8810

Echinoderm Fertilization and Development

Approved by Standard Methods Committee, 2011. Editorial revisions, 2021. Joint Task Group: 22nd Edition—Steven M. Bay (chair), R. Scott Carr, Paul Dinnel, Diane E.
Nacci, Patti L. TenBrook, Amy L. Wagner, Jack Q. Word.

8810  A. Introduction

1. Background 7. Okubo K, Okubo T. Study of the bio-assay method for the evaluation
of water pollution—II. Use of the fertilized eggs of sea urchins and
The Phylum Echinodermata encompasses a widely distrib- bivalves. Bull Tokai Reg Fish Res Lab. 1962;32:131–140.
uted and diverse group of marine animals. The class Echinoidea 8. Nacci D, Jackim E, Walsh R. Comparative evaluation of three rapid
marine toxicity tests: Sea urchin early embryo growth test, sea
includes sand dollars and sea urchins, organisms that are com-
urchin sperm cell toxicity test and Microtox. Environ Toxicol Chem.
mon inhabitants of rocky shores and ocean bottoms over all depth 1986;5(6):521–525.
ranges. Many echinoid species are maintained easily in the labo- 9. Dinnel PA, Link JM, Stober QJ, Letourneau MW, Roberts WE. Com-
ratory and are responsive to simple methods of spawning induce- parative sensitivity of sea urchin sperm bioassays to metals and pes-
ment. Gametes and embryos are easily obtained and reared in the ticides. Archiv Environ Contam Toxicol. 1989;18(5):748–755.
laboratory and have been the subject of scientific research for 10. Morrison G, Torello E, Comeleo R, Walsh R, Kuhn A, Burgess
more than 100 years.1 Numerous species have been used, and lab- R, Tagliabue M, Greene W. Intralaboratory precision of saltwater
oratory techniques have been developed that use various stages of short-term chronic toxicity tests. Res J Water Pollut Control Fed.
each organism’s life cycle.2–6 1989;61(11/12):1707–1710.
Toxicity tests involving short-term exposure of gametes or 11. Schimmel SC, Morrison GE, Heber MA. Marine complex effluent
toxicity program: Test sensitivity, repeatability and relevance to
embryos are of comparable or greater sensitivity to many contam-
receiving water toxicity. Environ Toxicol Chem. 1989;8(8):739–746.
inants than tests with other marine species and life stages.7–11 Echi- 12. Hose JE. Potential uses of sea urchin embryos for identifying toxic
noid toxicity tests can be performed on small volumes (≥2 mL) chemicals: Description of a bioassay incorporating cytologic, cyto-
over short periods (1–96 h), and under static conditions without genetic and embryologic endpoints. J Appl Toxicol. 1985;5(4):245–
feeding. These tests have been used successfully to evaluate the 254.
toxicity of effluents, receiving waters, chemicals, and sediments if 13. Carr RS, Chapman DC. Comparison of methods for conducting
the test samples’ salinity is near typical ocean levels (28–34 g/kg). marine and estuarine sediment porewater toxicity tests—extraction,
Recent adaptations of these test methods have expanded applica- storage, and handling techniques. Arch Environ Contam Toxicol.
tions to include evaluation of genotoxic effects,12 interstitial water,13 1995;28(1):69–77.
and toxicity identification evaluation (TIE) studies.14 Methods sim- 14. Bailey HC, Miller JL, Miller MJ, Dhaliwai BS. Application of tox-
icity identification procedures to the echinoderm fertilization assay
ilar to these have been proposed or recommended as components
to identify toxicity in a municipal effluent. Environ Toxicol Chem.
of regulatory programs.2,15–17 1995;14(12):2181–2186.
15. Pastorok RA, Anderson JW, Butcher MK, Sexton JE. West Coast
References marine species chronic protocol variability study. Final Report.
Olympia (WA): Washington Department of Ecology; 1994.
1. Hinegardner RT. Growth and development of the laboratory cultured 16. Chapman GA, Denton DL, Lazorchak JM. Short-Term Methods for
sea urchin. Biol Bull. 1969;137(3):465–475. Estimating the Chronic Toxicity of Effluents and Receiving Waters
2. Environment Canada. Biological Test Method: fertilization assay to West Coast Marine and Estuarine Organisms; EPA-600/R-95-136.
using Echinoids (sea urchins and sand dollars); Rep. EPS 1/RM/27. Cincinnati (OH): National Exposure Research Laboratory, U.S.
Ottawa (Ont): Environment Canada; 1992. Environmental Protection Agency; 1995.
3. E1563-98; Standard guide for conducting static acute toxicity tests 17. U.S. Environmental Protection Agency. Short-term methods for
with echinoid embryos. In: Annual Book of ASTM Standards, Vol. estimating the chronic toxicity of effluents and receiving waters to
11.06. West Conshohocken (PA): ASTM International; 2009. marine and estuarine organisms; EPA-821-R-02-014. Washington
4. Dinnel PA, Pagano GG, Oshida PS. A sea urchin test system for DC: Office of Water (4303T); 2002.
environmental monitoring. In: Burke RD, Mladenov PV, Lambert P,
Parsley RL, eds. Echinoderm biology. Rotterdam, The Netherlands: Bibliography
A.A. Balkema; 1988.
5. Bay S, Burgess R, Nacci D. Status and applications of echinoid (phy- Pagano G, Corsale G, Esposito GA, Dinnel PA, Romana LA. Use of sea
lum Echinodermata) toxicity test methods. In: Landis WG, Hughes urchin sperm and embryo bioassay for testing the sublethal toxicity
JS, Lewis MS, eds. Environmental Toxicology and Risk Assessment; of realistic pollutant levels. In: Grandjean E, ed. Carcinogenic, muta-
ASTM STP 1179. Philadelphia (PA): American Society for Testing genic, and teratogenic marine pollutants: impact on human health and
and Materials; 1993, p. 281. the environment. Houston (TX): 1989:153–163.
6. Kobayashi N. Bioassay data for marine pollution using echinoderms.
In: Chermisinoff PN, ed. Encyclopedia of environmental control
technology. Houston (TX): Gulf Publishing Co.; 1995, p. 539.

