1
TEST SPECIFIC CHECKLIST 1
Prepared: March 2007
Tests for M easuring Survival and Reproduction of
Springtails Exposed to Contaminants in Soil
Parameter
Specification
Sample Handling
Containers. . . . . . . . .
Labelling. . . . . . . . . . .
Transport. . . . . . . . . .
Holding Tim e. . . . . . .
Holding Conditions. . .
Sample Preparation
a) Field-Collected
Test Soil
Sieving. . . . . . . . . . . .
Hom ogenization. . . . .
TE & pH Adjustm ent
and Soil Equilibrium .
Characterization. . . . .
Moisture Content. . . .
1
Non-toxic m aterial for transport and storage (M ust). . . . . . . . . . . . . . . . . . . . . .
New or thoroughly cleaned, or lined with high-quality plastic (M ust). . . . . . . . . .
Sam ple containers sealed and labelled or coded im m ediately after filling for
field-collected soils and/or upon receipt in the lab for chem icals (M ust). . . . . . .
Labelling and accom panying records include a code or description that
identifies sam ple type (e.g., grab, core, com posite), source, precise location,
land use inform ation, replicate num ber, and date of collection (M ust); nam e
and signature of sam pler(s) should also be included. . . . . . . . . . . . . . . . . . . . . .
Sam ples to be kept from overheating during transport or storage. . . . . . . . . . . .
Sam ples m ust not freeze or partially freeze (unless they are frozen when
collected) or be allowed to dehydrate (unless they are saturated with excess
water upon arrival at the lab) during transport or storage(M ust). . . . . . . . . . . . .
Sam ples should be kept in the dark (i.e., light-tight or opaque containers). . . . .
Sam ples should rem ain cool (e.g., 7 ± 3EC) during transit. . . . . . . . . . . . . . . . .
Date sam ple(s) received at the laboratory recorded (M ust). . . . . . . . . . . . . . . .
Sam ple tem perature upon receipt at laboratory m easured and recorded. . . . . .
Test should start within 2 weeks, and m ust start within 6 weeks unless soil
contam inants are known to be stable (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sam ples stored for future use m ust be held in airtight containers (M ust). . . . . .
Store sam ples in dark at 4 ± 2EC; these conditions m ust be applied if sam ples
contain PAHs, other light-sensitive toxicants, or unstable volatiles (M ust). . . . .
Sam ple brought to room tem perature and thoroughly re-m ixed just before
analysis or testing (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sam ple sieved (e.g. 4 - 6 m m m esh) without water to rem ove oversize
m aterial, if necessary (e.g., debris and indigenous m acro-organism s). . . . . . . .
Soil and/or solid particulate waste for testing should be hom ogenized, unless
inappropriate (e.g., affects concentration or bioavailability of contam inants) . . .
Any m oisture that separates from a sam ple during its transport and/or storage
m ust be re-m ixed into it if possible (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Test soil prepared on day preceding test (Day -1)and held under test conditions
(i.e., 20 ± 2EC) overnight, prior to testing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
pH of field-collected soils not adjusted (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . .
Each soil (including negative control and reference soil) is analysed for particle
size distribution (%sand, silt, and clay), total organic carbon content (%),
organic m atter content (%), m oisture content (%), W HC (%), pH and
conductivity, as a m inim um (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Optional analyses of contam inants of concern (e.g., m etals, polycyclic arom atic
hydrocarbons (PAHs), pesticides). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
W ater Holding Capacity (W HC) of soils (artificial and site) are known and
determ ined using a recognized standard procedure (M ust). . . . . . . . . . . . . . . .
Optim al m oisture content of test soils (artificial and site) determ ined and
expressed as % W HC (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For soils with high peat content where W HC cannot be reliably determ ined
using the recom m ended m ethod, the optim al m oisture content can be
estim ated by eye (appropriate consistency) and m oisture content determ ined
thereafter and reported as such (i.e., as m oisture content instead of %W HC). .
W HC determ ined gravim etrically by drying subsam ple for ~24h at 105EC. . . . .
Test soil hydrated to optim al % of W HC after preparing test conc.. . . . . . . . . . .
Page 1 / 9
Met Specifics
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Checklist based on Environm ent Canada’s “Test for Measuring Survival and Reproduction
of Springtails Exposed to Contam inants in Soil”. See Endnote for references.
This checklist is a summary of the requirements and recommendations in the Environment Canada test method. As a summary, it will not contain all supplementary
information. If there is a discrepancy between the checklist and the Environment Canada test method, the test method is taken as the definitive source.
