Biological test method for measuring survival of springtails exposed to contaminants in soil: appendix F


Appendix F

Procedural Variations for Tests of Effects of Contaminated Soil on the Survival and Reproduction of Springtails, as Described in International Methodology Documents

Source documents are listed chronologically, by originating agency or by author(s).

W & K 1998 (Wiles and Krogh 1998) - this publication describes protocols for measuring survival, growth, and reproduction effects in three species of springtails including Folsomia candida (Willem, 1902), Isotoma viridis(Bourlet, 1839), and Folsomia fimetaria (Linné, 1758); published in 1998 in the “Handbook of Soil Invertebrate Toxicity Tests,” Løkke and van Gestel (eds.), West Sussex, England.

ISO 1999 - an international standard test method for measuring soil toxicity using a test for effects on reproduction of Folsomia candida, published in 1999 by the International Standard Organization in Geneva, Switzerland.

OECD 2005 - a draft proposal released by the National Environmental Research Institute in Denmark, to the Organization for Economic Cooperation and Development (OECD), for a new test guideline that assesses the effects of chemical-spiked soils on the reproduction of two species of Collembola (Folsomia fimetariaand Folsomia candida); under consideration for publication by the OECD in Paris, France.

1. Test Species, Test Type, and Test Duration

Document Species Test Type Test Duration
W & K 1998 Folsomia candida(Willem, 1902) static-renewalFootnote1 8 weeksFootnote2
W & K 1998 Isotoma viridis(Bourlet, 1839) static-renewalFootnote1 8 weeks
W & K 1998 Folsomia fimetaria (Linné, 1758) static 3 weeks
ISO 1999 Folsomia candida(Willem, 1902) static 28 days
OECD 2005 Folsomia fimetaria (Linné, 1758) static 3 weeks
OECD 2005 Folsomia candida(Willem, 1902) static 4 weeks

2. Specifics on Test Organisms at Start

Document Description of Organisms Used to Start Test Acclimation Conditions Age of Organisms at Test Start
W & K 1998 F. candida cultured juveniles, age-synchronized NIFootnote3 10 to 12 days
W & K 1998 I. viridis cultured juveniles, age-synchronized NI 5 to 7 days
W & K 1998 F. fimetaria cultured juveniles, age-synchronized NI 23 to 26 days
ISO 1999 cultured juveniles, age-synchronized NI 10 to 12 days
OECD 2005 cultured adults, age-synchronized NI adults

3. Test Vessels and Materials

Document Test Vessel Cover Type of Test SoilFootnote4 Amount of Soil/Container
W & K 1998 F. candida 5 × 2 cm glass tubes, closed tightly tight fitting lid AS, StS 4 ± 0.1 g per tube, dry wt (~5 g wet wt)
W & K 1998 I. viridis 5 × 2.5 cm glass tubes, closed tightly tight fitting lid AS, StS 4 ± 0.1 g per tube, dry wt (~5 g wet wt)Footnote5
W & K 1998 F. fimetaria cylinder; 6 cm in diameter, 5.5 cm high; 1 mm mesh bottom; closed lids at top and bottom AS, StS 30 g per container, wet wt
ISO 1999 100 mL glass container; 5 cm in diameter; closed tightly plastic or glass disk, or parafilm AS 30 g per container, wet wt
OECD 2005 partly transparent; transparent lids which reduce water evaporation but allow gas exchange glass or inert plastic AS, StS amount corresponding to 25 g dry wt; 3-4 cm deep

