Biological test method for measuring survival of springtails exposed to contaminants in soil: chapter 2


Section 2

Test Organisms

2.1 Species and Life Stage

The biological method described herein must be performed using laboratory-cultured Orthonychiurus folsomi Schäffer 1900, Folsomia candidaWillem 1902, Folsomia fimetaria Linnaeus 1758 or Proisotoma minuta Tyllberg 1871. P. minuta is recommended for use only with those soils collected from the boreal and taiga ecozones.Footnote2 The identification, distribution and life history of F. candida, O. folsomi, F. fimetaria and P. minuta are summarized in Section 1.2. Species identification must be confirmed and documentedFootnote3 by qualified personnel (e.g., taxonomist) experienced with identifying the intended species (see Section 1.2) of Collembola to be used in the toxicity test. Species identification may be made using the distinguishing taxonomic features described and illustrated in taxonomic keys, or using DNA- based taxonomic identification (i.e., barcoding). Cultures of F. candida, O. folsomi, F. fimetaria and P. minuta, held for a prolonged period at a testing laboratory, should be identified to species at least once every two years. The soil toxicity test described herein must be started using either 28- to 31-day-old O. folsomi, 10- to 12-day-old F. candida, 23- to 26-day-old F. fimetaria or 13- to 14-day-old P. minuta.

2.2 Source

Laboratory-cultured springtails (see Section 2.3) must be used as the source of the test organisms. Sources of Folsomia candida, Orthonychiurus folsomi, Folsomia fimetaria and Proisotoma minuta for establishing laboratory cultures may be government or private laboratories that are culturing these species of springtails for soil toxicity tests, or a commercial biological supplier.Footnote4

Breeding stock of Folsomia candida, Orthonychiurus folsomi, Folsomia fimetaria or Proisotoma minuta can be obtained by contacting the following Canadian source:

Method Development and Applications Unit
Science and Technology Branch
Environment Canada
335 River Road
Ottawa, Ontario  K1A 0H3
Email: methods@ec.gc.ca

All springtails used in a soil toxicity test must be derived from the same population. Springtails to be used as a source of breeding stock should be transported to the laboratory using a portion of the soil or other substrate to which they are adapted. Breeding stocks are best transported as a mixed-age culture in petri dishes containing the plaster of Paris substrate described in Section 2.3.5Footnote5 or in a small container of soil. Additional quantities of this substrate might be obtained for acclimation or culturing purposes, depending on culturing conditions and requirements (Section 2.3). Shipping and transport containers should be insulated to minimize changes in temperature during transit, and the temperature should be maintained at ~20°C. Live organisms should be transported quickly to ensure their prompt (i.e., within 24 h) delivery. Excessive crowding of animals during shipment or transport should be avoided to minimize stress in transit.

Upon arrival at the laboratory, organisms may be held in the substrate (i.e., soil or plaster of Paris) used in transit while temperature adjustments are made, or they may be transferred to new culturing substrate (Section 2.3.5). If the nature (including its texture and moisture content) of the substrate in which springtails were initially held (e.g., by a commercial supplier) or transported differs markedly from that in which they are to be cultured (Section 2.3.5), it is prudent to adapt the springtails to new substrate over several days.

Soil temperature should be adjusted gradually (e.g., ± 3°C per day) to the temperature to be used during culturing (Section 2.3.4). Guidance for handling Collembola given in Section 2.3.7 should be followed when transferring organisms from an outside source to culture vessels (Section 2.3.2). Other conditions during this interim holding period for acclimation of breeding stock or test organisms to laboratory conditions should be as similar as possible to those used for maintaining cultures (Section 2.3).

