Biological test method for measuring the inhibition of growth using freshwater macrophyte chapter 8

Section 8: Reporting Requirements

8.1 Minimum Requirements for a Test-Specific Report
8.2 Additional Reporting Requirements

Each test-specific report must indicate if there has been any deviation(s) from any of the must requirements delineated in Sections 2 to 7 of this biological test method, and, if so, provide details of the deviation(s). The reader must be able to establish from the test-specific report whether the conditions and procedures preceding and during the test rendered the results valid and acceptable for the use intended.

Section 8.1 provides a list of items that must be included in each test-specific report. Section 8.2 lists items that must either be included in the test-specific report, provided separately in a general report, or held on file for a minimum of five years. Specific monitoring programs, related test protocols, or regulations might require selected test-specific items listed in Section 8.2 (e.g., details about the test substance or material and/or explicit procedures and conditions during sample/subsample collection, handling, transport, and storage) to be included in the test-specific report, or might relegate certain test-specific information as “data to be held on file”.

Procedures and conditions common to a series of ongoing tests (e.g., routine toxicity tests for monitoring or compliance purposes) and consistent with specifications in this document, may be referred to by citation or by attachment of a general report that outlines standard laboratory practice.

Details pertinent to the conduct and findings of the test, which are not conveyed by the test-specific report or general report, must be kept on file by the laboratory for a minimum of five years so that the appropriate information can be provided if an audit of the test is required. Filed information might include:

a record of the chain-of-continuity for samples tested for regulatory or monitoring purposes;
a copy of the record of acquisition for the sample(s);
chemical analytical data on the sample(s) not included in the test-specific report;
bench sheets for the observations and measurements recorded during the test;
bench sheets and warning chart(s) for the reference toxicity tests;
detailed records of the source of the test organisms, their taxonomic confirmation, and all pertinent information regarding their culturing and health; and
information on the calibration of equipment and instruments.

Original data sheets must be signed or initialled, and dated by the laboratory personnel conducting the tests.

8.1 Minimum Requirements for a Test-Specific Report

The following lists items that must be included in each test-specific report.

8.1.1 Test Substance or Material

brief description of sample type (e.g., chemical or chemical substance, effluent, elutriate, leachate, or receiving water), if and as provided to the laboratory personnel;
information on labelling or coding for each sample/subsample;
date of sample/subsample collection;
date and time sample(s)/subsample(s) are received at the test facility;
dates or days during the test when individual sample(s) or subsample(s) were used;
measurement of the temperature of wastewater or receiving-water sample or, for multiple subsamples, measurement of the temperature for one (only) of the subsamples upon receipt at the test facility;
measurement of the pH of sample(s) or subsample(s) of wastewater or receiving water just before it is prepared and used in the toxicity test; and
date of elutriate generation and description of procedure for preparation; dates or days during an elutriate test when individual samples or subsamples were used.

8.1.2 Test Organisms

species, clone identification code (if known), and origin of culture;
age (i.e., 7 to 10 days) of test culture used to provide inocula of test organisms at start of test;
indication as to whether test culture is axenic;
growth medium used for culturing Lemna minor;
test medium in which Lemna were acclimated for the 18 to 24 hours before test start;
data showing increase in frond number in vessels setup to monitor health; and
any unusual appearance or treatment of the test culture, before it is used in the test.

8.1.3 Test Facilities and Apparatus

name and address of test laboratory;
name of person(s) performing the test; and
brief description of test vessels (size, shape, and type of material).

8.1.4 Control/Dilution Water

type of test medium used as control and dilution water;
type and source of water used to prepare test medium; and
type and quantity of chemical(s) used to prepare control/dilution water.

8.1.5 Test Method

citation of the biological test method used (i.e., as per this document);
indication as to whether test is performed with or without renewal of test solutions and, if static-renewal test, frequency of renewals;
design and description if specialized procedure (e.g., test performed with and without filtration of sample; test performed with and without adjustment of sample pH; preparation and use of elutriate; preparation and use of solvent and, if so, solvent control) or modification of standard test method;
brief description of frequency and type of observations and measurements made during test; and
name and citation of program(s) and methods used for calculating statistical endpoints.

8.1.6 Test Conditions and Procedures

design and description of any deviation(s) from or exclusion(s) of any of the procedures and conditions specified in this document;
number, concentration, volume, and depth of solutions in test vessels including controls;
number of fronds per plant and number of plants per test vessel at start of test;
number of replicates per treatment;
brief statement (including procedure, rate, and duration) of any pre-aeration of samples or test solutions before starting the test;
description of the procedure for sample filtration (i.e., pore size of filters, number of filtrations, type of filter paper, etc.), if applicable;
type and quantity of chemicals added to test sample before starting the test (i.e., nutrient-spiking);
brief description of any sample or test solutions receiving pH adjustment, including procedures;
all required (see Section 4.4) measurements of temperature and pH in test solutions (including controls), and measurements of light fluence rate made during the test; and
dates and times when test was started and ended;
brief statement indicating whether the reference toxicity test was performed under the same experimental conditions as those used with the test sample(s); and description of any deviation(s) from or exclusion(s) of any of the procedures and conditions specified for the reference toxicity test in this document.

