3. Effects on Fish and Fisheries Resources

3.1 Overview

3.2 Study Design Considerations

• 3.2.1 Selection of Reference and Exposure Areas
  • 3.2.1.1 Sampling of Exposure and Reference Areas
  • 3.2.1.2 Adequacy of the Reference Area
• 3.2.2 Confounding Factors
• 3.2.3 Marine Discharges
• 3.2.4 Historical Data

3.3 Selection of Sentinel Fish Species

• 3.3.1 Community Survey
• 3.3.2 Immature Fish
• 3.3.3 Small-bodied Fish Species
• 3.3.4 Live-bearers

3.4 Effect Indicators

• 3.4.1 Lethal Sampling
  • 3.4.1.1 Survival
  • 3.4.1.2 Energy Use (Growth and Reproduction)
  • 3.4.1.3 Energy Storage (Condition)
  • 3.4.1.4 Abnormalities
• 3.4.2 Non-lethal Sampling
  • 3.4.2.1 Survival (Size Distributions)
  • 3.4.2.2 Energy Use
  • 3.4.2.3 Condition
• 3.4.3 Wild Molluscs

3.5 Timing of Sampling

3.6 Verification of Fish Exposure

3.7 Power Analysis

• 3.7.1 Power and Significance Level
• 3.7.2 Effect-Size

3.8 Fish Sampling Methods

• 3.8.1 Bycatch
• 3.8.2 Remote Sensing
• 3.8.3 Alternative Methods

3.9 Fish Survey Quality Assurance and Quality Control

• 3.9.1 Field Practices to Improve Data Analysis and Interpretation
• 3.9.2 Quality Control in the Field
• 3.9.3 Determination of Sampling Effort
  • 3.9.3.1 Examples of Calculations of Sampling Effort
• 3.9.4 Consultation with Regional EEM Coordinators and Implementation in the Field
• 3.9.5 Data Entry
• 3.9.6 Quality Control in the Laboratory

3.10 Data Analysis

• 3.10.1 Statistical Analysis

3.11 Methods for Analysing Fish Usability

• 3.11.1 Selection of the Fish Species
• 3.11.2 Tissue Sample Collection and Preparation
• 3.11.3 Supporting Analyses: Lipid and Moisture Percentages
• 3.11.4 Guidance for Fish Tissue Analysis for Mercury Using Non-Lethal Methods
  • 3.11.4.1 Recommended Methodology

3.12 References

List of Tables

• Table 3-1: Required fish survey measurements, expected precision and summary statistics
• Table 3-2: Suggested aging structures for Canadian fish species
• Table 3-3: Fish survey effect indicators and endpoints for various study designs
• Table 3-4: Generalizations and suggested optimal sampling times for fish species in EEM
• Table 3-5: Fish species commonly used in EEM, aspects to consider during study design, and recommended sampling times
• Table 3-6: Suggested reporting format for the parameters (A) and the resulting regressions (B) required for the fish survey analysis

3. Effects on Fish and Fisheries Resources

3.1 Overview

Fish monitoring for the environmental effects monitoring (EEM) program may consist of a fish population survey and tissue analyses to determine if the mine effluent is having an effect on fish and fisheries resources. Detailed requirements and timelines are found in Chapter 1 and in the Metal Mining Effluent Regulations (MMER) (SOR/2002-222).

For the purposes of EEM, fish includes shellfish, crustaceans and marine animals, as per section 2 of the Fisheries Act, but excludes parts of these organisms (MMER Schedule 5, section (s.) 1).

The fish survey provides an assessment of whether there are differences in the growth, reproduction, condition and survival of the fish population between exposed and reference areas or within an exposure area along a gradient of effluent concentrations. Note that a mine is required to conduct a study of the fish population if the concentration of effluent in the exposure area is greater than 1% in the area located within 250 metres (m) of a final discharge point (MMER, Schedule 5, s. 9(b)).

In addition to the fish survey, biological monitoring studies may also include a study respecting fish tissue if, during effluent characterization conducted under paragraph 4(1)(d), a concentration of total mercury (inorganic and organic mercury) in the effluent is identified that is equal to or greater than 0.10 µg/L (MMER, Schedule 5, s. 9(c)). “Effect on fish tissue” is defined as measurements of concentrations of total mercury that exceed 0.5 µg/g wet weight in fish tissue taken in an exposure area and that are statistically different from and higher than the measurements of concentrations of total mercury in fish tissue taken in a reference area (MMER, Schedule 5, s. 1).

Top of Page

3.2 Study Design Considerations

General information regarding study designs is discussed in Chapter 2. The study design requirements and the definitions of effect for the fish population survey and fish tissue survey are discussed in Chapter 1.

To evaluate the effect of effluent on fish, the following questions should be answered:

• Is there an effect?
• Is the effect mine-related?
• Is the magnitude and extent of the effect known?
• Is the mine-related cause of the effect known?

Each mine’s EEM representatives or consultants should consult with the regional EEM authority to review the results of the previous phase’s site selection, species selection, fishing effort, etc., and to discuss the selection of the most appropriate options for the current phase. The results of previous phases, historical data, and local knowledge should be used to assess:

• the suitability and capture success of selected sentinel species
• the adequacy of the reference area
• the appropriateness of sampling methods and required equipment.

Mines may want to make changes between phases, including increasing sampling effort; changing sampling methods, equipment or fish species; selecting different exposure or reference areas; or using alternative monitoring techniques. Changes in the study design may need to be made for various reasons, including the following:

• The results indicate that power was insufficient in the previous phase due to collection of a low number of fish or high variability.
• The species characteristics were not measurable, not suitable, or there are concerns about the status of fish populations.
• It is uncertain if the fish were exposed to effluent.
• Reference sites were inappropriate.

Concerns raised about EEM studies (and field studies in general) can be separated into concerns about the adequacy of the reference sites, the potential impacts of confounding factors (e.g., potential influences of genetics on the variability of species characteristics), the ecological relevance of effect indicators used, the influences of natural variability, and concerns over statistical design issues. This guidance will attempt to provide input to deal with these issues.

Top of Page

3.2.1 Selection of Reference and Exposure Areas

The two main study designs are control-impact and gradient designs. The choice of reference area is the number one problem with control-impact field studies (Munkittrick et al. 2009). Ideally, a reference site would be located upstream, in similar habitat, and free of confounding influences, with a natural barrier that limits movement between sites. This situation is seldom available. The main issues cited regarding reference areas include whether the reference site is (a) comparable in terms of habitat; (b) free from the issue of concern (i.e., exposure) and from confounding influences (further discussed in Chapter 2); (c) open to movement of fish from the exposure area (fish in an upstream reference area could have been exposed previously or fish in the exposure area could be transient, reducing exposure to potential effects); and (d) whether the exposed fish were exposed to the effluent or stressor of interest.

No reference site is perfect. The ideal situation involves having data from before construction or initiation of the stressor of interest (e.g., before/after control-impact [BACI] design; outlined further in Chapter 4). Study sites that have barriers that prevent fish from moving between sites (e.g., dams, waterfalls, beaver dams) may be a good alternative, providing that the barrier does not alter the habitat. In open-water areas, choosing a species that has limited mobility improves the confidence that fish are not moving, but increases the potential influences of local differences in habitat. One difficult situation to interpret arises when there are no statistical differences in fish measurements between the areas, and there are no barriers restricting movement. In these cases, an indicator of exposure to the effluent is recommended, which can be chemical or physiological (e.g., liver enzyme induction, stable isotope signatures [Galloway et al. 2003; Dubé et al. 2006]).

If there are significant differences in fish characteristics between reference and exposure areas, there can be high confidence that fish are not moving between sites. Although differences are seen, variability in fish parameters (e.g., growth, weight, condition) is a function of a number of factors, not all of which will be related to the discharge of effluent. The selection of appropriate fish species for monitoring, survey timing and sampling gear will also facilitate the interpretation of any differences detected. Nevertheless, other natural and anthropogenic factors may influence effects on the fish and fish tissue and confound interpretation of the data. The requirement to confirm effects was developed to increase the confidence, over two phases, that effects are mine-related.

Top of Page

3.2.1.1 Sampling of Exposure and Reference Areas

The exposure area should be selected to ensure that the fish collected have been exposed to the effluent. It should be sampled first to determine which fish species are present, and their relative abundance within the area. The reference area can then be selected to provide fish of the same species that are available at the exposed site. Timing of sampling should be as close as possible between sites, to minimize temporal variability. The choice of time period for sampling will depend on factors such as time of year, stage of reproductive development, and potential habitat differences between sites (water temperature differences, etc.), but it is recommended that, if possible, all sampling be done within the same week. If a longer time period is required, reference samples should be collected before and after the collection of exposure samples, to allow comparison.

If fish are found in the reference area, but not in the exposure area where they are expected to occur (i.e., fish were historically found in the sampling area), the absence of fish in the exposure area should be reported as an effect. More information on reference site selection can be found in Chapter 2.

Top of Page

3.2.1.2 Adequacy of the Reference Area

It is now common in research programs to use a large number of reference sites. As an example, over the first 3 cycles of monitoring for the pulp and paper EEM program, there has been a trend toward using more reference sites. In Cycle 1, 3% of studies used multiple reference sites; in Cycle 2, 9%; and in Cycle 3, 25%. Including additional reference sites increases the ability to evaluate issues related to natural variability, ecological relevance and confounding factors, and improves the ability to evaluate the adequacy of the chosen reference site. Studies that use a gradient approach and multiple reference sites are statistically stronger than studies that depend only on a single reference site.

Other new approaches include reference condition approaches (Bailey et al. 1998), and using negative reference sites (using the exposed site as your reference). Regardless, the existence of consistent changes over two phases increases the level of confidence that changes are real. Follow-up studies must evaluate the adequacy of the reference site, especially if consistent results are not found.

Top of Page

3.2.2 Confounding Factors

In Cycle 2 of the pulp and paper EEM program, almost 90% of studies that detected effects also concluded that factors other than pulp mill effluent were responsible for such observations. Potential confounding factors exist at most sites and include other outfalls, habitat changes, historical uses and contamination, tributaries and non-point-source inputs. In highly confounded situations, alternative methods should be considered, but it should be emphasized that it is possible to obtain interpretable field results at most sites with adjustments to the study design. Given the complexity of certain situations, it is recommended that as much data as possible be gathered in order to demonstrate that other discharges or contaminant sources are primarily responsible for observed changes or an absence of observed changes. If changes are seen and determined to be influenced by confounding factors, the objective of subsequent study designs should be to eliminate the confounding factors or determine their significance.

Top of Page

3.2.3 Marine Discharges

Metal mines discharging into marine or estuarine receiving waters may face a number of problems and confounding factors that should be considered when developing an EEM fish survey study design. These problems may include the following:

• Some marine and estuarine areas are difficult to sample (e.g., tides, currents, high flushing rates or unsuitable habitat) and alternative approaches should be considered.
• There can be gradients for current, temperature and salinity, which can affect physical processes and the uptake of contaminants as well as have consequences for physiological changes within organisms.
• Selection of reference areas can be more difficult in marine situations.
• Different life stages of fish may utilize different habitats at different times of the year.
• Species availability can be low in marine environments. In many situations, small-bodied resident fish species are available and should be investigated. These species may be multiple spawners or live-bearers, or species for which there is little background information. However, this should not restrict or inhibit attempts to use these species, especially if they are abundant. The assumption inherent in an EEM program is that a fish community should be intact, with the normal abundant species present. The second priority, and underlying assumption, is that a fish population which shows a growth rate, reproductive development, and an age distribution indistinguishable from a reference area, is unaffected.

Potential solutions to these difficulties include using alternative species or caged bivalves, or mesocosms for confounded receiving environments. New facilities that will have collected baseline information prior to initiating effluent discharge will be in a better position to assess the effect of their effluent on the receiving environment compared to confounding factors.

Top of Page

3.2.4 Historical Data

Mines have the opportunity to submit historical data if there is previous biological monitoring information that could determine if there are effects on fish, fish tissue or the benthic invertebrate community. Historical data can be used to assist in the development of the first EEM study. See Chapter 13 for additional information on historical data.

