Biological test method for measuring the inhibition of growth using freshwater macrophyte: chapter 4


Section 4: Universal Test Procedures

Procedures described in this section apply to each of the toxicity tests for samples of chemical, wastewater, and receiving water described in Sections 5, 6, and 7. All aspects of the test system described in Section 3 must be incorporated into these universal test procedures. A summary checklist in Table 3 describes recommended universal procedures for performing growth inhibition tests with Lemna minor, as well as conditions and procedures for testing specific types of substances or materials.

Universal procedures are described herein for performing a 7-day toxicity test. They include the following two test options:

  1. a static test, where the test solutions are not renewed during the test; and
  2. a static-renewal test, where the test solutions are replaced at least every three days during the test.

The static-renewal option is recommended for test solutions where the concentration of the test substance (or a biologically active component) can be expected to decrease significantly during the test periodFootnote 38 due to factors such as volatilization, photodegradation, precipitation, or biodegradation (Institute of Applied Environmental Research (ITM), 1990; Organization for Economic Cooperation and Development (OECD), 1998, 2002).Footnote 39

Biological endpoints measured are the increase in frond number during the test, as well as the dry weight of fronds at the end of the test.

4.1 Preparing Test Solutions

All vessels, measurement and stirring devices, Lemna transfer apparatus (e.g., inoculating loops), and other equipment must be thoroughly cleaned and rinsed in accordance with standard operating procedures (see EC [1992a] for glassware cleaning procedures). Distilled or deionized water should be used as the final rinse for items that are to be used immediately in setting up the test. If items are to be stored, they should be rinsed in distilled or deionized water, oven dried, and covered to avoid contamination before use.

For a given test, the same control/dilution water (test medium) must be used for preparing the control and all test concentrations. Fresh control/dilution water should be prepared as outlined in Section 5.3 if testing chemicals, Section 6.3 if testing wastewaters, and Section 7.3 if testing receiving waters.

Table 3 Checklist of Recommended Test Conditions and Procedures for Conducting Toxicity Tests Using Lemna minor
Universal
Test type
  • static or static-renewal; 7-day test
Solution renewal
  • at least every three days for static-renewal option; none for static option
Control/dilution water
  • test medium (modified American Public Health Association (APHA) medium, Swedish Standards Institute (SIS) medium, or modified Steinberg medium); nutrient-spiked receiving water (spiked with the same nutrients used in test medium) to assess toxic effect at a specific location (for this option, there must be an additional control comprised of the test medium)
Test organisms
  • Lemna minor from 7- to 10-day old culture (test culture), acclimated for 18 to 24 hours in test medium; two, 3-frond plants/replicate
Number of concentrations
  • minimum of 7, plus control(s); recommend more (i.e., >8), plus control(s)
Number of replicates

For single-concentration test:

  • ≥3 replicates/treatment

For multi-concentration test:

