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Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Study conducted according to a non Guideline protocol; not GLP; no analytical monitoring; moisture level low; reference substance results not as expected; no information on test item equilibration with the soil.
Qualifier:
according to guideline
Guideline:
other: Donkin & Dusenberry (1993)
Deviations:
yes
Remarks:
(prolonged duration; method of test animals recovery from vials changes, lower soil moisture).
Principles of method if other than guideline:
The test method was modified to improve the recovery rate of test animals. The amount of soil requiring extraction was reduced, worms were extracted by adding 5ml Ludox HS40 to each vial then shaking the vial vigorously on a 'whirlmixer'. The Ludox/soil suspension were centrifuged for longer and at a higher speed (7000 rpm and for 15 minutes) in order to improve the clarity of the supernatant, which was sieved and the organisms rinsed with M9 salt solution and counted under a high power microscope. The test also reduced moisture content of soil respect to the OECD guideline, 15-20% rather than 20-30%, but the authors of the report considered that this would make no difference to the survival of organisms due to the short duration of the test.
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
yes
Details on preparation and application of test substrate:
- Method of mixing into soil: sand spike, using 10% of the total dry weight of soil. This involved adding an appropriate amount of compound to analar grade acetone to form a stock solution for the 1000 mg/kg dose. Aliquots of the stock solution were then diluted with acetone to prepare lower doses. Acetone solutions were added to silica sand in 500 ml amber glass jars. Mixtures were mixed thoroughly with a stainless steel spatula. The acetone was left to evaporate in a fume cupboard, leaving the test item coated onto the sand. Appropriate amount of moist soil was added to each of the glass jars. The dosed or undosed sand was incorporated into the bulk of the soil using a palette knife. Jars were then sealed with screw top lids containing aluminium foil inserts and tumbled overnight (ca. 16 h) on a rotary soil tumbler 50 rpm.

- Controls: One aliquot of sand was dosed with acetone only to serve as solvent control. A jar with an aliquot of sand only served as untreated control - both with the appropriate amount of moist soil added thereafter.

- Chemical name of vehicle: acetone

- Concentration of vehicle in test medium: acetone solutions was added between 1-7 ml to 5-35g of silica sand. The sand was left to evaporate the acetone completely before initial exposure, so that the concentration of vehicle would be negligible.

- Evaporation of vehicle before use: yes
Test organisms (species):
Caenorhabditis elegans
Animal group:
other: nematodes
Details on test organisms:
TEST ORGANISM

- Common name: nematode

- Source: Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge.

- Age at test initiation (mean and range, SD): adult nematodes, 2 days old.


ACCLIMATION

- Acclimation period: none, organisms were removed from mixed age populations and juveniles allowed to mature within 2 days.
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
72 h
Remarks:
The standard duration for a C. elegans mortality test is 1 day. Growth and reproduction is the normal endpoint assessed in a 3-day duration sublethal assay using this species.
Post exposure observation period:
none
Test temperature:
20 +/- 2 degC
pH:
5.4-5.5
Moisture:
18% +/- 1%
Details on test conditions:
TEST SYSTEM

- Test container (material, size): 50 ml clear glass vials

- Amount of soil or substrate: 10 g wet weight of sterilised soil

- No. of organisms per container (treatment): 10

- No. of replicates per treatment group: 3

- No. of replicates per control: 3

- No. of replicates per vehicle control: 3

The test was preformed with other species, all details of the test were similar, except for the exposure system in which the test were carried was different. See Table 2 for details.


SOURCE AND PROPERTIES OF SUBSTRATE

- Geographic location: Heath Farm, Leicester Lane, LandLook (Midlands), Leamington Spa, from Dr Graham Beard. OS map reference 32896926.

- Pesticide use history at the collection site: assumed to be pesticide free, as the field the soil was taken from land that had been set aside for 3-4 years.

- Collection procedures: the soil was sieved to 2 mm.

- Sampling depth (cm): 5-20cm to avoid surface root mat

- Soil texture

- % sand: 63.8%-64.8%

- Soil classification: sandy loam soil. The soil is described as soil series, Bromsgrove, standard soil number 33, variant.

