This section presents 14 worksheets documenting each step of a causal analysis conducted specifically for the Little Scioto River, Ohio as an example. The linked steps in the list below correspond to the individual worksheets for this example, so that either clicking on the list by name or number (tabs) will bring you to the same information.

1. Define the Case
2. List Candidate Causes
3. Assemble Data from the Case
4. Spatial/Temporal Co-occurrence
5. Evidence of Exposure or Mechanism
6. Causal Pathway
7. Stressor-Response Relationships from the Field
   • Sediment
   • Riffle/Pool
   • Dissolved Oxygen
   • Ammonia
   • Metals
8. Summary of Scores from the Case
   • Increased % DELT
   • Increased Relative Weight
   • Decreased % Mayflies and Increased % Tolerant Macroinvertebrates
9. Assemble Data from Elsewhere
10. Stressor-Response Relationships from Laboratory Studies
11. Mechanistically Plausible Cause
12. Summary of Scores from Elsewhere
    • Increased % DELT
    • Increased Relative Weight
    • Decreased % Mayflies and Increased % Tolerant Macroinvertebrates
13. Evaluating Multiple Lines of Evidence
    • Increased % DELT
    • Increased Relative Weight
    • Decreased % Mayflies and Increased % Tolerant Macroinvertebrates
14. Identify Probable Cause
15. Conclusion

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Worksheet 1. Define the Case

In Ohio, biological impairment is defined by standard multimetric indices including the IBI, and ICI, and the modified index of well being (Ohio EPA 1989). These indices were used to identify reaches of the Little Scioto River (see Figure 1) that had impaired aquatic assemblages in comparison to the criteria for warm-water habitat (WWH) or modified warm-water habitat (MWH) designated uses.

The Little Scioto River has both WWH and MWH sections. WWH is a designation shared by the majority of Ohio's rivers and streams, and is narratively defined as supporting a balanced, reproducing aquatic community. The minimum criteria required to be in attainment of WWH standards are defined as the 25th percentile values of reference condition scores for a given index, site type, and ecoregion. The MWH designation is a nonfishable aquatic life use, and is designed to protect streams that have been too physically impacted or modified to meet WWH standards.

Figure 1. A map highlighting the Little Scioto River (outlined in blue in the center of the map) generated using Enviromapper for Water.Of seven sites sampled in 1992, the highest IBI score was at an upstream site with a 33 out of a possible score of 60 (see Figure 2). The score translates into a fair ranking according to WWH standards. The remaining sites were described as severely impaired, with IBI scores between 25 and 12. Similarly, the ICI met WWH aquatic life use standards at the upstream site with a score of 38. The ICI score from RM 7.9 downstream was below MWH aquatic life use standards. Examination of the spatial distribution of the IBI, ICI and component metrics indicates that several distinct impairments occurred.

Figure 2. IBI and ICI scores on the Little Scioto River. River miles are listed in upstream to downstream order from left to right.

The Biological Impairment and its Basis

The Little Scioto River near Marion, Ohio, was listed as an impaired waterbody on the 1998 303(d) List of Impaired Waters. In 1992, Ohio EPA determined that the river was biologically impaired (see Figure 1).

Specific Effects

The IBI and ICI were disaggregated to identify more specific effects that were responsive to the environmental changes, had a sufficient range of variation, and appeared to indicate distinctive mechanisms (Figure 3).

Figure 3. Specific effects observed at the Little Scioto.

Specific effects associated with the impairment observed at RM 7.9 (Impairment A) include an increase in relative fish weight and DELT anomalies. For the invertebrates, the percentages of mayflies decreased, and the percentage of tolerant invertebrates increased (Table 1). The specific effects observed at RM 7.9 were sufficiently distinctive from the other locations on the Little Scioto to suggest that either additional or completely different causes may be influencing the assemblage at other sites on the stream. Those sites were not addressed in the current example, but are discussed in Norton et al. (2002), and Cormier et al. (2002).

Table 1. Specific effects observed at Sites A and B in the Little Scioto River.