TEST SPECIFIC CHECKLIST 1
Prepared: March 2007
Tests for M easuring Survival and Reproduction of
Springtails Exposed to Contaminants in Soil
Parameter
Sample Preparation
(continued)
Test Concentrations.
b) Chemical-Spiked
Test Soil
Chem ical
Characterization. . . . .
Preparation of
Mixtures. . . . . . . . . . .
Solvent. . . . . . . . . . . .
Test Soil Equilibrium .
Test Conditions
Test Facility. . . . . . . .
Specification
Each batch (i.e., treatm ent) is prepared in sufficient quantity for all replicates
and physicochem ical analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mix hom ogenized test soil with negative control soil or reference soil, if m ulticoncentration test to prepare each treatm ent/concentration using geom etric
series; ensure hom ogeneity (i.e., m ix until texture, colour, and m oisture are
hom ogeneous) and divide into replicates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Inform ation on chem ical or chem ical product(s) obtained before test starts
includes: stability, water solubility, vapour pressure, purity, estim ated toxicity to
test species and hum ans and biodegradability. . . . . . . . . . . . . . . . . . . . . . . . . .
Concentration of test chem ical in soil m easured at beginning and end of test, in
high, m edium and low concentrations, as a m inim um . . . . . . . . . . . . . . . . . . . . .
Procedure depends on nature of test substance(s), test design, and objectives;
test substance(s) m ay be prepared m anually or by m echanical agitation; test
substance(s) m ay be added as m easured quantities in solution (i.e., in water or
an organic solvent) or as a solid m aterial com prised partly or
com pletely of the test substance(s); ensure hom ogeneity. . . . . . . . . . . . . . . . . .
For each treatm ent, m ixing conditions (solution:soil ratio, m ixing and holding
tim e and TE) m ust be standardized (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Each batch (i.e., treatm ent) is prepared in sufficient quantity for all replicates
and physicochem ical analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Solvent control included in test (in addition to negative control) if organic
solvent used for test substance(s) that are not soluble in water (M ust) . . . . . . .
Solvent control from sam e batch used to m ake the stock solution of test
substance, contains the sam e concentration of solubilizing agent that is
present in the highest concentration of test chem ical, and is prepared using the
sam e procedure (M ust) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For any test that includes solvent control soil, test results for that soil are
com pared statistically with those for negative control soil (M ust). . . . . . . . . . . .
All test soil batches are hydrated, hom ogenized and placed in replicate test
vessels on the day prior to testing (Day -1). . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Replicate vessels covered (unperforated lids) and incubated overnight under
test conditions (i.e., 20 ± 2EC), prior to testing. . . . . . . . . . . . . . . . . . . . . . . . . . .
Isolated areas with tem perature & lighting control (e.g., environ. cham bers, or
equivalent); well ventilated & free of fum es; isolated from areas for organism
culturing, and for sam ple preparation/storage. . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipm ent, apparatus and construction m aterials m ade of non toxic m aterial
and m inim ize sorption of chem icals (e.g., borosilicate glass, nylon, high-density
polyethylene, high density polystyrene, polycarbonate, fluorocarbon plastics,
type 316 stainless steel) (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Instrum ents for routine m easurem ents (e.g., pH, tem p.) are available (M ust). .
Laboratory equipped for analysis of soil m oisture content. . . . . . . . . . . . . . . . . .
Other equipm ent includes: drying oven (capable of 105EC), a weighing balance
(accurate to 0.1 m g), and a pH m eter (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . .
Safety apparatus used when preparing m ixtures and test soils (M ust). . . . . . . .
All test vessels, equipm ent, and supplies that m ight contact site soils, test
soils, test (hydration) water, stock solutions, or test solutions, are clean and
rinsed with test water before being used (M ust). . . . . . . . . . . . . . . . . . . . . . . . .
Page 2 / 9
Met Specifics
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This checklist is a summary of the requirements and recommendations in the Environment Canada test method. As a summary, it will not contain all supplementary
information. If there is a discrepancy between the checklist and the Environment Canada test method, the test method is taken as the definitive source.
TEST SPECIFIC CHECKLIST 1
Prepared: March 2007
Tests for M easuring Survival and Reproduction of
Springtails Exposed to Contaminants in Soil
Parameter
Test Conditions
(continued)
Initial Tests. . . . . . . . .
Negative Control Soil.
Clean Field-Collected
Soil. . . . . . . . . . . . . . .
Artificial Soil. . . . . . . .
Positive Control Soil.
Reference Soil. . . . . .
Initial Hydration of
Test Soils. . . . . . . . . .