Top of Page

3.3 Selection of Sentinel Fish Species

The recommended method for carrying out the fish survey is to monitor adults of two species of relatively sedentary finfish that have been exposed to effluent over a long period of time. Sexually mature finfish are preferred, but where they are not available, it is possible to design a program using shellfish or juvenile fish, although it will not be possible to analyze all the same effect endpoints. If available, at least one of the species selected should be a benthivore. The most important factors when selecting fish species for the EEM program are exposure, abundance, relevance to the study area (Munkittrick et al. 2000; McMaster et al. 2002), and sensitivity to effluent. In selecting the two species, the species used in previous EEM studies at the site (if applicable) should be considered, and preference should be given to:

• resident (non-migratory) fish species identified in a site characterization
• sexually mature female and male fish species that are abundant in both the exposure and reference areas
• fish species for which fishing or sampling permits can be obtained
• fish species that have the highest exposure to effluent

At any given site, there may be limited choices of potential species for monitoring. It will often be necessary to obtain the advice of an experienced fisheries biologist with knowledge of fish species present in the study area. More than 60 species have been used as sentinels in EEM pulp and paper and metal mining programs to date, and mines and their consultants are encouraged to contact regional, federal and provincial government agencies for fisheries information and additional guidance.

Some receiving environments do not support adequate numbers of fish for sampling. In situations where it has been determined that fisheries resources may be impacted by a destructive fish survey, non-lethal sampling techniques may be used. In environments that do not support adequate numbers of fish to meet the recommended sample sizes or where there are not two suitable fish species for monitoring, the following options, in order of preference, may be considered:

• one sexually mature fish species and one sexually immature fish species
• two sexually immature fish species
• one sexually mature fish species
• one sexually immature fish species.

The mine should consider changing its study design (e.g., species, methods of collection) if the results from the previous phase suggest that the species is long-lived (> 30 years); that it was not possible to measure all survey parameters on the fish (e.g., age, liver and gonad weight); that an insufficient number of individuals were collected; and that the degree of variability was such that the numbers of fish required by power analysis for subsequent designs are unreasonable, and it is not possible to reduce this by selective sampling methods. If the fish species available at a site are present in the high effluent exposure (near-field) area only during certain times of the year or life-history stages, the life stage and sampling time should be selected to maximize exposure to effluent.

Some of the challenges with species selection may relate to attempts to design a single program for multiple purposes. Concerns about contamination of fishery resources for human consumption would direct the study design to collect a species that is long-lived (so that contaminants accumulate longer), is piscivorous (so that biomagnification is greater), matures late (to increase concentration), preferably focuses on male fish or species that do not spawn every year (so that elimination of contaminants through egg deposition is lessened), and are of importance for local consumption. To improve the sensitivity of detecting environmental impacts, it is preferable that species are benthic (because generally they will move less), are not commercially or recreationally important (because it obscures the determining cause), mature early, contribute much energy to reproduction (so that energy demands are high), and are short-lived (so that impacts are recent)--with a focus on female fish (environmental impacts are often more serious on female egg producers).

A number of other factors need to be considered when selecting a sentinel species (see Munkittrick and McMaster 2000; Munkittrick et al. 2000), including ensuring that the species are active participants in the local aquatic food web. Other life-history characteristics, such as spawning time and migration, need to be evaluated site-specifically, because the interaction between discharge site, spawning habitats, seasonal changes in flow and dilution can all influence results and potentially impact the sensitivity of the monitoring program.

A key consideration when selecting a species is the mobility and residence time of that species, as this determines effluent exposure. Species that are resident in the system for most or all of their life cycle and exhibit territorial behaviour or limited mobility relative to the size of the study area are preferred, because the observed responses of these species reflect their localized environment. Species that are migratory or spend only a small proportion of their life cycle in the system under investigation (e.g., anadromous salmonids, some marine fishes) are not suitable, because exposure to effluent is minimal or transient and difficult to determine. This is also true for species that are highly mobile and are likely to be moving in and out of the effluent exposure area. In some cases, it may be possible to select more mobile species (e.g., Mountain Whitefish) (Swanson 1993), due to physical constraints that limit movement (e.g., dams, natural barriers, changes in habitat). In general, the greater the likelihood that a fish species is exposed to effluent, the greater its value as a monitoring species.

Top of Page

3.3.1 Community Survey

If a mine is new or has no historical survey information available, a fish community survey should be done to aid in the selection of appropriate fish species. Fish community surveys evaluate whether there are differences between areas in the diversity and abundance of fish species present.

A change in fish community has occurred when species that are expected to be abundant from the collections conducted at reference areas are not present in the effluent discharge area. If the exposure areas do not support one or more of the abundant species found at the reference area, it will be necessary to document the geographical extent of this absence. When the fish community composition has changed because of the presence of an effluent, there will also likely be measurable changes in the fish populations that remain. Results from the EEM program should document this, and may help in determining whether other fish species are at risk of disappearing from the exposure area.

Fish communities often include a number of species that are not abundant for a variety of reasons that may be unrelated to the presence of mine effluent. Non-lethal techniques (e.g., electro-fishing) are preferred for the community survey where possible, and field sampling should be designed to limit mortality of the existing species.

Top of Page

3.3.2 Immature Fish

The recommended method for carrying out a fish survey is to monitor adults (sexually mature fish) of two species of relatively sedentary finfish that have been exposed to effluent over a long period. However, there have been situations where no adult fish can be collected in a receiving environment. For example, some areas may not be inhabited by adult finfish, but are nursery areas for their juveniles. If sexually mature fish do not reside in an effluent exposure area, the suitability of juvenile fish may be considered. When sexually immature species are used, there is no direct measurement of reproductive development. However, the relative abundance of young of the year (YOY) can be used as a measurement of reproductive success.

Relevant measures for juvenile fish would be similar to those of mature fish, but without gonad measurements: growth (length, weight, or weight-at-age, if possible); condition (length-at-body-weight relationships); liver-weight-to-body-weight ratio; abundance (YOY survival, percent composition of age classes); deformities associated with exposure to effluents, such as vertebral fusions and compressions, spinal curvatures including lordosis and scoliosis, and fin erosion; and growth in juveniles exposed to effluent compared to juveniles in the reference area. Methods for the collection of juvenile fish are well established and many juvenile fishes can be aged (e.g., Secor et al. 1995).

Top of Page

3.3.3 Small-bodied Fish Species

The trend toward the increasing use of small-bodied forage-fish species (Munkittrick et al. 2002) has continued, rising from their use in 10% of surveys in the pulp and paper Cycle 1, to 26% in Cycle 2 and 34% in Cycle 3. A small-bodied fish can be considered a fish species that has a maximum size of 150 mm or less. Their use has several advantages and disadvantages. On a practical level, small-bodied fish species are usually more abundant, easy to capture, and more sedentary than larger-bodied fish species. Small-bodied fish have also been shown to be more sensitive to environmental changes, such as pH (Shuter 1990). Their home-range size has been positively correlated with body size (Minns 1995), and many small-bodied species integrate local conditions very well.

On the other hand, small-bodied fish require more sensitive analytical balances and more careful measurements. They are more sensitive to microhabitat differences because they integrate the local habitat so well. They are also more sensitive to differences in timing of sampling (see section 3.5).

In addition, small-bodied fish often have a shorter life span, so if they are chosen as one or both of the fish species, an additional 20 sexually immature fish (0+ and 1+) should be collected to aid in size-at-age (growth) analysis. Also, because a small-bodied fish species may only have a life expectancy of 3 to 4 years, the 0+ and 1+ will constitute a significant portion of the population (e.g., the 0+ and 1+ Slimy Sculpin [Cottus cognatus] are up to 50–70% of the population). This measurement of the proportion of a sample composed of YOY fish does add another surrogate measurement for reproductive performance (Gray et al. 2002).

There are other considerations as well. The life history, biology, and reproductive characteristics of some small-bodied species are unknown, making it difficult to determine the best sample areas, times and methods. Some are multiple spawners, which means reproductive effort in these species is difficult to estimate from a single sample because the reproductive tissue can be turned over almost completely between clutches (i.e., most of the mass of ova in the ovary will be spawned, and then a new clutch of mature ova will be developed). The ovary will generally contain two or more class sizes of ova and the spawning season may last from several weeks to more than a month. The number of clutches produced during the spawning season becomes the important reproductive variable and is difficult to estimate for an individual female in the field, even with frequent sampling. It will be difficult to evaluate the significance of changes in egg production in multiple spawners if they show normal reproduction in the first clutches.

Species identification of small-bodied fish should be verified, especially for cyprinids, which can appear very similar without careful examination. Useful references for this purpose include Scott (1967), Scott and Crossman (1973), Roberts (1988), Nelson and Paetz (1992), Jenkins and Burkhead (1993) and Coad et al. (1995). The smaller organ size of these fish requires a more sensitive balance. Dissecting microscopes may be necessary for removing the organs properly and avoiding extraneous tissue or moisture, which could affect results. Dissection on recently collected, fresh fish is recommended. Differentiation among tissues and separation of the liver and gonads from intestinal tissue is easiest when the tissue is fresh. Dissection of frozen specimens of small fish can be difficult and lead to errors in organ measurements. Preservation in a formalin solution may give adequate results, but care must be taken to treat exposure and reference fish the same (e.g., duration of storage) in order to minimize preservation distortion.

Measurement of fecundity and egg weight requires special consideration. Many small species have few, large eggs. Gonadal estimates will be easier closer to spawning. The timing of sampling will also be affected by residency, and the two factors have to be optimized. The entire gonad should be preserved and fecundity counts conducted with the aid of a dissecting microscope.

Top of Page

3.3.4 Live-bearers

Live-bearers are not common in Canadian freshwater receiving environments, but if used, require special attention regarding measurement of reproductive variables. Live-bearing species have been used successfully for detecting responses in exposures to Swedish pulp mills (Larsson et al. 2000, 2002; Larsson and Forlin 2002). To estimate fecundity, the gonad must be preserved and the number of live and dead embryos counted. Proper sampling requires some preliminary data on spawning time and gonadal development so that sampling procedures can be optimized.

3.4 Effect Indicators

The effect indicators for the various types of study designs for the fish survey are listed in Table 3-3. For a much more detailed discussion on these topics, consult Munkittrick et al. (2009), where the authors re-emphasize the original purpose of the EEM program and discuss why the current EEM effect indicators are used in place of other levels of monitoring. Additional issues raised and addressed by Munkittrick et al. (2009) include the influence of natural variability (i.e., the tendency for parameter values to change from year to year, or potentially from site to site), genetic adaptation, and four important statistical design issues (site selection, pseudo-replication, power analysis, and concern over the number of comparisons made).

The EEM program focuses on parameters measurable in groups of individuals, for several reasons:

• The approach offers a compromise between the sensitivity and reversibility of biochemical approaches, and the relevance of community-level parameters.
• Monitoring at the community level will miss reversible, important effects at the population level.
• Changes to fish growth, reproduction, condition or survival puts fish at risk, and therefore, focusing on these population level parameters addresses the overall objective of the Fisheries Act, which is to protect fisheries resources.
• Knowing this level of risk is important to the management of ecosystems.

Top of Page

3.4.1 Lethal Sampling

In answer to the question “have fish been modified by the effluent?” effects on growth, reproduction, condition and survival of the fish population are examined. The program recommendation for the fish survey is that key indicators be measured in both sexes of adults of two species of fish. The precision for the measurements is listed in Table 3-1. The intention is to obtain estimates of age or size distribution, how well fish are using available energy for growth and reproduction, and the storage of energy as reserves. The required numbers of samples can be calculated from a statistical equation using the standard deviation (SD) of gonad sizes for the species and site (from previous samples), and a critical effect size (CES) of 25% (see section 3.7.1). The minimum sample size recommended for a lethal fish survey when there are insufficient data to calculate sample size by power analysis is 20 sexually mature males and 20 sexually mature females of 2 fish species, in each sampling area. The rationale for using 20 fish of each sex for lethal sampling is that there is little change in the 95% confidence limits with increasing sample size beyond 20 fish. For example, Munkittrick (1992) found that there was little improvement in White Sucker variance estimates with a sample size above 16.