Response	Impairment A: Change in assemblage at site A relative to upstream site	Impairment B: Change in assemblage at site B relative to site A
Relative fish weight	Increased	Decreased
DELT anomalies	Increased	Increased
% Mayflies	Decreased	Decreased
Tolerant taxa	Increased	Increased

 

The Investigation's Purpose

The purpose of the investigation is to inform decisions about how best to return the Little Scioto River to a condition that meets State biocriteria. As such, we considered stressors from both point and non-point sources including toxic contaminants in water and sediment, nutrients, and habitat degradation.

The Geographic Area Under Investigation

The Little Scioto River is located near Marion Ohio (see Figure 1). For the purposes of this example, the evaluation will be limited to identifying the probable cause of the biological impairment at RM 7.9 (Site A). The upstream site (RM 9.2) was used as a less-impaired reference site for comparison, and observations from six other downstream locations within the reach were brought in and analyzed as part of the case. For additional detail and an analysis of the biological impairments observed at other locations along the stream, see Norton et al. (2002) and Cormier et al. (2002).

References

• Cormier SM, Norton SB, Suter GW II, Altfater D, Counts B (2002) Determining the causes of impairments in the Little Scioto River, Ohio, USA: Part 2. Characterization of causes. Environmental Toxicology and Chemistry 21:1125-1137.
• Norton SB, Cormier SM, Suter GW II, Subramaniam B, Lin E, Altfater D, Counts B (2002) Determining probable causes of ecological impairment in the Little Scioto River, Ohio, USA: Part 1. Listing candidate causes and analyzing evidence. Environmental Toxicology and Chemistry 21:1112-1124.
• Ohio EPA (Environmental Protection Agency). (1989) Biological Criteria for the Protection of Aquatic Life. Volume III: Standardized Biological and Field Sampling and Laboratory Methods for Assessing Fish and Macroinvertebrate Communities. Ohio Environmental Protection Agency, Division of Water Quality Planning and Assessment, Ecological Assessment Section, Columbus OH. Procedure WQPA-SWS-3.

 

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Worksheet 2. List Candidate Causes

1. Increased fine sediments. Channel modification (i.e., narrowing, deepening and straightening of channel) leads to increased deposition of fine sediments.
2. Altered pools and riffles. Channel modification (i.e., narrowing, deepening and straightening of channel) leads to deeper pools and fewer riffle habitats.
3. Low dissolved oxygen (DO) concentration. Several pathways may contribute to low DO levels: decreases in large woody debris and loss of riffle habitats can decrease aeration; increased organic carbon loading (e.g., from wastewater inputs) can increase biological oxygen demand (BOD); and/or increases in nutrients (nitrogen and/or phosphorus) can stimulate algal production and thus BOD (due to respiration of living plants and/or decomposition of algal detritus).
4. Increased algal biomass. Nutrient inputs lead to moderate increases in algal biomass. These increases are insufficient to significantly reduce DO, but sufficient to stimulate secondary production in the system (i.e., lead to increased fish weights) and to alter invertebrate community structure.
5. Ammonia toxicity. Increases in nitrogen loading lead to increases in total ammonia within the system; this ammonia dissociates into un-ionized ammonia, which is toxic to aquatic organisms. This candidate cause can be significantly affected by pH levels and DO concentrations, as the relative abundance of unionized ammonia increases with increasing pH and decreasing DO (due to reduction of nitrate to ammonium). Because increases in photosynthesis can raise pH, increased nutrients can both directly and indirectly increase unionized ammonia concentrations.
6. Metal toxicity.
7. PAH toxicity.

Figure 4. Map of the Little Scioto river showing locations of tributaries, roads, and possible sources.

Figure 5. A conceptual model diagram of the six candidate causes for the Little Scioto stressor identification (revised August 2005).

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Worksheet 3. Assemble Data from the Case

Table 2. Measurements relevant to each candidate cause.

