Biological test method for determining toxicity of sediment using luminescent bacteria: chapter 6

Section 4: Procedure for Testing Sediment

• 4.1 Sample Collection
• 4.2 Sample Labelling, Transport, and Storage
• 4.3 Sample Manipulation and Characterization
• 4.4 Negative and Positive Control Sediment
• 4.5 Test Conditions
  • 4.5.1 Outline of Test
  • 4.5.2 Manipulations, Adjustments, and Corrections
  • 4.5.3 Temperature
  • 4.5.4 Timing of Events
  • 4.5.5 Test Array
• 4.6 Test Procedures
  • 4.6.1 Photometer, Water Bath, and Bench Sheet
  • 4.6.2 Subsamples for Moisture
  • 4.6.3 Primary Dilution
  • 4.6.4 Test Concentrations
  • 4.6.5 Reconstitution of Bacterial Reagent
  • 4.6.6 Inoculation and Incubation
  • 4.6.7 Preparing the Computer
  • 4.6.8 Filtration
• 4.7 Test Measurements and Observations
• 4.8 Criterion for a Valid Test

4.1 Sample Collection

Environment Canada (1994) provides guidance on field sampling designs and appropriate techniques for sample collection; this guidance document should be followed when collecting samples of sediment to be tested for toxicity using this reference method.

Procedures and equipment used for sample collection (i.e., core, grab, dredge, or composite) will depend on the study objectives or regulatory requirements, and on the nature of the material being sampled. Samples of dredged sediment should be taken at all depths of interest.

Each series of toxicity tests performed using this reference method should include one or more samples of reference sediment. Sites for collecting samples of reference sediment should be sought where the geochemical properties of the sediment, including grain size characteristics, are similar to those at the site(s) where samples of test (contaminated or potentially contaminated) sediment are collected. Ideally, reference sediment should be collected from a site uninfluenced by the source(s) of contamination but within the general vicinity of the site(s) where samples of test sediment are taken. It is recommended that reference sediment from more than one site be collected to increase the likelihood of a good match with grain size and other physicochemical characteristics of the test sediments.

Any toxicity test involving one or more samples of coarse-grained test sediment with less than 20% fines must include a sample of clean reference sediment or negative control sediment as part of the testing regime, for comparative purposes in judging sample toxicity (see Section 6.2). This reference or negative control sediment must have a percent fines content that does not differ by more than 30% from that of the sample(s) of test sediment against which it is compared (Section 6.2). Field-collected samples of clean reference sediment or negative control sediment may be used for this purpose if their percent fines meets this matching requirement, in which instance the guidance here for sample collection applies. Alternatively, the investigator may choose to formulate one or more samples of artificial negative control sediment for this purpose, to enable the close matching of its/their percent fines with that of the sample(s) of coarse-grained test sediment. Guidance on preparing artificial negative control sediment is provided in Sections 4.3 and 4.4.

The number of stations to be sampled at a study site and the number of replicate samples per station will be specific to each study. This will involve, in most cases, a compromise between logistical and practical constraints (e.g., time and cost), and statistical considerations. Environment Canada (1994) should be consulted for guidance with respect to the sampling design, including the recommended minimum number of field replicates. Additional guidance on sampling for disposal-at-sea applications is found in Environment Canada (1995a; 2002a). Applicants are encouraged to consult with their regional Environment Canada Ocean Disposal Office (see Appendix C for contact information), before sampling and testing.

Where practical and consistent with the study design and objectives, a minimum of five samples of sediment should be taken from each discrete sampling station and depth of interest. Where practical and appropriate (see Section 6), sample collection should also include ≥5 samples from each of one or more reference stations (i.e., sites where uncontaminated sediment, having physicochemical properties similar to that of the test sediments, can be found) within the vicinity. The objective of collecting replicate samples at each station (field replicates) is to allow for quantitative statistical comparisons within and among different stations (EC, 1994; 1998; 2002b). Accordingly, each of these “true replicate” samples of sediment should be tested for its acute toxicity to V. fischeri. Laboratory replicates, using subsamples of each field-collected sample of test and reference sediment after mixing and other manipulation (see Section 4.3), might also be included in a study in instances where sample homogeneity or precision of test results are in question.

