Biological test method for measuring the inhibition of growth using freshwater macrophyte: chapter 6


Section 6: Specific Procedures for Testing Samples of Effluent, Leachate, and Elutriate

This section gives specific instructions for collecting, preparing, and testing samples of effluent, elutriate, and leachate, in addition to the procedures described in Section 4.

6.1 Sample Collection, Labelling, Transport, and Storage

Containers for transporting and storing samples or subsamples of effluent, elutriate, or leachate must be made of nontoxic material. Collapsible polyethylene or polypropylene containers manufactured for transporting drinking water (e.g., Reliance™) are recommended. The volume of these containers can be reduced to fit into a cooler for transport, and the air space within can be minimized or eliminated if possible, when portions are removed in the laboratory for toxicity testing or chemical analyses. The containers must either be new or thoroughly cleaned, and rinsed with uncontaminated water. They should also be rinsed with the sample to be collected. Containers should be filled to eliminate any air space.

The requirements for volume of wastewater sample should be given serious consideration before undertaking the program. Generally, a 4-L sample of effluent or leachate is adequate for an off-site multi-concentration test and the associated routine sample analysis. Smaller amounts are required for single-concentration tests (see Section 4.5.3). Upon collection, each sample container must be filled, sealed, and labelled or coded. Labelling should include at least sample type, source, date and time of collection, and name of sampler(s). Unlabelled or uncoded containers arriving at the laboratory should not be tested nor should samples arriving in partially filled or unsealed containers be routinely tested, since volatile toxicants can escape into the air space. However, if it is known that volatility is not a factor, such samples might be tested at the discretion of the investigator. The chain-of-custody during sample collection, transport, and storage should be recorded along with any sample conditions (anomalies) that could effect test results.

An effort must be made to keep samples of effluent or leachate cool (1 to 7°C, preferably 4 ± 2°C) throughout transport. Upon collection, warm (>7°C) samples must be cooled to 1 to 7°C with regular ice (not dry ice) or frozen gel packs. As necessary, ample quantities of regular ice, gel packs, or other means of refrigeration must be included in the transport container in an attempt to maintain sample temperature within 1 to 7°C during transit. Samples must not freeze during transport or storage.

Upon arrival at the laboratory, the temperature of the sample or, if collected, one of the subsamples (with the remaining subsamples left unopened and sealed), must be measured and recorded. An aliquot of effluent or leachate required at that time may be adjusted immediately or overnight to the test temperature and used in the test. The remaining portion(s) of sample or subsamples required for subsequent solution renewal or held for possible additional testing must be stored in darkness, in sealed containers, without headspace, at 4±2°C. For elutriates, as well as for samples intended for aqueous extraction and subsequent testing of elutriate, transport and storage conditions should be as indicated for effluents and leachates.

Tests with effluent, leachate, or elutriate may be performed “off-site” in a controlled laboratory facility. The static test option is recommended for standard use with samples of effluent, elutriate, and leachate. If, however, the active component in the wastewater can be expected to decrease significantly during the test period, the static-renewal test option is recommended (see Section 4.3).

If the static test option is followed, a single sample of wastewater must be collected and used to prepare the test solutions at the beginning of the test. If the static-renewal test option is followed, samples must be collected using one of the following procedures and approaches:

  1. A single sample of wastewater may be used throughout the test, provided it is divided into three separate containers (i.e., three subsamples) upon collection.Footnote 72
  2. In instances where the toxicity of the wastewater is known or anticipated to change significantly if stored for up to 7 to 10 days before use, fresh samples must be collected on at least three separate occasions using sampling intervals of 2 to 3 days or less. These samples must be used consecutively during the test.Footnote 73

An alternative approach for unstable wastewater is to perform these tests on-site, using fresh wastewater and static-renewal conditions (see Section 4.3).

Testing of effluent and leachate samples should commence as soon as possible after collection. Use of any sample in a test should begin within 1 day whenever possible, and must begin no later than 3 days after sampling. If effluents or leachates are tested at on-site laboratories, samples should be used in the test within 1 day or less following their collectionFootnote 74 (United States Environmental Protection Agency (USEPA), 1989, 2002).

Samples of sediment or other solid material collected for aqueous extraction and subsequent testing of the elutriate should also be tested as soon as possible, following their collection and no later than 10 days following receipt in the laboratory. Procedures provided by Environment Canada (EC, 1994) for the preparation of elutriates should be followed. For the derived elutriates, aliquots of the prepared sample should be used on the same schedule as indicated for samples of effluent or leachate, if possible. The prolonged storage of elutriate samples is undesirable because the toxicity of the sample might not be stable. Testing of elutriates must commence within 3 days of their preparation, unless specified otherwise in a regulation or prescribed method.

