Biological test method for measuring survival of springtails exposed to contaminants in soil: 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-renewal^Footnote1	8 weeks^Footnote2
W & K 1998	Isotoma viridis(Bourlet, 1839)	static-renewal^Footnote1	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	NI^Footnote3	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 Soil^Footnote4	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 Soil^Footnote6
W & K 1998 F. candida	standard soil^Footnote7 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 soil^Footnote7, ^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 soil^Footnote7 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 soil^Footnote13 with added test substances (e.g., chemical in de-ionized water^Footnote14 or in organic solvent; if insoluble, test substance is mixed with fine quartz sand^Footnote15	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 capacity^Footnote18	NI^Footnote19
OECD 2005	blend dry constituents^Footnote20	hydrate to ~50% of the maximum water-holding capacity^Footnote21 (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 Characterization^Footnote22
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	NI^Footnote23	moisture content and pH after hydrating
ISO 1999	NI, NA^Footnote24	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. candida^Footnote25, ^Footnote26	10	NI^Footnote27	5	geometric series^Footnote28
W & K 1998 I. viridis^Footnote25, ^Footnote26	20	NI	5	geometric series^Footnote28
W & K 1998 F. fimetaria^Footnote25, ^Footnote29	20^{Footnote 3030}	1	4 + control	geometric series^{Footnote 3131}
ISO 1999^Footnote25, ^{Footnote 3232}	10	1	4 + control	geometric series^{Footnote 3131}
OECD 2005^Footnote25, ^{Footnote 3333} F. fimetaria	20^{Footnote 3030}	2	5 + control	geometric series^{Footnote 3434}
OECD 2005^Footnote25, ^{Footnote 3333} F. candida	10	2	5 + control	geometric series^{Footnote 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	NI^{Footnote 3535}, ^{Footnote 3636}
W & K 1998 I. viridis	20	≥ 3	≥ 5 + control	NI^{Footnote 3636}, ^{Footnote 3737}
W & K 1998 F. fimetaria	20^{Footnote 3838}	≥ 4	NI	NI ^{Footnote 3939}, ^{Footnote 4040}
ISO 1999^{Footnote 4141}	10	for NOEC: 5	≥ 5 + control	geometric series^{Footnote 4242}
		for ECx: ≥ 2 for treatments and 5 for controls	12 + control	geometric series^{Footnote 4343}
OECD 2005 F. fimetaria	20^{Footnote 3838}	≥ 4	NI	NI ^{Footnote 3939}, ^{Footnote 4040}
OECD 2005 F. candida	10	≥ 4	≥ 4 + control^{Footnote 4444}	NI^{Footnote 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	NI^{Footnote 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 Conditions^{Footnote 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 ± 0.5^{Footnote 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	Measurements^{Footnote 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 5252}and number of juveniles hatching from eggs collected from soil^{Footnote 5353}weekly and at the end of the test
W & K 1998 I. viridis	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 5252}weekly and at the end of the test
W & K 1998 F. fimetaria	W and pH at start and end of test^{Footnote 5151}, ^{Footnote 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 test^{Footnote 5555}, ^{Footnote 5656}	number of live adult and juvenile springtails per test vessel at the end of the exposure
OECD 2005	W and pH^{Footnote 5757} each treatment at start and end of test; weight of test vessels at start and weekly thereafter^{Footnote 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 extractor^{Footnote 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 counted^{Footnote 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 ECx^{Footnote 6161}
OECD 2005	Collembola are extracted from soil with a high gradient heat extractor1 offspring produced or by floatation^{Footnote 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	NI^{Footnote 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%	dimethoate^{Footnote 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 forte^{Footnote 6464}, ^{Footnote 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

Footnotes

Footnote 1

Collembola are placed in fresh soil at weekly intervals biological observations have been made.

Return to footnote1 referrer

Footnote 2

It is also feasible to carry out the test for 4 weeks at 20°C.

Return to footnote2 referrer

Footnote 3

NI = not indicated.

Return to footnote3 referrer

Footnote 4

AS = artificial soil; StS = standard soil.

