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


Appendix E

Procedural Variations for Culturing Springtails, as Described in International Guides and Test Methods for Measuring Soil Toxicity Using Various Species of Springtails

Source documents are listed chronologically, by originating agency of 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. Source of Brood Stock for Culture

Document Footnote1
Test Species
Initial Source
W & K 1998
Folsomia candida (Willem, 1902)
starter cultures may be obtained from numerous existing laboratory cultures
W & K 1998
Folsomia fimetaria (Linné, 1758)
may be obtained from soil samples collected from fields, meadows, or grasslands, after a heat/dry extraction of the soil
W & K 1998
Isotoma viridis (Bourlet, 1839)
may be collected from field sites (grassy habitats) by suction sampling Footnote2
ISO 1999
Folsomia candida (Willem, 1902)
OECD 2005
Folsomia candida (Willem, 1902) Footnote4
commercially available
OECD 2005
Folsomia fimetaria ( Linné , 1758) Footnote4
commercially available

2. Culture Vessels and Capacity

Document Vessel Type and Size Number of Units and Capacity
for Substrate or Springtail Production
W & K 1998
F. candidaand I. viridis
28 H 16 H 9 cm perspex containerFootnote a 1 cm of breeding substrate moistenedwith distilled water
 F. fimetaria 90 H 13 mm Petri dishes 0.5 cm breeding substrate
ISO 1999 400-mL commercial plasticcontainers, covered tightly 1 cm of breeding substrate withdeionized water to almost saturation
OECD 2005 90 H 13 mm Petri dishes 0.5 cm breeding substrate

3. Temperature and Lighting During Culturing

Document Temperature (°C) Lighting ConditionsFootnote b Humidity
W & K 1998 F. candida and I. viridis 15 ± 0.5Footnotec 16h L:8h D at <1000 lux NIFootnote d, Footnotee
F. fimetaria 20 ± 1 at <1000 lux 12h L:12h D NI
ISO 1999 20 to 22 continuous lighting at 400 to 800 lxFootnotef 70-80% RHFootnote g
OECD 2005 20 ± 1 or
20 ± 0.5
12h L:12h D or
16h L:8h D at <1000 luxFootnote h
NI

4. Culturing Substrate

Document Culturing Substrate pH Renewal Conditions
W & K 1998 F. candida and I. viridis plaster of Paris and charcoal, hydratedFootnoteI NIFootnoteJ stock cultures periodically moved to fresh plaster of Paris (e.g., every 2-3 months)
F. fimetaria plaster of Paris and charcoal, hydratedFootnoteI NI transfer to fresh breeding containers every 4-8 weeks
ISO 1999 8:1 mixture of plaster of Paris
and activated charcoal, hydratedFootnoteKFootnoteL
6.0-7.0 transfer to fresh breeding containers after 8 weeksFootnoteM
OECD 2005 plaster of Paris and activated charcoalFootnoteN NI transfer to fresh breeding containers every 4-8 weeksFootnoteOFootnoteP

5. Feeding During Culturing

Document Description of Food Used Quantity and Feeding Procedure Feeding Frequency
W & K 1998 F. candida and I. viridis dried baker’s yeastFootnoteQFootnoteR 10-30 mg placed on filter paper disks at least once or twice per week
F. fimetaria dried baker’s yeastFootnoteQ 15 mg once per week
ISO 1999 granulated dry yeast small amounts at frequent intervals at least once or twice per week
OECD 2005 granulated dry baker’s yeast 10-30 mg or mass needed once or twice per week

6. Culture Maintenance and Developmental

Document
Maintenance of age-synchronized cultures
W & K 1998 F. candida
transfer several hundred adult Collembola from stock cultures into fresh breeding containers, and supply with baker’s yeast; remove adults after 24-48 h; incubate eggs at 15°C (or 20°C, as required); observe daily and record date of hatching; feed
W & K I. viridis
prepare hatching tubes (2.5 X 5 cm) containing 1 cm depth of breeding substrate; transfer egg batches from stock cultures into tubes with dampened fine paint brush; incubate eggs at 15°C (or 20°C, as required); observe daily; brush eggs showing fungal contamination with distilled water; transfer any hatchlings to screw top jar (120 mL capacity) containing 1 cm breeding substrate; feed
W & K F. fimetaria
transfer 150-300 adults from a 4-8 week-old substrate to fresh breeding containers and feed; after 9 days carefully collect eggs with a needle and spatula and moved to an egg-paper (small piece of filter paper dipped in breeding substrate); place in fresh breeding container and maintain humidity; most eggs will hatch in 3 days; remove egg-paper from Petri dish to obtain age-synchronized cultures; feed
ISO 1999
avoid overcrowding Footnote5 ; transfer egg clusters Footnote6 from breeding containers to a freshly prepared breeding substrate using a fine spatula or hairbrush; after 48 h remove the egg clusters and feed the instars hatched from the eggs Footnote7
OECD 2005 F. candida
transfer several hundred adults from stock cultures into fresh breeding containers with a 1 cm layer of substrate, and supply with baker’s yeast; remove adults after 24-48 h; observe daily and record date of hatching; feed
F. fimetaria
transfer 150-300 adults from a 4-8 week-old substrate to fresh breeding containers with a 0.5 cm layer of breeding substrate and feed with 15 mg of baker’s yeast; after 9 days carefully collect eggs with a needle and spatula and moved to an egg-paper (small piece of filter paper dipped in breeding substrate); place in fresh breeding container and maintain humidity; most eggs will hatch in 3 days; remove egg-paper from Petri dish to obtain age-synchronized cultures; feed

7. Indices of Culture Health and Acceptability; Age of Springtails Used in Toxicity Tests; Transfer of Organisms to Test Vessels

Document Indices of Culture Health and Acceptability Age of Springtails Used in Toxicity Tests Transfer of Springtails to Test Vessels
W & K 1998 F. candida NIFootnoteS 10- to 12-day-old juveniles hand-held air aspirator
I. viridis NI 5- to 7-day-old juveniles  
F. fimetaria NI 23 to 26 days old  
ISO 1999 NI 10- to 12-day-old juveniles by tapping or with an exhaustorFootnoteT
OECD 2005 NI adults low-suction air-flow device
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