2.3 Culturing of Folsomia candida, Orthonychiurus folsomi, Folsomia fimetaria and Proisotoma minuta

2.3.1 General

General guidance and recommendations for culturing Folsomia candida, Orthonychiurus folsomi, Folsomia fimetaria and Proisotoma minuta in preparation for soil toxicity tests are provided here. In general, these four species are cultured under the same conditions and procedures. In keeping with the premise “What might work well for one laboratory might not work as well for another laboratory”(EC, 1997a, b, 2001; USEPA, 2000), explicit directions regarding many aspects of culturing, including the choice of culture vessel, number of organisms per vessel, soil-renewal conditions, culturing substrate, and food type and ration, are left to the discretion and experience of laboratory personnel, although guidance and recommendations are provided herein. Performance-based indicesFootnote6 are used to evaluate the suitability of the cultured organisms for tests and the acceptability of the test results. Cultures must have low mortalities to be suitable for use in tests, and the cultured organisms must appear healthy and behave and feed normally (see Section 2.3.9). Additionally, those used as controls in the test must have acceptably low mortality rates and meet all criteria for a valid toxicity test (see Section 4.4). The acceptability of the culture should also be demonstrated by concurrent or ongoing tests using a reference toxicant (see Section 4.9). If a culture of organisms fails to meet these criteria, its cause should be investigated. Care must be taken to ensure that each culture is not cross- contaminated with another Collembola species, since three of the four species recommended herein are difficult to distinguish from each other. Periodic (e.g., annual) taxonomic checks of the laboratory’s cultures are advisable to ensure that the springtail cultures maintained within the laboratory have not been cross- contaminated.

It is the responsibility of the laboratory to demonstrate its ability to obtain consistent, precise results using a reference toxicant, when initially setting up to perform soil toxicity tests with cultured F. candida, O. folsomi, F. fimetaria and P. minuta. For this purpose, intralaboratory precision, expressed as a coefficient of variation for the respective LC50 data, should be determined by performing five or more tests with different lots (groups) of test organisms from the same source, using the same reference toxicant and identical procedures and conditions for each test (see Section 4.9).

When routinely performing soil toxicity tests with F. candida, O. folsomi, F. fimetaria or P. minuta, reference toxicity tests should be conducted every two months with the laboratory’s cultures, using the conditions and procedures outlined in Section 4.9. If this bimonthly routine is not followed, the performance of individuals from the culture used to start a soil toxicity test should be evaluated in a reference toxicity test conducted concurrently. Additionally, the performance of any cultures that have been established recently using new breeding stock (Section 2.2) should be checked with a reference toxicity test, and the results determined to be acceptable (see Sections 2.3.9 and 4.9) before these cultures are used to provide test organisms.

Cultures of F. candida, O. folsomi, F. fimetaria and P. minuta should be observed frequently (e.g., once or twice per week). Ideally, records should be maintained documenting:

  • the date a culture is started with adults;
  • dates of substrate renewal;
  • feeding and watering regime (including type and quantity added on each occasion);
  • facility and substrate quality (e.g., air temperature, photoperiod and light quality, pH of substrate); and
  • observations of culture health (e.g., behaviour and appearance of springtails in culture, odour of substrate, location of springtails in the vessel, amount of uneaten food in vessel, presence of fungi).

A summary of the various conditions and procedures described in international methodology documents (Wiles and Krogh, 1998; ISO, 1999; OECD, 2009) for culturing various species of springtails is provided in Appendix E. A checklist of required and recommended conditions and procedures for culturing F. candida, O. folsomi, F. fimetaria and P. minuta to generate organisms for use in soil toxicity tests is given in Table 1.

2.3.2 Facilities and Apparatus

Springtails should be cultured in a controlled- temperature laboratory facility. Equipment for temperature control (i.e., an incubator or a room with constant temperature) should be adequate to maintain temperature within the recommended limits (Section 2.3.4). The culturing area must be isolated from any testing, sample storage or sample-preparation areas, to avoid contamination from these sources. It must be designed and constructed to prevent contamination of cultures (e.g., elimination of copper or galvanized piping or fixtures that could drip metal-contaminated condensation).

Table 1 Checklist of Required and Recommended Conditions and Procedures for Culturing Folsomia candida, Orthonychiurus folsomi, Folsomia fimetaria and Proisotoma minuta to Provide Test Organisms for Use in Soil Toxicity Tests