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 8810 ECHINODERM FERTILIZATION AND DEVELOPMENT - B. Selecting and Preparing Test Organisms

8810  B. Selecting and Preparing Test Organisms

1. Selecting Test Organisms freshly collected animals in spawning condition is recommended.

The spawning season can usually be extended to year-round by
In accordance with the criteria listed in Section 8010 E.1, the holding the organisms in the dark at constant temperature. Feed
recommended test species include (but are not restricted to) the sea urchins ad libitum brown macroalgae (e.g., Macrocystis spp.
following: or Egregia spp.). Substitute romaine lettuce4 or commercial fish
feed if fresh seaweed is unavailable. Rehydrated seaweed pur-
Approximate chased from food markets or dried overnight at 100 °C also has
Common Spawning been used successfully. In addition, a formulated diet based on a
Scientific Name Name Location Season combination of eggs, carrots, seawater, and agar has been used
Arbacia punctulata Atlantic sea Atlantic coast Summer with success.5 Vegetables from organic food sources are recom-
urchin Gulf coast Winter mended to reduce the possibility of introducing pesticides to the
Strongylocentrotus Green sea Northern Atlantic Winter holding system. Tripneustes gratilla will feed on sea grasses (e.g.,
droebachiensis urchin and Pacific Thalassia spp. and macroalgae), with the exception of Sargassum
coasts spp.6 Aquariums containing sand dollars should contain several
Strongylocentrotus Pacific purple Pacific coast Fall-spring centimeters of sand if animals will be held more than a few days.
purpuratus sea urchin Sand dollars feed on suspended or benthic materials (e.g., detritus
Dendraster Pacific eccentric Pacific coast Spring–summer or plankton); provide a source of unfiltered natural seawater or
excentricus sand dollar prepared food (e.g., powdered fish feed) if the animals will be
Tripneustes Collector urchin Indo-pacific Year-round held for long periods.
gratilla region Holding temperature varies with the species and should be sim-
ilar to that at the collection site. Recommended temperatures are
The use of the above species is encouraged to increase the com- 15 to 18 °C for A. punctulata, 12 to 16 °C for D. excentricus, 8
parability of interlaboratory results. Successful toxicity tests can to 15 °C for S. purpuratus, 8 to 12 °C for S. droebachiensis, and
be conducted with other species (e.g., the Hawaiian sea urchin 20 to 25 °C for T. gratilla. Hold animals at 28 to 34 g/kg salinity.
Echinometra spp.1 or Lytechinus spp.).2 Using alternate species
may be advantageous in certain regions, but modifications to the 4. Parasites and Diseases
test method may be necessary and results may not be comparable.
A variety of commensal organisms (e.g., annelids and crus-
2. Collecting Broodstock taceans) are often associated with sea urchins and sand dollars.
These organisms are neither harmful nor essential to the survival
Obtain test organisms (gametes or embryos) from broodstock of the broodstock.
collected from the field during their natural spawning season and Excessive microbial growth can result from the accumulation
held in the laboratory until needed. Collect in areas away from of feces in aquariums. These growths produce metabolites (e.g.,
obvious sources of pollution, with water quality similar to that hydrogen sulfide) that may be toxic or stress the animals. Clean
used for holding and testing. Organisms obtained from a commer- aquariums several times per week.
cial supplier may be used. Dendraster excentricus forms dense
aggregations in intertidal or subtidal sandy areas. Collect indi- 5. Gamete Preparation
viduals by hand at low tide, by diving or by dredge. Regular sea
urchin species inhabit rocky or sandy areas of the intertidal and Induce sea urchins or sand dollars to spawn just before the
subtidal zones. Gamete viability of Tripneustes gratilla can vary beginning of a test. Females and males of most echinoid species
depending on locality. It is best to collect urchins from different usually can be induced to spawn via injection of 0.5 M potassium
areas for each round of tests. Collect individuals by hand, either chloride (KCl). Tripneustes gratilla can also be induced to spawn
at low tide or by diving. by vigorously shaking, which is effective at stimulating spawning
Discard any individuals damaged during collection or subse- with or without KCl. Use a hypodermic syringe (20-gauge needle)
quent handling. Avoid sudden or extreme variations in tempera- to pierce the peristomial membrane surrounding the mouth and
ture, salinity, pressure, or other environmental factors during inject approximately 0.5 mL into the coelomic cavity (another 0.5 mL
collection and transport because they may induce premature may be injected if necessary). Use sterile needles and KCl to guard
spawning. Animals may be shipped by overnight mail service in against disease if the animals will be returned to laboratory aquar-
insulated containers containing an ice substitute. Take precau- iums. Usually, 2 injections of KCl are made on opposite sides of
tions to avoid oxygen-depletion stress during shipping to avoid the mouth. Place sea urchins upright (oral side down) and observe
premature spawning. Sea urchins have been successfully shipped for evidence of gamete release through the genital pores, located
in supersaturated oxygenated water or by wrapping animals in around the anus on the aboral surface. Sperm are milky white in
seaweed or towels soaked in seawater to maintain high humidity. color, while eggs are orange to red, depending on species.
Electrical stimulation is another spawning method that has
3. Culture Techniques been used with success on some species (primarily A. punctu-
lata). Place electrodes from a 12-V DC power source on either
Hold sea urchins and sand dollars in aquariums with either a side of the urchin’s anal pore for about 30 seconds to stimulate
flow-through seawater supply or recirculating filter system.3 Using the release of eggs from females and sperm from males.4 This