When there is background information available, it should be used to calculate adequate sample-size requirements prior to conducting the fish survey. It is important that sample size and variability be examined early in the study design phase so that the study can be redesigned if the variability estimates are sufficiently high for the survey not to achieve adequate power. Fish surveys benefit most by decreasing variability. When variability is so high that sample sizes are not justifiable or cost-effective, the first consideration should be to redesign the study to a) reduce variability, b) select alternative species that may be less variable, or c) consider an alternative method.

It is strongly recommended that sampled fish be processed and sexed immediately in the field on sample days to ensure the collection of fish with an equal sex ratio. Subsequent sexing of the fish in the lab using frozen samples may show a skewed sex ratio if it is assumed that fish sampled in the field displayed a 1:1 sex ratio.

It is important to identify immature fish (fish not developing to spawn) so that they can be excluded from the statistical analysis. There are three situations where gonadal development of fish is not uniform: a) situations with multiple spawning species where spawning is not synchronized; b) multiple spawning species where the number of spawns per year is influenced by fish size or age; and c) in northern populations, where fish may not acquire sufficient energy reserves to spawn each year. In all cases, fish should be analyzed within a group: comparisons should be conducted between fish developing to spawn and fish that are not. As well, the proportion of fish in each category can be analyzed. In situations where the existence of two or more groups is known before sampling, it may be possible to separate fish into categories during sampling based on condition or fish size.

The EEM program operates in an iterative fashion, so it is not necessary to develop a full assessment of the fish populations in a single sample, and the measurements are meant to act as surrogates to assist in the development of an assessment over more than one phase. Any effects in the fish survey must be confirmed in a subsequent phase, and be assessed against the CESs before studies progress (CESs are discussed in Chapter 1). While the measurements listed below are the required measurements, it may be necessary to provide alternative measurements due to site-specific or species-specific issues.

* If caudal fin is forked, use fork length (from the anterior-most part to the fork of the tail). Otherwise, use total length, and report type of length measurement conducted for each species. In cases where fin erosion is prevalent, standard length should be used.

** Fecundity can be calculated by dividing total ovary weight by weight of individual eggs. Individual egg weight can be estimated by counting the number of eggs in a sub-sample. The sub-sample should contain at least 100 eggs.

*** For small-size fish weights, use at least a 3-decimal scale.

Top of Page

3.4.1.1 Survival

Mean age is meant to give an assessment of the relative ages of the reference and exposed populations. If size-selective gear such as gillnets are used, and there is a significant difference in mean ages of fish sampled at both sites with identical gear, the difference indicates a need to further investigate the population and the reason for the difference in subsequent phases. More detailed information can be obtained through age distributions (or size distributions if aging is not possible), if adequate sample sizes are available or if aging is not possible. Furthermore, since many fish species have short life spans (< 4 years), it may be necessary to obtain immature fish and juveniles in order to conduct an appropriate assessment of this effect indicator. It is also very difficult to obtain a 25% difference in age when species are short-lived, and it may be possible to substitute a difference in average size (length) of 25% as a surrogate for age when species are short-lived.

A list of appropriate aging structures for a variety of potential sentinel species is provided in Table 3-2. In addition, there are many references that can be referred to for aging methods (e.g., Mackay et al. 1990). Methods of aging should be consistent at each sampling area and among phases, and appropriate quality assurance / quality control (QA/QC) procedures should be followed (e.g., independent confirmation). It is recommended that all aging structures be archived for future reference. If fish cannot be aged reliably or if it is not cost- or time-effective, the age can be determined by using size-frequency distributions. This may be especially useful when sampling small-bodied fish species or when conducting non-lethal sampling. It may also be possible to confirm the size-frequency distributions by aging representative sub-samples from each size class. For more information on size-frequency distributions, consult Nielsen and Johnson (1983).

Top of Page

3.4.1.2 Energy Use (Growth and Reproduction)

Growth and reproduction measures give an assessment of the ability of fish to use the food available to them. Growth is the change in size (weight or length) with time or age. In the case of growth, it may be helpful to collect information on other age classes, such as whether there are changes in growth of early life stages. This will assist in determining the magnitude of the effect. Subsequent phases should focus on confirming responses detected and examining the relevance of the changes to other size classes and species.

Reproduction is expressed as reproductive effort, fecundity, egg weight or gonad weight relative to body size. Reproduction may be the most sensitive measurement in resident fish. Changes in reproductive investment can be evident within a year, because the reproductive tissue is generally turned over annually. Fecundity and gonad weight are easy to measure if an appropriate sampling time is chosen. Confirmed changes in gonad size could lead to additional work related to magnitude, such as determining whether the change occurs at other times of the year (for multiple spawners) or whether the changes are present in other species in the same area.

Top of Page

3.4.1.3 Energy Storage (Condition)

Measures of energy reserves provide valuable information on the availability and quality of food to the fish. The EEM program uses condition (body-length-to-body-weight relationships) and liver size as indicators of energy reserves. As with other indicators, the consistency in response between indicators is important. Liver size can increase for several reasons, including storage of lipids and glycogen and enhanced detoxification activity.

Top of Page

3.4.1.4 Abnormalities

During the fish survey, a visual examination of fish is also conducted in order to identify the presence of any internal or external abnormalities, such as of body form, body surface, fins, eyes, lesions, tumours, neoplasms, scars or other abnormalities such as eroded, frayed or hemorrhagic fins, internal lesions, abnormal growths, parasites, and any other unusual observations. An area on the data sheet should also be included for other significant observations. Photographs can be a useful tool to document any obvious abnormalities.

It is recommended that a rough illustration of the selected fish species be incorporated into the data collection sheet for the recording of abnormalities in the external appearance. This information can then be used by others at a later date if significant differences exist between reference and exposure areas.

More information on fish anatomy can be found in general fish biology textbooks. Instructions on tumour descriptions are available in Gross Signs of Tumors in Great Lakes Fish: A Manual for Field Biologists (www.glfc.org/tumor/tumor1.htm).

* Fish survey effect endpoints used for determining effects as designated by statistically significant differences between exposure and reference streams. Other supporting endpoints can be used to support analyses.

Top of Page

3.4.2 Non-lethal Sampling

Non-lethal sampling should only be used in situations where it is warranted, i.e., where there is a concern about the potential impacts of sampling on small fish populations. Lethal sampling of adults is preferred where possible, although information on non-lethal samples can be valuable when large numbers of fish are collected during the sampling procedures. The indicators used for non-lethal sampling are contained in Table 3-3, and additional information on statistical analysis for the non-lethal sampling is contained in Chapter 8.

If the only option for a facility is to do a non-lethal sampling of fish in order to evaluate the effects of effluents on the fish population at a facility, a minimum of 100 fish older than YOY is recommended from each study site. The YOY acquired during the collection for the 100 non-YOY fish should also be retained and sampled (measured). YOY can usually be separated from older age-classes by size distributions; however, this may not be possible for species with extended spawning periods. The proportion of fish that are YOY should be estimated from the first 100 fish collected. If YOY abundance is extremely high (> 80-90%), sampling should then continue until 100 non-YOY are captured to calculate size-distributions of older fish. The collection of the additional non-YOY fish allows for a higher discrimination of the older fish classes to be achieved. The fish older than YOY that are collected should represent the whole range of fish sizes and be representative of the population (mature and immature). The recommended sample sizes in each area will give a good idea of the population distribution when plotting parameters such as the length or weight frequency. As well, when examining differences between the relative abundance of young versus mature fish, fairly good resolution is achieved (Gray et al. 2002).

When possible, sampling should be conducted when YOY are a catchable size in the gear being used. The same sampling gear should be used in both the exposure and reference areas; if it is not possible to use the same gear, or multiple gears must be used, the size distributions within a site should be compared between gears. If there are differences in the sizes of fish collected with different gear, comparisons between sites should be restricted within gear type. The sampling techniques and relative effort should be the same in all sampling areas. Pooling of data from different fish-sampling techniques should be avoided, and all methods used should be reported. If more than one gear type is used, the records of fish caught by each method should be reported, and any pooling of data clearly described. Fish should be measured for length (±1 mm), weight (± 0.01 g) (Gray and Munkittrick 2005), assessed for the presence of abnormalities, and external sex determination should be made, if possible. All fish should then be released. If possible, a small number of larger fish should be sacrificed to verify ages of older individuals. If only adults are used, the priority should be to sample prior to or at the start of the spawning season (see guidance on preferred sampling times in Table 3-5). However, if YOY are to be collected, the timing should move to the late fall, when it will be easier to measure YOY for most species. Fall sampling of YOY will be much more difficult if the fish are not single, synchronous spring spawners, as the size distributions of YOY fish will be broad.

A large number of areas can typically be sampled when conducting a non-lethal survey and the facility is encouraged to sample multiple exposure and reference areas. Programs that sample adults and YOY will allow for maximal assessment of effect indicators.

Species selection for non-lethal sampling can be difficult and is often based on availability. When choices are available, a synchronous spring spawner will offer the most advantages in terms of differentiating YOY from older year-classes. Discrimination of year-classes can also be affected by the longevity of the species. An annual species such as silverside will have a single year-class, eliminating the need to differentiate year-classes. A short-lived species (2-3 years) with fast growth and easily distinguished year-classes also offers advantages. However, these species are not always available.

When multiple species are available to choose from, it is recommended to collect initial samples and examine the ability to discriminate YOY and age-classes between species.

Top of Page

3.4.2.1 Survival (Size Distributions)

There are challenges to using age information on many short-lived species of fish. If a fish only lives 2-3 years, it will not be possible to measure a 25% difference in mean age. If non-lethal aging structures have not been validated for the sentinel species being used, size-distribution should be examined as a surrogate for differences in age.

Size distributions should be compared between exposure and reference areas with the Kolmogorov-Smirnov test, although this test is not very sensitive. Size comparisons should also examine distributions for YOY alone, for both sizes combined. If a site difference is present, subsequent phases should focus on understanding the difference and possible causes. When possible, verifying the ages of larger fish and YOY can be useful.

Top of Page

3.4.2.2 Energy Use

It should be possible at most sites to get estimates of growth and reproduction using non-lethal methods. Growth can be evaluated by the size of YOY at the end of the growing season and by the size of the 1+ fish. A comparison of the size of YOY fish between sites gives a good indicator of growth, as it is a direct indicator, in comparison to size-at-age, which is indirect. YOY are used because all of their growth is attributable to environmental conditions since the spawning time, and growth is not complicated by diverging energy into reproductive development. Differences between sites in spawning times will be integrated into this analysis. It is also possible to get a growth estimate by a shift in size distributions over time (e.g., repeating measurements 2 months apart at the same sites), or differences in average size (this would require a second sampling trip to determine). If the fish species chosen is externally sexually dimorphic, it is possible to examine whether there are gender-specific differences in growth rate.

Reproduction can be assessed using relative age-class strength or by the relative abundance of YOY individuals (Gray et al. 2002) or by YOY survival, which requires two sampling periods. A length-frequency distribution may be plotted as a surrogate of an age-frequency distribution. Size-frequency analysis can be used to examine size distributions and distributions of condition factors (using length and weight data), and can be used to infer age distributions and size-at-age data (if ages can be inferred) (Gray et al. 2002). It is recommended that, if possible, aging structures be collected from a sub-sample of each size-class, for situations where age may need to be verified (as in section 3.4.1.1, the utility of the age information is reduced in situations where the species is short-lived). In Slimy Sculpin, rapid growth of YOY fish in the spring can cause some overlap with the 1+ age-class, making resolution difficult (Gray et al. 2002). The ability to discriminate the YOY will depend on the duration of the spawning season, and the amount of time elapsed between the spawning time and sampling time. It may be easiest (for spring and early-summer spawning species) to examine length-frequency distributions using late summer and early fall data, when the YOY should be easiest to distinguish. To test for differences in relative abundance of YOY between the exposure and reference areas, a Kolmogorov-Smirnov test can be performed on length-frequency distributions with and without the YOY included. If inclusion of the YOY changes the interpretation of the significance of the difference (i.e., it is different with them included, and not different without them), there is then a difference in the relative abundance of YOY. Alternatively, replicate areas can be sampled to allow for the use of more statistical approaches, or the proportions of YOY can be tested using a Chi-squared test.