A benthic grab (i.e., Smith-MacIntyre, Van Veen, PONAR) or core sampler should be used to sample sediment rather than a dredge, to minimize disruption of the sample. Care must be taken during sampling to minimize loss of fines. The same collection procedure should be used for all field sites sampled.

The volume of sample required to perform a multi-concentration test for sediment toxicity using V. fischeri is small (see Section 4.6). A sample volume of ~100 mL should be submitted specifically for the performance of this test.^Footnote 6 A per-sample volume of at least 5 to 7 L of whole sediment is frequently required (EC, 1994), although this will depend on the study objectives/design and on the nature of the associated physicochemical analyses and the battery of toxicity tests to be performed. To obtain the required sample volume for a battery of toxicity tests, it is frequently necessary to combine subsamples retrieved using the sampling device. Guidance provided in Environment Canada (1994) for compositing subsamples in the field should be followed.

4.2 Sample Labelling, Transport, and Storage

In addition to the following, more detailed and useful guidance pertaining to sample labelling, transport, and storage is found in Environment Canada (1994). Persons undertaking these procedures should be familiar with this guidance document.

Containers for transporting and storing samples must be new or thoroughly cleaned, and rinsed with clean water. Environment Canada (1994) should be consulted for guidance in selecting suitable containers. Each sample container should be filled completely, to exclude air. Immediately after filling, each sample container must be sealed, and labelled or coded. Labelling and accompanying records made at this time must include at least a code which can be used to identify the sample or subsample. A cross-referenced record, which might or might not accompany the sample or subsample, must be made by the field personnel identifying the sample type (e.g., grab, core, composite), source, precise location (e.g., water body, latitude, longitude, depth), replicate number, and date of collection. This record should also include the name and signature of the sampler(s). Sediment sample collectors should also keep records describing:

• the nature, appearance, volume and/or weight of each sample;
• the sampling procedure and apparatus;
• any procedure used to composite or subsample grabs or cores in the field;
• the number of replicate samples taken at each sampling station;
• the sampling schedule;
• the types and numbers of containers used for transporting the samples;
• any field measurements (e.g., temperature, salinity, pH, dissolved oxygen) of the overlying water or sediment at the collection site; and
• procedures and conditions for cooling and transporting the samples.

Upon collection, warm (>7°C) samples should be cooled to between 1 and 7°C with regular ice or frozen gel packs, and kept cool (4 ± 3°C) in darkness throughout transport (EC, 1994; 1998). As necessary, gel packs, regular ice, coolers, or other means of refrigeration should be used to assure that sample temperatures range within 1 to 7°C during transit. Samples must not freeze or partially freeze during transport or storage, and must not be allowed to dry (EC, 1994).

Upon arrival at the laboratory, the sample temperature and date of receipt must be recorded on a bench sheet (see example, Appendix F). Samples to be stored for future use must be held in airtight containers and in darkness at 4 ± 2°C (EC, 1994; 1998). It is recommended that samples of sediment or similar particulate material be tested as soon as possible after collection. The sediment toxicity test should begin within two weeks of sampling, and preferably within one week; the test must start no later than six weeks after sample collection (EC, 1994; 1997b; 1997c; 1998).

4.3 Sample Manipulation and Characterization

Samples of field-collected test sediment and reference sediment must not be wet-sieved. Particles ≥2 mm should be removed along with large debris or large indigenous macro-organisms. Depending on the sample, this may be accomplished by using forceps or a gloved hand. Forceps or gloves contacting each sample should be rinsed or replaced thereafter, to prevent cross-contamination. If a sample contains a large number of particles ≥2 mm and/or a large number of indigenous macro-organisms which cannot be removed using forceps or a gloved hand, the sample may be press-sieved (not washed) through one or more suitably sized (e.g., ≥2 mm) mesh stainless steel screens. Such manipulation should include all portions of the sample used for physicochemical (including grain size) analyses as well as those used for solid-phase sediment toxicity tests with V. fischeri. Procedures used to manipulate each sample must be recorded on the bench sheet (see “Notes” in example, Appendix F).