Candidate cause	Relevant measurements
1. Increased fine sediments	Channel score, embeddedness subscore, siltation subscore
2. Altered pools and riffles	Channel score, pool score, riffle score
3. Low dissolved oxygen	Dissolved oxygen, biological oxygen demand of water, riffle score, channel score
4. Increased algal biomass	Nitrate and nitrite concentrations in water, total phosphorus concentrations in water
5. Ammonia Toxicity	Ammonia concentrations in water
6. Metal Toxicity	Metal concentrations in sediments, water and fish tissue
7. PAH Toxicity	PAH concentrations in sediments, bile metabolites in fish

 

Table 3. Summary of sampling locations for 1992 data collected in the Little Scioto River.

Sample medium	Parameter	Site designation and sample location (RM upstream from confluence with Scioto River)							Number of samples per location
		Upstream Site	Site A	Site B	Downstream Sites
Fish	IBI and Miwb metrics	9.2	7.9	6.5	5.7	4.4	2.7	0.3	2-3
Macroinvertebrates	ICI Metrics	9.2	7.9	6.5	5.7	4.4	2.1	0.4	1
Sediment	Volatile chemicals, semivolatile chemicals, metals and cyanide, PCBs and pesticides	9.5	7.9	6.5	5.8	4.4	2.7	0.4	1-3
Water	Metals, phenolic compounds, ammonia, nitrates and nitrites (NO x), total phosphorus (P), and BOD	9.2	7.9	6.5	5.8	4.4	2.7	0.4	1
Water	Dissolved oxygen	9.2	7.9		5.8	4.4	2.7	0.4	measured continuously over 3 days
Physical Habitat	QHEI metrics	9.2	7.9	6.5	5.7	4.4	2.7	0.3	1
Fish Tissue	Pesticides, PCBs, metals, semivolatile compounds, and % lipid	9.2		6.5			2.7		2-5 composite samples
White Sucker Bile	PAH metabolites		7.9	6.5	5.7	4.4	2.7	0.3	5-7

Table 4. Concentrations of chemicals in water. Units are in mg/L except where noted.

Chemical	Up-stream Site	Site A	Site B	Downstream sites (RM)
				5.8	4.4	2.7	0.4
BOD	1	1	2.3	4.7	4.2	3.5	2.2
Nitrate and Nitrite	1.22	1.44	0.81	8.1	6.6	4.5	4.47
Ammonia	ND	ND	0.12	1.16	1.44	2.1	0.58
Total phosphorus	0.06	0.07	0.09	2.17	1.96	1.8	1.34
Dissolved oxygen (minimum)	8.8 NA	5.7 2.8	NA 1.9	4.2 4.2	4.3 3.2	3.0 2.0	4.4 2.5
Coppera	ND	ND	15	ND	ND	ND	ND
Leada	ND	ND	3	3	ND	ND	ND
Zinca	ND	ND	ND	18	12	13	ND

a. Units are µg/L
ND. Not Detected

Table 5. Selected physical habitat quality scores.

Parameter	Up-stream Site	Site A	Site B	Downstream sites (RM)
				5.7	4.4	2.7	0.4
Substrate embeddednessa	2	1	1	1	1	1	1
Siltb	2	1	1	1	1	1	1
Channelc	17	10	10	10	10	10	7
Poold	11	8	8	8	6	8	8
Rifflee	6	0	0	0	0	0	0

a. Levels of embeddedness were scored from most to least (1=extreme, 2=moderate, 3=low, 4=none)
b. Levels of silt were scored from most to least (1=heavy, 2=moderate, 3=normal, 4=free)
c. Channels were scored from high to low quality (20=high quality)
d. Pools were scored from high to low quality (12=high quality)
e. Riffles were scored from high to low quality (8=high quality)

Figure 6. Bile metabolites (μg/mg protein) measured in white suckers from the Little Scioto River. The grey horizontal line shows the upper limits of concentrations considered background for the state of Ohio. The number of fish sampled is shown above each bar. Vertical lines are standard errors. BAP = benzo[a]pyrene; NAPH= naphthalene. Source: Lin et al. (1996) and Cormier et al. (2000).

