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6. Sublethal Toxicity Testing

6.1 Overview

6.2 Collection of Samples

6.3 Sampling Locations

6.4 Frequency and Reporting

6.5 Tabulation of Sublethal Toxicity Endpoints and Validation of Test Results

6.6 Data Interpretation in Relation to the Toxicity Objectives

6.7 Description of Freshwater and Marine Sublethal Toxicity Tests

6.8 Dilution Water in Freshwater Sublethal Toxicity Testing

6.9 Collection, Shipment and Storage of Samples for Sublethal Toxicity Testing

6.10 Use of Sublethal Toxicity Testing in Resolving Confounding Influences

6.11 References

List of Tables

6.1 Overview

There are two main uses for sublethal toxicity tests in the environmental effects monitoring (EEM) program: to compare processes and measure changes in effluent quality; and to contribute to the understanding of the relative contributions of the mine in multiple-discharge situations.

The purpose of sublethal toxicity testing in the metal mining EEM program is to provide an estimate of the potential effects on biological components (phytoplankton, zooplankton, benthic invertebrates, fish, macrophytes) in the exposure area, whether or not these components are being directly measured in the field.

To estimate the potential effects on biological components, sublethal toxicity testing shall be conducted by following the applicable methods referred to in the Metal Mining Effluent Regulations (MMER), section (s.) 5, subsections (ss.) 3 and 4 (as amended from time to time). Four freshwater sublethal toxicity tests (fish, invertebrate, algal and plant species) or three marine or estuarine sublethal toxicity tests (fish, invertebrate and algal species) shall be conducted, depending on the receiving environment type (MMER, Schedule 5, s. 5), and the results shall be recorded. The test chosen should primarily be based on the relevance of the species to the local receiving environment, and secondarily on the seasonal availability of test organisms.

Acceptable sublethal toxicity methods are outlined in Table 6-1. The following website contains all the biological test method documents published by Environment Canada’s Biological Methods Section. Test report checklists have been developed for assessing the validity of test results for each test option, which are available on the EEM website. Information on the relative sensitivity of the different sublethal toxicity tests can be found in ESG (1999).

For additional information on assessing changes in effluent over time, and other data-interpretation situations, see sections 6.6 and 6.10.

Table 6-1: Methodologies for effluent sublethal toxicity tests (text description)
Test Description Receiving Environment Test Species Methods
Fish early life stage development tests Marine Inland Silverside (Menidia beryllina) or Topsmelt (Atherinops affinis) US EPA (2002)
Freshwater Fathead Minnow (Pimephales promelas)1 or Rainbow Trout (Oncorhynchus mykiss) Environment Canada (1992a) or Environment Canada (1998)
Invertebrate reproduction tests Marine Echinoids (sea urchins or sand dollars) Environment Canada (1992b)
Freshwater Water Flea (Ceriodaphnia dubia) Environment Canada (2007a)
Plant and algae toxicity tests Marine - algae Barrel Weed (Champia parvula) US EPA (2002)
Freshwater - algae Green Algae (Pseudokirchneriella subcapitata) Environment Canada (2007b) or MDDEP (2007)2
Freshwater - plant Lesser Duckweed or Common Duckweed (Lemna minor) Environment Canada (2007c)

1 Where Fathead Minnows are not an indigenous species, Rainbow Trout will be used according to Environment Canada (1998).
2 In some jurisdictions, both Environment Canada (2007b) and the MDDEP (2007) testing requirements for Pseudokirchneriella subcapitata are acceptable for the EEM program.
Note: For all marine toxicity test procedures, it is recommended that the effluent salinity adjustment procedure by Environment Canada (2001) be followed. For all sublethal tests where the test organisms are purchased for sublethal toxicity testing, it is recommended that the test organism importation of Environment Canada (1999) be followed.

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6.2 Collection of Samples

Sublethal toxicity testing shall be conducted on the aliquots of effluent samples collected in accordance with ss. 4(2) of the MMER (Effluent Characterization) (MMER, Schedule 5, ss. 5(2)).

In choosing when effluent for toxicity tests should be collected, two aspects should be considered:

  1. when effluent poses the greatest potential for adverse environmental impact on the environment; and
  2. when biological monitoring is conducted to look at potential linkages with effects in the exposure area.

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6.3 Sampling Locations

To determine which outfall structure has potentially the most adverse environmental impact, the following should be taken into account:

In cases where it is not clear which discharge source has the greatest potential to affect the environment, mines may wish to use a series of single-concentration sublethal toxicity tests from each final discharge location to determine the source with the greatest sublethal response.

To estimate the potency of the response from each discharge source, the “time to response” can be observed and calculated as the sublethal toxicity test endpoint (e.g., Ceriodaphnia dubia adults are exposed to undiluted effluent samples from each different effluent discharge, and observations are made as to how long it takes to find a 25% or 50% response). The sublethal toxicity test endpoint would be an LT25 (time to 25% mortality) or LT50 (time to 50% mortality) if survival was the key observation. The single-concentration test would be the more cost-effective approach to screening effluent sources in order to determine the discharge point with the greatest potential to affect the receiving environment.

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6.4 Frequency and Reporting

Sublethal toxicity tests shall be conducted two times each calendar year for the first three years and once each year after the third year (MMER, Schedule 5, ss. 6(2)). The first effluent sample shall be collected, and sublethal toxicity testingconducted, not later than six months after the mine is subject to section 7 of the MMER (Schedule 5, ss. 6(1)).

The results of sublethal toxicity testing shall be submitted to the Authorization Officer as part of the Effluent and Water Quality Monitoring Report. Additional information on the content of the Effluent and Water Quality Monitoring report is discussed in Chapter 5. The reporting requirements of the sublethal toxicity results are described in the MMER (s. 23; and Schedule 5, s. 8). See Chapter 10 for information on electronic reporting of sublethal toxicity data.

The test methods in Table 6-1 can be referred to for reporting specifications for each test method.

The report should include the following:

In determining whether or not to use historical sublethal toxicity data as part of the EEM program, the mine should take the following factors into consideration:

Toxicity data submitted as part of the EEM program for the metal mining industry should be accompanied by a description of the materials and methods, and calculations for each test. Minimum reporting requirements are detailed in section 8 or 9 of the toxicity test method documents of Environment Canada. Test report checklists have been developed for assessing the validity of test results for each test option, which are available on the EEM website. The minimum reporting requirements for methods of the U.S. Environmental Protection Agency (EPA) (US EPA 2002) have been prescribed for the purpose of subsequent EEM phases. The U.S. EPA requirements generally conform to Environment Canada specifications.

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6.5 Tabulation of Sublethal Toxicity Endpoints and Validation of Test Results

Sublethal toxicity endpoints reported vary depending on the test being conducted (refer to test methods in Table 6-1). However, the IC25 will be discussed below for illustrative purposes. The geometric mean2 of all IC25s (GM-IC25) for a given species should be calculated for each phase.

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6.5.1 Validation of Test Results

Procedures for QA/QC will be followed by both the field crews collecting environmental samples and the laboratory carrying out the toxicity testing, as discussed in the required toxicity test method documents.

Therefore, a first step in the interpretation of toxicity data for EEM should be the resolution of any problems with QA/QC. In addition to the QA outlined in the individual sublethal toxicity test methods, further requirements and recommendations are as follows:

Data could be declared rejected if one or more essential elements of the test method were not followed (e.g., failure to meet organism health criteria, inappropriate manipulations of the sample, failure to conduct the minimum in-test monitoring, incorrect statistic used for sublethal toxicity endpoint calculation).

