Biological test method: fertilization assay using echinoids (sea urchins and sand dollars), chapter 6
Section 4: Universal Test Procedures
- 4.1 Preparing Test Solutions
- 4.2 Beginning and Performing the Exposure
- 4.3 Test Conditions
- 4.4 Test Observations and Measurements
- 4.5 Test Endpoints and Calculations
- 4.6 Reference Toxicant
- 4.7 Legal Considerations
Procedures described in this section apply to all the tests of particular chemicals, wastewaters, or receiving-water samples described in Sections 5, 6, and 7, and liquid samples derived from sediment (i.e., pore water) or similar solid materials, described in Section 8. All aspects of the test system described in the preceding Section 3 must be incorporated into these universal test procedures. The summary checklist of recommended conditions and procedures in Table 3 includes not only universal procedures but also those for specific types of test materials or substances.
There are some choices allowed within the general test procedures given in this report. Three options are available for duration of exposure. The shortest duration is a 10-min exposure of sperm, continued for an additional 10-min after eggs are added. That is the recommended standard exposure and it would minimize aging of gametes during a test or set of tests. The short exposure would also be most suitable for intensive programs involving many tests. For example, when attempting to identify toxic compounds in a complex effluent (TIE programs), successive manipulations of the effluent could be done before it aged appreciably.
A second option is a 20-min exposure of sperm plus 20 min of sperm plus eggs. That exposure might be used if it were desired to parallel certain existing methods or research results (Appendix D). The longest duration is 60 min of sperm plus 20 min of sperm plus eggs, an option that might be selected if maximum sensitivity were desired in the test. This longest exposure is also associated, however, with increased variation in results (see Section 4.2.4).
Three options are also available for the volume of test solution, which can be 10, 5, or 2 mL of each concentration of the sample. The 10-mL volume would be the usual standard choice and is preferred by Canadian investigators. The larger volume should be most convenient for manipulations by the operator and might improve the relative precision in handling small volumes. The smaller volumes require fewer adults to provide an assured supply of gametes, and can require less space in a water bath or constant temperature chamber. Small volume might be important for some investigations such as trials with pilot-plant outputs, perhaps as part of a TIE program.
Table 3 Checklist of Recommended and Required Test Conditions and Procedures
|Test type||static; standard sperm exposure of 10 min, continuing with 10-min exposure of both sperm and eggs to allow fertilization; alternative exposures 20 + 20 min, or 60 + 20 min|
|Control/dilution water||filtered (60 µm) uncontaminated laboratory seawater; reconstituted (artificial) seawater, filtered (60 µm) “upstream” receiving water to assess toxic impact at a specific location, with additional control of laboratory seawater; dissolved oxygen (DO) content 90 to 100% saturation at time of use; salinity 28 to 32 g/kg, preferably 30 g/kg, and pH 7.5 to 8.5, preferably 8.0 ± 0.2|
|Organisms||each replicate test vessel receives about 2000, 1000, or 400 eggs, depending on the selected volume of test solution; sperm:egg ratio is ascertained in a pre-test as that which targets an optimum of 80% fertilization under control conditions|
|Number of concentrations||minimum of 7, plus control(s); recommend more (i.e., ≥10), plus control(s)|
|Replicates||≥3 per treatment (recommend 5) for calculation of ICp; ≥4 per treatment if single-concentration (e.g. full strength) for hypothesis testing|
|Vessel/solution||standard volume of 10 mL test solution, with alternatives 5 or 2 mL; borosilicate glass vessels, capped or sealed|
|Temperature||for the native species 15 °C (green sea urchins, Pacific purple sea urchins, eccentric sand dollars), and 20 °C for Arbacia and white sea urchins; range for individual test vessels ± 1 °C of desired temperature|
|Salinity||standard test salinity 30 g/kg, limits 28 to 32 g/kg; each test solution in that range and also within 1 g/kg of the control; adjust salinity of sample or test solutions as necessary using hypersaline brine (HSB) with a salinity of 90 ± 1 g/kg, commercially-available dry ocean salts, reagent-grade salts or, if sample salinity >32 g/kg, deionized water; adjust salinity of control/dilution water as necessary using HSB at 90 ± 1 g/kg or dry salts; test requires second set of controls adjusted to 30 ± 2 g/kg and prepared by adding aged HSB (90 ± 1g/kg) or dry salts to deionized water, if HSB or dry salts added to sample/test solutions and if control/dilution water differs in any respect|
|Oxygen/aeration||no pre-aeration of aliquots of sample (e.g., effluent) or test solution unless DO is estimated to be <40% or >100% saturation in any concentration, in which case aerate an aliquot of the sample for ≤20 minutes through a plastic or glass tube with a small aperture (e.g., 0.5 mm ID) at a rate ≤100 bubbles/min, before making up concentrations and starting the test; no aeration during test|
|pH||regulatory or monitoring tests normally require no adjustment of pH of sample or solution; for other purposes, adjustment or a second (pH-adjusted) test might be required or appropriate; limits of pH 7.5 to 8.5, preferably 8.0 ± 0.2, apply for minimizing direct effects of pH on the gametes, and maximizing the potential for detecting toxic chemicals|
|Lighting||normal laboratory lighting or natural sunlight; variable photoperiod|
|Observations||percentage of fertilized eggs among 100 to 200 inspected microscopically for each test vessel|
|Measurements||temperature, salinity, pH, and DO at start of exposure, in aliquots of test solutions for high, middle, low concentrations and control|
|Endpoints||in multi-concentration tests, ICp for fertilization success; in single-concentration tests, percent fertilization and whether significantly lower than control; in porewater tests, percent fertilization and whether significantly lower than control or reference pore water at each treatment level (i.e., for each porewater dilution)|
|Reference toxicant||copper is recommended; determine ICp for fertilization success; perform within 14 days of the definitive test , or concurrently with definitive test for every new batch of adults if held ≤ 3d|
|Test validity||average success of fertilization in control must be ≥60% and <98%|
|Solvents||to be used only in special circumstances; maximum concentration 0.1 mL/L|
|Concentration||measurement at start is recommended, in aliquots of high, medium, and low strengths and control(s)|
|Control/dilution water||as specified and/or depends on intent; reconstituted seawater if high degree of standardization required; receiving water if concerned with local toxic impact; otherwise, uncontaminated laboratory seawater|
|Sample requirement||2 L should be adequate for the assay and for routine chemical analyses|
|Transport, storage||if warm (>7 °C), must cool to 1 to 7 °C with regular ice (not dry ice) or frozen gel packs upon collection; transport in the dark at 1 to 7 °C (preferably 4 ± 2 °C) using frozen gel packs as necessary; sample must not freeze during transit or storage; store in the dark at 4 ± 2 °C; use in testing should begin within 1 day and must start within 3 days of sample collection or elutriate extraction; extraction from sediment should occur within 2 weeks and must occur no later than 6 weeks after sampling|
|Control/dilution water||as specified and/or depends on intent; laboratory seawater or “upstream” receiving water for monitoring and compliance|
|Suspended solids||normally do not filter; filter effluent or leachate through 60 µm sieve if sample contains debris or indigenous organisms that could be confused with or attack the gametes or fertilized eggs; centrifuge elutriate|
|Sample requirement||as for effluents, leachates, and elutriates|
|Transport, storage||as for effluents, leachates, and elutriates|
|Control/dilution water||as specified and/or depends on intent; if studying local impact use “upstream” receiving water|
|Transport, storage||temperatures as for effluents and leachates; test should start within 2 weeks and must start within 6 weeks|
|Preparing/testing||aqueous samples derived from sediments should be treated as for effluents, leachates, and elutriates; solvent-based extracts should have balanced solvent concentrations; this is not a suitable assay for the solids themselves|
|Reference sediment||parallel test with clean sediment of similar physicochemical properties (uncontaminated sediment), if possible; otherwise use any clean (control) sediment|
|Control/dilution water||as for effluents, leachates, and elutriates|
4.1 Preparing Test Solutions
All test vessels, measurement devices, stirring equipment, and pails for transferring organisms must be thoroughly cleaned and rinsed in accordance with standard operational procedures. Control/dilution water should be the final rinse water for items which are to be used immediately in setting up the test; distilled or deionized water should be used as the final rinse for items which are to be stored after allowing them to dry.
