[No public or meaningful name is available]

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 February 2010 and 22 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C18 to C50

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Range-finding test
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.
In the range-finding test earthworms were exposed to a series of nominal concentrations of 10, 100 and 1000 mg/kg.
To prepare the test series, amounts of test item (5.0, 50 and 500 mg) were each added separately to 350 g of quartz sand in an amber glass jar and mixed using an industrial mixer. After 24 hours, the mixing was stopped and 100 g of clay and 50 g of air dried peat were added separately to each container prior to mixing for a further 24 hours to give the test concentrations of 10, 100 and 1000 mg/kg. After the mixing period each concentration was separately transferred to a Hobart A200N mixer prior to the addition of deionised reverse osmosis water (140 ml) and then mixed for 15 minutes to give a nominal moisture content of 30% of the dry weight.
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.
For the control and each test concentration 10 earthworms were added to each 1 litre glass beaker containing approximately 0.5 kg dry weight of substrate. The vessels were covered by plastic film with ventilation holes and maintained under continuous illumination (651 to 701 lux) in a temperature controlled room at 19C to 23ºC for a period of 14 days. The temperature was measured to be in excess of the 20 ± 2ºC range given in the study plan on 11 occasions during the test. This was considered to have no impact on the outcome or integrity of the test given that no adverse effects of exposure were observed throughout the duration of the test. After 7 and 14 days, the number of dead earthworms at each test concentration were determined.
Definitive test
Based on the results of the range-finding test a "limit test" was conducted at a concentration of 1000 mg/kg of soil to confirm that at the maximum concentration given in the OECD/EEC test guidelines no mortalities or sub-lethal effects of exposure were observed.
Experimental preparation
For the purpose of the definitive test the test item was prepared by direct addition to the basic soil substrate.
To prepare the test series, amounts of test item (3500 mg) was added directly to 2.45 kg of quartz sand in an amber glass jar and mixed using an industrial mixer. After 24 hours, the mixing was stopped and 0.70 kg of clay and 0.35 kg of air dried peat were added separately to each container prior to mixing for a further 24 hours to give the test concentration of 1000 mg/kg. After the mixing period each concentration was separately transferred to a Hobart A200N mixer prior to the addition of deionised reverse osmosis water (140 ml) and then mixed for 15 minutes to give a nominal moisture content of 30% of the dry weight.
An amount of soil (approximately 100 g) was taken from the control and the 1000 mg/kg test group for dry weight determination. The remaining control soil was dispersed to four replicate vessels and the test soil was dispersed to six replicate vessels.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation and given that the test item is classified as a UCVB and as such an appropriate analytical method of analysis in soil was not available. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Vehicle:

no

Details on preparation and application of test substrate:

Exposure conditions
As in the range-finding test 1 litre glass beakers were used. At the start of the test 10 earthworms were placed in each test and control vessel on the artificial soil, at random. The test vessels were covered with plastic film with ventilation holes and maintained in a temperature controlled room at 21°C to 23°C under continuous illumination at 532 to 649 lux. The earthworms were not individually identified and received no food during exposure.
The weight of each earthworm was recorded on Day 0 before addition to the test or control vessels. Each surviving earthworm was weighed on Day 14.
The control group was maintained under identical conditions but not exposed to the test item.
Any mortalities were recorded 7 and 14 days after the start of treatment and any sub-lethal effects are recorded daily. The criterion of death was taken to be the absence of any reaction to a physical stimulus at either end of the body.
hysico-chemical measurements
Room temperature and light intensity were recorded daily throughout the test. Soil moisture content and pH were recorded at the start and termination of the test. The pH was measured using a Model IQ150 portable pH meter.
Evaluation of data
An estimate of the LC50 values was given by inspection of the mortality data for the definitive test.
Statistical analysis of the earthworm weight data was performed using Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and a Students t-test. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Test organisms (species):

Eisenia fetida

Animal group:

nematods

Details on test organisms:

The test was carried out using earthworms of the species Eisenia foetida. Earthworms were obtained from Original Organics Limited, Langlands Business Park, Devon, UK and maintained in a 20 litre plastic trays at a temperature of 19ºC to 22ºC on a substrate consisting of 50% by volume peat and 50% by volume animal manure.
The test organisms were at least 2 months old with clitellum and were transferred into artificial soil (without test item) approximately 24 hours prior to the start of the test.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Post exposure observation period:

Any mortalities were recorded 7 and 14 days after the start of treatment and any sub-lethal effects were recorded daily. The criterion of death was taken to be the absence of any reaction to a physical stimulus at either end of the body.

Test temperature:

Room temperature remained at 21°C to 23°C throughout the test. The temperature during the definitive test was observed to be slightly in excess of the range given in the study plan of 20 ± 2°C on 10 occasions. This deviation was considered not to have affected the outcome or the validity of the test as no sub-lethal effects or mortalities were observed in the control worms over the test period.

pH:

The pH of the artificial soil was adjusted to 6.0 ± 0.5 by the addition of calcium carbonate. The pH was measured using a WTW pH 330 pH meter.

Moisture:

Moisture content of 30% of dry weight was considered appropriate for use during the study, as this level of moisture has been shown to ensure that a good crumb structure is formed within the soil.
The moisture content of the soil used in the definitive test was 30% of dry weight of basic substrate on Day 0 and 27% on Day 14.

Details on test conditions:

- Test container (material, size):
As in the range-finding test 1 litre glass beakers were used.

- Amount of soil or substrate:
100g of dried artificial soil.

- No. of organisms per container (treatment):
At the start of the test 10 earthworms were placed in each test and control vessel on the artificial soil, at random.

- No. of replicates per treatment group:
6

- No. of replicates per control:
6

- No. of replicates per vehicle control:
6


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)

EXAMPLE: A defined artificial soil was used as the basic test substrate with the following composition:
Industrial quartz sand 70% w/w
Kaolinite clay 20% w/w
Sphagnum moss peat 10% w/w
The pH of the artificial soil was adjusted to 6.0 ± 0.5 by the addition of calcium carbonate. Experience has shown that a moisture content of 35% of dry weight, as stated in the Test Guideline, may be too high for the preparation of the artificial soil, causing sludge formation. Therefore, a moisture content of 30% of dry weight was considered appropriate for use during the study, as this level of moisture has been shown to ensure that a good crumb structure is formed within the soil.
The moisture content of the soil used in the definitive test was 30% of dry weight of basic substrate on Day 0 and 27% on Day 14.

- Organic carbon (%):
Not stated

- Maximum water holding capacity (in % dry weigth):
Not stated

- CEC:
Not stated

- Pretreatment of soil:
Not stated

- Storage (condition, duration):
Not stated

- Stability and homogeneity of test material in the medium:
Not stated


OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination

- Light intensity:
651° to 701° lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
EXAMPLE: After 7 and 14 days, the number of dead earthworms at each test concentration were determined.

An estimate of the LC50 values was given by inspection of the mortality data for the definitive test.
Statistical analysis of the earthworm weight data was performed using Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) anda Students t-test. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).
The soil moisture content was determined as follows:
An amount of artificial soil (approximately 100 g) was dried at approximately 110ºC until a constant weight was obtained. The percentage moisture content of the artificial soil, expressed as percentage of dry weight, was calculated as follows:

% moisture content = where the weight of water in the soil is : weight of wet soil – weight of dried soil.



VEHICLE CONTROL PERFORMED:
EXAMPLE: yes


TEST CONCENTRATIONS
- Spacing factor for test concentrations:
Not applicable

- Justification for using less concentrations than requested by guideline:
Not applicable

- Range finding study

- Test concentrations:
In the range-finding test earthworms were exposed to a series of nominal concentrations of 100 and 1000 mg/kg.

- Results used to determine the conditions for the definitive study:
The results showed no significant mortalities at the test concentrations of 100 and 1000 mg/kg.

Nominal and measured concentrations:

In the range-finding test earthworms were exposed to a series of nominal concentrations of 10, 100 and 1000 mg/kg.
Based on this information, a single test concentration o fsix replicates, at 1000mg/kg was selected for the definitive test. This experiment design conforms t a 'limit test' to confirm that at the maximum test concentration given in the OECD/EEC test guidelines no mortalities or sub-lethal effects of exposure were observed.