Any pore water that has separated from the sample during shipment and storage must be mixed back into the sediment. To achieve a homogeneous sample, either mix it in its transfer/storage container, or transfer it to a clean mixing container. The sample should normally be stirred using a nontoxic device (e.g., stainless steel spoon or spatula), until its texture and colour are homogeneous. Alternatively, a mechanical method (EC, 1994; 1998) may be used to homogenize the sample. For each sample included in a test, mixing conditions including duration and temperature must be as similar as possible. If there is concern about the effectiveness of sample mixing, subsamples of the sediment should be taken after mixing, and analyzed separately to determine homogeneity.

Immediately following sample mixing, subsamples of test material required for this and other (e.g., EC, 1998) toxicity tests and for physicochemical analyses must be removed and placed in labelled test chambers, and in the labelled containers required for storage of samples for subsequent physicochemical analyses. Any remaining portions of the homogenized sample that might be required for additional toxicity tests using amphipods (EC, 1998) or other test organisms should also be transferred at this time to labelled containers. All subsamples to be stored should be held in sealed containers with no air space, and must be stored in darkness at 4 ± 2°C until used or analyzed. Just before it is analyzed or used in the toxicity test, each subsample must be thoroughly re-mixed to ensure that it is homogeneous.

Each sample (including all samples of reference sediment, negative control sediment, and positive control sediment) must be characterized by analyzing subsamples for at least the following (EC, 1998): for whole sediment--percent very coarse-grained sediment (i.e., particles >1.0 mm), percent sand (i.e., particles >0.063 to 2.0 mm), percent fines (i.e., particles ≤0.063 mm), percent water content, and total organic carbon content; for pore water--salinity and pH. Other analyses could include: total inorganic carbon, total volatile solids, biochemical oxygen demand, chemical oxygen demand, cation exchange capacity, acid volatile sulphides, metals, synthetic organic compounds, oil and grease, petroleum hydrocarbons, and porewater analyses for various physicochemical characteristics such as ammonia (total and un-ionized) or hydrogen sulphide. Recommended procedures for collecting pore water are described in Environment Canada (1994) and should be followed here. For disposal-at-sea applications, minimum information requirements are explained in two guideline documents by Environment Canada (i.e., EC, 1995a; 2002a).

Analyses for particle size distribution must be undertaken as soon as possible after sample collection, to enable the selection of the appropriate sample(s) of reference sediment and, if used, negative control sediment (see Sections 4.4 and 6.2).

Previous studies have found that V. fischeri is quite tolerant of elevated concentrations of ammonia in water (Qureshi et al., 1982) or pore water of sediment samples collected from the marine or estuarine environment (McLeay et al., 2001). These limited data indicate that elevated levels of ammonia in sediment pore water are not a major confounding factor influencing test results. However, investigators might wish to investigate the extent to which porewater ammonia might contribute to sample toxicity as determined using this reference method. In this instance, analyses for porewater pH, salinity, and ammonia should be undertaken within 24 h of the solid-phase test for sediment toxicity using V. fischeri, to enable determinations of the concentrations of total and un-ionized ammonia to which test organisms were exposed and the possible influence of this on test results (Section 6.2). Ammonia analyses must be conducted using a recognized and standardized procedure (for example, APHA et al., 1995; Standard Methods). Calculations of concentrations of un-ionized ammonia must be based on the test temperature and on the porewater pH and salinity of the sample (Trussell, 1972; Bower and Bidwell, 1978).