Return to footnote4 referrer

Footnote 5

Approximately 150 g dry weight of soil substrate is required for each concentration tested, to allow for eight weeks of assessment and extra soil (~50 g) to determine soil moisture and pH at the start and end of the test. At the onset of the study, soils for each test concentration are prepared in sufficient mass for the complete test. If, however, the test chemical is non-persistent, it is advisable to prepare a freshly treated portion of soil at each weekly assessment period.

Return to footnote5 referrer

Footnote 6

Percentages are expressed on a dry mass basis.

Return to footnote6 referrer

Footnote 7

Test soils may be artificial soil or LUFA 2.2 soil, which is a sandy soil with a pH of 6.0 ± 0.5 and a particle composition of 4% organic matter and 5.1% clay.

Return to footnote7 referrer

Footnote 8

Substances insoluble in water but soluble in organic solvents are dissolved in a volatile solvent (acetone or hexane) and mixed with a portion of the quartz sand or dry soil substrate. After evaporating the solvent (fume hood for 1 h), the remainder of the test substrate and the water are added and mixed thoroughly before introducing it into the test containers. Substances insoluble in water or organic solvents are mixed with 10 g of finely ground industrial quartz sand or 10 g of the dry soil substrate prior to adding the remainder of the soil substrate and water.

Return to footnote8 referrer

Footnote 9

The sphagnum peat is air dried, finely ground and sieved (mesh width of 1 mm).

Return to footnote9 referrer

Footnote 10

Test may be adapted for testing site soils (e.g., from remediated sites).

Return to footnote10 referrer

Footnote 11

Substances insoluble in water but soluble in organic solvents are dissolved in a volatile solvent (acetone or hexane) and mixed with a portion of the quartz sand required for the artificial soil formulation. After evaporating the solvent, the remainder of the artificial soil constituents and the water are added and mixed thoroughly before introducing it into the test containers. Substances insoluble in water or organic solvents are mixed with 10 g of finely ground industrial quartz sand prior to adding the remainder of the artificial soil constituents and water.

Return to footnote11 referrer

Footnote 12

The sphagnum peat is air dried, finely ground and with no visible plant remains.

Return to footnote12 referrer

Footnote 13

A natural standard soil such as LUFA Speyer is recommended.

Return to footnote13 referrer

Footnote 14

The test substance is applied by mixing it into the soil (this procedure is recommended in general) or by application to the soil surface after the Collembola have been added.

Return to footnote14 referrer

Footnote 15

Substances insoluble in water but soluble in organic solvents are dissolved in a volatile solvent (acetone) and mixed with 2.5 g of the quartz sand required for the artificial soil formulation. After evaporating the solvent, the remainder of the artificial soil constituents and the water are added and mixed thoroughly before introducing it into the test containers. Substances insoluble in water or organic solvents are mixed with 2.5 g of finely ground quartz sand prior to adding the remainder of the artificial soil constituents and water.

Return to footnote15 referrer

Footnote 16

The sphagnum peat is air-dried and finely ground (particle size of 2 ± 1 mm); check that soil prepared with a fresh batch of peat is suitable for Collembola before use in a test; recommend measuring the C/N ratio, pH, and CEC of the peat.

Return to footnote16 referrer

Footnote 17

The amount of sand (69-70%) depends on the amount of calcium carbonate (CaCO₃) needed (i.e., 0.3-1 % CaCO₃ is used depending on the quality and nature of the peat).

Return to footnote17 referrer

Footnote 18

The final water content (~40-60%) of the test substance is achieved either by adding the test substance in a sufficient amount of deionized water, or, if the test substance is added in a dry form, by adding deionized water.

Return to footnote18 referrer

Footnote 19

NI = not indicated.

Return to footnote19 referrer

Footnote 20

Artificial soil should be stored for two weeks in order to equilibrate/stabilise the acidity. If the soil is too acidic, more CaCO₃ is added; if it is too alkaline, the soil can be adjusted by adding more artificial soil mixture without the CaCO₃.

Return to footnote20 referrer

Footnote 21

Maximum water-holding capacity determined as described in the method.

Return to footnote21 referrer

Footnote 22

pH = hydrogen ion concentration.

Return to footnote22 referrer

Footnote 23

NI = not indicated.

Return to footnote23 referrer

Footnote 24

NA = not applicable (artificial soil prepared, hydrated, and then used).

Return to footnote24 referrer

Footnote 25

A preliminary (range-finding) test is optional.