Laboratories contracted by the metal mining industry to conduct sublethal toxicity testing should be accredited under the International Organization for Standardization standard ISO/IEC 17025:2005 entitled “General requirements for the competence of testing and calibration laboratories,” as amended from time to time.

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6.5.2 Tabulation of Sublethal Toxicity Endpoints

If the effluent does not cause a 25% sublethal inhibition or effect for any of the freshwater sublethal tests, an IC25/EC25 cannot be calculated and it is reported as > 100%.

Mortality in some of the concentrations might also prevent calculation of the IC25/EC25. For example, there might be no measured effects (mortality, growth or reproduction) in 32% concentration, but appreciable mortality in 56%. It would then be impossible to obtain a good estimate of the inhibition of growth or reproduction for the 56% concentration, and hence impossible to determine IC25/EC25. In such a situation, the IC25/EC25 should be assumed to be equal to the higher concentration, which in this case is 56%.

There should be IC25s reported for each of the test species. This information is summarized by calculating the geometric mean for each set of IC25s. For example, testing of a mine effluent on six occasions resulting in measured IC25 values of 10, 15, 17, 23, 25 and 30% would lead to a geometric mean of 19%.

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6.6 Data Interpretation in Relation to the Toxicity Objectives

6.6.1 Changes in Effluent Quality

The geometric mean of the IC25 (GM-IC25) for each species can be compared between phases to assess changes in the quality of effluent over time at each mine or between mines and mine types. Improvements are expected as a result of changes in process or effluent treatment.

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6.6.2 Understanding Multiple Discharge Situations

Comparison of mine effluent toxicity data from other nearby industrial and/or municipal discharges can help in understanding the relative contribution to the potential impact of each effluent source on the environment. Provided that relevant toxicity, flow and dispersion information is available for the other discharges, the inter-relation or overlap of the effluent fields may be better understood. Another method of comparing relative loading (toxic contribution) from each source is to calculate the TER (toxicity emission rate; = (100/GM-IC25) x flow). However, this calculation does not relate to the receiving environment, because the effluent dispersion and dilution are not taken into consideration. See section 6.10 for more information on confounding influences.

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6.6.3 Contributions to the Weight-of-Evidence Approach

Where sublethal toxicity data have an IC25 of less than 30%, it is recommended that mines calculate the geographic extent of the response in the exposure area and identify the zone where the concentration of effluent is comparable to the IC25. Data on effluent and receiving flow for the appropriate month are needed to complete this estimate. The estimation of the potential geographic extent can be effectively reported in map form and reported in the Interpretative Report.

A potential-effects zone may be interpreted as a rough indication of the extent of 25% inhibition by effluent in the environment. If, for example, a mine’s GM-IC25 for a particular test was 1% volume/volume (v/v), this would match the extent of the 1% zone. Invertebrates and plant IC25s are not expected to be similar, due to differing species sensitivities and test method sublethal toxicity endpoints, and therefore may have dissimilar potential-effects zones.

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6.6.4 Considerations for Integration of Toxicity Test Results

The following points should be considered when toxicity data are used to estimate a potential-effects zone.

(1) Laboratory results: Sublethal laboratory tests provide estimates of toxicity under strictly controlled laboratory conditions for each test species. These conditions do not replicate environmental conditions at the site under study. Chapman (2000) describes various abiotic and biotic modifying factors present in the uncontrolled receiving environment, which may affect an organism’s response to a toxicant.

(2) Species differences: Species differences in sensitivity to metal mining effluents will be taken into account when extrapolating results from laboratory sublethal toxicity tests to effects on indigenous biota.

(3) Background toxicity: The description above assumed that there were no other upstream contributions of toxicity. That assumption would be erroneous if there were overlapping plumes.

(4) Type of receiving water: Receiving-water pH, hardness, dissolved organic carbon (DOC) and other modifying factors could potentially increase or decrease toxicity of the effluent compared to tests with laboratory water.

(5) Plume uncertainties: Calculations of dilution might be difficult or inaccurate, or the position of the mixing zone might be variable. Where this uncertainty exists, estimating a zone of potential effect would have an equal level of uncertainty.

6.7 Description of Freshwater and Marine Sublethal Toxicity Tests

Table 6-2 provides short descriptions for the freshwater and marine sublethal toxicity tests included in the metal mining EEM program. Information on the relative sensitivity of the different sublethal toxicity tests can be found in ESG (1999).

For freshwater tests, laboratory or site water can be used as dilution/control water. For marine or estuarine environments, the mine has a choice of using uncontaminated sea water or artificial sea water produced from hyper-saline brine (HSB). The recommended procedures for adjusting the salinity of the effluent and dilution water and preparing the HSB are described in Environment Canada (2001).

Where applicable, the test organism importation methodology (Environment Canada 1999) should be referred to, where test organisms are purchased for immediate use in sublethal toxicity tests.

For QA/QC, detailed records of all aspects of the samples, test organisms, culture maintenance, test conditions, equipment and test results are validated and kept by the laboratory. A reference toxicant test is used to establish the validity of effluent toxicity data. Successive reference toxicant data are plotted on a control chart. If results are within expected limits, the performance of the batch of test organisms is ensured. The minimum level of reporting is outlined in each test method.

Technical personnel should be skilled in algae, macrophyte, invertebrate and fish culture, and in conducting toxicity tests following aseptic techniques.

For more detailed descriptions, please refer to the specific test method documents.

