Biological test method for measuring the inhibition of growth using freshwater macrophyte: chapter 2

2.1 Species and Life Stage
2.2 Source
2.3 Culturing

2.1 Species and Life Stage

Lemna minor Linnaeus (Arales:Lemnaceae) is the species that must be used in this biological test method. Landolt clones 8434 and 7730 are recommended for use in this test.^Footnote 6 A general description of L. minor and features that distinguish it from similar species are provided in Appendix E.

The test culture, comprised of plants isolated exclusively for obtaining test organisms, must be axenic and must be used to inoculate all vessels used in a given test.^Footnote 7 Inocula from these cultures must be 7- to 10-days old and consist of young, rapidly growing colonies^Footnote 8 without visible lesions before being used to set up a given test (see Figure 2).^Footnote 9

2.2 Source

All organisms used in a test must be from the same strain. Sources of plants required to establish cultures may be culture collections, government or private laboratories that culture L. minor for toxicity tests, or commercial biological suppliers. Upon initiating cultures using organisms from outside sources, species identification must be confirmed and documented by a qualified taxonomist, experienced in identifying aquatic macrophytes.^Footnote 10 It is also important to identify the L. minor clone being used (if possible), because it has been shown that different clones of the same species can have different sensitivities (Cowgill and Milazzo, 1989; Saskatchewan Research Council (SRC), 2003, 2005)^Footnote 11. Periodic (e.g., annual) taxonomic checks of the laboratory’s culture, or replacements (i.e., renewal) of the culture from a recognized culture collection, are also advisable to ensure that the laboratories L. minor culture hasn’t been contaminated with other Lemna species or clones, especially if the laboratory maintains several different Lemna cultures.

Axenic and non-axenic cultures of L. minor can be obtained from the following Canadian source:

University of Toronto Culture Collection^Footnote 12
Dept. of Botany, University of Toronto
25 Willcocks St., Toronto, Ontario
Canada, M5S 3B2

Telephone:(416) 978-3641
Facsimile: (416) 978-5878
e-mail: jacreman@eeb.utoronto.ca

Web site: Canadian Phycological Culture Centre

Lemna minor: University of Toronto Culture Collection (UTCC) 490^Footnote 13 and 492^Footnote 14.

Figure 2 General Appearance of Healthy and Unhealthy Lemna minor

(a) Normal control growth in plastic test cup containing modified American Public Health Association (APHA) medium, showing fronds with variable shades of green. (b) Test culture in Hoagland's medium (left) and acclimation culture in modified APHA medium (right), both “uncrowded”. (c) Colonies with “snake-bite” lesions from long-term iron deficiencies when cultured in the original (EC, 1999b) Hoagland’s E+ medium. (d) Cultures showing chlorosis (loss of chlorophyll/yellowing) of fronds in plastic test cup.

Long Description of Figure 2

This figure is comprised of four photographs of Lemna minor in various solutions and states of health. Picture A shows the normal growth of healthy plants in modified APHA medium. The fronds floating on the medium are variable shades of green. Photograph B shows more healthy plants in two containers. One of the containers contains Hoagland’s medium while the other contains APHA medium. The photo is attempting to show how “uncrowded” cultures appear. The plants in the culture cover most of the solutions surface but there are gaps between plants and there is little to no overlap of fronds. Photograph C is a close-up of three or four plants. The photo is illustrating “snake-bite” lesions on the fronds that are resultant from long-term iron deficiency. The lesions are small and appear as two white dots which are side by each on the otherwise green fronds. Photograph D is showing plants that are suffering from chlorosis (a loss of chlorophyll / yellowing). The structure of the plants is similar to what can be seen in photographs A or B but the fronds are distinctively less green and appear more yellow in colour.

2.3 Culturing

2.3.1 General

Recommended or required conditions and procedures for culturing L. minor are discussed here and summarized in Table 1. These are intended to allow some degree of inter-laboratory flexibility while standardizing those conditions which, if uncontrolled, might affect the health and performance of the test organisms. A large portion of Section 2.3 is derived from SRC (1997) and Organization for Economic Cooperation and Development (OECD) (1998, 2002).