Return to footnote25 referrer

Footnote 26

The preliminary acute mortality test may use the same methodology and equipment as the sublethal test but may be of shorter duration (e.g., one week exposure); only a single assessment of adult survival at the end of the test is required.

Return to footnote26 referrer

Footnote 27

NI = not indicated.

Return to footnote27 referrer

Footnote 28

It is recommended that the concentrations be spaced by a factor of 10.

Return to footnote28 referrer

Footnote 29

If a preliminary test is necessary to determine the range of concentrations for use in the final test, a preliminary one week acute test is performed.

Return to footnote29 referrer

Footnote 30

10 males and 10 females are added to each container.

Return to footnote30 referrer

Footnote 31

For example, 0, 1, 10, 100 and 1000 mg/kg, dry mass.

Return to footnote31 referrer

Footnote 32

If a preliminary test is necessary to determine the range of concentrations for a definitive test, perform an acute lethality test of two weeks duration. The test period can be extended to four weeks to obtain additional information for determining the concentration range of the final test (i.e., qualitative determination of concentrations at which effects on reproduction could be expected).

Return to footnote32 referrer

Footnote 33

The range-finding test is used in cases where sufficient information is not available for determining definitive test concentrations; mortality is the main endpoint accessed after two weeks of exposure.

Return to footnote33 referrer

Footnote 34

For example, 0.1, 1, 10, 100 and 1000 mg/kg, dry mass.

Return to footnote34 referrer

Footnote 35

NI = not indicated.

Return to footnote35 referrer

Footnote 36

The highest test concentration must be ≤ 1000 mg/kg dry wt of the test chemical.

Return to footnote36 referrer

Footnote 37

The range of test concentrations should be selected to obtain a concentration-response relationship for growth (i.e., adult mortality should be similar in the control and test treatments).

Return to footnote37 referrer

Footnote 38

10 males and 10 females are added to each container.

Return to footnote38 referrer

Footnote 39

The concentrations selected to provide the EC10 are based on the results of the preliminary test (see Table 8, this appendix).

Return to footnote39 referrer

Footnote 40

Substances do not need to be tested at concentrations higher than 1000 mg/kg dry wt of the test substance.

Return to footnote40 referrer

Footnote 41

Two experimental designs are proposed. Depending on that chosen, the statistical endpoints would differ as would the recommended number of test concentrations and the recommended number of replicates/treatment (including the number of control vessels).

Return to footnote41 referrer

Footnote 42

The concentrations should be spaced by a factor not exceeding 2.0 (e.g., = ~1.8).

Return to footnote42 referrer

Footnote 43

The spacing factor may vary, i.e., smaller at low concentrations and larger at high concentrations.

Return to footnote43 referrer

Footnote 44

For determination of the ECx, an adequate number of concentrations to cause at least four statistically significant different mean responses at these concentrations is recommended. The spacing factor should ensure that the majority of test concentrations are on the slope of the ECx curve.

Return to footnote44 referrer

Footnote 45

If no effects are observed at the highest concentration in the range-finding test (i.e., 1000 mg/kg), the reproduction test could be performed as a limit test, using a test concentration of 1000 mg/kg, in which case eight replicates should be used for both the treated soil and the control.

Return to footnote45 referrer

Footnote 46

NI = not indicated.

Return to footnote46 referrer

Footnote 47

L = light; D = dark.

Return to footnote47 referrer

Footnote 48

Test may be carried out at 20°C if required.

Return to footnote48 referrer

Footnote 49

W = percent water (moisture content); pH = hydrogen-ion concentration.

Return to footnote49 referrer

Footnote 50

Actual concentrations of the test chemical can be confirmed at the beginning and end of the test, where analytical techniques are available.

Return to footnote50 referrer

Footnote 51

An additional two replicates are set up for each concentration and control(s) to allow measurements of pH, moisture content, and actual test chemical concentrations. Water content and pH are determined in the presence of 1M potassium chloride (KCl) of the test substrate.

Return to footnote51 referrer

Footnote 52

Measurements of individual body lengths are made using a computerized image analysis system calibrated to measure lengths of between 0.5 and 3.5 mm.