4.4 Negative and Positive Control Sediment

Toxicity tests restricted to one or more samples of fine-grained sediment (i.e., samples with ≥20% fines) need not include a sample of negative control sediment or clean reference sediment, since the toxicity of such samples is not judged by comparison of test results with those for a sample of uncontaminated sediment with similar grain-size characteristics. However, any toxicity test involving one or more samples of test sediment with <20% fines must include a sample of clean sediment with a percent fines content that does not differ by more than 30% from that of the sample(s) of test sediment against which it is compared when judging toxicity (see Section 6.2). Field-collected clean sediment from an uncontaminated site may be used for this purpose. However, the use of laboratory-formulated (“artificial”) negative control sediment is recommended, since it can be prepared to closely match the percent fines of the test sediment(s). Artificial negative control sediment should be prepared in the laboratory using an appropriate mixture of commercially available kaolin clay and/or washed silica sand with grain sizes matching those of the test sediment(s). These ingredients should be mixed thoroughly in proportions similar to those of the test sediment(s). Preparation procedures and results for V. fischeri solid-phase toxicity tests using artificial negative control sediment are reported in Ringwood et al. (1997), Tay et al. (1998), and McLeay et al. (2001).

The percentage of fines (particles ≤0.063 mm) for the sample(s) of negative control sediment included in a particular toxicity test should be matched as closely as possible to that of the test sediment(s). If a series of test sediments with a wide range of percent fines is being measured consecutively for toxicity as part of a study, more than one negative control sediment, with percent fines matched as closely as possible to the range of percent fines for the test sediments, might be included in the study.

The use of one or more samples of positive control sediment is recommended for inclusion in each series of toxicity tests, to assist in interpreting test results (Section 6.2). The positive control sediment might be a standard contaminated sediment such as one available through the National Research Council of Canada’s Marine Analytical Chemistry Standards Program, Ottawa, ON (e.g., HS-3, Cook and Wells, 1996; HS-6, Tay et al., 1998). A second approach is to use a sample of clean sediment (e.g., artificial negative control sediment or field-collected clean reference sediment) that has been spiked experimentally with a toxic chemical (EC, 1995b). A third option is to use a highly contaminated sample of field-collected sediment shown previously to be toxic to V. fischeri in solid-phase testing; this option is not recommended unless the characteristics (including performance in a solid-phase test using V. fischeri) of this sediment are well known beforehand. Positive control sediment must be used as a reference toxicant when appraising the sensitivity of the test organisms and the precision and reliability of results obtained by the laboratory for that reference material (Section 5).

4.5 Test Conditions

4.5.1 Outline of Test

This solid-phase test for measuring the toxicity of samples of whole sediment involves the steps and associated apparatus outlined in Section 3.2.2.

Table 1 provides a checklist of the conditions that are required or recommended for this reference method. Further details are given in Sections 4.5.2 to 4.5.5.

4.5.2 Manipulations, Adjustments, and Corrections

• Test sediments, including the sample(s) of reference sediment recommended for inclusion in each test series, must not be wet-sieved and no adjustments of porewater salinity are permitted.^Footnote 7
• Sample pH must not be adjusted.
• No aeration of samples, test concentrations, or filtrates should be performed.
• Light-emission readings for concentrations of test sediments must not be adjusted or corrected using readings for concentrations of reference or other sediments.^Footnote 8
• The statistical endpoint for the test (i.e., IC50; see Section 6.1) must be normalized for the moisture content of the sample.

4.5.3 Temperature

• The test is conducted using the luminescent bacterial species, Vibrio fischeri, strain NRRL B-11177, which is reconstituted to an active state in pure non-toxic water and held at 5.5 ± 1.0°C until aliquots are transferred to each test concentration. Normally this temperature is met by placing the cuvette with the reconstituted bacterial solution in the specified well of the photometer if a Microtox^TM Model 500 Analyzer is used. Otherwise a temperature-controlled incubator must be used for this purpose.
• All concentrations of each sample of test sediment (including reference sediment) inoculated with bacteria must be incubated for 20 minutes at 15 ± 0.5°C. A temperature-controlled water bath or room would serve this purpose.
• Following incubation and filtration, all test solutions transferred to cuvettes must be held at 15 ± 0.5°C during the subsequent 10-minute period for stabilization of the filtrates. This temperature control is normally achieved within the wells of the photometer. Alternatively, the cuvettes containing test filtrates can be held within this temperature range in a cuvette holder placed in a temperature-controlled incubator or room.

