Biological test method: fertilization assay using echinoids (sea urchins and sand dollars), chapter 3
Section 1: Introduction
Aquatic toxicity tests are used within Canada and elsewhere to measure, predict, and control the discharge of substances or materials that might be harmful to aquatic life in the environment. Recognizing two decades ago that no single test method or test organism can be expected to satisfy a comprehensive approach to environmental conservation and protection, the Inter-Governmental Ecotoxicological Testing Group (see Appendix B) proposed the development and standardization of a set of single-species aquatic toxicity tests which would be broadly acceptable, and would measure different toxic effects using organisms representing different trophic levels and taxonomic groups (Sergy, 1987). A test based on fertilization success using gametes of sea urchins or sand dollars was one of several “core” aquatic toxicity tests which was then selected to help meet Environment Canada’s testing requirements.
The first edition of this biological test method was published by Environment Canada in December 1992 as Report EPS 1/RM/27 (EC, 1992c), and amended in November 1997. After 15 years of application by private and public sector testing laboratories, Environment Canada recognized that specific aspects of the test method needed to be changed. This revision began with the preparation and circulation of a questionnaire to Canadian and US toxicology testing laboratories with experience in conducting echinoid fertilization assays. The purpose of the questionnaire was to collect details on the echinoid species used in testing, the conditions for holding and acclimating organisms, and the spawning and fertilization techniques employed by the various laboratories using the method. Guidance derived from the feedback provided by the laboratories responding to the questionnaire has been included herein, where applicable. Revisions to sediment (pore water) testing procedures are largely based on the results of an inter-laboratory study which investigated the improvement (i.e., improved test sensitivity and reduced influence of confounding factors on test results) of the porewater testing component of this echinoid fertilization assay (Miller, 2008).
The current (second) edition includes numerous procedural improvements, updated and more explicit guidance, as well as instructions for the use of revised statistics (i.e., regression analyses) when calculating the test endpoint for fertilization inhibition.
Universal procedures for conducting a fertilization assay with echinoid gametes are described in this second edition of Environment Canada’s Report EPS 1/RM/27. Also presented herein are specific sets of test conditions and procedures, required or recommended when using the test to evaluate different types of substances or materials (e.g., samples of one or more chemicals, effluents, receiving waters, leachates, elutriates, or interstitial waters [pore waters] derived from sedimentor similar solid material; see Figure 1). Those procedures and conditions relevant to the conduct of the test are delineated and, as appropriate, discussed in explanatory footnotes.
In formulating these test conditions and procedures, an attempt was made to balance scientific, practical, and financial considerations, and to ensure that the results will be accurate and precise enough for the majority of situations in which they would be applied. It is assumed that the user has a certain degree of familiarity with aquatic toxicity tests. Guidance regarding test options and applications is provided here. Explicit instructions that might be required in a regulatory protocolor reference method are not provided, although the report is intended to serve as a guidance document useful for that and other applications.
For guidance on the implementation of this and other biological test methods and on the interpretation and application of the endpoint data, consult Report EPS 1/RM/34 (EC, 1999).
Figure 1 Diagram of Approach Taken in Delineating Test Conditions and Procedures Appropriate for Various Types of Materials or Substances
- Obtaining mature adults
- Holding adults
- Preparing test solutions
- Reference toxicants
- Test conditions (pH, DO, etc.)
- Water quality measurements
- Spawning to obtain gametes
- Beginning the test
- Validity of results
- Legal considerations
Items Covered in Specific Sections
- Chemical properties
- Labelling and storage
- Chemical measurements
- Choosing control/dilution water
Effluents, Elutriates, and Leachates
- Containers and labeling
- Sample transit and storage
- Preparation of solution
- Choosing control/dilution water
- Measurements at start
Sediments and Similar Solids
- Containers and labeling
- Sample transit and storage
- Preparing sample
- Observations on sample
- Control/reference sediments
- Preparing test material
- Choosing control/dilution water
1.2 General Aspects of Echinoids and Their Use in Tests
Sea urchins and sand dollars belong to the Phylum Echinodermata, Sub-phylum Echinozoa, and Class Echinoidea, and, therefore can collectively be called “echinoids”. Other members of the phylum, not included in this test method, are the sea stars (“starfish”), brittle and basket stars, sea cucumbers, and crinoids or sea lilies and feather stars. The phylum has worldwide marine distribution and about 6000 living species are known. Seven species of sea urchins and three species of sand dollars are commonly found in the coastal marine waters of Canada.
