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Toxicity to soil macroorganisms except arthropods

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Reference
Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: ASTM E2172-01 (Standard guide for conducting laboratory soil toxicity tests with the nematode Caenorhabditis elegans)
Version / remarks:
2001
Deviations:
not specified
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
not specified
Details on preparation and application of test substrate:
Tests were carried out with a range of concentrations of LAB in soil up to 20,000 mg/kg dry soil (2% w/w). The greatest concentration was mixed first, with lower concentrations being produced by diluting contaminated soil with uncontaminated soil. Soil was first adjusted to 15% moisture content to allow effective mixing with minimum dust.
Test organisms (species):
Caenorhabditis elegans
Animal group:
nematods
Details on test organisms:
TEST ORGANISM
- Common name: soil nematode
- Strain: Wild type strain N2
- Source: Cultured in Petri dishes of nutrient agar inoculated with a lawn of Escherichia coli OP50. Plates were inoculated and incubated at 37 ºC overnight prior to use. The dishes of worms were incubated at 20 °C. Test animals were taken from a synchronised culture of adult worms.
- Synchronised culture condition: Worms from a 9-day culture were suspended in 3 mL M9 buffer (6 g/L Na2HPO4, 3 g/L KH2PO4, 5 g/L NaCl, 0.25 g/L MgSO4.7H2O) from which 1 mL was removed and pipetted on top of 2 mL of Ficoll suspension (Ficoll 400 diluted to 15% (w/v) in 0.1 M NaCl) in a 15 mL centrifuge tube, taking care not to mix the two layers. The tube was left for 10 min, during which time the active adults migrated to the upper surface, while the relatively immotile dauer larvae sank into the lower layer. The top layer was removed and the dauer larvae washed 3 times with distilled water by centrifuging at 2000 rpm for 2 min and pouring off the supernatant. The dauer larvae were placed on E. coli OP50 plates and incubated at 20 °C for 48 h to mature. Adult worms were collected in 5 mL M9 buffer and placed in a 15 mL centrifuge tube. They were washed twice with dH2O and treated with 7 mL alkaline hypochlorite solution (2 mL Clorox + 5.0 mL 1 M NaOH) for 15 min to kill the gravid adults and lyse the body wall to release the eggs. The tubes were vortexed briefly every 2 min during the alkaline hypochlorite treatment to prevent hypoxia. Eggs were harvested by centrifuging at 1,000 rpm for 2 min and washing the pellet twice in 10 mL M9 buffer. The final pellet was resuspended in 0.5 mL M9 buffer and transferred onto NA plates inoculated with E. coli OP50.
- Synchronised culture time: These organisms were incubated at 20 °C for 4 days to obtain a synchronised culture of adult worms.
- Age at test initiation: 13 days (9 days culture + 4 days synhronised culture)
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
24 h
Test temperature:
20 °C
pH:
5.8
Moisture:
15%
Details on test conditions:
TEST SYSTEM
- Test container (material, size): 35 × 10 mm (diameter × depth) polystyrene Petri dishes
- Amount of soil: 2.33 g dry weight per replicate
- No. of organisms per replicate: 10
- No. of replicates per treatment group: 3
- No. of replicates per control: 3
- Introduce nematodes: The nematodes were removed under a dissecting microscope by lifting under the body using a flamed and cooled platinum wire. To prevent desiccation of the seed culture, 0.5 ml M9 buffer was pipetted onto the surface of the agar. This was supplemented as required with additional buffer. Individual worms were placed directly on the soil surface and observed to ensure that they burrowed into the soil. Where there was any doubt regarding the health of a worm, it was removed from the soil, the wire was flamed and a new worm was transferred.
- Recover nematodes: The worms were recovered from the soil by differential density flotation in 2 mL colloidal silica (50 mL Ludox HS-40, 50 mL dH2O, adjusted to pH 7 with conc. HCl).