4. Description of Test Soils, Including Composition of Artificial Soil

Document Description of Test Soil(s) Composition of Artificial SoilFootnote6
W & K 1998 F. candida standard soilFootnote7 with added test substance (e.g., chemical in deionized water or organic solvent; if insoluble, test substance mixed in fine quartz sand)Footnote8 and distilled or deionized water 10% sphagnum peat,Footnote9 20% kaolinite clay with ≥ 30%  kaolinite, and 70% industrial sand with > 50% particles 50-200 μm adjust to pH 6.0 ± 0.5 using CaCO3,
W & K 1998 I. viridis standard soilFootnote7, Footnote10with added test substance (e.g., chemical in deionized water or organic solvent; if insoluble, test substance mixed in fine quartz sand)3 and distilled or deionized water 10% sphagnum peat,Footnote9 20% kaolinite clay with ≥ 30% kaolinite, and 70% industrial sand with > 50% particles 50-200 μm, adjust to pH 6.0 ± 0.5 using CaCO3
W & K 1998 F. fimetaria standard soilFootnote7 with added test substance (e.g., chemical in deionized water or organic solvent; if insoluble, test substance mixed in fine quartz sand)Footnote8 and distilled or deionized water 10% sphagnum peat,Footnote12 20% kaolinite clay with ≥ 30% kaolinite, and 70% industrial sand with > 50% particles 50-200 μm, adjust to pH .0 ± 0.5 using CaCO3
ISO 1999 artificial soil with added test substance (e.g., chemical in deionized water or organic solvent; if insoluble, test substance mixed in fine quartz sand)Footnote11 10% sphagnum peat,Footnote12 20% kaolinite clay with ≥ 30% kaolinite, and 70% ndustrial sand with > 50% particles 50-2003μm, adjust to pH 6.0 ± 0.5 using CaCO3
OECD 2005 artificial soil or standard soilFootnote13 with added test substances (e.g., chemical in de-ionized waterFootnote14 or in organic solvent; if insoluble, test substance is mixed with fine quartz sandFootnote15 5 or 10% sphagnum peat,Footnote16 20% kaolin clay with > 30% kaolinite, and 69-70%Footnote17 air- dried industrial sand with > 50% particles 50-2003μm, adjust to pH 6.0 ± 0.5 using CaCO3

5. Manipulation of Artificial Soil Before Use in Test

Document Mixing Hydration pH Adjustment
W & K 1998 F. candida blend dry constituents in correct proportions and mix with some deionized water; hydrate hydrate to crumbly structure; normally to 40-60% of total water-holding capacity none when acidic or basic substances are tested
W & K 1998 I. viridis blend dry constituents in correct proportions and mix with some deionized water; hydrate hydrate to crumbly structure; normally to 40-60% of total water-holding capacity none when acidic or basic substances are tested
W & K 1998 F. fimetaria blend dry constituents in correct proportions and mix with some deionized or distilled water; hydrate hydrate to porous soil texture one week before initiating test none when acidic or basic substances are tested
ISO 1999 blend dry constituents in correct proportions and mix with some deionized water; hydrate hydrate to crumbly structure; normally to 40-60% of total water-holding capacityFootnote18 NIFootnote19
OECD 2005 blend dry constituentsFootnote20 hydrate to ~50% of the maximum water-holding capacityFootnote21 (corresponding to 50 ± 10% moisture dry mass) moisture content should be optimized to loose porous texture NI

6. Negative Control Soil

Document Description of Negative Control Soil Number of Control Vessels (replicates)
W & K 1998 F. candida prepare in the same way as that for test soils, but without the test substance; use additional controls if auxillary substances other than water are used to dissolve test substance (e.g., organic solvent, quartz sand) ≥ 3
W & K 1998 I. viridis prepare in the same way as that for test soils, but without the test substance; use additional controls if auxillary substances other than water are used to dissolve test substance (e.g., organic solvent, quartz sand) ≥ 3
W & K 1998 F. fimetaria prepare in the same way as that for test soils, but without the test substance; use additional controls if auxillary substances other than water are used to dissolve test substance (e.g., organic solvent, quartz sand) ≥ 4
ISO 1999 prepare in the same way as that for test soils, but without the test substance; use additional controls if auxillary substances other than water are used to dissolve test substance (e.g., organic solvent, quartz sand) 5
OECD 2005 prepare in the same way as for test soils, but without the test substance; apply organic solvents, quartz sand or other vehicles to additional controls in amounts consistent with those used in treatments ≥ 4