References

• Cormier SM, Lin ELC, Fulk FA, Subramanian B (2000) Estimation of exposure criteria values for biliary polycyclic aromatic hydrocarbon metabolite concentration in white suckers (Catostomus commersoni). Environmental Toxicology and Chemistry 19:1120-1126.
• Lin ELC, Cormier SM, Torsella JA (1996) Fish biliary polycyclic aromatic hydrocarbon metabolites estimated by fixed-wavelength fluorescence: comparison with HPLC-fluorescent detection. Ecotoxicology and Environmental Safety 35:16-23.

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Worksheet 4. Spatial/Temporal Co-occurrence

Consider whether measurements of particular stressors (sediment, riffles/pools, dissolved oxygen, ammonia, metals, and PAHs) co-occur with observed biological impairment.

Data

Single observations of habitat quality were collected from the upstream site and the impaired site in 1992. PAH and metal concentrations were determined from fine sediments up to a depth of 15cm at each site. Dissolved oxygen and ammonia were measured in the water column.

Analysis

Table 6. Comparison of stressor measurements at Site A and an upstream site.

Candidate Cause	Measurement	Upstream site	Site A	Exposure increased at Site A?
Increased fine sediments	Substrate embeddedness	2	1	Yes
	Silt	2	1	Yes
Altered riffles	Pool score	11	8	Yes
	Riffle score	6	0	Yes
Dissolved oxygen (mg/L)	Dissolved oxygen	8.8	5.7	Yes
Metals (mg/kg)	Chromium	7.34	13.6	Yes
	Copper	7.44	17.2	Yes
	Lead	12.1	19.2	Yes
	Zinc	3.06	79	Yes
PAHs (mg/kg)	Anthracene	ND	ND	No
	Benzo[a]anthracene	ND	ND	No
	Benzo[ghi]perylene	ND	ND	No
	Chrysene	ND	ND	No
	Fluoranthene	ND	ND	No

Discussion

PAHs were not detected at the site, so PAH toxicity was refuted as a potential cause. Ammonia was not detected at the site, but water column measurements of ammonia are highly variable. Spatial co-occurrence for all other stressors was compatible with the stressor being the cause.

Table 7. Evidence scoring table for spatial/temporal co-occurrence.

Finding	Interpretation	Score
The effect occurs where or when the candidate cause occurs OR the effect does not occur where or when the candidate cause does not occur.	This finding somewhat supports the case for the candidate cause, but is not strongly supportive because the association could be coincidental.	+
It is uncertain whether the candidate cause and the effect co-occur.	This finding neither supports nor weakens the case for the candidate cause, because the evidence is ambiguous.	0
The effect does not occur where or when the candidate cause occurs OR the effect occurs where or when the candidate cause does not occur.	This finding convincingly weakens the case for the candidate cause, because causes must co-occur with their effects.	---
The effect does not occur where or when the candidate cause occurs OR the effect occurs where or when the candidate cause does not occur, and the evidence is indisputable.	This finding refutes the case for the candidate cause, because causes must co-occur with their effects.	R

Table 8. Spatial/temporal co-occurrence scores for candidate causes.

Candidate Cause	Result	Score
Sediment	Elevated sediment co-occurs with impairment	+
Pool/riffle	Poor pool/riffle condition co-occurs with impairment	+
Dissolved oxygen	Reduced DO co-occurs with impairment	+
Ammonia	Elevated ammonia not detected at site	---
Metals	Elevated metals concentrations observed at impairment	+
PAHs	Elevated PAHs not detected at site	R

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Worksheet 5. Evidence of Exposure or Mechanism

Consider whether evidence exists for exposure to PAHs.

Data

Bile metabolites measured in white suckers from the Little Scioto River.

Figure 7. Bile metabolites (ug/mg protein) measured in white suckers from the Little Scioto River.

Analysis/Discussion

Bile metabolites show evidence of exposure to PAHs at the site. Concentrations are low but no measurements at upstream site are available for comparison.

Table 9. Evidence scoring table for evidence of exposure.