Reference substance (positive control):

yes

Remarks:

chloroacetamide

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 other: mg/kg

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CL not stated

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

1 000 other: mg/kg

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CL not stated

Details on results:

Range-finding Test
Cumulative mortality data from the exposure of Eisenia foetida to the test item during the range-finding test are given in Table 1 (see in any other information on results).
The results showed no mortalities at any of the test concentrations employed in the range-finding test.
Based on this information, a single test concentration of six replicates, at 1000 mg/kg was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the OECD/EEC test guidelines no mortalities or sub-lethal effects of exposure were observed.
Definitive Test
Mortality data
Cumulative mortality data from the exposure of Eisenia foetida to the test item during the definitive test are given in Table 2 (see in any other information on results).
Inspection of the mortality data based on the nominal test concentrations gave the following results:
Time (days) LC50 (mg/kg)
7 > 1000
14 > 1000
There were no mortalities or sub-lethal effects of exposure in 60 earthworms exposed to a test concentration of 1000 mg/kg for a period of 14 days.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/kg.
There were no observed sub-lethal effects of exposure of Eisenia foetida to the test item.
Earthworm weight data
Statistical analysis using Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and a Students t-test was carried out on the earthworm weight data (see Table 3 and Appendix 1 and Appendix 2) see in any other information on results.
There were significant differences (P>0.05) between the control and 1000 mg/kg test groups in terms of weight on Day 0.
There were no significant differences (P>0.05) between the control and all the test groups in terms of weight on Day 14.
The worm weights for the control at Day 0 were found to be significantly heavier (P>0.05) than the 1000 mg/kg test group, however as there were no significant differences in terms of weight on Day 14 it was considered that the differences observed on Day 0 did not affect the outcome or validity of the test.
No observed effect concentration
The No Observed Effect Concentration (NOEC) was 1000 mg/kg on the basis that no mortalities were observed after 14 days exposure and additionally no sub-lethal effects on weight or behaviour were observed at 1000 mg/kg.
Physico-chemical measurements
The measured pH values and soil moisture content in the control and test cultures are given in Appendix 3 (see in any other information on results). There were no significant changes in pH or moisture content over the test period. pH values ranged from 6.1 to 6.3 at initiation and 6.3 to 6.5 at termination. The soil moisture contents were 29% to 30% at initiation and 25% at termination.

Results with reference substance (positive control):

Positive Control
Cumulative mortality data from the exposure of Eisenia foetida to chloroacetamide during the positive control are given in Table 4 (see in any other information on results section). The relationship between mortality and concentration after 7 and 14 days is given in Figures 1 and 2 (see attached section).
Analysis of the mortality data by the trimmed Spearman-Karber method (Hamilton et al 1977) on Days 7 and 14 gave the following results based on nominal test concentrations:
Time (days) LC50 (mg/kg) 95% Confidence limits (mg/kg)
7 44 42 – 45
14 43 41 – 45
The No Observed Effect Concentration was 18 mg/kg.
The results from the positive control with chloroacetamide are within the normal range for this reference item of 10 to 50 mg/kg.

Reported statistics and error estimates:

The LC50 values and associated confidence limits were calculated by the probit method (Finney 1971) on Day 7 and by the trimmed Spearman-Karber method (Hamilton et al 1977) on Day 14 using the ToxCalc computer software package (ToxCalc 1999).

Table 1: Cumulative Mortality Data in the Range-finding Test

Nominal Concentration (mg/kg)	Cumulative Mortality (Initial Population: 10)		% Mortality
	Day 7	Day 14	Day 7	Day 14
Control	0	0	0	0
10	0	0	0	0
100	0	0	0	0
1000	0	0	0	0

Table 2: Cumulative Mortality Data in the DefinitiveTest

Nominal Concentration (mg/kg)		Cumulative Mortality (Initial Population : 10)		% Mortality
		Day 7	Day 14	Day 7	Day 14
Control	R1	0	0	0	0
	R2	0	0	0	0
	R3	0	0	0	0
	R4	0	0	0	0
1000	R1	0	0	0	0
	R2	0	0	0	0
	R3	0	0	0	0
	R4	0	0	0	0
	R5	0	0	0	0
	R6	0	0	0	0

R₁– R₆= Replicates 1 to 6

 Table 3: Mean Earthworm Weights in the Definitive Test

Nominal Concentration (mg/kg)		Mean Earthworm Weight (g)
		Day 0		Day 14
		Mean	SD	Mean	SD
Control	R1	0.46	0.08	0.36	0.06
	R2	0.48	0.08	0.40	0.06
	R3	0.53	0.06	0.45	0.07
	R4	0.41	0.05	0.35	0.04
1000	R1	0.41	0.09	0.36	0.10
	R2	0.43	0.07	0.35	0.07
	R3	0.46	0.10	0.39	0.10
	R4	0.39	0.05	0.36	0.04
	R5	0.42	0.09	0.37	0.06
	R6	0.45	0.08	0.40	0.07

SD= Standard deviation

R₁– R₆= Replicates 1 to 6

 Appendix 1: Individual Worm Weights (g) – Day 0

Nominal Concentration (mg/kg)		Individual Worm Weights (g)
		1	2	3	4	5	6	7	8	9	10
Control	R1	0.53	0.60	0.58	0.42	0.39	0.40	0.37	0.40	0.50	0.44
	R2	0.56	0.50	0.37	0.39	0.52	0.42	0.51	0.41	0.55	0.57
	R3	0.60	0.53	0.43	0.57	0.50	0.60	0.53	0.54	0.45	0.59
	R4	0.49	0.37	0.42	0.37	0.40	0.38	0.44	0.39	0.49	0.38
1000	R1	0.40	0.42	0.37	0.59	0.35	0.30	0.38	0.42	0.34	0.55
	R2	0.38	0.39	0.39	0.45	0.53	0.54	0.42	0.39	0.30	0.48
	R3	0.54	0.39	0.47	0.38	0.47	0.31	0.38	0.45	0.59	0.60
	R4	0.40	0.42	0.34	0.39	0.48	0.38	0.35	0.36	0.31	0.44
	R5	0.35	0.49	0.30	0.42	0.39	0.55	0.37	0.38	0.56	0.40
	R6	0.33	0.49	0.38	0.52	0.42	0.58	0.47	0.38	0.48	0.40

R₁– R₆= Replicates 1 to 6

Appendix 2: Individual Worm Weights (g) – Day 14

Nominal Concentration (mg/kg)		Individual Worm Weights (g)
		1	2	3	4	5	6	7	8	9	10
Control	R1	0.36	0.39	0.32	0.31	0.33	0.30	0.37	0.47	0.44	0.35
	R2	0.44	0.38	0.50	0.38	0.37	0.39	0.46	0.31	0.33	0.43
	R3	0.49	0.48	0.53	0.56	0.46	0.38	0.36	0.40	0.40	0.43
	R4	0.40	0.31	0.37	0.36	0.34	0.30	0.39	0.39	0.30	0.31
1000	R1	0.35	0.50	0.55	0.30	0.41	0.30	0.30	0.33	0.30	0.27
	R2	0.27	0.31	0.49	0.44	0.28	0.31	0.36	0.41	0.32	0.30
	R3	0.52	0.40	0.36	0.36	0.40	0.30	0.31	0.40	0.59	0.28
	R4	0.38	0.36	0.38	0.29	0.37	0.39	0.29	0.41	0.35	0.35
	R5	0.45	0.32	0.32	0.44	0.36	0.34	0.36	0.46	0.29	0.36
	R6	0.34	0.41	0.47	0.40	0.42	0.34	0.52	0.34	0.33	0.46

R₁– R₆= Replicates 1 to 6

Appendix 3: Soil Moisture Content and pH Values

i)         Soil Moisture Content

Nominal Concentration (mg/kg)	Soil Moisture Content % (Expressed as % of Dry Weight)
	Day 0	Day 14
Control	29	25
1000	30	25

 

ii)        pH Values

Nominal Concentration (mg/kg)		Day 0	Day 14
Control	R1	6.3	6.3
	R2	6.3	6.3
	R3	6.4	6.3
	R4	6.3	6.3
1000	R1	6.2	6.5
	R2	6.2	6.4
	R3	6.2	6.3
	R4	6.2	6.3
	R5	6.2	6.3
	R6	6.1	6.3

R₁– R₆= Replicates 1 to 6

Table 4: Cumulative Mortality Data in the Positive Control with Chloroacetamide

Nominal Concentration (mg/kg)	Cumulative Mortality (Initial Population: 40)		% Mortality
	Day 7	Day 14	Day 7	Day 14
Control	0	0	0	0
5.6	0	0	0	0
10	0	0	0	0
18	0	0	0	0
32	0	1	0	2.5
56	36	36	90	90

The light intensity was 532 to 649 lux (see Appendix 4 see below).