Test W ater. . . . . . . . .
Specification
$5 control perform ance tests and $5 reference toxicity tests with candidate
sam ples of artificial or natural negative control soil intended for routine use,
should be undertaken by laboratory personnel to confirm acceptable
perform ance of test species using procedures & conditions in test m ethod. . . .
Conditions and procedures for initial control perform ance test should follow
those described for conducting definitive tests.. . . . . . . . . . . . . . . . . . . . . . . . . .
Conditions and procedures for initial reference toxicity tests should be identical
to those described for routine reference toxicant tests. . . . . . . . . . . . . . . . . . . .
Each test should be perform ed using a different lot (group of age-synchronized
organism s) of test organism s of the sam e species, from the sam e source.. . . .
Data from initial control perform ance test shows that criteria for test validity can
be m et (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Data from initial reference toxicity tests should be com pared by calculating and
appraising the m agnitude of the coefficient of variation (CV) of the derived
LC50s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Natural clean field-collected soil or artificial soil for which previous tests with
the chosen test species dem onstrated that the test validity criteria could be
regularly m et; recom m end artificial soil for tests with chem icals or chem ical
products spiked in soil. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Negative control soil included as a treatm ent in every toxicity test (M ust). . . . .
Natural soil collected from a clean (uncontam inated) site; free of pesticide or
fertilizer for $5 years. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Laboratory dem onstrates experim ental evidence that natural soil from a given
source has m et test validity criteria before being used as negative control soil
in a definitive test (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Soil analysed for recom m ended physicochem ical characteristics (see Section
3.3.1 in EPS 1/RM/47). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Natural soil can be air-dried (10 - 20% m oisture content), coarse-screened (4 6 m m ), transferred to clean plastic pails, and stored in darkness at 4 ± 2EC. . .
10% Sphagnum sp. peat, air dried and sieved (2-m m m esh); 20% kaolin clay
(with particle size < 40ìm ); and 70% silica sand (grade 70); m ixed dry. . . . . . .
Add reagent-grade calcium carbonate to dry m ixture to adjust pH to 6.0 - 7.5 . .
Hydrate using test water to ~28% of W HC and adjust pH as necessary with
m ore calcium carbonate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Artificial soil stored in the dark at 20 ± 2EC for $3 days before use in toxicity
test; thereafter soil can be stored at 4 ± 2EC. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Should be included in each series of soil toxicity tests; m ay be a negative
control soil spiked with a reference toxicant or with one or m ore toxic
chem icals of concern; or a highly contam inated sam ple of field-collected soil. .
One or m ore sam ples for tests with field-collected soil, ideally taken from
site(s) presum ed to be clean but near sites of test soil collection. . . . . . . . . . . .
Physicochem ical characteristics including organic carbon, organic m atter,
particle size distribution, texture, and pH are sim ilar to test soils. . . . . . . . . . . . .
Tests involving sam ples of reference soil m ust also include a sam ple of
negative control soil (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Field-collected soils are hydrated with test water to the optim al percentage of
its W HC (i.e., soil is a hom ogenous, crum bly consistency; clum ps 3 - 5 m m );
artificial soils are hydrated to ~70% of W HC. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Deionized or distilled water or better, such as reagent-grade water produced by
a system of reverse osm osis, carbon and ion exchange cartridges (M ust). . . .
Page 3 / 9
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This checklist is a summary of the requirements and recommendations in the Environment Canada test method. As a summary, it will not contain all supplementary
information. If there is a discrepancy between the checklist and the Environment Canada test method, the test method is taken as the definitive source.
TEST SPECIFIC CHECKLIST 1
Prepared: March 2007
Tests for M easuring Survival and Reproduction of
Springtails Exposed to Contaminants in Soil
Parameter
Test Conditions
(continued)
Glassware Cleaning.
Measurem ents
During Test
Moisture Content. . . .
pH. . . . . . . . . . . . . . . .
Tem perature.. . . . . . .
Conductivity. . . . . . . .
Chem ical Analyses. . .
Reference Toxicity
Tests.. . . . . . . . . . . . .
W arning Chart. . . . . .
Test Type. . . . . . . . . .
Test Duration. . . . . . .
Test TE. . . . . . . . . . . .
Light Quality. . . . . . . .
Light Intensity. . . . . . .
Page 4 / 9
Specification
Met Specifics
Y
N NA
Soak; detergent wash; 2 tap water rinses; acid wash (e.g., 10% nitric or
hydrochloric acid, m etal-free grade) to rem ove scale, m etals and bases; 2
rinses with test water; pesticide-free acetone wash to rem ove organic
com pounds and HPLC-grade hexane wash for oily residues; allow organic
solvent to volatilize and rewash with detergent; 3 rinses with test water. . . . . . .