Finding	Interpretation	Score
Data show that exposure or the biological mechanism is clear and consistently present.	This finding strongly supports the case for the candidate cause, but is not convincing because it does not establish that the level of exposure or mechanistic action was sufficient to cause the effect.	+ +
Data show that exposure or the biological mechanism is weak or inconsistently present.	This finding somewhat supports the case for the candidate cause.	+
Data show that exposure or the biological mechanism is uncertain.	This finding neither supports nor weakens the case for the candidate cause.	0
Data show that exposure or the biological mechanism is absent.	This finding strongly weakens the case for the candidate cause, but is not convincing because the exposure or the mechanism may have been missed.	- -
Data show that exposure or the biological mechanism is absent, and the evidence is indisputable.	This finding refutes the case for the candidate cause.	R

 

Table 10. Evidence of exposure scores for candidate causes.

Candidate Cause	Result	Score
PAHs	Evidence of PAH exposure observed	+

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Worksheet 6. Causal Pathway

Consider whether support exists for the causal pathway leading to dissolved oxygen.

Data

Total phosphorus (TP) and biochemical oxygen demand (BOD) were measured at the site

Analysis

Table 11. Steps in the causal pathway leading from increased nutrients to decreased DO.

	Data confirmed at site?
Increased nutrients (N and/or P)	Yes
Increased algae	No data
Increased BOD	No

Table 12. Steps in the causal pathway leading from channel modification to decreased DO.

	Data confirmed at site?
Channel modified	Yes
Decreased woody debris	No data
Increased BOD	No

Discussion

Pathway linking nutrients to DO requires elevated BOD. Since BOD is not elevated, this causal pathway seems unlikely. Pathway linking channel modification to DO requires data on woody debris. Since data are missing, evidence is ambiguous.

Table 13. Evidence scoring table for causal pathway.

Finding	Interpretation	Score
Data show that all steps in at least one causal pathway are present.	This finding strongly supports the case for the candidate cause, because it is improbable that all steps occurred by chance; it is not convincing because these steps may not be sufficient to generate sufficient levels of the cause.	+ +
Data show that some steps in at least one causal pathway are present.	This finding somewhat supports the case for the candidate cause.	+
Data show that the presence of all steps in the causal pathway is uncertain.	This finding neither supports nor weakens the case for the candidate cause.	0
Data show that there is at least one missing step in each causal pathway.	This finding somewhat weakens the case for the candidate cause, but is not strongly weakening because it may be due to temporal variability, problems in sampling or analysis, or unidentified alternative pathways.	-
Data show, with a high degree of certainty, that there is at least one missing step in each causal pathway.	This finding convincingly weakens the case for the candidate cause, assuming critical steps in each pathway are known, and are not found at the impaired site after a well-designed, well-performed, and sensitive study.	- - -

Table 14. Causal pathway scores for candidate causes.

Candidate Cause	Result	Score
Dissolved oxygen	Evidence of some steps are present for one pathway.	+

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Worksheet 7. Stressor-Response Relationships from the Field

Sediment | Riffle/Pool | Dissolved Oxygen | Ammonia | Metals

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Sediment

Data

Data downstream from Site A were used to characterize the stressor-response relationship. Sediment levels are characterized with a qualitative substrate score.

Analysis

Mean substrate scores were plotted against the three specific variables. Where appropriate a curve was fit to the observations as a visualization aid. The point corresponding to observed substrate score and the biological response at Site A was overlaid on the plot (shown in red).

Figure 8. Comparisons of site conditions to stressor-response curves for the sediment candidate cause.

Discussion

None of the observed biological responses exhibited definitive trends across the observed range of substrate. However, uncertainty in the assessment of these trends is large, given that only six data points were available.

Table 15. Evidence scoring table for stressor-response relationships from the field.