Appendix 4: Room Temperature and Light Intensity Records

Day	Room Temperature (°C)		Light Intensity (Lux)
	Min	Max
0	22	23	645
1	22	23	649
2	22	23	638
3	22	23	642
4	22	23	648
5	22	23	643
6	22	23	649
7	22	23	599
8	22	22	603
9	22	22	600
10	21	22	609
11	21	22	535
12	22	23	601
13	22	22	627
14	21	23	532

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item, to the earthworm Eisenia foetida, in an artificial soil test has been investigated and gave a 14-Day LC50 value of greater than 1000 mg/kg. Correspondingly the No Observed Effect Concentration was 1000 mg/kg.

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test item ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’ to the earthworm (Eisenia foetida) in an artificial soil. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 207 "Earthworm, Acute Toxicity Tests" and Commission Regulation (EC) No. 440/2008.

Methods.

Following a preliminary range-finding test, 60 earthworms (six replicates of 10 worms) were exposed to a single concentration of 1000 mg/kg of soil for a period of 14 days at a temperature of 21ºC to 23ºC. The number of mortalities were determined after 7 and 14 days exposure. A positive control using chloroacetamide, conducted approximately every 6 months, was reported for reference purposes.

Results.

The 14-Day LC₅₀ for the test item to earthworms (Eisenia foetida) based on nominal test concentrations was greater than 1000 mg/kg. The No Observed Effect Concentration was 1000 mg/kg.

These results are based on the soil dry weight.

The result of the positive control gave a 14-Day LC₅₀ for chloroacetamide of 43 mg/kg with 95% confidence limits of 41 - 45 mg/kg. The No Observed Effect Concentration was 18 mg/kg.

Exposure of the earthworm (Eisenia foetida) to the reference item gave 14-Day LC₅₀ values within the accepted range.

Conclusion.

The acute toxicity of the test item, to the earthworm Eisenia foetida, in an artificial soil test has been investigated and gave a 14-Day LC₅₀value of greater than 1000 mg/kg. Correspondingly the No Observed Effect Concentration was 1000 mg/kg.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 09-Aug-2010 Experimental Completion Date: 07-Oct-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C18 to C50

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 11268-1 (Effects of Pollutants on Earthworms. 1. Determination of Acute Toxicity Using Artificial Soil Substrate)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Please see Principles of method if other than guideline section

Deviations:

no

Principles of method if other than guideline:

Biologische Bundesanstalt für Land- und Forstwirtschaft Bundesrepublik Deutschland: Richtlinien für die Prüfung von Pflanzenschutzmitteln im Zulassungsverfahren Part VI, 2-2: „Auswirkungen von Pflanzenschutzmitteln auf die Reproduktion und das Wachstum von Eisenia fetida/Eisenia andrei“, January 1994.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

- Concentrations: 10, 32, 100, 320 and 1000 mg/kg dry soil
- Sampling method: Not applicable
- Sample storage conditions before analysis: Not applicable

Vehicle:

no

Details on preparation and application of test substrate:

Please see "Test Substrate" in "Any other information on materials and methods incl. tables" sections.

Test organisms (species):

Eisenia fetida

Animal group:

nematods

Details on test organisms:

Test System
The study was performed with earthworms of the species Eisenia fetida. The worms were obtained from a synchronized culture maintained at Harlan Laboratories. During breeding and keeping, they were fed with suitable food (i.e., horse manure and potatoes).

For one day prior to the test start, the test organisms were acclimatized to the artificial soil and test temperature. The individual body wet weight of the test organisms at the start of the test ranged from 308 to 594 mg (the worms were weighed individually at the test start, see attached Appendix I). The worms used were 4-5 months old adults with a clitellum. The age of the worms from the synchronized culture did not differ by more than 4 weeks.

The method of application and test species are recommended by the international test guidelines.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Remarks:

Adult's were exposed in treated soils for 4 wks. Adults were then removed from vessels the survival/growth rate were determined. Cocoons juvenile's remained in vessels for further 4 wks. The reproduction rate was determined by counting hatched offspring

Test temperature:

At the test start and at the end of the test, the pH of the soil was determined for all treatment groups and the control (please see Table 5). For this purpose, the soil samples from all four test vessels per treatment group were mixed. Then, the pH was determined in the samples of this mixed soil. Soil samples of about 10 g were shaken with 25 mL of a KCl solution (1 M) for 30 minutes and then the pH was measured in the suspensions by means of a pH electrode.

The test temperature (room temperature) was continuously monitored by a digital temperature recorder.

pH:

At the test start and at the end of the test, the pH of the soil was determined for all treatment groups and the control (please see Table 5). For this purpose, the soil samples from all four test vessels per treatment group were mixed. Then, the pH was determined in the samples of this mixed soil. Soil samples of about 10 g were shaken with 25 mL of a KCl solution (1 M) for 30 minutes and then the pH was measured in the suspensions by means of a pH electrode.
The test temperature (room temperature) was continuously monitored by a digital temperature recorder.

Moisture:

The water holding capacity (WHC) was determined in two samples of the artificial soil (method as mentioned in the ISO guideline 11268-2) and the moisture of the test substrate was adjusted as described in Section “Test Substrate”. At the start of the test, the soil moisture was checked in samples from the treatments.

The test vessels were covered with transparent lids to reduce evaporation. Once per week, water evaporation losses were compensated for by adding purified water up to the initial weight of the test vessels (taking account of added food amounts).
At the end of the test, the soil moisture was checked again for all treatment groups and the control ( please see Table 5). Soil samples from all four test vessels per treatment group were mixed. Then, the soil moisture was determined in the mixed soil samples.

At all moisture determinations, soil samples (approx. 10g each) were dried at 105°C for 4 hours. Then, the dry weight of the soil samples was determined and the soil moisture content was calculated.

Details on test conditions:

Experimental Conditions
Plastic boxes (length about 16.5 cm, width about 12 cm, height about 6.5 cm, area about 200 cm2) were used as test vessels. Each test vessel contained a defined amount of dry artificial soil (see Section “Test Substrate”), corresponding to a layer depth of approximately 5-6 cm. The test vessels were covered by transparent lids to prevent worms from escaping and to reduce evaporation during the test period. The lids were perforated to allow air exchange. The test vessels were labeled with the study number and all necessary additional information to ensure unique identification.
During the whole test period, they were kept in a temperature-controlled room set at 20±2 °C under a 16-hour light to 8-hour darkness photoperiod (light intensity within 683-761 Lux).
For each treatment, four test vessels (replicates) with ten earthworms each were prepared. Thus, in total 40 test organisms were tested per treatment. However, for the control group, eight test vessels (replicates) with ten earthworms each were prepared, i.e. a total of 80 test organisms.
To demonstrate satisfactory test conditions, a positive control with the reference item Derosal® with the active ingredient Carbendazim was performed.
Immediately after soil treatments (see Section “Dosage and Concentrations”), ten of the weighed test organisms were randomly placed on the soil surface of each test vessel.

Nominal and measured concentrations:

10, 32, 100, 320 and 1000 mg/kg dry soil

Reference substance (positive control):

yes

Remarks:

Derosal® for full details please see "Any other information on materials and methods incl. tables"

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: survival, growth and reproduction rates

Remarks on result:

other: Survival, growth & reproduction of the adult’s the highest concentration without toxic effects (NOEC) was at least 1000 mg/kg ds. The lowest concentration with toxic effects (LOEC) could not be quantified, but was higher than 1000 mg/kg ds.

Details on results:

Survival of Adults and Adverse Effects
In the control group, the mortality rate of the adult test organisms after 28 days was 0% (Table1). Also at all test item concentrations up to and including 1000 mg/kg dry soil, the mortality rate was 0%.
Thus, the survival rate of the earthworm Eisenia fetida after 28 days of exposure was not affected up to and including the highest concentration of 1000 mg test item/kg dry soil. Moreover, no adverse effects were observed in the adult test organisms at any test concentration.