- Organic carbon (%): 1.3%

- Maximum water holding capacity: ca. 40%

- Pretreatment of soil: the soil was treated with gamma radiation (27 KGy) in order to kill the indigenous nematodes and potential predators. However observation from extracted fresh soil indicated that interference from the indigenous population was likely to be insignificant. Consequently, the definitive test were carried out using unsterilised soil.

- Storage: stored in closed black plastic bags at 4 deg C until required.


OTHER TEST CONDITIONS

- Photoperiod: vials were placed in the dark.


EFFECT PARAMETERS MEASURED: number of test organisms at the end of the test, i.e. after 72 h (comparison with solvent control)


VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 3

- Range finding study

- Test concentrations: 10, 100, 1000 mg/kg

- Results used to determine the conditions for the definitive study: 72h EC50 = 100-1000 mg/kg dw soil
Nominal and measured concentrations:
Nominal concentrations: 0, 10, 30, 100, 300, 1000 mg/kg
Reference substance (positive control):
yes
Remarks:
dimethoate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
98 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: reproduction and survival
Remarks on result:
other: (95% CL 89-100 mg/kg soil dw)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
30 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: reproduction and survival
Details on results:
- Mortality at end of exposure period: at the end of the exposure (72 h) the effect level at the two highest concentrations (300 and 1000 mg/kg dw) was 100%. See table 1 for details.

The effect level was calculated by comparing the total number of nematodes seen in treated samples with the number seen in solvent controls.

See table 2 for details on results with other species.
Results with reference substance (positive control):
- Results with reference substance valid? no effect on nematode population was observed following exposure to dimethoate at 10, 100 or 1000 mg/kg soil dw. Organisms were considered to be tolerant by the authors of the report.
Reported statistics and error estimates:
Probit analysis (Finney 1971) was used to determine EC50 values and their corresponding 95% intervals in definitive tests.

Table 1. Result of C. elegans exposure to decanol, number of surviving animals and offspring combined at 72 h.

 Compound  Dose (mg/kg dw soil)  Number of C. elegans found in extracts (average)
 Control  -  36
 Solvent control  -  40
 Dimethoate        100  33
 300  23
 1000  23
 C10              10  49
 30  47
 100  19
 300  0
 1000  0

Solvent control is acetone used (up to 7 ml of acetone was used to prepare stock solutions with sand, then added to soil, however the acetone was left to evaporate before exposure).

The EC value should be normalised to a soil with 2% organic carbon according to the following equation.

 

NOEC or L(E)C50 (standard) = NOEC or L(E)C50 (exp) * [Focsoil(standard)/ Focsoil(exp)]

 

Where Focsoil = fraction organic carbon in soil

 

So 72h EC50 98 mg/kg dw * [2.0%/1.3%] = 98 * 1.54 = 151 mg/kg dw

and 72 h NOEC 30 mg/kg dw * 1.54 = 46 mg/kg dw

Table 2. Results of the test conducted with species: Eisenia foetida, Fulsomia candida, Heterocypris incongruens.

 Species  Duration (d)  EC50 (mg/kg dw)
 Eisenia foetida  7  170
 Fulsomia candida  7  320
 Heterocypris incongruens  6  150

Results are based on reproduction and survival.