  • ≥4 replicates/treatment for equal replicate test design; or
  • regression design; unequal replicates among test treatments:
    • 6 replicates for control(s)
    • 4 replicates for lowest 3-5 test concentrations
    • 3 replicates for highest 4-5 test concentrations
Vessel/solution
  • test vessels should be disposable polystyrene cups or Erlenmeyer flasks; may be crystallizing dishes, petri dishes, or glass beakers; require no overlapping of Lemna fronds in controls at test end; volume ≥100 mL, preferably 150 mL; covered
  • test vessels should be placed on a non-reflective dark background for test duration
Temperature
  • daily mean of 25 ± 2°C throughout the test
Filtration
  • none for wastewater samples, unless algae present; receiving-water samples must be filtered through glass fibre filters (pore size ~1µm); additional filtration through 0.22 µm filters is optional.
Nutrient spiking
  • test samples are spiked with the same nutrients, at the same concentrations as those in the test medium; receiving-water samples or wastewater samples containing algae are spiked following sample filtration (if sample filtration is required)
Aeration
  • wastewater and receiving-water samples must be gently pre-aerated for 20 minutes at a minimal rate (e.g., 100 bubbles per min.) before test initiation or renewal of test solutions
pH
  • no adjustment if pH of test solution is in the range 6.5 to 9.5 for tests with modified APHA medium, 6.0 to 8.0 for SIS medium and 5.0 to 8.0 for modified Steinberg medium; a second (pH-adjusted) test might be required or appropriate, for pH beyond this range
Lighting
  • Full-spectrum (fluorescent or equivalent); light must be continuous, and selected light fluence rate must be 64 to 90 µmol/(m2 · s) at surface of test solution; fluence rate in the entire test area should be within ±15% of the selected light intensity
Observations
  • number of fronds and appearance at test start and test end (Day 7); dry weight at test end; optional counting of fronds on two other occasions during the test for growth rate calculation
Measurements
  • temperature measured daily in representative vessels; for static test, pH measured at start and end of the test in representative concentrations; for static-renewal test, pH measured at start and end of test and before and after each test solution renewal in representative concentrations; light fluence rate measured at several locations in the test area once during the test
Endpoints
  • growth based on increase in the number of fronds during the test and dry weight at the end of the test; if multi-concentration test, ICp (inhibiting concentration for a percent effect)
Reference toxicant
  • Ni or KCl; 7-day test for ICp (growth) started within 14 days of test-period, following the same procedure (modified APHA, SIS, or modified Steinberg) as the definitive test
Test validity
  • invalid if the number of fronds in controls at the end of the 7-day test period is <8-times the original number of fronds (i.e., the mean number of fronds per control vessel is <48 at test end)
Chemicals
Solvents
  • only in special circumstances; maximum concentration 0.1 mL/L; a second control with solvent is required
Concentration
  • recommended measurements are at the beginning and end of exposure, in high, medium, and low strengths and in the control(s) for the static option; and at the beginning and end of each renewal period, in high, medium, and low strengths and in the control(s) for the static-renewal option
Control/dilution water
  • SIS or modified Steinberg medium; APHA medium if metals are being tested; nutrient-spiked receiving water can be used if the objective is to assess local toxic effect(s)
Effluents, Elutriates, and Leachates
Sample requirement
  • for static tests performed off-site, a single sample is collected (or prepared, if elutriate); for static-renewal tests performed off-site, either 3 subsamples from a single sampling or ≥3 separate samples are collected (or prepared, if elutriate) and handled as indicated in Section 6.1; for on-site tests, samples are collected every 3 days and used within 24 h; volumes of ≥1 L (single concentration test) or ≥4 L (multiple-concentration test)
Transport and storage
  • If warm (> 7°C), must cool to 1 to 7°C with regular ice (not dry ice) or frozen gel packs upon collection; transport in the dark at 1 to 7°C (preferably 4 ± 2°C) using regular ice or frozen gel packs as necessary; sample must not freeze during transit or storage; store in the dark at 4 ± 2°C; use in testing should begin as soon as possible after collection and must start within 3 days of sample collection or elutriate extraction
Control/dilution water
  • modified APHA medium; nutrient-spiked receiving water may be used for monitoring and compliance
Receiving water
Sample requirement
  • as for effluents, leachates, and elutriates
Transport and
  • as for effluents, leachates, and elutriates storage
Control/dilution water
  • modified APHA medium; nutrient-spiked “upstream” receiving water for estimating local effect(s)

The characteristics of the control/dilution water used throughout the test period should be uniform. If the static-renewal option is used, uniformity is improved in a sample if a volume of control/dilution water sufficient to complete the test is properly stored and aliquots used for the periodic renewal of test solutions (Section 4.3).

If receiving or upstream water is used as control/dilution water to simulate local situations such as effluent discharge, a chemical spill, or pesticide spraying, a second control solution must be prepared using test medium (modified APHA medium, SIS medium, or modified Steinberg medium; see Sections 5.3, 5.6, 6.3, and 6.6). Upstream or receiving water cannot be used, however, if it is clearly toxic and produces an invalid result in the control according to the criteria of this growth test.Footnote 40 In such a case, test medium should be used as control/dilution water.

The temperature of the control/dilution water and the sample or each test solution must be adjusted as necessary to within ± 2°C of the test temperature, before starting the test. Sample or test solutions may be adjusted to the test temperature by heating or chilling in a water bath, but must not be heated by immersion heaters, since this could alter chemical constituents and toxicity.

If a sample requires filtration (i.e., receiving-water sample or wastewater sample containing algae), then it is filtered through a glass fibre filter (pore size ~ 1µm, e.g., Whatman GF/C filters) before testing (see Sections 6.2 and 7.2). The pH of the sample is then recorded. An aliquot of each of the same nutrient stock solutions used to prepare the modified APHA medium (i.e., stock solutions A, B, and C) is then added to the wastewater or receiving-water sample at a ratio of 10 mL aliquot per 1000-mL sample. This dilutes the sample to 97%, which is the maximum concentration of wastewater or receiving water (or any sample that requires a v:v dilution) that can be tested. The nominal concentrations of the solutions corrected for the volume of nutrient stock (or for chemicals, measured concentrations; see Section 5.4) are adopted as the test concentrations.

