Biological test method for measuring the inhibition of growth using freshwater macrophyte: chapter 1


1.1 Background

Aquatic toxicity tests are used within Canada and elsewhere to determine and monitor the toxic effects of discrete substances or materials that might be harmful to aquatic life in the environment. The results of toxicity tests can be used to determine the need for control of discharges, to set effluent standards, for research, and for other purposes. Recognizing that no single test method or test organism can be expected to satisfy a comprehensive approach to environmental conservation and protection, Environment Canada and the Inter-Governmental Environmental Toxicity Group (IGETG) (Appendix A) proposed that a set of standardized aquatic toxicity tests be developed, that would be broadly acceptable for use in Canada. It was decided that a battery of federally approved biological test methods was required that would measure different acute and chronic toxic effects using different test substances or materials and organisms representing different trophic levels and taxonomic groups (Sergy, 1987). As part of this ongoing undertaking, a toxicity test for determining the effect of contaminants on the inhibition of growth of the aquatic macrophyte, Lemna minor, was recommended for standardization. The first edition of this method was used in Environment Canada’s regional laboratories (Appendix B), as well as in provincial and private laboratories, to help meet Environment Canada’s metal mining effluent regulations and other testing requirements. The current (second) edition includes numerous procedural improvements, updated and more explicit guidance, and instructions for the use of revised statistics (i.e., regression analyses) when calculating the test endpoint for growth inhibition.

Universal procedures and conditions for conducting aquatic toxicity tests that measure growth inhibition of the aquatic macrophyte, L. minor, are described in this second edition. Also presented are specific sets of conditions and procedures required or recommended when using the test to evaluate different types of substances or materials (e.g., samples of one or more chemicals, effluents, receiving waters, leachates, or elutriates) (see Figure 1). Some details of methodology are discussed in explanatory footnotes.

This biological test method has been developed following a review of variations in specific culturing and test procedures indicated in existing Canadian, American, and European methodology documentsFootnote 1 that describe how to prepare for and conduct phytotoxicity tests using Lemna spp. A summary of these culturing and testing procedures is found in Appendix C. A summary of various media used for culturing and testing Lemna spp. in existing or past procedures is found in Appendix D. The biological endpoints for this method are: (a) increased number of fronds during the 7-day test; and (b) dry weight (as an indication of growth) at the end of the test.Footnote 2 The test method is intended for use in evaluating samples of:

  1. single chemicals, commercial products, or known mixtures of chemicals;
  2. freshwater industrial or urban effluents, elutriates, or leachates; and
  3. freshwater surface or receiving waters.

In formulating these procedures, an attempt was made to balance scientific, practical, and cost considerations, and to ensure that the results would be accurate and precise enough for most situations in which they would be applied. It is assumed that the user has a certain degree of familiarity with aquatic toxicity tests. Guidance regarding test options and applications is provided here. Explicit instructions that might be required in a regulatory protocol are not provided, although this report is intended as a guidance document useful for this and other applications.

For guidance on the implementation of this and other biological test methods and on the interpretation and application of the endpoint data, consult the Environment Canada report (EC, 1999a).

Figure 1 Considerations for Preparing and Performing Toxicity Tests Using Lemna minor with Various Types of Test Materials or Substances

Universal Procedures

  • Culturing organisms
  • Choosing control/dilution water
  • Preparing test solutions
  • Test conditions (lighting, temperature, etc.)
  • Beginning the test
  • Observations and measurements during the test
  • Test endpoints and calculations
  • Validity of results
  • Reference toxicity tests
  • Legal considerations

Items Covered in Specific Sections

Chemicals

  • Chemical properties
  • Labelling and storage
  • Chemical measurements
  • Choosing control/dilution water
  • Preparation of solutions
  • Observations during tests
  • Measurements during tests
  • Endpoints

Effluents, Elutriates, and Leachates

  • Containers and labelling
  • Sample transit and storage
  • Choosing control/dilution water
  • Preparation of solutions
  • Observations during tests
  • Measurements during tests
  • Endpoints

Receiving Waters

  • Containers and labelling
  • Sample transit and storage
  • Choosing control/dilution water
  • Preparation of solutions
  • Observations during test
  • Measurements during test
  • Endpoints

1.2 Species Description and Historical Use in Tests

Lemna minor, commonly referred to as lesser duckweed or common duckweed, is a small, vascular, aquatic macrophyte belonging to the family Lemnaceae. Members of the family Lemnaceae are free-floating, monocotyledonous angiosperms which are found at, or just below, the surface of quiescent water (Hillman, 1961). There are four genera (Spirodela, Lemna, Wolffiella, and Wolffia) and approximately 40 Lemna (i.e., duckweed) species world wide (Wang, 1990). The two species commonly used in toxicity tests, L. minor and L. gibba, are well represented in temperate areas (Organisation for Economic Cooperation and Development (OECD), 1998, 2002).