Soil m oisture content in each treatm ent/concentration at test start and end
(M ust).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Moisture content determ ined gravim etrically (see EPS 1/RM/47). . . . . . . . . . .
Moisture content calculated on a dry wt. basis (M ust). . . . . . . . . . . . . . . . . . . .
Soil pH in each treatm ent/concentration at test start and end (M ust). . . . . . . .
Soil pH m easured using a m odified CaCl 2 Slurry Method (see EPS 1/RM/47).
Air tem perature in test facility, daily or continuously (M ust).. . . . . . . . . . . . . . .
Conductivity m easured at test start and end when test soil is suspected of
having a high salt content.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Norm ally m easure at beginning and end of test, in high, m edium , and low
strengths as a m inim um . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Static, 7-day (for O. folsomi, and F. fimetaria) or 14-day (for F. candida) m ulticoncentration test (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conditions the sam e as those for a m ulti-concentration definitive test except for
test duration, # of test concentrations, # of organism s per vessel for O. folsomi,
and F. fimetaria, and test validity criteria (M ust). . . . . . . . . . . . . . . . . . . . . . . . .
Use age-synchronized springtails derived from the sam e population (i.e.,
culture) of springtails used to produce age-synchronized organism s for the
definitive tests (M ust).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Recom m end using a portion of the sam e age-synchronized culture used to
start the definitive test, if possible. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Perform once every 2 m onths, or in conjunction with definitive test(s) with soil
sam ples (M ust); use boric acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare and test $5 concentrations plus a negative control (M ust), using
artificial soil. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Calculate m ean (± SD) % survival in each treatm ent at test end (Day 7 or Day
14) (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reference toxicity test invalid if m ean survival of adults in negative control soil
is <80% at test end (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Determ ine 7- or 14-day LC50 and 95% confidence lim its (M ust); express as
m g boric acid/kg dry wt.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Recom m end 21-day (for F. fimetaria) or 28-day (for O. folsomi & F. candida)
tests with boric acid be perform ed according to Section 4 of EPS 1/RM/47, at
least twice a year or in conjunction with definitive test. . . . . . . . . . . . . . . . . . . . .
Prepared and updated with all com parable LC50s for each species and
reference toxicant (i.e., all com parable LC50s plotted successively on a
warning chart) (M ust).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Separate warning chart prepared and updated for each dissim ilar procedure
(e.g., species of test organism s, reference toxicant, test duration) (M ust). . . . .
Static; whole soil (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
21 days for F. fimetaria; 28 days for O. folsomi and F. candida (M ust). . . . . . . .
Air tem perature: 20 ± 2EC daily average (M ust); 20 ± 3EC instantaneous
(M ust).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Incandescent or fluorescent (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
400 to 800 lux; $400 lux (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
This checklist is a summary of the requirements and recommendations in the Environment Canada test method. As a summary, it will not contain all supplementary
information. If there is a discrepancy between the checklist and the Environment Canada test method, the test method is taken as the definitive source.
TEST SPECIFIC CHECKLIST 1
Prepared: March 2007
Tests for M easuring Survival and Reproduction of
Springtails Exposed to Contaminants in Soil
Parameter
Test Conditions
(continued)
Photoperiod. . . . . . . .
Vessel Size & Type.
Soil Mass. . . . . . . . . .
Vessel Labelling. . . . .
Vessel Position. . . . . .
# Replicates/Conc.. . .
# Test Conc.. . . . . . . .
# Springtails/Vessel.
Organism Selection.
Feeding Regim e. . . . .
Test Soil Hydration. . .
Test Soil Aeration. . . .
Test Validity Criteria.
Specification
Fixed daily photoperiod (M ust) (i.e., 16 h light: 8 h dark or 12 h light:12 h
dark). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
100- to 125-m L wide-m outhed glass jars (~ 5 to 8 cm diam ) (M ust); covered
(plastic or m etal lid). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Test vessels are inert to test and reference substances or contam inant
m ixtures (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
All test units are cleaned thoroughly and rinsed with test water before use
(M ust) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
30 g wet weight of test soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Clearly labelled/coded: test substance, concentration, and replicate # (M ust). .
Date and tim e of test initiation on labels or data sheets (M ust). . . . . . . . . . . . . .
Test containers are positioned random ly within test facility and m oved during
test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
$3 replicates/treatm ent; $5 replicates/control for m ulti-concentration test; $5
replicates/treatm ent and control soil for single-concentration test (M ust). . . . . .