Table 6-2: Descriptions of the freshwater and marine sublethal toxicity tests included in the metal mining EEM program (text description)
Test Purpose and Results Description Biological Test Method and  Cost
Larval growth and survival assay using Inland Silverside (Menidiaberyllina) Evaluate effects of effluent exposure on fish larvae. Result is expressed as the concentration at which larval growth is reduced by 25% (IC25). If mortality is significant, it may be possible to calculate the lethal concentration for 50% of the test population (statistical endpoint is a LC50). The Inland Silverside is a small fish that populates a variety of habitats and tolerates a wide range of temperature (2.9 to 32.5°C) and salinity (0 to 58 g/kg). The Inland Silverside is a multiple spawner, and spawning can be induced by diurnal interruption in the circulation of water in the culture tanks. The eggs adhere to vegetation in the wild or to filter floss in laboratory culture tanks. The larvae hatch in 6 to 7 days when incubated at 25°C and maintained in seawater with salinity ranging from 5 to 30 g/kg. Seven- to 11-day-old larvae are exposed to a minimum of 5 concentrations of the effluent sample and a control for 7 days in a static renewal system at 25°C. During the 7 days, the larvae are fed brine shrimp once or twice per day and the solutions are replaced once each day. At the end of the 7-day exposure period, the surviving larvae are counted and individual replicate weights are measured to calculate the growth changes, which are compared statistically between exposure concentrations and the controls. For a valid test, average dry weight of control larvae will be ≥ 0.50 mg and control survival will be ≥ 80%. The test requires approximately 40 L of effluent. Short Term Methods for Estimating Chronic Toxicity of Effluent and Receiving Waters to Marine and Estuarine Organisms (3rd Edition) (Reference Method EPA-821-R-02-014), October 2002, published by the U.S. EPA.
* For the U.S. EPA method, the minimum reporting outlined in Environment Canada test methodologies should be followed.
Larval growth and survival assay using Topsmelt (Atherinopsaffinis) Evaluate effects of effluent exposure on fish larvae. Result is expressed as the concentration at which larval growth is reduced by 25% (IC25). If mortality is significant, it may be possible to calculate the lethal concentration for 50% of the test population (statistical endpoint is a LC50). Topsmelt are small fish which occur from the Gulf of California to Vancouver Island. Topsmelt reproduce from May through August, depositing eggs on benthic algae in the upper ends of estuaries and bays. Off-season spawning has been successful in a laboratory-held population. Spawning is induced by a combination of three environmental cues: lighting, tidal cycle and temperature. Nine- to 15-day-old larvae are exposed to a minimum of 5 concentrations of the effluent sample and a control for 7 days in a static renewal system at 20°C. During the seven days, the larvae are fed brine shrimp twice per day and the solutions are replaced once each day. At the end of the seven-day exposure period, the surviving larvae are counted and individual weights are measured to calculate the growth changes, which are compared statistically between exposure concentrations and the controls. For a valid test, average dry weight of control larvae will be ≥ 0.85 mg and control survival will be ≥ 80%. The test requires approximately 40 L of effluent. Short Term Methods for Estimating Chronic Toxicity of Effluent and Receiving Waters to West Coast Marine and Estuarine Organisms (1st Edition) (Reference Method EPA/600/R-95-136), August 1995, published by the U.S. EPA.
* For the U.S. EPA method, the minimum reporting outlined in Environment Canada test methodologies should be followed.
Fathead Minnow (Pime-
phales promelas)Growth and survival test		 Evaluate effects of effluent exposure to an early life stage of fish. Results are expressed as the concentration at which larval growth/survival is reduced by 25% (IC25). If mortality is significant, it may be possible to calculate the lethal concentration for 50% of the test population (statistical endpoint is LC50). Fathead Minnows are small, warm-water fish found across North America in ponds and slow-moving water areas of rivers. The female lays her eggs on the underside of hard surfaces, where the male cares for the eggs until hatching. Fathead Minnow larvae, less than 24 hours old, are exposed to a minimum of 7 concentrations of the effluent sample and a control for seven days at 25°C. During the seven days, the larvae are fed brine shrimp 2 or 3 times per day and the test/control solutions are replaced once each day. At the end of the seven-day exposure period, the surviving larvae are counted and individual replicate weights are measured to calculate the growth changes, which are compared statistically between exposure concentrations and the controls. The test requires approximately 40 L of effluent. Test of Larval Growth and Survival Using the Fathead Minnows (Reference Method EPS 1/RM/22), February 1992, Amended in September 2008, published by Environment Canada.
Rainbow Trout (Oncorhyn-
chus mykiss) Embryo develop-
ment Test		 Evaluate effects of effluent exposure to an early life stage of fish. Results are expressed as the concentration at which embryo viability is reduced by 25% (statistical endpoint is an EC25). Rainbow Trout are common in clean, cold-water streams in North America. In some areas, they are not a native species but have been introduced to the watershed. Rainbow Trout are cultured throughout the country by commercial hatcheries. Adult Rainbow Trout migrate to shallow water to spawn in clean gravel. They bury their eggs in the rocks and gravel, where the young live until the yolk sac is absorbed. The embryo development test involves exposing recently fertilized Rainbow Trout eggs to a series of concentrations of the effluent sample for seven days at 14°C. Test exposure solutions are renewed every day. Dead embryos are counted and removed during the test. At the end of the test, the embryos’ viability is assessed and numbers of healthy embryos are counted for statistical comparison between test concentrations and the control. The test requires approximately 80–90 L of effluent. Toxicity Tests Using Early Life Stages of Salmonid Fish (Rainbow Trout) (Reference Method EPS 1/RM/28), July 1998, published by Environment Canada.
Fertilization assay using echinoids (sea urchins and sand dollars) Evaluate effects of effluent exposure on egg fertilization success of echinoids. Results are expressed as the concentration at which the fertilized egg number is reduced by 25% (statistical sublethal toxicity endpoint is the IC25). Echinoids are considered to be structurally advanced and complex invertebrates. Seven species of sea urchins and three species of sand dollars are commonly found in the coastal marine waters of Canada. Mature and gravid male and female echinoids are stimulated to spawn by injecting potassium chloride. Semen from at least 3 males is pooled and numbers are adjusted to the desired sperm:egg ratio. Eggs from at least 3 females are pooled and numbers are adjusted to 2000 eggs/millilitre (ml). Sperm is exposed for 10, 20 or 60 minutes (depending on the test option chosen) to a series of concentrations of the effluent sample. Eggs are then added to the test vessels for a 10- or 20-minute additional exposure. Adding formalin terminates the test. Preserved eggs are counted (in the range of 100 to 200 eggs) and classified as either fertilized or not fertilized, under a microscope at 100x magnification. For a valid test, the fertilization rate in the controls will be ≥ 50%, but < 100% and a positive and logical dose-effect curve should be obtained. The test requires approximately 1 litre (L) of effluent. Fertilization Assay using Echinoids (Sea Urchins and Sand Dollars) (Reference Method EPS 1/RM/27), December 1992, amended in November 1997, published by Environment Can
Ceriodaph-
nia dubia Repro-
duction and survival test		 Evaluate effects of effluent exposure on the reproduction of an invertebrate. Results are expressed as the concentration at which the average number of young per female is reduced by 25% (IC25). If mortality is significant, it may be possible to calculate the lethal concentration for 50% of the test population (statistical sublethal toxicity endpoint is an LC50). Ceriodaphnia is a species of zooplankton abundant in lakes, ponds and quiescent sections of streams and rivers throughout North America. In the test, Ceriodaphnia are separated so that there is 1 female adult animal per test vessel and 10 replicates per concentration. Young ceriodaphnids, less than 24 hours old, are exposed to a minimum of 7 effluent concentrations and a control, at 25°C. The test is completed when at least 60% of the surviving control organisms have had 3 broods of neonates or at the end of 8 days, whichever occurs first. During each day of the test, adult survivorship is assessed, all young produced are removed and counted, and the test solutions are renewed. At the end of the test, the number of surviving adults and the number of young produced per adult in 3 broods are compared statistically between exposure concentrations and the controls. The test requires 3–4 L of effluent. Test of Reproduction and Survival using the Cladoceran Ceriodaphnia dubia (Reference Method EPS 1/RM/21), 2nd edition, February 2007, published by Environment Canada.
Sexual reproduction assay using the red macroalga Champia parvula Evaluate effects of effluent exposure on the sexual reproduction of a marine red macroalga. Result is expressed as the concentration at which the number of cystocarps is reduced by 25% (statistical sublethal toxicity endpoint is IC25). Mature plant body of Champia parvula is hollow, septate and highly branched. New cultures can be propagated asexually from excised branches, making it possible to maintain clonal material indefinitely. Two sexually mature male and 5 female branches of Champia parvula are exposed in a static system for 2 days to a series of concentrations of the effluent sample, followed by a 5-7–day recovery period in control medium. The recovery period allows time for the development of cystocarps on the female branches resulting from fertilization during the exposure period. For a valid test, the female control mortality must be <20%, and the average number of cystocarps per female control plants is ≥ 10. The test requires approximately 2 L of effluent. Short Term Methods for Estimating Chronic Toxicity of Effluent and Receiving Waters to Marine and Estuarine Organisms (3rd Edition) (Reference Method EPA-821-R-02-014), October 2002, published by the U.S. EPA. For the U.S. EPA method, the minimum reporting outlined in Environment Canada test methodologies should be followed.
Algal growth inhibition test using Pseudo-
kirchner-
iella subcapitata		 Evaluate effects of effluent exposure on the growth of a unicellular freshwater alga. Result is expressed as the concentration at which the number of cells is reduced by 25% (statistical sublethal toxicity endpoint is an IC25). Pseudokirchneriella subcapitata is a non-motile, unicellular, crescent-shaped (40-60 micrometres3 [µm3]) green alga found in most freshwaters in North America. Its uniform shape makes it ideal for enumeration with an electronic particle counter. Clumping seldom occurs, because Pseudokirchneriella is free of complex structures and does not form chains. Growth is sufficiently rapid to accurately count cell numbers after 72 hours. Axenic (i.e., aseptically prepared stock cultures containing only the test species), exponentially growing Pseudokirchneriella are exposed to the test solutions in a static, 96-well microplate. The algae are exposed to a dilution series of filtered effluent sample over several generations under constant temperature (24°C), with continuous light for 72 hours. The number of algal cells in the test concentrations is compared with the number in the control solutions. An effluent is considered toxic when a statistically significant, dose-dependent inhibition of algal growth occurs. The test requires < 1 L of effluent. Growth Inhibition Test using a Freshwater Algae (Reference Method EPS 1/RM/25), 2nd edition, March 2007, published by Environment Canada.
Or: Détermination de la toxicité : inhibition de la croissance chez l’algue Pseudokirchneriella subcapitata(Reference Method MA. 500-P.sub 1.0, Rév. 1, 2007), September 1997, published by the Centre d’expertise en analyse environnementale du Québec, Ministère du Développement durable, de l’Environnement et des Parcs du Québec.
Macrophyte growth inhibition test using Lemna minor Evaluate effects of effluent exposure on the growth of a freshwater plant. Results are expressed as the concentration at which frond number and frond dry weight is reduced by 25% (statistical endpoint is an IC25). Lemna minor (Lesser Duckweed or Common Duckweed) is a small vascular, macrophyte plant, found at or just below the surface in freshwater (ponds, lakes, stagnant waters and quiet areas of streams and rivers). It is a common macrophyte with nearly worldwide distribution from tropical to temperate zones and grows in most regions of Canada. Its growth is rapid and occurs by lateral branching. Seven- to 10-day-old, rapidly growing plants (typical size of frond is 1 cm) are exposed to a series of concentrations of the effluent sample diluted with growth medium for 7 days. During the test, the plants are incubated at 25°C under continuous light and static conditions. Plants are acclimated to the test media for 18–24 hours before testing. The leaves are counted and weighed at the end of the test and growth is compared statistically to the controls. For a valid test, the number of leaves on control plants will increase by 8-fold at the end of the test. The test requires approximately 1–2 L of effluent. Test for Measuring the Inhibition of Growth using the Freshwater Macrophyte, Lemna minor(Reference Method EPS 1/RM/37), 2nd edition, January 2007, published by Environment Canada.