4.5.4 Timing of Events

• The lyophilized bacteria should be reconstituted immediately before inoculating the test concentrations. This bacterial solution should be used within 2 h, and must be used within 3 h of reconstitution. The time of reconstitution should be logged on a bench sheet (see example, Appendix F).
• Test concentrations must be allowed to equilibrate to 15 ± 0.5°C for a minimum of 10 minutes before inoculation with bacterial solution. Inoculation should proceed as quickly as possible; all test concentrations should be inoculated within a total time span of ≤4 minutes. Record the time of the first inoculation as the start of the test, on the bench sheet (Appendix F).
• All test concentrations must be incubated for 20 minutes. Once the test concentrations are filtered and transferred to cuvettes, the filtrates must be incubated in cuvettes for 10 minutes before their light output is measured.^Footnote 9
• Total lapsed time for the transfer of filtrates to cuvettes and for reading the luminescence of the test filtrates should be similar to that (≤4 min) spent inoculating the test concentrations with bacteria.

4.5.5 Test Array

• The test array consists of three controls (comprised of dilution water only) and twelve test concentrations.
• The maximum test concentration is normally 197 000 mg/L (19.7%, wet wt:v), with each successive concentration normally being 50% of the previous one.

4.6 Test Procedures

This biological test method involves the simultaneous incubation of a minimum of three control solutions (comprised of an inoculum of reconstituted V. fischeri in Diluent) together with 12 differing concentrations of test material in Diluent.^Footnote 10 After a prescribed incubation period, the incubated solutions (held in test tubes at a controlled temperature) and test concentrations are filtered, and the resulting filtrates are transferred to cuvettes. After a brief period for stabilization of holding conditions for the filtrates, the light production by the test organisms remaining in the filtrate is measured by a photometer.

In this section, the procedures applied to a photometer assume the use of a Microtox^TM Model 500 Analyzer or another photometer with similar features. Since the Microtox^TM Model 500 Analyzer has 30 wells for holding cuvettes containing filtrates of test concentrations, the laboratory technician using this photometer has the option of duplicating a test, or performing two tests simultaneously on different samples. The following procedural description follows a single sample of sediment through the various steps. Depending on the study design and the nature and source of the test sediments, one or more samples of field-collected reference sediment should also be included in each study (see Sections 4.1 and 6.1). Toxicity tests involving one or more samples of coarse-grained (i.e.,<20% fines) sediment must include a negative control sediment (artificial or natural) or a reference sediment, with a percent fines content that does not differ by more than 30% from that of the test sediment(s) (see Section 6.2). The use of a positive control sediment (Section 4.4) as part of each series of toxicity tests is also recommended. In order to include one or more samples of test sediment along with reference sediment, negative control sediment, and/or positive control sediment in a single study, it is necessary to run each sample consecutively.

4.6.1 Photometer, Water Bath, and Bench Sheet

• Switch on the computer, photometer, and balance.
• For the Model 500 Analyzer, ensure that the temperature selector switch at the back is set to “Microtox Acute”.
• Place 15 cuvettes in the first three rows (A-C) of wells. These will be maintained at 15 ± °C. The incubated wells are arrayed in a grid of rows labelled A to C and columns numbered 1 to 5. They are referenced as A1 to C5.
• Place one cuvette in the reagent well, and pipette 1.0 mL of Reconstitution Solution into it. This will be maintained at 5.5 ± °C.
• Switch on the water bath incubator. Allow the water temperature to stabilize at 15 ± 0.5°C.
• Stir the sample to homogenize it, using a stainless steel spoon.
• Fill out a bench sheet (see Section 7.2.7 and Appendix F). Place 15 SPT tubes (see Appendix E, Table 5) into a rack.

4.6.2 Subsamples for Moisture

• For each test sediment, label and weigh three empty vials (e.g., 20-mL glass scintillation vials or other suitable containers for drying and weighing), and record the weights to the nearest 0.01 g. Enter the data, if a spreadsheet is used when converting the IC50 to a value based on dry weight (Section 6.1).
• Add 5.0 ± 0.2 g of sediment to the vials and record the weights to the nearest 0.01 g (or enter them into the spreadsheet).
• Dry the subsamples by putting the vials into an oven at 100 ± 5°C for 24 h. Record the oven temperature.
• Record the dry weights to the nearest 0.01 g.