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 8810 ECHINODERM FERTILIZATION AND DEVELOPMENT - C. Echinoderm Fertilization Test

method has the advantage of permitting a check of gamete type Carefully monitor the holding times of activated sperm and pref-
and quality by applying the electrodes briefly. Neither spawning erably standardize in the laboratory. Longer holding times (e.g.,
method kills the animal, which may be respawned in 30 to 60 d if 60 min) can be used successfully if sperm are chilled on ice and
held under appropriate conditions. higher sperm densities are used in the test. The final sperm den-
If spawned animals are returned to the culture system, they sity needed for the test varies according to test type, species, and
should be isolated from unspawned individuals. The presence of maturation stage. Determine this value from trial fertilization
recently spawned animals may stimulate unintended spawning by tests (see 8810 C.4d) or previous experience.
other sea urchins. Gently wash eggs once by allowing them to settle in the spawn-
Invert the females oral side up and place on a beaker filled to ing beaker and decanting excess seawater. Gently resuspend eggs
the brim with seawater at the appropriate temperature. The eggs in fresh seawater and let them settle again. Use care when wash-
will fall to the bottom of the beaker after extrusion. Collect sperm ing sand dollar eggs. Each egg is surrounded by a thick jelly coat
in the “dry” condition (without contacting seawater, which acti- that is easily disturbed leading to adverse fertilization effects.
vates the sperm). Remove sea urchin sperm from the gonopore Prepare a working stock solution of known egg density (depen-
area with a glass transfer pipet or automatic pipet (with enlarged dent on test volume and test type). Check density by mixing the
tip) and place in a small conical test tube. Dry sperm collection stock solution well, removing a small subsample, diluting it 1:100
is impractical for sand dollars; the sperm should be collected via with seawater, and counting the number of eggs in a known vol-
minimal mixing with seawater until needed. Collect sand dollar ume on a Sedgwick–Rafter cell. Using a perforated plunger (plas-
sperm by inverting males over 5- to 10-mL beakers of seawater. tic disk containing numerous holes, attached to a plastic rod) is
The sand dollar sperm fall to the bottom with little dilution and strongly recommended to provide a homogeneous suspension of
can be removed easily via pipet. Use care to avoid transferring gametes and embryos for this and other steps of the procedure.
fecal material with gametes. Adjust the density by adding or removing seawater. Store the
Gametes for most species may be stored for several hours in working stock at or below the exposure temperature (depending
an ice bath or refrigerator. Do not allow gametes to freeze. Keep on species) and use within 2 h if possible.
eggs from each female separate until evaluated for quality. Store
A. punctulata and T. gratilla eggs at the culture temperature. References
Examine subsamples of sperm and eggs from each animal under
a compound microscope to evaluate their quality. Eggs should be 1. Dinnel PA. Adaptation of the sperm/fertilization bioassay protocol to
round, and a germinal vesicle (clear spot) should not be visible. Hawaiian sea urchin species. Final Report to State of Hawaii Depart-
A germinal vesicle indicates immature eggs. Viability also can ment of Health, Honolulu; 1988.
be checked by removing a subsample, adding sperm, and deter- 2. Environment Canada. Biological test method: fertilization assay
mining fertilization. Use eggs from a more mature female if more using echinoids (sea urchins and sand dollars); Rep. EPS 1/RM/27.
than a few percent of immature eggs are present. Evaluate sperm Ottawa (ONT): Environment Canada; 1992.
3. Leahy PS, Tutschulte TC, Britten RJ, Davidson EH. A large-scale
quality by diluting a subsample in seawater containing eggs to
laboratory maintenance system for gravid purple sea urchins (Stron-
check for motility and fertilization. gylocentrotus purpuratus). J Exper Zool. 1978;204(3):369–380.
Good quality gametes increase the test’s sensitivity and suc- 4. Weber CI, Horning WB II, Klemm DJ, Neiheisel TW, Lewis PA,
cess rate. Individual batches of gametes should be evaluated for Robinson EL, Menkedick J, Kessler F, eds. Short-term methods for
quality using a pretest in which several combinations of sperm estimating the chronic toxicity of effluents and receiving waters
and egg batches are evaluated at various sperm:egg ratios. The to marine and estuarine organisms; EPA-600/4-91/003. Cincinnati
combination yielding the most viable gamete combination at the (OH): Environmental Monitoring and Support Laboratory, U.S.
optimal sperm:egg ratio to maximize test sensitivity is selected. Environmental Protection Agency; 1994
Alternatively, if it is infeasible to evaluate batches individually, 5. Urchin cookie recipe. Sea Urchins in Education. Stanford University
gametes from several individuals may be pooled to minimize the [accessed 29 March 2021]. https://seaurchineducation.stanford.edu/
cookie
influence of variable gamete quality.
6. Klumpp DW, Salita-Espinosa JT, Fortes MD. Feeding ecology and
Store sperm in an ice bath and avoid additional dilution until trophic role of sea urchins in a tropical seagrass community. Aquat
just before testing. Activate sperm via dilution in seawater and Bot. 1993;45(2-3):205–229.
use within 30 min. Sperm have limited energy reserves and their
viability declines exponentially within minutes of activation.

8810 C. Echinoderm Fertilization Test

1. General Procedures avoid cross-contamination of egg and sperm solutions (e.g., use
separate pipets and glassware for each sex). Seemingly minute
Conduct exploratory tests (see Section 8010 D) first if the con- amounts of sperm are sufficient to fertilize an egg stock prema-
centration range to be tested is unknown. Prepare dilution water turely and invalidate an entire test. Second, enlarge the opening
and toxicant solutions and introduce them into test containers as of disposable pipet tips used to dispense gametes. Trim the tip
described in Section 8010 F. with a razor blade to produce a 1- to 2-mm opening when trans-
Observe the following general precautions in procedures ferring eggs. Preferably also enlarge pipet tips used for concen-
that involve handling gametes. First, take stringent measures to trated sperm solutions to facilitate transfer of this highly viscous

