Metal mining technical guidance: sublethal toxicity checklist, chapter 2
Report Assessment Checklist for the Pulp and Paper and Metal Mining EEM Program: Fertilization Assay using Echinoids (Sea Urchins and Sand Dollars)*
(Revised: May 2016)
Effluent Sample Identification
Client Name/Location: _______________________
Testing Lab Name/Location: _______________________
Instructions for Completion of Checklist
- Column one of the table lists reporting & method requirements (including the Regulations Amending the Pulp and Paper Effluent Regulations and Schedule 5 of the Metal Mining Effluent Regulations). Reporting requirements are specified in regular type, while “must” requirements are indicated in bold type.
- In column two of the table, mark under the Y if data have been reported, or under the N if data have not been provided as required by the minimum reporting of the test result.
- For column 3, this reported information can be either method minimum reporting or an EEM program requirement which is needed to evaluate whether “must” requirements have been met. If data meet the “must” requirements specified, mark under the Y in the third table column or under the N if it is not the case. Items which have no associated method or EEM “must” requirement have been hard-coded with an X under the NA.
| Reporting & Method Requirements and EEM Requirements | Reported Data? | Met “must” Requirement? | |||
|---|---|---|---|---|---|
| Y | N | Y | N | NA | |
| Effluent Sample | |||||
| Effluent type identified (e.g., process effluent, final effluent) | X | ||||
| Information on labelling/coding of sample | X | ||||
| Temperature of sample upon arrival at lab | X | ||||
| Date for sample collection and date/time for sample receipt at lab | X | ||||
| Date for test start (within 3 d of sample collection) | |||||
| Test Organisms | |||||
| Species is one of the following: Strongylocentrotus droebachiensis, Strongylocentrotus purpuratus, Dendraster excentricus, Arbacia punctulata, Lytechinus pictus | |||||
| Source (all adults used to provide gametes for a test be from the same batch and source) | X | ||||
| Brief description of holding time and conditions for adults | X | ||||
| Any unusual appearance, behaviour, or treatment of adults or gametes, before test start | X | ||||
| Any deviations from test-method-specific “must” requirements for culture/holding conditions for echinoids held in lab for >3 days or for echinoids held for immediate use (≤3 d) | |||||
| Health Criteria | |||||
| Weekly % of mortalities among the adults being acclimated and held for >3 d (≤2% per day averaged over 7d preceding collection of gametes, cumulative mortality over the same 7-d period ≤20%) | |||||
| Percentage of mortality for adults shipped and held briefly (≤3 d) (cumulative mortality ≤20% for 7-d period prior to shipment) | |||||
| Test Conditions and Facilities | |||||
| Test method (EPS 1/RM/27 and acknowledgement of February 2011 2nd Edition ) and options selected (recommend 10 min sperm + 10 min sperm & egg; options include a 20 min +20 min and a 60 min + 20 min exposure) | |||||
| Statement of test duration | X | ||||
| Description of test vessels (size, shape, type of material) | X | ||||
| Person performing test | X | ||||
| Rate of pre-aeration of sample, if required, before starting the test (≤100 bubbles/min; minimal and controlled) | |||||
| Duration of pre-aeration of sample (only if D.O. is estimated to be <40% or >100% saturation in any conc., in which case pre-aerate the sample or an aliquot of sample for ≤20 min and attaining 40% of air saturation; test initiated at this point regardless of whether D.O. of 40-100% saturation was achieved) | |||||
| Procedure, if any, for pH adjustment of sample (recommend no adjustment if pH of test solution within 6.5-8.5; pH adjusted outside this range is an option or parallel test with pH adjusted solution) | X | ||||
| Procedure, if any, for sample filtration (recommend none; if indigenous organisms, filter through a sieve with 60 µm mesh openings before use) | |||||
| Statement that EC guidance document (December 2001) on salinity adjustment has been followed | |||||
| Salinity 28-32 g/kg, adjusted using hypersaline brine, dry ocean salts, reagent-grade salts or deionized water | |||||
| After dry salt addition, was the stabilization (aging) period of 16 to 24 hours respected (4 ± 2 °C in darkness and in sealed container with minimum air space)? | |||||