L. minor is ubiquitous in nature, inhabiting relatively still fresh water (ponds, lakes, stagnant waters, and quiet streams) and estuaries ranging from tropical to temperate zones (American Public Health Association (APHA) et al., 1992). It is a cosmopolitan species whose distribution extends nearly world wide (Godfrey and Wooten, 1979). In North America, L. minor is one of the most common and widespread of the duckweed species (Arber, 1963; APHA et al., 1992). The fronds of L. minor occur singly or in small clusters (3 to 5) and are flat, broadly obovate to almost ovate, 2- to 4-mm long, green to lime green, and have a single root that emanates from the centre of the lower surface (Hillman, 1961; Godfrey and Wooten, 1979; Newmaster et al., 1997). Vegetative growth in Lemna spp. is by lateral branching, and is rapid compared with other vascular and flowering plants (Hillman, 1961; APHA et al., 1992). Further details on the taxonomy, description, distribution and ecology, and reproductive biology of this species are provided in Appendix E.

Duckweeds have been used as test organisms for the detection of phytotoxicity since the 1930s. They were among the species used to define the effects of the earliest phenoxy herbicides on plants (Blackman and Robertson-Cumminghame, 1955). In 1979, the United States Environmental Protection Agency (USEPA) proposed that L. minor be classified as a “representative” aquatic macrophyte, useful in the environmental safety assessment of chemicals (Federal Register, 1979 in Bishop and Perry, 1981). In the past several years, there has been increasing interest in the use of vascular plants for environmental monitoring and assessment, including laboratory phytotoxicity tests (Wang and Freemark, 1995). Besides being an essential component of aquatic ecosystemsFootnote 3, aquatic macrophytes have a key role in assessing the effects of herbicides on vegetation in aquatic environments through phytotoxicity testing (Wang and Freemark, 1995).

Many important environmental legislation and guidelines developed under different authorities have included phytotoxicity testing as part of environmental monitoring and assessment (Wang and Freemark, 1995). The USEPA requires phytotoxicity testing under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), including a duckweed growth test. Duckweed testing can also be required in the USEPA’s Toxic Substances Control Act (TSCA) and is optional for National Pollution Discharge Elimination System (NPDES) permits under the U.S. Water Quality Act, 1987 (Wang and Freemark, 1995).

A duckweed growth inhibition test developed for the Organization for Economic Cooperation and Development (OECD, 1998, 2002) underwent interlaboratory validation (Sims et al., 1999). The international ring test included the participation of 37 testing laboratories from Europe, North America, and the Far East. The key performance characteristics of the draft test method that were assessed included compliance with the critical quality criteria, repeatability of the method within laboratories, and reproducibility between laboratories. The results of the ring test, which included testing of two Lemna species (Lemna minor and Lemna gibba), indicate that the requirements of the draft OECD Lemna growth inhibition guideline were successfully met by most of the data sets submitted (Sims et al., 1999). Other findings from the ring test apply to the use of 3,5-dichlorophenol and potassium dichromate as reference toxicants.

Duckweed test methods currently available and used in North America and abroad include those by: the American Public Health Association et al. (APHA et al., 1995); the American Society for Testing and Materials (ASTM, 1991); the United States Environmental Protection Agency (USEPA, 1996); the Association Française de Normalisation (AFNOR, 1996); the Swedish Standards Institute (SIS, 1995); and the Institute of Applied Environmental Research (ITM, 1990). More recently, the International Organization for Standardization (ISO) has also developed a Lemna minor growth inhibition test method (2005).

Duckweed species have many attributes that make them advantageous for use in laboratory toxicity tests and assessments of freshwater systems. These include their:

  • small sizeFootnote 4;
  • relative structural simplicity; and
  • rapid growthFootnote 5 (Hillman, 1961; Smith and Kwan, 1989).

