Metal mining technical guidance: sublethal toxicity checklist, chapter 8
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 method “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 | ||||
| Measurement of temperature of the sample or of one subsample upon receipt at the laboratory | X | ||||
| Measurement of pH before preparation and use in test | X | ||||
| Date for sample collection and date/time for sample receipt at lab | X | ||||
| Date for test start (≤ 3 d of sample collection)1 | |||||
| Test Organisms | |||||
| Species (Pseudokirchneriella subcapitata, formerly Selenastrum capricornutum) | |||||
| Strain number and origin of culture | X | ||||
| Age of known-age culture used to provide inocula of test organisms, at the start of the test 3 - 7 d old | |||||
| Inoculum prepared ≤ 3 hrs before microplate incubation | |||||
| Initial cell density of the inoculum (10 000 ± 1000 cells/mL) | |||||
| Any unusual appearance or treatment of culture, before its use in the test | X | ||||
| Culture Health | |||||
| Algae growth curve starting with an inoculum from the algal stock culture determined over an 8 to 10 day period using an Erlenmeyer flask (2 times per year to confirm logarithmic growth phase). Note: this is not a reporting requirement but it is a method “must”. | |||||
| Test Conditions and Procedures | |||||
| Test method (EPS 1/RM/25, 2nd edition, March 2007) | |||||
| Date for test end and statement of duration (72 h) | |||||
| Person performing test | X | ||||
| Mean test temperature (24 ± 2°C, monitored throughout the test) | |||||
| Procedure/rate/duration of aeration of sample, if any, before start of test | X | ||||
| Procedure, if any, for pH adjustment (recommend no adjustment if pH of test solution within 6.5 - 8.5; pH adjusted outside this range is an option or parallel testing with and without pH adjusted solution) | X | ||||
| Final volume in each well is 220 µL | |||||
| pH of sample before any dilution, at start of test | X | ||||
| pH in one standard control well at the start and one standard control well at the end of test (e.g., D6 and D7); should not differ by more than 1.5 pH units | X | ||||
| Procedure for sample filtration (through preconditioned 0.45 µm pore diameter membrane) | |||||
| Type(s) and source(s) of control/dilution water (same water used for preparing control and test solutions; all field collected dilution water is filtered through 0.45 µm filter before the addition of stock nutrients) | |||||
| An additional control row must be set up if control/dilution water is not reagent water | |||||
| Type and quantity of any chemical(s) added to control/dilution water | |||||
| # of test concentrations (≥ 7 plus a control, ≥ 10 recommended. Note: less than 7 concentrations are enumerated only if : (1) cell counts of lower test concentrations show a large effect (i.e., >>IC50) has been reached, then counts at higher concentrations are not required, or (2) if cell counts show there is no effect, then only 6 concentrations are required for enumeration and the highest test concentration must be enumerated) | |||||
| # of replicates per concentration (≥3 per test concentration and 10 for control); 8 wells are enumerated for controls and minimum 3 replicates enumerated per test concentration. If counts are inconsistent (i.e., highly variable) then the additional replicates must be counted; 2 remaining control wells are used for measuring pH | |||||
| For metal mining effluents, the final amount of Na2EDTA•2H20 must be reduced by 25% (final concentration of 46.9 µg/L in test medium) | |||||
| If absorbance used, cell conc. (direct count) in the 3 wells containing high/medium/low test conc. and their corresponding values estimated using the absorbance method | X | ||||
| Anything unusual about the test, any deviation from the test method, any problems encountered, any remedial measures taken | |||||
| Test Endpoints and Calculations | |||||
| Nominal concentrations of test solutions are corrected for the volume of nutrient stock & algal inoculum and reported as the test concentrations (i.e., no 100%) | |||||
| Cell conc. in each replicate (including control) at 72 h | |||||
| Mean cell yield (± SD) for each treatment (including control) at 72 h with orresponding CV (test is invalid if # of algal cells in control wells have not increased by a factor of > 16 in 72 h or if cell yield estimated in control wells are not homogenous (CV > 20%) or if an inhibitory gradient is detected across the control wells) | |||||
| Established relationship between cell yield and absorbance or fluorescence if these measurements are being used as a surrogate for cell yield; absorbance/fluorescence measurements are made only after cells are centrifuged and resuspended in clear solution | |||||
| IC25 (and 95% confidence limits); using concentrations corrected for the volume of algal inoculum & nutrient stock; indication that regression analysis was used (ICPIN can still be used to derive an ICp if the data do not allow regression statistics); program name & citation of statistical method(s) used; details regarding any weighting techniques applied to the data | |||||
| Endpoints generated by regression analysis must be bracketed by test concentrations (i.e., extrapolation beyond the highest test concentrations is not acceptable) | |||||
| Data exhibiting hormesis can be entered directly, there is no trimming of data points which show a hormetic response (i.e., growth stimulation at low concentrations followed by growth inhibition at higher test concentrations) | |||||
| If the data exhibited hormesis and ICPIN is used, control responses must be entered for those concentrations which demonstrated hormesis | |||||
| Any outliers identified and the justification for their removal | X | ||||
| % Stimulation: Any findings of significant growth stimulation (i.e., enhanced growth at one or more higher concentrations tested or at all concentrations tested), expressed as % stimulation at any concentration(s) | |||||
| If microplate includes both a standard reagent control and a sample control (where receiving water is used as control/dilution water in the test) a statistical comparison for significant differences of means is performed | |||||
| Results of the reference toxicant test(s) (recommend that the result fall within the warning limits (± 2 SD) of the historic reference toxicant mean) | X | ||||
| Was a reference toxicant test started within 14 d before or after the algae test on effluent was started or during it? | |||||
| Was it conducted under the same experimental conditions? | |||||
| EEM Program Specific Requirements | |||||
| Were quantitative endpoints for effluent and reference toxicant tests provided (i.e., no less than values reported)? | - | - | |||
| Were the test endpoints bracketed by at least one test concentration (except for >91%)? | - | - | |||
| For the Pulp and Paper EEM Program: was the report submitted within 90 d of test completion? | |||||
* Covers reporting and method requirements outlined in the growth inhibition test using a freshwater alga, amended in 2007, and the Amended Pulp and Paper Effluent Regulations, and Schedule 5 of the Metal Mining Effluent Regulations (June 2002).
1 For more information on test start date, please refer to Section 7.4 of the Metal Mining Guidance Document and Section 6.3 of the Pulp and Paper EEM Technical Guidance Document.
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