Samples of effluent, elutriate, leachate, and receiving water must then be pre-aerated before they are used to set up test solutions. Pre-aeration of spiked wastewater and receiving-water samples serves to equilibrate the sample with the added nutrients and stabilize the sample pH after the addition of the nutrient stock solutions. Oil-free compressed air should be dispensed through airline tubing and a disposable plastic or glass tube (e.g., capillary tubing or a pipette with an Eppendorf tip) with a small aperture (e.g., 0.5 mm ID). The rate of aeration should not exceed 100 bubbles/minFootnote 41, and the duration of pre-aeration must be 20 minutes.Footnote 42

Adjustment of sample/solution pH might be necessary (see Section 4.3.1). Solutions of hydrochloric acid (HCl) or sodium hydroxide (NaOH) at strengths ≤1 N should normally be used for all pH adjustments. Some situations (e.g., effluent samples with highly buffered pH) could require the use of higher strengths of acid or base.

For any test that is intended to estimate the ICp (see Section 4.5), at least seven concentrations plus a control solution (100% test medium) must be prepared, and more (>8 plus a control) are recommended to improve the likelihood of bracketing each endpoint sought. An appropriate geometric series may be used in which each successive concentration is about a factor of 0.5 of the previous one (e.g., 100, 50, 25, 12.5, 6.3, 3.1, 1.6 or, in the case of wastewater and receiving-water samples, 97, 48.5, 24.3, 12.1, 6.1, 3.0, 1.5). Test concentrations may be selected from other appropriate dilution series (e.g., 100, 75, 56, 42, 32, 24, 18, 13, 10, 7.5; see column 7 in Appendix G). Usually, there is not a great improvement in precision of the test from the use of concentrations closer together than those obtained with the 50% dilution. In routine tests, concentrations should not be more widely spaced than those obtained using a factor of 0.3, because this leads to poor precision of the toxicity endpoint estimate. If there is considerable uncertainty about the toxic levels, more concentrations should be used to obtain a greater spread, rather than using a lower dilution factor for wider spacing.

Test dilutions can be prepared directly in the test vessels. First, the appropriate volumes of control/dilution water are pipetted into the individual test vessels. Nutrient-spiked, pre-aerated test sample is then added to each test vessel, and the mixtures thoroughly mixed to achieve the desired test concentrations. Alternatively, test dilutions can be prepared in volumetric flasks and then distributed to the replicate test vessels. Test vessels are left at room temperature for 1 h to allow equilibration of the medium and toxicant.

In cases of appreciable uncertainty about sample toxicity, it is beneficial to run a range-finding (or screening) test for the sole purpose of choosing concentrations for the definitive test. Conditions and procedures for running the screening test should be identical to the definitive test; however, the experimental design might differ. A wide range of concentrations (e.g., ≥2 orders of magnitude) should assist in selection of the concentrations for the definitive test.

Single-concentration tests used for regulatory purposes (e.g., pass/fail), would normally use full-strength (or 97% in the case of this method) effluent, leachate, receiving water, elutriate, or an arbitrary or prescribed concentration of chemical. Use of controls would follow the same rationale as multi-concentration tests. Single-concentration tests are not specifically described herein, but procedures are evident, and all items apply except for testing only a single concentration and a control.

For a single-concentration test, a minimum of three replicate test vessels and three replicate control vessels must be set up. For a multi-concentration test, either equal or unequal replication across treatments can be used. If replication is equal across treatments, at least four replicate test vessels must be set up for each treatment. Alternatively, unequal replication across treatments (i.e., regression design) may be used when historical data is available and/or the laboratory has experience with the dose response.Footnote 43 If replication is unequal across treatments, six replicate vessels should be prepared for the control(s), four replicate vessels should be prepared for the lowest 3-5 test concentrations, and three replicate vessels should be prepared for the highest 4-5 test concentrations.

4.2 Beginning the Test

Once the test solutions have been prepared and any permitted and/or required adjustments made for temperature, pH, and filtration (see Sections 4.1, 6.2, and 7.2), the test should be initiated.