Growth of Adults
The mean body wet weights of the worms were determined per replicate at test start and of the surviving worms after the exposure period of 28 days are listed in Table 2. The individual wet weights of all test organisms at test start are given in the attached Appendix I.
The mean body weight of the adult worms in the control decreased during the 4-week exposure period on average by 8 mg per worm (-2% of the mean initial weight). At all test concentrations, the mean increase in body weight (0 to 4%) was statistically not significantly different than in the control (results of a Dunnett t-test, one-sided smaller, a = 0.05). The results for the growth of the test organisms correspond to the observation that no prominent difference in food consumption was determined between the control and all treatments up to and including the highest test concentration of 1000 mg test item/kg dry soil (Table 3).

Reproduction
The total number of living offspring, produced by all adults within 28 days of continuous exposure, is shown in Table 4 (expressed per test vessel and per surviving adult).

In the control, an average of 112 juvenile worms per test vessel were found (corresponding to a mean reproduction rate of 11.2 juveniles per surviving adult). The coefficient of variance of the reproduction rate per test vessel and per surviving individual in the control was 12%. Thus, the validity criteria of the test guidelines (at least 30 juveniles per test vessel, coefficient of variance of reproduction £30%) were well fulfilled.

At all test item concentrations up to and including 1000 mg/kg dry soil, the mean reproduction rates per surviving adult worm corresponded to 87-105% of the control value without a concentration-effect relationship. According to the results of a Dunnett t-test (one-sided smaller, a = 0.05), the mean reproduction rates were not statistically significantly reduced up to and including the highest test item concentration of 1000 mg/kg dry soil.
NOEC and LOEC
Taking into account the survival, consumption rates, growth and reproduction rates of the exposed adult test organisms, the highest concentration of the test item without toxic effects (NOEC) on Eisenia fetida after the test period was determined to be at least 1000 mg/kg dry soil. The NOEC might even be higher, but concentrations in excess of 1000 mg/kg dry soil were not tested. The lowest concentration with toxic effects (LOEC) could not be quantified, but was higher than 1000 mg/kg dry soil.

Soil Moisture, pH, and Temperature
The soil moisture content was sufficiently constant (39-40% at test start and 46-47% at test termination after 8 weeks, Table 5). The pH value in the untreated artificial soil was 6.2 at the start of the test. At the test end, the pH in the test replicates was between 6.2 and 6.4. (Table 5) During the entire test period, the room temperature was in the range of 20 to 22 °C.

Results with reference substance (positive control):

Reference Item Derosal
At the single concentration of the reference item Derosal (8.33 mg formulation /kg dry soil = 5 mg Carbendazim/kg dry soil), the mortality rate of the adult worms was 2.5% (Table 1).
The mean decrease of 6% in body wet weight of the earthworms in the positive control during the exposure period was not statistically significantly different to the control (results of a Student-t-test, one-sided smaller, a = 0.05, Table 2).
The reproduction rate of Eisenia fetida in the positive control was almost completely inhibited and statistically significantly reduced in comparison to the control (Welch t-test, one-sided smaller, a = 0.05, Table 4).

Therefore, the results of the positive control demonstrated satisfactory test conditions.

TABLES

Table 1 Number of Living Adult Earthworms and % Mortality after 4 Weeks

Nominal test item concentration (mg/kg dry soil)	Vessel No.	Number of earthworms exposed	Number of living worms after 4 weeks	Sum of dead earthworms	% mortality
	1	10	10
	2	10	10
	3	10	10
Control	4	10	10
	5	10	10
	6	10	10
	7	10	10
	8	10	10	0	0
	1	10	10
10	2	10	10
	3	10	10
	4	10	10	0	0
	1	10	10
32	2	10	10
	3	10	10
	4	10	10	0	0
	1	10	10
100	2	10	10
	3	10	10
	4	10	10	0	0
	1	10	10
320	2	10	10
	3	10	10
	4	10	10	0	0
	1	10	10
1000	2	10	10
	3	10	10
	4	10	10	0	0
Derosal 8.33 mg/kg dry soil	1	10	10
	2	10	9
	3	10	10
	4	10	10	1	2.5

 Table 2 Mean Body Wet Weights of Adult Earthworms at the Test Start and after 4 Weeks of Exposure

Nominal test item concentration	Vessel	Test start (Day 0)				After 4 weeks (Day 28)			Difference to test start
		Weight of all worms		No. of worms	Mean body weight/worm	Weight of all worms	No. of worms	Mean body weight/worm	Mean difference per worm
(mg/kg dry soil)	No.	(mg)			(mg)	(mg)		(mg)	(mg)	%	stat.^#
	1	4410		10	441	3984	10	398	-43	-10%
	2	4483		10	448	4422	10	442	-6	-1%
	3	4158		10	416	4130	10	413	-3	-1%
Control	4	4158		10	416	4162	10	416	0	0%
	5	4482		10	448	4174	10	417	-31	-7%
	6	4101		10	410	4080	10	408	-2	-1%
	7	4201		10	420	4372	10	437	17	4%
	8	3882		10	388	3939	10	394	6	1%
	Mean:				423			416	-8	-2%	¾
	SD:				21			17	20
	1	4492		10	449	4288	10	429	-20	-5%
10	2	4534		10	453	4480	10	448	-5	-1%
	3	3765		10	377	4067	10	407	30	8%
	4	4214		10	421	4368	10	437	15	4%
	Mean:				425			430	5	1%	n.s.
	SD:				35			17	22
	1	4691		10	469	4616	10	462	-8	-2%
32	2	4169		10	417	4235	10	424	7	2%
	3	4349		10	435	4587	10	459	24	5%
	4	3989		10	399	4420	10	442	43	11%
	Mean:				430			446	17	4%	n.s.
	SD:				30			18	22
	1	3871		10	387	3678	10	368	-19	-5%
100	2	4118		10	412	3945	10	395	-17	-4%
	3	3632		10	363	3730	10	373	10	3%
	4	4014		10	401	4310	10	431	30	7%
	Mean:				391			392	1	0%	n.s.
	SD:				21			29	23
	1	4377		10	438	4516	10	452	14	3%
320	2	4567		10	457	4496	10	450	-7	-2%
	3	4042		10	404	4242	10	424	20	5%
	4	4423		10	442	4647	10	465	22	5%
	Mean				435			448	12	3%	n.s.
	SD:				22			17	13
	1	4829		10	483	4602	10	460	-23	-5%
1000	2	4589		10	459	4577	10	458	-1	0%
	3	4098		10	410	4242	10	424	14	4%
	4	4052		10	405	4135	10	414	8	2%
	Mean:				439			439	0	0%	n.s.
	SD:				38			24	16
Derosal 8.33 mg/kg dry soil	1	4570		10	457	4372	10	437	-20	-4%
	2	4320		10	432	3413	9	379	-53	-12%
	3	3763		10	376	3657	10	366	-11	-3%
	4	3685		10	369	3509	10	351	-18	-5%
	Mean:				408			383	-25	-6%	n.s.^a
	SD:				43			38	19

Note: Calculations were performed with a commercial computer program (EXCEL). The results given in the table are rounded values.

^#Statistical comparison of the changes in mean body wet weight of the treatments with the control (absolute mean differences from test start to test end): results of a Dunnett t-test (one-sided,a= 0.05).

n.s.: Mean value not statistically significantly different compared to the control.

n.s.^a: Mean value not statistically significantly smaller than the control (results of a Student t-test, one-sided smaller,a= 0.05).

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, taking into account the survival, growth and reproduction rates of the exposed adult test organisms, the highest concentration of the substance without toxic effects on Eisenia fetida (NOEC) after the test period was determined to be at least 1000 mg/kg dry soil. The lowest concentration with toxic effects (LOEC) could not be quantified, but was higher than 1000 mg/kg dry soil.
The reproduction rate of Eisenia fetida in the positive control was almost completely inhibited. Thus, the results of the positive control demonstrated satisfactory test conditions.

Executive summary:

The toxic effects of the substance ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’ on survival, growth, and reproduction of the earthworm Eisenia fetida were assessed during a test period of eight weeks based on the OECD Guidelines for Testing Chemicals, Test No.222 (April 2004), the International Standards ISO 11268-2:1998(E) and the BBA Guideline “Richtlinien für die Prüfung von Pflanzenschutzmitteln im Zulassungsverfahren“ Part VI, 2-2, January 1994.

The test material was homogeneously mixed into artificial soil at the following test concentrations: 10, 32, 100, 320, and 1000 mg per kg dry soil. Additionally, a control and a reference item treatment were tested in parallel.