Finding	Interpretation	Score
A strong effect gradient is observed relative to exposure to the candidate cause, at spatially linked sites, and the gradient is in the expected direction.	This finding strongly supports the case for the candidate cause, but is not convincing due to potential confounding.	++
A weak effect gradient is observed relative to expsoure to the candidate cause, at spatially linked sites, OR a strong effect gradient is observed relative to exposure to the candidate cause, at non-spatially linked sites, and the gradient is in the expected direction.	This finding somewhat supports the case for the candidate cause, but is not strongly supportive due to potential confounding or random error.	+
An uncertain effect gradient is observed relative to exposure to the candidate cause.	This finding neither supports nor weakens the case for the candidate cause, because the evidence is ambiguous.	0
An inconsistent effect gradient is observed relative to exposure to the candidate cause, at spatially linked sites, OR a strong effect gradient is observed relative to exposure to the candidate cause, at non-spatially linked sites, but the gradient is not in the expected direction.	This finding somewhat weakens the case for the candidate cause, but is not strongly weakening due to potential confounding or random error.	-
A strong effect gradient is observed relative to exposure to the candidate cause, at spatially linked sites, but the relationship is not in the expected direction.	This finding strongly weakens the case for the candidate cause, but is not convincing due to potential confounding.	--

Table 16. Stressor-response from the field scores for sediment.

Candidate Cause	Specific Effect	Result	Score
Sediment	Relative weight	No apparent gradient	0
	Proportion DELT	No apparent gradient	0
	Proportion mayflies	No apparent gradient	0

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Riffle/Pool

Data

Data downstream from Site A were used to characterize the stressor-response relationship. Riffle quality is characterized with a qualitative riffle score.

Analysis

Mean riffle scores were plotted against the three specific variables. Where appropriate a curve was fit to the observations as a visualization aid. The point corresponding to observed channel score and the biological response at Site A was overlaid on the plot (shown in red).

Figure 9. Comparisons of site conditions to stressor-response curves for the riffle/pool candidate cause.

Discussion

None of the observed biological responses exhibited definitive trends across the observed range of channel scores. However, uncertainty in the assessment of these trends is large, given that only six data points were available.

Table 17. Stressor-response from the field scores for riffle/pool.

Candidate Cause	Specific Effect	Result	Score
Riffle/pool	Relative weight	No apparent gradient	0
	Proportion DELT	No apparent gradient	0
	Proportion mayflies	No apparent gradient	0

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Dissolved Oxygen

Data

Data downstream from Site A were used to characterize the stressor-response relationship. Dissolved oxygen was measured continuously at each location over a 3-d period with Datasonde continuous monitors.

Analysis

Observed minimum DO were plotted against the three specific variables. Where appropriate a curve was fit to the observations as a visualization aid. The point corresponding to observed minimum DO and the biological response at Site A was overlaid on the plot (shown in red).

Figure 10. Comparisons of site conditions to stressor-response curves for the dissolved oxygen candidate cause.

Discussion

The observed proportion of mayflies and DELT seem reasonably consistent with the trends exhibited across the observed range of minimum DO values. However, uncertainty in the position of the fitted curve is large, given that only six data points were available. The observed value of relative weight is much larger than observed at other sites, and falls in the middle of the observed range of DO values.

Table 18. Stressor-response from the field scores for dissolved oxygen.

Candidate Cause	Specific Effect	Result	Score
Dissolved oxygen	Relative weight	No apparent gradient	0
	Proportion DELT	Consistent with gradient	+
	Proportion mayflies	Consistent with gradient	+

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Ammonia

Data

Data downstream from Site A were used to characterize the stressor-response relationship.

Analysis

Ammonia concentrations were plotted against the three specific variables. Where appropriate a curve was fit to the observations as a visualization aid. The point corresponding to observed ammonia concentration and the biological response at Site A was overlaid on the plot (shown in red).

Figure 11. Comparisons of site conditions to stressor-response curves for the ammonia candidate cause.

Discussion

All of the biological responses exhibited definitive trends across the observed range of ammonia concentration. However, ammonia concentration was lower at the site than at other locations without the biological impairment (i.e., spatial co-occurrence was scored negatively).

Table 19. Stressor-response from the field scores for ammonia.

Candidate Cause	Specific Effect	Result	Score
Ammonia	Relative weight	Field stressor-response not applicable because of negative spatial co-occurrence score	NA
	Proportion DELT	Field stressor-response not applicable because of negative spatial co-occurrence score	NA
	Proportion mayflies	Field stressor-response not applicable because of negative spatial co-occurrence score	NA

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Metals

Data

Data downstream from Site A were used to characterize the stressor-response relationship. Metals concentrations were characterized with Zn concentration.