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 8810 ECHINODERM FERTILIZATION AND DEVELOPMENT - C. Echinoderm Fertilization Test

suspension. Modified pipet tips are not required for sand dollar ST = desired test salinity after adjustment,
sperm shed into seawater. Finally, avoid inadvertently warming SS = initial salinity of sample, and
the gamete or embryo solutions during preparation steps because SB = salinity of brine.
this may greatly hasten degradation during storage. Use a tem- Check the pH of adjusted samples because some brine-prepa-
perature-controlled room or chilled water baths to ensure that all ration methods may alter pH. Allow samples to equilibrate with
preparatory steps are conducted at or below test temperature. the atmosphere to stabilize the pH. Because of the risk of chem-
ical contamination, only add dilute hydrochloric acid or sodium
2. Water Supplies hydroxide to adjust the pH when necessary, to correct deviations
likely to confound test results.
Maintain dilution water salinity within 2 g/kg of holding salin-
ity and at a pH that is within the species’ tolerance range. Dilution
3. Exposure Chambers
water quality should be sufficient to produce ≥70% fertilization
in control samples.
Conduct tests in glass culture tubes or vials with 10 to 20 mL of
a. Artificial seawater: See Section 8010 E.4b2. Avoid commer-
capacity. Cover chambers loosely to prevent contamination during
cial sea salt mixes because they are often toxic to echinoid gam-
test. Sealed chambers may be used if acceptable control perfor-
etes and embryos. However, some seawater formulations based
mance is obtained. Disposable glass tubes, scintillation vials, or
on reagent-grade chemicals have been used successfully.1,2 Con-
shell vials make convenient exposure chambers that can be dis-
duct preliminary tests to determine the suitability of each batch of
carded after test. Ensure that all test chambers and equipment used
artificial salts before use.
to prepare test solutions are clean and noncontaminating. Dispos-
b. Natural seawater: Choose a source of natural seawater that
able test chambers should be rinsed or soaked in reagent water or
is free of contamination and of uniform quality. Pass the water
seawater before use. Avoid using detergent and hypochlorite solu-
through a filter with an effective pore size ≤1.0 μm to remove
tions to clean other equipment because they could be toxic to test
sediment and organisms that might interfere with the test. Addi-
organisms. In multipurpose laboratories, use glassware dedicated
tional treatment (e.g., aeration, additional filtration, sterilization,
solely to toxicity tests.
or activated carbon treatment) may be needed to obtain accept-
able water quality, especially during storage. Avoid prolonged
storage of seawater (e.g., > 4 days) and store in the dark at 4 °C, if 4. Conducting the Test
possible, to minimize water quality degradation. Seawater stored
for extended periods should be filtered to ≤5.0 μm, continuously a. Setting up test chambers: Set up the test as described in
circulated, aerated, and held in the dark. Section 8010 D. Prepare all solutions and equilibrate to test tem-
c. Salinity adjustment: Echinoids have limited osmoregulation perature before beginning to spawn animals. Prepare at least 4
ability. Adjust the salinity of test samples that deviate by more replicates of each solution to facilitate statistical analyses, such as
than 2 g/kg from the culture environment to eliminate potential determining the NOEC or LOEC (see Section 8010 B).
interferences. Use hypersaline brine (HSB) or an artificial sea salt b. Duration and type of test: In the fertilization test, add a prede-
mixture to adjust the salinity. Dry reagent-grade sea salts may be termined number of sperm to the test solution and expose for 20 min
added directly to a sample when necessary to avoid diluting the (60 min for T. gratilla). Other times ranging from 5 to 120 min
sample due to brine addition. Exercise caution in selecting the have been used. Then add eggs to produce a specific sperm-to-egg
material used to adjust salinity so sample toxicity is not altered ratio and allow 20 min for fertilization to occur. Preserve sam-
by the introduction of such chemicals as chelators (e.g., EDTA) ples by adding buffered formalin and examine under a compound
or toxic contaminants (e.g., metals). microscope. Toxic effects are manifested by an impaired ability of
Slow evaporation is an effective method of preparing HSB sperm to fertilize eggs, indicated by lack of an obvious fertiliza-
in sufficient quantities for fertilization or embryo development tion membrane around the egg.
tests.3 Freezing and partially thawing seawater is also a conve- c. Test organisms: Fertilization tests can be conducted with all
nient method of preparing HSB.3 Freezing (−10 to −20 °C) one recommended species.
or two 4-L containers (glass or plastic) overnight for 6 to 12 h d. Performing the test:
provides sufficient 80 to 100 g/kg brine for most tests. Filter HSB 1) Preparation—Arrange replicate 5-mL samples of test solu-
through a filter with an effective pore size of >1.0 μm to remove tion in random order by assigning random numbers to individual
any particulates that may interfere with the test. Either HSB test chamber numbers (i.e., replicate chambers of the first treat-
preparation method could introduce toxicity into the experiment ment group will have unrelated numbers, such as 16, 31, and 4,
via the use of inappropriate materials or methods. Preliminary instead of sequential numbers); then arrange chambers in a rack
experiments should be conducted to verify HSB quality before in numerical order.
using it in experiments. HSB salinity must not exceed 100 g/kg. Other test volumes may be used if preferred (e.g., 2 mL or 10 mL),
Use the following formula to determine the volume of brine but adjustments to the following instructions will be necessary to
(VB) to be added to the sample: maintain the desired number of eggs and sperm in test chambers.
Measure the water quality of each test substance concentration on
additional samples of test material. A single initial measurement
VB = VS ×(ST − SS ) / (SB − ST )
is sufficient for most parameters unless the test material is highly
unstable. Using the proper sperm-to-egg ratio is critical to obtain
where: acceptable test sensitivity and precision. Because a fixed num-
VS = volume of test sample to be added (mL), ber of eggs is used in the test, sperm-to-egg ratios are altered by

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 8810 ECHINODERM FERTILIZATION AND DEVELOPMENT - C. Echinoderm Fertilization Test