Table 1 Checklist of Recommended Conditions and Procedures for Culturing Lemna minor
Source	culture collection, biological supply house, government laboratories, or private laboratories; species confirmed taxonomically and clone identified (if possible)
Culture medium	modified Hoagland’s E+ medium (see Table 2); subcultured weekly in fresh medium
Temperature	within the range 25 ± 2°C
pH	4.4 to 4.8
Lighting	continuous, full-spectrum fluorescent or equivalent; 64 to 90 µmol/(m² · s) at surface of culture media; within 15% of the selected light fluence rate throughout culture area
Test culture	5 to 10 plants transferred from a week-old test tube culture to sterile, modified Hoagland’s E+ medium and incubated for 7 to 10 days under test conditions
Acclimation	7- to 10-day old plants from test culture transferred to fresh test medium for 18 to 24 hours before testing
Health criteria	in order for the test culture to be acceptable for use in the test, the frond number must increase to ≥8-times (i.e., ≥24 fronds) the original frond number in 7 days in a culture set up for monitoring organism health; plants in test culture must appear healthy

If organisms are obtained from an outside laboratory or culture collection, plants must be cultured in the laboratory for a minimum of 3 weeks before being used.

Axenic stock cultures can be maintained by the weekly subculture of 1 plant into approximately 25 mL of sterile modified Hoagland’s E+ medium (SRC, 2003) in 25 × 150 mm test tubes with Kimcaps™. Lemna is aseptically transferred into test tubes containing fresh modified Hoagland’s E+ medium and incubated on an angle under controlled light and temperature.

Cloudy medium in a Lemna stock culture indicates bacterial contamination, whereas contamination with mould may not be clearly evident until large colonies appear in the medium or a slime layer develops on the vessel. Contaminated Lemna cultures (e.g., with algae, protozoa, fungi, or bacteria) must be discarded or sterilized (see Section 2.3.7).

Cultures used for toxicity tests (i.e., test cultures) should be initiated 7 to 10 days before starting the test. For best harvest of plants having 2 to 3 fronds, prepare one or more test cultures. Aseptically transfer 5 to 10 plants from a week-old test tube culture into a 150 mm diameter petri dish (or other sterile, shallow containers) filled with sterile Hoagland’s E+ medium to a depth of at least 1 to 1.5 cm (≥100 mL), and incubate under test conditions. Test cultures should not be crowded at the end of the 7- to 10-day incubation. Cultures are considered crowded if plants cover more than two thirds of the medium surface.

For determining whether the test culture meets the health criteria outlined in Section 2.3.8, one or more vessels containing approximately 100 mL of test medium (modified APHA, Swedish Standards Institute (SIS), or modified Steinberg medium, whichever will be used in the test), is prepared each time a test culture is initiated.

Multiple subcultures of an axenic Lemna culture should be made to ensure the availability of at least one sterile culture, in case of contamination.^Footnote 15 The maintenance of a clean laboratory, good sterile technique, and the proper use of a laminar flow hood are all essential for axenic culturing of Lemna minor (Acreman 2006; see Appendix F).

A single, three-frond Lemna plant is placed into each vessel. Assuming that the cultures appear healthy (see footnote 8 and Figure 2), the culture is considered acceptable for use in the test if the number of fronds (or mean number of fronds if several vessels are used) in the vessel(s) set up for monitoring the health of the culture has increased to ≥8-times the original number of fronds in the test vessel(s), in 7 days (i.e., ≥24 fronds) (Section 2.3.8).

Cultures older than 10 days become crowded and the plants are smaller in size; such cultures should not be used for testing. The test culture is easily contaminated if exposed to non-sterile air or equipment. If the medium becomes cloudy, indicating bacterial contamination, the Lemna cannot be used and must be replaced with an uncontaminated culture (see Section 2.3.7).