7. Storage and Characterization of Test Soil

Document Storage Conditions Soil CharacterizationFootnote22
W & K 1998
F. candida
store mixed test soils in closed glass vessels at 15°C until required moisture content and pH after hydrating
W & K 1998
I. viridis
store mixed test soils in closed glass vessels at 15°C until required moisture content and pH after hydrating
W & K 1998
F. fimetaria
NIFootnote23 moisture content and pH after hydrating
ISO 1999 NI, NAFootnote24 moisture content and pH after hydrating
OECD 2005 NI, NA water-holding capacity and pH

8. Preliminary Test - Number of Organisms per Vessel, Number of Replicates per Treatment, Number of Concentrations per Sample, and Recommended Dilution Factor

Document Number of Organisms per Vessel Number of Replicates per Treatment or Concentration Number of Concentrations per Sample or Test Material Recommended Dilution Factor
W & K 1998
F. candidaFootnote25Footnote26
10 NIFootnote27 5 geometric seriesFootnote28
W & K 1998
I. viridisFootnote25Footnote26
20 NI 5 geometric seriesFootnote28
W & K 1998
F. fimetariaFootnote25, Footnote29
20Footnote 3030 1 4 + control geometric seriesFootnote 3131
ISO 1999Footnote25, Footnote 3232 10 1 4 + control geometric seriesFootnote 3131
OECD 2005Footnote25, Footnote 3333 F. fimetaria 20Footnote 3030 2 5 + control geometric seriesFootnote 3434
OECD 2005Footnote25, Footnote 3333 F. candida 10 2 5 + control geometric seriesFootnote 3434

9. Definitive Test - Number of Organisms per Vessel, Number of Replicates per Treatment, Number of Concentrations per Sample, and Recommended Dilution Factor

Document Number of Organisms per Vessel Number of Replicates per Treatment or Concentration Number of Concentrations per Sample or Test Material Recommended Dilution Factor
W & K 1998
F. candida
10 ≥ 3 ≥ 5 + control NIFootnote 3535, Footnote 3636
W & K 1998
I. viridis
20 ≥ 3 ≥ 5 + control NIFootnote 3636, Footnote 3737
W & K 1998 F. fimetaria 20Footnote 3838 ≥ 4 NI NI Footnote 3939, Footnote 4040
ISO 1999Footnote 4141 10 for NOEC: 5 ≥ 5 + control geometric seriesFootnote 4242
    for ECx: ≥ 2 for treatments and 5 for controls 12 + control geometric seriesFootnote 4343
OECD 2005 F. fimetaria 20Footnote 3838 ≥ 4 NI NI Footnote 3939, Footnote 4040
OECD 2005 F. candida 10 ≥ 4 ≥ 4 + controlFootnote 4444 NIFootnote 3535, Footnote 4545

10. Feeding and Aeration During Test

Document Type of Food Recommended Feeding Quantity, Procedure, and Frequency Aeration of Test Vessels
W & K 1998
F. candida
granules of baker’s yeast add 3 granules to each test tube at the start of the test and at each weekly transferral NIFootnote 4646
W & K 1998
I. viridis
granules of baker’s yeast add 3 granules to each test tube at the start of the test and at each weekly transferral NI
W & K 1998
F. fimetaria
dried baker’s yeast add 15 mg at the beginning of the test and after 14 days NI
ISO 1999 granulated dry yeast add ~2 mg of granulated dry yeast to each test vessel at the beginning of the test and after a period of 14 days; cover vessels tightly after feeding test containers are opened briefly two times per week to allow for aeration
OECD 2005 granulated dry baker’s yeast add 15 mg at the beginning of the test and after each 14-day interval NI

11. Temperature and Lighting During Test

Document Temperature (°C) Lighting ConditionsFootnote 4747
W & K 1998 F. candida 15 ± 0.5 12h L:12h D or 16h L:8h D; intensity <1000 lux
W & K 1998 I. viridis 15 ± 0.5Footnote 4848 16h L:8h D; intensity <1000 lux
W & K 1998 F. fimetaria 20 ± 1 12h L:12h D; intensity 400-800 lux
ISO 1999 20 ± 2 12h L:12h D or 16h L:8h D; intensity 400-800 lux at substrate surface
OECD 2005 20 ± 2 16h L:8h D preferred; intensity of 400-800 lux in the area of the test vessels