varying the number of sperm added to test chambers. The proper 4) Egg stock preparation—Add a sufficient volume of washed
number of sperm is the least amount that produces 80% to 95% eggs to seawater to make 100 to 500 mL of a stock solution con-
fertilization. Although controls outside this range do not automat- taining 4000 to 5000 eggs/mL. Determine the density of eggs as
ically disqualify a test, particularly if a valuable dose response is directed in 8810 B.5. Adjust the density to a proper range by add-
generated, test sensitivity is reduced by fertilization rates exceed- ing or removing seawater. Verify that stock volume is sufficient
ing 95% and good dose responses may be difficult to obtain with for the number and size of test chambers.
less than 80% fertilization in controls. Density of the sperm solu- 5) Trial fertilization test—Although a trial fertilization test is
tion or sperm:egg ratios should be determined with this goal in optional, it allows for the evaluation of sperm condition and via-
mind. bility. To conduct a trial fertilization test, determine the density of
2) Sperm density measurement—Use a portion of the concen- pooled sperm as directed in paragraph d2 above. Prepare the egg
trated pooled sperm to determine the density (be sure to reserve stock solution as directed in paragraph d4 above. Calculate the vol-
sufficient sperm to conduct the test and a possible trial fertiliza- ume of seawater needed to dilute 0.025 mL pooled sperm and pro-
tion). Use the following procedures to aid in accurately pipet- duce a trial stock solution such that a sperm-to-egg ratio of 3000:1
ting the highly viscous concentrated sperm: Enlarge the pipet results when 0.05 mL of stock is added to test chamber (e.g., if
tip opening to about 2 mm, wipe off any sperm adhering to the 1000 eggs are in each chamber and 0.05 mL sperm stock is added
outside of the pipet tip before delivering sample (take care not to to the test sample, then a sperm stock density of 6.0 × 107 sperm/mL
wick away sperm from inside the tip), and repeatedly rinse the is needed). Prepare duplicate test chambers for each sperm-to-egg
pipet tip with dilution water after sample delivery until all sperm ratio to be tested (including 3000:1), each containing 5 mL seawa-
inside has been removed. ter at correct temperature. Prepare trial sperm stock solution. Pre-
Add 0.025 mL sperm to approximately 180 mL seawater in a pare several dilutions of stock that will produce the desired range
graduated cylinder. Bring the mixture up to 200 mL using 10% of sperm-to-egg ratios in test chambers.
acetic acid (kills sperm) to produce an 8000× dilution. For T. A suggested dilution series is as follows:
gratilla, use a 0.1% acetic acid solution to kill the sperm; using
a more concentrated solution causes sperm to clump. Cover the Sperm-to-Egg Ratio Trial Stock (mL) Seawater (mL)
cylinder, mix well by inversion, and let bubbles dissipate. Add a
3000:1 No dilution —
sample of the mixture to each side of a hemocytometer. Alternate 1304:1 5 6.5
dilution volumes (e.g., 1 mL sperm solution in 100 mL) may be 545:1 2 9.0
more suitable if a less dense sperm solution is used. 231:1 1 12.0
Let sperm settle for about 10 min. Examine a sufficient num- 100:1 0.5 14.5
ber of small squares on the slide so about 100 sperm are counted.
Examine the same number of squares on the opposite side of Add 0.05 mL trial stock or dilution to appropriate test cham-
the hemocytometer. If the two counts are within 20%, use the bers. After 20 min, add 1000 eggs. Add formalin preservative
mean to calculate the density according to the equation below. after 20 additional min. Determine the percentage fertilized in
Reload the hemocytometer and repeat the counts if the variabil- a subsample of 100 eggs from each replicate. Select the lowest
ity exceeds 20%. sperm-to egg ratio producing ≥80 to ≤95% fertilization. A refer-
ence toxicant dilution series may also be included in the pretest
to verify test sensitivity at various sperm densities. If using this
(dilution factor )(count )(4000 squares/mm3 )(1000 mm3 /mL )
sperm/mL = approach, then the sperm dilution selected for use corresponds to
No. squares counted the lowest sperm-to-egg ratio producing 80% to 95% fertiliza-
tion and a reference toxicant EC50 within the laboratory control
Alternatively, use a ratio turbidimeter with a 1-cm-diam cuvette chart confidence limits. It may be necessary to determine the final
to determine sperm density rapidly.4 The relationship between tur- sperm-to-egg ratio by interpolation. Sperm viability for some
bidity and sperm density is linear but may vary with species, indi- species may decline between the trial fertilization test and actual
vidual, season, or type of instrument. Use hemocytometer counts test, necessitating the use of a higher sperm-to-egg ratio to obtain
for the initial calibration of turbidimeter, regression equation, and the desired control fertilization value. Verify the sperm density in
verification of method suitability. the stock dilution corresponding to the chosen sperm-to-egg ratio
3) Sperm-to-egg ratio selection—The sperm-to-egg ratio for via a hemocytometer count.
the test usually is based on the control’s performance in previ- 6) Sperm stock preparation—Calculate the sperm stock solu-
ous tests. The appropriate ratio may vary, depending on species tion density required to produce the desired sperm-to-egg ratio in
and time of year. Conduct a trial fertilization test (just before the the test chambers (e.g., a stock containing 5.0 × 106 sperm/mL is
actual test) if adequate data are not available to determine the needed to produce a sperm-to-egg ratio of 250:1 when 1000 eggs
sperm-to-egg ratio (see ¶ d5 below). For purple sea urchins, use are present). Remove 0.025 mL pooled sperm and dilute with sea-
sperm-to-egg ratios ≤500:1 if fertilization in controls is accept- water to produce a stock solution of the desired density. Mix the
able. Higher ratios may reduce test sensitivity and are only appro- solution well and use within 30 min.
priate when prior experiments or trial fertilization results indicate 7) Sperm addition—Use an automatic pipet to add 0.05 mL
that the ratio is needed to obtain acceptable control fertilization sperm stock to each test chamber. Mix stock frequently during
(≥70%). Sperm-to-egg ratios above 3000:1 indicate unacceptable inoculations. Add sperm in a steady rhythm, with each addition at
quality of purple sea urchin sperm; use additional animals or a intervals of about 5 s.
different species (in better spawning condition). Optimum sperm- 8) Egg addition—Add eggs after a 20-min exposure period
to-egg ratios are species-specific.1,5,6 (other sperm exposure times of 5 to 60 min can be used for

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 8810 ECHINODERM FERTILIZATION AND DEVELOPMENT - C. Echinoderm Fertilization Test

Figure 8810:1. Early development stages of sea urchins and sand dollars. A—unfertilized egg; B—fertilized egg; C, D, E—early cleavage; F—blastula
with arrow indicating abnormal example; G—gastrula with arrows indicating abnormal examples; H—prism; I—frontal and lateral views of normal pluteus.