4.1.1 Control/Dilution Water
The same control/dilution water must be used for preparing the control and all test concentrations. Each test solution must be well mixed with a glass rod, TeflonTM stir bar, or other device made of non-toxic material.
The temperature of the control/dilution water and the sample or each test solution must be adjusted as necessary to within ± 1 °C of the test temperature, before starting the test. Sample or test solutions must not be heated by immersion heaters, since this could alter chemical constituents and toxicity. It might be necessary to adjust the salinity or pH of the sample of test substance or the test solutions (see Sections 4.3.2 and 4.3.4), or to provide preliminary aeration (Section 4.3.3).
Control/dilution water may be the laboratory’s online supply of uncontaminated natural seawater, “upstream” water (i.e., receiving water) from a specific location under investigation, or reconstituted (artificial) seawater (see Section 2.3.4 and 3.4). As necessary, quantities of dry ocean salts (e.g. Instant OceanTM, Red Sea SaltTM), reagent-grade salts (e.g., modified GP2; see Bidwell and Spotte, 1985 or Table 2 in USEPA, 1994 or USEPA, 1995), natural hypersaline brine, artificial hypersaline brine, or deionized water should be added to seawater to adjust it to the test salinity (30 ± 2 g/kg). Any HSB, dry ocean salts, or reagent-grade salts used must be from the same source as that used to adjust the salinity of the test sample or test solutions (see Sections 5.2 and 6.2).
If any HSB is added to the test sample/solutions to adjust salinity, the toxicity test must include a set of controls (HSB controls) prepared using only this HSB and deionized water, adjusted to the test salinity (30 ± 2 g/kg). Likewise, if any commercially-available dry ocean salts or reagent-grade salts are added to the sample or test solutions, the toxicity test must include a set of controls (i.e., salt controls) which is prepared using the same source, batch, and concentration of dry salts as that added to the test sample. A second set of controls (i.e., dilution-water controls), comprised of 100% dilution water, is required if any water used to dilute the sample differs in any respect from the HSB controls or salt controls (e.g., natural seawater with or without HSB or dry salts added; natural fresh water with HSB or dry salts added, etc.) (see Section 4.1.4).
If natural seawater must be stored, it should be held at the test temperature or cooler, and should be used in three days or less.
Portions of seawater (i.e., control/dilution water or control/reference pore water) used for determining sperm density (see Section 4.2.2) and the appropriate sperm-to-egg ratio to be used in the test (see Section 4.2.3) should be filtered to remove solids that might interfere with sperm counts. Filtration is particularly important for natural seawater. A filter of pore size approximately 60 µm (USEPA, 1994) is recommended for this purpose. Filtered water should be used in three days or less.
Receiving water may be used as control/dilution water to simulate local situations such as effluent discharge, a spill of chemical, or pesticide spraying. If that is done, a second control solution must be prepared using the laboratory seawater in which adults were kept (see Section 4.1.4). “Upstream” receiving water cannot be used, however, if it is clearly toxic and produces an invalid result in the control according to the criteria of this fertilization assay.Footnote 15 In such a case, reconstituted seawater (Section 2.3.4) or the laboratory’s natural seawater should be used as control/dilution water. The laboratory water could also be used if the collection and use of receiving water is impractical.Footnote 16
For any test that is intended to estimate the ICp by regression analysis (see Section 4.5.2), at least seven test concentrations plus a control solution (100% control/dilution water) must be prepared, and more treatments (≥10 plus a control) are recommended. An appropriate geometric dilution series might be used, in which each successive concentration is about 0.5 of the previous one (e.g., 100, 50, 25, 12.5, 6.3, 3.1, 1.6, etc.). Test concentrations may also be selected from other appropriate dilution series (e.g., 100, 75, 56, 42, 32, 24, 18, 13, 10, 7.5; see column 7 in Appendix F). A dilution factor as low as 0.3 (e.g., concentrations 100, 30, 9, etc.) is not recommended for routine use because of poor precision of the estimate of toxicity; however, it might be used if there is considerable uncertainty about the range of concentrations likely to be toxic.
Each desired concentration is prepared and the standard volume selected (10.0 mL, 5 mL, or 2 mL) is added to the replicate test vessels. These nominal concentrations of the solutions (or measured concentrations, see Section 5.4) are adopted as the concentrations of the test. The slight decrease in concentration upon addition of the aliquot of sperm suspension is neglected.Footnote 17 The nominal concentration during the exposure of sperm is adopted as the concentration of the entire test. There is a concentration decrease of about 9% in the final part of the test, after the suspension of eggs is added, but for purposes of characterizing the test, the initial concentrations for sperm exposure are used.Footnote 18
In cases of appreciable uncertainty about sample toxicity, it is beneficial to run a range-finding or screening test for the sole purpose of choosing concentrations for the definitive test. Conditions and procedures for running the test can be relaxed. A wide range of concentrations (e.g., ≥2 orders of magnitude) should assist in selection for the full test.
Single-concentration tests could be used for regulatory purposes (e.g., pass/fail). They would normally use full-strength effluent, leachate, receiving water, elutriate or other liquid (i.e., pore water) from a sediment or similar solid, or an arbitrary or prescribed concentration of chemical. Use of controls would follow the same rationale as multi-concentration tests. Single-concentration tests are not specifically described here, but procedures are evident, and all items apply except for testing only a single concentration and a control.
If a multi-concentration test is conducted and an ICp is determined, each treatment including the control(s) must include a minimum of three replicate test vessels, and more than three (i.e., five) are recommended. If a single-concentration test (or multiple full-strength solutions) is conducted and hypothesis testing is usedFootnote 19 each treatment including the control(s) must include a minimum of four replicate vesselsFootnote 20 and more than four is recommended.
A control exposure which employs the same control/dilution water (dilution-water control) that is used to make up the test concentrations is required for all tests. A separate set of controls comprised solely of hypersaline brine (HSB) or dry salts in deionized water at a salinity of 30 ± 2 g/kg (Sections 2.3.4 and 4.1.1) is required if HSB or dry salts are added to the test sample or test solutions (Section 4.3.2), and if the dilution water differs from this HSB control or salt control in any respect. Each control must have the same number of replicates (i.e., at least 3) as for each of the other test solutions. The results for each dilution-water control, HSB control, or salt control used in a toxicity test must be examined to determine if they independently meet the test-specific criterion or criteria for test validity (see Section 4.5.1). In instances where two sets of control solutions are used (i.e., HSB controls or salt controls as well as dilution-water controls), the results for the toxicity test are considered to be valid and acceptable only if each set of control solutions independently meets the respective validity requirements (see EC, 2001 and EC, 2005 as well as Section 4.5.1). If, and only if, both sets of controls have met the validity criteria of the test, and the results of the two sets of controls are not statistically different from one another, then the results of the two sets of control solutions may be pooled (if desired) before calculating any statistical endpoints of the findings for each set of test concentrations versus those for the control solutions. Pooling control data for these two sets of controls before determining if the test results are valid or not is not acceptable (EC, 2001). If the controls are statistically different from each other by t-test (EC, 2005), then the two sets of data must not be pooled and the most applicable of the controls are used to calculate any statistical endpoints.
A set of salinity controls should be run if test salinity is, for any reason, outside the required range of 28 to 32 g/kg. If samples which were essentially fresh water (salinity ≤5 g/kg) were tested without adjusting salinity, salinity controls should be prepared by adding deionized or distilled water to a series of test vessels, at the same concentrations as used for the test liquid. The salinity controls indicate the effect of low salinity acting alone, but do not indicate any increased effect caused by interaction of low salinity with toxic substances or materials in the sample (see Section 4.3.2).
If a solvent is used in testing a chemical that is sparingly soluble, then a “solvent control” must be run with replicates, and must contain the solvent at the highest concentration present in any test concentration.
If receiving water is used as the control/dilution water, a second set of controls must be run using the laboratory seawater (artificial or natural) that was used for holding the adults (see Section 2.3.4).
