Biological test method for measuring the inhibition of growth using freshwater macrophyte: chapter 3

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

3.1 Facilities and Apparatus

The Lemna minor growth inhibition test must be conducted in a constant-temperature room, incubator, environmental chamber, or equivalent facility with good temperature control and acceptable lighting (see Section 3.2). The test facility must be capable of maintaining the daily mean temperature of all test solutions at 25 ± 2°C (see Section 4.3). Test conditions (e.g., light quality, light fluence rate, and temperature) should be uniform throughout the environmental chamber. The facility should be well ventilated, and isolated from physical disturbances or any contaminants that could affect the test organisms. The test facility should also be isolated from the area where Lemna are cultured. Dust and fumes should be minimized within the test and culturing facilities.

Any construction materials and equipment that might contact the test material, test solutions, or control/dilution water must not contain any substances or materials that can be leached into the solutions at concentrations that could cause toxic effects, or that increase sorption of the test substances or materials (see Section 2.3.2). The laboratory must have the instruments to measure the basic variables of water quality (temperature, conductivity, dissolved oxygen, pH), and it should be prepared to undertake prompt and accurate analysis of other variables such as hardness, alkalinity, ammonia, and residual chlorine.

All instruments used for routine measurements of the basic chemical, physical, and biological variables must be maintained properly and calibrated regularly.

Disposal facilities should be adequate to accommodate laboratory-generated waste, as well as any bench covering, lab clothing, or other contaminated materials (United States Environmental Protection Agency (USEPA), 1996).

3.2 Lighting

Lighting conditions to which test organisms are subjected should be the same as those defined in Section 2.3.4. Full-spectrum fluorescent or equivalent lighting is recommended (see footnote 23). Continuous light is required for the duration of the test, and the light fluence rate must be 64 to 90 µmol/(m² · s) (approximately 4000 to 5600 lux; see footnote 24) at the level of the Lemna in the test.^Footnote 28 The light fluence rate measured at several points in the test area, at the level of the medium, should not vary by more than ±15% of the selected light fluence rate.^Footnote 29

3.3 Test Vessels

Test vessels recommended for use in the test include disposable polystyrene cups or Erlenmeyer flasks. Crystallizing dishes, petri dishes, or glass beakers may also be used^Footnote 30; however, a standard type and size should be selected and used within a laboratory.^Footnote 31 Glass vessels should be used for chemical testing (Section 5). The test vessels must be wide enough for the fronds in the control vessels to grow without overlapping at the end of the test. It does not matter if the roots reach the bottoms of the test vessels; however, a minimum depth of 4 cm of test solution is recommended. The vessel must contain at least 100 mL of solution during the test and 150 mL is recommended.^Footnote 32

The test vessels should be covered to avoid potential contamination from the air and loss of volatile components. Polystyrene lids that fit plastic test cups, or petri dish lids or bottoms placed on top of Erlenmeyer flasks are recommended; however, other suitable covers may be used.^Footnote 33 For a given test, all test vessels and covers (i.e., type, size, shape) as well as solution depth and volume must be identical.

Test vessels should be placed on a non-reflective dark background (e.g., black poster board) for the duration of the test.^Footnote 34 Any new test system (e.g., vessel, cover, lighting and temperature conditions) should be tested by conducting a non-toxicant test in which all test vessels contain only test medium. The coefficient of variation (CV) for frond number and dry weight at the end of test should be < 20%.

3.4 Control/Dilution Water

For a given test, the same water must be used to prepare sample dilutions and controls. The choice of control/dilution water will depend on the test substance or material and objectives, and on the logistics, practicality, and costs of sample collection (see Sections 5 to 7). Accordingly, these factors might lead to the selection of a specific type of control/dilution water that is best suited for a particular situation. The control/dilution water recommended for use is test medium which is deionized or glass-distilled water to which reagent-grade chemicals (i.e., nutrients for growing Lemna) have been added.

Three different test media are recommended for use in this biological test method, and the selection will depend on the type of substance being tested. For wastewaters (see Section 6.3) and receiving waters (see Section 7.3), a modified American Public Health Association (APHA) growth medium (Saskatchewan Research Council (SRC), 1997)^Footnote 35 must be used as control/dilution water. For chemicals, commercial products, or known mixtures (see Section 5.3), a modification of the Swedish Standard (SIS) growth medium (Organization for Economic Cooperation and Development (OECD), 1998, 2002), or a modified Steinberg medium (International Organization for Standardization (ISO), 2005) should be used.^Footnote 36