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 8810 ECHINODERM FERTILIZATION AND DEVELOPMENT - C. Echinoderm Fertilization Test

comparability with other laboratories). Use egg stock to add 1000 correct interpretation of test results. Sea urchin gametes and
eggs (e.g., 0.20 mL stock) to each chamber using the same order embryos are relatively sensitive to ammonia, pH, and dissolved
and rhythm as for sperm. Use a perforated plunger or equivalent organic carbon. Unmeasured variations in these and other water
device to gently mix egg stock thoroughly during additions. characteristics may confound test results.7 Measure basic water
9) Test termination—Stop the test by adding buffered forma- quality parameters (e.g., pH, dissolved oxygen, salinity, tempera-
lin (pH >7.0; see Section 10200 B.2a2)c) to each tube 20 min ture) on representative samples of controls and test materials.
after egg addition to produce a 5% final concentration. Glutaral- Because ammonia can be highly toxic to marine organisms, mea-
dehyde may be used as a preservative instead of formalin. Lugols sure total ammonia with a sensitive method (e.g., 4500-NH3 E)
solution [Section 10200 B.2a2)a] also is an effective preservative and report the concentration of un-ionized ammonia (NH3).
and has the advantage of being less toxic to analysts. However, Additional controls or adjustments may be needed to avoid the
formalin and more concentrated glutaraldehyde negatively affect confounding effects of variations in water quality characteristics
fertilization membranes of T. gratilla. Use only a 0.002% final (e.g., pH). For example, reduced fertilization and embryo devel-
concentration of glutaraldehyde to stop T. gratilla tests. Cap the opment in S. purpuratus has been observed at pH <7.5 or pH
test chambers securely, store at room temperature, and determine >8.3.8
fertilization within 48 h or as soon as practical. The samples can Include additional controls or blanks in the experimental design
be stored indefinitely, but the appearance of the egg or fertiliza- to verify that special treatments (e.g., storage, centrifugation, pH
tion membrane may change during storage, making the endpoint adjustment, carrier solvent addition, salinity adjustment) do not
more difficult to detect. Be extremely careful not to contaminate produce unanticipated effects. A “no sperm” control—consisting
test equipment or laboratory furniture with preservative. of several exposure chambers containing just the test sample and
10) Sample evaluation—Examine eggs in an exposure vial eggs—should be included to account for artificial parthenogene-
using an inverted compound microscope or transfer a represen- sis and verify that the eggs were not accidentally fertilized before
tative subsample to a Sedgwick-Rafter cell for use with a con- conducting the test. This control is recommended when conduct-
ventional microscope. It is often convenient to concentrate the ing tests with unknown substances.7
eggs before transfer by removing most of the overlying water Use reference toxicant tests to provide a measure of test preci-
with a pipet. Mix the remaining sample well before transfer to a sion and possible organism condition.3 The reference toxicant test
counting chamber. If membranes are difficult to discern, adding usually consists of replicate exposures to 3 to 5 concentrations
1 or 2 drops of HSB to the slide just before observation shrinks of a stable chemical (e.g., Cu, Cd, sodium dodecyl sulfate) that
the eggs momentarily, resulting in more discernable membranes. are sufficient to calculate a point estimate of effect (e.g., EC50).
Discard formalin-contaminated exposure chambers promptly Preferably include a reference toxicant test with each experiment,
(do not reuse chambers). To protect analysts from exposure to or test at least monthly. Plot cumulative mean and confidence lim-
toxic chemicals, provide adequate ventilation when transferring its on a control chart to identify outlier values. Outliers indicate
or examining samples containing formalin or glutaraldehyde in potential problems with the technique or test organisms. It is rec-
unsealed containers. ommended that test organisms be replaced if repeated reference
Examine at least 100 eggs (40× to 100× magnification) from toxicant EC50 values fall outside the laboratory quality control
each replicate and score for presence or absence of an elevated confidence limits.
fertilization membrane. Avoid bias by counting all eggs in sub-
samples transferred to counting chambers. Newly fertilized eggs References
usually have a completely elevated membrane around the egg
(Figure 8810:1, A and B). The fertilization membranes of T. gra- 1. Weber CI, Horning WB II, Klemm DJ, Neiheisel TW, Lewis PA,
tilla eggs are not as elevated as in other species and should be Robinson EL, Menkedick J, Kessler F, eds. Short-term methods for
examined under a phase contrast microscope. The fertilization estimating the chronic toxicity of effluents and receiving waters to
membrane may change in appearance with prolonged storage, marine and estuarine organisms; EPA-600/4-87-028. Cincinnati
partially collapsing or touching a portion of the egg. Conse- (OH): Environmental Monitoring and Support Laboratory, U.S.
quently, count eggs showing any elevation of the fertilization Environmental Protection Agency; 1988.
2. Neiheisel TW, Young ME. Use of three artificial sea salts to main-
membrane as fertilized. Exclude unusually small, immature, or
tain fertile sea urchins (Arbacia punctulata) and to conduct fertiliza-
abnormally shaped eggs from counts. tion tests with copper and sodium dodecyl sulfate. Environ Toxicol
Calculate the percentage of fertilized eggs in each sample. Chem. 1992;11(8):1179–1185.
3. Chapman GA, Denton DL, Lazorchak JM. Short-term methods for
5. Statistical Analysis estimating the chronic toxicity of effluents and receiving waters to
west coast marine and estuarine organisms; EPA-600/R-95-136. Cin-
Assemble, analyze, evaluate, and report data as described in cinnati (OH): National Exposure Research Laboratory, U.S. Envi-
Section 8010 G. ronmental Protection Agency; 1995.
4. Hall TJ, Haley RK, Battan KJ. Turbidity as a method of preparing
sperm dilutions in the echinoid sperm/egg bioassay. Environ Toxicol
6. Quality Assurance Chem. 1993;12(11):2133–2137.
5. Dinnel PA, Link JM, Stober QJ. Improved methodology for a sea
Success in conducting this toxicity test depends on an over- urchin sperm cell bioassay for marine waters. Archiv Environ Con-
all effort to maintain and improve laboratory techniques and tam Toxicol. 1987;16(1):23–32.
equipment.3 Accurate background information regarding sample 6. Wagner A, Nacci D. Tropical collector urchin, Tripneustes gratilla,
characteristics and test organism condition is necessary to enable fertilization test method; EPA/600/R-12/022. Narragansett (RI):

https://doi.org/10.2105/SMWW.2882.174 7
 8810 ECHINODERM FERTILIZATION AND DEVELOPMENT - D. Echinoderm Embryo Development Test