Return to footnote52 referrer

Footnote 53

The soil from which individuals have been removed is collected and placed into a tube. It is then incubated at 15°C for 21 days or at 20°C for 14 days until egg hatch is complete. After the incubation period, the number of juveniles present is assessed by flotation in water.

Return to footnote53 referrer

Footnote 54

The water content of the soil substrate is maintained during the test period by reweighing the test containers periodically and if necessary replenishing lost water. At the end of the test, the water content should not differ by more than 10% from the water content at the beginning of the test.

Return to footnote54 referrer

Footnote 55

After two weeks, the water content is checked by reweighing. If water loss exceeds 2% of the initial water content, then water is added to compensate for loss.

Return to footnote55 referrer

Footnote 56

To facilitate checking of pH and moisture content of the test substrate, it is recommended that an additional vessel be set up for each concentration.

Return to footnote56 referrer

Footnote 57

pH is measured in a 1M KCl solution.

Return to footnote57 referrer

Footnote 58

Vessels are weighed to check soil humidity; weight loss is replenished by the addition of an appropriate amount of deionized water (note: weight loss can be reduced by maintaining a high air-humidity, > 80%).

Return to footnote58 referrer

Footnote 59

A controlled temperature gradient extractor is used to collect Collembola from the soil. The heat coming from a heating element at the top of the extraction box is regulated through a thermistor placed on the surface of a soil sample. The temperature in the cooled liquid surrounding the collecting vessel is regulated through a thermistor situated at the surface of the collecting vessel. The thermistors are connected to a programmable controlling unit which raises the temperature according to pre-programmed schedules. At the end of the test, the animals are collected either in saturated aqueous benzoic acid with a few drops of detergent added, or at 2°C on a smooth plaster/charcoal surface. Heat development during extraction may be increased every 12 h beginning with 25°C and ending with 40°C. Extraction onto plaster/charcoal surface makes chemical analysis and enumeration using digital image processing possible.

Return to footnote59 referrer

Footnote 60

Juveniles may be counted manually with a counting grid or with image analysis. When using an estimation technique, the average error of counting should not exceed 10%.

Return to footnote60 referrer

Footnote 61

NOEC and LOEC (expressed in mg/kg dry mass soil); EC10 and EC50 are optional additional endpoints.

Return to footnote61 referrer

Footnote 62

Test soil is emptied into a 250 mL vessel to which 200 mL of distilled water is added. The soil is gently agitated with a fine paintbrush to allow Collembola to float to the water surface. A small amount, approximately 0.5 mL of black Kentmere photographic dye (nontoxic) may be added to the water to aid counting by increasing the contrast between the water and the white Collembola. Counts may be carried out by eye, under a light microscope, or by photographing the surface of each vessel and later counting the Collembola on the enlarged prints or projected slides. Counts may also be performed using digital image processing.

Return to footnote62 referrer

Footnote 63

A concentration series from 0 to 4 mg dimethoate (a.i.)/kg LUFA 2.2 soil will produce a decreasing survival and reproduction. EC50 for reproduction should range within 1.5 to 2.5 mg/kg in the LUFA 2.2 soil.

Return to footnote63 referrer

Footnote 64

Betanal plus (a.i. 160 g/L Phenmedipham); E 605 forte (a.i. 507.5 g/L Parathion).

Return to footnote64 referrer

Footnote 65

For Betanal plus, effects on reproduction (α = 0.05) were observed at concentration of between 100 mg and 200 mg of the product per kg dry mass of the substrate. For E 605 forte, effects on mortality and reproduction were observed at concentrations of between 0.18 mg and 0.32 mg product and between 0.1 mg and 0.18 mg product per kg dry mass of the substrate, respectively.

Return to footnote65 referrer

Footnote 66

NI = not indicated.

Return to footnote66 referrer

Table of contents

Date modified:: 2017-06-13

Language selection

Search

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

1. Test Species, Test Type, and Test Duration

2. Specifics on Test Organisms at Start

3. Test Vessels and Materials

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

5. Manipulation of Artificial Soil Before Use in Test

6. Negative Control Soil

7. Storage and Characterization of Test Soil

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

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

10. Feeding and Aeration During Test

11. Temperature and Lighting During Test

12. Measurements and Biological Observations During Test

13. Terminating Test, Biological Endpoints, and Statistical Endpoints

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

Page details