Validity criteria fulfilled:
not applicable
Remarks:
The test is not supported by analysis and there are some deviations from the Donkin & Dusenbury mortality test protocol.
Conclusions:
A 72 h EC50 value of 98 mg/kg dw soil and NOEC of 30 mg/kg dw soil have been determined for the effects of the test substance on population numbers of the nematode C. elegans. The EC50 and NOEC normalised for standard soil organic carbon content 2% would be 151 mg/kg dw soil and 46 mg/kg dw soil respectively. The test is not supported by analysis. Therefore the results are considered to be indicative only and not suitable for quantitative application in the chemical safety assessment.
Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Study conducted according to a modified Guideline protocol with several deviations; not GLP; no analytical monitoring; no information on test item equilibration with the soil.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
yes
Remarks:
(method of test animals recovery from vials changes; lower soil moisture; juvenile worms instead of adults were used; test duration 7d rather than 14d)
Principles of method if other than guideline:
The test method was modified to improve the recovery rate of test animals. The amount of soil requiring extraction was reduced, worms were extracted by adding 5ml Ludox HS40 to each vial then shaking the vial vigorously on a 'whirlmixer'. The Ludox/soil suspension were centrifuged for longer and at a higher speed (7000 rpm and for 15 minutes) in order to improve the clarity of the supernatant, which was sieved and the organisms rinsed with M9 salt solution and counted under a high power microscope. The test also reduced moisture content of soil respect to the OECD guideline, 15-20% rather than 20-30%, but the authors of the report thought that this would make no difference to the survival of organisms due to the short duration of the test.
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
yes
Details on preparation and application of test substrate:
- Method of mixing into soil: sand spike. This involved adding an appropriate amount of compound to analar grade acetone to form a stock solution for the 1000 mg/kg dose. Aliquots of the stock solution were then diluted with acetone to prepare lower doses. Acetone solutions were added to silica sand in glass jars. Mixtures were mixed thoroughly with a stainless steel spatula. The acetone was left to evaporate in a fume cupboard, leaving the test item coated onto the sand. Appropriate amount of moist soil was added to each of the glass jars. The dosed or undosed sand was incorporated into the bulk of the soil using a paletted knife. Jars were then sealed with screw top lids containing aluminium foil inserts and tumbled overnight (ca. 16 h) on a rotary soil tumbler 50 rpm.

- Controls: One aliquot of sand was dosed with acetone only to serve as solvent control. A jar with an aliquot of sand only served as untreated control - both with the appropriate amount of moist soil added thereafter.

- Chemical name of vehicle: acetone

- Concentration of vehicle in test medium: acetone solutions was added between 1-7 ml to 5-35g of silica sand. The sand was left to evaporate the acetone completely before initial exposure, so that the concentration of vehicle would be negligible.

- Evaporation of vehicle before use: yes
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM

- Common name: earthworm

- Source: Blades Biological, Cowden, Edenbridge, Kent, UK.

- Age at test initiation: adults

- Weight at test initiation (mean and range, SD): 0.076 (s.d. +/- 0.034 d)

- Length at test initiation: 2-3 cm


ACCLIMATION

- Acclimation period: not specified.

- Acclimation conditions (same as test or not): similar to test
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
7 d
Post exposure observation period:
none
Test temperature:
20 +/- 2 degC
pH:
range: 5.8-6.1
Moisture:
range: 17.6 to 19%
Details on test conditions:
TEST SYSTEM

- Test container (material, size): sealed jars (with screw cap top)

- Amount of soil or substrate: 10 g wet weight of sterilised soil

- No. of organisms per container (treatment): 10

- No. of replicates per treatment group: 3

- No. of replicates per control: 3

- No. of replicates per vehicle control: 3


SOURCE AND PROPERTIES OF SUBSTRATE

- Geographic location: Heath Farm, Leicester Lane, LandLook (Midlands), Leamington Spa, from Dr Graham Beard. OS map reference 32896926.

- Pesticide use history at the collection site: assumed to be pesticide free, as the field the soil was taken from land that had been set aside for 3-4 years.

- Collection procedures: the soil was sieved to 2 mm.

- Sampling depth (cm): 5-20cm to avoid surface root mat

- Soil texture

- % sand: 63.8%-64.8%

- Soil classification: sandy loam soil. The soil is described as soil series, Bromsgrove, standard soil number 33, variant.

- Organic carbon (%): 1.3%

- Maximum water holding capacity: ca. 40%

- Pretreatment of soil: n/a.

- Storage: stored in closed black plastic bags at 4 degC until required.