6.8 Dilution Water in Freshwater Sublethal Toxicity Testing

6.8.1 Dilution Water Selection

The sublethal toxicity test methods required for the metal mining EEM program clearly define the culture conditions and test procedures that need to be followed (Environment Canada 1992a, 1992b, 1998, 2007a, 2007b, 2007c; US EPA 1994a, 1994b, 1995, 2002). Some testing decisions are left to the discretion of the individual laboratories, as long as the standard test acceptability criteria can be achieved. For example, standard methods for testing Ceriodaphnia, Fathead Minnows, Pseudokirchneriellaand Rainbow Trout allow the use of uncontaminated ground or surface water, dechlorinated tap water or reconstituted water as a source for the culture or the test control/dilution water, as long as the water of choice supports a healthy culture and provides a valid test result.

Most laboratories in Canada use “standard laboratory” water for routine culturing and testing requirements. This water is generally supplied to the laboratory through a natural groundwater system (well) or a local municipal water source, which must be dechlorinated and may be buffered to meet acceptable culturing criteria. Deionized water reconstituted to targeted water quality parameters is also used. Advantages of using laboratory water include the following:

During the metal mining EEM program, most sublethal toxicity tests will likely be performed using laboratory water as control/dilution water, in order to attain comparable results among different laboratories and over time. It is also likely that in many sublethal toxicity tests where there is measurable effluent toxicity, this toxicity can be attributed to inorganic substances such as metals and ammonia, and the toxicity of the effluent may also be affected by site-specific characteristics such as pH, alkalinity and hardness. These characteristics can be controlled and reproduced in the laboratory for cases in which test results, reflective of the site conditions, are desired. However, a mine may decide to test its effluent using unexposed surface water (as control and dilution water), providing the sample is not exposed to effluent. Alternatively, a reference area of similar physicochemical characteristics to a mine site could be used to supply control/dilution water.

The use of unexposed surface waters can be especially helpful in obtaining the following information.

Estimating the mitigating or stimulatory effect of unexposed surface site water as dilution water on theexpression of toxicity from the effluent discharge or effluent-exposed surface water

Although parallel testing of effluents and effluent-exposed surface waters using site water and hardness-adjusted laboratory water have produced similar results (BEAK 1998, 1999), it is impossible to simulate all the physicochemical characteristics of site water using laboratory water. Therefore, if characteristics of site water, other than hardness, alkalinity and pH, are suspected to influence in the expression of toxicity, it may be useful to perform toxicity testing using site water in order to account for site-specific effects.

Unexposed surface water includes mine-site-collected water that has been collected upstream of a mine effluent discharge or from a nearby reference area. Unexposed surface water from the mine site area may vary in physical, chemical and biological characteristics over time.

Disadvantages of using unexposed surface water as dilution/control water include the following:

In spite of its practical technical disadvantages as control/dilution water, unexposed surface water may provide more site-specific toxicity information. The advantages include the following:

In Canada, this practice has been logistically constraining due to the large volumes of water needed to be shipped far distances. However, recently it has been shown that individual sites can identify 1 or 2 test organisms most likely to detect site-specific changes in effluent quality, so tests using receiving water could be conducted on just 1 or 2 species (Taylor et al. 2010). For example, the Pseudokirchneriella test requires smaller volumes of water, making this test an ideal candidate for evaluating the effects of receiving-water chemistry on effluent toxicity (Taylor et al. 2010). Sites should determine which type of water best suits their study objectives.

It should be noted that when using site water, problems may arise with reference water exhibiting sublethal responses. Beak International Inc. (BEAK 1998) attributed this problem to indigenous populations of micro-organisms infecting the laboratory organisms. BEAK staff found that boiling the water prior to use before testing was successful in reducing mortality.

Sprague (1997) prepared an extensive review of studies that compared toxicity test results to receiving-water impact, and concluded that effects measured in sublethal toxicity tests correlate with environmental effects most of the time, especially if water collected upstream of the effluent discharge is used as the control/dilution water.

When the purpose of a sublethal toxicity test is to estimate site-specific effects of contaminants, unexposed surface water from the vicinity of the mine site is recommended for use as control/dilution by Environment Canada, the U.S. EPA and the American Society for Testing and Materials (ASTM) in their method and associated guidance documents (Environment Canada 1992a, 1992b, 1998, 2007a, 2007c; US EPA 1994a; ASTM 1998).