Additional kinds of controls are not required, but are recommended to improve the ability to judge quality of results. A “low-sperm” control would use only half the number of sperm in order to check for “over-sperming”, which is a common imperfection in this assay. If the normal control achieved >90% fertilization and the low-sperm control was not 5% lower than the rate in the normal control, over-sperming is indicated, with associated poor sensitivity of the test. A “toxicant/egg control”, or “egg blank” uses a high concentration of the toxicant, but no sperm; it can indicate whether the sample being tested causes false fertilization membranes. A “control blank” with eggs but no sperm can reveal accidental contamination of stocks of eggs with sperm (Chapman, 1991).
4.2 Beginning and Performing the Exposure
Semen containing sperm is collected from several echinoids by forced spawning. Semen from each individual can be pooled before use. Eggs are collected, and can be pooled in the same fashion. Sperm are exposed to the test substance or material in each test vessel for either 10, 20, or 60 min. Then an appropriate number of eggs is added to each vessel, and exposure continues for 10 to 20 min to allow fertilization. Preservative is added to each vessel to end the exposure.
4.2.1 Collecting Gametes for the Test
Ideally, the sperm should represent three or more male adult echinoids of the selected species, and the eggs should represent three or more adult females. Since it is possible that sperm or eggs from one adult might be particularly sensitive or particularly tolerant, an attempt should be made to achieve homogeneity of the experimental units (i.e., to avoid any differences among vessels that are related to the parent). The only practical way to do this is to pool the male or female gametes from different parents before transferring them to the test vessels, however, pooling good quality gametes, with poor quality gametes can result in poor fertilization success. Therefore, a gamete check (see following paragraph) of individual males and females must be performed to ensure that only good-quality gametes are being selected for use in the test. If good-quality gametes from three adults of each sex cannot be obtained (see following paragraph), and/or if in addition to the gamete check a pre-test is carried out with individual gamete combinations of at least 2 males and 2 females (as described in Section 4.2.3), it is permissible to use gametes from only one adult from each sex (i.e., 1 male and 1 female whose gametes yield good fertilization success when combined in a gamete check and pre-test).
A gamete check is required to ensure that a subsample of gametes from each of the several adult males and females chosen as likely sources of sperm and eggs to be used in the test have a high degree of viability. In this procedure, three to five females and at least three males are selected for microscopic examination of each individual’s gametes. Each of these individuals is spawned, and their gametes placed in a separate container. The semen from each male are stored separately on ice. A small portion of each male’s sperm is then diluted with control/dilution water on a microscope slide, so that the motility of the sperm can be judged. Eggs from each individual female are similarly examined under a microscope. Poor quality eggs are small in size, irregular in shape, and display vacuolization. Small aliquots of eggs from each female having “good-quality” eggs are then placed in several scintillation vials. Separate groups of eggs representing each “good-quality” batch are then fertilized with a few drops of diluted sperm from one of each of the “good-quality” batches of sperm. For example, if gametes from four females and three males are being examined, three vials of eggs are prepared for each female (i.e, for every female spawned, one vial of eggs is prepared for each male spawned). Each vial is fertilized with 5 to 7 drops of slightly diluted sperm (i.e., 20 - 50 µL of concentrated or “dry” sperm in 10 mL of filtered seawater) from one of the three different males (i.e., each vial of eggs is fertilized by the sperm from a different male). After 10 minutes each mixture of sperm and eggs in each vial are observed under a microscope. Sperm quality is assessed by looking at motility, activity, clumping, and fertilization success. Egg quality is assessed by looking at shape, colour, size, and fertilization success.
The number of eggs fertilized in each vial should also be examined. If the proportion of eggs fertilized is high (i.e., 95-100%) in a particular vial, and a pre-test (see Section 4.2.3) is carried out on the same batch (i.e., gametes from the individual male and female being stored on ice) of gametes to determine the optimal sperm:egg ratio, then the original batches of sperm from the male and the eggs from the female for which combined aliquots (subsamples) showed a high fertilization rate, can be used in the definitive test. If a laboratory chooses not to run a pre-test to determine the “optimal” sperm:egg ratio, then good quality gametes pooled from at least three males and three females, as determined in a gamete check, must be used. Only good-quality gametes are pooled and then used in the test.Footnote 21 If good-quality gametes are not available from three males and three females, fewer adults may be used, however a pre-test must be carried out to determine the optimal sperm:egg ratio for a given batch of gametes, prior to the definitive test, thereby improving the likelihood of successful control fertilization. “It is more important to use high quality [gametes] than it is to use a pooled population of [gametes]” (Chapman, 1992a).
The adults are stimulated to spawn by injecting potassium chloride.Footnote 22 Sea urchins are injected with 0.5 to 1.0 mL of 0.5 M KCl through the peristomial membrane (i.e., between Aristotle’s lantern and the test or the hard outer shell) on an angle pointing toward the outer shell into the coelom (Figure 2).Footnote 23 The KCl injection can be divided and injected in several different locations around Aristotle’s lantern, and/or the sea urchin can be gently shaken to distribute the KCl within the organism. Sand dollars are injected with 0.5 mL of the same solution at an angle through the mouth. A tuberculin syringe with 25 gauge needle is satisfactory for this manoeuvre. An alternate method, that appears to work only with Arbacia, is stimulation of the shell for 30 seconds by electrodes supplied with 12 volts D.C.Footnote 24
The preferred and recommended technique for collecting semen from male sea urchins is called “dry spawning”. Once sperm is wetted, it has limited viability (see footnote 25), so in order to complete both a gamete check and a pre-test (see Section 4.2.3), and still have viable sperm for use in the definitive test, sperm should be collected “dry”. Care must be taken when collecting “dry” sperm from the males to avoid the sperm becoming contaminated with water or KCl solution from the animal while spawning. One technique for dry-spawning male urchins is to place an individual in a dry beaker or petri dish, with its aboral surface down. Semen is then collected from the bottom of the container (as opposed to from the surface of the animal). Another technique is to place the animal in a beaker with its aboral surface up, and with control/dilution water covering only the lower half of the test or shell. Extruded semen which accumulates on the animal’s surface by the pores is gathered with a micropipet, transferred to a small capped or covered tube, and stored on ice. Care must be taken to ensure that the surface onto which the sperm is extruded (i.e., the bottom of a petri dish or the surface of the sea urchin) is dry, in order to avoid wetting the sperm and thereby activating it. Similar techniques may be used for collecting eggs from females, if desired, but they should be washed and stored as indicated below.
Male eccentric sand dollars might produce insufficient volumes of sperm when spawned “dry”. Sand dollars can be spawned in a minimal amount of seawater (5 mL)Footnote 25; however, they should be suspended over the water column. (Experience indicates that sand dollars won’t spawn if placed in a seawater-rinsed petri dish with their aboral surface in direct contact with the bottom of the dish).
For the alternative “wet spawning” method, each sea urchin or sand dollar is placed aboral side down on a small beaker, 50 to 250 mL or other size as appropriate, filled to the brim with control/dilution water at the test temperature. After spawning is terminated, decant as much water as possible from the gametes. Alternatively, females can be placed aboral side up in a vessel with just enough control/dilution water to cover the test (shell) of the urchin by about 1 cm. Eggs can be collected off the surface of the test and placed in a small beaker or other appropriate vessel.
If there is no spawning in 5 or 10 minutes, a second injection may be used, however this might cause the organisms to extrude gametes that are immature and of poorer quality. Semen or eggs should be produced by the adults in a steady stream, within half an hour of the final injection, as a maximum. Semen appears as a compact white string when shed into water, and eggs will appear as somewhat granular material, usually with a pastel colour (pinkish in sand dollars). Coloured products are sometimes extruded before or during the spawning, and should not be mistaken for gametes.
Collection of spawn should be terminated within 15 min of the start of steady spawning. Enough gametes should be collected from the same individuals for the gamete check, the pre-test, and the definitive test. Multiple collections of gametes from the same adult are normally pooled using a pipette. For manipulations of eggs, many investigators use a standard 1-mL plastic micropipette with 2 to 3 mm cut off by means of a scalpel, to provide a bore diameter of approximately 1 mm and reduce damage to the eggs.