It may not be possible to distinguish YOY in species that spawn multiple times, in northern areas where YOY may emerge later in the year, or in situations where there are habitat-preference differences that are age-dependent in a species. In those cases it will not be possible to easily infer potential reproductive impacts. Some professional judgement will be required. If the species lives multiple years and immature fish can be distinguished non-lethally (condition near spawning time can be used in many situations for this), the proportion of immature fish can be used as a substitute. In cases where this is not possible, interpretation will need to be made based on size distributions alone, and care must be exercised to be conscious of the potential impacts of adult mortality on interpretation.

It is important to remember that a difference in water temperature between sites will affect spawning time. End-of-summer differences in size distributions could as easily result from differences in spawning time due to temperature as other potential causes. If there are temperature differences between sites that are suspected to be a major cause in the differences in size distribution observed, then subsequent studies should determine whether these site differences are a consequence of the facility or an inadequacy in choosing reference sites.

If it is possible to make multiple sampling trips, it may also be possible to measure changes in condition before and after spawning as an indicator of reproductive investment. For some small-bodied species, spawning females are very easy to distinguish by condition factor. Differences in condition factor of females between sites before spawning, or an indication of the change before and after spawning in females, could be used to infer reproductive investment, if females can be distinguished after spawning.

Top of Page

3.4.2.3 Condition

Condition factor (k) can be evaluated by the relationship (k = 100 000* (wt/l³)) of the fish examined (where wt = weight [in grams] and l = length [in mm]). The appropriate analysis for final interpretation is an analysis of covariance (ANCOVA) of weight versus length, by site.

Top of Page

3.4.3 Wild Molluscs

Where there are no appropriate finfish present, collection of wild molluscs, such as oysters or mussels, may be considered. Shellfish are included under the definition of fish in the Fisheries Act and they have been used by some pulp and paper mills in the EEM program. However, there are some drawbacks, including difficulties in aging individuals and in estimating reproductive investment in some species. Crabs and lobsters are not suitable species because they cannot be reliably aged at the present time (Environment Canada 1997). Currently, guidance is available on the relative gonad index (mantle somatic index) for bivalves (see mesocosm guidance in Chapter 9).

Molluscs are a diverse taxonomic group that include bivalves and gastropods, and are widely distributed throughout Canada. Molluscs possess many qualities that a species for monitoring should exhibit:

• they are relatively sedentary, although some species (i.e., unionids) may migrate short distances (metres) within their habitat;
• they are widely distributed across Canada and are identified with limited taxonomic expertise;
• most unionid bivalves are large enough to provide sufficient tissue for analyses;
• several bivalve species have been shown to readily accumulate many chemicals from a variety of pathways (water, sediment, food) and show sublethal effects associated with exposure; and
• bivalve growth is relatively easy to measure and has been shown to be as sensitive or more sensitive than mortality in other standard assays on species such as Daphnia, Fathead Minnow and Rainbow Trout (see Salazar and Salazar 2001).

In general, reproductive periods for molluscs and patterns of abundance are related to climate and the abundance of food supply. For most freshwater lotic or lentic habitat types, sampling is best conducted during the fall when the majority of taxa will be present and/or are large enough to be easily collected. In marine environments, sampling should be conducted in late summer or fall, as populations with spring recruits have stabilized by this time.

3.5 Timing of Sampling

A variety of factors need to be considered when deciding on a time to sample, including potential migratory behaviour of the sentinel species, water conditions (e.g., flow, turbidity, wave action), accessibility, and the cycle of gonadal development for the sentinel species. Where historical data exist, it would be useful to examine the data and, if appropriate, conduct the survey during similar periods so that the surveys can be compared.

The timing of sampling should be synchronized with the development of sufficient gonadal tissue so that effects on the reproductive function can be assessed. However, such information is unavailable for many species of fish. Species for which there exists extensive background information on their biology and life history characteristics should be preferred as sentinel species in order to ensure that sampling can be synchronized with sufficient gonadal tissue development.

Recent research has been conducted to evaluate the optimal timing for interpreting gonadal development, using seasonal collections from a variety of species. Five types of fish categorized by spawning characteristics have been identified, and Table 3-4 provides the recommended sampling time based on the following background collection studies: background collections followed Canadian freshwater species that were synchronous spawners (such as Slimy Sculpin; Gray et al. 2005; Brasfield 2007), multiple spawners with few spawnings per year (such as Blacknose Dace [Rhinichthys atratulus]; Galloway and Munkittrick 2006; Hicks and Munkittrick, unpublished data), multiple spawners with many spawns per season (such as Redbelly Dace [Chrosomus eos]; Carroll 2007), and asynchronous spawners (every few days, such as Mummichog [Fundulus heteroclitus; McMullin et al. 2009). There is a fifth type of freshwater species that has asynchronous development, where individuals may take a year off from spawning because of cold temperatures or low food availability. This variability has a major impact on power and sample size requirements.

Examination of these data confirms that there are specific times when power is higher for detecting differences, and when gonadal development is adequate for detecting impacts. The generalizations in Table 3-4 may not apply to all species or all regions; the regional EEM contact should be consulted for any available updates to regional guidance.

Synchronous spawners show a difference in timing of gonadal development between males and females. For synchronous spring spawners, adequate data can usually be obtained as late as possible in the fall, or prior to spawning in the spring. If previous data are available for a site, the reproductive strategy can usually be estimated from the magnitude of the correlation coefficient (R²) between gonad weight and body weight, if the previous collections were done at a time when the gonads were well developed.

Evaluation in multiple-spawning species is complicated by the duration of the spawning period. In the case of such species, the frequency distribution of age- or length-classes may provide valuable complementary information on the reproductive success.

Multiple spawners with few spawns should be sampled at least 6 weeks prior to the initiation of the spawning season (for information on spawning temperatures, consult references such as Scott 1967; Scott and Crossman 1973; Roberts 1988; Nelson and Paetz 1992; Jenkins and Burkhead 1993; Coad 1995) due to an increased variability in the gonad-weight-to-body-weight relationship as the spawning season approaches, because of a lack of synchronization in timing for the second clutch of eggs (Galloway and Munkittrick 2006). Multiple spawners with many spawns, and asynchronous spawners, should be sampled close to the start of the spawning period because of the rapid development of the gonads in both species.

The consequences of sampling at an inappropriate time have been examined using data from the pulp and paper EEM program (cycles 1 to 3). For large-bodied species, fish were sampled outside of the optimal window in more than 33% of previous studies, but interpretation was not strongly affected when optimal and suboptimal studies were compared. However, small-bodied species were sampled at suboptimal times more than 75% of the time, and data collected outside of the optimal windows failed to detect significant effects on gonad or liver size (Barrett and Munkittrick, unpublished data).

Top of Page

Reproductive Strategies:
S, single spawner; M, multiple spawner (few spawning events); MM, multiple spawner (many spawning events); K, exhibit ''skip'' spawning; G, guard nests and (or) provide some form of parental care to their eggs or young; (GSI), strategy was decided based on GSI data over a reproductive cycle; (I), strategy implied or some evidence supporting a particular strategy (e.g., duration of spawning season); ?, data were unavailable to support a reproductive strategy, the strategy was predicted based on observations by the authors of ova sizes in mature ovaries.
Spawning time:
Integers from 1 to 12 to indicate the months in which the species is known or is believed to spawn in Canada. Ranges correspond to all months in that range (e.g., 5-7 corresponds to May, June, and July).
Spawning temperature:
Single temperature in combination with > or < signs, threshold at which a species has been known to initiate spawning activities; Single temperature without > or < sign simply corresponds to a single spawning temperature provided in the literature; Range of temperatures, range at which spawning activities has been observed; ?, spawning temperature data were unavailable or values were predicted based on data for other species of the same genus.
Sampling times:
Late fall, as late as possible before ice cover; 4-6 weeks pre-spawn, four to six weeks before the first spawning event; Spawning, close to the first seasonal spawning event.

a Reproductive strategy as per Barrett and Munkittrick 2010 is S(I). However, there is evidence from data collections in New Brunswick in 2011 and 2012 that common shiners are multiple spawners (Barrett, pers. comm., April 2013).

b Reproductive strategy, spawn time and recommended sampling time were modified from Barrett and Munkittrick 2010 following availability of data from a more recent study conducted in New Brunswick (Barrett, pers. comm., April 2013).

3.6 Verification of Fish Exposure

It is crucial that studies be designed to maximize the possibility of detecting effects if they are present. This can be accomplished by sampling at the proper time of year, with appropriate gear, at appropriate reference areas and during the period of residence in the effluent area. If fish exposure to the mine effluent is uncertain, redesigning the survey (selecting different species, using tracers, changing sampling time or changing exposure or reference areas) or using alternative monitoring methods should be considered for the subsequent phase.

Controversy arises when fish show no differences in characteristics among sites, and there are no indicators of exposure. In this case, it is difficult to determine whether the fish at both sites belong to the same population. In order to verify the exposure of fish to effluent in the exposure areas, and to verify the lack of exposure at reference areas, it may be necessary to select a tracer which accumulates in fish tissue. The selection of a tracer depends on the type of mine involved and the complexity of the receiving environment.

It is possible to infer exposure by examining metal levels in indicator tissues. The indicators and the tissues will vary with the mine type and species being used. In general, gills, liver and kidney have the greatest potential for estimating exposure and bioavailability of metals. Mercury is the only metal element of concern that has been found to accumulate in muscle tissue, so if mercury is a contaminant of concern, dorsal muscle tissue should be analyzed. Blood and bone tissue may reflect exposure to lead, and might be considered if lead is the primary element of concern (Hodson et al. 1984). Bone concentrations are expected to be most indicative of long-term metal exposure, while blood concentrations are indicative of short-term exposure (AETE 1998). For larger species, samples of liver or kidney can be collected. The tissues should be frozen for later preparation and analysis. For small species (< 10 cm), whole body levels can be examined, or levels in the carcass after removing the digestive tract. See section 3.11 for fish usability methods.

Large statistical differences between sites in whole-organism characteristics in a number of parameters give some confidence that the samples are from different populations of fish. If there are no differences between sites, it may be that fish are moving or that there is no impact. Stable isotopes of carbon and nitrogen can be used to document that there are differences in fish residence times, provided that the stressors in question locally alter stable isotopes (i.e., Farwell 1999; Galloway et al. 2004), or there are local geochemical differences that alter stable isotopic signatures and that can be used to demonstrate local residency (i.e., Gray et al. 2004). However, the stable isotopes are not always sufficiently different between sites to be useful, and their suitability has to be evaluated on a site-specific basis (Dubé et al. 2006).

By selecting a sampling time and fish species that have life history habits that may increase the likelihood of exposure, potential exposure can be maximized. For example, for species having spawning movements that take them away from or temporarily into the effluent exposure area, a survey conducted during the spawning season would be ineffective. Thus, for spring-spawning freshwater species, a fall survey would be appropriate. For fall spawners, a spring or summer survey is appropriate. This may not apply to fish in which ova mature rapidly; for example, as some late-spring-spawning cyprinids should be sampled in early spring, rather than in fall when ova may still be immature, it is pertinent to have some background biological information, if possible.

The timing of sampling and the choice of fish species should be made according to normal operation of the facility to ensure that the effluent is present in the environment. Sampling when effluent has not been discharged for long periods (months) should be avoided. However, the selected sampling gear, flow conditions and effluent conditions may limit the preferred season for the survey.

If no fish are captured (or they are captured in reduced density) and there are no fish resident in the exposure area, it could be interpreted that fish are avoiding the exposure area. The suitability of fish species should be evaluated at the end of each monitoring phase, based on the site-specific nature of the results and the site-specific concerns about residency and exposure.