Echinoids and other members of the phylum are considered to be structurally advanced and complex invertebrates. They have many sophisticated features and many similarities to chordate animals including the basic pattern of embryonic development and some biochemical processes. The apparent radial arrangement of the body in five parts around a central axis is superimposed on a primary bilateral organization. There is a true internal skeleton covered by a thin epidermis. The skeleton is of small jointed calcareous plates, which in sea urchins and sand dollars are fused together into a solid test, or “shell”, the latter term being used in this report for convenience. There is a well-developed coelom or internal body cavity, most of which surrounds the internal organs (Figure 2). Another part of the coelom is a tube-like water vascular (“hydraulic”) system running to all parts of the body, used to manipulate small tube feet for locomotion, and to perform other functions.
Sea urchins are spherical and covered in spines, while sand dollars are flattened on the oral-aboral axis and generally disk-shaped (Figure 2). The oral surface is oriented downwards. A peristomial membrane surrounds the mouth in sea urchins, and injection of a chemical solution through that membrane and into the coelom is part of the procedure in these tests. For sand dollars, injection has to be through the mouth opening. The anus of sea urchins is on the aboral (upper) surface, but in sand dollars it is on the same surface as the mouth.
The sexes are separate but cannot be distinguished externally. There are large internal gonads (Figure 2) with outlets on the aboral surface, as five genital pores in urchins and four in sand dollars. One of the pores of urchins is in the madreporite, an obvious large plate of the shell, which is a terminus of the animal’s water vascular system.
The gametes (sperm and eggs) are simply passed through the pores to the sea for fertilization.
The early development of sea urchins from egg to late larval stage (“pluteus” stage) is of great embryological interest, and more than 5000 papers were published on the topic by 1980 (NRC, 1981). This background has led to the use of young stages of urchins in toxicity tests over many decades (Lillie, 1921; Drzewina and Bohn, 1926; Bougis, 1959), with a particularly thorough study of metal toxicity using fertilization in a sea urchin completed in the first quarter of the last century (Hoadley, 1923). Both sea urchins and sand dollars are now frequently used as standard organisms in toxicity testing (reviewed in Dinnel et al., 1987, 1988), and an extensive background of toxicological data has accumulated (Kobayashi, 1984).
The echinoid fertilization assay is sensitive. A major effect on egg fertilization, for example, was caused by municipal effluents at concentrations which were one-tenth of those causing 50% mortality of fathead minnows in a four-day test (Oshida et al., 1981). It was the second to third most sensitive among six sublethal tests (marine and freshwater) used in an inter-laboratory survey of effluent toxicity in California (Anderson et al., 1991). The 80-min echinoid fertilization assay was more sensitive to the effluent from a municipal wastewater treatment plant than were 48-hour tests with oyster and crab embryos and larvae (Dinnel and Stober, 1987). Variable results were obtained in a comparison of the toxicities of metal and organic compounds using the fertilization assay, a bacterial luminescence assay, and acute lethality tests with fish and crustaceans. Sometimes the echinoid test was one, two, or three orders of magnitude more sensitive, and sometimes an order of magnitude less sensitive (Nacci et al., 1986). Results from echinoid fertilization assays were similar in sensitivity to those from embryo-larval tests with crab, squid, and fish, and were quite sensitive to metals, but much less so to pesticides than were tests of acute lethality using marine fish (Dinnel et al.,1989). For pulp mill wastes, NCASI (1992) cites work of Johnson et al. (1990) that embryo- larval tests with oysters were approximately an order of magnitude more sensitive than the echinoid fertilization assay. In turn, the echinoid assay was about as sensitive as a reproductive test using red alga, and was more sensitive, often by an order of magnitude, than other sublethal marine tests on growth and development of larval fish (silversides minnows and sheepshead minnows) or juvenile mysid shrimps (Schimmel et al., 1989).