Semen collected “dry” may be held on iceFootnote 26 for 4 h before “activation” in seawater, then used in a test in the subsequent 30 to 120-min period.Footnote 27 If sperm are collected in beakers of seawater, they should be used to start the test in a period ≥0.5 h to ≤2 h after collection is completed. In the interim, they are to be stored in a minimum amount of control/dilution water, on ice.
The collected eggs are washed three times by diluting with 100 mL of control/dilution water, mixing, settling for 10 minutes, and decanting. If pigmented substance is obtained with the eggs, it might be important to rinse them soon after collection, since the substance might be toxic to the Pacific purple sea urchin and perhaps with other species.Footnote 28 Eggs may be held in the final addition of control/dilution water, at the test temperature, for 4 h until use. It is recommended that eggs be aerated gently during holding.
4.2.2 Preparing Standard Suspensions of Gametes
Semen from the male sea urchins or sand dollars, chosen following the gamete check (see Section 4.2.1), is pooled to produce a concentrated suspension of quality sperm. If sperm were collected in beakers of water, pipette them from the bottom of the water and combine sperm from the various beakers. Semen should be transferred by drawing it slowly (without cavitation) into a micropipette (orifice ≥1 mm), and delivering by multiple expulsions and refills, to rinse it into the water receiving it.
Sperm density in the initial suspension is estimated with a hemocytometer or other counting cell under 400 × magnification.Footnote 29 Dilute a small sample (0.1 to 1 mL) of the mixed suspension 100-fold to 10,000-fold (depending on concentration of sperm), using 10% glacial acetic acid made up with control/dilution water. Mix by inverting ten times and allow bubbles to clear for a minute or two. Add a drop of the mixture to the hemocytometer counting chamber and let the sperm settle for 15 minutes. Count the sperm in the middle 400 small squares. Calculate the number of sperm per mL in the initial suspension. This is done by multiplying: (dilution factor) × (number of sperm counted) × (hemocytometer conversion factor) × (conversion of mm3 to mL) ¸ (the number of squares counted). For a standard hemocytometer (Neubauer), the formula becomes:
No. sperm/mL = 100 × (No. of sperm counted) × 4000 × 1000 ÷ 400
Adjust the initial suspension of sperm to the desired concentration in a “standard sperm suspension”, using control/dilution water.Footnote 30 The concentration of this standard sperm suspension is determined by the sperm:egg ratio that is selected (Section 4.2.3).
An alternative counting technique that may be used, is turbidity or optical density as an indication of the number of sperm/mL, without a hemocytometer count. The advantage is a saving of time, since the measurement takes only one minute compared to 20 to 30 minutes with a hemocytometer (NCASI, 1992). That in turn allows tests to start sooner after collection of gametes. The concentrated collection of sperm is mixed with control/dilution water in a 1-cm spectrophotometer tube, just before starting the test. Standard turbidity meters designed for analysis of water samples may be used. NCASI (1992) reports that a range of 2.0 to 4.0 Nephelometric Turbidity Units (NTU) usually yields the desired numbers of sperm. A count of 2.5 million sperm/mL would be associated with about 3.0 NTU for the eccentric sand dollar, and about 2.7 NTU for the Pacific purple sea urchin. The turbidimetric technique can have precision that is almost as good as that obtained by counting. NCASI (1992) found an average CV of about 9% for repeated hemocytometer counts of single dilutions of sperm, and a CV of 12% for repeated hemocytometer counts of dilution to 5.0 NTU of sperm from three males. No evaluations of the turbidimetric method are available from other laboratories at the time of writing. The final criterion of whether turbidimetric assessment of sperm density was satisfactory would be the fertilization rate achieved in the control, during the test, compared to the validity criterion of ≥60%, and <98% fertilization (Section 4.5.1).
There are three options for initial test volume, the standard of 10 mL and of 5 or 2 mL. The concentrations of the gamete suspensions are the same for each. The amount of gamete suspension to be added is scaled down proportionally for the smaller test volumes. In the largest test volume (10 mL), there is 0.1 mL of sperm suspension added, and 1.0 mL of egg suspension. (See Table 4 for summary of numbers of gametes and volumes of gamete suspensions for the three sizes of test).
|Initial test volume
|Number of eggs||Volume of egg suspension||Number of sperm (millions) at usual sperm:egg ratios of 200:1||Number of sperm (millions) at usual sperm:egg ratios of 2500:1||Volume of sperm suspension added
|Usual range of concentration in sperm suspension
|10||2000||1.0||0.4||5||0.1||4 to 50|
|5||1000||0.5||0.2||2.5||0.05||4 to 50|
|2||400||0.2||0.08||1||0.02||4 to 50|
The numbers of sperm in columns 4/5 and 7 are governed by the sperm:egg ratios of 200:1 and 2500:1 selected as examples.
The numbers of gametes and procedures are given here for a test with initial volumes of 10 mL. The required strength of the sperm suspension must be calculated first. About 2000 eggs are used in the 10-mL test, and the ratio of sperm to eggs is often in the range 50:1 to 2500:1 (Section 4.2.3), although it might sometimes be higher, to 20 000:1 or more. Within the range 50:1 to 2500:1, the required number of sperm would be from 100 000 to 5 million. Since 0.1 mL of the sperm suspension is added in the test, the concentration of sperm required in the standard suspension will usually be in the range of one million to 50 million per mL.Footnote 31
Calculations of proper dilution are easily done by the following standard chemistry formula:
C1 × V1 = C2 × V2
“concentration one × volume one = concentration two × volume two”.
If a count of 125 million sperm/mL were obtained for the initial suspension, and if 5 mL of standard sperm suspension of 40 million/mL were desired, then the volume of initial suspension to be made up to 5 mL would be calculated as V1:
125 × V1 = 40 × 5
therefore, V1 = 1.6 mL
Determine the density of the mixed suspension of eggs by counting and adjust to 2000 eggs/mL.
Counting can be done by adding to a Sedgwick-Rafter cell, 1 mL or less of the mixed suspension as required, then observing at 20 to 100 × magnification. It is often useful to dilute an aliquot 10-fold, 100-fold, or, in some instances, 1000-fold, for the purpose of counting. With experience, the original suspension can be diluted according to its appearance, to a few hundred eggs/mL, then a count is made with 0.5 mL. Other techniques of counting may be used if they are effective. Adjust the suspension to 2000 eggs/mL by adding control/dilution water to reduce the density, or settling eggs and decanting water to increase the density.
For a test with an initial volume of 5 mL, exactly the same procedures are followed except that smaller volumes of the gamete suspensions are added to the test vessels (Table 4). The volume of sperm suspension added would be 0.05 mL (usually containing 2 to 25 million sperm, depending on the sperm:egg ratio required), and the volume of egg suspension added would be 0.5 mL (containing 1000 eggs).
For a test with an initial volume of 2 mL, proportionally smaller volumes of gamete suspensions are used. The added volume of sperm suspension would be 0.02 mL (usually containing 0.8 to 10 million sperm), and the added volume of egg suspension would be 0.2 mL (containing about 400 eggs).
4.2.3 Ratio of Sperm to Eggs
The optimum sperm-to-egg ratio should be determined by pre-test in each laboratory, as that which targets an optimum rate of 80% fertilization under control conditions.Footnote 32 Very low fertilization rates in the control would mean that effects of a toxicant on fertilization might be difficult to distinguish from the generally poor and variable background performance. Rates that are high indicate an excess of sperm that might mask an effect by compensating for part of the toxicity, thus reducing the sensitivity of the test and raising the IC25.Footnote 33 Several options are available for determining a suitable sperm:egg ratio, since the final criterion of a satisfactory test will be the actual rate of fertilization achieved in the control, which must be between the control limits of ≥60% and <98% for a valid test (Section 4.5.1).