The day before the test is to be set up, sufficient L. minor (7- to 10-day old uncrowded culture in modified Hoagland’s E+ medium) are rinsed twice in test medium (see Section 3.4) by replacing the spent modified Hoagland’s E+ medium with fresh test medium (modified APHA medium, SIS medium, or modified Steinberg medium). The Lemna should then be transferred into a shallow container containing ≥2 cm fresh test medium.^Footnote 16 Lemna should not be crowded (i.e., Lemna should not be overlapping and at least one third of the surface area of the medium should be free of Lemna fronds). Incubate these acclimation cultures under test conditions for 18 to 24 hours before being used. Although the Lemna stock culture is maintained under aseptic conditions, acclimation and testing are not carried out in sterile medium. Reasonable care should be taken to avoid algal contamination of the culture and therefore, it is recommended that Lemna be handled in a laminar flow cabinet (see Appendix F).

2.3.2 Facilities and Apparatus

Lemna are to be cultured in facilities with controlled temperature and lighting (constant-temperature room, incubator, or environmental chamber).^Footnote 17 The culture area should be well ventilated to prevent the occurrence of a local temperature increase underneath the illumination equipment (Institute of Applied Environmental Research (ITM), 1990), and the air supply should be free of odours and dust. Ideally, the culturing facility should be isolated from the test facility to reduce the possibility of culture contamination by test substances or materials. Cultures should also be isolated from regions of the laboratory where stock or test solutions are prepared, effluent or other test material or substance is stored, or equipment is cleaned.

Vessels and accessories in contact with the Lemna cultures and culture media must be made of nontoxic, chemically inert material, and where necessary, should be sterile. Materials such as borosilicate glass (e.g., Pyrex™), stainless steel, porcelain, nylon, high density polystyrene, or perfluorocarbon polyethylene plastics (e.g., Teflon™), may be used to minimize leaching and sorption. Plastic vessels may be used only if duckweeds do not adhere to the walls^Footnote 18 and the test substance does not sorb to the plastic more than it does to the glass (American Society for Testing and Materials (ASTM), 1991). Materials or substances such as copper, brass, galvanized metal, lead, and natural rubber must not contact the culture vessels or media, test samples, test vessels, dilution water, or test solutions.

Items made of materials or substances other than those mentioned herein should not be used unless it has been shown that their use does not adversely affect the quality of the Lemna cultures. All culture vessels and accessories should be thoroughly cleaned and rinsed with culture water between uses. New and previously used glassware must be chemically cleaned and sterilized before use (EC, 1992a). All culture and test vessels should be covered with appropriate transparent covers to exclude dust and minimize evaporation (see Section 3.3).

Equipment recommended for the maintenance of axenic Lemna cultures includes: disposable inoculating loops, for the aseptic transfer of Lemna; an autoclave, for sterilizing glassware and media; and a sterile transfer hood (laminar flow hood) for maintaining axenic conditions (see Appendix F).^Footnote 19

2.3.3 Growth Medium

Modified Hoagland’s E+ (SRC, 2003) is the medium required for culturing L. minor that are to be used for tests involving wastewater (e.g. effluents, elutriates, leachates) or receiving water.^Footnote 20 The chemical composition of modified Hoagland’s E+ medium is presented in Table 2.

Table 2 Chemical Composition of Nutrient Stock Solutions for Preparing Modified Hoagland’s E+ Medium (SRC, 2003), Used for Culturing Lemna minor
Stock	Substance	Concentration Stock Solution (g/L)	Concentration Medium^{Table note a} (mg/L)
A^{Table note b}	Ca(NO₃)₂ · 4H₂O KNO₃ KH₂PO₄	59.00 75.76 34.00	1180.0 1515.2 680.0
B	Tartaric Acid	3.00	3.00
C^{Table note c}	FeCl₃ · 6H₂O Na₂EDTA · 2H₂O^{Table note d}	1.21 3.35	24.20 67.00
D	MgSO₄ · 7H₂O	50.00	500.0
E	H₃BO₃ ZnSO₄ · 7H₂O Na₂MoO₄ · 2H₂O CuSO₄ · 5H₂O MnCl₂ · 4H₂O	2.86 0.22 0.12 0.08 3.62	2.86 0.22 0.12 0.08 3.62
---	Sucrose	---	10.00 g/L
---	Yeast extract	---	0.10 g/L
---	Bactotryptone	---	0.60 g/L