Lemna fronds used in the test must be from cultures that satisfy the requirements indicated in Section 2.3 and the health criteria given in Section 2.3.8. For multi-concentration tests, 3-frond plants, of identical (or as identical as possible) size and condition,Footnote 44 are selected from the acclimated culture for use in setting up the test. The plants may be transferred directly from the acclimated culture into the test cups. Alternatively, 3-frond plants may be selected from the acclimated culture and transferred to a shallow dish containing fresh test medium before being transferred to the test cups. This latter procedure is particularly useful, since the investigator can ensure that there are an adequate number of Lemna plants, of identical quality, before initiating the test (Moody, 1998).

An identical number of fronds must be added to each test vessel. To begin the test, two, 3-frond Lemna plants are randomly assigned or transferred to each test vessel (for a total of 6 fronds per test vessel) using a disposable plastic sterile inoculating loop. The plants are submersed briefly in the test solution. Care must be taken to not contaminate the Lemna designated for use in the test while transferring the plants to their individual test cups. If the plants are being selected directly from the acclimated culture or from a single dish of washed Lemna allocated for use in the test (see above), a separate inoculating loop for each plant should be used or the inoculating loop should be rinsed in distilled/deionized water before it is returned into the dish of washed Lemna. Alternatively, enough Lemna plants can be placed into a shallow dish filled with test medium, designated for division between the replicates in a single test concentration. A single inoculating loop can then be used to transfer the Lemna plants into each test cup at a given test concentration. Care must be taken to ensure that the plant does not adhere to the side of the cup and that the roots are inside the cup. Any plants that break apart during the transfer process must be replaced.

In carrying out these procedures, there must be formal random assignment of organisms to test vessels. The group of replicate vessels representing a particular treatment (e.g., a specific test concentration) must also be in randomized positions in the environmental chamber or test area. The test vessels must be coded or labelled to enable proper identification of the sample and its concentration. The date and time that the test is started must be recorded on separate data sheets dedicated to the test.

Lemna transfers should be done in a clean, draft-free area, as quickly as possible, to minimize contamination of the colonies. Once the plants have been placed into the test vessels, care should be taken not to swirl or agitate the vessels as plants may adhere to the sides of the vessel. The day the Lemna plants are initially exposed to solutions of test substance is designated Day 0. Day 7, therefore, is the day the test is terminated.

4.3 Test Conditions

The duration of the L. minor growth inhibition test is 7 days. The test can be a static type, or, in the case of degradable test substances or materials or chemicals, a static-renewal test. The test solutions are not changed for the duration of the test if a static test is done.

If the static-renewal option is chosen, each test solution must be replaced every 3 days (i.e., on Days 3 and 5), or more frequently, during the test (see Sections 5.2 and 6.1).Footnote 45 Replacement solutions and test vessels should be prepared, as described in Section 4.1. Lemna colonies must be transferred carefully, with an effort to minimize contamination, to respective vessels containing fresh test solutions. The transfer of Lemna to new test solutions must be done in random order across the replicates within a concentration and should follow procedures for handling the plants (see Section 4.2). The physical/chemical characteristics of the old solutions should be determined (see Section 4.4) and then the test solutions should be discarded (following provincial and federal regulations) or stored if additional chemical determinations are required (see Section 5.4).

Tests are initiated using two Lemna plants per 100-mL (or 150-mL) volume of test solution in each replicate test vessel (see Sections 3.3 and 4.1).

The test must be conducted at a daily mean temperature of 25 ± 2°C. Light conditions must be as described in Section 3.2. Test solutions must not be aerated during the test, and the test must end seven days after initiation.

The test must be considered invalid if the mean number of fronds in the controls has not increased to ≥8-times the original number fronds by the end of the test (i.e., the mean number of fronds per control test vessel must be ≥48 at the end of the test, for the test to be valid).

4.3.1 pH

Toxicity tests should normally be conducted without adjustment of pH. However, if the sample of test substance causes the pH of any test solution to be outside the range 6.5 to 9.5 for tests with modified APHA medium, 6.0 to 8.0 for SIS medium and 5.0 to 8.0 for modified Steinberg medium, and the toxicity of the test substance rather than the deleterious or modifying effects of pH is being assessed, the pH of the test solutions or sample should be adjusted, or a second, pH-adjusted test should be conducted concurrently. For this second test, the initial pH of the sample, the test solutions, or of each fresh solution before renewal (static-renewal tests) may, depending on the objectives, be neutralized (adjusted to pH 7.0) or adjusted to within ± 0.5 pH units of that of the control/dilution water, before Lemna exposure. Another acceptable approach for this second test is to adjust the pH of the sample upwards to pH 5.0 to 7.0 (if the sample has/causes a pH < 5.0), or downward to pH 9.0 to 9.5 (if the sample has/causes a pH > 9.5). Solutions of hydrochloric acid (HCl) or sodium hydroxide (NaOH) at strengths ≤1 N should normally be used for all pH adjustments. Some situations (e.g., effluent samples with highly buffered pH) might require higher strengths of acid or base.