12. Measurements and Biological Observations During Test

Document MeasurementsFootnote 4949 Biological Observations
W & K 1998 F. candida W and pH, each treatment, at start and end of test Footnote 5050, Footnote 5151 number of live adults per test vessel, measurements of individual body lengths of surviving adults,Footnote 5252and number of juveniles hatching from eggs collected from soilFootnote 5353weekly and at the end of the test
W & K 1998 I. viridis W and pH, each treatment at start and end of testFootnote 5050, Footnote 5151 number of live adults per test vessel, measurements of individual body lengths of surviving adults,Footnote 5252weekly and at the end of the test
W & K 1998 F. fimetaria W and pH at start and end of testFootnote 5151Footnote 5454 number of live adults per test vessel, number of offspring per test container at the end of the test
ISO 1999 W and pH, each treatment, at start and end of test; reweigh test containers periodically throughout testFootnote 5555Footnote 5656 number of live adult and juvenile springtails per test vessel at the end of the exposure
OECD 2005 W and pHFootnote 5757 each treatment at start and end of test; weight of test vessels at start and weekly thereafterFootnote 5858  

13. Terminating Test, Biological Endpoints, and Statistical Endpoints

Document Terminating Test Biological Endpoints Statistical Endpoints
W & K 1998
F. candida
Collembola are extracted from soil in the tubes at weekly intervals during the exposure and at the end of the test; the soil from each tube is put into a larger vessel and Collembola are collected with a hand-held air aspirator mean percent survival, mean body size of individuals, and mean number of juveniles produced at each concentration and each assessment interval EC10 and EC50 for growth and reproduction (mg/kg dry wt of soil)
W & K 1998
I. viridis
Collembola are extracted from soil in the tubes at weekly intervals during the exposure and at the end of the test; the soil from each tube is put into a larger vessel and Collembola are collected with a hand-held air aspirator mean percent survival and mean body size of individuals produced at each concentration and each assessment interval EC10 and EC50 for growth (mg/kg dry wt of soil)
W & K 1998
F. fimetaria
At the end of 3 weeks, the organisms are extracted from each test container with a high gradient extractorFootnote 5959 mortality, percent loss or increase of adult biomass, and number of offspring produced EC10, EC50, and LC50 for chronic mortality and growth (mg/kg dry wt of soil)
ISO 1999 4 weeks after introducing parental springtails onto the test and control substrates, the test substrate is poured into a 500-600 mL container and water is added; the suspension is gently stirred;the adults and juveniles floating on the water surface are countedFootnote 6060 mean number of adults and juveniles for each concentration; percent mortality of adults per concentration; mean number of offspring produced for each concentration after 28 days NOEC and ECxFootnote 6161
OECD 2005 Collembola are extracted from soil with a high gradient heat extractor1 offspring produced or by floatationFootnote 6262 mortality and number of offsprings produced LC50, ECx, and NOEC

14. Requirements for Valid Test; Use of Reference Toxicity Test

Document Requirements for Valid Test Reference Toxicant(s) Procedures and Conditions for Reference Toxicity Test
W & K 1998
F. candida
≥ 10 juveniles/surviving parental adult in control soil each week; percent mortality of adults in control(s) < 20% none recommended at present NIFootnote 6666
W & K 1998
I. viridis
percent mortality of adults in control(s) < 50% none recommended at present NI
W & K 1998
F. fimetaria
> 200 juveniles/control vessel; percent mortality of adults in control(s) < 20% dimethoateFootnote 6363 and reproduction determine effects on survival
ISO 1999 ≥ 100 instars/control vessel; CV for control reproduction ≤ 30%; percent mortality of adults in control(s) ≤ 20% Betanal plus; and E 605 forteFootnote 6464Footnote 6565 determine effects on survival and reproduction once or twice per year
OECD 2005 F. fimetaria > 200 juveniles/control vessel; percent mortality of adults in control(s) < 20%; CV of juveniles in controls < 25% to be determined determine effects on survival and reproduction
OECD 2005 F. candida ≥ 100 juveniles/control vessel; percent mortality of adults in control(s) < 20%; CV of juveniles in controls < 25% to be determined determine effects on survival and reproduction
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