Define the Case

1. Hip Pocket Guide to SI Detect or Suspect Biological Impairment Stressor Identification Define the Case List Candidate Causes Decision-maker and Stakeholder Involvement As Necessary: Acquire Data, and Iterate Process Evaluate Data from the Case Evaluate Data from Elsewhere Identify Probable Cause Identify and Apportion Sources Management Action: Eliminate or Control Sources, Monitor Results Biological Condition Restored or Protected CADDIS 2007 www.epa.gov/caddis

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3. Evaluate Data from the Case • The most powerful evidence is based on cause-effect relationships developed from data obtained from the place where the impairment occurs. • Use data from the case to develop evidence that allows you to confidently eliminate very improbable causes. • Use symptoms to refute or diagnose a cause. • -Use data from the case to build a body of evidence for those candidate causes that cannot be refuted or diagnosed. This evidence is used to identify the most probable causes. The more types of evidence and the more characteristics that support a candidate cause, the more likely that it IS the true cause. The more types of evidence that weaken the case for a candidate cause the more likely that it IS NOT the true cause. • Refuted causes require no further analysis, but are revisited when summarizing the overall conclusions. • Confirmed diagnoses by definition are probable causes and require no further analysis, but are also revisited when describing probable causes in the final summary. • All other candidate causes are further evaluated by comparison among remaining candidate causes after also developing evidence using data from other studies or locations. Introduction U.S. EPA's Stressor Identification (SI) Guidance Document (2000) describes a formal and rigorous process to identify stressors causing biological impairments in aquatic ecosystems, and a structure for organizing the scientific evidence supporting the conclusions. The SI process has been updated on the website www.epa.gov/caddis. The Stressor Identification process consists of five steps depicted within the yellow box on the cover of this guide. The hip pocket guide is intended as a convenient source of brief descriptions, illustrations, and scoring of the types of evidence used to evaluate candidate causes of biological condition. For more detail please consult the CADDIS website. 3 4

4. EVIDENCE FROM THE CASE Spatial/Temporal Co-Occurrence Through Time Spatial/Temporal Co-Occurrence with positive reference sites The biological effect must be observed where and when the cause is observed, and must not be observed where and when the cause is absent. The biological effect must be observed where and when the cause is observed, and must not be observed where and when the cause is absent. Refutes Spatial/Temporal Co-occurrence Through Time at a Site, Refutes The impairment (dead fish) occurs whether the causal agent occurs (the effluent is being discharged) or not (no discharge). Spatial/Temporal Co-occurrence with Positive Reference Sites, Refutes The impairment (dead fish) does NOT occur in similar ecosystems exposed to the same causal agent (effluent from the same industry) at another site. Supports Supports Spatial/Temporal Co-occurrence Through Time at a Site, Supports The impairment (dead fish) occurs when the causal agent occurs (the effluent is being discharged) but not when it does not occur (no discharge). Spatial/Temporal Co-occurrence with Positive Reference Sites, Supports The impairment (dead fish) occurs in similar ecosystems exposed to the same causal agent (effluent from the same industry) at another site 5 6

5. Spatial/Temporal Co-Occurrence Upstream Downstream Comparison Temporal Sequence The cause must precede the biological effect. The biological effect must be observed where and when the cause is observed, and must not be observed where and when the cause is absent. Refutes Refutes Temporal Sequence, Refutes The impairment (dead fish) occurred before the candidate causal agent (the effluent). Spatial/Temporal Co-occurrence with Upstream/Downstream Comparisons, Refutes The impairment (dead fish) occurs both upstream and downstream of the source of the causal agent (effluent). Supports Supports Temporal Sequence, Supports The impairment (dead fish) occurred only after initiation of the candidate causal agent (the effluent). Spatial/Temporal Co-occurrence with Upstream/Downstream Comparisons, Supports The impairment (dead fish) occurs downstream of the source of the causal agent (effluent) but not upstream. 7 8