Table notes

Footnote a

Concentration of substance in medium

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Footnote b

Add 6 mL of 6N HCl to stock solution A

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Footnote c

Add 1.2 mL of 6N KOH to stock solution C

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Footnote d

Na₄EDTA · 2H₂O can be used instead of Na₂EDTA · 2H₂O. If Na₄EDTA · 2H₂O is used, the concentrations in the stock solution and the test medium are 3.75 g/L and 75 mg/L, respectively, and KOH should not be added to stock solution C (see footnote c above)

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To prepare 1 L of modified Hoagland’s E+ medium, the following are added to 900 mL of glass-distilled, deionized water (or equivalent):

Solution A 20 mL Solution B 1 mL Solution C 20 mL Solution D 10 mL Solution E 1 mL Sucrose 10 g Yeast Extract 0.10 g Tryptone (Bactotryptone) 0.6 g

Chemicals must be reagent-grade. The medium is stirred until all the contents are dissolved. Adjust the pH to within the range of 4.4 to 4.8 with NaOH or HCl and bring the volume up to 1 L with distilled water. Autoclave for 20 minutes at 121°C and 124.2 kPa (1.1 kg/cm²). Stock solutions should be stored in the dark (i.e., dark amber or covered bottles) due to potential photosensitivity. Individual stock solutions (i.e., A, B, C, etc.) may be stored in the refrigerator (4°C) for up to one month, provided they are isolated from solvents or other potential contaminants. Once autoclaved, prepared modified Hoagland’s E+ medium can be stored for up to one month at room temperature in the dark.^Footnote 22

Other nutrient-rich media (i.e., SIS medium or Steinberg medium) can be used for maintaining cultures of L. minor to be used for chemical tests only, as long as the Lemna cultures meet the health criteria of organisms to be used in the test (Section 2.3.8).

2.3.4 Lighting

Organisms being cultured should be illuminated using continuous full-spectrum fluorescent or equivalent lighting.^Footnote 23 The light fluence rate, measured at the level of the culture medium, should be 64 to 90 µmol/(m² · s) (approximately 4000 to 5600 lux).^Footnote 24 Since light intensity tends to vary in a given space, it should be measured at several points within the culture area (at the level of the culture medium) and should not vary by more than ± 15% of the selected light fluence rate.

2.3.5 Temperature

L. minor should be cultured at a temperature of 25 ± 2°C.^Footnote 25 If cultures are maintained outside this temperature range, temperature must be adjusted gradually (≤ 3°C/day) to within the range of 25 ± 2°C, and held there for a minimum of two weeks before the test is initiated. If temperature in the culture vessels (or in one or two extra vessels set up for the purpose of monitoring water temperature) is based on measurements other than those in the vessels themselves (e.g., in the incubator or controlled temperature room within the vicinity of the culture vessels) the relationship between the readings and the temperature within the culture vessels must be established and periodically checked to ensure that the plants are being cultured within the desired temperature range.

2.3.6 pH

Lemna cultures should be at a pH range of 4.4 to 4.8. The pH of modified Hoagland’s E+ medium is around 4.6 and therefore Lemna plants will be at that pH when transferred into fresh medium. The pH, however, drifts up towards a pH of 7 to 8 as the culture ages for 7 to 10 days in modified Hoagland’s E+ medium. (Moody, 1998). The pH of Lemna cultures should not be adjusted.

2.3.7 Culture Maintenance

Several stock cultures should be prepared each week in modified Hoagland’s E+ medium, to maintain the laboratory’s stock culture in a rapidly growing state (see Section 2.3.1). Lemna that has not been subcultured on a weekly basis must be subcultured in fresh medium at least twice during the 14 days immediately preceding the test, to allow the recovery to its fast growth rate. Lemna should be subcultured each time a test is set up so that an adequate number of test organisms will be available and acclimated.