4.6.3 Primary Dilution

• Weigh 7.00 ± 0.05 g of homogenized sample into a glass or disposable 50-mL plastic beaker.
• Add a 2.5-cm Teflon-coated magnetic stir bar and 35.0 mL of Solid-Phase Diluent to the beaker.
• Stir for 10 min on a magnetic stirrer at a rate sufficient to create a vortex that reaches 1/2 the height of the liquid level.

4.6.4 Test Concentrations

• Dispense 1.50 mL Solid-Phase Diluent (Section 3) into the first 14 tubes in the rack (Section 4.6.1). Tube 15 will be the highest concentration, taken from the primary dilution.
• Following the 10-minute stirring of the primary dilution, use a large-bore macropipette tip to transfer 1.50 mL of sample suspension from the 50-mL beaker, while it is still stirring, to each of SPT tubes 15 and 14. To do this, insert the pipette tip near the side of the beaker, at about half the depth of the stirring sample. Avoid plugging the tip while aspirating the sample.
• Beginning at SPT tube 14, which now has 3.00 mL of solution, make 1:2 serial dilutions as follows. Mix the contents three times with the macropipette, and then quickly draw up a volume of 1.50 mL from about one third depth (to help draw some of the heavier sand grains). Transfer this aliquot to tube 13. Repeat this mixing and transferring from tube 13 to tube 12, and continue consecutively thereafter from tube 12 to tube 11, tube 11 to tube 10, etc. until 1.5 mL of the 1:2 serial dilutions is transferred into tube 4. Finally, discard 1.5 mL from tube 4. Tubes 1-3 contain Diluent only, and serve as the controls.
• Place the rack with SPT tubes containing all of the test concentrations (including controls) into the water bath at 15 ± 0.5°C. Leave it for 10 min to allow the temperature to equilibrate. The water level of the bath should be just above the liquid level in the SPT tubes.

4.6.5 Reconstitution of Bacterial Reagent

• Take a vial of freeze-dried bacteria (Microtox Acute Reagent) from storage.
• Open the vial and reconstitute its contents by quickly pouring the Reconstitution Solution from the cuvette in the reagent well of the Model 500 Analyzer (or other photometer) into the vial, swirling three times and pouring the rehydrated bacteria into the same cuvette.
• Replace the cuvette in the reagent well.
• Using a 500-µL pipette, aspirate any remaining Reconstituted Bacterial Reagent from the vial, add it to the cuvette, and mix 10 times using the same pipette and tip.
• Record the reagent lot number, expiry date, and time of reconstitution on the bench sheet (see Section 7.2.7 and Appendix F).

4.6.6 Inoculation and Incubation

• Prepare the following three pipettes: (a) a repeat pipette (such as an Eppendorf or Oxford Nichiryo) with a 0.5-mL syringe fitted with an ultramicro tip; (b) a macropipette (e.g., Oxford 1-5 mL); and (c) a 500-µL pipette.
• Following the 10-min temperature equilibration of the SPT tubes containing the test concentrations (Section 4.6.4), set a timer for 20 min but do not start it.
• Make sure the repeat pipette is set to dispense 20 µL per ejection. Place the tip below the surface of the reconstituted bacteria (Reconstituted Bacterial Reagent), and draw up sufficient Reagent for at least 18 ejections.
• Holding the tip above the Reagent and against the cuvette wall, eject two times. Carefully wipe the tip with a clean wiper (e.g., using a KimWipe^TM) after drawing up the Reagent. Then, immerse the tip in a tube or beaker containing Solid-Phase Diluent and make another ejection (to rinse the outside of the tip). Discard the contents of this tube or beaker.
• Start the 20-min timer, record the time as the “Start” of the test on the bench sheet, and immediately eject 20 µL of Reagent into each of the SPT tubes, starting with tube 1 (first control solution) and continuing consecutively to and including tube 15. Resting the collar of the ultramicro tip on the top edge of each tube will assure the tip will be at the surface of the sample and not on the bottom of the tube.
• Remove and discard the ultramicro tip. Eject the remaining Reagent from the syringe into the holding cuvette in the reagent well.
• If a second test (i.e., a test with either a second sample or a duplicate test with the same sample) is to be performed concurrently using the remaining 15 wells of the Analyzer, refit the syringe with a clean tip, refill it with Reagent, and inoculate the next series of test concentrations. This second (concurrent) test may be performed using another sample of test (contaminated or potentially contaminated) sediment, reference sediment, negative control sediment, or positive control sediment.
• With the macropipette set at 1.5 mL, mix each tube twice, beginning with tube 1 (first control) and proceeding consecutively through to and including tube 15.
• Insert a filter column (see footnote 5 in Section 3.2.3) in each tube, with its lower end positioned ~1 cm above the surface of the liquid. Do not wet the filter, since this might adversely affect filtration of the sample.