$2 additional replicate for each treatm ent is recom m ended for conducting
physicochem ical analyses on Day 0 and at test end. . . . . . . . . . . . . . . . . . . . . .
1, plus controls for single-concentration test. . . . . . . . . . . . . . . . . . . . . . . . . . . .
$7, plus controls for m ulti-concentration test (M ust); m ore recom m ended ($10,
plus controls); geom etric series. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For O. folsomi: 15 organism s/vessel (10 fem ales and 5 m ales)(M ust). . . . . . . .
For F. candida: 10 organism s/vessel (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . .
For F. fimetaria: 20 organism s/vessel (10 fem ales and 10 m ales)(M ust). . . . . .
Springtails are transferred to test vessels on the day after the soil equilibration
period (Day 0). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Excess num ber of springtails than those required for testing are available from
age-synchronized culture vessels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For O. folsomi and F. fimetaria, the appropriate num ber of fem ales (larger with
round abdom ens) and m ales (sm aller, m ore slender) are transferred to each
vessel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For F. candida, the required num ber of individuals (asexual) are transferred to
each vessel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For each replicate, springtails are selected and m oved to a transfer container,
given a final observation to confirm num ber, sex, and health, and gently
transferred as a group to the soil surface of the test vessel.. . . . . . . . . . . . . . . .
The order of adding springtails to each vessel are random ly allocated with
respect to treatm ent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Granulated dry yeast; for O. folsomi: ~5m g/vessel on Days 0, 7, 14, and 21; for
F. candida: ~10 m g/vessel on Day 0 and ~20 m g/vessel on Day 14; and for F.
fimetaria: ~10 m g/vessel on Days 0 and 14; if yeast not consum ed from
previous feeding, no further yeast is added(M ust); yeast not rem oved if
unconsum ed. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Soil m oistened with de-ionized water weekly during aeration, as necessary. . . .
Lids of each test vessel rem oved $once/week for aeration (M ust). . . . . . . . . . .
Test invalid if m ean survival of adults in negative control soil at test end is
<70% for F. fimetaria and O. folsomi; and for F. candida <70% in natural soil
and <80% in artificial soil; or reproduction rate for adult springtails in negative
control soil is <100 live progeny per control vessel at test end for all three
species (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Negative control soil used to judge validity of test regardless of whether the
reference or negative control soils are used for statistical com parisons (M ust).
Page 5 / 9
Met Specifics
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This checklist is a summary of the requirements and recommendations in the Environment Canada test method. As a summary, it will not contain all supplementary
information. If there is a discrepancy between the checklist and the Environment Canada test method, the test method is taken as the definitive source.
TEST SPECIFIC CHECKLIST 1
Prepared: March 2007
Tests for M easuring Survival and Reproduction of
Springtails Exposed to Contaminants in Soil
Parameter
Test Conditions
(continued)
Biological
Observations. . . . . . .
Biological Endpoint. . .
Statistical Endpoint. . .
Calculation of ICp. . . .
Test Organisms
Species.. . . . . . . . . . .
Source. . . . . . . . . . . .
Source of Brood
Stock for Culture.. . . .
Age at test start. . . . .
Specification
Condition, appearance, and # live springtails placed in each test vessel on Day
0 (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
W eekly observations of any excessive growth of bacteria or fungi, any feeding
activity, and the presence and quantity of any uneaten food. . . . . . . . . . . . . . . .
# live adult springtails and # of live progeny in each test vessel on Day 21 for F.
fimetaria or Day 28 for O. folsomi and F. candida (M ust). . . . . . . . . . . . . . . . . .
Springtails extracted using floatation or heat extraction. . . . . . . . . . . . . . . . . . . .
Springtails enum erated directly (i.e., m anually), through digital analysis, or with
im age analysis software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Missing adults are counted as dead (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . .
# live springtail progeny in each test vessel at test end (M ust). . . . . . . . . . . . . .
# live adult springtails in each replicate at test end (M ust). . . . . . . . . . . . . . . . .
Mean (± SD) % survival of adults in each treatm ent on Day 21 for F. fimetaria
and Day 28 for O. folsomi and F. candida (M ust). . . . . . . . . . . . . . . . . . . . . . . .
Mean (± SD) # live progeny in each treatm ent on Day 21 for F. fimetaria and
Day 28 for O. folsomi and F. candida (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . .
For m ulti-concentration test: 21- or 28-day LC50 for adult springtails and 21- or
28-day ICp for reproductive inhibition based on num bers of live progeny
produced in each concentration during 21- or 28-day test (M ust). . . . . . . . . . . .