Adult earthworms were exposed in treated soils for a period of four weeks. 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.

Summary of effects on earthworms exposed to the test substance:

Parameters	Control	test substance (mg/kg dry soil)					Derosal^® (mg/kg dry soil)
		10	32	100	320	1000	8.33
Mortality (%) after a 4-week exposure	0	0	0	0	0	0	2.5
Mean change in body fresh weight(%) after a 4-week exposure	-2	1	4	0	3	0	-6
Mean reproduction rate (juveniles per vessel)	112	117	112	97	114	106	0.75^a
Reproduction rate (% of control)	---	105	101	87	102	95	0.34

a: Statistically significant different from the control (Welch t-test, one-sided smaller, a=0.05).

In conclusion, taking into account the survival, growth and reproduction rates of the exposed adult test organisms, the highest concentration of the substance without toxic effects on Eisenia fetida (NOEC) after the test period was determined to be at least 1000 mg/kg dry soil. The lowest concentration with toxic effects (LOEC) could not be quantified, but was higher than 1000 mg/kg dry soil.

The reproduction rate of Eisenia fetida in the positive control was almost completely inhibited. Thus, the results of the positive control demonstrated satisfactory test conditions.

Reference 3

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 February 2010 and 22 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C8 to C26

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Please see "GLP COMPLIANCE STATEMENT" in "Any other information on materials and methods incl. tables" section.

Specific details on test material used for the study:

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

Analytical monitoring:

no

Details on sampling:

- Concentrations:
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. In the range-finding test earthworms were exposed to a series of nominal concentrations of 10, 100 and 1000 mg/kg.

Based on the results of the range-finding test a "limit test" was conducted at a concentration of 1000 mg/kg of soil to confirm that at the maximum co centration given in the OECD/EEC test guidelines no mortalities or sub-lethal effects of exposure were observed.

- Sampling method:
Not applicable.
- Sample storage conditions before analysis:
Not applicable.

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation and given that the test item is classifiedas a UVCB and as such an appropriate analytical method of analysis in soil was not available. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Vehicle:

yes

Details on preparation and application of test substrate:

Range-finding test
To prepare the test series, amounts of test item (5.0, 50 and 500 mg) were each added separately to 350 g of quartz sand in an amber glass jar and mixed using an industrial mixer. After 24 hours, the mixing was stopped and 100 g of clay and 50 g of air dried peat were added separately to each container prior to mixing for a further 24 hours to give the test concentrations of 10, 100 and 1000 mg/kg. After the mixing period each concentration was separately transferred to a Hobart A200N mixer prior to the addition of deionised reverse osmosis water (140 mL) and then mixed for 15 minutes to give a nominal moisture content of 30% of the dry weight.

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.
For the control and each test concentration 10 earthworms were added to each 1 L glass beaker containing approximately 0.5 kg dry weight of substrate. The vessels were covered by plastic film with ventilation holes and maintained under continuous illumination (651 to 701 lux) in a temperature controlled room at 19 ºC to 23 ºC for a period of 14 days. The temperature was measured to be in excess of the 20 ± 2 ºC range given in the study plan on11 occasions during the test. This was considered to have no impact on the outcome or integrity of the test given that no adverse effects of exposure were observed throughout the duration of the test. After 7 and 14 days, the number of dead earthworms at each test concentration were determined.

Experimental preparation
For the purpose of the definitive test the test item was prepared by direct addition to the basic soil substrate.
To prepare the test series, amounts of test item (3500 mg) was added directly to 2.45 kg of quartz sand in an amber glass jar and mixed using an industrial mixer. After 24 hours, the mixing was stopped and 0.70 kg of clay and 0.35 kg of air dried peat were added separately to each container prior to mixing for a further 24 hours to give the test concentration of 1000 mg/kg. After the mixing period each concentration was separately transferred to a Hobart A200N mixer prior to the addition of deionised reverse osmosis water (140 mL) and then mixed for 15 minutes to give a nominal moisture content of 30% of the dry weight.
An amount of soil (approximately 100 g) was taken from the control and the 1000 mg/kg test group for dry weight determination. The remaining control soil was dispersed to four replicate vessels and the test soil was dispersed to six replicate vessels.

Test organisms (species):

Eisenia fetida

Animal group:

nematods

Details on test organisms:

Test Species
The test was carried out using earthworms of the species Eisenia foetida. Earthworms were maintained in a 20 L plastic trays at a temperature of 19 ºC to 22 ºC on a substrate consisting of 50% by volume peat and 50% by volume animal manure.
The test organisms were transferred into artificial soil (without test item) approximately 24 hours prior to the start of the test.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Post exposure observation period:

Any mortalities were recorded 7 and 14 days after the start of treatment and any sub-lethal effects are recorded daily. The criterion of death was taken to be the absence of any reaction to a physical stimulus at either end of the body.

Test temperature:

Room temperature remained at 21 ºC to 23 ºC throughout the test. On 10 occasions the room temperature during the test was observed to be above the 20 ± 2 ºC range given in the study plan. This deviation was considered not to have affected the outcome or validity of the test as no adverse effects of exposure were observed throughout the duration of the test.

pH:

The pH of the artificial soil was adjusted to 6.0 ± 0.5 by the addition of calcium carbonate. The pH was measured using a WTW pH 330 pH meter.

Moisture:

Moisture content of 30% of dry weight was considered appropriate for use during the study, as this level of moisture has been shown to ensure that a good crumb structure is formed within the soil.
The moisture content of the soil used in the definitive test was 30% of dry weight of basic substrate on Day 0 and 27% on Day 14.

Details on test conditions:

- Test container (material, size):
As in the range-finding test 1 L glass beakers were used.

- Amount of soil or substrate: 100g of dried artificial soil.

- No. of organisms per container (treatment):
At the start of the test 10 earthworms were placed in each test and control vessel on the artificial soil, at random.

- No. of replicates per treatment group:
6

- No. of replicates per control:
6

- No. of replicates per vehicle control:
6


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
A defined artificial soil was used as the basic test substrate with the following composition:
Industrial quartz sand 70% w/w
Kaolinite clay 20% w/w
Sphagnum moss peat 10% w/w
The pH of the artificial soil was adjusted to 6.0 ± 0.5 by the addition of calcium carbonate. Experience has shown that a moisture content of 35% of dry weight, as stated in the Test Guideline, may be too high for the preparation of the artificial soil, causing sludge formation. Therefore, a moisture content of 30% of dry weight was considered appropriate for use during the study, as this level of moisture has been shown to ensure that a good crumb structure is formed within the soil.
The moisture content of the soil used in the definitive test was 29 to 30% of dry weight of basic substrate on Day 0 and 25% on Day 14.


- Organic carbon (%):
Not stated

- Maximum water holding capacity (in % dry weigth):
Not stated

- CEC:
Not stated

- Pretreatment of soil:
Not stated

- Storage (condition, duration):
Not stated

- Stability and homogeneity of test material in the medium:
Not stated


OTHER TEST CONDITIONS
- Photoperiod:
Continuous illumination

- Light intensity:
651 to 701 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 7 and 14 days, the number of dead earthworms at each test concentration were determined.

An estimate of the LC50 values was given by inspection of the mortality data for the definitive test.
Statistical analysis of the earthworm weight data was performed using Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and a Students t-test. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).
The soil moisture content was determined as follows:
An amount of artificial soil (approximately 100 g) was dried at approximately 110 ºC until a constant weight was obtained. The percentage moisture content of the artificial soil, expressed as percentage of dry weight, was calculated as follows:

% moisture content = where the weight of water in the soil is : weight of wet soil – weight of dried soil.



VEHICLE CONTROL PERFORMED:
yes


TEST CONCENTRATIONS
- Spacing factor for test concentrations:
Not applicable

- Justification for using less concentrations than requested by guideline:
Not applicable

- Range finding study:
Yes, The test concentration to be used in the definitive test was determined by a preliminary range-finding test.


- Test concentrations:
In the range-finding test earthworms were exposed to a series of nominal concentrations of 100 and 1000 mg/kg.

- Results used to determine the conditions for the definitive study:
The results showed no significant mortalities at the test concentrations of 100 and 1000 mg/kg.

Based on this information, a single test concentration of six replicates, at 1000 mg/kg was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the OECD/EEC test guidelines no mortalities or sub-lethal effects of exposure were observed.