If sample pH is to be adjusted, it is done so after the addition of the nutrient stock solutions and pre-aeration (see Section 4.1). If adjustment of the pH by more than 0.5 units is required, a further 30-minute period of aeration followed by another pH adjustment is recommended (Saskatchewan Research Council (SRC), 1997). Abernethy and Westlake (1989) provide useful guidelines for adjusting pH. Aliquots of samples or test solutions receiving pH-adjustment should be allowed to equilibrate after each incremental addition of acid or base. The amount of time required for equilibration will depend on the buffering capacity of the solution/sample. For effluent samples, a period of 30 to 60 minutes is recommended for pH adjustment (Abernethy and Westlake, 1989). Once the test is initiated, the pH of each solution is monitored, but not adjusted. Volumes of nutrient spikes, and NaOH and HCl used in pH adjustment, must be recorded and used to calculate the nominal concentration of the test substance at the beginning of the test.

If the purpose of the toxicity test is to gain an understanding of the nature of the toxicants in the test substance, pH adjustment is frequently used as one of a number of techniques (e.g., oxidation, filtration, air stripping, addition of chelating agent) for characterizing and identifying sample toxicity. These “Toxicity Identification Evaluation” (TIE) techniques provide the investigator with useful procedures for assessing the physical/chemical nature of the toxicant(s) and their susceptibility to detoxification (United States Environmental Protection Agency (USEPA), 1991a; 1991b).

4.4 Test Observations and Measurements

The fronds in each vessel must be observed and counted at the beginning and end of the test (Day 0 and Day 7).Footnote 46 Control solutions must receive identical treatment. Observation is improved if a magnifying glass, dissecting microscope, or other magnifying device is used to observe plants and a light is directed into the side or bottom of the cup.

The number of fronds in each test vessel must be counted and recorded at each observation. The count must include every frondFootnote 47 and every visible protruding bud. Observations of the following should also be made and recorded for each test vessel: chlorosis (loss of pigment); necrosis (localized dead tissue on fronds, which appears brown or white); yellow or abnormally sized fronds; gibbosity (humped or swollen appearance); colony destruction (single fronds); root destruction; and loss of buoyancy (see Figure 2).

Temperature must be monitored throughout the test. As a minimum, temperature must be measured daily in representative test vessels (i.e., in at least the high, medium, and low concentrations plus the control solutions in a multi-concentration test). Extra test vessels may be prepared for the purpose of measuring water temperature during the test. If temperature records are based on measurements other than in the test vessels (e.g., in the incubator or controlled-temperature room within the vicinity of the test vessels), the relationship between these readings and temperature within the vessels must be established. Continuous recordings or daily measurement of the maximum and minimum temperatures are acceptable options.

For both static and static-renewal exposures, the pH must be measured at the beginning of the test, before the Lemna plants are added and at the end of the test, in at least the high, medium, and low test concentrations and in the control(s). For static-renewal exposures, the pH must also be measured immediately before and immediately after each test solution renewal (i.e., in fresh solutions and those to be discarded) in at least the high, medium, and low test concentrations and in the control(s).

Light fluence rate must be measured at least once during the test period at points approximately the same distance from the light source as the Lemna fronds and at several locations in the test area.

The general appearance of test samples and any changes that occur during the preparation of the test solutions should be noted and recorded as well as any changes in the appearance of test solutions observed during the test period (see Sections 5.4, 6.4, and 7.4).

The number of fronds are recorded for each replicate of the control and the various concentrations of the test substance at the beginning and end of the 7-day exposure. Vessels that have fronds or colonies accidentally removed or stuck (and dried) to their sides during the test should be removed from the test and that replicate should be eliminated from endpoint calculations.