National Health and Environmental Effects Laboratory, U.S. Envi- Bibliography

ronmental Protection Agency; 2012.
7. Carr RS, Biedenbach JM, Nipper M. Influence of potentially con- Environment Canada. Biological test method: fertilization assay using
founding factors on sea urchin porewater toxicity tests. Archiv Envi- echinoids (sea urchins and sand dollars); Rep. EPS 1/RM/27. Ottawa
ron Contam Toxicol. 2006;51(4):573–579. (ONT): Environment Canada; 1992.
8. Bay SM, Anderson BS, Carr RS. Relative performance of porewa- Phillips BM, Nicely PA, Hunt JW, Anderson BS, Tjeerdema RS, Palmer
ter and solid-phase toxicity tests: Characteristics, causes, and con- FH. Tolerance of five west coast marine toxicity test organisms to
sequences. In: Carr RS, Nipper M, eds. Porewater toxicity testing: ammonia. Bull Environ Toxicol Contam. 2005;75(1):23–27.
biological, chemical, and ecological considerations. Pensacola (FL):
Society of Environmental Toxicology and Chemistry (SETAC);
2003, p. 11.

8810 D. Echinoderm Embryo Development Test

1. General Procedures chambers may be used if control performance is acceptable.

Scintillation or shell vials make convenient exposure chambers
Conduct exploratory tests (see Section 8010 D) first if the con- that can be discarded after the test. Clean all equipment for pre-
centration range to be tested is unknown. Prepare dilution water paring test solutions before use. Clean test chambers by soaking
and toxicant solutions and introduce them into test containers as in fresh water or seawater. Avoid detergent or hypochlorite solu-
described in Section 8010 F. Take precautions to avoid gamete tions because they could be toxic to test organisms.
temperature stress (see 8810 C.1). The procedure described below
uses many of the same techniques described for the fertilization 4. Conducting the Test
test (8810 C) but has been optimized for use with embryos. Some
laboratories conduct both tests on a sample to gain more informa- a. Setting up test chambers: See 8810 C.4a.
tion. The fertilization test also may be extended into an embryo b. Duration and type of test: Various volumes of test solution
development test by including additional replicate chambers. (5 to 1000 mL) may be used. Add embryos to test solution and
Such an approach requires modification of the test methods and let develop under static conditions for 48 to 96 h until the pluteus
may reduce the precision of embryo development results because stage is reached. Preserve subsamples (or entire sample if vials
of variable fertilization rates. An embryo development test has not are used) with formalin and examine under the microscope. Toxic
yet been developed for T. gratilla. effects are indicated by embryo mortality or abnormal develop-
ment.
2. Water Supplies c. Test organisms: Embryo development tests can be conducted
with all the recommended species.
Maintain dilution-water salinity within 2 g/kg of the holding d. Performing tests:
salinity and at a pH that is within the species’ tolerance range. 1) Preparation—Test preparation is the same as for the fertil-
Dilution water quality should be sufficient to produce ≥70% nor- ization test (8810 C.4d1) with 2 exceptions. First, the test may be
mal survival (relative to initial number of embryos) in control conducted in larger volumes (up to 1000 mL) if desired. Using
samples. large volumes provides no distinct advantage in test sensitivity
a. Artificial seawater: See Sections 8010 E.4b2 and 8810 C.2. or precision, but does allow the exposure chamber to be subsa-
Avoid commercial sea salt mixes because they often are toxic to mpled to measure water quality or determine effects at various
echinoid gametes and embryos. times or developmental stages. Second, measure both initial and
b. Natural seawater: Choose a source of natural seawater that is final water quality for each treatment group. Include one addi-
free of contamination and of uniform quality. Pass water through tional replicate test chamber in each treatment group for final
a filter with an effective pore size ≤1.0 μm to remove sediment water quality measurements when tests are conducted in small
and organisms that might interfere with the test. Additional treat- volumes (e.g., 10 mL).
ment (e.g., aeration, activated carbon treatment) may be needed to Quantify a toxic response by either a complete count or relative
obtain acceptable water quality. count. For a complete count, calculate the percentage of normal
c. Salinity adjustment: The sea urchin development test usually pluteus larvae at the end of the exposure, based on counts of all
is more sensitive to deviations in salinity than is the fertilization preserved test organisms and the number of embryos added ini-
test. Use the methods described in 8810 C.2c to adjust the salinity tially. This method provides the most comprehensive assessment
of samples that deviate by more than 2 g/kg. of effects because all instances of embryo mortality and aberrant
development are included in the percentage. A relative count
3. Exposure Chambers requires counts of only a subsample of organisms at the end of the
test. Both embryo mortality and aberrant development are reflected
Preferably, use glass chambers with a capacity between 10 mL in this method as well, but toxic effects may be underestimated
and 1 L. Maintain the recommended density of test organ- if the test solution causes rapid decomposition of dead embryos
isms, regardless of volume. Cover chambers loosely to prevent and a consequent failure to detect them during microscopic exam-
contamination and reduce evaporation during the test. Sealed ination. Penicillin G in a concentration of 100 units/mL has been