Source of brood stock for culture
adults obtained from a government, private, or commercial culture; identification to species confirmed
Acclimation
gradually, for temperature (recommend ≤ 3°C/day) and substrate differences upon arrival
Culture vessels
for O. folsomi , F. candida and P. minuta : breeding boxes of ~1−6 L capacity are suitable (e.g., plastic trays measuring ~15 x 23 x 8 cm to ~20 x 33 x 11 cm), covered with solid or perforated lids; sides and/or lid transparent or translucent to enable light to contact surface of culturing substrate for F. candida ; for F. fimetaria : Polystyrene Petri dishes (10 cm diam x 1.5 cm high), covered
Air temperature
daily average, 20 ± 2°C; instantaneous, 20 ± 3°C
Lighting
for O. folsomi , F. fimetaria and P. minuta : continuous darkness; or incandescent or fluorescent; intensity, 400−800 lux at surface of culture vessel; fixed photoperiod (e.g., 16h L:8h D or 12h L:12h D); for F. candida : incandescent or fluorescent; intensity, 400−800 lux at surface of culture vessel; fixed photoperiod (e.g., 16h L:8h D or 12h L:12h D)
Type of substrate
8:1 mixture of plaster of Paris and activated charcoal; recommended minimum depth, 1 cm; for O. folsomi and P. minuta , plaster of Paris caps or pie-shaped pieces of filter paper coated with plaster of Paris placed on the surface of the substrate; or a very thin layer of artificial soil placed on substrate surface for O. folsomi
Hydration of substrate
hydrated with test water; moisture content sufficient to keep surface of substrate moist but with no standing water on the surface of the culture vessel
pH of substrate
6.0−7.0
Renewal of substrate
as required, and at least once every 1−2 months; transfer springtails to fresh breeding vessels manually; mix adults between culture vessels of same species for O. folsomi , F. fimetaria and P. minuta
Monitoring culture
air temperature of culture facility monitored weekly; pH measured on new batches of plaster of Paris
Maintaining culture
vessels aerated at least once/week by removing lids from culture vessels for several minutes; moisture level of substrate observed for each culture vessel at time of aeration; add several drops of test water to maintain humidity; record condition of culture; maintain loading density of springtails at ~2 to 3 organisms/ cm 2  for O. folsomi and F. candida , ~5 to 6 organisms/cm 2  for F.fimetaria , and ~6 to 8 organisms/cm 2  for minuta ; recommend a minimum of 2 females/male for O. folsomi and F. fimetaria
Feeding
granulated dry yeast (e.g., Fleischmann’s™); divided into two or three piles or sprinkled onto substrate surface; feed twice/week; for O. folsomi and F. candida : ~100 mg (for 15 x 23 x 8 cm culture vessels); for P. minuta : ~200 mg (for 15 x 23 x 8 cm culture vessels); for F. fimetaria : ~10 mg (for 10-cm Petri dishes)
Maintenance of age-synchronized cultures
for F.candida , F. fimetaria and P. minuta : place 200−300 adults from existing culture onto new substrate to stimulate oviposition; feed; monitor daily for eggs; for F. candida , O. folsomi , F. fimetaria and P. minuta : monitor existing cultures for large egg clutches; 7 days after the first egg clutches appear in new culture vessels or large egg clutches appear in existing cultures, transfer egg clusters to hatching vessels (i.e., Petri dish ~10 cm diam. and ≥ 1 cm high) containing fresh substrate; feed; monitor daily for eclosion; remove unhatched eggs 24 h after appearance of juveniles for P. minuta , 48 h after appearance of juveniles for F.candida , and 72 h after appearance of juveniles for O. folsomi and F. fimetaria
Age/size for test
for O. folsomi : 28- to 31-day-old adults; for F. candida : 10- to 12-day-old juveniles; for F. fimetaria : 23- to 26-day-old adults; for P. minuta : 14-day-old juveniles (with optional addition of 13-day-old juveniles)
Indices of culture health
considered healthy if (1) springtails move actively over the surface of the substrate, and (2) results for reference toxicity tests using springtails from the culture fall within historic warning limits

For P. minuta, efforts should be made to minimize exposure of the cultures to any source of vibration since vibrations caused by nearby equipment or construction can disturb their reproduction (EC, 2013b).

All equipment, vessels, and accessories that might contact the organisms or substrate within the culturing facility must be clean, rinsed as appropriate, and made of nontoxic materials (e.g., glass, TeflonTM, type 316 stainless steel, nylon, NalgeneTM, porcelain, polyethylene, polypropylene). Toxic materials including copper, zinc, brass, galvanized metal, lead and natural rubber must not come in contact with this apparatus and equipment, or the culturing substrate or water.