A sample of receiving water or upstream water (collected adjacent to the source of contamination but removed from it, or upstream from the source), spiked with the same reagent-grade nutrients and at the same concentration as those used to make up the modified APHA growth medium (nutrient-spiked receiving water), may also be used as control/dilution water for testing effluents (see Section 6.3) or receiving waters (see Section 7.3).^Footnote 37 In instances where the toxic effect of a specific chemical or chemical compound in a particular receiving water is to be appraised, receiving water spiked with the same concentration of nutrients as those used to prepare the SIS medium or the modified Steinberg medium may be used as control/dilution water (see Section 5.3). In either case, if nutrient-spiked receiving water is used, it must first be filtered through glass fibre filters (approximately 1µm, e.g., Whatman GF/C filters) to reduce the possibility of contamination of the test by algae, and may be further filtered through 0.22µm filters to eliminate any remaining potential for algal or bacterial contamination (SRC, 1997). Conditions for collection, transport, and storage of surface water should be as described in Section 6.1.

The test medium or nutrient-spiked receiving water (used for control and dilution water) must be prepared as outlined in Sections 5, 6, and 7, and adjusted to 25 ± 2°C before use (see Section 4.1).

Footnotes

Footnote 28

The type of photo-receiver (collector) used to measure the light fluence rate can influence the measured value. Spherical photo-receivers (which respond to diffuse and reflected light of all angles below and above the measured plane) and hemispherical receivers (which respond to light of all angles only above the measured plane) are preferable to unidirectional receivers and give a higher value for non-punctual light sources (Association Française de Normalisation (AFNOR), 1996).

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Footnote 29

Light intensity, and the control thereof, can be as important, if not more so, than pH and temperature for plant testing. The light fluence rate in the entire test area should be checked before initiating the test. Cheesecloth can be used to reduce the lighting in specific areas of the test facility in order to achieve the appropriate light conditions (Staveley, 1998). Alternatively, the portion of the test area that is within 15% of the selected light fluence rate can be “mapped out” to designate the boundaries of adequate versus inadequate light fluence rate (Moody, 1998).

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Footnote 30

The use of beakers should be approached with caution as frond numbers and health were reduced significantly in tests carried out using beakers (SRC, 2003).

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Footnote 31

Variations in size of test vessel might affect the results of the test through changes in relative depths, relative surface area of the test solution, and other variables, in ways that are as yet unrecognized.

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Footnote 32

Jonczyk and Gilron (1996) determined that larger test vessels (100 mL) yielded improved growth over smaller test vessels (50 mL).

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Footnote 33

Transparent covers will allow the illumination of test organisms, while minimizing evaporation of test solutions and reducing their contamination. However, the use of watch glasses as covers for tests vessels is not recommended due to excessive losses of test medium via evaporation and the possible increase in light reflection (SRC, 2003).

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Footnote 34

In a series of studies carried out to determine the impact of methodology differences between the draft ISO standard and the Environment Canada L. minor test method, Moody determined that frond appearance and general health (i.e., number, colour and uniformity) were improved when test vessels were placed on a black background for the duration of the test. The black background reduced the amount of reflective light to which the fronds were exposed, thereby enabling frond exposure to higher light intensities (i.e., those recommended by ISO) (SRC, 2003).

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Footnote 35

The modified APHA medium differs from the medium described in the American Public Health Association (APHA et al., 1992) L. minor test method (SRC, 1995). The modifications include the addition of potassium chloride (KCl), the omission of EDTA, and the stabilization of medium pH by aeration (SRC, 1995) (see Appendix D, Table 9).

The addition of KCl roughly doubled the potassium content of the original APHA medium, resulting in increased rate and reproducibility of frond growth and reproducibility of reference toxicant results. EDTA was omitted since it can potentially interact with certain substances or materials (e.g., metals) in the test sample resulting in altered toxicity. The pH drift, observed in the original APHA medium, was eliminated (pH stabilized) by including a 1- to 2-hour aeration period following medium preparation (SRC, 1995).

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Footnote 36

The Swedish Standard (SIS) growth medium is recommended for substance testing with L. minor in the draft OECD Lemna growth inhibition test (OECD, 1998, 2002). The modified Steinberg medium is recommended for testing substances or materials that do not contain predominantly metals in the draft ISO Lemna minor growth inhibition test (ISO, 2005).

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Footnote 37

Receiving water may be used as the control/dilution water in certain instances where site-specific information is required regarding the toxic effect(s) of an effluent, elutriate, or leachate on a particular receiving water. “Upstream” water may be used as control/dilution water for receiving-water samples collected in the vicinity of a wastewater discharge, chemical spill, or other point-source of possible contamination.

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Biological test method for measuring the inhibition of growth using freshwater macrophyte: chapter 3

Section 3: Test System

3.1 Facilities and Apparatus

3.2 Lighting

3.3 Test Vessels

3.4 Control/Dilution Water

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