6. Causal Pathway Stressor-Response Relationships from the Field Steps in the pathways linking sources to the cause can serve as supplementary or surrogate indicators that the cause and the biological effect are likely to have co-occurred. As exposure to the cause increases, intensity or frequency of the biological effect increases; as exposure to the cause decreases, intensity or frequency of the biological effect decreases. Refutes Weakens Causal Pathway, Refutes The impairment (dead fish) occurs downstream of the source of a precursor (phosphate) of the candidate causal agent (dissolved oxygen) but the proposed causal pathway is interrupted by turbulence which aerates the water, and the lack of periphyton on the cobble substrate confirms that there is no eutrophication, so another step is absent. Stressor-Response Relationships from the Field, Refutes The impairment (dead fish) increases as the causal agent (the effluent) is diluted, and the impairment is absent where the agent is most concentrated as well as upstream. Supports Supports Causal Pathway, Supports The impairment (dead fish) occurs downstream of the source of the precursor (phosphate) and high algal production is sufficient to cause high early morning respiration resulting in observed low dissolved oxygen (the causal agent). Turbulence, which might have aerated the water, is absent. Stressor-Response Relationships from the Field, Supports The impairment (dead fish) is greatest where the causal agent (the effluent) is most concentrated, and the impairment diminishes (more live fish) as the agent is diluted. 9 10

7. Evidence of Exposure or Biological Mechanism Manipulation of Exposure Field experiments or management actions that increase or decrease exposure to a cause must increase or decrease the biological effect. Measurements of the biota show that relevant exposure to the cause has occurred, or that other biological mechanisms linking the cause to the effect have occurred. Measurements of the biota show that relevant exposure to the cause has occurred, or that other biological mechanisms linking the cause to the effect have occurred. Refutes Supports Manipulation of Exposure, Refutes The impairment (dead fish) occurs when the causal agent is present (the effluent) and when it is removed (the effluent is shut down). Weakens Supports Evidence of Exposure or Biological Mechanism The candidate cause (low dissolved oxygen) of the impairment (dead fish) causes surviving fish to swim at the surface and gulp. Observations of that behavior strengthen the candidate cause and observations that surviving fish are swimming below the surface weakens the candidate cause. Manipulation of Exposure, Supports The impairment (dead fish) occurs when the causal agent (the effluent) is present but not when it is removed (the effluent is shut down). 11 12

8. Laboratory Tests of Site Media Supports Weakens Verified Predictions Controlled exposure in laboratory tests to causes (usually toxic substances) present in site media should induce biological effects consistent with the effects observed in the field. Knowledge of a cause's mode of action permits prediction and subsequent confirmation of previously unobserved effects. Supports Laboratory Tests of Site Media, Supports The impairment (dead fish) occurs in water that is toxic (dead fish in beaker of effluent diluted in site water). Water from an unimpaired reference site with the same industry is nontoxic. Verified Predictions A clever assessor predicts that if the impairment (dead fish) is due to a cholinesterase inhibiting pesticide, he would find that crustaceans would also be dead, but organisms without cholinergic systems would be alive. If he then searches for evidence at the site and finds dead crayfish and live rotifers (upper right panel), that supports the candidate cause. If the crayfish are alive (lower right panel), that weakens the candidate cause. Weakens Laboratory Tests of Site Media, Refutes The impairment (dead fish) occurs in water that is not toxic (live fish in beaker of effluent diluted in site water). Water from an unimpaired reference site with the same industry is also nontoxic. 14 13

9. Diagnose or Supports Weakens Symptoms Biological measurements (often at lower levels of biological organization than the effect) can be characteristic of one or a few specific causes. Evaluate Data from Elsewhere Most candidate causes cannot be diagnosed or eliminated. Further evaluation is required that uses knowledge gained from laboratory studies and from past experiences and observations in other waterbodies. For example, one of the most useful types of evidence from elsewhere uses the stressor-response relationships developed from laboratory studies. Familiar examples are single chemical, single species toxicity tests. -Use data from elsewhere to weaken or support a particular candidate cause. Symptoms The impairment (low fish production) may be caused by many agents, but only a few cause the specific symptom (spinal deformity). Observation of that symptom supports candidate causes that produce that symptom (e.g., selenium) and weakens others. 15 16

10. EVIDENCE FROM ELSEWHERE Stressor-Response Relationships from Other Field Studies At the impaired sites, the cause must be at levels sufficient to cause similar biological effects in other field studies. Mechanistically Plausible Cause The relationship between the cause and biological effect must be consistent with known principles of biology, chemistry and physics, as well as properties of the affected organisms and the receiving environment. Weakens Weakens Supports Stressor-Response Relationships from Other Field Studies Field studies from the region that associate measurements of the candidate cause (orange dots) with the intensity of the effect (proportion of dead fish) can be used to generate an exposure-response model. If concentrations in site water at the time of the fish kill (the impairment) are in concentration range A, the candidate cause is weakened. If they are in range B it is strengthened. Mechanistically Plausible Cause If the impairment is a fish kill with dermal lesions, the candidate cause copper toxicity is not mechanistically plausible, because copper does not cause dermal lesions. 18 17