Calculation of endpoints by entering concentrations as logarithm s (M ust). . . . .
Linear and/or nonlinear regression procedures used for calculation of ICps and
95% confidence lim its (M ust).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Initial plot of raw data against log concentration.. . . . . . . . . . . . . . . . . . . . . . . . .
All requirem ents for regression analysis outlined in Section 4.8.2 of EPS
1/RM/47 are m et (M ust).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Endpoints generated by regression analysis are bracketed by test
concentrations (i.e., extrapolation of endpoints beyond the highest test
concentration is not acceptable) (M ust).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ICPIN analyses used only if regression analyses fail to provide m eaningful
ICps. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Page 6 / 9
Met Specifics
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Mixed-age cultures from governm ent, private, or com m ercial culture. . . . . . . . .
O. folsomi: 28 to 31 days old (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
F. candida: 10 to 12 days old (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
F. fimetaria: 23 to 26 days old (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
...
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Controlled-tem perature laboratory facility.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Culture area isolated from testing, sam ple storage, or sam ple-preparation
areas; designed and constructed to prevent culture contam ination (M ust). . . . .
Culture practices such that each culture is not cross-contam inated with another
Collem bola species (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
...
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Laboratory cultured Orthonychiurus folsomi, Folsomia candida, or Folsomia
fimetaria (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Species identification confirm ed and docum ented by qualified personnel
(M ust).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cultures held in a testing laboratory should be identified to species every 2
years, as a m inim um . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Test organism s m ust be cultured in testing laboratory (M ust).. . . . . . . . . . . . . .
All organism s used in a test are derived from the sam e population (M ust).. . . .
Culture Conditions
Facilities. . . . . . . . . . .
This checklist is a summary of the requirements and recommendations in the Environment Canada test method. As a summary, it will not contain all supplementary
information. If there is a discrepancy between the checklist and the Environment Canada test method, the test method is taken as the definitive source.
TEST SPECIFIC CHECKLIST 1
Prepared: March 2007
Tests for M easuring Survival and Reproduction of
Springtails Exposed to Contaminants in Soil
Parameter
Culture Conditions
(continued)
Apparatus. . . . . . . . . .
Culture Substrate. . . .
Hydration. . . . . . . . . .
Aeration. . . . . . . . . . .
Tem perature of
Substrate. . . . . . . . . .
pH. . . . . . . . . . . . . . . .
Lighting. . . . . . . . . . . .
Acclim ation. . . . . . . . .
Culture Maintenance.
Substrate Renewal. . .
Substrate Monitoring.
Feeding.. . . . . . . . . . .
Specification
All equipm ent, vessels, and accessories that m ight contact organism s, test
water or substrate is clean, rinsed and m ade of non-toxic m aterial (e.g., glass,
Teflon™ , type 316 stainless steel, nylon, Nalgen™ , porcelain, polyethylene,
polypropylene)(M ust) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Culture vessels (e.g., plastic trays or breeding boxes) of 1-6-L capacity are
recom m ended for O. folsomi, and F. candida; transparent or translucent sides
and/or lid for F. candida; 10 cm polystyrene Petri dishes are recom m ended for
F. fimetaria; m inim um substrate depth of 1 cm ; solid or perforated lids; wood is
not recom m ended. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Copper, zinc, brass, galvanized m etal, lead, and natural rubber m ust not be
used (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plaster of Paris and charcoal recom m ended; fertilizer-free potting soil m ay be
used for m aintaining m ass or back-up cultures. . . . . . . . . . . . . . . . . . . . . . . . . .
Hydrate with distilled or de-ionized water; re-hydrate 1-2 tim es/week to
m aintain m oisture (i.e., water just begins to rem ain on surface). . . . . . . . . . . . .
Vessels aerated (i.e., rem ove lid for $ 1 m in.) once/week (M ust); twice/week
recom m ended for F. fimetaria and other species if history of fungal growth. . . .
Daily average, 20 ± 2EC; instantaneous, 20 ± 3EC. . . . . . . . . . . . . . . . . . . . . . .
6.0 - 7.0; verified for each new batch of substrate with pH paper on wet
substrate surface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Incandescent or fluorescent; 400 to 800 lux at substrate surface; fixed daily
photoperiod (e.g., 16 h light: 8 h dark or 12 h light:12 h dark); avoid
overheating cultures; O. folsomi and F. fimetaria can be cultured in com plete
darkness. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gradually (recom m end #3EC/day) for tem perature differences upon arrival. . . .
