 1.0, 10 and 100 μg product/bee

Reference substance (positive control):

yes

Remarks:

Dimethoate 400 g/L EC

Key result

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

> 100 other: μg product/bee

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: oral test

Key result

Duration:

48 h

Dose descriptor:

other: NOEL

Effect conc.:

100 other: μg product/bee

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: oral test

Key result

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

> 100 other: μg product/bee

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: contact test

Key result

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

100 other: μg product/bee

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: contact test

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: not reported
- No. of offspring produced: n/a
- No. of unhatched cocoons: n/a
- Morphological abnormalities: n/a
- Behavioural abnormalities: none reported
- Other biological observations: none reported

Results with reference substance (positive control):

The LD50 values were determined statistically to be;
Contact: 0.3 μg a.i./bee
Oral: 0.3 μg a.i./bee
The LD50 values agree with the expected concentration range of 0.10-0.35μg/bee for oral dose and 0.1-0.3 μg/bee for contact dose.

Reported statistics and error estimates:

none

Table 3: Mean oral treatment mortality

Oral Test
Test Rate μg product/bee	Control (0)*	0.01	0.1	1	10	100
4-hour Mortality (%)	0	0	0	3.4	3.3	0
24-hour Mortality (%)	0	0	0	3.4	3.3	0
48-hour Mortality (%)	0	0	0	3.4	3.3	0

* Control and Solvent Control combined

All test doses were consumed in the control and treatment groups within 4 hours of dose addition. One honeybee from the 1.0 μg/bee vessels was accidentally trapped and killed during the oral consumption check at 1 hour; this bee was therefore excluded from the results.

At 4, 24 and 48 hours exposure, one honeybee in each of the 1.0 and 10 μg/bee vessels was recorded as ‘dead’.

Table 4: Mean contact treatment mortality

Contact Test
Test Rate μg product/bee	Control (0)*	0.01	0.1	1	10	100
4-hour Mortality (%)	0	0	0	0	0	3.3
24-hour Mortality (%)	0	0	0	0	0	3.3
48-hour Mortality (%)	0	0	0	0	0	3.3

* Control and Solvent Control combined

At 4, 24 and 48 hours exposure a single honeybee in the 100 μg product/bee test rate was recorded as ‘dead’.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on mortality of honeybees has been investigated in an acute oral toxicity test and an acute contact toxicity test. The results are as follows:
Oral Test
48 hour LD50: >100 μg product/bee
48 hour NOEL: 100 μg product/bee
Contact Test:
48 hour LD50: >100 μg product/bee
48 hour NOEL: 100 μg product/bee

Executive summary:

Introduction

Two studies were performed to determine the potential effects of the test substance on mortality of honeybees.

30 bees per treatment group and 20 bees per control group were exposed to the test substance, dispersed in the solvent Tween-80, via oral toxicity and contact toxicity routes. This study was conducted according to the procedures specified in OECD (1998) OECD Guidelines for the testing of chemicals, 213: Honeybees, Acute Oral Toxicity Test, and OECD (1998) OECD Guidelines for the testing of chemicals, 214: Honeybees, Acute Contact Toxicity Test.

Methods…….

A definitive test was conducted at the following test concentrations: 0 (control), 0 (solvent control), 0.01, 0.1, 1.0, 10 and 100 μg product/bee, for a period of 48 hours.

Due to the insolubility of the test item in RO water, a dispersing agent, Tween-80, was used to absorb the test substance.

Three replicate vessels were prepared for each test concentration and two replicate vessels were prepared for each control, each replicate containing 10 bees per test vessel.

All vessels of honeybees were examined at approximately 4, 24 and 48 hours after treatment. Details of all toxic effects were recorded including lethal and sub-lethal effects.

 

Results…….

The percentage mortality was calculated for each vessel and a mean per treatment dose was determined for the test.

As the 50% mortality level was not reached in either the oral or contact test, no statistical analysis was used when estimating the LD₅₀ value.

The results are as follows:

Oral Test

48 hour LD₅₀: >100 μg product/bee

48 hour NOEL: 100 μg product/bee

Contact Test:

48 hour LD₅₀: >100 μg product/bee

48 hour NOEL: 100 μg product/bee

Description of key information

A 48-hour LD₅₀ value of >100 μg product/bee and 48 -hour NOEL value of 100 μg product/bee (nominal concentration) (highest concentration tested) have been determined in an oral test for the effects of the test substance on mortality of Apis mellifera.

A 48-hour LD₅₀ value of >100 μg product/bee and 48-hour NOEL value of 100 μg product/bee (nominal concentration) (highest concentration tested) have been determined in a contact test for the effects of the test substance on mortality of Apis mellifera.

Additional information

Measured toxicity data are available for Shell GTL Solvent GS270 (Hydrocarbons, C15-C19, n-alkanes, isoalkanes, <2% aromatics) with the terrestrial arthropod Apis mellifera (Shell, 2015). The test was conducted in accordance with OECD Guideline 213 (Honeybees, Acute Oral Toxicity Test) and OECD Guideline 214 (Honeybees, Acute Contact Toxicity Test).

Due to the insolubility of Shell GTL Solvent GS270 in RO water, a dispersing agent, Tween-80, was used to absorb the test substance during preparation for the oral test; this was then topped up using 50% (w/v) sucrose solution. For the contact test, solutions were prepared in RO water and, as with the oral dose, due to the insolubility of Shell GTL Solvent GS270 in RO water, the dispersing agent, Tween-80, was used to absorb the test substance. Five test substance concentrations of control (0), solvent control, 0.01, 0.1, 1.0, 10 and 100 μg product/bee were tested. No analysis of test concentrations was carried out.

After 48 hours, no effects on mortality were observed, in either the oral or contact test. As the 50% mortality level was not reached in either the oral or contact test, no statistical analysis was used when estimating the LD₅₀ value.

The results are as follows:

Oral Test

48-hour LD₅₀: >100 μg product/bee

48-hour NOEL: 100 μg product/bee

Contact Test:

48-hour LD₅₀: >100 μg product/bee

48-hour NOEL: 100 μg product/bee