4.6.7 Preparing the Computer

• Prepare the computer to receive data from the photometer.
• Start the appropriate software program and menus for the solid phase test.
• Follow the on-screen instructions.
• Refer to the user's manual for additional information.
• Information requested by the software might include the number of controls (3), number of dilutions (12), initial concentration (197 000 mg/L), dilution factor (2), and the test time (30 min).

4.6.8 Filtration

• When the 20-min timer sounds, respond to the computer software as appropriate.
• Reset the timer for 10 min and start it. If using Strategic Diagnostics Inc. software (Omni^TM Version 1.18 or equivalent), this period is automatically initiated. Otherwise, program the computer software to initiate this 10-min period automatically at the touch of a key.
• Gently push the filter in tube 1 (first control) down far enough to obtain slightly greater than 500 µL of filtrate in the tube. Then, using the 500-µL pipette, transfer 500 µL of filtrate to the cuvette in well A1 of the photometer.
• Repeat this step for all 15 tubes using the same pipette tip, ending with 500 µL of filtrate being transferred from tube 15 to cuvette C5.
• Take note of the time required to complete all the transfers.
• Often with sediments having a high proportion of fines, the filter in the highest test concentration (tube 15) will become plugged. Obtain what can be recovered, and transfer it to the designated cuvette (C5). Usually the luminescence from these samples will have decreased to zero before you test the highest concentration. Make a note of such problems on the bench sheet (see Section 7.2.7 and Appendix F).
• At the end of all transfers, respond to the computer if required. Usually this will involve reading light production (Section 4.8), taking approximately the same amount of time as was taken to filter and transfer the filtrate to the cuvettes.
• The data is often sent from the photometer to the computer, and received by the software which builds a data file.

4.7 Test Measurements and Observations

Section 4.3 should be consulted for requirements and recommendations related to sample characterization (e.g., measurements of grain size and other physicochemical characteristics of the test material).

The procedure to measure light production of the bacteria in the test concentrations will vary depending on the photometer and software used. For the Model 500 Analyzer, place the first control (cuvette A1) into the read well and press the “set” button. The instrument lowers the cuvette into the well (sometimes 2 or 3 times) to set the zero (dark) and control reading at about 95 and thereby establish the appropriate sensitivity range for light measurements. After the green “ready” light appears, press the “read” button. Read the cuvette, then remove it from the read well and replace it in the incubator block. If the software program does not collect the light measurement data, record it on the bench sheet. Proceed to read and record the light emission from all the cuvettes, taking approximately the same average time per cuvette as was taken to do the filtering and transferring (see Section 4.6.8). If using Strategic Diagnostics Inc. software (Omni^TM Version 1.18 or equivalent), this timing is performed by the computer and prompts occur indicating when each cuvette should be read.

If a problem arises, refer to the user manuals for whatever photometer and software you are using.

4.8 Criterion for a Valid Test

The following criterion must be met for a valid sediment toxicity test, performed using this reference method:^Footnote 11

• The coefficient of variation representing the mean light reading measured for the filtrates of the three control solutions included in the test must be ≤12%.