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 8810 ECHINODERM FERTILIZATION AND DEVELOPMENT - D. Echinoderm Embryo Development Test

used in the embryo stock solution to retard bacterial degradation 10-mL subsample to a vial, and add formalin. Glutaraldehyde (e.g.,
of dead or dying embryos during the test, thus allowing for more 0.02% final concentration) may also be used as a preservative.
complete counts upon termination. Choose the evaluation method 8) Test evaluation—Examine preserved embryos and larvae
before the test begins so all required information is obtained. with a compound microscope at a magnification of 100×. Con-
2) Egg density adjustment—Add a sufficient volume of washed centrate test organisms by removing overlying water from the
eggs to seawater to make 100 to 500 mL of a stock solution con- storage vial and transfer to a Sedgwick-Rafter counting chamber
taining 1000 eggs/mL. It may be more convenient to prepare a for examination with a conventional microscope. Alternatively,
more concentrated solution (e.g., 10 000 eggs/mL) if test vol- use an inverted microscope to examine organisms in the storage
umes larger than 50 mL are used. Determine the density of eggs vial and eliminate losses due to transfer.
as directed in 8810 B.5. Adjust the density to the desired value Normally developing embryos develop synchronously through
by adding or removing seawater. Verify that the stock volume is a series of characteristic stages, including early cleavage, blastula,
more than sufficient (approximately 50% greater) for the number gastrula, prism, and pluteus (Figure 8810:1, B–I). The appearance
and size of test chambers used. of pluteus larvae varies with species, but all normal plutei should
3) Sperm stock preparation—Prepare a sperm stock by adding have the following features:
about 0.025 mL dry sperm to 50 mL seawater. If the volume of • a pyramid shape supported by a framework of skeletal rods,
stock solution needed to fertilize eggs is not known from prior • an internal gut that is attached to the body wall at both ends
experience, determine the density of sperm stock with a hemocy- and consists of 3 distinctive regions, and
tometer (see 8810 C.4d). • at least 1 pair of post-oral arms (Figure 8810:1).
4) Egg fertilization—Add a sufficient volume of sperm stock Compare sample larvae to the control to determine abnormal
to egg stock to produce a sperm-to-egg ratio of 200 to 1000:1. development. The length of the post-oral arms varies among spe-
Mix well and examine a subsample after about 10 min to assess cies. Count as abnormal
fertilization percentage. Add more sperm if less than 90% of eggs • all grossly deformed pluteus larvae,
are fertilized. A fertilization rate of less than 90% after the second • deformed embryos,
addition of sperm indicates poor quality gametes. Spawn addi- • mostly normal-appearing embryos that have not attained the
tional animals to obtain better gametes if possible. Add embryos pluteus stage (inhibited development), and
to test containers as soon as possible (generally within 2 h but no • uncleaved fertilized eggs.
later than 4 h after fertilization). Do not count unfertilized eggs.
5) Embryo addition—Use an automatic pipet to add sufficient Determine the percentage of normal pluteus larvae for each rep-
embryo stock solution to each test chamber to result in a count of licate using either the complete or relative count method (below).
at least 100 embryos in the sample at the end of the test. Typically, a) Complete count method—Count all embryos in a preserved
a density of 25 to 400 embryos per milliliter is used, depending on sample. Preferably use an inverted microscope to minimize count-
the test volume and method used to evaluate embryos. It is import- ing errors. If a conventional compound microscope is used, use a
ant to add the same number of embryos to each test chamber. Use consistent and efficient method to transfer embryos to the counting
a perforated plunger to mix the stock solution thoroughly during chamber because lost embryos (remaining in vial or stuck to transfer
embryo addition. If using the complete count method to evaluate pipet) are assumed to have died. Variability in recovering larvae from
toxic effects, add embryos to at least 5 additional test chambers the storage vial may introduce experimental error that reduces the
containing control water. Intersperse these chambers through- ability to detect statistically significant effects. Calculate the percent-
out the experimental array and add embryos in the same manner age of embryos developing to normal pluteus larvae, Pn, as follows:
used for the other chambers. Examine these additional chambers
promptly to estimate the actual number of embryos added. Pn = 100 ( En /Ei )
6) Exposure—Loosely cover test chambers and leave undis-
turbed for 48 to 96 h under static conditions. The optimum expo-
sure time varies with species and test temperature. Exposure time where:
should be long enough to allow embryos to develop to the pluteus En = number of normal larvae at end of test, and
stage, yet short enough (≤96 h) that internal food reserves are not Ei = number of embryos at start of test.
exhausted. The following exposure conditions are recommended
to provide consistency with results from other laboratories: b) Relative count method—It is just as effective to determine
• A. punctulata, 48 h at 20 °C; the percentage of normal development in a representative sample
• D. excentricus, 72 h at 15 °C; of at least 100 embryos and larvae at the end of the test. To use the
• S. purpuratus, 72 h at 15 °C or 96 h at 12 °C; and relative count method when using an inverted microscope, begin
• S. droebachiensis, 96 h at 12 °C. at one side of the vertical center of the vial and move across to
These are target times; a few extra hours may be allowed to the opposite site, scoring eggs as normal or abnormal. Counting is
help ensure that most control larvae (>90%) have attained the nor- complete if at least 100 embryos have been scored. If fewer have
mal pluteus stage. Ambient laboratory light levels and photoperi- been counted, move the stage down one field of view and score all
ods are adequate for all species. embryos encountered in a second pass across the vial. Repeat this
7) Test termination—Preserve organisms for later microscopic process until at least 100 embryos have been scored. Calculate the
examination by adding sufficient buffered formalin (pH >7.0, see percentage of normally developed embryos as follows:
Section 10200 B.2a2)c) to produce a 5% concentration. Add forma-
lin directly to the test chamber if disposable vials or culture tubes are Pn = 100  En / ( En + Ea )
used. Otherwise, thoroughly mix test chamber contents, transfer a

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 8810 ECHINODERM FERTILIZATION AND DEVELOPMENT - D. Echinoderm Embryo Development Test

where: where:
Ea = number of abnormal embryos/larvae, and other terms are as Padj = normalized value,
defined above. Pn = percent normal or fertilized for the sample, and
M = mean percent normal or fertilized for controls.
5. Statistical Analysis
6. Quality Assurance
See Section 8010 G.2 for general information.
Control performance may vary among tests because of factors See 8810 C.6.
such as variations in test temperature and gamete condition. Nor-
malize the response data to the control performance before statis- Bibliography
tical evaluation (e.g., EC50) and to facilitate comparisons among
tests as follows: ASTM E-1563-98(2004). Standard guide for conducting static acute
toxicity tests with echinoid embryos. In: Annual Book of ASTM
Standards, Vol. 11.06. West Conshohocken (PA): ASTM Interna-
Padj = 100 ( Pn /M )
tional; 2006.

Published Online: August 27, 2018

Revised: June 3, 2021
https://doi.org/10.2105/SMWW.2882.174 10