11. Stressor-Response Relationships from Laboratory Studies Stressor-Response Relationships from Ecological Simulation Models Within the case, the cause must be at levels associated with related biological effects in laboratory studies. Within the case, the cause must be at levels associated with effects in mathematical models simulating ecological processes. Supports Stressor-Response Relationships from Ecological Simulation Models If the impairment (dead planktivorous fish) is thought to be due to starvation rather than direct toxicity, a mathematical model can show how the loss of zooplankton is a function of exposure to the candidate cause (chloronaphthol) and the starvation of fish is a function of zooplankton abundance. Weakens Supports Stressor-Response Relationships from Laboratory Studies The short-term lethality to fish of zinc (a candidate cause) has been tested in the laboratory and the results used to develop a concentration-response relationship. If concentrations in site water at the time of the fish kill (the impairment) are in concentration range A, the candidate cause is weakened. If they are in range B, it is strengthened. 19 20

12. Manipulation of Exposure at Other Sites Analogous Stressors At similarly impacted locations outside the case sites, field experiments or management actions that increase or decrease exposure to a cause must increase or decrease the biological effect. Agents similar to the causal agent at the impaired site should lead to similar effects at other sites. Weakens Manipulation of Exposure Elsewhere, Weakens The impairment (dead fish) occurs when the causal agent is present (an effluent of the same type as at the impaired site) and when it is removed (the effluent is shut down). Supports Analogous Stressors If evidence is lacking for the candidate cause (17B Tremblone) evidence for a similar agent (Tremblone) may be used to strengthen or weaken that candidate cause. Supports Manipulation of Exposure Elsewhere, Supports The impairment (dead fish) occurs when the causal agent (an effluent of the same type as at the impaired site) is present but not when it is removed (the effluent is shut down). 21 22

13. Consistency of Evidence Weakens Weakens Supports Confidence in the argument for or against a candidate cause is increased when many types of evidence consistently support or weaken it Evaluating Multiple Lines of Evidence as a Form of Evidence There are two types of evidence that are not directly taken from the data. Rather they concern a types of evidence that evaluate how other types of evidence relate to one another. Consistency evaluates if all the types of evidence support or weaken the argument for a cause. Explanation of the Evidence evaluates if an inconsistent type of evidence is credible and if there is a reason to place less weight on that piece of evidence. Consistency of the Evidence A candidate cause is strongly supported if all available types of evidence are consistently supportive. It is greatly weakened if all available types of evidence are consistently weakening. It is weakened if some types of evidence support and others weaken the candidate cause. 23 24

14. Supports Weakens Explanation of the Evidence • Scoring Evidence • The score for the evidence from the case and from elsewhere is influenced by the quantity and quality of the evidence. The magnitude of the score is based on the likelihood of observing the effect due to the true cause rather than from a chance outcome. Therefore, the highest scores are given to the types of evidence that: • Use data from the case, • Are based on more than one piece of evidence, • Effectively link the causal agent with the effect. • All of the evidence is evaluated for consistency by looking at the overall pattern of scores. Confidence in the argument for or against a candidate cause is increased when it is supported by many types of evidence. Confidence in the argument for a candidate cause is increased when a post hoc mechanistic, conceptual, or mathematical model reasonably explains any inconsistent evidence. R refutes D diagnoses +++ convincingly supports - - - convincingly weakens ++ strongly supports - - strongly weakens + somewhat supports - somewhat weakens 0 neither supports nor weakens NE no evidence Reasonable Explanation of the Evidence If recovery has not occurred one month following elimination of the candidate cause (the effluent) then spatial/temporal co-occurrence is not complete. However, it may be explained by the fact that recolonization is blocked by a weir and enough time has not elapsed for recovery by reproduction, which might be observed a year later (upper panel). That explanation supports the candidate cause. There is no such explanation for the continuation of the impairment (more dead fish) one month after elimination of exposure to the candidate cause (lower panel). 24 25

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19. CADDIS 2007 www.epa.gov/caddis