Nominal and measured concentrations:

In the range-finding test earthworms were exposed to a series of nominal concentrations of 10, 100 and 1000 mg/kg.
Based on this information, a single test concentration of six replicates, at 1000 mg/kg was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the OECD/EEC test guidelines no mortalities or sub-lethal effects of exposure were observed.

Reference substance (positive control):

yes

Remarks:

chloroacetamide

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CL not stated

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CL not stated

Details on results:

Range-finding Test
Cumulative mortality data from the exposure of Eisenia foetida to the test item during the range-finding test are given in Table 1.
The results showed no mortalities at any of the test concentrations employed in the range-finding test.
Based on this information, a single test concentration of six replicates, at 1000 mg/kg was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the OECD/EEC test guidelines no mortalities or sub-lethal effects of exposure were observed.

Definitive Test

Mortality data
Cumulative mortality data from the exposure of Eisenia foetida to the test item during the definitive test are given in Table 2.
Inspection of the mortality data based on the nominal test concentrations gave the following results:
Time (days) LC50 (mg/kg)
7 > 1000
14 > 1000
There were no significant mortalities or sub-lethal effects of exposure in 60 earthworms exposed to a test concentration of 1000 mg/kg for a period of 14 days.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/kg.
There were no observed sub-lethal effects of exposure of Eisenia foetida to the test item.

Earthworm weight data
Statistical analysis using Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and a Students t-test was carried out on the earthworm weight data (see Table 3 and Appendix 1 and Appendix 2).
There were significant differences (P≥0.05) between the control and 1000 mg/kg test groups in terms of weight on Day 0.
There were no significant differences (P≥0.05) between the control and all the test groups in terms of weight on Day 14.
The worm weights for the control at Day 0 were found to be significantly heavier (P≥0.05) than the 1000 mg/kg test group, however as there were no significant differences in terms of weight on Day 14 it was considered that the differences observed on Day 0 did not affect the outcome or validity of the test.

No observed effect concentration
The No Observed Effect Concentration (NOEC) was 1000 mg/kg on the basis that no significant mortalities were observed after 14 days exposure and additionally no sub-lethal effects on weight or behaviour were observed at 1000 mg/kg.

Physico-chemical measurements
The measured pH values and soil moisture content in the control and test cultures are given in Appendix 3. There were no significant changes in pH or moisture content over the test period. pH values ranged from 6.3 to 6.5 at initiation and 6.3 to 6.5 at termination. The soil moisture contents were 29% to 30% at initiation and 25% at termination. These measured values were within the acceptable limits given in the OECD guidelines.
Room temperature remained at 21ºC to 23ºC throughout the test and the light intensity was 659 to 706 lux (see Appendix 4).
On 10 occasions the room temperature during the test was observed to be above the 20 ± 2 ºC range given in the study plan. This deviation was considered not to have affected the outcome or validity of the test as no adverse effects of exposure were observed throughout the duration of the test.

Results with reference substance (positive control):

Positive Control
Cumulative mortality data from the exposure of Eisenia foetida to chloroacetamide during the positive control are given in Table 4 (see in any other information on results section).
Analysis of the mortality data by the trimmed Spearman-Karber method (Hamilton et al 1977) on Days 7 and 14 gave the following results based on nominal test concentrations:
Time (days) LC50 (mg/kg) 95% Confidence limits (mg/kg)
7 44 42 – 45
14 43 41 – 45
The No Observed Effect Concentration was 18 mg/kg.
The results from the positive control with chloroacetamide are within the normal range for this reference item of 10 to 50 mg/kg.

Reported statistics and error estimates:

The LC50 values and associated confidence limits were calculated by the probit method (Finney 1971) on Day 7 and by the trimmed Spearman-Karber method (Hamilton et al 1977) on Day 14 using the ToxCalc computer software package (ToxCalc 1999).

Table 1 Cumulative Mortality Data in the Range-finding Test

Nominal Concentration (mg/kg)	Cumulative Mortality (Initial Population: 10)		% Mortality
	Day 7	Day 14	Day 7	Day 14
Control	0	0	0	0
10	0	0	0	0
100	0	0	0	0
1000	0	0	0	0

Table 2              Cumulative Mortality Data in the DefinitiveTest

Nominal Concentration (mg/kg)		Cumulative Mortality (Initial Population : 10)		% Mortality
		Day 7	Day 14	Day 7	Day 14
Control	R1	0	0	0	0
	R2	0	0	0	0
	R3	0	0	0	0
	R4	0	0	0	0
1000	R1	0	0	0	0
	R2	0	0	0	0
	R3	0	0	0	0
	R4	1*	1	10	10
	R5	0	0	0	0
	R6	0	0	0	0

R₁– R₆= Replicates 1 to 6

*A single worm was unaccounted for on Day 7, this was considered not to affect the outcome or validity of the test as the overall mean mortality value was less than 10% and no mortalities were observed throughout the duration of the test.

Table 3              Mean Earthworm Weights in the Definitive Test

Nominal Concentration (mg/kg)		Mean Earthworm Weight (g)
		Day 0		Day 14
		Mean	SD	Mean	SD
Control	R1	0.46	0.08	0.36	0.06
	R2	0.48	0.08	0.40	0.06
	R3	0.53	0.06	0.45	0.07
	R4	0.41	0.05	0.35	0.04
1000	R1	0.44	0.09	0.35	0.08
	R2	0.43	0.06	0.35	0.06
	R3	0.46	0.09	0.38	0.10
	R4	0.43	0.08	0.36	0.07
	R5	0.45	0.07	0.40	0.07
	R6	0.40	0.04	0.34	0.05

SD= Standard deviation

R₁– R₆= Replicates 1 to 6

Appendix 1      Individual Worm Weights (g) – Day 0

Nominal Concentration (mg/kg)		Individual Worm Weights (g)
		1	2	3	4	5	6	7	8	9	10
Control	R1	0.53	0.60	0.58	0.42	0.39	0.40	0.37	0.40	0.50	0.44
	R2	0.56	0.50	0.37	0.39	0.52	0.42	0.51	0.41	0.55	0.57
	R3	0.60	0.53	0.43	0.57	0.50	0.60	0.53	0.54	0.45	0.59
	R4	0.49	0.37	0.42	0.37	0.40	0.38	0.44	0.39	0.49	0.38
1000	R1	0.37	0.58	0.60	0.47	0.42	0.41	0.38	0.39	0.34	0.42
	R2	0.40	0.43	0.35	0.55	0.38	0.43	0.48	0.37	0.46	0.43
	R3	0.48	0.48	0.44	0.47	0.51	0.59	0.35	0.37	0.33	0.59
	R4	0.50	0.59	0.41	0.35	0.38	0.38	0.46	0.37	0.37	0.46
	R5	0.40	0.44	0.54	0.51	0.41	0.31	0.42	0.53	0.43	0.49
	R6	0.42	0.42	0.40	0.41	0.41	0.34	0.40	0.37	0.48	0.36

R₁– R₆= Replicates 1 to 6

Appendix 2      Individual Worm Weights (g) – Day 14

Nominal Concentration (mg/kg)		Individual Worm Weights (g)
		1	2	3	4	5	6	7	8	9	10
Control	R1	0.36	0.39	0.32	0.31	0.33	0.30	0.37	0.47	0.44	0.35
	R2	0.44	0.38	0.50	0.38	0.37	0.39	0.46	0.31	0.33	0.43
	R3	0.49	0.48	0.53	0.56	0.46	0.38	0.36	0.40	0.40	0.43
	R4	0.40	0.31	0.37	0.36	0.34	0.30	0.39	0.39	0.30	0.31
1000	R1	0.35	0.34	0.30	0.46	0.28	0.34	0.34	0.50	0.25	0.35
	R2	0.30	0.42	0.35	0.30	0.32	0.39	0.31	0.47	0.37	0.29
	R3	0.39	0.47	0.57	0.39	0.33	0.30	0.32	0.45	0.25	0.28
	R4	0.27	0.35	0.33	0.41	0.40	0.49	0.35	0.29	0.36	-
	R5	0.41	0.52	0.40	0.36	0.28	0.46	0.35	0.48	0.36	0.33
	R6	0.30	0.34	0.40	0.33	0.31	0.28	0.32	0.46	0.38	0.31

