EPA Region 10 Guidance for Conducting Effluent Toxicity Tests
Using West Coast Sea Urchins and Sand Dollars
Effluent toxicity tests using west coast sea urchins and sand dollars
that are conducted in compliance with NPDES permit requirements should be
performed following the sperm cell test protocol described by Dinnel, Link,
and Stober, 1987. Additional technical and statistical guidelines for
conducting these tests are provided in the EPA "Chronic Marine Toxicity Test
Manual" (U.S. EPA, 1988). However, the following summary of recommended
conditions for conducting sperm cell tests with west coast echinoderms is to
provide interim guidance that is more consistent with the present EPA protocol
describing a sperm fertilization .test using the east coast sea urchin,
Arbacia punculata (U.S. EPA, 1988).
EPA test development efforts are expected to produce a draft "West Coast
Echinoderm Sperm Fertilization Test" protocol later this year that*will
replace Region 10's guidance.
References:
Dinnel, P.A., J.M. Link, and Q.J. Stober. 1987. Improved methodology
for a sea urchin sperm cell bioassay for marine waters. Arch.
Environ. Contam. and Toxicol., 16: 23-32.
U.S. EPA. 1988: Short-term methods for estimating the chronic toxicity
of effluents and receiving waters to marine and estuarine
organisms. Environmental Monitoring and Support Laboratory, U.S.
Environmental Protection Agency, Cincinnati, Ohio, EPA/600/4-87/028.
Other Sources of Information:
U.S. Environmental Protection Agency, Environmental Research Laboratory,
Hatfield Marine Science Center, Newport, Oregon
U.S. Environmental Protection Agency, Environmental Research Laboratory,
Narragansett, Rhode Island
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Summary of Recommended Test Conditions for Conducting Sperm Fertilization
Tests Using the Purple Sea Urchin, Stronqvlocentrotus purpuratus. Green Sea
Urchin, Stronqvlocentrotus droebachiensis. Red Sea Urchin, Stronqvlocentrotus
franciscannus. and Sand Dollar, Dendraster excentricus.
1. Test Type
2. Salinity
3. Temperature
4. Light Quality and Intensity
5. Test Vessel Size
6. Test Solution Volume
7. No. of Spawning Adults
8. Sperm Collection and
Storage Procedure
9. Number of Eggs per Test Vessel
10. Sperm:Egg Ratio
Static
30 o/oo ± 2 o/oo
12 ± 1°C
Ambient laboratory illumination,
10-20 uE/m2/s (50-100 ft-c).
Disposable (Glass) Liquid
Scintillation Vials (20-mL); Not
Pre-cleaned.
10 mL
Two to four males and females,
with sperm and eggs being pooled
separately.
"Dry" sperm collection
technique8, with pooled sperm
stored on ice. Sperm must be
used within one hour of being
collected.
2000 eggs
Depends on species. (Note:' Each
control and effluent treatment
should be tested using several
sperm:egg ratios, with
fertilization being scored only
at that ratio yielding 70% - 90%
fertilization success in the
seawater controls. Tests
achieving less than 50%
fertilization success in the
seawater controls are considered
invalid.)
11. Number of Replicates per Treatment 4 (minimum of 3)
12. Dilution Water
13. Dilution Factor
14. Test Duration
Uncontaminated natural seawater;
deionized water mixed with
hypersaline brine or artificial
sea salts.
0.3 or 0.5
1 hr and 20 min (60 min sperm
exposure plus 20 min
fertilization period).
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15. Aeration
16. Effect Measured
17. Number of Treatments per Test
18. Reference Toxicant
19. Egg Control
20. Egg-Effluent Control
None
Fertilization success as
indicated by the presence of a
fertilization membrane.
Minimum of five effluent
concentrations and a control.
The toxicity (EC50) of a
reference toxicant must be
determined concurrently with each
test.
An egg control (eggs minus sperm)
should be included with each test
to reveal any contamination of
the eggs with sperm or the
presence of false positives, i.e.
eggs that appear to be fertilized
in the absence of sperm.
An egg-effluent control (eggs
plus effluent, minus sperm)
should be included with each test
to reveal the presence of fal se
positives, i.e. eggs that appear
to be fertilized in the presence
of effluent, but in the absence
of sperm.
Dry" Sperm Collection Technique:
Sea urchins and sand dollars are induced to spawn by KC1-injection
as described by Dinnel, et al_., 1987. Eggs are also collected and washed
as described in the Dinnel protocol, however, sperm are collected by the
"dry" (seawater-free) sperm^collection technique outlined below.
Spawning males are removed immediately from the seawater-filled
beakers and placed, right-sided-up, in a shallow tray containing
seawater. The males should be partially immersed in the seawater, taking
care that the water does not cover the gonophores located at the top of
their tests (shells). Sperm are then collected from the gonophores using
a pipet or syringe. The "dry" sperm are stored on ice until diluted (and
activated) with seawater for counting and testing. The sperm must be
used in a toxicity test within one hour of collection.
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