SOURCE AND PROPERTIES OF SUBSTRATE
- Geographic location: Scottish Agricultural College site on the Bush Estate, Edinburgh
- Collection procedures: The soil was collected by removing the turf and topsoil to a depth of approximately 20 cm. Subsoil (20 - 35 cm depth) was dug and kept refrigerated in sealed polythene bags until required for use.
- % sand: 44%
- % silt: 39%
- % clay: 17%
- Test of moisture content: Moisture content of the soil was determined by accurately weighing 3-g soil samples before and after drying at 105 °C for 24 h or to constant weight.
- Organic carbon (%): 5.8% w/w
- pH: 5.8
- Equilibrate period: 7 days (The soil surface was smoothed with a stainless steel spatula to facilitate the introduction of the nematodes. Once prepared, the containers were placed in a sealed container lined with damp absorbent paper. This was stored in the dark at 20 °C for 7 d to allow the soils to equilibrate.)

OTHER TEST CONDITIONS
- Test in darkness: Yes

EFFECT PARAMETERS MEASURED
The contents of each test chamber were transferred to a clean 50 mlLcentrifuge tube. The bulk of the soil was carefully transferred using a clean spatula. Soil adhering to the test chamber was rinsed with the Ludox suspension. The tubes were capped and vortexed gently in order to suspend the soil, then centrifuged at 2,000 rpm for 2 min and left to settle for 15 to 30 min in order to allow the nematodes to migrate to the liquid surface.
The liquid was withdrawn using a 5-mL pipette and placed in a clean Petri dish with a grid ruled on the base to facilitate the search for test organisms. It was inspected under a dissecting microscope and worms were removed using a 200 μL pipette. If the worms were seen to be moving before removal, they were counted as living. If there was any doubt, they were deposited on a lawn of E. coli OP50. If after several minutes the worms did not react to gentle stimulation of the anterior end with a platinum wire they were counted as dead, as were any worms not recovered.


Nominal and measured concentrations:
six concentrations of LAB in soil up to 20,000 mg/kg dry soil (2% w/w), plus an uncontaminated control.
Reference substance (positive control):
no
Key result
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
7 960 mg/kg soil ww
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 C.L.: 4060 - 10980 mg/kg soil dw
Details on results:
Overview of the results is provided in Table 1 in 'Any other information on results incl. tables'.

- Mortality: At least 80% of test animals were recovered from all treatments, with a clear increase of mortality with concentration. Approximately 70% test organisms were survived in the uncontaminated soil. A slightly higher than 20% organisms were survived in 10,000 mg/kg soil d.w. while less than 10 % organisms were survived in 20,000 mg/kg soil d.w. Mean mortality in the negative control treatments was 30%, which is higher than the 10% recommended in the standard method (ASTM 2001).

- LAB recovery: The oil content and isomeric composition analysis by GCMS. Verified that the soil samples were homogeneous and that no measurable biodegradation of LAB occurred during the 24 h exposure period. LAB recovery was 71.3% (standard deviation = 6.99, n = 9).

Reported statistics and error estimates:
The mortalities for each of the 3 replicates of each treatment were pooled and the LC50 (C. elegans, 24 h) was calculated by Probit analysis using the MS-DOS probit.exe program from the USEPA (EPA 2003). The LAB content of the soil remaining in the centrifuge tubes was verified by GC-MS of a hexane extraction with 1-(dodecyl)benzene internal standard. This was done both to verify the relative oil contents of the different concentrations and, by analysis of the isomeric composition, to ensure that no appreciable biodegradation had occurred during the equilibration period

Table 1.Probit analysis - concentrations of linear alkylbenzene required to achieve a range of lethalities in C. elegans after 24 h exposure in a loam soil, with upper and lower 95% confidence limits (CL).

LC

LAB concentration, mg/kg dry weight soil (%)

Lower 95 % CL, mg/kg

Upper 95 % CL, mg/kg

1

1,550 (0.155)

80

3,360

5

2,500 (0.255)

260

4,600

10

3,230 (0.323)

480

5,450

15

3,840 (0.384)

740

6,140

50

7,960 (0.796)

4,060

10,980

85

6,520 (0.652)

11,920

36,800

90

19,630 (1.963)

13,810

54,610

95

25,360 (2.536)

16,770

100,290

99

40,980 (4.098)

23,370

323,790

Validity criteria fulfilled:
yes
Conclusions:
Based on the findings, the LC50 was determined to be 7960 mg/kg soil dry weight (with 95% confidence limit of 4060 - 10980 mg/kg soil dry weight).
Executive summary:

Acute toxicity of LAB was tested on the soil nematode,Caenorhabditis elegans, according to ASTM E2172-01 guideline (2001). 9-day old of the test organisms were incubated at 20 °C for 4 days to obtain a synchronised culture of adult worms. Three replicates of 2.33 g (dry weight) of each of six concentrations of LAB (up to 20,000 mg/kg dry soil) in hydrated soil, plus uncontaminated controls, were placed in individual 35×10 mm (diameter×depth) polystyrene Petri dishes. A loam soil (44% sand, 39% silt, 17% clay, total organic carbon 5.8% w/w, pH 5.8) was used in this study. The soil surface was smoothed with a stainless steel spatula to facilitate the introduction of the nematodes.Once prepared, the containers were placed in a sealed container lined with damp absorbent paper. This was stored in the dark at 20 °C for 7 d to allow the soils to equilibrate.Each replicate was populated with 10 nematodes from 3- to 4-d old synchronised cultures. Tests were performed in darkness for 24 hours.

After 24 hours exposure, at least 80% of test animals were recovered from all treatments, with a clear increase of mortality with concentration. Approximately 70% test organisms were survived in the uncontaminated soil. A slightly higher than 20% organisms were survived in 10,000 mg/kg soil d.w. while less than 10 % organisms were survived in 20,000 mg/kg soil d.w. Mean mortality in the negative control treatments was 30%, which is higher than the 10% recommended in the standard method (ASTM 2001). The oil content and isomeric composition analysis by GCMS. Verified that the soil samples were homogeneous and that no measurable biodegradation of LAB occurred during the 24 h exposure period. LAB recovery was 71.3% (standard deviation = 6.99, n = 9).

Based on the findings, the LC50 was determined to be 7960 mg/kg soil dry weight (with 95% confidence limit of 4060 - 10980 mg/kg soil dry weight).

Description of key information

A 24-hour acute toxicity test of the test subtsance to soil nematode (Caenorhabditis elegans) was conducted according to ASTM E2172 -01 guideline (2001). The LC50 was determinded to be 7960 mg/kg soil dry weight (with 95% confidence limit of 4060 - 10980 mg/kg soil dry weight).

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
7 960 mg/kg soil dw

Additional information

Acute toxicity of the test substance was tested on the soil nematode,Caenorhabditis elegans, according to ASTM E2172-01 guideline (2001). 9-day old of the test organisms were incubated at 20 °C for 4 days to obtain a synchronised culture of adult worms. Three replicates of 2.33 g (dry weight) of each of six concentrations of LAB (up to 20,000 mg/kg dry soil) in hydrated soil, plus uncontaminated controls, were placed in individual 35×10 mm (diameter×depth) polystyrene Petri dishes. A loam soil (44% sand, 39% silt, 17% clay, total organic carbon 5.8% w/w, pH 5.8) was used in this study. The soil surface was smoothed with a stainless steel spatula to facilitate the introduction of the nematodes.Once prepared, the containers were placed in a sealed container lined with damp absorbent paper. This was stored in the dark at 20 °C for 7 d to allow the soils to equilibrate.Each replicate was populated with 10 nematodes from 3- to 4-d old synchronised cultures. Tests were performed in darkness for 24 hours.

After 24 hours exposure, at least 80% of test animals were recovered from all treatments, with a clear increase of mortality with concentration. Approximately 70% test organisms were survived in the uncontaminated soil. A slightly higher than 20% organisms were survived in 10,000 mg/kg soil d.w. while less than 10 % organisms were survived in 20,000 mg/kg soil d.w. Mean mortality in the negative control treatments was 30%, which is higher than the 10% recommended in the standard method (ASTM 2001). The oil content and isomeric composition analysis by GCMS. Verified that the soil samples were homogeneous and that no measurable biodegradation of LAB occurred during the 24 h exposure period. LAB recovery was 71.3% (standard deviation = 6.99, n = 9).

Based on the findings, the LC50 was determined to be 7960 mg/kg soil dry weight (with 95% confidence limit of 4060 - 10980 mg/kg soil dry weight)

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