8.1.7 Test Results

number of fronds and frond appearance in each test vessel as noted during each observation period over the 7-day exposure;
for each treatment including the control treatment(s): the mean ± SD (standard deviation) for the increase in frond number, as determined at test end;
for each treatment including the control treatment(s): the mean ± SD for the dry weight of Lemna fronds determined at test end;
any ICp (inhibiting concentration for a specified percent effect) (together with its 95% confidence limits) determined for the growth (i.e., increase in frond number during the test and frond dry weight attained at test end) using concentrations corrected for the volume of nutrient stock; details regarding any weighting techniques applied to the data; and indication of quantitative statistic used;
any outliers and the justification for their removal;
the results and duration of any toxicity tests with the reference toxicant(s) performed within 14 days of the test, together with the geometric mean value (± 2 SD) for the same reference toxicant(s), test species and clone, and test medium as derived at the test facility in previous tests using the procedures and conditions herein;
any findings of significant growth stimulation, expressed as % stimulation, at any concentration(s); and
anything unusual about the test, any problems encountered, any remedial measures taken.

8.2 Additional Reporting Requirements

The following list of items must be either included in the test-specific report or the general report, or held on file for a minimum of five years.

8.2.1 Test Substance or Material

identification of person(s) who collected and/or provided the sample/subsample;
records of sample/subsample chain-of-continuity and log-entry sheets; and
conditions (e.g., temperature, in darkness, in sealed container, etc.) of samples/subsamples upon receipt, during storage, and just before use.

8.2.2 Test Organisms

name of person(s) who identified the organisms and the taxonomic guidelines used to confirm species;
history of laboratory culture;
description of culture conditions and procedures including: lighting (fluence rate, quality, and photoperiod) and temperature conditions; composition of culture medium; and procedures and conditions for preparation and storage of culture medium;
frequency of culture renewal;
procedures, observations, and records related to the purity of stock cultures; and
records of all Lemna growth curves performed to monitor culture health and performance.

8.2.3 Test Facilities and Apparatus

description of system for regulating light and temperature within the culturing and test facilities; and
description of procedures used to clean, rinse, and sterilize test apparatus.
records of maintenance and performance checks conducted on apparatus (e.g., laminar air flow hoods, growth cabinets, meters, scales, pipettes).

8.2.4 Control/Dilution Water

sampling and storage details if the control/dilution water was “upstream” receiving water;
details regarding any water pretreatment (i.e., procedures and conditions for filtration, sterilization, aeration; adjustment of temperature and/or pH);
any ancillary water-quality variables measured before and/or during the toxicity test; and
storage conditions and duration before use.

8.2.5 Test Method

description of previous experience the laboratory has had with this biological test method for measuring toxicity using L. minor;
procedure used in preparing and storing stock and/or test solutions of chemicals; description and concentration(s) of any solvent used;
methods used (with citations) for chemical analyses of sample or test solutions (including details on sampling, sample/solution preparation and storage, before chemical analyses); and
use and description of preliminary or range-finding test.

8.2.6 Test Conditions and Procedures

photoperiod, light source, and fluence rate adjacent to the surface of test solutions;
appearance of sample and test solutions before and after sample filtration and any change in appearance noted during test;
water quality measurements for culture/control/dilution water;
any other physical or chemical measurements on sample, stock solutions, or test solutions (e.g., concentrations of one or more specific chemicals before and/or at time of the test);
conditions, procedures, frequency, dates, and times for toxicity tests with reference toxicant(s) using L. minor; and
chemical analyses of concentrations of chemical in test solutions of reference toxicant.

8.2.7 Test Results

results for any range-finding test(s) conducted;
results for any statistical analyses conducted both with outliers and with outliers removed; for regression analyses, hold on file information indicating sample size (e.g, number of replicates per treatment), parameter estimates with variance or standard error, any ANOVA (analysis of variance) table(s) generated, plots of fitted and observed values of any models used, results of outlier tests, and results of tests for normality and homoscedasticity;
growth curves, if generated;
control/warning chart showing the most recent and historic results for toxicity tests with the reference toxicant(s);
graphical presentation of toxicity data; and
original bench sheets and other data sheets, signed and dated by the laboratory personnel performing the test and related analyses.

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