Biological test method for measuring the inhibition of growth using freshwater macrophyte: chapter 7

Section 7: Specific Procedures for Testing Receiving-Water Samples

• 7.1 Sample Collection, Labelling, Transport, and Storage
• 7.2 Preparing Test Solutions
• 7.3 Control/Dilution Water
• 7.4 Test Observations and Measurements
• 7.5 Test Endpoints and Calculations

Instructions for testing samples of receiving water, in addition to those provided in Section 4, are provided in this section.

7.1 Sample Collection, Labelling, Transport, and Storage

Procedures for collecting, labelling, transportation, and storing samples are found in Section 6.1. Testing of receiving-water samples/subsamples should commence as soon as possible after collection, preferably within 24 hours of sampling, but no later than 3 days after sampling.

7.2 Preparing Test Solutions

Samples in the collection container(s) should be agitated before pouring to ensure their homogeneity.

Each receiving-water test sample must be filtered through a glass fibre filter (approximate pore size of 1 µm, e.g., Whatman GF/C filters) before being used, to reduce the possibility of test contamination by algae. Receiving waters may be subsequently filtered through 0.22 µm filters to prevent the growth of algae (Saskatchewan Research Council (SRC), 1997). A second, unfiltered test should be run concurrently if there is concern about the effect of filtration on toxicity (see Section 6.2).

Receiving-water test samples are then spiked with modified American Public Health Association (APHA) nutrient stock solutions and gently pre-aerated for 20 minutes (see Sections 4.1 and 6.2).

7.3 Control/Dilution Water

For samples of surface water collected in the vicinity of a wastewater discharge, chemical spill, or other point-source of contamination, “upstream” water may be sampled concurrently and used as control/dilution water for the downstream sample (see footnote 37 and Section 6.3). This control/dilution water should be collected as close as possible to the contaminant source(s) of concern, but upstream or outside of the zone of influence. Such surface water must be filtered to remove organisms, as described in Section 7.2.

If “upstream” water is used as control/dilution water, a separate control solution must be prepared using the modified APHA medium that is normally used for testing L. minor. Test conditions and procedures for preparing and evaluating each control solution should be identical, and as described in Sections 4, 5.3, and 6.3. Results of test exposures must be statistically compared with those for the control that used receiving water (see Section 4.5).

Logistic constraints, expected toxic effects, or other site-specific practicalities might prevent or rule against the use of upstream water as the control/dilution water. In such cases, modified APHA medium should be used as the control water and for all dilutions (see Section 6.3).

7.4 Test Observations and Measurements

The primary observations on test organisms should be as described in Section 4.4. In addition, there should be observations of sample and solution colour, turbidity, foaming, precipitation, etc., as described in Section 6.4, both during the preparation of test solutions and during the tests.

Each receiving-water sample should be characterized chemically. Depending on the suspected nature of the toxicants, measurements might include pH, conductivity, hardness, alkalinity, colour, chemical oxygen demand, biochemical oxygen demand, and concentrations of specific toxicants (e.g., resin acids, chlorophenolic compounds, dissolved metals, chlorine, chloramine, ammonia, etc).

7.5 Test Endpoints and Calculations

Endpoints for tests with samples of receiving water should be consistent with the options and approaches identified in Sections 4.5, 6.5, and 6.6.

Tests with receiving water could be multi-concentration or single concentration. Tests of regulatory compliance would normally include three or more replicates containing “full-strength” (or 97%, in the case of this test) sample and three or more replicate control solutions to determine the growth inhibition obtained for L. minor exposed to 97% receiving water for 7 days (Section 4.5). Single-concentration tests are often cost-effective for determining the presence of measurable toxicity, and also for screening a large number of samples (e.g., from various locations within the receiving water). Statistical testing and reporting of results for such tests should follow the procedures outlined in Section 4.5.3.

If receiving-water samples are predicted to be toxic, and information is desired concerning the degree of dilution necessary to permit normal duckweed growth, a multi-concentration test to determine the IC25 for growth should be conducted, as outlined in Section 4. Any multi-concentration test should include the “full strength”, nutrient-spiked receiving water (97%) as the highest concentration in the series tested.

Certain sets of tests might use a series of samples such as surface waters from a number of locations, each tested at “full strength” (97%) only. Statistical testing and reporting of results for such tests should follow the procedures outlined in Section 4.5.3.