Metal mining technical guidance: sublethal toxicity checklist, chapter 7
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 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 (within 3 d of sample collection)1 | |||||
| Test Organisms | |||||
| Species (Lemna minor; clone identification 8434 or 7730) | |||||
| Origin of culture (culture collection, commercial biological supplier, government, or private laboratories); if of outside origin, cultured for ≥ 3 weeks before use | |||||
| Sterile culture (Axenic culture) | |||||
| Culture medium (i.e., must be sterile modified Hoagland’s E+ medium for wastewater or receiving water tests) | |||||
| Age of culture is 7-10 d old; acclimation period in test medium is 18 to 24 h and ≥2 cm fresh test medium before testing; two, 3-frond plants randomly transferred to each test vessel (i.e., a total of 6 fronds/vessel) | |||||
| Record any unusual appearance or treatment of known-age culture, before its use in the test | X | ||||
| Health Criteria | |||||
| Frond number increased by ≥ 8-times (i.e., ≥24 fronds) in 7 days in same test medium set up as a culture test for monitoring organism health | |||||
| Test Conditions and Facilities | |||||
| Test method (EPS 1/RM/37, 2nd edition, January 2007) | |||||
| Type of test (static or static-renewal); solution renewal frequency (at least every 3 d for static- renewal) | |||||
| Date for test end and statement of duration (7 d) | |||||
| Name and address of test laboratory; name of person(s) performing test | X | ||||
| Test vessel type (large enough so that there is no overlapping of Lemna fronds in controls at test end); ≥100 mL of test solution, preferable 150 mL; covered; minimum depth of 4 cm of test solution; description of test vessel (size, shape, type of material) | |||||
| Control/Dilution Water | |||||
| Type of test medium used as control and dilution water (must be the same medium); modified APHA growth medium for testing wastewaters & receiving waters (note: SIS or Steinberg medium is used for testing chemicals), or receiving water spiked with same nutrients used in test medium (with an additional control comprised of test medium and culture dilution water) | |||||
| Type and source of water used to prepare test medium | X | ||||
| Type and quantity of chemical(s) used to prepare control/dilution water | X | ||||
| Test Conditions and Procedures | |||||
| ≥3 replicates (i.e., 6 fronds/replicate) per concentration plus controls (for single concentration test); or ≥4 replicates per concentration plus control (for multi- concentration test with equal replicates); can have unequal replicate design with 6 reps for control, 4 replicates for lowest 3-5 test concentrations and 3 replicates for highest 4-5 test concentrations; randomised position of replicates in test area | |||||
| Number of test concentrations (minimum ≥7 plus controls) | |||||
| No filtration for effluents unless algae is present; procedure for filtering should be through ~1 µm glass fibre filter, then filter through 0.22 µm if necessary (same procedure to be used if receiving water is used as dilution water before nutrients are added) | |||||
| Type and quantity of nutrient added to test samples before start of test | X | ||||
| Duration of pre-aeration (20 min) and minimum rate (e.g., 100 bubbles/min) | |||||
| Procedure, if any, for pH adjustment (no adjustment if pH of test solution is in the range 6.5 to 9.5) | X | ||||
| Static test - pH: (pH of sample at the start (Day 0) and at the end (Day 7) of test minimally for the low, medium and high test concentrations, and the control) | |||||
| Static-renewal - pH: (pH of sample at the start (Day 0) and at the end (Day 7) of test and before and after test solution renewal minimally for the low, medium and high test concentrations, and the control) | |||||
| Daily temperature (adjusted to 25 ± 2 °C before test start; no immersion heaters). As a minimum, measured daily in representative test vessels (i.e., at least the high, medium, and low concentrations, plus the control) | |||||
| Lighting: continuous (fluorescent or equivalent); fluence rate: 64 to 90 µmol/m2 •s-1 at surface of test solution; test area within ±15% of selected light fluence rate throughout test area; (light measured at several locations in the test area at least once during the test approximately same distance from light source as the test fronds) | |||||
| Anything unusual about the test, any deviation from the test method, any problems encountered, any remedial measures taken | X | ||||
| Test Observations and Measurements | |||||
| Number and appearance of fronds in each test vessel (including control) at Day 0 and Day 7; dry weight in each vessel at Day 7 | |||||
| Mean ± SD of the increase in frond number in each treatment and control(s) at test end (controls must have ≥ 8 times increase, i.e., ≥ 48 fronds) | |||||
| Mean ± SD for dry weight of fronds in each treatment and control(s) at test end | |||||
| Other observations if present (i.e., chlorosis, necrosis, appears brown or white, yellow or abnormally-sized fronds, gibbosity, colony destruction, root destruction, loss of buoyancy, other) |
X | ||||
| Test Endpoints and Calculations | |||||
| Nominal concentrations of test solutions are corrected for the volume of nutrient stock and reported as the test concentrations (i.e., no 100%) | |||||
| Frond Number: (IC25 and 95% confidence limits); using concentrations corrected for the volume of 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 and citation of statistical method(s) used; details regarding any weighting techniques applied to the data | |||||
| Frond dry Weight: (IC25 and 95% confidence limits); using concentrations corrected for the volume of 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 and citation of statistical method(s) used; details regarding any weighting techniques applied to the data | |||||
| Endpoints generated by regression analysis are bracketed by test concentrations (i.e., extrapolation beyond the highest test concentration is not acceptable) | |||||
| Endpoints generated by regression analysis included (i.e., no trimming) any hormetic data (i.e., growth stimulation at low concentrations followed by growth inhibition at higher test concentrations) | |||||
| 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 (frond number or dry weight), at any concentration(s) | |||||
| Results and duration of test with reference toxicant(s) with geometric mean for frond number ± 2 SD (for the same test species, clone, light, medium, vessel) | |||||
| Was a reference toxicant test started within 14 d before or after the Lemna test on effluent was started or during it? | |||||
| Was the ref tox test conducted under the same experimental conditions (same procedure and test medium)? | |||||
| Record reference toxicant used (Ni or KCl recommended) | |||||
| EEM Program Specific Requirements | |||||
| Were quantitative endpoints for effluent and ref tox tests provided (no less than values reported)? | |||||
| Were the test endpoints bracketed by at least one test concentration (except for >97%)? | - | - | |||
* Covers reporting and method requirements outlined in the inhibition test using the freshwater macrophyte, Lemna minor, amended in 2007 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 EEM Guidance Document.
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