Once the Lemna fronds are counted, they are dried and weighed. For each vessel of test solution, dry weight is determined for the Lemna fronds as a group. Colonies in the respective vessels (including the roots) are collected, blotted dryFootnote 48, and dried immediately in a drying oven in small tared and numbered weighing boats, at either 100°C for six hours or at 60°C for 24 hours. Upon removal from the oven, the boats must be moved immediately to a desiccator. Thereafter, the boats should be individually and randomly removed from the desiccator, and weighed on a balance that measures consistently to 0.01 mg. To avoid excessive and inconsistent absorption of water vapour, rapid weighing and standard timing among boats is necessary. Trays should be removed in random order for weighing, and the first one weighed should be replaced in the desiccator and weighed again at the end as a check on gain of water by the last trays weighed. The change should not be >5%. If it is, the trays should be re-dried for 1 to 2 hours and then re-weighed. A few weighing boats should be tared, dried, and weighed without plants, and results should conform to the laboratory’s quality control standards. The total dry weight of fronds in each test vessel (i.e., in each replicate of each test concentration and the control) must be determined.

4.5 Test Endpoints and Calculations

The endpoints of the test are based on the adverse effects of test materials or substances on the growth of L. minor, assessed by comparison with the controls. There are two biological endpoints for the test, the first is based on the reduction of the increase in the number of fronds compared to the control, and the second is based on a decrease in the final dry weight of the fronds compared to the control. The increase in frond number is calculated by subtracting the initial number of fronds in a given test vessel from the final number of fronds in the same test vessel. The biological endpoint for frond dry weight measures the total dry weight of Lemna fronds compared to the control at the end of the test (Day 7). This is essentially a measurement of growth, except that no determination of initial weight is made.

4.5.1 Validity of Test

Assuming that all the recommended procedures and conditions were followedFootnote 49, the mean number of fronds in the controls must have increased to ≥8-times the original number of fronds by the end of the 7-day test period in order for the test to be valid (i.e., mean number of fronds in the controls must be ≥48 per test vessel at the end of the test, for the test to be valid).

4.5.2 Multi-Concentration Tests

In a multi-concentration test, the required statistical endpoint for growth data (frond number, frond dry weight) is an ICpFootnote 50,Footnote 51 and its 95% confidence limits. A separate ICp and its 95% confidence limits must be calculated for each of the two biological endpoints (i.e., one for reduction of increase in frond number and one for reduction of total dry weight). For derivation of ICp and the 95% confidence limits, the quantitative measurement endpoints are used directly (i.e., increase in frond number and total dry weight). Environment Canada (2005) provides direction and advice for calculating the ICp, including decision flowcharts to guide the selection of appropriate statistical tests. All statistical tests used to derive endpoints require that concentrations be entered as logarithms and if applicable, that concentrations be corrected for the volume of nutrient stock (i.e., 97% dilution).

An initial plot of the raw data (increase in frond number, dry weight) against the logarithm of concentration is highly recommended, both for a visual representation of the data, and to check for reasonable results by comparison with later statistical computations.Footnote 52 Any major disparity between the approximate graphic ICp and the subsequent computer-derived ICp must be resolved. The graph would also show whether a logical relationship was obtained between log concentration (or, in certain instances, concentration) and effect, a desirable feature of a valid test (EC, 2005).

Regression analysis is the principal statistical technique to be used for calculation of the ICp. A number of models are available to assess growth data (using a quantitative statistical test) via regression analysis. Use of regression techniques requires that the data meet assumptions of normality and homoscedasticity. Weighting techniques may be applied to achieve the assumption of homoscedasticity. The data are also assessed for outliers using one of the recommended techniques (see Section 10.2 in EC, 2005). Any statistical analyses conducted without outliers should also be conducted with the outliers. Any outliers and the justification for their removal must be reported. Finally, the model with the best fitFootnote 53 must be chosen as the most appropriate for generation of the ICp and associated 95% confidence limits. Endpoints generated by regression analysis must be bracketed by test concentrations; extrapolation of endpoints beyond the highest test concentration is not an acceptable practice.

The ability to mathematically describe hormesis (i.e., a stimulatory or “better than control” response occurring only at low exposure concentrations) in the dose-response curve has been incorporated into recent regression models for quantitative data (see Section 10.3 in EC, 2005). Data exhibiting hormesis can be entered directly, as the model can accommodate and incorporate all data points; there is no trimming of data points which show a hormetic response.

In the event that the data do not lend themselves to regression analysis, linear interpolation (e.g., ICPIN; see Section 6.4.3 in EC, 2005) can be used in an attempt to derive an ICp. The same decision-making for statistical analysis must be followed for each of the two Lemna minor test endpoints (i.e., frond increase and frond dry weight) independently. For example, if frond increase data cannot be analyzed by regression, and the analyst defaults to ICPIN, regression analysis must still be attempted on the frond dry weight data. The fact that the first endpoint examined is analyzed by ICPIN does not preclude regression analysis for the second endpoint.