Analysis

Mean Zn concentrations were plotted against the three specific variables. Where appropriate a curve was fit to the observations as a visualization aid. The point corresponding to observed Zn concentrations and the biological response at Site A was overlaid on the plot (shown in red).

Figure 12. Comparisons of site conditions to stressor-response curves for the metals candidate cause.

Discussion

The proportion of mayflies exhibited a strong response to Zn. The change in percent DELT and relative weight also seemed correlated with Zn concentration, but observations at the site did not seem consistent with the overall gradient.

Table 20. Stressor-response from the field scores for metals.

Candidate Cause	Specific Effect	Result	Score
Metals	Relative weight	No apparent gradient	0
	Proportion DELT	No apparent gradient	0
	Proportion mayflies	No apparent gradient	+

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Worksheet 8. Summary of Scores from the Case

Increased % DELT | Increased Relative Weight | Decreased % Mayflies and Increased % Tolerant Macroinvertebrates

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Increased % DELT

Table 21. Scores for increased % DELT, for evidence that uses data from the case.

Types of Evidence	Algae	Ammonia	Dissolved Oxygen	Metals	PAHs	Riffles/Pools	Sediment
Spatial/Temporal Co-occurrence	NE	---	+	+	R	+	+
Evidence of Exposure or Biological Mechanism	NE	NE	NE	NE	0	NE	NE
Causal Pathway	+	NA	+	NA	NA	NE	NA
Stressor-Response Relationships from the Field	NE	NA	0	0	NA	0	0
Manipulation of Exposure	NE	NE	NE	NE	NE	NE	NE
Laboratory Tests of Site Media	NE	NE	NE	NE	NE	NE	NE
Temporal Sequence	NE	NE	NE	NE	NE	NE	NE
Verified Predictions	NE	NE	NE	NE	NE	NE	NE
Symptoms	0	0	0	0	0	0	0

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Increased Relative Weight

Table 22. Scores for increased relative weight, for evidence that uses data from the case.

Types of Evidence	Algae	Ammonia	Dissolved Oxygen	Metals	PAHs	Riffles/Pools	Sediment
Spatial/Temporal Co-occurrence	NE	---	+	+	+	R	+
Evidence of Exposure or Biological Mechanism	NE	NE	NE	NE	NE	0	NE
Causal Pathway	+	NE	NE	NE	NE	NE	NE
Stressor-Response Relationships from the Field	NE	NA	0	0	NA	0	0
Manipulation of Exposure	NE	NE	NE	NE	NE	NE	NE
Laboratory Tests of Site Media	NE	NE	NE	NE	NE	NE	NE
Temporal Sequence	NE	NE	NE	NE	NE	NE	NE
Verified Predictions	NE	NE	NE	NE	NE	NE	NE
Symptoms	0	0	0	0	0	0	0

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Decreased % Mayflies and Increased % Tolerant Macroinvertebrates

Table 23. Scores for decreased % mayflies and increased % tolerant macroinvertebrates, for evidence that uses data from the case.

Types of Evidence	Algae	Ammonia	Dissolved Oxygen	Metals	PAHs	Riffles/Pools	Sediment
Spatial/Temporal Co-occurrence	NE	---	+	+	+	R	+
Evidence of Exposure or Biological Mechanism	NE	NE	NE	NE	NE	0	NE
Causal Pathway	+	NE	NE	NE	NE	NE	NE
Stressor-Response Relationships from the Field	NE	NA	0	0	NA	0	0
Manipulation of Exposure	NE	NE	NE	NE	NE	NE	NE
Laboratory Tests of Site Media	NE	NE	NE	NE	NE	NE	NE
Temporal Sequence	NE	NE	NE	NE	NE	NE	NE
Verified Predictions	NE	NE	NE	NE	NE	NE	NE
Symptoms	0	0	0	0	0	0	0

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Worksheet 9. Assemble Data from Elsewhere

Table 24. Threshold Effects Levels and Probable Effects Levels available for PAHs and metals measured in sediments from the Little Scioto River. Values are in mg/kg, normalized to sediment dry weight. Source: U.S. EPA (1996).