A variety of culture vessels, such as plastic trays or breeding boxes of 1- to 6-litre capacity (e.g., white polystyrene rectangular boxes with dimensions of ~15 x 23 x 8 cm to ~20 x 33 x 11 cm), are suitable for culturing O. folsomi, F. candida and P. minuta. For F. candida, the sides and/or lid should be translucent or transparent, to enable light to contact the surface of the culturing substrate (see Section 2.3.3). Each vessel should have a lid, which can be solid, to minimize drying of the surface substrate and the risk of contamination, or perforated (e.g., holes covered with fibreglass mesh screening) to allow air exchange and prevent the springtails from escaping. Polystyrene Petri dishes (10 cm diam. x 1.5 cm high) are recommended for culturing F. fimetaria. Table 2 of Appendix E provides details of the type and size of various vessels recommended by international agencies for culturing several species of springtails in the laboratory, and to generate organisms for soil toxicity tests. The use of culture vessels constructed of wood is not recommended, due to the possible presence of toxic contaminants (e.g., plywood glues, antisapstain chemicals, or wood extractives such as resin acids, juvabiones, etc.).

The choice of size and numbers of culture vessels required might be influenced by the number of adult springtails required by the testing facility for one or more series of soil toxicity tests. Each culture vessel should accommodate a minimum depth of 1 cm of culturing substrate.

2.3.3 Lighting

Cultures of O. folsomi, F. fimetaria and P. minuta can be cultured in complete darkness (e.g., inside a closed drawer or opaque breeding vessel), or with incandescent or fluorescent light and a regulated photoperiod (e.g., 16-h light:8-h dark or 12-h light:12-h dark). For F. candida, incandescent or fluorescent lights should illuminate the cultures, and the photoperiod should be regulated (e.g., 16-h light:8-h dark or 12-h light:12-h dark). Light intensity adjacent to the top of the culture vessels should range within 400−800 lux. This range is equivalent to a quantal flux of 5.6−11.2 μmol/(m2·s) for cool-white fluorescent, 6.4−12.8 μmol/(m2 ·s) for full-spectrum fluorescent, or 7.6−15.2 μmol/(m2 ·s) for incandescent. The lights should be positioned sufficiently far from the culture vessels to prevent evaporation caused by heat buildup.

2.3.4 Temperature

All four test species should be cultured in a facility with an air temperature of 20 ± 2°C, as a daily average. Additionally, the instantaneous temperature of the facility should be 20 ± 3°C.

2.3.5 Culturing Substrate

Various substrates have been used for culturing springtails in preparation for soil toxicity tests (see Table 4 of Appendix E). The choice of substrate for culturing these species is left to the discretion and experience of laboratory personnel; however, the following culture substrate is proven and recommended for all four test species.

A substrate comprised of 8 parts plaster of Paris (Stucco)Footnote7 and 1 part charcoal (e.g., analytical- grade activated charcoal 375 μm mesh; e.g., Fisher cat # 35-474) is recommended by Wiles and Krogh (1998), ISO (1999), Greenslade and Vaughan, (2003), EC (2007a), and OECD (2009) and for culturing F. candida, F. fimetaria, O. folsomi and P. minuta, and is also recommended here. Working in a chemical fume hood, the culture substrate is prepared in a 1-L glass or plastic bottle. First, 120 g of plaster of Paris and 15 g of charcoal are put into the 1-L bottle using a funnel and the bottle is shaken vigorously for approximately 30 seconds. Once the solids are homogenized, 130 mL of test water (ultra pure, e.g., MilliQ® water is recommended for use when preparing substrate for F. fimetaria) is added, and the bottle is closed and shaken for another 30 seconds. The amount of water needed can vary depending on the type of plaster used. Once prepared, the plaster of Paris mixture is then poured into the culture vessel(s) to a depth of 1 cm (Becker-van Slooten et al., 2003; Stämpfli et al., 2005).Footnote8, Footnote9 This should be done fairly quickly to prevent the substrate from hardening before being poured into the breeding vessels. The vessels are gently tapped on the sides and on the laboratory bench top to release any air bubbles that may have formed during mixing, as well as to evenly distribute the culture substrate and to create a flat substrate surface.Footnote10 The culture vessels should be placed on a level surface and allowed to air-dry flat for at least three hours. Once hardened, test water is added to the culture vessels to almost saturation (i.e., there should be no standing water on the substrate surface). If the prepared culture vessels are not being used immediately, they may be stored at room temperature for up to three weeks. Before storage, the substrate should be saturated with test water (i.e., slowly add ~1 cm of test water on top of the set surface) to prevent it from drying out during storage. Over-drying will result in the substrate shrinking away from the edges of the vessel, thereby creating a gap. If a gap is created between the sides of the vessels and the substrate due to over-drying, the substrate should be discarded since the springtails will reside and lay eggs down the sides and at the bottom of the vessel (i.e., where they are inaccessible). The substrate should be rinsed with test water before springtails are added. Approximately 1 cm of test water is added to the substrate and the edges and surface gently rubbed with a gloved fingertip to remove any sharp or uneven edges. The substrate should be rinsed three times. Excess water may be poured off, the surface lightly blotted with paper towel, and the vessels sealed with lids, after which the vessels are ready to use.