Ratios that have been reported in the literature to give satisfactory fertilization range from 50:1 to 2500:1 for the various test species (Appendix D). The following sperm-to-egg ratios have been reported by Canadian and US laboratories in a recent survey (see Section 1.1) to achieve a fertilization range of 70 to 90%: green sea urchin, 2000:1 up to 5000:1Footnote 34; Pacific purple sea urchin, commonly 100 to 500:1 but as low as 2:1 and as high as 2000:1; eccentric sand dollar, often about 200:1 to 400:1 but also reliably reported in the range 50:1 to 6000:1; white sea urchin, typically 20,000:1; and Arbacia, 2500:1. Such general guidance cannot, however, be depended on to yield satisfactory test results in any given laboratory or season. Canadian interlaboratory tests, for example, found that some sperm:egg ratios had to be an order of magnitude higher than values mentioned above (Miller et al., 1992).
Ideally, the appropriate sperm:egg ratio should be determined just before each test, and with the gametes to be used in that test. The pre-test may be shortened and simplified to use one or two sperm:egg ratios that are thought to be low. Results could be used to position the gametes that are to be used on a “curve of fertilization success” from past experience in the laboratory, allowing an appropriate ratio to be selected for the real test.
An alternative pre-test procedure may be used to determine the sperm:egg ratio to be used in order to target 80% fertilization in the controls (Carr and Chapman, 1995).Footnote 35 This pre-test uses two replicates of control/dilution water and one replicate of each of three concentrations of a reference toxicant, tested with each of several sperm:egg ratios (i.e., 5) in order to determine the “optimum” sperm:egg ratio to be used in the test. The sperm:egg ratios used in the pre-test, should cover a wide range (e.g., 10-fold difference in sperm concentration). The pre-test is performed like a regular test, with the addition of an appropriate aliquot of sperm to each vial, and then eggs added after the appropriate exposure time. After counting the % fertilization in all of the sperm:egg ratios for each treatment, a sperm:egg ratio is chosen based on the % fertilization results in the control/dilution water (targeting 80% fertilization), and that which maximizes the potential for the reference toxicant result to fall within the warning limits of a control chart. Using this method, a sperm:egg ratio can be chosen which demonstrates the appropriate sensitivity at the targeted optimum fertilization rate in the controls.
For porewater testing, this pre-test method is recommended, and should include two replicates of a control pore water (see Section 8.1.4), in addition to the two replicates of control/dilution water and one replicate of each of three concentrations of a reference toxicant, previously described.Footnote 36 This pre-test can be combined with an extended gamete check by performing the test on specific combinations of gametes from individual males and females (e.g., sperm from each of several males is tested separately with the eggs from each of several females to determine the best quality gamete combination; see Section 4.2.1). As such, the gametes from individual males and females, which, when combined at the right sperm:egg ratio, result in the ideal percent control fertilization, and the appropriate sensitivity to a reference toxicant (i.e., will yield results that fall within the warning limits of a control chart). These gametes can then be chosen for use in the definitive test and the appropriate sperm:egg ratio is known.
In practice, experience at a given laboratory can establish a “standard” ratio that usually gives the desired results for a particular species. However, the routine use of a “standard” ratio risks lowering the quality of testing. If the standard ratio yielded < 60% fertilization in the control, or ≥98% fertilization (Section 4.5.1), the test would be invalid and would have to be repeated using a different ratio. Other tests might lose sensitivity because of “over-sperming”. The sperm:egg ratio might require adjustments with season, and 10-fold changes in requirements due to season are not unknown (Chapman, pers. comm., 1992b).
Because of the normal variation in percent fertilization for controls, a pre-test is highly recommended. Investigators familiar with the echinoid fertilization assay find that the time spent in a pre-test for each definitive test has, in the long run, saved considerable time, money, and sometimes irreplaceable samples.
An alternative approach to circumvent control pre-tests is to include replicates of two or three sperm:egg ratios for each concentration used in the test including controls. Results for the ratio that yielded a fertilization rate of 80% in the controls would be used in calculating the ICp (Section 4.5.2). NCASI (1992) points out that this actually requires less of the investigator’s time than running a pre-test and then a test, and has a further advantage of avoiding any changes of sperm activity during the interval from pre-test to test.
If the sperm:egg ratio was determined by a pre-test, or arbitrarily selected, the strength of the suspension of sperm is also fixed (Section 4.2.2). For example, if a sperm:egg ratio of 2000:1 were required for the 2000 eggs to be added, then 4 million sperm would be needed in the 0.1 mL of added suspension, or 40 million sperm per mL in the suspension.
4.2.4 Exposure of Gametes
Individual vessels are positioned for the exposure in a test tube rack or other rack, held in the water bath or other temperature-control facility. Vessel positions in the rack must be either completely randomized, or randomized in “columns” of the rack, each column representing one replicate of each concentration and control.Footnote 37 Each vessel must be clearly labelled or positions coded so that concentrations and replicates can be identified.
The temperature, salinity, dissolved oxygen, and pH levels in representative (i.e., controls plus at least the high, medium, and low concentrations if a multi-concentration test) aliquots of the test solutions must be measured when they are prepared. If required or permitted, values must/should be adjusted to acceptable levels (Section 4.3) before adding the solutions to the test vessels.
The test has three options for duration of exposure, options which are otherwise identical in their procedures. Obviously, only one of these options can be used in a given test, and for comparative tests. The shortest option is the standard exposure for normal testing and monitoring. It is 10 min of sperm exposure, with the addition of eggs at that time and an exposure that continues for a further 10 min of sperm plus eggs, i.e., the 20-min test. For Arbacia, however, the time for development of a fertilization membrane is slower than that for the other four test species, and therefore longer exposures (i.e., 20 + 20 min or 60 + 20 min) are recommended for this species.
Either of two longer exposures might also be used for special purposes such as research or comparison with other data. The second option is 20 min of sperm exposure followed by 20 min sperm plus eggs, the 40-min test. The longest option is exposure of the sperm for 60 min, plus 20 min, an 80-min test.Footnote 38
The three options for volume of test solution are independent of the options for duration (thus nine options for test procedure). The option for an initial test volume of 10 mL is the usual standard and is described here.Footnote 39 The procedures for the smaller test volumes of 5 and 2 mL would be identical except that proportionally smaller volumes of gamete suspensions would be added (Table 4).
The solution of sperm is mixed, and to start the test, 0.1 mL is added to each test vessel, which already contains 10.0 mL of test solution (Section 4.1.2). At the end of the sperm exposure, the egg preparation is mixed and 1.0 mL is added to each test vessel. Automatic dispensing micro-pipettes are needed to accomplish these steps within narrow time limits. Care must be taken when adding sperm and eggs to the vessels; all of the fluid delivered from a pipette must enter the test solution rather than striking the side of the vessel, and the pipette tip must not touch the test solution. The suspension of gametes should be mixed after every second or third vessel is filled. After sperm have been added to all vessels, and again after eggs have been added, all vessels should be thoroughly mixed by swirling, in-and-out pipetting, or brief use of a vortex mixer.
A timing procedure should be used for adding sperm to vessels in sequence, for example one vessel every 5 seconds. The eggs should be added to the vessels in the same sequence (order of vessels) and with the same timing interval as was used for sperm, in order to equalize exposure periods. Termination of the test should again be done in the same sequence with the same timing. Additions to test vessels should not be done according to magnitude of concentration, but by replicate, i.e., the first set of replicates, then the second, then the third (Chapman, 1992a).
At the end of the sperm-plus-eggs exposure, the test is terminated by adding either 2 mL or less of 1% glutaraldehyde, or 2 mL or less of 10% buffered formalin to each test vessel.Footnote 40 (The amounts of preservative are divided by 2 and by 5 for the two smaller-volume test options). Preserved eggs should be counted within three days of test completion. During storage, vessels containing eggs should be sealed (e.g., using plastic film).
4.3 Test Conditions
This a static test without aeration and without renewal of test solutions. The test is carried out at 15 °C for the four native species, and at 20 °C for the listed non-native species. Salinities in all test vessels are normally within 1 g/kg of the control, in the range 28 to 32 g/kg. An attempt is made, if necessary, to raise the dissolved oxygen of all test solutions above 40% saturation before the test is started.