Duckweeds also have several characteristics that make them uniquely useful for toxicity tests:

  • their vegetative reproduction and genetically homogenous populations enable clonal colonies to be used for all experiments, and eliminate effects due to genetic variability (Hillman, 1961; Bishop and Perry, 1981; Smith and Kwan, 1989);
  • duckweeds can be disinfected and grown in a liquid medium as well as on agar, autotrophically or heterotrophically (Hillman, 1961);
  • duckweeds cultured in the laboratory can grow indefinitely and controlled conditions of temperature, light, and nutrition are far easier to maintain than for most other angiosperms (Hillman, 1961; Wang, 1987);
  • they have a high surface area to volume ratio, and little, if any, cuticle present on the underside of the frond that is in contact with the test solution (Bishop and Perry, 1981);
  • they are excellent accumulators of a number of metallic elements, making them good candidates for use in water quality monitoring and in laboratory tests for toxicity and uptake studies (Jenner and Janssen-Mommen, 1989; Smith and Kwan, 1989);
  • duckweeds are especially susceptible to surface-active substances, hydrophobic compounds, and similar substances that concentrate at the air-water interface (Taraldsen and Norberg-King, 1990; ASTM, 1991); and
  • unlike algal toxicity tests, test solutions can be renewed, and coloured or turbid wastewater or receiving-water samples can be tested (Taraldsen and Norberg-King, 1990; Forrow, 1999).

Since Lemna spp. were first used for comparative phytotoxicity studies, a number of test procedures have been described. Plant growth has been quantified by various procedures including frond count, dry weight, growth rate, doubling time, percent inhibition, frond area, root length, chlorophyll content, and photosynthesis (Lockhart and Blouw, 1979; Bishop and Perry, 1981; Cowgill and Milazzo, 1989; Wang, 1990; Greenberg et al., 1992; Huang et al., 1997). Examples of Lemna species that have been used for testing include: Lemna aequinoctialis, Lemna major, Lemna minor, Lemna gibba, Lemna paucicostata, Lemna perpusilla, Lemna trisulca, and Lemna valdiviana (OECD, 1998, 2002). Numerous test options, including test duration, type (static, static-renewal, flow-through), test and culture media, light intensity, and temperature have been investigated and reviewed (see Appendices C and D).

The Lemna minor growth inhibition test, developed by the Saskatchewan Research Council (SRC) Water Quality Section (SRC, 1997) is a modification of the “8211 Duckweed (Proposed)” toxicity test procedure published by APHA et al. (1995). The major modifications include changes to the medium composition (potassium added, pH stabilized, and EDTA removed), pre-cultivation methods, and the use of axenic cultures, as well as the establishment of a requirement for a greater biomass increase during the test. The method developed by the SRC has been used successfully in assessing single-metal solutions, as well as metal mine wastewaters (SRC, 1997).

Precision of the test appears to be satisfactory. The SRC has demonstrated within-laboratory coefficients of variation (CVs) for mean percent inhibition of biomass, using chromium (Cr) as a reference toxicant, of <10% (Moody, 1998).

The purpose of the biological test method herein is to provide a “standardized” Canadian methodology for estimating the toxicity of various substances or materials in fresh water using L. minor. Whereas the application of other published methods (see Appendix C) for performing this test might have been restricted to certain types of substances or materials, this report is intended for use in evaluating the sublethal toxicity of chemicals, effluents, leachates, elutriates, and receiving waters. The generic culture and test conditions and procedures herein are largely those developed by SRC (1997), with incorporation of useful test modifications and harmonization with OECD (1998, 2002), ISO (2005) and elsewhere. The rationale for selecting certain approaches is provided in the document.

This method is intended for use with freshwater-acclimated L. minor, with fresh water as the dilution and control water, and with effluents, leachates, or elutriates that are essentially fresh water (i.e., salinity ≤10 g/kg) or are saline but are destined for discharge to fresh water. Its application may be diverse but includes instances where the effect(s) or potential effect(s) of substances or materials on the freshwater environment is under investigation. Other tests, using other species acclimated to seawater, may be used to assess the effect(s) or potential effect(s) of substances or materials in estuarine or marine environments, or to evaluate wastewaters having a salinity >10 g/kg.

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