Biological test method: fertilization assay using echinoids (sea urchins and sand dollars), chapter 11

Section 9: Reporting Requirements

Each test-specific report must indicate if there has been any deviation from any of the “must” requirements delineated in Sections 2 to 8 of this biological test method, and, if so, provide details as to the deviation. 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 9.1 provides a list of the items which must be included in each test-specific report. Section 9.2 gives a list of those items which 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 or related test protocols might require selected test-specific items listed in Section 9.2 to be included in the test-specific report, or might relegate certain test-specific information (e.g., details regarding the test material and/or explicit procedures and conditions during sample collection, handling, transport, and storage) as “data to be held on file”.

Procedures and conditions that are 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 which 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);
  • certain chemical analytical data on the sample(s);
  • 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 and health of the breeding stock; and
  • information on the calibration of equipment and instruments.

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

9.1 Minimum Requirements for Test-Specific Report

Following is a list of items that must be included in each test-specific report.

9.1.1 Test Substance or Material

  • Brief description of sample type (e.g., chemical or chemical substance, effluent, elutriate, leachate, receiving water, or liquid extracted from sediments or similar solids) and volume or weight (if dry chemical), if and as provided to the laboratory personnel;
  • information on labelling or coding for each sample;
  • date of sample collection; date and time sample received at test facility;
  • for effluent or leachate, measurement of temperature of sample upon receipt at test facility;
  • measurements of pH and dissolved oxygen of sample of wastewater or receiving water, just before its preparation and use in toxicity test; and
  • for a test with elutriate or any liquid extracted from sediments or similar solids, dates for sample generation and use; description of procedure for preparation.

9.1.2 Test Organisms

  • species and source;
  • brief description of holding time and conditions, for adults;
  • percentage of mortalities among adults shipped and held briefly (i.e., ≤3 d); and/or weekly percentage of mortalities among the adults being acclimated and held for longer periods (i.e., >3 d); and
  • any unusual appearance, behaviour, or treatment of adults or gametes, before the test is started.

9.1.3 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, type of material).

9.1.4 Control/Dilution Water

  • type(s) and source(s) of water used as control and dilution water;
  • type(s),source(s), and collection procedure of control and/or reference pore water used, if any; and
  • type and quantity of any chemical(s) added to control or dilution water.

9.1.5 Test Method

  • citation of biological test method used (i.e., as per this document), and options selected;
  • design and description if specialized procedure (e.g., test performed with and without filtration of sample; preparation and use of elutriate; preparation and use of solvent and, if so, solvent control);
  • brief description of procedure for preparation of hypersaline brine and duration of aging;
  • description of procedure(s), products used, and duration of aging for any salinity adjustments of control/dilution water, sample, or test solutions;
  • description of procedure(s) if any sample or test solutions received filtration and/or pH adjustment;
  • brief description of frequency and type of all observations and all measurements made during test; and
  • name and citation of programs and methods used for calculating statistical endpoints.

9.1.6 Test Conditions and Procedures

  • design and description if any deviation from or exclusion 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 replicates per treatment;
  • brief statement indicating that the gamete viability check was performed, and whether a pre-test was performed;
  • estimated number of sperm per vessel and sperm:egg ratio;
  • number of males and females used to pool sperm and eggs;
  • brief statement concerning aeration (if any, give rate, duration) of sample or test solutions before starting the test;
  • measurements of temperature, salinity, pH, and dissolved oxygen (mg/L and percent saturation) in aliquots of test solutions (including controls), at the start of the test;
  • period of time test vessels (i.e., preserved eggs) are stored prior to enumerating results;
  • date when test was started; statement of test duration; and
  • date when the reference toxicity test was performed and brief statement indicating whether it was performed under the same experimental conditions as those used with the test sample(s); and description of any deviation from or exclusion(s) of any of the procedures and conditions specified for the reference toxicity test in this document.

9.1.7 Test Results

  • for each replicate test solution (including each of the control replicates): the number of fertilized and unfertilized eggs counted in each vessel at the end of the test;
  • mean (± SD) percent fertilized eggs or proportion fertilized for each test vessel and each treatment (e.g., each concentration), including the controls;
  • ICp (together with its 95% confidence limits) for the percent fertilization or proportion fertilized data; details regarding any weighting techniques and indication of the quantitative statistics used;
  • any outliers, and the justification for their removal;
  • the duration and results of any toxicity tests with the reference toxicant(s) performed at the same time or within 14 days of the test, together with the geometric mean value (± 2 SD) for the same reference toxicant(s) as derived at the test facility in previous tests with the same species; and
  • anything unusual about the test, any problems encountered, any remedial measures taken.

9.2 Additional Reporting Requirements

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

9.2.1 Test Substance or Material

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

9.2.2 Test Organisms

  • records of taxonomic confirmation of species; all supplier’s records provided with each shipment, including number of test organisms shipped, as well as date and time of shipment; temperature and dissolved oxygen concentration of any water in shipment container(s) (or of shipment container(s) if adults are shipped dry) when shipped and upon arrival;
  • detailed description of holding conditions and procedures for adults, including: facilities and apparatus; lighting; water source and quality; water pre-treatment; water exchange rate and procedure for replacement; density of adults in tanks; temperature in those tanks;
  • type and source of food for adults in tanks; procedures for preparation and storage of food; feeding procedures, frequency, and ration;
  • incidence of diseased adults; details regarding any treatment of adults for disease;
  • records of checks and findings for maturation of gonads, spawning success and time, fecundity, and fertilization success rates before test; and
  • procedures and conditions for inducing spawning and collecting gametes, and or adding them to test vessels.

9.2.3 Test Facilities and Apparatus

  • description of systems for regulating light and temperature within the test facility; and
  • description of procedures used to clean or rinse test apparatus.

9.2.4 Control/Dilution Water

  • sampling and storage details if the control/dilution water was “upstream” receiving water;
  • details regarding any water pre-treatment (i.e., procedures and conditions for salinity adjustment, filtration, sterilization, temperature adjustment, de-gassing, aeration); and
  • measured water quality variables (Section 2.3.4) before and/or at time of starting the test.

9.2.5 Test Method

  • description of laboratory’s previous experience with this biological test method for measuring toxicity using the same procedures, conditions, and test species;
  • procedures 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; details concerning sampling, sample/solution preparation and storage, before chemical analyses; and
  • use and description of preliminary or range-finding test.

9.2.6 Test Conditions and Procedures

  • photoperiod, light source, and intensity adjacent to surface of test solutions;
  • conditions, procedures, and frequency for toxicity tests with reference toxicant(s);
  • water quality measurements for water supply used for holding and control/dilution water (see Section 2.3.4);
  • any other chemical measurements on sample, stock solutions, or test solutions (e.g., concentrations of one or more specific chemicals; suspended solids content), before and/or at time of the test; and
  • appearance of sample or test solutions; changes in appearance noted during test.

9.2.7 Test Results

  • results for any range-finding test(s) conducted in conjunction with the definitive test;
  • results of the gamete viability check and pre-test;
  • graphical presentation of dose-response data;
  • results for any statistical analyses conducted both with outliers and with outliers removed; for regression analyses, file information indicating sample size (e.g., number of replicates per treatment), parameter estimates with variance or standard error, any ANOVA table(s) generated, plots of fitted and observed values of any models used, results of outlier tests, results of tests for normality and homoscedasticity, and how the model of best fit was chosen;
  • warning chart showing the most recent and historic results for toxicity tests with the reference toxicant(s);
  • any other observed effects; 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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