Table 6-3: Specifications of the Environment Canada test methods and recommendations for collection, storage and use of site-collected dilution waters(text description)
Criteria Ceriodaphniadubia Fathead Minnow Pseudokirchneriellasubcapitata Lemnaminor Rainbow Trout embryo
Acceptable Dilution Water
For Culturing Uncontaminated groundwater, surface water, dechlorinated water, reconstituted water, dilute mineral water, or receiving water Uncontaminated groundwater, surface water or dechlorinated water Growth medium Hoagland’s E+ medium Groundwater, surface water, reconstituted water, dechlorinated water or receiving water
For Testing Reconstituted, dechlorinated, uncontaminated groundwater or surface water, receiving water Reconstituted, dechlorinated, uncontaminated groundwater or surface water, receiving water Reagent water, uncontaminated receiving water, groundwater, surface water or reconstituted water Modified APHA growth medium, SIS growth medium, receiving water Groundwater, surface water, reconstituted water, dechlorinated water or receiving water
Site Water
Collection Point Upstream from or adjacent to source but removed from effluent exposure Upstream from or adjacent to source but removed from effluent exposure Upstream from or adjacent to source but removed from effluent exposure Upstream from or adjacent to source but removed from effluent exposure Upstream from or adjacent to source but removed from effluent exposure
Collection Procedure As for effluent As for effluent As for effluent As for effluent As for effluent
Acclimation Procedure Recommends acclimating at least 2 generations of brood organisms before collecting neonates for tests Recommends acclimation of breeding stock prior to testing None Recommends placing plants in site-collected dilution water 18 to 24 hours prior to testing None
Acclimation Rationale Recommended hardness ± 20% of culture water range, alkalinity range ± 20% of culture water When outside hardness, alkalinity range ± 20% of culture water recommended N/A Needs to be done for all types of test medium N/A
Treatments Recommend filter 60 µm, boiling if necessary Recommend filter 60 µm, boiling if necessary Filter 0.45 µm Filter 1 µm, then filter 0.22 µm, nutrient-spiked Recommend filter 60 µm or boiling if necessary
Storage Preferably no more than 14 days; maximum of 1 month at 4ºC with no headspace Preferably no more than 14 days; maximum of 1 month at 4ºC with no headspace Preferably no more than 14 days; maximum of 1 month at 4ºC with no headspace Preferably no more than 14 days; maximum of 1 month at 4ºC with no headspace Preferably no more than 14 days; maximum of 1 month at 4ºC with no headspace
During Toxicity Testing Include lab water control. If screening test shows impairment, treat water by boiling. If impairment remains, use hardness-adjusted lab water. Include lab water control. If screening test shows impairment, treat water by boiling. If impairment remains, use hardness-adjusted lab water. Include reagent water control Include lab water control Include lab water control. If screening test shows impairment, treat water by boiling. If impairment remains, use hardness-adjusted lab water.

The widespread acceptance of unexposed surface dilution water for predicting site-specific effects is based on knowledge regarding the interaction of contaminants with water quality characteristics. For example, metal toxicity is well known to be influenced by the physicochemical characteristics of water, such as pH, alkalinity, hardness (reviewed by Wang 1997 and Sprague 1995). However, studies comparing results of toxicity tests on effluents and effluent-exposed surface waters from 4 different mine sites indicated that a similar estimation of toxicity could be obtained from tests using unexposed surface water or laboratory water for dilution, especially if the laboratory water was adjusted to the hardness, alkalinity and pH of the site water (BEAK 1998, 1999). This finding indicates that the use of site-collected dilution water may not always be necessary, because laboratory waters can be prepared to reflect site-water characteristics such as hardness, pH and alkalinity.

Naturally elevated levels of organic carbon is one important aspect of some site waters; organic carbon is known to mitigate the effects of metal toxicity by reducing metal bioavailability. Ranges of dissolved organic carbon (DOC) have been measured as high as 58 mg/L in some Ontario lakes (Neary et al. 1990, as cited in Welsh et al. 1993), and such waters may influence the expression of metal toxicity in effluent or effluent-exposed surface water matrices. However, in parallel toxicity tests of mine effluent conducted using site water of moderately high organic carbon (total organic carbon of 9.4 mg/L) and hardness-adjusted laboratory water, there was no significant difference in organism response (BEAK 1999). Of note is that high organic carbon content may not always result in reduced metal bioavailability. In waters of high hardness, concentrations of calcium and magnesium may be high enough to bind with humic acid (which makes up the majority of organic carbon). Therefore, humic acid binding sites would be limited and unavailable to tie up free metal ions. In this situation, decreases in effluent sublethal toxicity due to high receiving water DOC would not occur (Winner 1985). In addition, the type of organic matter will also influence metal bioavailability (i.e., more allochthonous-like organic matter decreases Cu toxicity better than autochthonous-like natural organic matter; Schwartz et al. 2004), which complicates the interpretation of DOC’s role in reducing metal toxicities to aquatic biota.

Comparing toxicity of the discharge or effluent-exposed surface water relative to an impaired upstream water

If upstream water is contaminated by nonpoint or upstream-point sources of pollution that are unrelated to the mine operation, a mine may decide to use that water for test dilution purposes in toxicity testing in order to provide an appropriate comparison of test organism responses, as long as the upstream water can support the health of the test organisms. If the upstream water cannot support health of the test organisms, it could be tested separately in a dilution series to quantify its effects with uncontaminated reference site or regular laboratory water used for test control and dilutions.

6.9 Collection, Shipment and Storage of Samples for Sublethal Toxicity Testing

The procedures for the collection, shipment and storage of site-collected dilution water are outlined in each of the Environment Canada and EPA test methods (Environment Canada 1992a, 1992b, 1998, 2007a, 2007b, 2007c; US EPA 1994a, 1994b). Table 6-4 provides estimates of the volumes of site water needed for performing a suite of EEM tests, and includes estimates for effluents or effluent-exposed surface water volume. As recommended by the U.S. EPA (1994a), site-collected dilution water samples should be representative of the water body and be unaffected by recent runoff or erosion events that may cause the water to have a higher total suspended solids concentration.

Table 6-4: Dilution/control water and corresponding effluent volumes for sublethal toxicity tests* (text description)
Test Dilution Water Volume (L) Effluent Volume (L)
Fathead Minnow 45 21
Rainbow Trout (embryo test) 300 125
Ceriodaphnia dubia 10 4
Pseudokirchneriella subcapitata 1 1
Lemna minor 5 for static,
12 for static-renewal		 2 for static,
5 for static-renewal
Testing Series Dilution Water Volume (L) Effluent Volume (L)
Fathead, Ceriodaphnia, Pseudokirchneriella, Lemna(static) 65 30
Fathead, Ceriodaphnia, Pseudokirchneriella, Lemna(static-renewal) 72 35
Rainbow Trout embryo, Ceriodaphnia, Pseudokirchneriella, Lemna (static) 325 140
Rainbow Trout embryo, Ceriodaphnia, Pseudokirchneriella, Lemna (static-renewal) 330 150

All volumes are calculated assuming 1 control and 7 test concentrations, except for the Rainbow Trout embryo test where volumes are calculated assuming 1 control and 5 test concentrations. Fathead Minnow assumes 500 ml test volume and 3 replicates, and Lemna minor assumes 150 ml test volume and 4 replicates.
* Estimated effluent volumes for marine/estuarine sublethal toxicity tests are outlined in Table 6-2.

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6.9.1 Test Organism Acclimation

Pre-acclimation of culture organisms is recommended prior to exposure to site water. As the purpose of using site water for test control and dilutions is to more accurately predict receiving-water impact, the most accurate prediction should be achieved using organisms adapted to the physicochemical conditions of the receiving environment. For example, Lloyd (1965) found that fish cultured in hard water need to lose calcium before they are as sensitive to metals as fish cultured in soft water. Therefore, if the site water to be used for test dilution is softer than the lab water, pre-acclimating fish to the site water conditions would provide enough time for loss of calcium prior to test initiation. Alternatively, if site-collected water is higher in hardness, then pre-acclimation would allow fish species to accumulate calcium prior to testing.