6.2 Preparing Test Solutions

Each sample or subsample in a collection or storage container must be agitated thoroughly just before pouring to ensure the re-suspension of settleable solids. The pH of each sample or subsample must be measured just before being used.

Filtration of samples or subsamples is normally not required nor recommended. However, if the wastewater samples are mixed with, or contain receiving water (e.g., effluent collected from a mixing zone in a lake, stream, river, etc.) they may contain algae and sample filtration may be required to reduce the possibility of contamination (i.e., excessive algae growth) during the test. All wastewater samples should be checked under the microscope for the presence of algae. If algae is present then the sample should be filtered through glass fibre filters (pore size of approximately 1 µm; e.g., Whatman GF/C (glass filter grade C) filters) to reduce the risk of algal contamination. Samples may be subsequently filtered through 0.22 µm filters to eliminate any remaining potential for algal contamination (Saskatchewan Research Council (SRC), 1997). Such filtration could remove some suspended solids that are characteristic of the sample and might otherwise contribute part of the toxicity or modify the toxicity. In instances where there is concern about the effect of this filtration on sample toxicity, a second (concurrent) test should be conducted using portions of the unfiltered sample/subsample, but procedures should otherwise be identical.

A sample of wastewater must then be spiked with the same nutrients as those used to prepare the modified American Public Health Association (APHA) growth medium (nutrient-spiked wastewater) (see Section 6.3; Table 5). An aliquot of each of three nutrient stock solutions (A, B, and C) are added to the wastewater sample in the ratio of 10 mL aliquot per 1000 mL sample diluting the samples to 97%. The spiked wastewater sample is then gently pre-aerated for 20 minutes (see Section 4.1) before being distributed to replicate test vessels.

6.3 Control/Dilution Water

Tests conducted with samples of effluent or leachate, intended to assess compliance with regulations, must use modified APHA medium (Table 5) or a sample of the receiving water spiked with modified APHA nutrient stock solutions (nutrient-spiked receiving water) as the control/dilution water. The objectives of the test must be defined before selecting the appropriate control/dilution water because the results could be different for the two sources of water. Difficulties and costs associated with the collection and shipment of receiving-water samples for use as control/dilution water should also be considered.

The APHA (modified) test medium is prepared with 3 stock solutions, as outlined in Table 5. The stock solutions are prepared using reagent-grade chemicals in glass-distilled, deionized water, or equivalent. To prepare 1 L of medium, 10 mL of each stock solution (A, B, and C) are added to 970 mL of distilled water in a 1 L media bottle. The medium is aerated vigorously for at least 1 to 2 hours. If a larger volume (>4 L) of media is prepared, overnight aeration of the medium is recommended to stabilize the pH of the medium. Immediately before testing, the pH of the test medium is adjusted to 8.3 ± 0.1 using 0.5N NaOH and 0.5N HCl.Footnote 75 The medium is not sterilized. Stock solutions A, B, and C can be stored as separate solutions in a refrigerator (4 ± 2°C) for up to one month.

Modified APHA medium is the control/dilution water required for standard use with samples of effluent, elutriate, and leachate. The use of receiving water as the control/dilution water, however, might be desirable in certain instances where site-specific information is required on the potential toxic effect of an effluent, leachate, or elutriate on a particular receiving water (see footnote 40 and Section 4.1). An important example of such a situation would be testing for sublethal effect at the edge of a mixing zone, under site-specific regulatory requirements. Conditions for the collection, transport, and storage of such receiving-water samples should be as described in Section 6.1.

An aliquot of the receiving water, to be used as control/dilution water, is filtered through glass fibre filters (approximate pore size of 1 µm, e.g., Whatman GF/C filters), before being used, to reduce the possibility of the test being contaminated by algae. Receiving waters may be subsequently filtered through 0.22 µm filters to prevent the growth of algae (SRC, 1997). The receiving-water sample must then be enriched with the same levels of nutrients as the modified APHA medium (10 mL of each stock solution (A, B, and C) per 1000 mL of receiving water). Once enriched, the receiving-water samples should be aerated vigorously for 1 to 2 hours (or longer for larger volumes), without pH adjustment, to stabilize the pH of the nutrient-spiked receiving water.Footnote 76 The pH of the aerated, spiked, receiving water is recorded before testing.

If a sample of upstream receiving water is to be used for control/dilution water, a separate control solution must be prepared using the modified APHA medium. Test conditions and procedures for evaluating each control solution should be identical and as described in Sections 4 and 5.3.