There are some situations where fish may move freely in and out of the exposure area, and no species spend significant periods of time in the effluent. In these cases, the sampling should be designed to maximize exposure time in the effluent area and possibly during periods of optimal gonadal development.

There are two main issues dealing with residency: whether the fish from reference and exposure areas were mixing; and whether the fish captured in the exposure area were indeed exposed. If fish demonstrate exposure, are collected in the exposure area, and demonstrate differences from reference-area fish, there should be no controversy. Follow-up studies can examine other species to see if they demonstrate effects.

If fish demonstrate exposure, are collected in the exposed area, and show no differences, it is outside the scope of the EEM to determine why exposure-area effects are not seen. If subsequent monitoring phases confirm the absence of demonstrated effects and the study design was adequate, it would be concluded that the conditions of the area allow for fish that are exposed to effluent not to be affected, using the current design.

Top of Page

3.7 Power Analysis

The purpose of defining an effect-size and power level is to determine if the sampling program is collecting sufficient information for decisions to be made. The statistical power of a comparison is a function of the sample size, the variability and the target difference set between areas. To determine the sample size for detecting a specific difference, some knowledge is needed about the statistical power level that is acceptable for the decision-making process and the variability of the population.

Top of Page

3.7.1 Power and Significance Level

Earlier cycles of the pulp and paper EEM program set the power (1-beta [β]) at 0.80 and alpha (α) at 0.05. The EEM program now encourages setting α and β equal to one another. If values are set at α = β = 0.10, the sample sizes required to detect the same effect are approximately the same as in earlier cycles. Where possible, provided sample sizes determined by the power analysis are not unreasonably large, mines are encouraged to reduce α = β = 0.05 (the traditional level for alpha). In many statistical programs, the default β is 0.20, and needs to be adjusted. Again, these recommendations are to help ensure that studies are designed to provide a reasonably high probability of statistically detecting a predetermined effect size if it has occurred, (i.e., the power of the test [1-β] should be high). Refer to Chapter 8 for the rationale for setting α and β at equal levels.

It is important to understand that variability and power will vary with the parameter being studied. Fish are not equally variable across all of their characteristics. Reproductive variables are usually as changeable, or even more on a relative scale, than parameters such as length, weight and liver weight (Environment Canada 1997). If effect sizes are also expressed on a relative scale (i.e., as percent differences), any study that can detect a ± 25% difference in relative gonad size can detect similar or smaller differences in other important parameters.

Top of Page

3.7.2 Effect-Size

It is recommended that the EEM program be designed to detect a difference of 20-30% in gonad size, using a recommended power level of 0.90 (1-β). The magnitude of the difference that could be detected for other parameters would be fixed based on the sample size for determining an effect on gonad size. The power for detecting differences in other parameters should be reviewed during study design to ensure that reasonable power is achieved for as many variables as possible. The same approach used to identify a target effect-size for relative gonad weight should be applied to other variables. Sensitivity analyses using population models should be used to explore the consequences of the effect-size chosen for any and all variables (Environment Canada 1997).

An extensive literature review has shown that CESs that have been defined in other programs are often consistent with a CES of around 25% or 2 SDs for many biological or ecological monitoring variables. This value appears to be reasonable for use in a wide variety of monitoring programs and with a wide variety of variables (Munkittrick et al. 2009). Barnthouse et al. (1989) argue that a 10% change in variables would be societally and ecologically significant, although they were concerned primarily with laboratory toxicity tests and not field surveys. Their proposed effect-size was deliberately conservative (small) because of concerns about the uncertainty in extrapolating laboratory results to the field.

When preliminary analyses show that power will be insufficient given reasonable sample sizes, the assessments should be redesigned. Studies are designed site-specifically, and priority should be given to reducing variability rather than increasing sample size. As variability will also vary between sampling campaigns, the target effect-size should not be a fixed number, but rather should be a range of changes that you wish to detect, such as 20-30% difference. Sample sizes can be calculated using methods described in Green (1989); sample size calculators can also be downloaded from the Internet, such as the common one that can be found at http://biostat.mc.vanderbilt.edu/wiki/Main/PowerSampleSize.

A priori power calculations and CES calculations are described in section 8.6.2.1 of Chapter 8.

Top of Page

3.8 Fish Sampling Methods

Sampling methodology should be chosen site-specifically, and capture gear and effort should be focused on methods shown to be successful. The same sampling methods can be used for population and community surveys. The difference lies with the selectivity of the fishing gear. During a community survey, the gear should be as non-selective and non-destructive as possible. For the population survey, which focuses on one or two species, the gear will be more selective. For example, trap netting may be preferred during a community survey, while a one-size mesh gillnet of the appropriate size could be appropriate for a population survey.

Standardized sampling is a priority. Therefore, in situations where sentinel species are the same as for a previous phase, and the sampling techniques used previously were sufficient to capture the target number of each sentinel species, these same sampling techniques should be retained unless good reasons for change, such as unacceptable bycatch, are documented. The sampling techniques and relative effort should be the same in all sampling areas. Pooling of data from different fish-sampling techniques should be avoided, and all methods used should be reported. If more than one gear type is used, the records of fish caught by each method should be reported, and any pooling of data clearly described.

A number of good guidance documents fully describe fish collection methods (Schneider 2000; Portt et al. 2006). Portt et al. (2006) describe the use and efficacy of 1) gillnets; 2) beach seines; 3) hoop, fyke and trap nets; 4) electro-fishing; 5) underwater observation; 6) Gee or minnow traps; and 7) enclosure (drop, pop and throw) traps. However, methods will usually have to be developed and optimized site-specifically.

Top of Page

3.8.1 Bycatch

It may be possible to obtain samples using the bycatch of commercial, research or other fisheries operations in either marine or freshwater situations. The investigator is responsible for ensuring and documenting that sampling procedures and conditions are met (QA/QC), and that fish are exposed. Capture techniques also have to be standardized between sites.

Top of Page

3.8.2 Remote Sensing

Fish abundance near outfalls can be monitored using video or still cameras mounted on remotely operated vehicles. This technique may be particularly effective in rocky and steep areas where use of fishing gear may be difficult. Camera surveys may also be useful in reconnaissance surveys of bottom conditions before trawls or traps are deployed for fishing. Any proposed methods should be clearly outlined in the study design for review.

Top of Page

3.8.3 Alternative Methods

There may be situations where conducting the fish survey is not suitable. The reasons for this are site-specific, but the most common reasons are the presence of hazardous conditions (e.g., strong currents) or the presence of confounding factors such as other effluent discharges in the exposure area, which will make it difficult or impossible to isolate any effects attributable to the effluent being monitored. Under these circumstances, the mine may select an alternative option to the fish survey and/or the fish usability survey. Recommended alternative monitoring methods for the fish survey are mesocosm studies and caged bivalves. Detailed guidance on how to conduct the alternative monitoring methods and interpret the data can be found in Chapter 9.

Top of Page

3.9 Fish Survey Quality Assurance and Quality Control

3.9.1 Field Practices to Improve Data Analysis and Interpretation

The quality of data collected in the field influences the ease of data analysis and interpretation. The preparation of data recording sheets beforehand will save time in the field, and the use of waterproof paper is encouraged. Field conditions, habitat, gear used and information for catch-per-effort calculations should be recorded. The use of the same balance and measuring device for all measurements, and having the same person taking the measurements, will reduce measurement error. If the person taking the measurements is reporting the data to a person recording the measurements, avoid the use of decimal points and report all measurements as digits and not numbers to avoid transcription errors (e.g., report 14.5 cm as 1-4-5 and use units of mm); some numbers can be easily confused when reported orally, such as “fourteen” vs. “forty.”

It is essential that the sampling gear be consistent between the sampling areas, because most sampling methods select for certain age- or size-classes, and thus inconsistent sampling gear between sampling sites could result in detecting false differences (e.g., in age or size).

Top of Page

3.9.2 Quality Control in the Field

This is the first stage of data collection. QA/QC procedures for the fish survey should be outlined during the development of the study plan and should be followed precisely in order to maintain high-quality data. While a QA/QC plan for field sampling can have many components, some of the main procedures are as follows:

• initiate and maintain communication with local government agencies (e.g., fishing licence, dates of fish collection, location of collection, endangered species, etc.);
• all personnel involved in field sampling should have appropriate education and/or training and be familiar with the written standard operating procedures for the survey;
• all safety measures should be identified, understood and adhered to;
• fish collection methods and equipment should be appropriate for the specific water body and fish species;
• habitat descriptions, including possible modifying factors (water depth and current, dissolved oxygen concentration, temperature, substrate classification, evidence of pollution [discolouration, odour, residues], salinity, conductivity, etc.);
• date and time of collection;
• collection methods need to be consistent throughout the study;
• location of sampling areas and fish collection areas documented (geographic coordinates); photograph the collection location;
• record of the number of fish species and incidental species caught per collection stations;
• estimate of catch per unit effort;
• samples from fish (e.g., ovaries, age structures, stomach content) should be placed in appropriate containers;
• suitable preservatives/fixatives (e.g., ovaries--frozen or formalin) should be used;
• all samples should have appropriate labelling;
• all measurements will be taken using appropriate equipment of acceptable accuracy and precision (this should be documented);
• instruments should be calibrated and maintained in good working order (records and methods should be available);
• detailed field notes should be maintained in a bound notebook; and
• chain-of-custody forms and appropriate shipping and storage procedures should be used.

Top of Page

3.9.3 Determination of Sampling Effort

To aid in assessment of expected effort requirements at individual sites, the study plan submitted to Environment Canada should include details on how fish sampling will be performed.

The following are performance-based criteria and guidance to determine a “reasonable level of fishing effort.” Each site is unique. It is uncertain whether fishing success will be achieved at a site just because a certain level of sampling effort has been successful in the past at other sites or even at the same site at other times.

1. The study design should document all details on how the adult fish sampling will be performed, to aid in assessment of effort. Details to include in the study design (where applicable) are:
   
   
   • how and why the sentinel species were selected;
   • who was consulted on the locations and techniques chosen to collect the proposed sentinel species;
   • contingency plans regarding alternative gear and sentinel species;
   • scheduling of dates for work that will be performed so that EEM contacts can be available for consultation;
   • type, location(s) and dimensions of gear (e.g., gillnet, trapnet, hoopnet, fish trap, trawl; in some cases more than one type of gear may be advisable);
   • mesh type (e.g., nylon, cotton fibre or wire, knotted or knotless) and size;
   • proposed level of gear / fishing effort;
   • sampling time (i.e., time of day);
   • sampling duration (i.e., time interval between gear placement and retrieval); and
   • frequency of checks.

   Any preliminary fish survey results or observations made during pre-design activities should be provided where they have guided selection of sentinel species or procedures. The regional EEM contact will review these data and may request further information to clarify sampling procedures.

2. Proper operating procedures should be used. These include use of gear as outlined in the study plan. Gear should be checked at a frequency that ensures the recovery of sentinel species in useful condition and the release of non-target (especially protected and endangered) species. The use of non-lethal and/or selective techniques should be a consideration. A record of the identity and estimated numbers of non-target fish may be a useful addition to contingency plans. The mill and consultant should have a good understanding of the habitat, the characteristics of the species, and the gear being considered.
   
3. Consultation with local experts (e.g., provincial and federal fisheries personnel, Aboriginal groups, individuals and associations involved in local sport and commercial fisheries, the public, and others with knowledge of local fisheries resources) should be conducted to ascertain that the selection of sentinel species, location of nets, timing of collections, etc., are optimal.
   
4. Personnel tasked with the fish collection and sampling procedures should have documented experience.
   
5. Licences for collecting should be obtained from local fisheries agencies.
   
6. Records should be kept that document the operating procedures used (e.g., mesh size, sampling time, location, frequency of checks, etc.). These records may be required in order to properly assess the manner in which the study was conducted.

Although not required, it is recommended that an estimate of catch per unit effort (CPUE) be provided for each sampling area (e.g., number of fish caught per unit of time or area or net). The CPUE information is useful in documenting the effort expended in situations where collection of the minimum number of fish may be difficult.