R₁– R₆= Replicates 1 to 6

- no weight as worm unaccounted for at Day 7

Appendix 3      Soil Moisture Content and pH Values

i)         Soil Moisture Content

Nominal Concentration (mg/kg)	Soil Moisture Content % (Expressed as % of Dry Weight)
	Day 0	Day 14
Control	29	25
1000	30	25

 

ii)        pH Values

Nominal Concentration (mg/kg)		Day 0	Day 14
Control	R1	6.3	6.3
	R2	6.3	6.3
	R3	6.4	6.3
	R4	6.3	6.3
1000	R1	6.5	6.5
	R2	6.4	6.5
	R3	6.4	6.4
	R4	6.4	6.5
	R5	6.4	6.4
	R6	6.4	6.4

R₁– R₆= Replicates 1 to 6

 

Table 4              Cumulative Mortality Data in the Positive Control with Chloroacetamide

Nominal Concentration (mg/kg)	Cumulative Mortality (Initial Population: 40)		% Mortality
	Day 7	Day 14	Day 7	Day 14
Control	0	0	0	0
5.6	0	0	0	0
10	0	0	0	0
18	0	0	0	0
32	0	1	0	2.5
56	36	36	90	90

Validity criteria fulfilled:

yes

Remarks:

This study is classified as acceptable and satisfies the guideline requirements for this study end point.

Conclusions:

The acute toxicity of the test item, to the earthworm Eisenia foetida, in an artificial soil test has been investigated and gave a 14-Day LC50 value of greater than 1000 mg/kg. Correspondingly the No Observed Effect Concentration was 1000 mg/kg.

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test item to the earthworm (Eisenia foetida) in an artificial soil. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 207 "Earthworm, Acute Toxicity Tests" and Commission Regulation (EC) No. 440/2008.

Methods.

Following a preliminary range-finding test, 60 earthworms (six replicates of 10 worms) were exposed to a single concentration of 1000 mg/kg of soil for a period of 14 days at a temperature of 21 ºC to 23 ºC. The number of mortalities were determined after 7 and 14 days exposure. A positive control using chloroacetamide, conducted approximately every 6 months, was reported for reference purposes.

Results.

The 14-Day LC₅₀ for the test item to earthworms (Eisenia foetida) based on nominal test concentrations was greater than 1000 mg/kg. The No Observed Effect Concentration was 1000 mg/kg.

These results are based on the soil dry weight.

The result of the positive control gave a 14-Day LC₅₀ for chloroacetamide of 43 mg/kg with 95% confidence limits of 41 - 45 mg/kg. The No Observed Effect Concentration was 18 mg/kg.

Exposure of the earthworm (Eisenia foetida) to the reference item gave 14-Day LC₅₀ values within the accepted range.

CONCLUSION.

The acute toxicity of the test item, to the earthworm Eisenia foetida, in an artificial soil test has been investigated and gave a 14-Day LC₅₀ value of greater than 1000 mg/kg. Correspondingly the No Observed Effect Concentration was 1000 mg/kg.

Reference 4

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-07-21 - 2011-06-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C8 to C26

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 11268-1 (Effects of Pollutants on Earthworms. 1. Determination of Acute Toxicity Using Artificial Soil Substrate)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Biologische Bundesanstalt für Land- und Forstwirtschaft Bundesrepublik Deutschland: Richtlinien für die Prüfung von Pflanzenschutzmitteln im Zulassungsverfahren Part VI, 2-2: „Auswirkungen von Pflanzenschutzmitteln auf die Reproduktion und das Wachstum

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

- Concentrations: 10, 32, 100, 320 and 1000 mg/kg dry soil
- Sampling method: Not applicable
- Sample storage conditions before analysis: Not applicable

Vehicle:

no

Details on preparation and application of test substrate:

The artificial soil was prepared according to the guidelines. The air dry components of the artificial soil (without calcium carbonate and food) were intensively mixed in a batch of 20 kg (dry weight) in a laboratory mixer to prepare a substrate mixture with the composition of peat, kaolinite clay, and sand. For each test vessel, 457 g (corresponding to 450 g dry weight) of the prepared artificial soil was thoroughly mixed with 50 g sand+artificial soil+test item mixture, 2.6 g calcium carbonate, 5 g food, and 194 mL purified water in a laboratory mixer. The amount of 700 g test substrate (corresponding to 500 g dry weight) was filled into each test vessel resulting in a soil layer of approx. 5-6 cm. With the addition of 194 mL purified water to the test substrate of each test vessel, the soil moisture content was adjusted to 40%, corresponding to approximately 54% of the total water holding capacity (WHC) of the artificial soil. The WHC amounted in average to 73.63 g water per 100 g dry soil. In the positive control with Derosal®, 500 g (dry weight) of the same artificial soil was used. However, the reference item was mixed into the artificial soil as aqueous application solution.

Test organisms (species):

Eisenia fetida

Animal group:

nematods

Details on test organisms:

Test System
The study was performed with earthworms of the species Eisenia fetida. The worms were obtained from a synchronized culture. During breeding and keeping, they were fed with suitable food (i.e., horse manure and potatoes).

For one day prior to the test start, the test organisms were acclimatised to the artificial soil and test temperature. The individual body wet weight of the test organisms at the start of the test ranged from 312 to 591 mg (the worms were weighed individually at the test start). The worms used were 3-4 months old adults with a clitellum. The age of the worms from the synchronized culture did not differ by more than 4 weeks.

The method of application and test species are recommended by the international test guidelines.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Remarks:

Adult's were exposed in treated soils for 4 wks, then removed from vessels the survival/growth rate were determined. Cocoons & juvenile's remained in vessels for further 4 wks. The reproduction rate was determined by counting hatched offspring.

Test temperature:

The test temperature (room temperature) was continuously monitored by a digital temperature recorder.

pH:

At the test start and at the end of the test, the pH of the soil was determined for all treatment groups and the control. For this purpose, the soil samples from all four test vessels per treatment group were mixed. Then, the pH was determined in the samples of this mixed soil. Soil samples of about 10 g were shaken with 25 mL of a KCl solution (1 M) for 30 minutes and then the pH was measured in the suspensions by means of a pH electrode.

Moisture:

The water holding capacity (WHC) was determined in two samples of the artificial soil (method as mentioned in the ISO guideline 11268-2) and the moisture of the test substrate was adjusted. At the start of the test, the soil moisture was checked in samples from the treatments.

The test vessels were covered with transparent lids to reduce evaporation. Once per week, water evaporation losses were compensated for by adding purified water up to the initial weight of the test vessels (taking account of added food amounts).
At the end of the test, the soil moisture was checked again for all treatment groups and the control. Soil samples from all four test vessels per treatment group were mixed. Then, the soil moisture was determined in the mixed soil samples.

At all moisture determinations, soil samples (approx. 10g each) were dried at 105°C for 4 hours. Then, the dry weight of the soil samples was determined and the soil moisture content was calculated.

Details on test conditions:

Experimental Conditions
Plastic boxes (length about 16.5 cm, width about 12 cm, height about 6.5 cm, area about 200 cm2) were used as test vessels. Each test vessel contained a defined amount of dry artificial soil, corresponding to a layer depth of approximately 5-6 cm. The test vessels were covered by transparent lids to prevent worms from escaping and to reduce evaporation during the test period. The lids were perforated to allow air exchange. The test vessels were labeled with the study number and all necessary additional information to ensure unique identification.
During the whole test period, they were kept in a temperature-controlled room set at 20±2 °C under a 16-hour light to 8-hour darkness photoperiod (light intensity within 574 -743 Lux).
For each treatment, four test vessels (replicates) with ten earthworms each were prepared. Thus, in total 40 test organisms were tested per treatment. However, for the control group, eight test vessels (replicates) with ten earthworms each were prepared, i.e. a total of 80 test organisms.
To demonstrate satisfactory test conditions, a positive control with the reference item Derosal® with the active ingredient Carbendazim was performed.
Immediately after soil treatments, ten of the weighed test organisms were randomly placed on the soil surface of each test vessel.

Nominal and measured concentrations:

10, 32, 100, 320 and 1000 mg/kg dry soil

Reference substance (positive control):

yes

Remarks:

Derosal® with the active ingredient Carbendazim

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: survival and reproduction

Remarks on result:

other: Survival, growth & reproduction of the adult’s the highest concentration without toxic effects (NOEC) was at least 1000 mg/kg ds. The lowest concentration with toxic effects (LOEC) could not be quantified, but was higher than 1000 mg/kg ds.