The Environment Canada methods for Ceriodaphnia dubiaand Fathead Minnows recommend that cultures be maintained in water of similar hardness, alkalinity and pH (i.e., within 20%) to the site water used for test dilution (Environment Canada 1992a, 2007a). Since many Canadian mines are located beside rivers or lakes of low hardness, it is likely that some acclimation of laboratory cultures would be necessary. BEAK developed a pre-acclimation procedure during the 1997 Aquatic Effects Technology Evaluation (AETE) Program study, later refined in the 1999 study, for Fathead Minnows and Ceriodaphnia dubia(BEAK 1998, 1999). Based on the expected hardness, alkalinity and pH of the site water, cultures are gradually introduced to laboratory water of decreasing hardness over several days until the appropriate hardness is reached. This procedure was adapted from that used by B.A.R. Environmental Inc. (BAR) during its 1996 AETE Program study, in which cultures were gradually acclimated to hardness-adjusted laboratory water and site water if screening of un-acclimated organisms showed impairment to laboratory organisms (BAR 1997).

For pre-acclimation of cultures, laboratory water of reduced hardness may be prepared by diluting standard laboratory water with deionized water. Hardness can be increased by adding salts, used for the preparation of reconstituted water in the appropriate amounts (Table 6-5). When hardness-adjusting water, it is important to keep the alkalinity level appropriate to the hardness, because alkalinity affects the speciation of metals (US EPA 2002; Laurén and McDonald 1986). Appropriate hardness and alkalinity relationships are available in Table 6-5, and additional values may be interpolated (US EPA 1994b).

Detailed procedures for pre-acclimation of Ceriodaphnia dubia and Fathead Minnow are described below. References to hardness assume a corresponding change in alkalinity and pH. No pre-acclimation procedures are described for the Rainbow Trout test, since eggs are delivered from the hatchery and used in testing within 24 hours (Environment Canada 1998). Similarly, Pseudokirchneriella and Lemna cultures are maintained in standard culture media that are different from standard testing media, although Lemna does allow for some pre-acclimation of cultures, since plants are transferred to the test medium 18 to 24 hours prior to initiation of testing (Environment Canada 2007c).

Table 6-5: Preparation of different hardness/alkalinities (text description)
Water Type Reagent Added (mg/L)1 Final Water Quality
NaHCO3 CaCSO4-2H20 MgSO4 KCL pH2 Hardness3 Alkalinity3
Very soft 12.0 7.5 7.5 0.5 6.4–6.8 10–13 10–13
Soft 48.0 30.0 30.0 2.0 7.2–7.6 40–48 30–35
Moderately hard 96.0 60.0 60.0 4.0 7.4–7.8 80–100 60–70
Hard 192.0 120.0 120.0 8.0 7.6–8.0 160–180 110–120
Very hard 384.0 240.0 240.0 16.0 8.0–8.4 280–320 225–245

Source: US EPA (1994a)
1 Add reagent-grade chemicals to deionized water.
2 Approximate equilibrium pH after 24 hours of aeration.
3 Expressed as milligrams mg) CaCO3/L.

Ceriodaphnia dubia

Ceriodaphnia cultures are initiated and maintained according to the Environment Canada standard method. To pre-acclimate cultures to the hardness of the site-collected dilution water, a brood of neonates, less than 24 hours old, is initiated in water, reduced in hardness by 20% from that of laboratory water by addition of deionized water. Each day, organisms are transferred to new solutions, decreased by a further 20-30% in hardness. Once the desired hardness is reached (within approximately 1 week) and the culture organisms pass the health criteria of the Environment Canada method (i.e., production of at least three broods, total neonate production of at least 15 per adult, less than 20% adult mortality), a new culture is initiated. The second-generation cultures are maintained in the hardness-adjusted water until organisms pass the health criteria (approximately 1 week). Selenium and vitamin B12 are added to the hardness-adjusted culture water if low in hardness, as recommended by the standard method.

Fathead Minnows

Acclimation and pre-acclimation procedures carried out by BAR and BEAK during the 1996 and 1997 AETE studies used breeding tanks of Fathead Minnows, gradually changed in hardness and alkalinity to that of the applicable site water (hardness-adjusted laboratory water or HALW) (BAR 1997; BEAK 1998). Eggs were collected from the adjusted-water tanks and reared at the HALW until hatching. However, preliminary work completed by BEAK in early 1999 showed that eggs could be hatched out in HALW without prior acclimation of the breeding tanks. By omitting breeding tanks from the pre-acclimation procedure, the volume of HALW needed for pre-acclimation is reduced while maintaining a supply of eggs for hatching in a number of different water types. Eggs are hatched according to the Environment Canada standard method, with fresh water renewal every 24 hours.

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6.9.2 Screening Tests

Once organisms are pre-acclimated to the physicochemical conditions of the site water, they may be exposed to the site water in screening tests. If organisms exposed to the site water meet the test method control acceptability criteria, the site water may be considered suitable for use as dilution water provided the site water meets the control validity criteria for the test method. Comparison of the site-water response to the laboratory response in a screening test may reveal a statistically significant reduction in reproduction and/or survival, even though the site-water-exposed group is within the control acceptability criteria for the test. As long as the site water meets the control validity criteria for the test method, it may be considered suitable for testing. For additional information, consult the Environment Canada test methods.

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6.9.2.1 Screening Test Impairment

If organisms are given an adequate opportunity to gradually acclimate to the physicochemical characteristics of the site water (by exposure to hardness-adjusted laboratory water), impairment observed during screening tests should not be due to shock exposure to different water quality characteristics such as hardness, alkalinity or pH. Therefore, impairment would likely be due to the presence of harmful biological agents or toxicants.

Special attention should be paid to any 100% site-water exposures showing significant mortality in one or more replicates. Microbiological organisms present in the site water may impair the health of test organisms, and anecdotal evidence from several ecotoxicity laboratories indicates that impairment by indigenous micro-organisms usually occurs after a few days of exposure. For example, this has been manifested in Fathead Minnow tests conducted at BEAK as a sudden onset of significant mortality in the site-water control, often in one or two replicates only. Occasionally, evidence of fungal or bacterial growth may be observed in the test vessels. If such contamination is indicated by a screening test, acclimation of the organisms will most likely not result in a removal of impairment. Therefore, either the site water should be treated by a suitable means to remove the impairment (i.e., boiling or ultraviolet treatment--see below), or hardness-adjusted laboratory water should be used as a surrogate.

Limited laboratory trials of site water showed that impairment could be removed by boiling site water gently for 10 minutes and cooling it prior to use in testing. Other treatments have been reported in the literature, such as ultraviolet light and 0.45-µm filtration (Grothe and Johnson 1996; Kszos et al. 1997). If site water is collected during late spring to early fall, some form of biological contamination should be expected (unless experience with a particular site water indicates otherwise), and precautions such as boiling should be taken.

If the impairment is due to chemical contamination, the suitability of the site water for use as a dilution and control water is questionable, even though cultures may be acclimated to naturally high levels of metals in site water (see section 6.9.1). If the cultures are exposed to higher levels of contaminants, post-acclimation can result in either higher or lower sensitivity of laboratory organisms, depending on the contaminant, organism and the water characteristics, and the utility of the post–screening test acclimation procedure becomes questionable. If impairment is detected in a screening test, the recommended procedure is to attempt treatment by boiling or to use hardness-adjusted laboratory water as a surrogate dilution water. If it is suspected that site water is contaminated, including a boiled-site-water exposure in screening tests would resolve the question of the effectiveness of that treatment for the site water of interest.