During age-synchronizing period, organism s are acclim ated in the lab to
tem perature and food to be used in test (M ust).. . . . . . . . . . . . . . . . . . . . . . . . .
Exam ine contents of culture vessels at least once/week; record condition of
culture (organism s and substrate); m aintain loading density of ~2 to 3
adults/cm 3 for O. folsomi and F. candida, & ~ 5 to 6 adults/cm 3 for F. fimetaria.
As required, and at least once every 1 - 2 m onths, regardless of loading
densities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare new culture vessels and transfer springtails into new vessels by
tapping the old vessel over the new one; transfer only a portion to reduce the
population; m ix organism s between independent culture vessels to avoid
inbreeding.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
For O. folsomi and F. fimetaria new cultures should contain a m ixture of m ales
and fem ales. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Air tem p.of culture facility m easured once per week; m oisture level observed at
tim e of weekly aeration; adjust as necessary. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Activated dry yeast (e.g., Fleischm ann’s™ ); quantity based on previous food
consum ption; recom m end ~100 m g for O. folsomi and F. candida (in a 15 x 23
x 8 cm vessel) and ~10 m g for F. fimetaria (in a 10 cm Petri dish); twice/week
at tim e of aeration and re-hydration; place food in 2-3 piles or sprinkle over
m oist (i.e., to activate yeast) substrate surface after rem oving excess
(uneaten) food .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Avoid excessive fungal and bacterial growth (especially for F. fimetaria).. . . . . .
Page 7 / 9
Met Specifics
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This checklist is a summary of the requirements and recommendations in the Environment Canada test method. As a summary, it will not contain all supplementary
information. If there is a discrepancy between the checklist and the Environment Canada test method, the test method is taken as the definitive source.
TEST SPECIFIC CHECKLIST 1
Prepared: March 2007
Tests for M easuring Survival and Reproduction of
Springtails Exposed to Contaminants in Soil
Parameter
Culture Conditions
(continued)
Indices of Culture
Health. . . . . . . . . . . . .
Age-Synchronized
Cultures. . . . . . . . . . .
Handling. . . . . . . . . . .
Test Report
Test Substance. . . . .
Test Organism s. . . . .
Test Facilities. . . . . . .
Test Method. . . . . . . .
Test Conditions. . . . .
Specification
Cultures have low m ortalities, appear healthy, and behave and feed norm ally
(M ust).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Considered healthy if: (1) springtails are m oving actively over the substrate
surface, and (2) results for reference toxicity tests using age-synchronized
springtails derived from the sam e population (i.e., source) as the agesynchronized springtails used to start a definitive test fall within historic warning
lim its (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lab follows age-synchronization procedures described in EPS 1/RM/47. . . . . .
Age-synchronization procedures produce the required num ber of healthy test
organism s of the required age (i.e., 28-31 days old for O. folsomi, 10-12 days
old for F. candida, and 23-26 days old for F. fimetaria) and of sim ilar size
(M ust).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Age-synchronized cultures m eet specific health and perform ance-related
indices (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Handling m inim ized; springtails are transferred using a m oist, fine-tipped
paintbrush; a low suction exhaustor; or by gently tapping one vessel over
another. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Springtails that are injured or appear stressed are discarded and m ust not be
used in a test (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sam ple type or coding as provided to laboratory personnel (M ust). . . . . . . . . .
Inform ation on labelling or coding of each sam ple (M ust). . . . . . . . . . . . . . . . . .
Date of sam ple collection (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Date and tim e sam ple(s) received at test facility (M ust). . . . . . . . . . . . . . . . . . .
Species and source of brood stock (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Age-range of test organism s, at start of test (M ust). . . . . . . . . . . . . . . . . . . . . .
Any unusual appearance, behaviour, or treatm ent of the organism s before the
test (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nam e and address of test laboratory (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nam e of person(s) perform ing the test (or each com ponent of the test)(M ust)
Citation of biological test m ethod used (i.e., as per EPS 1/RM/47) (M ust). . . . .
Design and description if specialized procedure(s) (e.g., preparation of
m ixtures of spiked soil; preparation and use of solvent and, if so, solvent
control) or m odification(s) of the standard test m ethod (M ust). . . . . . . . . . . . . .
Brief description of frequency and type of all m easurem ents and all
observations m ade during test (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nam e and citation of program (s) and m ethods used for calculating statistical
endpoints (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Design and description of any deviation(s) from , or exclusion of, any of the
procedure and conditions specified in EPS 1/RM/47 (M ust). . . . . . . . . . . . . . . .