Top of Page

3.9.3.1 Examples of Calculations of Sampling Effort

Some examples of fishing methods that have been successful in collecting fish in a timely manner are provided below. These are provided as examples to guide consultants in the development and implementation of their study design, and to indicate when it may be advisable to consult with the Environment Canada regional EEM coordinator.

1. Data from two Ontario lakes indicated that 40 individuals of any of 6 warm-water fish species were collected in 1-6 sets for 24 hours duration. The equipment included a 6-x-6-foot trap net. Details of the process are presented in the Ontario Ministry of Natural Resources Fisheries Assessment Unit Newsletter (FAU Update Issue 94-1, OMNR 1994).
   
2. During the Assessment of the Abundance of Cold Waters Ontario Fish Communities Program, the fishing effort recommended to collect 40 Lake Trout in 7 lakes varied from approximately 12 to 120 hours. The equipment included a 46-m gillnet gang with 3 panels of 15.2 m. Details of the process are in the Ontario Ministry of Natural Resources Fisheries Assessment Unit Newsletter (FAU Update Issue 94-2, OMNR 1994). Mesh size should be consistent and selected according to the target species.
   
3. Experience has shown that gillnets made up of 4 panels of 50 m of net, set 24 hours/day for 5 days (equivalent to 24 000 metre-hours of effort) in freshwater systems, should allow for 20 fish of each sex to be collected. This is contingent on correct deployment of the panels and shifting of the panels to cover areas inhabited by the fish. Mesh size should be consistent and selected according to the target species.
   
4. Alternatively, a good strategy would be to initially set a minimal amount of net to decrease bycatch (< 400 m). If fishing is selective enough, and the amount of bycatch acceptable, up to 2 km of a single mesh size has been necessary in small unproductive rivers.
   
5. In marine situations, experience has shown that 48 hours of beam trawling, long-lining using a variety of hook sizes or other methods including traps (alone or in combination), should provide the 20 fish of each sex.
   
6. Consultation with users of electro-fishing technique (large rivers using boat-mounted apparatus) indicates that all fish can be obtained in one day. Procedures enhancing success include operating at dusk or night, passing over the same area at least three times, and using intermittent pulses of current (since a continuous field may actually chase fish away). In small streams, lakes and rivers, more time will often be necessary for sampling because of the difficulties encountered in moving through these environments.
   
7. In 1999, a consultant was collecting fish for an East Coast paper mill located on a tidal estuary. The target species were Mummichogs and the consultants used a 15-x-1.5-m beach seine with 0.5-cm mesh. One end of the beach seine was extended about 10 m out from shore by a technician wearing chest waders, while a second technician held the other end of the seine along the shore. The technicians towed the seine perpendicular to the shore for a distance of 20-30 m and then the outer end was towed into the shore to close the net. Once both ends of the seine were secured on the shore, the upper and lower lines were carefully retrieved to capture any fish enclosed in the net. The consultants found that fishing was most successful at slack high tide. A total of 108 Mummichogs were captured and retained over 4 days of sampling. Total fishing time was 12.5 hours. Many more Mummichogs were captured and released due to the need to balance male and female numbers. In addition, 11 other species of fish were captured and released (Final Report, Repap New Brunswick Inc. Kraft Mill, Second Cycle Aquatic EEM Study, Jacques Whitford Environment Limited, April 2002).

Although the above techniques and gear may apply to a variety of species, these examples are not all-inclusive because each site is unique and the examples are provided as suggested effort only. Local expertise can serve as further advice. The previous examples are adequate guidelines toward catching a minimum of 20 fish per sex, species and area.

Top of Page

3.9.4 Consultation with Regional EEM Coordinators and Implementation in the Field

If all of the above criteria are met and a mine/consultant is having problems meeting the minimum data requirements of the adult fish survey, the owner or operator may deviate from the study design but is required to inform the Authorization Officer without delay of those circumstances and of how the study was or will be conducted. All reasonable efforts should be made to collect target sample sizes of two species of fish and demonstrate due diligence on the part of the mine.

Possible outcomes and options of the consultation with the EEM coordinator are as follows:

1. Continue – Advice on the following situations will depend on site-specific conditions. Set further consultation dates if required.

1. Absence of target species at reference area:
   • continue with current gear and technique;
   • continue at alternative reference area identified in contingency plan;
   • continue at existing reference area with alternative target species, gear and/or technique identified in contingency plan.
2. Absence of target species at exposure area:
   • continue with current gear and technique;
   • continue at alternative exposure area identified in contingency plan;
   • continue at existing exposure area with alternative target species, gear and/or technique identified in contingency plan.
3. Absence of target species at reference and exposure areas:
   • continue with alternative areas, target species, gear and/or technique identified in contingency plan.

2. Postpone (not to continue) – Existing dangerous conditions; sampling conditions (e.g., weather, cold) will not allow collection of fish; alternative gear is not available; no further contingencies are available (e.g., no further alternative species; further investigation is needed):

• design new sampling plan in consultation with regional EEM contact;
• redeploy at a later date with original or alternative areas, target species, gear or technique, but under more favourable conditions;
• set dates for further consultation.

3. Discontinue – If the full complement of fish is not obtained, the absence (or paucity) of fish will be considered a result that will be thoroughly explained in the study findings, taking all possible contributing factors into account. If the minimum number of fish is not caught, this could result in inflated variance estimates. The decision on whether to continue will be influenced first by safety considerations. In all scenarios refer to the contingency plan where appropriate, and set date(s) for further discussion. Field technicians should speak directly with the regional EEM contact. Sentinel species choices will apply to both reference and exposure areas. Pooling of data from different seasons is not valid.

Top of Page

3.9.5 Data Entry

Data entry and preparation of analysis is discussed in Chapter 8, and reporting is discussed in Chapter 10.

Top of Page

3.9.6 Quality Control in the Laboratory

Although much of the survey information is collected while in the field, variables such as fecundity, egg weight, and age are usually determined later in the laboratory. With each measurement, the primary concern of the laboratory QA/QC program is to ensure consistency (precision) and accuracy of the data. The following issues should be considered as part of the measurement procedures:

• all personnel involved in sample processing and analyses should have appropriate education and/or training;
• measurements should be conducted using recognized protocols and methods (these should be documented), and all instruments used should be properly calibrated and maintained (records, methods available);
• keep fish measurements recorded for each fish (target species);
• keep a record of external lesions, tumours, parasites, etc;
• fecundity data, including methods and sub-sampling precision (if applicable);
• aging data, including methods and independent confirmation of estimates;
• maintain records that describe the sample, measurement, and responsible personnel; if possible, a minimum number of individuals should conduct a particular measurement to maintain consistency and reduce measurement error (especially for age determination);
• if sub-sampling is necessary (e.g., fecundity, egg weight), information describing the efficiency and accuracy of the sub-sampling technique should be documented; this information should also be used to calculate appropriate correction or scaling factors (if needed) to minimize possible differences in methods and efficiency;
• all data should be verified; for example, measurements such as fecundity and egg weight should be replicated to ensure precision and accuracy; a recognized expert should verify estimates of age;
• literature and taxonomic keys used for fish identification should be documented;
• archive samples and voucher specimens; and
• maintain detailed sample processing and laboratory notes in a bound notebook.

Top of Page

3.10 Data Analysis

QA/QC concerns regarding data analysis include data verification and validity, repeatability and robustness of statistical analyses, and rigour and defensibility of analyses. For the most part, validation and verification of data depends on the success of QA/QC procedures during field sampling, sample processing, and laboratory analyses (see above). However, there are other considerations regarding the data verification and analyses:

• conduct screening techniques to identify possible transcription errors, outliers and other potentially questionable data points;
• maintain tabular summaries of the general descriptive statistics (sample size, mean, minimum, maximum, standard error, and SD) of fish measurements (e.g., see Table 3-6);
• provide results of assessing assumptions of normality and homogeneity of variance;
• maintain a record of transformation used;
• provide parameter estimates of variability (analysis of variance [ANOVA] mean square error [MSE], ANCOVA MSE, SD for age-to-maturity);
• provide calculations of sample size requirements for each parameter;
• provide a summary of adherence to data quality objectives, standard operating procedures and identification of any QA/QC problems, which should incorporate considerations related to laboratory and field QA/QC;
• to allow reproduction of analyses and results, provide all raw data in an appendix and archive computer data files for an approved period of time after the analyses are published in a report;
• document in detail the methods used for analyses;
• verify that statistical software packages used produce the same output and results as other packages;
• evaluate the robustness of the analyses, (i.e., the results and conclusions should be similar);
• take note of whether outliers are included or excluded, and whether transformations are used, etc.; the objective is to ensure that results are not a function of some manipulation or assumption prior to or during analyses; and
• maintain detailed notes regarding the analyses of the survey data.

Top of Page

3.10.1 Statistical Analysis

The standard statistical assumptions required for many parametric statistical tests are those of independence, normality, and homogeneity of variances. These three assumptions and additional information on data assessment and interpretation are discussed in Chapter 8.

Note: The percentage difference should be reported as exposed relative to reference site. Statistical significance should be given as p-value.
Legend: Diff = difference, stat sign = statistical significance (p-value), ref = reference, exp = exposed, adj = adjusted, sign interax = significant interaction.

3.11 Methods for Analysing Fish Usability

The objective of the question “has there been a change in fish usability due to effluent?” is to determine whether effluent has altered fish in such a way as to limit their use by humans. Fish usability can be affected by altered appearance, altered flavour or odour, or contaminant levels that exceed consumption guidelines for human health and are statistically different from levels measured in the reference area. This section examines fish usability with respect to contaminant levels of mercury.

Mercury is the only metal for which there is a standard Health Canada tissue consumption guideline for humans, and therefore is a pollutant of national concern. Health Canada recently completed a study on mercury and reaffirmed the standard (maximum limit) of 0.5 µg/g with the exception of fresh/frozen tuna, shark, swordfish, escolar, marlin and orange roughy. Provincial and territorial governments are responsible for implementing fish consumption advisories for sport fisheries with the exception of federal parks. Consumption restrictions for sport fish begin at levels above 0.45 mg/g total mercury.

Biological monitoring studies consist of a study respecting fish tissue, if during effluent characterization conducted under paragraph 4(1)(d) a concentration of total mercury in the effluent is identified that is equal to or greater than 0.10µg/L (MMER, Schedule 5, s. 9(c)).

An effect on fish tissue means measurements of concentrations of total mercury that exceed 0.5 µg/L wet weight in fish tissue taken in an exposure area and that are statistically different from and higher than the measurements of concentrations of total mercury in fish tissue taken in a reference area (MMER, Schedule 5 s. 1). At some mine sites there may be reference areas that have levels of total mercury in fish tissue higher than the guideline (e.g., northern Quebec – Schetagne et al. 1997; Schetagne and Verdon 1999); therefore to be considered an effect in fish tissue, there must be a statistical difference between the areas and an exceedance of the guideline using a one-tailed statistical test.

As discussed in Chapter 5, the method detection limit for mercury in effluent has been changed to 0.01 µg/L (0.00001 mg/L) so that the concentration of 0.1 µg/L specified in Schedule 5, s. 9(c) of the Metal Mining Effluent Regulations can be detected with confidence. Analytical methodologies suitable to achieve this level of detection include cold vapour atomic absorption spectrometry (CVAA), cold vapour atomic fluorescence spectrometry (CVAFS), and inductively coupled plasma mass spectrometry (ICP-MS).

In the Guide to Eating Ontario Sportfish, it is suggested that other metals--lead, copper, nickel, zinc, cadmium, magnesium, chromium, arsenic and selenium (Pb, Cu, Ni, Zn, Cd, Mg, Cr, As and Se, respectively)--may be found in fish tissue, but not at levels for consumption restrictions. On a site-specific basis these metals may be identified as a concern if there are human health consumption guidelines from another regulatory agency (e.g., provincial or territorial), that are applicable to the region where the study is being conducted and if the metal for which there is a consumption guideline is present in the effluent. Local consumption and commercial fisheries should be considered to determine which edible tissues (liver, kidney, bones, flesh or even entire fish) should be analyzed. It is recommended that other metals in fish tissue be analyzed where there are site-specific concerns. The Guide to Eating Ontario Sportfish is available at the following website: http://www.ene.gov.on.ca/en/water/fishguide/index.php.