A test temperature of 15 °C should be used for green sea urchins, Pacific purple sea urchins, and eccentric sand dollars. The test temperature should be 20 °C for the non-native Arbacia and white sea urchins. Temperatures of all test solutions should be within 1 °C of the intended value as determined by measurements in aliquots or test vessels without gametes (dedicated to temperature monitoring). Temperatures must be measured in aliquots of the control(s), high, medium, and low concentrations before beginning the test.
The test temperatures recommended here are 3 ° to 7 °C higher than the values recommended for holding the adults of the same species, but within the biokinetic ranges. These somewhat elevated temperatures should make the test more sensitive in detecting some toxicants.Footnote 41 Some of the recommended temperatures conform with those previously used in Canadian methods or U.S. standard methods, but they necessarily diverge from some other methods, because of the variety employed elsewhere (Appendix D).
A standard test should be carried out at a salinity of 30 g/kg. All test solutions should be in the 28 to 32 g/kg range, and they should also be within 1 g/kg of the salinity of the control.Footnote 42
If a chemical is being tested, it should be made up to the test concentration(s) using a control/dilution water which has a salinity in the required range (see Sections 4.1.1, 5.2, and 5.3). The salinity of aqueous samples (e.g., chemical products or formulations made up in water; effluents; leachates) or test solutions should be measured before the test and, if outside the range 28 to 32 g/kg, should be adjusted to within this range using one of two approaches: (1) sample salinity may be adjusted by the direct addition of dry salt to the effluent or other material (e.g., leachate or elutriate); or (2) sample salinity may be adjusted by the addition of hypersaline brine (following guidance in EC, 2001 and in Section 2.3.4). Deionized water can be used to reduce the salinity of test samples. The sample must not be warmed to the test temperature before this salinity adjustment, rather, the temperature during salinity adjustment should approximate either that of the sample when received, or in instances when the sample is stored overnight in a refrigerator at 4 ± 2°C, that of the sample when it is removed from the refrigerator (EC, 2001).
If the first approach is chosen, either a mixture of commercially-available dry ocean salts (e.g., Instant OceanTM , Red Sea SaltTM) or reagent-grade salts (e.g., modified GP2; see Bidwell and Spotte, 1985 or Table 2 in USEPA, 1994 or USEPA, 1995) may be added to the undiluted sample, in a quantity sufficient to raise sample salinity to 30 ± 2 g/kg. Any sample to which dry salts are added directly must be aged for a period of no more than 16 to 24 hours before its use in a toxicity test (EC, 2001). To age the sample, the required quantity of salt must be added while stirring the effluent; thereafter, the salinity-adjusted (30 ± 2 g/kg) sample must be held for 16 to 24 h at 4 ± 2°C in the dark and within a sealed container with minimal air space (and without any aeration). Sample pH should be measured and recorded before salt addition and after salt addition but before aging. Following this aging period, the effluent sample should be stirred, warmed to the test temperature, its pH checked and recorded, test concentrations prepared, and the toxicity test started (EC, 2001).
If the second approach is chosen, sample salinity must be adjusted to the test salinity (30 ± 2 g/kg) by the addition of the required amount of hypersaline brine (and, as necessary, deionized water). HSB must be used for this purpose and it should have salinity of 90 ± 1 g/kg . Guidance provided in EC 2001 and Section 2.3.4 for preparing HSB must be followed. If HSB with a salinity of 90 g/kg is used to adjust the salinity of a freshwater sample to 30 g/kg (see Sections 3.4 and 4.1.1), the maximum concentration of the sample that could be tested would be 67%.
Samples of effluent, leachate, receiving water, elutriate, produced water, or other aqueous extract from sediment could also be tested without adjusting salinity of the sample, if it were desired to assess the total effect, including divergent salinity. It should be realized that if the sample is essentially fresh water (salinity <5 g/kg) or is a brine (e.g., produced water), the results of the toxicity test will probably reflect unfavourable salinity rather than any toxic substance(s) in the sample. If an unadjusted sample were tested, it would be desirable to run a set of salinity controls using parallel concentrations of distilled water (Section 4.1.4), or to conduct a second test with salinity of the sample adjusted, or both, in order to understand the contribution of salinity to toxicity.
4.3.3 Dissolved Oxygen and Aeration
If (and only if) calculations from the dissolved oxygen measured in the sample to be tested indicated that one or more of the test concentrations would be outside the 40 to 100% range of air saturation, the sample or an aliquot of sample should be aerated before starting the test (“pre-aeration”). To achieve this, oil-free compressed air should be dispensed through airline tubing and a disposable plastic or glass tube of small aperture (e.g., capillary tubing or a pipette with an Eppendorf tip, with an opening of about 0.5 mm). The rate of pre-aeration must be at a minimal and controlled rate, which should not exceed 100 bubbles/min. Duration of pre-aeration must be the lesser of 20 minutes and attaining 40% saturation (or 100% saturation, if supersaturation is evident).Footnote 43 Any pre-aeration must be discontinued at ≤20 minutes and the test initiated, whether or not 40 to 100% saturation was achieved in the aliquot of sample, or would be expected in all test solutions. Dissolved oxygen must then be recorded for the start of the test in representative aliquots of the test solutions including the highest concentration. Any pre-aeration must be reported, including the duration and rate (Section 9).
If oxygen in one or more test vessels is below 40% of saturation, the test becomes invalid as an assessment of the toxic quality, per se, of the material or substance being tested. The test would still be a valid assessment of the total effect of the material (e.g., effluent) or substance (e.g., chemical) including its deoxygenating influence.Footnote 44 The required use of oxygen-saturated control/dilution water will, in most instances, result in dissolved oxygen levels that should not have a large influence on test results.
The pH must be measured in aliquots of the control(s), high, medium, and low concentrations before beginning the test.
Toxicity tests for regulatory or monitoring purposes would normally be carried out without adjustment of pH. However, if the sample of test material or substance causes the pH of any test solution to be outside the 7.5 to 8.5 range, results might reflect effects due to pH alone.Footnote 45 If it is desired to assess toxic chemical(s) per se rather than the deleterious or modifying effects of pH, then the pH of the solutions or sample should be adjusted, or a second, pH-adjusted test should be conducted concurrently.Footnote 46 For an adjusted test, the initial pH of the sample, or of each test solutionFootnote 47 could, depending on objectives, be adjusted to within ± 0.5 pH units of that of the control/dilution water, before exposure of the gametes. Another acceptable approach for this second, pH-adjusted test is to adjust each test solution, including the control, upwards to pH 7.5 to 8.0 (if the solution has pH <7.5), or downwards to pH 8.0 to 8.5 (if the solution has pH >8.5). Solutions of hydrochloric acid (HCl) or sodium hydroxide (NaOH) at strengths ≤1 Nshould normally be used for all pH adjustments. Some situations (e.g., effluent samples with highly buffered pH) might require higher strengths of acid or base.Footnote 48
In some circumstances it might be desired to carry out the most sensitive test possible for detecting toxic chemicals, rather than including pH as part of the total effect of a chemical, effluent, leachate, elutriate, or liquid extracted from sediments or other solid materials (such as pore water). In such a case, any effect of low or high pH, in changing viability of gametes and success of fertilization, should be eliminated by adjusting pH of test solutions as necessary, to the preferred range of 8.0 ± 0.2.Footnote 49
Abernethy and Westlake (1989) provide useful guidelines for adjusting pH. Aliquots of samples or test solutions receiving pH adjustment should be allowed to equilibrate after each incremental addition of acid or base. The amount of time required for equilibration will depend on the buffering capacity of the solution/sample. For effluent samples, a period of 30 to 60 min is recommended for pH adjustment (Abernethy and Westlake, 1989). For an echinoid test, the adjustment would be made on aliquots used to prepare test concentrations, the pH in each would be recorded (Section 4.4), and the test started with no further attempt at adjustment.
If the purpose of the toxicity test is to gain an understanding of the nature of the toxicants in the test substance or material, pH adjustment is frequently used as one of a number of techniques (e.g., oxidation, filtration, air stripping, addition of chelating agent, etc.) for characterizing and identifying sample toxicity. These “Toxicity Identification Evaluation” (TIE) techniques provide the investigator with useful methods for assessing the physical/chemical nature of the toxicant(s) and its (their) susceptibility to detoxification (USEPA, 1991a, 1991b).