For each test concentration including the control treatment(s), the following calculations must be performed and reported: (i) the mean (± SD) of the increase in frond number in each treatment, including control(s) as determined at test end, and (ii) the mean ± SD for dry weight of Lemna fronds in each treatment, including control(s) as determined at test end.

4.5.3 Single-Concentration Tests

In single-concentration tests, the response in the test concentration is compared with the control responseFootnote 54. If frond number and dry weight (quantitative data) are assessed at a single test site and control site, a t-testFootnote 55 is normally the appropriate method of comparing the data from the test concentration with that for the control. In situations where more than one test site is under study, and the investigator wishes to compare multiple sites with the control, or compare sites with each other, a variety of Analysis of Variance (ANOVA) (or non-parametric equivalent) tests exist (Section 3.3. in EC, 2005). Choice of the test to use depends on:

  1. the type of comparison that is sought (e.g. complete a series of pairwise comparisons between all sites or compare the data for each location with that for the control only);
  2. if a chemical and/or biological response gradient is expected, and
  3. if the assumptions of normality and homoscedasticity are met.

As with multi-concentration tests, other calculations which must be performed and reported when performing a single-concentration test include: (i) the mean (± SD) of the increase in frond number in each treatment, including control(s) as determined at test end, and (ii) the mean ± SD for dry weight of Lemna fronds in each treatment, including control(s), as determined at test end.

4.5.4 Stimulatory Effects

A stimulatory effect (increased response at all concentrations or at high concentrations) must be reported for all concentrations in which significant stimulation was observed. If a stimulatory effect was observed, statistical comparison with controls is performed using ANOVA analysis, followed by appropriate pairwise comparisons with control (see Section 3.3 and 7.5 of EC, 2005). This analysis will identify which concentrations show a stimulatory effect that is significantly different from controls. The percent stimulation for these concentrations must be reported, using the following calculation (USEPA, 2002)Footnote 56:

    

equation - see long description below

Where:

  • S(%) = percent stimulation
  • T = average increase in frond number, or average total dry weight of fronds at test end in test vessel
  • C = average increase in frond number; or average total dry weight of fronds in the controls
Long description of equation

S(%) = [(T-C)/C]×100

This formula is describing how to quantify the percent stimulation that may manifest during a test. To calculate the percent stimulation the average increase in frond number in the control replicates is subtracted from the average increase in frond number in the test replicates. This number is then divided by the average increase in frond number in the control replicates and multiplied by 100. It is also possible to calculate the percent stimulation on the basis of dry weights instead of frond number.

4.5.5 Other Test Designs and Purposes

Average specific growth rate (or relative growth rate)Footnote 57 and/or area under the curveFootnote 58 can also be calculated based on frond numbers in each replicate; however, measurements at intervals during the test (e.g., Days 3 and 5) are required for both average specific growth rate and area under the curve estimate (ASTM, 1997; OECD, 1998, 2002).Footnote 59

4.6 Reference Toxicant

The routine use of a reference toxicant or toxicants is practical and necessary to assess, under standardized conditions, the relative sensitivity of the culture of Lemna being used, and the precision and reliability of data produced by the laboratory for the selected reference toxicant (EC, 1990). Sensitivity of Lemna to reference toxicant(s) must be evaluated within 14 days of the toxicity test (i.e., the reference toxicity test must be started within 14 days of the period over which the test was conducted). The same test culture (7- to 10-days old) may be used for tests with both the reference toxicant and sample(s). The reference toxicity test must be performed under the same experimental conditions as those used with the test sample(s).

Criteria used in recommending the appropriate reference toxicants for this test include:

  • chemical readily available in pure form;
  • stable (long) shelf life of chemical;
  • highly soluble in water;
  • stable in aqueous solution;
  • minimal hazard posed to user;
  • easily analyzed with precision;
  • good dose-response curve for L. minor; and
  • knowledge of the degree and type of any influence of pH on toxicity of chemical to test organism.

Reagent-grade nickel (Ni)Footnote 60 and/or potassium chloride (KCl)Footnote 61 are recommended for use as the reference toxicant(s) for this test. If Ni is used as the reference toxicant(s), it is recommended that the appropriate Material Safety Data Sheets be carefully consulted, and all necessary safety precautions be followed.