Figure 2 General Appearance of Echinoids
a. Cut-away view of a typical sea urchin, Arbacia, showing location of genital pores on the aboral (upper) side. Only two or three of the numerous spines and tube feet are indicated. b. Oral side (normally down) of a typical sand dollar. c. Aboral side of a sand dollar showing location of the genital pores. [Drawings by M.A. White, after Storer et al. (1979) and Barnes (1974)].
Long Description of Figure 2
Three diagrams illustrating: a cut-away of a mature sea urchin, the oral side of a sand dollar, and the aboral side of a sand dollar. These diagrams are intended primarily to illustrate the location of the genital pores on these two organisms. In the sea urchin the genital pores (five total) are located on the aboral (typically upper) side forming a ring around the organisms’ anus. In sand dollars the genital pores (four total) are located on the aboral (typically upper) side near the centre of the animal.
The fertilization assay is a sensitive sublethal test. Gametes of echinoids are either the most sensitive of the developmental stages, or are among the most vulnerable stages of the entire life cycle, when tested using various toxicants (Kobayashi, 1980, 1984). The fertilization assay is not a chronic test, however, because of its very short duration relative to the life spans of the species (some years). The fertilization assay described in this report is not intended to replace chronic toxicity tests using echinoids, because it might not estimate the effects of longer exposures. However, this test can be expected to yield results closer to such chronic tests than would conventional lethality tests with marine or freshwater species (e.g., EC, 1990a, 1990b, 1990c).
Precision of the test appears to be satisfactory. The USEPA (2002) determined that within- laboratory coefficients of variation (CV) using reference toxicants and one species of sea urchin (A. punctulata) for IC50s were 23% to 48%, and for IC25s were 29% to 55%. A CV of 74% was found for IC50s of copper tested by six laboratories using four species of echinoids in an effluent testing program, compared to CVs of 29% to 38% obtained with sublethal tests on single species (Ceriodaphnia reproduction, and early life stages of fathead minnows and oysters, Anderson and Norberg-King, 1991). In five single-species comparisons among Canadian laboratories, the CVs were 62%, 65%, 75%, 82% and 110% for IC25s of copper (tests involved three species of sea urchins with total exposure times of 20 minutes). IC50s from the same tests showed lower CVs, with values 23%, 48%, 57%, 80% and 94% (Miller et al., 1992). These interlaboratory CVs, averaging 79% for IC25s and 63% for IC50s, are similar to the precision for chemical analyses, e.g., an average CV of 30 to 60% found in an interlaboratory comparison of chemical analyses of priority pollutants (Rue et al., 1988; Gossett et al., 2003). Unpublished results for an interlaboratory round-robin sponsored by the USEPA are apparently similar, with CVs of 57% for 40-min fertilization assays and 86% for 80-min assays (NCASI, 1992).
Prior to 1992, the echinoid fertilization assay had been used in several Canadian aquatic toxicity laboratories, both governmental and industrial. Standard test methods had been described in British Columbia (B.C. MOE, 1990; van Aggelen, 1988), and by consulting companies (Beak, 1988; EVS, 1989). At the national level, a trial of methods had been carried out by certain Environment Canada laboratories (see Appendix C), under the sponsorship of a federal-provincial body (IGATG, 1991). Additional interlaboratory trials, involving federal, provincial (B.C. Ministry of Environment), and private testing facilities, were done the following year (Miller et al., 1992). Echinoid tests were reviewed and recommended by an Environment Canada scientist (Wells, 1982, 1984), but prior to 1992, no standard method had been published by a Canadian federal government agency.