Sterilization of Lemna cultures in the event of culture contamination (e.g., with algae, protozoa, fungi, or bacteria) should be avoided if possible. It is strongly recommended that cultures showing signs of contamination be discarded rather than treated. This might be a feasible approach if several cultures are held separately. If the use of cultures having undergone sterilization cannot be avoided, a minimum 8-week period must follow sterilization before use in tests. Records (including date of sterilization, sterilization procedure applied, chemicals and quantity applied, and reason for treatment) must be kept for any cultures treated for contamination.^Footnote 26

2.3.8 Health Criteria

Individual test cultures of L. minor to be used in toxicity tests must meet the following health criteria:

the number of fronds in the vessel(s) set up for monitoring culture health must have increased to ≥8-times the original number of fronds by the end of 7 days in order for the test cultures to be valid for use in setting up a test (i.e., mean number of fronds in the vessel(s) set up for the purpose of determining culture health must be ≥24 per vessel at the end of 7 days).

This can be determined by preparing individual test vessels^Footnote 27 containing 100 mL of the test medium (modified APHA, SIS, or modified Steinberg medium) that will be used in a given test, each time a test culture is initiated (see Section 2.3.1). A single 3-frond Lemna plant is transferred from the stock culture into each vessel and incubated for 7 days. The number of Lemna fronds in each vessel are counted at the end of 7 days and if the mean number of fronds per vessel have increased to ≥8-times the original number of fronds (i.e., ≥24 fronds), then the test culture is considered acceptable for use in the test. Lemna plants from the vessels set up for monitoring culture health must not be used in the toxicity test.

The general appearance of the test culture (in modified Hoagland’s E+) must also be taken into consideration. The culture must consist of young, rapidly growing colonies without visible lesions (see Section 2.1, footnote 8, and Figure 2). Plants that appear in good condition must be used to set up the test. Characteristics indicative of good plant health include: bright green fronds with no discoloured areas.

Reference toxicity tests should be conducted monthly with the Lemna culture(s), when toxicity tests are being conducted on a regular basis in the laboratory, using the conditions and procedures outlined in Section 4.6. Alternatively, a reference toxicity test should be performed in conjunction with the toxicity test. Related criteria used to judge the health and sensitivity of the culture, according to the findings of this and earlier reference toxicity tests, are given in Section 4.6.

Footnotes

Footnote 6

The Landolt 8434 Lemna minor clone was collected from the Niagara Peninsula, Ontario in 1977, and isolated in axenic cultures in Zürich, Switzerland. The Landolt 7730 Lemna minor clone was collected from Elk Lake, British Columbia in 1973 and isolated in axenic cultures in Zürich, Switzerland. Both L. minor clones are available from the University of Toronto Culture Collection (see Section 2.2).

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Footnote 7

For greater standardization, a culture grown from a single isolated plant can be used to inoculate all the flasks used in a given test (United States Environmental Protection Agency (USEPA), 1992; 1996).

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Footnote 8

Good quality cultures are indicated by a high incidence of colonies comprised of at least two fronds (2-4 fronds). A large number of relatively small single fronds (with or without two unsatisfactorily developed fronds) is indicative of environmental stress, e.g., nutrient limitation, and plant material from such cultures must not be used for testing. L. minor in its most intensive growth phase (younger plants) are lighter in colour, have shorter roots, and consist of two to three fronds of different size (ITM, 1990; OECD, 1998, 2002).

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Footnote 9

SRC (1995) growth curves indicate that the most intensive growth phase for L. minor in modified Hoagland’s E+ medium is between Days 7 and 10. USEPA (1992; 1996) and Association Française de Normalisation (AFNOR) (1996) recommend cultures < 2 weeks old be used as test inocula.