4.4 Test Observations and Measurements
At the end of the exposure, preserved eggs are taken from each test vessel after mixingFootnote 50, and an equal number from each vessel, in the range of 100 to 200 eggs, is counted and classified as either fertilized or not fertilized (Figure 3).Footnote 51 The count is made under a microscope at 100 × magnification, preferably by phase-contrast microscopy. A counting cell such as a Sedgwick-Rafter chamber might be useful, although the count can be made using an etched petri plate. Microscopic technique is important, and can affect the accuracy of the counts. Consistency of counting should therefore be checked by trials, especially among different people who might be involved in counting.
The criterion of fertilization is a raised fertilization membrane, and this includes full, partial or collapsed membranes (see Figure 3), none of which are seen in unfertilized eggs (NCASI, 1991).Footnote 52
Eggs of Arbacia, when fertilized, have much smaller fertilization membranes than those of the other 4 species. This could lead to greater uncertainty in the counting of fertilized vs. unfertilized eggs, even by experienced analysts. Adding several drops of a 150 - 200 g/kg NaCl brine to the microscope slide containing the test eggs causes the eggs to shrink temporarily, leaving a greater space between the fertilization membrane and the egg and allowing the fertilization membrane, if present, to be more easily discerned.
Artifacts such as partial collapse of membrane or movement of the egg to one side of the hyaline sphere, can occur during preservation after the test. Clearly abnormal eggs, or dead ones, are simply omitted from the count, whether they are fertilized or not. The counts are recorded for each test vessel.
Figure 3 Discriminating Between Fertilized and Unfertilized Eggs
Outlines of eggs as seen under a dissecting microscope. The three drawings in the upper row represent a sea urchin such as the green sea urchin. The egg on the left is not fertilized. The middle egg has a fertilization membrane that is partially raised and is considered fertilized. The right-hand egg has a completely raised fertilization membrane. The Pacific purple sea urchin is similar but within the outer fertilization membrane, an inner hyaline membrane might be evident. The three drawings in the lower row represent the eccentric sand dollar, from left to right, unfertilized, fertilized with a partially raised membrane, and with a completely raised membrane. The jelly-like coating of the sand dollar contains pigment granules and usually disappears during later development of the egg. Drawn by M.A. White, from prepared slides from McGibbon and Moldan (1986), and from drawings of Kelley Battan of NCASI, Anacortes, Washington.
Long description for Figure 3
Illustrations of eggs which are: unfertilized, newly fertilized, and exhibiting complete signs of fertilization are presented for both sea urchins and sand dollars. In both organisms fertilization is recognized through the formation of a fertilization membrane which surrounds the egg. This membrane may be complete or, in the case of newly fertilized eggs, only partially raised. Eggs with a fully only partially raised membrane are both considered to be fertilized.
4.5 Test Endpoints and Calculations
The biological endpoint of the test is adverse effect on success of fertilization, assessed by comparison with the controls. Percent fertilization is calculated for each test vessel.
The inhibiting concentration for a specified percent effect (ICp) is the required endpoint for a multi-concentration test. Regression analysis must be used to for the calculation of the ICp, if possible, following the guidance provided in Section 4.5.2 and in EC, 2005. The 95% confidence limits must be given for any ICp reported.
4.5.1 Validity of Test
The test is invalid if the mean fertilization rate for all replicates of the control water is <60%, or ≥98%.Footnote 53 Also, a positive and logical dose-effect curve should have been attained, for the results to be considered valid, i.e., the effect on fertilization must become generally greater at higher concentrations.
If dissolved oxygen in one or more test vessels was less than 40% saturation, the test should be considered an invalid assessment of the toxic quality, per se, of the substance or material being tested. The test would still be a valid assessment of the total effect of the test substance or material (Section 4.3.3).
4.5.2 Multi-Concentration Tests
Echinoid fertilization data presents a unique case in toxicity data analysis, for the following reasons:
- While the data is, by nature, binomial (an egg is either fertilized or not fertilized), because the number of replicates is 100, the data often meet the assumption of normality.Footnote 54
- One of Environment Canada’s test validity criterion limits the control response to ≥ 60%, or < 98%. As a result, control response will not be 100% (by design), and this needs to be accounted for in the data analysis. In addition, because the control response will not be maximized, there is the possibility that fertilization may be enhanced (stimulated) at low doses of test substances (i.e., hormesis may occur).
Probit analysis would be the usual choice for multi-concentration binomial data; however, non-linear regression techniques (specifically, the parameter estimate procedure used) provide several advantages over probit analysis.Footnote 55 These include:
- The ability to directly estimate the control response (Abbott's correctionFootnote 56 is not needed)
- A wider variety of model choice, including the potential to model hormesis,Footnote 57 if it exists
- Avoiding the unjustified rejection of analysis based on the chi-square heterogeneity testFootnote 58
Non-linear regression is usually applied to continuous data, however, weighting techniques can be used to accommodate the binomial nature of the data and correct variance heterogeneity.Footnote 59 Arcsin square root transformation, which has historically been used to transform binomial data for analysis, is not recommended.Footnote 60
Given the rationale above, in a multi-concentration test, the required statistical endpoint for percent fertilization is an ICp and its 95% confidence limits via non-linear regression analysis.
An initial plot of the raw data (percent fertilization) against the logarithm of concentration is highly recommended, for a visual representation of the data, to check for reasonable results by comparison with later statistical computations, and to assess for outliers. Any major disparity between the approximate graphic ICp and the subsequent computer-derived ICp must be resolved. The graph would also show whether a logical relationship was obtained between log concentration (or, in certain instances, concentration) and effect, a desirable feature of a valid test (EC, 2005).
Regression analysis is the principal statistical technique and must be used for the calculation of the ICp, provided that the assumptions below are met (Figure 4). A number of models are available to assess fertilization data via regression analysis. Use of regression techniques requires that the data meet assumptions of normality and homoscedasticity. For this test, binomial weighting techniques must be applied to all data. The data are also assessed for outliers using one of the recommended techniques (see Section 10.2 in EC, 2005). An attempt must be made to fit more than one model to the data. Finally, the model with the best fitFootnote 61 must be chosen as the one that is most appropriate for generation of the ICp and associated 95% confidence limits. The lowest residual mean square error (or alternate measure of fit, such as AIC or BICFootnote 62) is recommended to determine best fit. Endpoints generated by regression analysis must be bracketed by test concentrations; extrapolation of endpoints beyond the highest test concentration is not an acceptable practice.
Figure 4 Flowchart summarizing steps in statistical analysis of a multi-concentration test to derive an ICp
Long description for Figure 4
Statistical analysis of a multi-concentration test to derive an ICp begins with plotting of the data (e.g. scatterplot) to aid in the selection of potential models. If outliers are present the analysis should be completed both with and without the outliers to determine if they should be included or removed from the final analysis. If no outliers are present in the data the next step in the analysis is to apply binomial weighting and fit the selected potential models to the data set. At this point, the data should be assessed to ensure that any assumptions of normality or homoscedasticity made by the models are met. If the chosen model assumes a normal distribution in the data and such a distribution is determined to be present (through the use of the Shapiro-Wilks test and normal probability plots) then the model of best fit can be chosen (e.g. the model with the lowest residual mean square). If the data is not normally distributed then alternate models or a linear interpolation analysis should be performed. If the chosen model assumes homoscedasticity in the data and this is determined to be the case (through the use of Levene’s test and examination of residual plots) then the model of best fit can be chosen. If the data is not homoscedastic then alternate models or a linear interpolation analysis should be performed.
With some highly toxic test materials or substances, it is possible to record zero or near-zeroFootnote 63 percent fertilization at one or more exposure concentration(s). In these cases, the results from the high test concentration(s) provide no further information on the response of the organism, and the repetitive zeroes may interfere with regression assumptions of normality and homoscedasticity.Footnote 64 The lowest test concentration inducing zero or near-zero percent fertilization is kept in the data set, but data from any subsequent high test concentration(s) must be removed before the regression analyses.