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6.9.3 Effluent or Effluent-Exposed Surface Water Toxicity Tests

Table 6-3 summarizes the recommendations for collecting, storing and using site-collected dilution waters in sublethal toxicity testing.

Once site water has been deemed suitable for testing, toxicity tests may be initiated as per the appropriate Environment Canada testing method. In addition to the site-water control, an additional control, using water from the laboratory culture, needs to be included in testing to serve as a check of culture health and site-water quality. No additional control is necessary when the test-control dilution water is the same as the culture water. In the case of Lemna minor and Pseudokirchneriella subcapitatatests, the laboratory control would be the standard-test growth medium, specified in the standard method.

If site-collected water is deemed unsuitable for use as test control and dilution water, a treatment such as boiling should be attempted in order to remove the impairment. If successful, the treated site-collected dilution water should be used in testing. However, boiling large volumes of water may not be practical for tests such as the Rainbow Trout test, and hardness-adjusted laboratory water may be a more suitable alternative. If no practical treatment can be found to remove impairment to test organisms caused by the site-collected dilution water, hardness-adjusted water should be used as the dilution water with pre-acclimated organisms. Care should be taken to match the pH of the characteristics of the site-collected dilution water as closely as possible.

6.10 Use of Sublethal Toxicity Testing in Resolving Confounding Influences

Sublethal toxicology data also have the potential utility to aid in the resolution of confounding factors. A multistakeholder group on metal mining toxicology has elaborated on this third use for sublethal toxicity data.

Estimating the relative contribution of mine effluent releases and other natural and/or anthropogenicinfluences on sublethal toxicity in the same receiving water body

During any phase of the EEM program, sublethal toxicity test data can be used to deal with situations where there are confounding influences. Sometimes the site characteristics do not permit full determination of the mine’s effluent effects even with an adapted study design. Information from sublethal toxicity testing may then help in the interpretation of field results. The choice of when to use sublethal toxicity tests in this application is up to the mine operator and the nature of the confounding influences. However, the confounding influence scenario where sublethal toxicity testing would be most relevant is the multiple-point-source discharge and/or nonpoint-source input situation. Sublethal tests or frequency monitoring should be determined based on the site-specific nature of confounding influence situations.

Estimates of sublethal toxicity can help in understanding the relative contribution of diverse industrial or municipal discharges to effects on aquatic organisms in the receiving water, whether the discharges are from upstream point or nonpoint sources (e.g., municipal landfill leachate, agricultural runoff) or the mine’s property. The upstream contribution of an observed environmental effect can be estimated, given surface water sublethal toxicity data, discharge flow, and features of dispersion into the receiving environment. If plumes from different discharges at a mine site overlap, more effort is necessary to distinguish the toxic contributions of the mine’s discharge sources vs. upstream sources. Samples of surface water from key locations in the high effluent exposure areas could be tested, to estimate the combined toxic contribution of the sources.

The following is a 3-step procedure for assessing the relative contribution from different sources of sublethal toxicity to the high effluent exposure receiving environment:

Testing samples of surface water, which receives discharges or toxicants from multiple sources, should be done synoptically and ideally during low-flow or worst-case periods. At a minimum, sampling should be carried out over as short a period of time as possible (e.g., 1 or 2 days). Repeated rounds of sampling and testing would be desirable if the toxicity of the discharges were variable. The above guidance on conducting toxicity assessment studies to estimate the contribution of multiple-discharge sources to instream effects is based on the 8 site investigations conducted under the U.S. EPA Complex Effluent Toxicity Testing Program (CETTP). Detailed reports on these studies were prepared by Mount and Norberg-King (1985, 1986), Mount et al. (1984, 1985, 1986a, 1986b, 1986c) and Norberg-King and Mount (1986). The results of CETTP testing, including independent critiques and re-analyses, were reviewed by Sprague (1997) during a project commissioned by the Aquatic Effects Technology Evaluation (AETE) Program. Sprague concluded that the U.S. EPA CETTP studies provided valid findings that should be considered by Canadian metal mining companies when designing aquatic environmental monitoring programs at their mine sites.

Mines may elect to conduct additional investigations where the most sensitive species in the effluent produced an IC25of less than 30%. Below are additional recommended investigations. At a minimum, the most sensitive test species can be used to estimate the geographic extent of the potential response. Alternatively, the results of the toxicity test(s) may lead to or trigger other recommended laboratory or field monitoring tools.

A tiered approach to resolving the confounding influences is recommended, starting with these additional recommended investigations:

  1. re-testing with the sublethal test that provided the most sensitive IC25 result, using upstream or reference-site water for test control and dilutions; or
  2. receiving-water toxicity testing with samples collected from the area where a sublethal response is predicted.

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6.11 References

[ASTM] American Society for Testing and Materials. 1998. Standard guide for conducting acute toxicity tests on aqueous ambient samples and effluents with fishes, macroinvertebrates, and amphibians, designation E1192-97. Conshohochen (PA): Annual book of ASTM standards. Section 11: Water and environment technology.

[BAR] B.A.R. Environmental Inc. 1997. Toxicity assessment of mining effluents using upstream or reference site waters and test organism acclimation techniques. Aquatic Effects Technology Evaluation Program. AETE Project 4.1.2a.

[BEAK] Beak International Incorporated. 1998. Additional tool evaluations. Aquatic Effects Technology Evaluation Program. Beak Reference 20776.1.

[BEAK] Beak International Incorporated. 1999. Final report: effects of dilution water on the results of sublethal toxicity tests. Report to Natural Resources Canada. Beak reference 33940.

Chapman PM. 2000. Whole effluent toxicity testing – usefulness, level of protection, and risk assessment. Environ Toxicol Chem 19(1):3-14.

Environment Canada. 1992a. Biological test method: test of larval growth and survival using fathead minnows. Ottawa (ON): Environmental Technology Centre. Report EPS 1/RM/22, February 1992, Amended in September 2008.

Environment Canada. 1992b. Biological test method: fertilization assay with echinoids (sea urchins and sand dollars). Ottawa (ON): Environmental Technology Centre. Report EPS 1/RM/27, December 1992, Amended in November 1997.

Environment Canada. 1998. Biological test method: toxicity tests using the early life stages of salmonid fish (rainbow trout). Report EPS 1/RM/28, 2nd ed., July. Ottawa (ON): Environmental Technology Centre.

Environment Canada. 1999. Recommended procedure for the importation of test organisms for sublethal toxicity testing. Ottawa (ON): Environmental Technology Centre.

Environment Canada. 2001. Revised procedures for adjusting salinity of effluent samples for marine sublethal toxicity testing conducted under environmental effects monitoring (EEM) programs. Ottawa (ON): Method Development and Applications Section, Environmental Technology Centre.

Environment Canada. 2007a. Biological test method: test of reproduction and survival using the Cladoceran Ceriodaphnia dubia. Ottawa (ON): Environmental Technology Centre. Report EPS 1/RM/21, 2nd edition, February.

Environment Canada. 2007b. Biological test method: growth inhibition test using a freshwater alga. Ottawa (ON): Environmental Technology Centre. Report EPS 1/RM/25, 2nd edition, March 2007.

Environment Canada. 2007c. Biological test method: test for measuring the inhibition of growth using the freshwater macrophyte Lemna minor. Ottawa (ON): Environmental Technology Centre. Report EPS 1/RM/37, 2nd edition, January 2007.

