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1.1 Submersed rooted aquatic macrophytes are important components of aquatic systems. They contribute to primary productivity, improve water quality, cycle nutrients, generate oxygen, affect flow patterns, provide habitat and food for other organisms, and stabilize the sediment. These plants can be adversely affected when pesticides are sprayed to control aquatic weeds and algal blooms or when phytotoxic chemicals enter the waterway through atmospheric fallout, soil erosion, industrial effluent, sewage discharge, spills or drift from aerial or ground applications.

1.2 This guide is designed to give guidance for assessing the potential phytotoxicity of a test material added to a sterile liquid growth medium on a species of freshwater submersed macrophyte (Myriophyllum sibiricum Komarov) during a 14-day static exposure. A sterile system is recommended to determine the direct effect of the test chemical upon individual parameters of the submersed macrophyte because there is no degradation of the test item by micro-organisms. For similar reasons, other aquatic plant testing, such as those of, Lemna and algae, is commonly conducted in an axenic fashion. Overall environmental impact can not be directly determined. The main other disadvantage of the axenic test system is the difficulty in preventing accidental contamination. These procedures could possibly be useful for conducting toxicity tests with other species of submersed macrophytes, although modifications might be necessary (1-8)².

1.3 The procedures in this guide are applicable to most chemicals, either individually or in formulations, commercial products, or known mixtures. These procedures might be used to conduct tests for dependency on temperature, light, nutrients and pH. With appropriate modification, these procedures might be used to conduct tests for contaminated surface waters and aqueous effluents (see Guide E1192). This static, axenic toxicity test might not be applicable to materials that contain microorganisms unless the sample can be filter sterilized without removing the toxicant. If the test materials are highly volatile, care should be taken to ensure that the test chambers are isolated. It might be necessary to replace the test material on a regular basis if the test material is rapidly biologically or chemically transformed in aqueous solution, or is removed from the test solutions in substantial quantities by the test chambers or organisms during the test. This toxicity test is not suitable for testing interactions between aquatic plants and other organisms, such as plant pathogens.

1.4 Results from the toxicity test outlined in this guide can be reported in terms of a 14-day IC25, IC50, or NOEC. This parameter may be based on several endpoints including inhibition of plant growth during the 14-day period, inhibition of shoot length, inhibition of root number and length, inhibition of fresh or dry weight (see Guide E1415), inhibition of oxygen production, change in membrane permeability, and change in chlorophyll a, chlorophyll b and carotenoid content extracted from sections of the plants (see Practice D3731 and Guide E1218) (9-18). All or some of these endpoint parameters may be examined depending upon the mode of phytotoxic action or researcher preference. It might be necessary to conduct the toxicity test at only one concentration to determine whether or not that specific concentration is inhibitory to plant growth and development.

1.5 This guide is arranged as follows:

1.6 The values stated in SI units are to be regarded as the standard.

1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. This standard may involve hazardous materials, operations, and equipment. See Section 9 for specific hazard statements.

5.1 Protection of aquatic areas is currently being emphasized by several agencies including the U.S. Environmental Protection Agency and Environment Canada. For pesticide registration, these agencies are beginning to require data regarding the toxicity of test chemicals to aquatic rooted vascular plants (25-28).

5.2 Toxicological research with terrestrial and aquatic vascular plants has been initiated (29) including the development of a protocol for testing with emergent macrophytes (Guide E1841) (30). However, protocols for phytotoxicity testing with freshwater submersed plants still require development. Toxicological research has been conducted using submersed macrophytes (1-8, 31-35) but standardization of the methods is required.

5.3 This guide is designed to assess the phytotoxic effects of chemicals upon a selected freshwater species of submersed aquatic macrophyte, Myriophyllum sibiricum Komarov. This species is an ecologically important submersed aquatic dicotyledon with a north temperate distribution. It is readily cultured in test tubes in the laboratory (36). Lower temperatures in autumn initiate the formation of turions on lateral branches that develop into new plants when environmental conditions become favorable (19-23). Toxicological testing with this species has demonstrated that it is an ideal species for laboratory testing since it grows readily under laboratory conditions, the toxic response is reproducible and there is very little variation between experimental replicates (9-15).

5.4 It is a common practice to use sterile plant culture when testing the direct effects of test materials upon a plant species. Sterile plant culture and toxicity testing have been conducted with algae (Practice D3978, 37-39), floating aquatic macrophytes (Guide E1415, 37, 40) and submersed aquatic plant species (2-5). An axenic testing system is designed to determine the direct effect of the test material upon the test species. There is nothing except the plant within the test system that could degrade or otherwise change the test chemical. Hydrolysis or phytolysis may occur but degradation studies can determine the rate of degradation by these means. Axenic tests are especially valuable during the initial stages of examining a new compound (for example, pesticide evaluation and registration (Tier 1 and Tier 2)) (25-27). In studies with other species of aquatic macrophytes, it has been shown that the presence of filamentous algae can cause a reduction in new shoot growth, fresh weight and chlorophyll a content of the macrophytes when compared to macrophytes grown in the absence of algae (41). The test tubes are recommended for testing because they require a small incubation area, small amount of plant tissue, small volume of test material and allow for the maintenance of a sterile culture (2, 3, 36). Furthermore, test tubes permit height measurements in situ (36).

5.5 There are numerous possible physiological and morphological endpoints that can be utilized to assess the toxicity of chemicals to this aquatic plant species. The test material effect may be assessed as a change in total plant height, growth rate, fresh or dry weight, number and total length of roots, chlorophyll a, chlorophyll b, carotenoids, membrane integrity or oxygen evolution, or any combination of these parameters. Peroxidase activity (31-35) and chlorophyll fluorescence (42) might be other endpoints that could be explored. Some endpoints have been compared (43, 44), but selected appropriate endpoint(s) based upon mode of action or route of exposure.

5.6 This toxicity test may be utilized during the pesticide registration process, to provide an early warning of potential ecosystem problems, identify hazardous chemicals before contamination of aquatic systems occurs, and help establish “margins of safety” for specific chemicals within wetlands (see Guide E1023). Plants cultured using this method have also been used to determine the effects of toxicants on plants in microcosms (45).

5.7 This test is not designed to replace field assessments of test material damage or other aquatic testing procedures, but should be used as a screening tool. It should compliment other testing so that a more complete environmental assessment is possible. It is difficult to interpret effects observed in the lab in reference to those that could be found in the environment. Currently, there is a need for additional field data to validate the results obtained in laboratory plant toxicity tests. Since this toxicity test can detect non-lethal physiological endpoints as well as morphological changes, this toxicity test could act as an early warning system for possible environmental effects. If effects are noted in this toxicity test, it could indicate that further lab and field testing may be required.