Since its publication in 1992, and formal amendment in 1997, Environment Canada’s Fertilization Assay Using Echinoids (Sea Urchins and Sand Dollars) (EPS 1/RM/27) has been used extensively in two important programs falling under the authority of the Canadian Environmental Protection Act (CEPA), and has also been applied under specific regulations of the Canadian Fisheries Act. Under the Disposal at Sea Program, this sublethal test uses echinoid fertilization on sediment pore water to help evaluate the suitability of dredged material for disposal at sea (CEPA, 1999; Government of Canada, 2001). Under the Environmental Effects Monitoring Program, the sublethal toxicity of pulp and paper, and mining effluent discharged to the marine environment is assessed using echinoid fertilization (DFO, 1992, 2002).
In the United States, several groups have provided methods for conducting sublethal toxicity tests using echinoids. The United States Environmental Protection Agency has developed authoritative procedures for species of echinoids indigenous to their Atlantic (USEPA, 1988, 1994, 2002) and Pacific (Chapman, 1991, 1992a; USEPA, 1995) coasts. Fertilization assays were also being developed by the American Society for Testing and Materials (ASTM, 1990), however, only the echinoid embryo test was formalized and published (ASTM, 2002). A critique of methodology was provided by NCASI (1992), with special relevance to pulp and paper effluents. In addition, a number of consulting companies and other marine laboratories have written procedures for their own organizations (see Appendix D).
Numerous papers have been published by various authors and groups of authors who use standard techniques. Notable among these papers are those of Kobayashi, Dinnel and co-authors, and Pagano and fellow-workers. Some of their papers are in the reference list, and many others are in the bibliography (Appendix E).
There are several reasons for choosing an echinoid fertilization test as a method of assessing sublethal toxicity in Canadian marine locations. In general, the test is quick, sensitive, and relatively simple. Some advantages are:
- Much of the biology and life history of major species are documented.
- The organisms are commonly and widely distributed on the three Canadian coasts.
- Adult sea urchins and sand dollars are easily collected in shallow waters.
- Adults are readily held in the laboratory and conditions can be manipulated to lengthen their spawning season.
- Gametes of consistent quality and sensitivity can be obtained.
- Success of fertilization is a sensitive and fundamental sublethal effect to measure.
- The fertilization assay is rapid and economical because it is small-scale, easy to do, and uses ordinary facilities and supplies.
- Echinoid eggs are already haploid when released, unlike those of most animals, and so the need for a mandatory waiting period before use is avoided.
- The test has a relatively simple and objective endpoint.
- Echinoids are available worldwide, and are frequently used as standard marine species for regulatory and research purposes. They can be easily shipped, and used at inland laboratories.
(NRC, 1981; Dinnel and Stober, 1985; Esposito et al., 1986; Dinnel et al., 1987).
In addition to general toxicity testing in a marine venue, the echinoid fertilization test would seem suitable for identifying the sublethally toxic components of complex effluents, using the “Toxicity Identification Evaluation” or TIE procedures described by the USEPA (1991a, 1991b).
The purpose of this “generic” report is to provide standardized Canadian methods for testing the sublethal toxicity of various substances or materials using echinoid gametes. Preferred choices are given among the alternatives available within a standard framework, for choice of species, exposure times, single-concentration (pass-fail) test versus multi-concentration test, test volumes, and type of water used for dilution and the controls. The echinoid test procedures in existing documents vary in their coverage of endpoints, and also differ in issues such as pH adjustment, alternative procedures for various objectives, selection of control/dilution water, and how to deal with samples that contain appreciable solids or floating material. This report is intended for evaluation of sublethal toxicity in samples of chemical, effluent, leachate, elutriate, receiving water, and liquid derived from sediment and similar solid materials. The rationale for selecting certain approaches is given.
The method is meant for use with seawater-acclimated animals and seawater as the dilution and control water. Depending on the test objectives, this seawater may be reconstituted or natural, but should approach the salinity of full-strength seawater. Other tests, using freshwater-acclimated fish or other sensitive freshwater organisms, are available for evaluating the lethal and sublethal toxicity of chemicals or wastewaters that are destined for, discharged to, or within the freshwater environment (See Appendix A).
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