[ESG] ESG International Inc. 1999. AETE synthesis report of selected technologies for cost-effective environmental monitoring of mine effluent impacts in Canada. AETE Project 4.1.4, March 1999. Ottawa (ON): Canadian Centre for Mineral and Energy Technology, Natural Resources Canada.

Grothe DR, Johnson DE. 1996. Bacterial interference in whole effluent toxicity tests. Environ Toxicol Chem 15(5):761-764.

Kszos LA, Stewart AJ, Sumner JR. 1997. Evidence that variability in ambient fathead minnow short-term chronic tests is due to pathogenic infection. Environ Toxicol Chem 16(2):351-356.

Laurén DJ, McDonald DG. 1986. Influence of water hardness, pH and alkalinity on the mechanisms of copper toxicity in juvenile rainbow trout, Salmo gairdneri. Can J Fish Aquat Sci 43:1488-1496.

Lloyd R. 1965. Factors that affect the tolerance of fish to heavy metal poisoning. In: Biological problems in water pollution, third seminar, 1962. Washington (DC): U.S. Public Health Service. Publ. 999-WP-25. p. 181-187.

[MDDEP] Ministère du Développement durable, de l’Environnement et des Parcs du Québec. 2007. Détermination de la toxicité : inhibition de la croissance chez l’algue Pseudokirchneriella subcapitata. Centre d’expertise en analyse environnementale du Québec. MA. 500-P.sub 1.0, Revised in September 2007).

Mount DI, Norberg-King TJ, editors. 1985. Validity of effluent and ambient toxicity tests for predicting biological impact, Scippo Creek, Circleville, Ohio. Washington (DC): U.S. Environmental Protection Agency. EPA 600/3-85/044.

Mount DI, Norberg-King TJ, editors. 1986. Validity of effluent and ambient toxicity tests for predicting biological impact, Kanawha River, Charleston, West Virginia. Washington (DC):U.S. Environmental Protection Agency. EPA 600/3-86/006.

Mount DI, Thomas NA, Norberg-King TJ, Barbour MT, Roush TH, Brandes RW. 1984. Effluent and ambient toxicity testing and instream community response on the Ottawa River, Lima, Ohio. Washington (DC): U.S. Environmental Protection Agency. EPA 600/3-84/080.

Mount DI, Steen AE, Norberg-King TJ. 1985. Validity of effluent and ambient toxicity testing for predicting biological impact on Five Mile Creek, Birmingham, Alabama. Washington (DC): U.S. Environmental Protection Agency. EPA 600/8-85/015.

Mount DI, Norberg-King TJ, Steen AE. 1986a. Validity of effluent and ambient toxicity tests for predicting biological impact, Naugatuck River, Waterbury, Connecticut. Washington (DC): U.S. Environmental Protection Agency. EPA 600/8-86/005.

Mount DI, Steen AE, Norberg-King TJ. 1986b. Validity of effluent and ambient toxicity tests for predicting biological impact, Back River, Baltimore Harbor, Maryland. Washington (DC): U.S. Environmental Protection Agency. EPA 600/8-86/001.

Mount DI, Steen AE, Norberg-King TJ. 1986c. Validity of ambient toxicity tests for predicting biological impact, Ohio River near Wheeling, West Virginia. Washington (DC): U.S. Environmental Protection Agency. EPA 600/3-85/071.

Neary BP, Dillon PJ, Munro JR, Clark BJ. 1990. The acidification of Ontario lakes: an assessment of their sensitivity and current status with respect to biological damage. Toronto (ON): Ontario Ministry of the Environment. 171 p.

Norberg-King TJ, Mount DI, editors. 1986. Validity of effluent and ambient toxicity tests for predicting biological impact, Skeleton Creek, Enid, Oklahoma. Washington (DC): U.S. Environmental Protection Agency. EPA 600/8-86/002.

Schwartz ML, Curtis PJ, Playle RC. 2004. Influence of natural organic matter source on acute copper, lead, and cadmium toxicity to rainbow trout (Oncorhynchus mykiss). Environ Toxicol Chem 23(12):2889-2899.

Sprague J. 1995. Factors that modify toxicity. In Rand G, editor. Fundamentals of aquatic toxicology. 2nd edition. Washington (DC): Taylor and Francis.

Sprague J. 1997. Review of methods for sublethal aquatic toxicity tests relevant to the Canadian metal-mining industry. Ottawa (ON): Aquatic Effects Technology Evaluation Program, Canadian Centre for Mineral and Energy Technology, Natural Resources Canada.

Taylor LN, Van der Vliet LA, Scroggins RP. 2010. Sublethal toxicity testing of Canadian metal mining effluents: national trends and site-specific uses. Hum Ecol Risk Assess 16(2):264-281.

[US EPA] United States Environmental Protection Agency. 1994a. Short-term methods for estimating the toxicity of effluents and receiving water to freshwater organisms. Third edition. Cincinnati (OH): Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency. EPA /600/4-90/027.

[US EPA] United States Environmental Protection Agency. 1994b. Interim guidance on determination and use of water-effect ratios for metals Office of Water, U.S. Environmental Protection Agency. EPA 823-B-94-001.

[US EPA] United States Environmental Protection Agency. 1995. Short-term methods for estimating the toxicity of effluents and receiving water to West Coast organisms. First edition. Cincinnati (OH): Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency. EPA 600/R-95/136.

[US EPA] United States Environmental Protection Agency. 2002. Short-term methods for estimating chronic toxicity of effluent and receiving waters to marine and estuarine organisms. Third edition. Cincinnati (OH): Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency. EPA/821/R-02/014, October 2002.

Wang W. 1997. Factors affecting metal toxicity to (and accumulation by) aquatic organisms - an overview. Environ Internat 13:437-457.

Welsh PG, Skidmore JF, Spry DJ, Dixon DG, Hodson PV, Hickie BE. 1993. Effect of pH and dissolved organic carbon on the toxicity of copper to larval fathead minnows (Pimephales promelas) in natural lake waters of low alkalinity. Can J Fish Aquat Sci 50:1356-1362.

Winner RW. 1985. Bioaccumulation and toxicity of copper as affected by interactions between humic acid and water hardness. Water Res 19:449-455.

Tables

Table 6-1 outlines methodologies for effluent sublethal toxicity tests. Test descriptions--which include fish early life stage development tests, invertebrate reproduction tests, and plant and algae toxicity tests--are linked to receiving environment, test species, and methods.

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Table 6-2 offers descriptions of the freshwater and marine sublethal toxicity tests included in the metal mining EEM program. Each test is aligned with its purpose and results, a description, and the biological test method and cost.

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Table 6-3 provides the specifications of the Environment Canada test methods and recommendations for collection, storage and use of site-collected dilution waters. Criteria for acceptable dilution water and site water are identified and aligned with two examples: a fathead minnow, and a rainbow trout embryo.

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Table 6-4 outlines dilution/control water and corresponding effluent volumes for sublethal toxicity tests. Tests and testing series are identified, and aligned accordingly with their dilution water volumes (in litres), and their effluent volumes (in litres).

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Table 6-5 illustrates the preparation of water with different hardness and alkalinities. Water types include very soft, soft, moderately hard, hard, and very hard. Each water type is aligned with reagent added (mL/L) and the final water quality.

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Footnotes

Footnote 1

IC25 is defined as the effluent concentration where a 25% inhibition is observed in the exposed test organisms.

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Footnote 2

The geometric mean may be calculated as the nth root of n numbers multiplied together. Alternatively, the logarithms of the n IC25s (EC25s or LC50s) may be added together, the sum divided by n, and the antilog of the result is the geometric mean.

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