The pH of each new batch of substrate is verified by placing pH paper on the wet substrate surface. Substrate pH should be between 6.0 and 7.0. Culture vessels should be re-hydrated with test water once or twice/week to maintain the humidity (e.g., optimum humidity is provided by keeping the plaster of Paris moist). This is accomplished by adding several drops of test water with a pipette or by gently spraying the sides of the vessel using a fine mist spray bottle or squeeze bottle until the water just begins to remain on the surface. Care should be taken not to damage the springtails or to blow organisms out of the culture vessel during the re-hydration process.

The vessels must be aerated a minimum of once/week; however, twice/week is recommended if there is a history of fungal problems in the cultures, or for cultures of F. fimetaria, which are more sensitive to fungal growth. Aeration can be achieved during the weekly re-hydration process by simply removing the lids for ≥ 1 minute.

A mixture of fertilizer-free potting soil (manure/peat/loam blend), Sphagnum sp. peat moss, and artificial soilFootnote11 has also proven to be a suitable culturing substrate for O. folsomi (ESG and AquaTerra Environmental Ltd., 2003), F. fimetaria(J.I. Princz, personal communication, Soil Toxicology Laboratory, Environmental Science and Technology Centre, Ottawa, ON, 2006) and P. minutaFootnote12 (C. Fraser, personal communication, Soil Toxicology Laboratory, Environmental Science and Technology Centre, Ottawa, ON, 2013), and can be used for maintaining mass or back-up cultures in the laboratory.Footnote13

2.3.6 Food and Feeding

Various types of food and feeding regimes have been used for culturing springtails in preparation for soil toxicity tests (see Table 5; Appendix E). Success in culturing the four species described in this test method document has been achieved using activated dry yeast (AquaTerra Environmental Ltd., 1998; Wiles and Krogh, 1998; ISO, 1999; AquaTerra Environmental Ltd. and ESG, 2000; ESG 2000, 2001, 2002; ESG and AquaTerra Environmental Ltd., 2002, 2003; Greenslade and Vaughan, 2003; Becker-van Slooten et al., 2003, 2005; Stämpfli et al., 2005; EC, 2006a; EC, 2013b).

Activated dry yeast, to be used as food for cultures, can be purchased from the grocery store. Fleischmann’s™ is recommended for this purpose, since this brand has been used successfully during the development of this method (AquaTerra Environmental Ltd., 1998; EC, 2006a, 2007a,b; C. Fraser, personal communication, Soil Toxicology Laboratory, Environmental Science and Technology Centre, Ottawa, ON, 2013). The quantity of food added to each culture vessel depends on springtail density and developmental stage and, therefore, should be based on observations and records of food consumed or not consumed, during preceding weekly feedings. The quantities are species-specific as follows:

  • for O. folsomi and F. candida, ~100 mg, (i.e., for a culture vessel that is ~ 15 x 23 x 8 cm);
  • for P. minuta, ~200 mg (i.e., for a culture vessel that is ~15 x 23 x 8 cm); and
  • for F. fimetaria, ~10 mg (i.e., for a 10-cm diam. Petri dish).