OTHER TEST CONDITIONS

- Photoperiod: continuous light


EFFECT PARAMETERS MEASURED: mortality (immobilisation) at the end of the test


VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 3

- Range finding study

- Test concentrations: 10, 100, 1000 mg/kg

- Results used to determine the conditions for the definitive study: 7d EC50 100-1000 mg/kg
Nominal and measured concentrations:
Nominal concentrations: 0, 10, 30, 100, 300, 1000 mg/kg
Reference substance (positive control):
yes
Remarks:
dimethoate
Duration:
7 d
Dose descriptor:
LD50
Effect conc.:
170 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival
Remarks on result:
other: (95% CL 100-300 mg/kg soil dw)
Details on results:
- Mortality at end of exposure period: 100% at 300 and 1000 mg/kg
Results with reference substance (positive control):
- Results with reference substance valid? yes, 67 mg/kg soil dw in the definitive test.
Reported statistics and error estimates:
Probit analysis (Finney 1971) was used to determine EC50 values and their corresponding 95% intervals in definitive tests.

Table 1: Result of E. fetida exposure to decanol, results of definitive test

 Compound  Dose (mg/kg dw soil) Number of E. fetida: live (average)   Number of E. fetida (average): dead / missing (average)
 Control  -  10  0
 Acetone control  -  10  0
 Dimethoate  10  10  0
   30  6.33  3.67
   100  4.67  5.33
   300  1  9
   1000  0  10
 C10  30  10  0
   100  10  0
   300  0  10
   1000  0  10

Solvent control is acetone (up to 7 ml of acetone was used to prepare stock solutions with sand, then added to soil, however the acetone was left to evaporate before exposure).

 

The EC value should be normalised to a soil with 2% organic carbon according to the following equation.

 

NOEC or L(E)C50 (standard) = NOEC or L(E)C50 (exp) * [Focsoil(standard)/ Focsoil(exp)]

 

Where Focsoil = fraction organic carbon in soil

 

So 7 d LD50 170 mg/kg dw * [2.0%/1.3%] = 170 * 1.54 = 262 mg/kg dw

Table 2. Results of the tests conducted with species: Eisenia fetida, Folsomia candida, Heterocypris incongruens.

 Species  Duration (d)  EC50 (mg/kg dw)
 Caenorhabditis elegans  72  98
 Folsomia candida  7  320
 Heterocypris incongruens  6  150

Results are based on reproduction and survival.

Validity criteria fulfilled:
yes
Conclusions:
A 7-d LC50 value of 170 mg/kg soil dw has been determined for the effects of the test substance on survival of the earthworm Eisenia foetida. The LC50 normalised for standard soil organic carbon content (2%) is 262 mg/kg soil dw.
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Data waiving:
other justification
Justification for data waiving:
other:

Description of key information

No reliable, standard tests of soil macroorganisms are available. Effects on E. foetida (earthworm): LC50 (7 d) 170 mg/kg soil dw is available from a study with some deficiencies. Effects on C. elegans (nematode): EC50 (72 h) 98 mg/kg soil dw and NOEC 30 mg/kg soil dw is available from a non-guideline study with some methodological deficiencies.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
98 mg/kg soil dw
Long-term EC10, LC10 or NOEC for soil macroorganisms:
30 mg/kg soil dw

Additional information

Short-term toxicity 

A 72 h EC50 value of 98 mg/kg soil dw and NOEC of 30 mg/kg soil dw (1.3% organic carbon) has been determined for the effects of decan-1-ol on population numbers of the nematode C. elegans. When normalised for standard organic carbon content, these results are equivalent to EC50 of 151 and NOEC of 46 mg/kg soil dw. The study reflects the lowest value that is available for this endpoint. Although a relatively short exposure duration, 72 h is sufficient to cover a full life cycle of this species. The normal duration for a mortality study with this species is 24 h.

 

A 7-d LC50 value of 170 mg/kg soil dw has been determined for the effects of decan-1-ol on survival of the earthworm Eisenia foetida. The LC50 normalised for standard soil organic carbon content (2%) is 262 mg/kg soil dw. 

 

Long-term toxicity 

In accordance with Column 2 of REACH Annex X, further long-term toxicity testing with terrestrial invertebrates (required in Section 9.4.4) is not needed as the chemical safety assessment according to Annex I indicates that this is not necessary. Moreover, considerable technical difficulties would be expected in the conduct of such a test, due to the very rapid biotic removal of the substance from the test system. Please refer to discussion of the long-term aquatic invertebrate and fish studies, and evidence of rapid removal in non-sterilised soils during method development for the adsorption/desorption study with natural soils in the environmental fate section.