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Footnote 10

The taxonomy of Lemna species is complicated by the existence of numerous phenotypes. Also, taxonomic keys are based mainly on the flowering and fruiting characteristics of Lemna and contain relatively few diagnostic vegetative characteristics. Since flowering and fruiting are rarely observed in Lemna species, positive taxonomic identification can be extremely challenging. L. minor, for example, can only be positively differentiated from another closely related species Lemna turionifera by the lack of overwintering turions and the lack of reddish anthocyanin blotches on the ventral side of L. minor. These characteristics are produced only under culturing conditions that differ substantially from those commonly used to culture Lemna in laboratories.

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Footnote 11

Cowgill and Milazzo (1989) tested four different clones of L. minor in modified Hoagland’s medium with various concentrations of selenium (Se), vanadium (V), cobalt (Co), and tin (Sn), to determine the optimum levels of these elements in culture medium for plant growth. They found that the clones varied in their responses. Clone 6591 showed no increase in growth with Sn and Co added to the Hoagland’s medium and their biomass (dry weight) peaked with 8.4 µg/L of Se and 12.8 µg/L of V. Clone 7102 achieved peak biomass at 8.4 µg/L of Se, 685 µg/L of Sn, and 10.2 µg/L of Co added to the medium. Clone 7101 also achieved peak biomass at 8.4 µg/L of Se and 685 µg/L of Sn added to the medium, but showed no increase in growth on addition of V and Co. Clone 7136, however, performed best with no Sn, V, Se, or Co added to the modified Hoagland’s medium.

In more recent studies (SRC, 2003, 2005), the sensitivity of various L. minor clones (UTCC 490, 492, and 620) differed, depending on the toxicant to which they were exposed and the methodology followed. IC25 values for zinc (Zn), cadmium (Cd), copper (Cu), and nickel (Ni) were not significantly different between UTCC strains 490 and 492 (SRC, 2003). In addition, there was no significant difference in IC25s for UTCC strains 490, 492, and 620 exposed to potassium chloride (KCl) (SRC, 2005). IC25s for Ni based on frond count, however, were 4 times higher for UTCC 620 compared to UTCC 492 using the Environment Canada method (EPS 1/RM/37), whereas the IC25s for Ni were not significantly different between UTCC clones 492 and 620 using the methodologies outlined in the ISO draft standard (SRC 2005).

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Footnote 12

Certificates of taxonomic confirmation should be obtained upon acquisition of the Lemna culture for future reference and evidence of culture integrity.

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Footnote 13

UTCC 490: Axenic culture; Landolt clone 8434; Niagara Peninsula, Ontario, Canada.

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Footnote 14

UTCC 492: Axenic culture; Landolt clone 7730; Elk Lake, British Columbia, Canada.

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Footnote 15

Larger vessels (e.g., 250-mL or 125-mL Erlenmeyer flasks containing 100 mL or 50 mL of modified Hoagland’s E+ medium) can be used to sustain well-growing healthy cultures as long as sterility is maintained.

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Footnote 16

The SRC (1995) attempted a longer acclimation in modified APHA medium (test medium); however, they observed increasing deterioration of control growth with longer cultivation in the medium, particularly at test loading. Good quality plants could be obtained up to 7 days, but thereafter the plants deteriorated and grew poorly in the test. The SRC (1995) concluded that it is better to culture Lemna in “rich” media, such as modified Hoagland’s E+, followed by a defined pre-cultivation period in the test medium before testing in “lean” medium is carried out.

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Footnote 17

Water baths are not acceptable because they prevent proper illumination of the culture vessels (ASTM, 1991).

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Footnote 18

Plastic cups may be soaked in clean water before use to reduce the static charge and therefore the possibility of plants sticking to the sides of the vessels.