The food can be divided into two or three piles, or sprinkled over the surface of the culture substrate. The yeast should be placed onto the surface of the substrate of each vessel twice/week at the time of aeration and re- hydration.Footnote14 The old, unconsumed yeast is removed (if remaining) before the new yeast is added.Footnote15Care should be taken to avoid excessive fungal and bacteria growth in the culture vessels, for F. fimetaria and P. minuta especially, since these species are more sensitive to fungi and bacteria.Footnote16 In order to activate the yeast, it should be added after the substrate has been hydrated. Alternatively, the yeast may be activated by hydrating it with a few drops of test water.

2.3.7 Handling Organisms and Maintaining Cultures

Springtails should be handled as little as possible, to avoid damage and undue stress. When handling is necessary, it should be done gently, carefully and quickly to minimize stress to the animals. The use of a moist, fine-tipped paintbrush is suitable for moving springtails to and from the culture or test vessels; however, care must be taken to avoid damaging the organisms or their eggs. A low suction exhaustor system, described by ISO (1999), may also be used for transferring springtails. A glass Pasteur pipette, fitted with a suction bulb, or a water-based aspiration system (see Appendix I) also function well in this capacity. The suction strength should be modified to a very gentle level to prevent damage to the springtails, especially for P. minuta, which is very sensitive to excessive vibrations (EC, 2013b). Collembola can also be transferred by gently tapping one vessel over another. When handled, any animals that are injured or appear stressed should be discarded, and must not be used for testing.

Table 6 of Appendix E summarizes useful guidance for maintaining cultures of various species of springtails, found in international guides and methodology documents on soil toxicity tests using these Collembola.

It is recommended that the contents of each culture vessel be inspected just before each weekly feeding, to determine the apparent condition of the springtails and the culture substrate. Records should be kept of the apparent condition of the culture (organisms and substrate) noted during each observation period (Section 2.3.1).

The loading density of springtails in each culture vessel should be restricted to prevent overcrowding and the resulting adverse effects on springtail growth, reproduction and culture health (ISO, 1999). A loading density of ~ 2 to 3 adult Collembola per cm2 is suggested for O. folsomi and F. candida (G.L. Stephenson, personal communication, AquaTerra Environmental Ltd., Orton, ON, 2006; K. Becker-van Slooten, personal communication, Laboratory of Environmental Chemistry and Ecotoxicology, ENAC-ISTE, Ecolé polytechnique fédérale de Lausanne, Lausanne, Switzerland, 2004); ~ 5 to 6 adult Collembola per cm2 is suggested for F. fimetaria (J.I. Princz, personal communication, Soil Toxicology Laboratory, Environmental Science and Technology Centre, Ottawa, ON, 2007); and ~ 6 to 8 adult Collembola per cm2 is suggested for P. minuta (C. Fraser, personal communication, Soil Toxicology Laboratory, Environmental Science and Technology Centre, Ottawa, ON, 2013).

The substrate in each culture vessel should be renewed as required and every one to two months, regardless of springtail densities. This can be achieved by preparing new culture vessels, and by transferring the springtails into the new vessels by tapping the old vessel over the new one. To reduce the population of springtails in a crowded culture vessel, only a portion of the total culture (e.g., 75% of individuals) are transferred. The change of substrate will stimulate oviposition (Wiles and Krogh, 1998; C. Fraser, personal communication, Soil Toxicology Laboratory, Environmental Science and Technology Centre, Ottawa, ON, 2013). For O. folsomi, F. fimetaria and P. minuta, it is important that new cultures contain a mixture of males and females (i.e., a minimum of two females to every male) and that the organisms are mixed between independent culturing vessels to avoid inbreeding (Stämpfli et al., 2005). P. minuta will lay eggs on fresh substrate for one to three weeks (depending on the density of the population). After this time, egg production will slow and the organisms will need to be transferred to fresh substrate in order for egg production to continue (C. Fraser, personal communication, Soil Toxicology Laboratory, Environmental Science and Technology Centre, Ottawa, ON, 2013).Footnote17

The air temperature of the culture facility should be monitored weekly and the moisture level of the culture substrate should be observed at the time of weekly aeration. Adjustments should be made as and if necessary (see Sections 2.3.4 and 2.3.5).