Details on results:

Survival of Adults and Adverse Effects
In the control group, the mortality rate of the adult test organisms after 28 days was 0% (Table1). Also at all test item concentrations up to and including 1000 mg/kg dry soil, the mortality rate was 0%.
Thus, the survival rate of the earthworm Eisenia fetida after 28 days of exposure was not affected up to and including the highest concentration of 1000 mg test item/kg dry soil. Moreover, no adverse effects were observed in the adult test organisms at any test concentration.

Growth of Adults
The mean body wet weights of the worms were determined per replicate at test start and of the surviving worms after the exposure period of 28 days.
The mean body weight of the adult worms in the control had decreased during the 4-week exposure period on average by 17 mg per worm (-4% of the mean initial weight). At all test concentrations of "Distillates (Fischer-Tropsch), C8-C26 -branched and linear", the mean decrease in body weight (0 to -3%) was statistically not significantly different than in the control (results of a Dunnett t-test, one-sided smaller, α = 0.05). The results for the growth of the test organisms correspond to the observation that no prominent difference in food consumption was determined between the control and all treatments up to and including the highest test concentration of 1000 mg test item/kg dry soil.

Reproduction
In the control, an average of 117 juvenile worms per test vessel was found (corresponding to a mean reproduction rate of 11.7 juveniles per surviving adult). The coefficient of variance of the reproduction rate per test vessel and per surviving individual in the control was 12%. Thus, the validity criteria of the test guidelines (at least 30 juveniles per test vessel, coefficient of variance of reproduction ≤30%) were well fulfilled.
At all test item concentrations up to and including 1000 mg/kg dry soil, the mean reproduction rates per surviving adult worm corresponded to 86-100% of the control value without a concentration-effect relationship. According to the results of a Dunnett t-test (one-sided smaller, α = 0.05), the mean reproduction rates were not statistically significantly reduced up to and including the highest test item concentration of 1000 mg/kg dry soil.

Soil Moisture, pH, and Temperature
The soil moisture content was sufficiently constant (38-39% at test start and 44-46% at test termination after 8 weeks). The pH value in the untreated artificial soil was 6.3 at the start of the test. At the test end, the pH in the test replicates was between 6.0 and 6.2. During the entire test period, the room temperature was in the range of 20 to 22 °C.

Results with reference substance (positive control):

Reference Item Derosal
At the single concentration of the reference item Derosal (8.33 mg formulation /kg dry soil = 5 mg Carbendazim/kg dry soil), the mortality rate of the adult worms was 0%.
The mean decrease of 8% in body wet weight of the earthworms in the positive control during the exposure period was not statistically significantly different to the control (results of a Student-t-test, one-sided smaller, a = 0.05).
The reproduction rate of Eisenia fetida in the positive control was almost completely inhibited.
Therefore, the results of the positive control demonstrated satisfactory test conditions.

Number of Living Adult Earthworms and % Mortality after 4 Weeks

Nominal test item concentration (mg/kg dry soil)	Vessel No.	Number of earthworms exposed	Number of living worms after 4 weeks	Sum of dead earthworms	% mortality
	1	10	10
	2	10	10
	3	10	10
Control	4	10	10
	5	10	10
	6	10	10
	7	10	10
	8	10	10	0	0
	1	10	10
10	2	10	10
	3	10	10
	4	10	10	0	0
	1	10	10
32	2	10	10
	3	10	10
	4	10	10	0	0
	1	10	10
100	2	10	10
	3	10	10
	4	10	10	0	0
	1	10	10
320	2	10	10
	3	10	10
	4	10	10	0	0
	1	10	10
1000	2	10	10
	3	10	10
	4	10	10	0	0
Derosal 8.33 mg/kg dry soil	1	10	10
	2	10	10
	3	10	10
	4	10	10	0	0

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, taking into account the survival, growth and reproduction rates of the exposed adult test organisms, the highest concentration of 'Distillates (Fischer-Tropsch), C8-C26 -branched and linear' without toxic effects on Eisenia fetida (NOEC) after the test period was determined to be at least 1000 mg/kg dry soil. The lowest concentration with toxic effects (LOEC) could not be quantified, but was higher than 1000 mg/kg dry soil.
The reproduction rate of Eisenia fetida in the positive control was completely inhibited. Thus, the results of the positive control demonstrated satisfactory test conditions.

Executive summary:

The toxic effects of Distillates (Fischer-Tropsch), C8-C26 -branched and linear on survival, growth, and reproduction of the earthworm Eisenia fetida were assessed during a test period of eight weeks based on OECD 222 (April 2004), the International Standards ISO 11268-2: 1998(E) and the BBA Guideline “Richtlinien für die Prüfung von Pflanzenschutzmitteln im Zulassungsverfahren“ Part VI, 2-2, January 1994.

Distillates (Fischer-Tropsch), C8-C26 -branched and linear was homogeneously mixed into artificial soil at the following test concentrations: 10, 32, 100, 320, and 1000 mg per kg dry soil. Additionally, a control and a reference item treatment were tested in parallel.

Adult earthworms were exposed in treated soils for a period of four weeks. 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.

Summary of effects on earthworms exposed to Distillates (Fischer-Tropsch), C8-C26 -branched and linear:

Parameters	Control	test substance (mg/kg dry soil)					Derosal^® (mg/kg dry soil)
		10	32	100	320	1000	8.33
Mortality (%) after a 4-week exposure	0	0	0	0	0	0	0
Mean change in body fresh weight(%) after a 4-week exposure	-4	0	-1	-2	-1	-3	-8
Mean reproduction rate (juveniles per vessel)	117	111	108	113	116	100	0.0
Reproduction rate (% of control)	---	95	92	97	100	86	0.0

In conclusion, taking into account the survival, growth and reproduction rates of the exposed adult test organisms, the highest concentration of 'Distillates (Fischer-Tropsch), C8-C26 -branched and linear' without toxic effects on Eisenia fetida (NOEC) after the test period was determined to be at least 1000 mg/kg dry soil. The lowest concentration with toxic effects (LOEC) could not be quantified, but was higher than 1000 mg/kg dry soil.

The reproduction rate of Eisenia fetida in the positive control was completely inhibited. Thus, the results of the positive control demonstrated satisfactory test conditions.

Description of key information

* OECD 207 (same results for test mat. 'Distillates (Fischer-Tropsch), C8-26 - branched and linear' and 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'):

- LC50 (14-d): >1000 mg/kg dw test mat. (nominal) based on: mortality

- NOEC (14-d): ≥1000 mg/kg dw test mat. (nominal) based on: mortality;

* OECD 222 (test mat. 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'):

- LC50 (4-w): >1000 mg/kg dw test mat. (nominal) based on: growth, reproduction and mortality

- NOEC (4-w): ≥1000 mg/kg dw test mat. (nominal) based on: growth, reproduction and mortality.

* OECD 222 (test mat. 'Distillates (Fischer-Tropsch), C8-26 - branched and linear'):

NOEC (28-d): ≥1000 mg/kg dw test mat. (nominal) based on: survival and reproduction;

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

1 000 mg/kg soil dw

Long-term EC10, LC10 or NOEC for soil macroorganisms:

1 000 mg/kg soil dw

Additional information

No toxicity studies to soil macroorganisms are available for the substance 'Paraffin waxes (Fischer–Tropsch), full-range, C15–C50 branched and linear':

A short-term earthworm toxicity study is available for the closely related substance GTL Base Oil Distillates covering the carbon range from C18 to C50 (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.

An acute earthworm toxicity study is also available for the supporting substance GTL Gasoil covering the carbon range from C8 to C26 (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-d acute LC50 for earthworms was therefore determined to be greater than 1000 mg/kg dw.

Moreover, 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 LC50and a LOEC could not be determined and a NOEC of at least 1000 mg/kg dw has been determined.

An earthworm reproduction study, conducted in accordance with OECD 222 and GLP has been performed with GTL Gasoil as well (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.

Conclusion:

Short and long-term soil macroorganism toxicity data are available for two GTL-derived substances in the relevant carbon number range for 'Paraffin waxes (Fischer–Tropsch), full-range, C15–C50 branched and linear'.

In the absence of adverse effects in any of these studies, based on weight of evidence it can be concluded that the NOEC for the registered substance is ≥1000 mg/kg dw soil.