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Footnote 19

The following procedures are recommended for laboratories that are not equipped with a laminar flow cabinet. A small pre-sterilized space with minimal air flow is recommended for handling and/or transferring Lemna. This can be done by building an opaque Plexiglass™ hood, equipped with a UV light for pre-sterilization of the work space within the hood. The light can be left on when the hood or transfer room is not in use but must be turned off when the hood is in use (exposure to UV light is highly dangerous to skin and eyes). A bunsen burner and a gas source (or a portable, gas bunsen burner) is needed to conduct aseptic culturing techniques (i.e., for flaming the mouths of culture test tubes and media vessels, etc.). Handling of the plant should be minimal and transfers should be carried out as quickly as possible (Acreman, 1998).

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Footnote 20

The SRC (1995) found that the highest quality Lemna plants can be obtained from fast growing cultures in Cowgill and Milazzo’s (1989) Hoagland’s E+ medium. This medium contains high levels of organic and inorganic nutrients and trace metals. Subsequent research at SRC (2003), resulted in further modifications of Hoagland’s E+ medium (now recommended herein) for improved long-term health of L. minor cultures. These modifications included the replacement of separate iron (Fe) and EDTA solutions with a combined solution (Stock C) containing increased amounts of Fe and EDTA (SRC, 2003). Modified APHA medium is required only as a test medium since it produces fronds of excellent quality in the short-term; however, it is unsuitable for long-term cultivation of Lemna (SRC, 1995).

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Footnote 21

The use of BDH #7213 Peptone from casein trypsin-digested is an acceptable alternative to Bacto-tryptone (SRC, 1997).

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Footnote 22

A large batch of modified Hoagland’s E+ can be prepared, autoclaved as smaller aliquots (i.e., in 1 L bottles), and stored for future use. Each aliquot of medium should be used up within a short period of time after opening (i.e., not re-stored for future use), in order to reduce the risk of contamination of the medium. Any stocks or prepared media that contain precipitates or algae, or that show any signs of deterioration should not be used.

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Footnote 23

Both warm- and cool-white fluorescent lights have been used for culturing L. minor (Appendix C). Full-spectrum light, which is recommended for both culturing and testing in this method, is more representative of natural light conditions than cool-white light, and is being used with increased frequency for photosynthetic plant testing (SRC, 1995).

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Footnote 24

This conversion of µmol/(m² · s) to lux assumes an average wavelength of 550 nm, which is the average wavelength of many common laboratory light sources for visible light (e.g., cool-white fluorescent).

However, if the light source has a spectral quality that is not centred at 550 nm (e.g., outside the 400 to 700 nm range), the assumed wavelength for conversion of µmol/(m² · s) to lux will have to be adjusted (see ASTM, 1995).

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Footnote 25

To reduce the frequency of culture maintenance, e.g., when no Lemna tests are planned for a period, plants can be held under reduced illumination and temperature (4 to 10°C). Under these conditions, subculturing may be conducted less frequently. Intervals of up to three months have been found to be acceptable (OECD, 1998, 2002). According to the Swedish method (ITM, 1990), stock cultures can be stored at a temperature of 8 to 10°C in subdued lighting (e.g., 2 × 10 Watt warm-white fluorescent tubes).

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Footnote 26

Surface sterilization can be used to eliminate contaminating organisms (e.g., algae) from a stock culture. A sample of contaminated plant material is taken, and the clonal clusters separated from each other. The individual plants should then be dipped into a 0.5% (v:v) sodium hypochlorite solution for at least 1 min. The plants may be treated with bleach for varying amounts of time to ensure that at least one culture is both sterile and alive. The plant material is then rinsed several times with sterile water or medium and transferred into fresh culture medium. Many fronds will die as a result of this treatment, especially if longer exposure periods are used, but some of those surviving will usually be free of contamination. Properly sterilized plants will have a small green area in the bud zone along the center of the frond. Surviving plants can then be used to re-inoculate new cultures (see Appendix F) (AFNOR, 1996; OECD, 1998, 2002; Acreman, 2006).

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Footnote 27

Different types of test vessels (i.e., plastic cups, Erlenmeyer flasks, beakers) produce significantly different performance in the controls. Laboratories can assess the suitability of their choice of test vessel as well has the health of the culture if the test vessel used to set up the health test is the same as that to be used for substance/material testing.

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