2.3.8 Age-synchronized Cultures for Toxicity Tests

To be successful, the culturing procedures used must produce the required number of healthy test organisms of a known developmental stage, and of similar age and size. Additionally, the cultured organisms must meet specific health- and performance-related indices (Section 2.3.9). The following paragraphs describe procedures that should be followed to obtain age- synchronized test organisms (i.e., 28−31 days old for O. folsomi, 10−12 days old for F. candida, 23−26 days old for F. fimetaria, 14 days old for P. minuta) for use in the toxicity tests described in this method document.

For F. candida, F. fimetaria and P. minuta, new cultures can be created to begin the process of age-synchronization.Footnote18 At least two new large cultures (large culture vessels described for O. folsomi, F. candida and P. minuta in Section 2.3.2) or several small ones (Petri dishes described for F. fimetaria in Section 2.3.2) should be prepared in order to obtain enough springtails for a toxicity test. This can be accomplished by transferring 200 to 300 mature F. candida, F. fimetaria, or P. minuta from an existing culture by gently tapping the vessel of the existing culture and allowing the springtails to gently drop onto the newly prepared culture substrate of a culture vessel (see Section 2.3.5). Avoid transferring more than 300 individuals, since this would result in over-crowding and the inhibition of reproduction. Cultures are fed by adding 100−200 mg of yeast to each new culture vessel. The number of organisms transferred and the amount of yeast added to the cultures depends on the size of the culture vessels being used. These new cultures should be monitored daily for eggs. The springtails should begin to lay the first egg clutches within 24−48 hours of being transferred to new culture substrate.

For O. folsomi, existing cultures should be used to produce sufficient numbers of eggs for age-synchronization. Existing cultures may also be used to produce eggs for F. candida, F. fimetaria or P. minuta age-synchronization, in addition to the procedure of setting up new cultures, described previously.

Seven days after the first egg clutches appear in new cultures (for P. minuta, eggs will have just begun to hatch), or a large number of egg clutches appear in existing cultures, several (or all, if possible) egg clutches should be transferred onto moistened coated filter paperFootnote19 which is then placed into new culture vessels or smaller hatching vessels.Footnote20,Footnote21 The eggs can be transferred using a fine spatula or a slightly dampened paint brush. The egg clutch should be “swept” by gently rolling the paint brush under and through the clutch and then tapping the paintbrush gently to deposit the eggs onto the moistened coated filter paper or plaster substrate in the hatching vessels. The plaster substrate in the new hatching vessels and the coated filter paper should be sufficiently moist, or the eggs will dehydrate  (i.e., the plaster of Paris substrate is sufficiently moist when a droplet of water will stay on the surface and only very slowly absorb into the substrate). For O. folsomi and F. fimetaria, a few adult females (i.e., ~6 of the biggest organisms) can be introduced into each hatching vessel to improve the rate of hatching. For P. minuta, it is useful to collect ~30% more eggs than is required for setting up a test to ensure sufficient adults are available for easier selection of the appropriate number of males and females.

Three to five grains of activated dry yeast (≈0.0003 g) should be added to the moistened substrate of each hatching vessel. The hatching vessels should then be sealedFootnote22 and monitored daily for the appearance of juveniles. The substrate and coated filter paper should be continuously kept moist with test water. P. minuta eggs should hatch within 24 hours, whereas F. candida eggs hatch within 2 to 3 days following their transfer (i.e., ~10-days after oviposition), and F. fimetaria eggs hatch within 3 to 4 days following their transfer. The coated filter papers containing any unhatched eggs should be removed 24 hours after they are first placed in the hatching vessels for P. minuta, 48 hours after the appearance of the first juveniles for F. candida, and 72 hours after the appearance of the first juveniles for O. folsomi and F. fimetaria, and placed into fresh hatching vessels. The process is repeated for the production of more age-synchronized test organisms. Since juvenile springtails tend to stay under or crawl on top of the coated filter paper, the filter paper should be tapped or brushed with a dry paint brush (being careful not to remove eggs from the filter paper in the process) before removing the filter paper. If adult female F. fimetaria were placed into the hatching vessels with the eggs, they should be removed when the coated filter paper is removed. F. candida juveniles can be used for testing 10 days after removal of the remaining unhatched eggs (i.e., F. candida used to start toxicity tests must be 10−12 days old); F. fimetaria adults can be used for testing 23 days after the removal of the remaining unhatched eggs (i.e., F. fimetaria used to start toxicity tests must be 23−26 days old); O. folsomiadults can be used for testing 28 days after the removal of remaining unhatched eggs (i.e., O. folsomi used to start toxicity tests must be 28−31 days old); and P. minuta juveniles should be used 14 days after the removal of remaining unhatched eggs. If, however, there is an insufficient number of 14-day-old P. minuta available for testing, a secondary group of 13- day-old organisms may be used in combination with the 14-day-old age-synchronized test organisms to provide an adequate number of test organisms (i.e., P. minuta used to start the test must be 13−14 days old; however, the use of only 14-day-old test organisms is recommended).Footnote23