Lemna sensitivity must be evaluated by standard tests following the procedures and conditions given herein to determine the ICp for the reference toxicant(s) chosen. If nickel is chosen, nickel sulphate (NiSO4 · 6H2O) should be used to prepare the stock solutions. Fresh stock solutions should be prepared for each reference toxicity test. The concentration of nickel should be expressed as mg Ni/L. Stock solutions of KCl should be prepared on the day of testing. The control/dilution water should be appropriate for the reference toxicant used (i.e., modified APHA medium for tests with Ni and modified APHA, SIS, or modified Steinberg medium for KCl).

Concentrations of reference toxicant in all stock solutions should be measured chemically using appropriate methods (e.g., APHA et al., 1995). Upon preparation of test solutions, aliquots should be taken from at least the control, low, middle, and high concentrations, and analyzed directly or stored for future analysis, in case the ICp is outside the warning limits. If stored, sample aliquots must be held in the dark at 4 ± 2°C and preserved if necessary (see APHA et al., 1995). Stored aliquots requiring chemical measurement should be analyzed promptly upon completion of the toxicity test. It is desirable to measure concentrations in the same solutions at the end of the test after completing biological observations. Calculations of ICp should be based on measured concentrations if they are appreciably (i.e., ≥20%) different from nominal ones, and if the accuracy of the chemical analyses is satisfactory.

Once sufficient data are available, a warning chart, which plots ICp values for frond number must be prepared and updated for each reference toxicant used (EC, 1990; 2005). A separate warning chart must be prepared for each L. minor clone used in toxicity testing since the clones can differ in their sensitivity to toxicants (see Section 2.2; footnote 11). A separate warning chart must also be prepared for each medium used in reference toxicant testing (i.e., a separate chart for testing in each of modified APHA, SIS, and modified Steinberg medium). Successive ICps are plotted on this chart and examined to determine whether the results are within ± 2 SD (= warning limits) of values obtained in previous tests using the same reference toxicant and test procedure. The mean and standard deviation of available log ICps are recalculated with each successive test until the statistics stabilize (EC, 1990; 2005). The warning chart should plot logarithm of ICp on the vertical axis against date of the test (or test number) on the horizontal axis.

The logarithm of concentration (log ICp) must be used in all calculations of mean and standard deviation. This simply represents continued adherence to the assumption by which each ICp was estimated on the basis of logarithms of concentrations. The warning chart may be constructed by plotting the logarithms of the mean and its limits on arithmetic paper, or by plotting arithmetic values on the logarithmic scale of semi-log paper. If it were definitely shown that the ICps failed to fit a log-normal distribution, an arithmetic mean and limits might prove to be more suitable.

Each new ICp for the reference toxicant should be compared with the established warning limits for frond number. The ICp is considered to be acceptable if it falls within the warning limits. If a particular ICp falls outside the warning limits, the sensitivity of the Lemna culture and the performance and precision of the test are suspect. Since this might occur 5% of the time due to chance alone, an outlying value does not necessarily mean that the sensitivity of the Lemna culture or the precision of the toxicity data produced by the laboratory are in question. Rather, it provides a warning that this might be the case. A thorough check by laboratory personnel of all culturing and test conditions and procedures is required at this time. Depending on the findings, it might be necessary to repeat the reference toxicity test, and/or to prepare a new Lemna culture before undertaking further toxicity tests with the test organisms.

Results that remained within the warning limits would not necessarily indicate that a laboratory was generating consistent results. Extremely variable data for a reference toxicant would produce wide warning limits; a new data point could be within the warning limits but still represent undesirable variability. For guidance on reasonable variation among reference toxicant data (i.e., warning limits for a warning chart), please refer to Section 2.8.1 and Appendix F in EC, 2005.

If an ICp fell outside the control limits (mean ± 3 SD), it would be highly probable that the test was unacceptable and should be repeated, with all aspects of the test being carefully scrutinized. If endpoints fell between the control and warning limits more than 5% of the time, a deterioration in precision would be indicated, and again the most recent test should be repeated with careful scrutiny of procedures, conditions, and calculations.

4.7 Legal Considerations

Care must be taken to ensure that samples collected and tested with a view to prosecution will be admissible in court. For this purpose, legal samples must be: representative of the substance or material being sampled; uncontaminated by foreign substances or materials; identifiable as to date, time, and location of origin; clearly documented as to the chain of custody; and analyzed as soon as possible after collection. Persons responsible for conducting the test and reporting the findings must maintain continuity of evidence for court proceedings (McCaffrey, 1979), and ensure the integrity of the test results.

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