Biological test method for determining toxicity of sediment using luminescent bacteria: chapter 5

Section 3: Facilities, Equipment, and Supplies

• 3.1 Facilities
• 3.2 Apparatus
  • 3.2.1 Cleaning Procedure
  • 3.2.2 Outline of Test and Associated Apparatus
  • 3.2.3 Equipment
  • 3.2.4 Supplies

3.1 Facilities

The test can be conducted in a normal, clean laboratory with standard lighting. The need for any special facilities would be governed by the degree of hazard associated with the samples that were to be tested, and by the risk of sample and apparatus contamination. Facilities must be well ventilated, free of fumes, and isolated from physical disturbances or airborne contaminants that might affect the test organisms. The testing facilities should also be isolated from areas where test sediments are prepared, and removed from areas where equipment is cleaned.

3.2 Apparatus

3.2.1 Cleaning Procedure

All equipment and supplies that might contact test sediment or water must be clean and dry. All nondisposable materials should be washed after use. The following cleaning procedure (EC, 1997b, c) is recommended.

1. Soak in tap water for 15 minutes, then scrub with detergent or clean in an automatic dishwasher.
2. Rinse twice with tap water.
3. Rinse carefully with fresh, dilute (10%, v:v^Footnote 3) nitric (HNO₃) or hydrochloric acid (HCl) to remove scale, metals, and bases.
4. Rinse twice with deionized water.
5. Rinse once with full-strength, pesticide-grade acetone to remove organic compounds (use a fume hood or canopy). Use hexane for oily residues.
6. Rinse three times with high-quality deionized water.

3.2.2 Outline of Test and Associated Apparatus

This solid-phase test for measuring the toxicity of samples of whole sediment involves the following steps:

• preparation of a series of concentrations of the sample by serial dilutions in water;
• mixing this series with an inoculum of test organisms (reconstituted V. fischeri) and incubation for 20 minutes in test tubes held in a water bath at 15 ± 0.5°C;
• immediately thereafter, filtration of the contents of each test tube;
• the subsequent stabilization of the filtrate at 15 ± 0.5°C for 10 minutes in a series of cuvettes held within wells of a photometer; followed by
• the photometric reading of light produced by the luminescent bacteria remaining in the filtrate.

3.2.3 Equipment

The equipment and supplies required to achieve these steps are described briefly here. The supplier of specialty items should be consulted for further details. Equipment and supplies which contact sediments or water must not contain substances that can be leached or dissolved in amounts that adversely affect the test organisms, and should be chosen carefully to minimize sorption of materials from water.

Equipment for performing the solid-phase test for sediment toxicity includes:

• a Microtox^TM Model 500 Analyzer or equivalent temperature-controlled photometer (15 ± 0.5°C for ≥ 15 cuvettes with test solutions; 5.5 ± 1°C for single cuvette holding reconstituted bacteria in Reagent well) capable of reading light output at a wavelength of 490 ± 100 nm (ASTM, 1995)
• refrigerated water bath with temperature controlled at 15 ± 0.5°C
• test tube rack or incubator block for incubating test tubes containing concentrations of test material and V. fischeri in the water bath
• freezer (not self-defrosting or “frost free” type) for storing lyophilized bacteria (Bacterial Reagent)
• pipettors for delivering volumes of 20, 500, 1000, and 1500 µL, with disposable plastic tips^Footnote 4
• disposable polystyrene SPT tubes (15.5 × 56 mm, 7.5-mL capacity, hemispherical bottom) or equivalent test tubes
• disposable glass cuvettes (borosilicate, 3-mL capacity, 50 mm length × 12 mm diameter, flat bottom)
• disposable filter columns for SPT or equivalent test tubes^Footnote 5
• volumetric borosilicate glassware (acid washed as per Section 3.2.1) for processing small aliquots of samples
• countdown timer or stopwatch
• magnetic plate mixer with Teflon^TM stir bar
• a balance, accurate to 0.01 g
• a drying oven (100 ± 5°C)
• weighing vessels for dry weight determination
• metal spoon or spatula for sample homogenization

3.2.4 Supplies

The supply of test organisms is purchased as a standardized culture of freeze-dried bacteria (“Bacterial Reagent”, see Section 2). Storage should be in a freezer at -20°C (EC 1992; ASTM 1995).

Non-toxic distilled or deionized water is used to activate a vial of Bacterial Reagent (see Section 4.6). This water is frequently referred to as Reconstitution Solution, and can either be purchased as such (see Table 11; Appendix E), or taken from a laboratory supply and used after testing to confirm that it does not decrease light production by V. fischeri. This testing is accomplished by using a portion of the water to be used as Reconstitution Solution as the control water and for preparing test concentrations of the reference toxicant(s) used routinely at the laboratory for evaluating test performance with V. fischeri. Accordingly, the light production of V. fischeri achieved using the intended Reconstitution Solution should be evaluated in a water-only test with one or more reference toxicants, according to the procedures and conditions given in Environment Canada (1990) as well as those provided in Section 4 “Universal Procedures” of Environment Canada’s biological test method for performing liquid-phase toxicity tests using V. fischeri (EC, 1992).

A supply of Solid-Phase Diluent (see Table 5; Appendix E), comprised of 3.5% sodium chloride (NaCl), is required for diluting each sample of test sediment (see Section 4.6). This Diluent can either be purchased or prepared by dissolving 35.0 g NaCl (reagent grade) in 1000 mL of the Reconstitution Solution (i.e., non-toxic distilled or deionized water).