The ability to mathematically describe hormesis (i.e., a stimulatory or “better than the control” response occurring only at low exposure concentrations) in the dose-response curve has been incorporated into recent regression models (see Section 10.3 in EC, 2005). Data exhibiting hormesis can be entered directly, as the model can accommodate and incorporate all data points; there is no trimming of data points which show a hormetic response.
In the event that the data do not lend themselves to regression analysis (i.e., assumptions of normality and homoscedasticity cannot be met), linear interpolation (e.g., ICPIN; see Section 6.4.3 in EC, 2005) can be used to derive an ICp. In this case, the log values of the concentration data would be used, but binomial weighting is not applied. If the data exhibited hormesis and ICPIN is used, control responses must be entered for those concentrations which demonstrated hormesis (Option 4, Section 10.3.3 in EC, 2005).
For each test concentration, including the control treatment(s), the mean (± SD) percent fertilization as determined at the end of the test, must be reported.
4.5.3 Single Concentration Tests
The most commonly-used test design with echinoid fertilization is a multi-concentration test design. If investigators wish to consider a single-concentration test (e.g., to evaluate sediments from different sample locations), the echinoid contact test method (in preparation, “Biological Test Method : Reference Method for Determining Sublethal Toxicity of Embryo/Larval Echinoids (Sea Urchins or Sand Dollars) in Contact with Sediment”) may be more appropriate, as sediment is incorporated into this test design.
In a single-concentration test, the response in one or more full-strength test solutions (e.g., from multiple sites) are compared with the control response. Echinoid fertilization data presents a unique case in toxicity data analysis, since although the data is, by nature, binomial (an egg is either fertilized or not fertilized), because the number of replicates is 100, the data often meet the assumption of normality.Footnote 65 Accordingly, the recommendations made here emphasize techniques for quantitative (continuous) data.
If percent fertilization is assessed for a single test solution and the control, a t-testFootnote 66 is normally the appropriate statistical test. In situations where more than one test site is under study, and the investigator wishes to compare multiple treatments with the control, or compare treatments with each other, a variety of multiple comparison tests exist (Section 3.3. in EC, 2005). Choice of the test to use depends on:
- the type of comparison that is sought (e.g. complete a series of pairwise comparisons between all sites or compare the data for each location with that for the control only);
- if a chemical and/or biological response gradient is expected, and
- if the assumptions of normality and homoscedasticity are met.
Correction of fertilization using Abbott’s formula will, in most cases, not be necessary.Footnote 67
For each test solution or treatment, including the control treatment(s), the mean (± SD) percent fertilization as determined at the end of the test, must be reported.
4.6 Reference Toxicant
The routine use of a reference toxicant or toxicants is required to assess the relative sensitivity of the batches of gametes that are used, under standardized test conditions, and the precision and reliability of data produced by the laboratory for the selected reference toxicant(s) (EC, 1990d).
For adults that are gradually acclimated to test conditions and held in the laboratory for an extended period of time (i.e., >3 days), sensitivity of gametes to the reference toxicant(s) must be determined by performing a reference toxicity test within 14 days before or after the date that the definitive toxicity test is performed, or by performing this test concurrently with the definitive one. When a reference toxicity test is performed at the same time as the definitive toxicity test, the same batch of gametes must be used for each of these two tests.
If gametes are collected from adults on the day of arrival or within 3 days of arrival at the laboratory, a portion of the gametes collected for use in a definitive test must be tested for its tolerance to the reference toxicant(s). The reference toxicant test must be performed under the same experimental conditions as those used with the test sample(s). Testing of the reference toxicant must be performed concurrently with the actual toxicity test.
Criteria considered in recommending appropriate reference toxicants for this test include:
- chemical readily available in pure form;
- stable (long) shelf life of chemical;
- highly soluble in water;
- stable in aqueous solution;
- minimal hazard posed to user;
- easily analyzed with precision;
- good dose-response curve for echinoid gametes;
- known influence of pH on toxicity of the chemical, in this test; and
- known influence of salinity on toxicity of the chemical, in this test.
Copper is recommended for use as the reference toxicant for this test.Footnote 68 Gamete sensitivity must be evaluated by tests following the standard methods and conditions given in this document, to determine the ICp for copper. As for all multi-concentration tests described herein, regression analysis must be used for the calculation of the reference toxicant ICp and its 95% confidence limits, if possible, following the guidance provided in Section 4.5.2 and in EC, 2005. Copper sulphate or copper chloride should be used for preparing stock solutions, which should be acidic (pH 3 to 4), and may be used when prepared, or stored in the dark at 4 ± 2 °C for several weeks before use. Concentration of copper should be expressed as mg Cu++/L.
Natural or reconstituted seawater is to be used for controls and dilution. To provide a high degree of standardization for this reference toxicity test, the salinity of the control/dilution water should be adjusted to a consistent value that is favourable to the gametes, in the range 28 to 32 g/kg, preferably 30 g/kg.
Concentrations of reference toxicant in all stock solutions should be measured chemically by appropriate methods (e.g., APHA et al., 1989, 2005). Upon preparation of the test solutions, aliquots should be taken from at least the control, low, middle, and high concentrations, and analyzed directly or stored for future analysis, in case the ICp is outside warning limits. If stored, sample aliquots must be held in the dark at 4 ± 2 °C. Copper solutions should be preserved before storage (APHA et al., 1989, 2005). Stored aliquots requiring chemical measurement should be analyzed promptly upon completion of the toxicity test. Calculations of ICp should be based on measured concentrations if they are appreciably (i.e., ≥ 20%) different from nominal ones and if the accuracy of the chemical analyses is satisfactory.
Once sufficient data are available (EC, 1990d), a warning chart must be prepared and updated for each reference toxicant used. Successive ICps are plotted on this chart and examined to determine whether the results are within ± 2 SD of values obtained in previous tests. The geometric mean ICp together with its upper and lower warning limits (± 2 SD calculated on a logarithmic basis)Footnote 69 are recalculated with each successive ICp until the statistics stabilize (USEPA, 1989, 2002; EC, 1990d).
If a particular ICp falls outside the warning limits, the sensitivity of the gametes and the performance and precision of the test are suspect. Since this might occur 5% of the time due to chance alone, an outlying value does not necessarily mean that the sensitivity of the batch of gametes or the precision of the toxicity data produced by the laboratory are in question. Rather, it provides a warning that this might be the case. A thorough check of all holding and test conditions is required at this time.
One check that might be made in such circumstances is the fertilization success for various sperm:egg ratios, compared with the range of values previously obtained. That assessment should provide a useful indication of decreasing viability of gametes, as might occur, perhaps, at the end of a spawning season. Depending on the findings, it might be necessary to repeat the reference toxicity test with new gametes, and/or a new batch of adults, before undertaking further toxicity tests.
Test results that usually fall within warning limits do not necessarily indicate that a laboratory is generating consistent results. A laboratory that produced extremely variable data for a reference toxicant would have wide warning limits; a new datum-point could be within the warning limits but still represent undesirable variation in results obtained in the test. For guidance on reasonable variation among reference toxicant data (i.e., warning limits for a warning chart), please refer to Section 2.8.1 and Appendix F in Environment Canada, 2005.
If an ICp fell outside the control limits (mean ± 3 SD), it would be highly probable that the test was unacceptable and should be repeated, with all aspects of the test being carefully scrutinized. If endpoints fell between the control and warning limits more than 5% of the time, a deterioration in precision would be indicated, and again the most recent test should be repeated with careful scrutiny of procedures, conditions, and calculations.
4.7 Legal Considerations
Care must be taken to ensure that samples collected and tested with a view to prosecution will be admissible in court. For this purpose, legal samples must be: representative of the material or substance being sampled; uncontaminated by foreign substances or materials; identifiable as to date, time, and location of origin; clearly documented as to the chain of custody; and analyzed as soon as possible after collection. Persons responsible for conducting the test and reporting the findings must maintain continuity of evidence for court proceedings (McCaffrey, 1979), and ensure the integrity of the test results.
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