Biological test method: fertilization assay using echinoids (sea urchins and sand dollars), chapter 5

Section 3: Test System

• 3.1 Facilities and Apparatus
• 3.2 Lighting
• 3.3 Test Vessels
• 3.4 Control/Dilution Water

3.1 Facilities and Apparatus

Tests are to be conducted in a facility isolated from general laboratory disturbances. If a separate room is unavailable, the test area should be subject to minimal dust and fumes.

Construction materials and any equipment that might contact the test solutions or control/dilution water should not contain any substances that can be leached into the solutions or increase sorption of test substance or material (see Section 2.3.2). The laboratory must have the instruments to measure the basic variables of water quality (temperature, salinity/conductivity, dissolved oxygen, pH), and must be prepared to undertake prompt and accurate analysis of other variables such as ammonia.

All test solutions should be maintained within ± 1 °C of the desired temperature. This can be achieved using various types of equipment such as a temperature-controlled water bath in which test vessels are immersed.

3.2 Lighting

Normal laboratory lighting is satisfactory for the test.

3.3 Test Vessels

The three options for initial volume of test solution are 10.0 mL, 5.0 mL, and 2.0 mL; vessels must be suitable for containing the selected volume. These volumes span the usual range used in other written methods (Appendix D). The 10-mL volume is normally standard, and the smaller volumes are used for special purposes (see introduction to Section 4).

Borosilicate glass vials or tubes are to be used as test vessels. A capacity of about 20 mL is recommended for use with 10 mL of test solution, as is common practice (Appendix D, item 8). For smaller volumes of test solution, size of the vessels should be scaled down, to about double the volume of solution or somewhat more, e.g., vessels of 5 mL capacity for 2 mL of test fluid. However, larger vials may be used if desired, and vials up to 13 mL are sometimes used for 2 mL of test solution (Appendix D). A standard size should be selected and used within a laboratory.^Footnote 12

The test vessels should have caps or some other seal, to avoid potential contamination from the air and loss of volatile components. The seal could be a sheet of plastic film which covers all the vessels in a test. The vessels should normally be of the disposable type, new and unwashed before use. An option is to reuse tubes after thorough washing and rinsing, but that technique has been known to result in measurable toxicity,^Footnote 13 and is not recommended.

Considerable latitude is allowed in the design and shape of test vessels. For a given test, however, every treatment must use containers of identical type, size, and shape. Plastic vessels are not to be used since there is evidence of deleterious effects on fertilization success (Dinnel et al., 1987). In descriptions of existing procedures (Appendix D) the vessels are mostly disposable tubes of one kind or another, with caps, and made of borosilicate glass (such as Pyrex^TM). They are variously described as scintillation vials, culture tubes, test tubes, or simply as tubes or vials, and vessels of those designs would seem satisfactory, if of the appropriate size.

3.4 Control/Dilution Water

Depending on the test material or substance and intent (Sections 5 to 8), the control/dilution water may be uncontaminated natural seawater, reconstituted (artificial) seawater, or a sample of receiving water collected beyond the influence of the zone of contamination. Artificial seawater can be made up to the test salinity by adding the appropriate amount of commercially-available dry ocean salts (e.g., Instant Ocean^TM, Red Sea Salt^TM) or reagent-grade salts (e.g., modified GP2; see Bidwell and Spotte, 1985 or Table 2 in USEPA, 1994 or USEPA, 1995) to deionized water, or by adding appropriate quantities of natural or artificial hypersaline brine to deionized water (following guidance in EC, 2001 and Section 2.3.4). A supply of uncontaminated natural seawater with a lower (i.e., <28 g/kg) or higher (i.e., >32 g/kg) salinity can be mixed with the appropriate amount of dry ocean salts, reagent-grade salts, natural or artificial HSB, or deionized water sufficient to adjust its salinity to within the test range. During prolonged storage (>1 day), natural or artificial seawater prepared for use as dilution water should be refrigerated (4 ± 2°C) to minimize microbial growth (EC, 2001). If receiving water is used, conditions for collection, transport, and storage should be as described in Section 6.1. Control/dilution water used in any given test must be from the same source (if natural seawater) or the same batch (if reconstituted water), and artificial seawater should not be used after 14 days following its preparation (EC, 2001). All marine waters used as a source of control/dilution water, including the laboratory supply of natural seawater, should be passed through a filter with a pore size of approximately 60 µm (USEPA, 1994).

Salinity of control/dilution water should be 30 g/kg and must be in the range 28 to 32 g/kg. Lower salinities should be adjusted upwards using aged hypersaline brine with a salinity of 90 ± 1 g/kg (see Section 2.3.4), and higher ones should be adjusted downwards with deionized water, distilled water, or uncontaminated fresh water.^Footnote 14

The pH of control/dilution water must be in the range 7.5 to 8.5, and should normally be 8.0 ± 0.2. Those values would usually be obtained because of the natural buffering capacity of seawater. If not, adjustment should be made with acid or base (Section 4.3.4).

Control/dilution water must be adjusted to the test temperature before use. It should not be supersaturated with excess gases (see Section 2.3.4), and must contain dissolved oxygen at 90 to 100% of the air-saturation value before use. If necessary, achieve that level by aerating vigorously with oil-free compressed air passed through air stones.