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

NI ^Footnote3

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 container^{Footnote 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 Conditions^{Footnote b}	Humidity
W & K 1998 F. candida and I. viridis	15 ± 0.5^Footnotec	16h L:8h D at <1000 lux	NI^{Footnote 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 lx^Footnotef	70-80% RH^Footnote g
OECD 2005	20 ± 1 or 20 ± 0.5	12h L:12h D or 16h L:8h D at <1000 lux^{Footnote h}	NI

4. Culturing Substrate

Document	Culturing Substrate	pH	Renewal Conditions
W & K 1998 F. candida and I. viridis	plaster of Paris and charcoal, hydrated^FootnoteI	NI^FootnoteJ	stock cultures periodically moved to fresh plaster of Paris (e.g., every 2-3 months)
F. fimetaria	plaster of Paris and charcoal, hydrated^FootnoteI	NI	transfer to fresh breeding containers every 4-8 weeks
ISO 1999	8:1 mixture of plaster of Paris and activated charcoal, hydrated^FootnoteK, ^FootnoteL	6.0-7.0	transfer to fresh breeding containers after 8 weeks^FootnoteM
OECD 2005	plaster of Paris and activated charcoal^FootnoteN	NI	transfer to fresh breeding containers every 4-8 weeks^FootnoteO, ^FootnoteP

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 yeast^FootnoteQ, ^FootnoteR	10-30 mg placed on filter paper disks	at least once or twice per week
F. fimetaria	dried baker’s yeast^FootnoteQ	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	NI^FootnoteS	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 exhaustor^FootnoteT
OECD 2005	NI	adults	low-suction air-flow device

Footnotes

Footnote 1

See preceding page.

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Footnote 2

April to May and September to October are optimum times of the year for collection in temperate climates.

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Footnote 3

NI = not indicated.

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Footnote 4

Other collembolan species may also be suitable, e.g., I. viridis or O. folsomi. If other species of Collembola are used, they must be clearly identified and the rationale for the selection of the species should be reported.

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Footnote A

Larger stocks may be held in large plastic food containers (30 x 30 x 20 cm); note: some types of plastic may emit toxic organic compounds, which could influence the culturing and testing.

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Footnote B

L = light; D = dark.

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Footnote C

May be kept at 20°C, if required.

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Footnote D

NI = not indicated.

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Footnote E

Cultures are to be kept moist at all times; however, for I. viridis, it is advisable to avoid a layer of moisture being formed on the surface of the plaster of Paris as this tends to increase hatchling mortality.

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Footnote F

A light:dark cycle of 16h:8h is also suitable.

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Footnote G

RH = relative humidity.

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Footnote H

Containers are kept moist at all times.

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Footnote I

260-300 mL of water is added to 450 g of the plaster charcoal mixture.

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Footnote J

NI = not indicated.

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Footnote K

Higher ratios (9:1 or 10:1) of plaster and charcoal may also be used.

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Footnote L

60 to 100 g of water is added to 100 g of the plaster and charcoal mixture; however, the amount of water added depends on the type of plaster. The presence of water on the saturated substrate surface is essential for breeding springtails.

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Footnote M

Transfer Collembola by tapping or blowing individuals off the plaster of Paris onto fresh substrate.

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Footnote N

20 mL of activated charcoal, 200 g of plaster of Paris and 200 mL of distilled water or 50 g of activated pulverized charcoal, 400 g plaster of Paris, and 260-300 mL of distilled water.

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Footnote O

Stock cultures periodically moved (e.g., every 2-3 months) to fresh plaster of Paris.

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Footnote P

Dead individuals and stale food are removed from container.

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Footnote Q

Alternative food stuffs include green algae (Pleurococcus spp.), Tetramin fish food, shredded carrots mixed with oatmeal, Drosophila food flakes, and potato chunks.

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Footnote R

For I. viridis a small amount of defaunated field soil (5 g sieved and heated to 70°C) may be placed at one end of the containers.

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Footnote 5

Overcrowding the containers may lead to reduced growth and as a consequence the 10- to 12-day-old organisms used for the test may be too small and not yet able to produce a sufficient number of eggs to meet the requirements of the test.

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Footnote 6

The egg clusters can be easily removed if placed on small pieces of breeding substrate or cover glasses are laid on the breeding substrate.

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Footnote 7

An alternative method of obtaining age-synchronized test organisms is to place a number of adult springtails in a small container with plaster of Paris in the base and allow them to lay eggs over a two-day period. The adults are then removed and the juveniles used 12 days after they first emerge from the eggs. It is advisable to check the containers for egg production before removing the adults since in some cases the adults do not start laying eggs immediately. If only a few eggs are produced in two days, the adults are kept in the containers for an additional day or more.

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Footnote S

NI = not indicated.

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Footnote T

Springtails are sucked individually through a pipette tip to a small covered container. Care shall be taken to ensure that the suction of the pump is low to avoid damage to the organisms. After removing the cover, springtails provided for one test container are transferred onto the substrate surface of the test chamber. A manual exhaustor may also be used.

Return to footnoteT referrer

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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

1. Source of Brood Stock for Culture

2. Culture Vessels and Capacity

3. Temperature and Lighting During Culturing

4. Culturing Substrate

5. Feeding During Culturing

6. Culture Maintenance and Developmental

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

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