Molluscs can accumulate metals (Cd, Cu, Zn, Pb, Ni, mercury [Hg], As, silver [Ag], and Cr). Field studies suggest that the relationship between mollusc tissue metal concentrations and ambient metal concentrations are influenced by a number of biological, physical and chemical parameters that need to be taken into account. Ultimately, the relationship is metal-specific and depends on the availability of the metal from the dissolved and particulate phase (AETE 1997).

Below is the EEM protocol for fish tissue analysis. Other protocols may be used provided they meet the minimum EEM standards. For example, the Hydro Quebec protocol for the monitoring of mercury levels in fish (Tremblay et al. 1998) has been widely used in Quebec, as this protocol provides an examination of mercury in different age-classes of fish. The protocol can be found on the EEM website.

Top of Page

3.11.1 Selection of the Fish Species

In selecting the location of the sampling areas for the fish tissue sampling, the same factors considered for the fish survey should be taken into consideration. The species selected for tissue analyses should be, if present, sport, subsistence and/or commercial species, including molluscs and crustaceans, where relevant. The fish species used for the tissue analysis may or may not be the same as the species used in the fish survey. On a site-specific basis, the tissue used for the analysis should be chosen based on the portion of the fish constituting the edible portion locally consumed, including the muscle, liver, eggs, hepatopancreas (crustaceans), bone or any other relevant portion. For molluscs, whole soft body parts should be collected, and it may be necessary to produce a composite sample from more than ten specimens to create an adequate sample weight. For lobster or crab, edible tissue (e.g., muscle, eggs, hepatopancreas) should be collected.

Top of Page

3.11.2 Tissue Sample Collection and Preparation

Tissue analyses should be conducted on 8 samples (to achieve 95% power) of a single species from each of the exposure area and reference area, for a minimum of 16 samples. The 8 samples may be tissue from 8 individual fish, or each sample may be a composite of a number of fish; however, tissue from an individual fish should be used in one sample only. If possible, the samples should be of one sex- and age-class. The sex of each fish making up the sample should be reported. If fish are not of the same age-class, the age-classes of the fish should be consistent among sampling areas. Although the largest (oldest) fish of a similar size are preferred, the size specifications set by the responsible authority for fishing regulations in the jurisdiction where the study is undertaken should be respected.

The amount of tissue collected should be appropriate for the analytical method being used. Fish should be used independently in a sample and not mixed between samples. Tissues collected for analysis should be handled in such a way as to avoid contamination from sources such as boat fuel. Each sample should be clearly labelled, sealed in an appropriate contaminant-free container, frozen and forwarded to the analytical laboratory. The individual samples should be homogenized separately and sub-sampled for mercury analysis.

Top of Page

3.11.3 Supporting Analyses: Lipid and Moisture Percentages

Monomethylmercury (MeHg) comprises almost all (95% or greater) of the total mercury found in muscle tissue of fish regardless of the composition of diet sources and exposure water (Bloom 1992). Because of its strong affinity for sulfhydryl groups of proteins, the relative ease with which it passes through the digestive wall and slower depuration rate relative to inorganic mercury, MeHg is accumulated and retained in biological tissues (Clarkson 1994; Saouter et al. 1993).

Lipid concentration has been used to normalize tissue residues among species or within species between seasons, as well as being a key variable in modelling bioaccumulation. Lipid extraction methods by Randall et al. (1991) and the chloroform-methanol extraction method are recommended. Lipid analysis should only be completed when the contaminant being tested is known to be lipophilic.

Percent lipid and percent moisture determinations should be provided for every sample submitted for total metal analysis. Also, percent lipid values should be reported for the replicates analyzed in the same batch with the submitted sample. The percent lipid precision for the replicate samples should be ± 30% for tissues containing more than 2% and ± 60% for tissues with less than 2% lipid. The method for the lipid determinations would be reported and the solvents used clearly specified.

Top of Page

3.11.4 Guidance for Fish Tissue Analysis for Mercury Using Non-Lethal Methods

Tissue analysis for mercury has been traditionally conducted by extracting a fillet from fish. Non-lethal harvesting methods can produce accurate and reliable measures of fish muscle mercury concentrations provided appropriate analytical techniques are used (Tyus et al. 1999; Baker 2002; Baker et al. 2004; Peterson et al. 2005). The use of non-lethal methodologies for mercury analysis are particularly attractive at sites where destructive sampling methods would be detrimental to fish populations, for example, at sites where fish density is low. The purpose of this section is to describe appropriate non-lethal methodologies for tissue sampling and analysis.

Currently, it is recommended that tissue analysis be conducted on 8 samples (to achieve 95% power) from the exposure area and 8 samples from the reference area of a single species from one sex and age class during a lethal sampling study. This guidance should also be followed in a non-lethal survey with the exception of determining sex. It will not be possible to determine for most species if non-lethal sampling is used. However, several studies failed to find differences in mercury concentrations between males and females, although they can differ in energy requirements (Lange et al. 1994; Henderson et al. 2003; Ward and Neumann 1999).

Baker et al. (2004) demonstrated that small tissue quantities collected with two different types of non-lethal biopsy tools (dermal punch and a Tru-Cut™ biopsy needle) provided accurate and precise estimates of mercury concentration in fish muscle relative to benchmark values from the traditional, fillet-style methods and did not reduce survival of recaptured Northern Pike. Tyus et al. (1999) examined survival of Rainbow Trout and Razorback Sucker subjected to tissue collection using dermal punches, fin punches or liver punches and found no significant differences in growth or survival in any of the treated fish.

Top of Page

3.11.4.1 Recommended Methodology

Reliability of the non-lethal technique can depend on the biopsy tool, analytical methodology and tissue sample weight (Baker et al. 2004). The following recommended methodology for extraction of fish muscle tissue using a non-destructive approach is based on the work of Baker (2002) and Baker et al. (2004).

1. Practice – If at all possible, attempt to collect tissue from archived material or incidental mortalities before trying this method on a living fish. Becoming familiar with a technique will minimize possible handling and sampling stress.
   
2. Capture and anaesthetize fish – Prepare two holding containers, one with well-oxygenated water and another containing an anaesthetic (e.g., MS222). Capture fish by non-lethal means such as angling, short-set gill nets or electrofishing and place in the holding container. Transfer fish to the container containing the anaesthetic, one at a time, as necessary.
   
3. Obtain external fish measurements – Once anaesthetized, weigh and measure the fish, and obtain an aging structure (such as a pelvic fin ray) if appropriate.
   
4. Tissue extraction – Two tools currently available for harvesting small tissue samples include dermal punches or the Tru-Cut™ biopsy needle.
   • Tru-Cut™. Remove two or three scales from the dorsal region of the fish just below the dorsal fin using a sterilized needle. The outer barrel is then inserted to a depth of about 1 cm into the fish muscle tissue beneath the scale at an oblique angle (to minimize penetration depth). The 2-cm-long notched needle (inner barrel) is then extended into the flesh. The containment cover (i.e., sharp outer barrel) slides over the extended needle to cut the tissue and capture it within the notch. The needle is then withdrawn, the barrel opened and tissue slug removed with stainless steel (which should be acid washed between samples) or disposable plastic tweezers and placed in a small labelled vial. Samples obtained are approximately 25 mg. At least two tissue samples should be harvested and composited per fish to obtain a sufficient quantity to permit analysis. Baker et al. (2004) indicate that this procedure requires about 10 seconds for an experienced person to harvest a single sample.
   • Dermal punch. The dermal punch harvests a larger quantity of tissue and, for this reason it is the recommended harvesting method if only cold vapor atomic absorption spectrophotometry (CVAAS) is available for tissue analysis. This method can be used on fish greater than 200 mm in size. A few scales are removed and the dermal punch is placed on the skin. Moderate pressure and twisting action is applied to penetrate the epaxial musculature to harvest a small slug of tissue (approximately 60 mg of tissue). As with the biopsy approach, two samples should be harvested per fish and composited.
     
5. Sample preservation – Samples should be frozen using dry ice or liquid nitrogen to prevent decomposition during storage and transport to an analytical laboratory. Samples should be freeze-dried and weighed prior to analysis.
   
6. Infection prevention – Tissue extraction methods, particularly the dermal punch, leaves an open wound that may lead to an increased likelihood of infection. Sterile crazy glue, such as Nexaband™, which acts like a waterproof bandage, should be used to close the wounds to decrease the chance of infection.
   
7. Monitor and reintroduce fish – Once the tissue samples are harvested, return the fish to the holding container until it appears to have recovered and swims normally. The fish is then released back into the receiving water body.
   
8. Chemical Analysis – Selection of an analytical method must consider the accuracy of chemical measurement for small tissue quantities. CVAAS requires a minimum of 100 mg sample weight. Cold vapour atomic fluorescence spectrophotometry has lower detection limits and is better suited to determining mercury concentrations in small tissue quantities. Combustion atomic absorption spectrometry with gold amalgamation is a simplified and rapid procedure for analyzing small tissue quantities for total mercury (Cizdziel et al. 2002).

Top of Page

3.12 References

[AETE] Aquatic Effects Technology Evaluation. 1997. Technical evaluation of molluscs as a biomonitoring tool for the Canadian mining industry. Prepared by Robin Stewart and Diane F. Malley for Aquatic Effects Technology Evaluation (AETE) Program, CANMET, Natural Resources Canada. March 1997.

[AETE] Aquatic Effects Technology Evaluation. 1998. Technical evaluation of fish methods in environmental monitoring for the mining industry in Canada. Prepared by EVS Environment consultants for Aquatic Effects Technology Evaluation (AETE) Program, CANMET, Natural Resources Canada. Draft, July 1998.

Bailey RC, Kennedy MG, Dervish MZ, Taylor RM. 1998. Biological assessment of freshwater ecosystems using a reference condition approach: comparing predicted and actual benthic invertebrate communities in Yukon streams. Freshwat Biol 39:765-774.

Baker R. 2002. Fish mercury database summary – 2001, British Columbia. Prepared by the Aqualibrium Environmental Consulting Group (now the Azimuth Consulting Group, Vancouver BC) for BC Hydro.

Baker RF, Blanchfield PJ, Paterson MJ, Flett RJ, Wesson L. 2004. Evaluation of nonlethal methods for the analysis of mercury in fish tissue. Trans Am Fish Soc 33:568-576.

Barnthouse LW, Suter II GW, Rosen AE. 1989. Inferring population-level significance from individual-level effects: an extrapolation from fisheries science to ecotoxicology. In Suter II GW, Lewis MA, editors. Aquatic toxicology and environmental fate, 11th edition, ASTM STP 1001. Philadelphia (PA): American Society for Testing and Materials. p. 289-300.

Barrett TJ, Munkittrick KR. 2010. Seasonal reproductive patterns and recommended sampling times for sentinel fish species used in environmental effects monitoring programs in Canada. Environ Rev 18:115-135.

Bloom NS. 1992. On the chemical form of mercury in edible fish and marine invertebrate tissue. Can J Fish Aquat Sci 49:1010-1017.

Brasfield SM. 2007. Investigating and interpreting reduced reproductive performance in fish inhabiting streams adjacent to agricultural operations [doctoral dissertation]. Saint John (NB): University of New Brunswick.

Carroll L. 2007. The reproductive cycle of the redbelly dace (Phoxinus eos) [honours thesis]. Saint John (NB): University of New Brunswick, Department of Biology.

Cizdziel JV, Hinners TA, Heithmar EM. 2002. Determination of total mercury in fish tissues using combustion atomic absorption spectrometry with gold amalgamation. Water Air Soil Pollut 135:355-370.

Clarkson TW. 1994. The toxicology of mercury and its compounds. In Watras CJ, Huckabee JW, editors. Mercury pollution: integration and synthesis. Boca Raton (FL): Lewis Publishers.