If a high degree of standardization is required, modified APHA medium should be used for all dilutions and as the control water, since the use of a specific medium increases the probability of reducing the modifying influences attributable to different chemical compositions of dilution water. Situations where such use is appropriate include investigative studies intended to interrelate toxicity data for various effluent, leachate, or elutriate types and sources, derived from a number of test facilities. In such instances, it is desirable to minimize any modifying influence of dilution-water chemistry.

Table 5 Chemical Composition of Nutrient Stock Solutions for Preparing Modified APHA Medium, Nutrient-Spiked Wastewater, and Nutrient-Spiked Receiving Water, for Testing Samples of Effluent, Elutriate, Leachate, or Receiving Water, Using Lemna minor
Stock Substance Concentration
Stock Solution
(g/L)
Concentration
MediumTable note a
(mg/L)
A NaNO3 25.5 255
A NaHCO3 15.0 150
A K2HPO4 1.04 10.4
A KCl 1.01 10.1
BTable note b CaCl2 · 2H2O 4.41 44.1
B MgCl2 · 6H2O 12.17 121.7
B MnCl2 · 4H2O 0.4149 4.149
B FeCl3 · 6H2O 0.16 1.6
CTable note c MgSO4 · 7H2O 14.7 147
C H3BO3 0.186 1.86
C Na2MoO4 · 2H2O 0.00726 0.0726
C ZnCl2 0.00327 0.0327
C CoCl2 · 6H2O 0.0014 0.014
  CuCl2 · 2H2O 1.5 × 10-5 1.5 × 10-4

6.4 Test Observations and Measurements

There are certain observations and measurements that should be made during tests with effluents, elutriates, and leachates in addition to those described in Section 4.4.

Colour, turbidity, odour, and homogeneity (the presence of floating or settled solids) of the effluent, leachate, or elutriate sample should be observed and recorded before and after the sample is filtered. Any changes that occur during the preparation of the test sample should be recorded (e.g., precipitation, flocculation, change in colour or odour, release of volatiles, etc.), as well as any changes in the appearance of test solutions during the test period (e.g., foaming, settling, flocculation, increase or decrease in turbidity, colour change, etc.).

For effluent samples with appreciable solids content, it is desirable to measure total suspended and settleable solids (APHA et al., 1995) upon receipt, as part of the overall description of the effluent, and as sample characteristics that might influence the results of the toxicity test. Additional measurements that would help characterize each sample of effluent, leachate, or elutriate should also be made. These could include pH, conductivity, hardness, alkalinity, colour, chemical oxygen demand, biological oxygen demand, dissolved oxygen, and concentrations of specific toxic contaminants (e.g., resin acids, chlorophenolic compounds, dissolved metals, chlorine, chloramine, ammonia, etc.).

6.5 Test Endpoints and Calculations

The endpoints for tests performed with samples of wastewater will normally be IC25s based on increase in frond number during the test and frond dry weight attained at test end, as indications of growth. Tests for monitoring or regulating effluents, leachates, or elutriates must use the standard options and endpoints defined in Section 4.

Tests for monitoring and compliance with regulatory requirements should normally include, as a minimum, three or more replicate solutions of the undiluted sample/subsamples (or a specified dilution thereof), and three or more replicate control solutions. Depending on the specified regulatory requirements, tests for compliance might be restricted to a single concentration (e.g., “full-strength” sample, which is 97% using this test method, unless otherwise specified) or might require a series of concentrations (i.e., a multi-concentration test) (see Section 4.5.2). Single-concentration tests are often cost-effective for determining the presence of measurable toxicity, and also for screening a large number of samples.

Specific adaptations of the standard toxicity test could be adopted for special purposes such as locating in-plant sources of toxicity, or assessing the effectiveness of in-plant process changes or of effluent treatment. The tests could be multi-concentration or single-concentration (97% or an appropriate dilution, plus a control). Endpoints would depend on the objectives of the undertaking, but could include arbitrary “pass/fail” limits or percent reduction in growth at a specified concentration (Section 4.5.3).

Section 4.5.3 provides relevant instructions on statistical analysis and reporting for sets of tests with different samples, each tested at only one concentration.

6.6 Interpretation of Results

For any test that uses a water source other than modified APHA medium for the control/dilution water, particular attention should be given to a comparison of Lemna growth in the control/dilution water with that in the standard controls using modified APHA medium. A statistical comparison is necessary to determine whether the control/dilution water is phytotoxic (see Section 4.5.3). Any enhanced growth in test solutions, relative to that in the control solutions, must be reported and considered when interpreting the findings (see Sections 4.5.2 and 4.5.4).

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