An alternative method for obtaining age-synchronized springtails is to place a number of adult springtails into many small vessels containing the plaster of Paris/charcoal substrate (see Section 2.3.5). The adults should be allowed to lay eggs over a 24-hour period (for P. minuta) or a 48-hour period (for F. candida, F. fimetaria and O. folsomi). Adults are then removed 24 hours (for P. minuta) or 48 hours (for F. candida, F. fimetaria and O. folsomi) after the first egg clutch appears. The small vessels should be monitored for the appearance of juveniles and organisms should be fed with 2 to 3 grains of active dry yeast, as necessary. Unhatched eggs are removed 24 hours after the appearance of the juveniles for P. minuta, 48 hours after the appearance of juveniles for F. candida, and 72 hours after the appearance of juveniles for O. folsomi and F. fimetaria. The organisms can then be used in a toxicity test 10, 14, 23 and 28 days after the first juveniles have emerged from the eggs, for F. candida, P. minuta, F. fimetaria and O. folsomi, respectively.

Any laboratory-cultured F. candida, O. folsomi, F. fimetaria or P. minuta used to start a toxicity test (including that with a reference toxicant) for effects on survival and reproduction should be acclimated in the laboratory as much as possible to conditions representing those in this toxicity test (Section 4.3). During the age-synchronizing period, temperature conditions must be the same as those to be used in the toxicity test, and springtails must be fed dry yeast (see Sections 2.3.4, 2.3.6 and 4.3).

2.3.9 Health and Performance Indices

Each culture vessel should be checked at least once per week, during which time culture performance should be monitored and recorded (see Sections 2.3.1, 2.3.6 and 2.3.7). Procedures and conditions used to maintain each culture should be evaluated routinely, and adjusted as necessary to maintain or restore the health of the culture. If the culture appears unhealthy or atypical during any weekly (or more frequent) check, it should be checked daily to make sure that “cascade mortality” (i.e., rate of death increasing exponentially over time) is not occurring. Cultures are considered healthy if springtails of differing sizes are moving actively over the substrate surface.

One or more reference toxicity tests (14-day test for F. candida and P. minuta, and 7-day test for O. folsomi and F. fimetaria) must be conducted using a portion of the population of age-synchronized springtails taken from a particular culture to start a definitive soil toxicity test (see Section 4.9). Ideally, a reference toxicity test should be performed together with each soil toxicity test. Laboratories routinely undertaking soil toxicity tests using cultured Collembola may choose instead to routinely conduct one or more reference toxicity tests (i.e., at least once every two months), using age-synchronized springtails derived from the same culture(s) from which the test organisms for the soil toxicity test(s) are obtained. All tests with the reference toxicant(s) should be performed using the conditions and procedures outlined in Section 4.9. Test-related criteria used to judge the validity of a particular soil toxicity test (and, indirectly, the health of the culture), based on the performance of test organisms in the negative control soil, are given in Section 4.4. A laboratory that routinely (e.g., once per month or more) performs toxicity tests with springtails might find it useful to monitor the data on number of progeny produced in negative control soil, as a measure of culture health and performance. A plot of such data over time can show problems with respect to reproductive success that are attributable to diet or other conditions to which cultures are exposed (G.L. Stephenson, personal communication, Stantec Consulting Ltd., Guelph, ON, 2004).

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