Coad BW, Waszczuk H, Labignan I. 1995. Encyclopedia of Canadian fishes. Canada Museum of Nature and Canadian Sportfishing Productions Inc. 928 pp.

Dubé MG, Benoy GA, Wassenaar LI. 2006. Contrasting pathways of assimilation: Stable isotope assessment of fish exposure to pulp mill effluents. J Environ Qual 35:1884-1893.

Environment Canada. 1997. Fish Survey Working Group final report. Recommendations from Cycle 1 review. EEM/1997/6.

Farwell A. 1999. Stable isotope study of riverine benthic food webs influenced by anthropogenic developments [doctoral dissertation]. Guelph (ON): University of Guelph, Dept. Environ. Biol.

Galloway BJ, Munkittrick KR. 2006. Influence of seasonal changes in relative liver size, condition, relative gonad size and variability in ovarian development in multiple spawning fish species used in environmental monitoring programmes. J Fish Biol 69:1788-1806.

Galloway BJ, Munkittrick KR, CurrieS, Gray MA, Curry RA, Wood CS. 2003. Examination of the responses of slimy sculpin (Cottus cognatus) and white sucker (Catostomus commersoni) collected on the Saint John River (Canada) downstream of pulp mill, paper mill, and sewage discharges. Environ Toxicol Chem 22:2898-2907.

Galloway BJ, Munkittrick KR, Curry RA, Wood CS, Dunn S. 2004. Identifying a suitable fish species for monitoring multiple effluents in the Upper Saint John River, Canada. In Borton DL, Hall TJ, Fisher RP, Thomas JF, editors. Pulp and paper mill effluent environmental fate and effects. Lancaster (PA): DesTech Publications. p. 169-181.

Gray MA, Munkittrick KR. 2005. An effects-based assessment of slimy sculpin (Cottus cognatus) populations in agricultural regions of northwestern New Brunswick. Water Qual Res J Can 40:16-27.

Gray MA, Curry RA, Munkittrick KR. 2002. Non-lethal sampling methods for assessing environmental impacts using a small-bodied sentinel fish species. Water Qual Res J Can 37:195-211.

Gray MA, Cunjak RA, Munkittrick KR. 2004. Site fidelity of slimy sculpin (Cottus cognatus): insights from stable carbon and nitrogen analysis. Can J Fish Aquat Sci 61:1717-1722.

Gray MA, Curry RA, Munkittrick KR. 2005. Impacts of nonpoint inputs from potato farming on populations of slimy sculpin (Cottus cognatus). Environ Toxicol Chem 24:2291-2298.

Green RH. 1989. Power analysis and practical strategies for environmental monitoring. Environ Res 50:195-205.

Henderson BA, Collins N, Morgan GE and Vaillancourt A. 2003. Sexual size dimorphism of walleye (Stizostedion vitreum vitreum). Can J Fish Aquat Sci 60:1345-1352.

Hodson PV, Blunt BR, Whittle DM.1984. Monitoring lead exposure of fish. In Cairns VW, Hodson PV, Nriagu JO, editors.Contaminant effects of fisheries. New York (NY): John Wiley and Sons. p. 87-98.

Jenkins RE,. Burkhead NM. 1993. Freshwater fishes of Virginia. Bethesda (MD): American Fisheries Society.

Lange TR, Royals HE, Connor LL. 1994. Mercury accumulation in largemouth bass (Micropterus salmoides) in a Florida lake. Arch Environ Contam Toxicol 27:466-471.

Larsson DGJ, Forlin L. 2002. Male-biased sex ratios of fish embryos near a pulp mill: Temporary recovery after a short-term shutdown. Environ Health Perspect 110:739-742.

Larsson DGJ, Hallman H, Forlin L. 2000. More male fish embryos near a pulp mill. Environ Toxicol Chem 19:2911-2917.

Larsson DGJ, Mayer I, Hyllner SJ, Forlin L. 2002. Seasonal variations of vitelline envelope proteins, vitellogenin, and sex steroids in male and female eelpout (Zoarces viviparus). Gen Comp Endocrinol 125:184-196.

Mackay WC, Ash GR, Norris HJ, editors. 1990. Fish ageing methods for Alberta. Edmonton (AB): R.L. & L. Environmental Services Ltd. in assoc. with Alberta Fish and Wildl. Div. and Univ. of Alberta.

McMaster ME, Frank M, Munkittrick KR, Riffon R, Wood C. 2002. Follow-up studies addressing questions identified during cycle one of the adult fish survey of the pulp and paper EEM program. Wat Qual Res J Can 37(1):133-153.

McMullin VA, Munkittrick KR, Methven DA. 2009. Latitudinal variability in lunar spawning rhythms: absence of a lunar patter in the Northern Mummichog (Fundulus heteroclitus macrolepidotum Walbaum). J Fish Biol 75(4):885-900.

Minns CK. 1995. Allometry of home range size in lake and river fishes. Can J Fish Aquat Sci 52:1499-1508.

Munkittrick KR. 1992. A review and evaluation of study design considerations for site-specificity in assessing the health of fish populations. J Aquat Ecosys Health 1:283-292.

Munkittrick KR, McMaster ME. 2000. Assessment of multiple stressors in aquatic ecosystems by directed assessment of cumulative effects using fish populations. In Ferenc SA, Foran JA, editors. Multiple stressors in ecological risk and impact assessment: approach to risk estimation.Pensacola (FL): SETAC Press. p. 27-65.

Munkittrick KR, McMaster M, Van Der Kraak G, Portt C, Gibbons W, Farwell A, Gray M. 2000. Development of methods for effects-based cumulative effects assessment using fish populations: Moose River Project. Pensacola (FL): SETAC Press.

Munkittrick KR, McGeachy SA, McMaster ME, Courtenay SC. 2002. Overview of freshwater fish studies from the pulp and paper environmental effects monitoring program. Water Quality Res J Can. 37:49-77.

Munkittrick KR, Arens CJ, Lowell RB, Kaminski GP. 2009. A review of potential methods for determining critical effect size for designing environmental monitoring programs. Environ Toxicol Chem 28:1361-1371.

Nelson JS, Paetz MJ. 1992. The fishes of Alberta. Calgary (AB): The University of Calgary Press.

Nielsen LA, Johnson DL. 1983. Fisheries techniques. Bethesda (MD): American Fisheries Society.

[OMNR] Ontario Ministry of Natural Resources. 1994a. Nearshore community index netting (NSCIN): indexing the abundance of the warm water fish community. FAU Update 94-1, Fisheries Assessment Unit Newsletter. Sutton West (ON): Lake Simcoe Fisheries Assessment Unit.

[OMNR] Ontario Ministry of Natural Resources. 1994b. Spring littoral index netting (SLIN): indexing the abundance of the coldwater fish community. FAU Update 94-2, Fisheries Assessment Unit Newsletter. Bracebridge (ON): Muskoka Lakes Fisheries Assessment Unit.

Peterson SA, Van Sickle J, Hughes RM, Schacher JA, Echols SF. 2005. A biopsy procedure for determining filet and predicting whole-fish mercury concentration. Arch Environ Contam Toxicol 48:99-107.

Portt CB, Coker GA, Ming DL, Randall RG. 2006. A review of fish sampling methods commonly used in Canadian freshwater habitats. Canadian Technical Report of Fisheries and Aquatic Science 2604. Catalogue number Cat. No.Fs97-6/2604E.

Randall RC, Lee II H, Ozretich RJ, Lake JL, Pruell RJ. 1991. Evaluation of selected lipid methods for normalizing pollutant bioaccumulation. Environ Toxicol Chem 10:1431-1436.

Roberts WE. 1988. The sculpins of Alberta. Alberta Naturalist 18:121-127.

Salazar M, Salazar S. 2001. Standard guide for conducting in-situ field bioassays with caged marine, estuarine and freshwater bivalves. Philadelphia (PA). American Society for Testing and Materials (ASTM). 2001 Annual Book of ASTM Standards.

Saouter E, Hare L, Campbell PGC, Boudou A, Ribeyre F. 1993. Mercury accumulation in the burrowing mayfly Hexagenia rigida (Ephemeroptera) exposed to CH3HgCl or HgCl2 in water and sediment. Water Res. 27:1041-1048.

Schetagne R, Verdon R. 1999. Mercury in fish of natural lakes of northern Quebec. In Lucotte M, Schetagne R, Thérien N, Langlois C, Tremblay A, editors. Mercury in the biogeochemical cycle: natural environments and hydroelectric reservoirs of northern Quebec. Springer-Verlag. p. 115–30.

Schetagne R, Doyon J-F, Verdon R. 1997. Summary report: evolution of fish mercury levels at the La Grande Complex, Québec (1978–1994). Montréal (QC): Joint report of the Direction principale, Communication et Environnement, Hydro-Québec, and Groupe conseil Genivar Inc.

Schneider, JC, editor. 2000. Manual of fisheries survey methods II: with periodic updates. Fisheries Special Report 25. Ann Arbor (MI): Michigan Department of Natural Resources.

Scott WB. 1967. Freshwater fishes of Eastern Canada. Toronto (ON) University of Toronto Press.

Scott WB,. Crossman EJ. 1973. Freshwater fishes of Canada. Fisheries Research Board of Canada Bulletin 184. Ottawa (ON): Fisheries and Oceans Canada.

Secor DH, Henderson AR, Zapalo A, Piccoli PM. 1995. Can otolith microchemistry chart patterns of migration and habitat utilization in anadromous fishes. J Exp Mar Biol Ecol 192:15-33.

Shuter BJ. 1990. Population-level indicators of stress. Amer Fish Soc Symposium 8:145-166.

Swanson SM. 1993. Wapiti/Smoky river ecosystem study. Prepared for Procter and Gamble Ltd./Weyerhaeuser Canada Ltd., Grand Prairie, Alta. by Sentar Consultants Ltd. Calgary (AB).

Tremblay G, Doyon JF, Schetagne R. 1998. Réseau de suivi environnemental du complexe La Grande. Démarche méthodologique relative au suivi des teneurs en mercure des poissons. Rapport conjoint direction principale Communication et Environnement d’Hydro-Québec et Groupe-conseil Génivar inc.

Tyus HM, Starnes WC, Karp CA, Saunders III JF. 1999. Effects of invasive tissue collection on rainbow trout, razorback and bonytail chub. Nor Am J Fish Manage 19:848-855.

Ward SM, Neumann RM. 1999. Seasonal variation in concentrations in mercury in axial muscle tissue of largemouth bass. Nor Am J Fish Manage 19:89-96.

Top of Page

Tables 

Table 3-1 outlines the expected precision and summary statistics of fish survey measurements. Measurement requirements to be assessed include length, total body weight, age, gonad weight, egg size, fecundity, weight of liver or hepatopancreas, abnormalities, and sex. Each measurement requirement is accompanied by its expected precision, and a reporting of summary statistics.

Return to the table

Table 3-2 provides the suggested aging structures for Canadian fish species. Fish species are categorized based on common structure. Comments are offered regarding the relationships between the aging structures and the fish species.

Return to the table

Table 3-3 outlines the fish survey effect indicators and endpoints for various study designs. The primary effect indicators include survival, growth, reproduction, and condition. Each effect indicator is accompanied by the identification of lethal effect and supporting endpoints; non-lethal effect and supporting endpoints; and sentinel mollusc effect and supporting endpoints.

Return to the table

Table 3-4 exhibits generalizations and suggested optimal sampling times for fish species in EEM. Sample times are identified based on reproduction type. Contingent on the reproduction type and its sample time, the relationship between gonad weight and body weight for reference-site females is provided.

Return to the table

Table 3-5 displays the fish species commonly used in EEM, aspects to consider during study design, and recommended sampling times. Fish are identified by family, species, and scientific name.

Return to the table

Table 3-6 outlines the suggested reporting format for the parameters and the resulting regressions required for fish survey analysis in two parts. Table A offers the suggested format for parameter summaries, while Table B shows the suggested format for regression analyses. The percentage difference should be reported as exposed relative to reference site. Statistical significance should be given as p value.

Return to the table