| Type(s) and source(s) of control/dilution water (same water used for preparing control and test solutions) | |||||
| Type and quantity of any chemical(s) added to control/dilution water | X | ||||
| Test temperature 15 ± 1°C for green sea urchin, Pacific purple sea urchins and eccentric sand dollars; 20 ± 1°C for Arbacia and white sea urchins | X | ||||
| # and conc. of test solutions (≥7 conc. plus control) | |||||
| Volume and depth of solution in each test vessel (recommend 10mL; options are 5 and 2 mL) | X | ||||
| # of replicates per conc. (≥3 replicates/ conc. including controls, control has same # of replicates as for each test solution) | |||||
| If HSB or salts added to adjust salinity, test include set of controls using same source, batch and conc. | |||||
| If uncontaminated receiving water used as control/dilution, additional control run using lab seawater previously shown to achieve valid test results | |||||
| Any test using dilution water that differs from HSB or salt control include separate set of controls prepared using this same water | |||||
| # of gametes (2000 eggs per vessel for 10 mL test volume; options include 1000 eggs for 5 mL test volume and 400 eggs for 2 mL test volume) | X | ||||
| Estimated # of sperm per vessel and sperm:egg ratio | X | ||||
| Sperm should represent ≥3 males and eggs ≥3 females; perform gamete check to ensure only good quality gametes selected for test | |||||
| If good quality gametes not available from ≥3 males and ≥3 females, fewer used if pre-test is done to determine optimal sperm:egg ratio for a given batch | |||||
| If no pre-test, gametes pooled from ≥3 males and ≥3 females as determined in gamete check | |||||
| D.O. and pH of sample just before its use | X | ||||
| Temperature, salinity, D.O. and pH in aliquot of test solutions and controls at the start of the test | X | ||||
| Anything unusual about the test, any deviation from the test method, any problems encountered, any remedial measures taken | X | ||||
| Test Endpoints and Calculations | |||||
| # of fertilized and unfertilized eggs counted for each replicate test solution (including each control replicate) at the end of the test and mean (±SD) % fertilized eggs for | |||||
| each test vessel (including controls) (test is invalid if the mean fertilization rate for all replicates of controls is <60% or >98%) | |||||
| Positive and logical dose effect curve should be obtained | X | ||||
| IC25 (and 95% confidence limits) for fertilization success via non-linear regression analysis; data meets assumptions for normality and homoscedasticity; (ICPIN can be used to derive ICp if data do not allow regression statistics) | |||||
| Details regarding any weighting techniques used and indication of quantitative statistic used | |||||
| Name and citation of programs and methods used for calculating statistical endpoints | |||||
| Endpoints generated by regression analysis are bracketed by test concentrations; endpoint not extrapolated beyond highest test concentration | |||||
| Outliers (if any) are identified and their removal justified | |||||
| Results and duration of the reference toxicant test(s) (recommend that the result fall within the warning limits (± 2 SD) of the historic reference toxicant mean) | X | ||||
| For adults held >3 d, reference toxicant test conducted within 14 d of effluent test or concurrently using same batch of gametes | |||||
| For adults held ≤3 d, reference toxicant test performed concurrently with effluent test | |||||
| The reference toxicant test conducted under same experimental conditions | |||||
| EEM Program Specific Requirements | |||||
| Was the test endpoint bracketed by at least 1 test conc. (except for “>100%”)? | |||||
| For the Pulp and Paper EEM Program: Was report submitted within 90 d of test completion? | |||||
* Covers reporting and method requirements outlined in the fertilization assay using echinoids (sea urchins and sand dollars), second edition published in 2011, and the Regulations Amending the Pulp and Paper Effluent Regulations and Schedule 5 of the Metal Mining Effluent Regulations (June 2002).
EC (Environment Canada). “Revised Procedures for Adjusting Salinity of Effluent Samples for Marine Sublethal Toxicity Testing Conducted under Environmental Effects Monitoring (EEM ) Programs”, Unpublished Report, December 2001, 10 p. Method Development and Applications Section, Environmental Technol. Centre, Ottawa, ON (2001).
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