Num ber of discrete sam ples per treatm ent (M ust). . . . . . . . . . . . . . . . . . . . . . .
Num ber of replicate test vessels for each treatm ent (M ust).. . . . . . . . . . . . . . .
Num ber and description of treatm ents in each test including the control(s); test
concentrations (if applicable) (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Volum e and/or m ass of soil in each test vessel (M ust). . . . . . . . . . . . . . . . . . . .
Num ber of organism s per test vessel and treatm ent (M ust). . . . . . . . . . . . . . . .
Dates when test was started and ended (M ust). . . . . . . . . . . . . . . . . . . . . . . . .
Page 8 / 9
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This checklist is a summary of the requirements and recommendations in the Environment Canada test method. As a summary, it will not contain all supplementary
information. If there is a discrepancy between the checklist and the Environment Canada test method, the test method is taken as the definitive source.
TEST SPECIFIC CHECKLIST 1
Prepared: March 2007
Tests for M easuring Survival and Reproduction of
Springtails Exposed to Contaminants in Soil
Parameter
Specification
Test Conditions. . . . .
(continued)
Feeding regim e and ration during test (M ust)). . . . . . . . . . . . . . . . . . . . . . . . . .
For each soil sam ple: any m easurem ents of soil particle size, m oisture content,
water holding capacity, pH and conductivity (if done)(M ust). . . . . . . . . . . . . . . .
For each com posite sam ple of subsam ples taken at the sam e tim e from all
replicates of each treatm ent: all m easurem ents of tem perature, pH, m oisture
content, and water holding capacity (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method used for extracting the Collem bola from the soil (i.e., flotation or heat
extraction) at test end (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method used for enum erating the Collem bola (i.e., m anual, digital analysis, or
im age analysis) at test end (M ust).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mean (± SD) percent survival of adult Collem bola in each treatm ent, including
controls on Day 21 for F. fimetaria, and Day 28 for F. candida and O. folsomi
(M ust).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mean (± SD) num ber of surviving juveniles in each treatm ent, including
controls on Day 21 for F. fimetaria, and Day 28 for F. candida and O. folsomi
(M ust).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Any LC50 (including the associated 95% confidence lim its and, if calculated,
the slope) determ ined (M ust).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Any additional LCx (e.g., LC20) calculated (M ust). . . . . . . . . . . . . . . . . . . . . . .
Any ICp (with its 95% confidence lim its) determ ined for the data on
reproductive success (i.e., num ber of surviving juvenile Collem bola in each
treatm ent at test end) (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Details regarding any transform ation of data, and indication of quantitative
statistical m ethod used or procedures applied to the data (M ust). . . . . . . . . . . .
For a m ulti-concentration test with chem ical-spiked soil, indication as to
whether results are based on nom inal or m easured concentrations of
chem ical(s) or chem ical product(s)(M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
All values for m easured concentrations (M ust). . . . . . . . . . . . . . . . . . . . . . . . . .
Results for any 7-day LC50 (including its 95% confidence lim its) for O. folsomi,
or F. fimetaria or 14-day LC50 (including its 95% confidence lim its) for F.
candida perform ed with the reference toxicant in conjunction with the definitive
soil toxicity test, using the sam e lot (group) of test organism s(M ust). . . . . . . . .
Geom etric m ean value (± 2 SD) for the sam e reference toxicant and test
species, as derived at the test facility in previous 7- or 14-day LC50 tests using
the procedures and conditions for reference toxicity tests described in EPS
1/RM/47 (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Anything unusual about the test, any problem s encountered, and any rem edial
m easures taken (M ust). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Original data sheets m ust be signed or initialled, and dated by the laboratory
personnel conducting the tests (M ust).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Test Results. . . . . . . .
Original Data Sheets.
Info. Kept on-File
Do lab SOPs indicate that the inform ation on Section 7.2 of the EPS 1/RM/47
m ethod m ust be kept on file for $5 years? (M ust). . . . . . . . . . . . . . . . . . . . . . . .
Page 9 / 9
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For details of this inform ation, see Section 7.2 of EPS 1/RM/47.
Environm ent Canada, “Biological Test Method: Test for Measuring Survival and Reproduction of Springtails
Exposed to Contam inants in Soil”, Method Developm ent and Applications Section, Environm ent
Canada, Ottawa, ON, Report EPS 1/RM/47 (2007).
This checklist is a summary of the requirements and recommendations in the Environment Canada test method. As a summary, it will not contain all supplementary
information. If there is a discrepancy between the checklist and the Environment Canada test method, the test method is taken as the definitive source.