Chemical	Threshold Effects Level	Probable Effects Level
Benzo(a)pyrene	0.03	0.32
Fluoranthene	0.04	0.32
Benzo[a]anthracene	0.03	0.28
Chrysene	0.02	0.41
Benzo(g,h,i)perylene	0.01	0.25
Chromium	32.3	119.4
Copper	28	101.2
Lead	37.2	81.7
Zinc	98.1	544

Table 25. Available criteria values.

Chemical	Criterion (units)	Source
Ammonia	1.27 mg/L at pH 8.0 0.57 mg/L at pH 8.5	U.S. EPA 1998
Dissolved oxygen	3.0 mg/L for Modified Warmwater Habitat	Ohio EPA 1996
Nitrate-nitrite	1.6 (mg/L)	Ohio EPA 1999
Total phosphorus	0.28 (mg/L)	Ohio EPA 1999
Copper	21 (μg/l)	U.S. EPA 1987 (hardness of 200 mg/l)
Lead	7.7 (μg/l)	U.S. EPA 1987 (hardness of 200 mg/l)
Zinc	190 (μg/l)	U.S. EPA 1987 (hardness of 200 mg/l)

References

• Ohio EPA (Environmental Protection Agency) (1999) Association between nutrients, habitat, and the aquatic biota in Ohio rivers and streams. Ohio Environmental Protection Agency, Columbus OH. Technical Bulletin MAS/1991-1.
• U.S. EPA (1987) Quality Criteria for Water 1986. U.S. Environmental Protection Agency, Office of Water Regulations and Standards, Washington DC. EPA 440/5-86-001.
• U.S. EPA (1996) Calculation and evaluation of sediment effects concentrations of the amphipod Hyalella azteca and the midge Chironomus riparius. Assessment and Remediation of Contaminated Sediments (ARCS) Program. Great Lakes National Program Office, Chicago IL. EPA 905-R96-008.
• U.S. EPA (1999) 1999 Update of Ambient Water Quality Criteria for Ammonia. U.S. Environmental Protection Agency, Washington DC. EPA-822-R-99-014. 153 pp.

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Worksheet 10. Stressor-Response Relationships from Laboratory Studies

Do observed concentrations of metals exceed probable effect levels determined from laboratory studies?

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Data

Fine-grained sediments were collected in the upper 15 cm of bottom material with decontaminated steel scoops. Samples were analyzed for metals. Concentrations of toxics were compared to sediment screening values, expressed as probable effect levels, above which toxicity to the test animal Hyallela azteca have been found to occur frequently.

Analysis

Table 26. Comparison of stressor measurements with probable effect levels.

Candidate Cause	Measurement	Observed value	Probable effect level	Exceedance?
Metals (mg/kg)	Cr	13.6	119.4	No
	Cu	17.2	101.2	No
	Pb	19.2	81.7	No
	Zn	79	544	No

Discussion

None of the measured metals exceeded probable effect levels.

Table 27. Evidence scoring table for stressor-response relationships from laboratory studies.

Finding	Interpretation	Score
The observed relationship between exposure and effects in the case agrees quantitatively with stressor-response relationships in controlled laboratory experiments.	This finding strongly supports the case for the candidate cause, but is not convincing because the correspondence could be coincidental due to confounding or differences in organisms or conditions between the case and the laboratory.	++
The observed relationship between exposure and effects in the case agrees qualitatively with stressor-response relationships in controlled laboratory experiments.	This finding somewhat supports the case for the candidate cause, but is not strongly supportive because the correspondence is only qualitative, and the degree of correspondence could be coincidental due to confounding or differences in organisms or conditions between the case and the laboratory.	+
The agreement between the observed relationship between exposure and effects in the case and stressor-response relationships in controlled laboratory experiments is ambiguous.	This finding neither supports nor weakens the case for the candidate cause.	0
The observed relationship between exposure and effects in the case does not agree with stressor-response relationships in controlled laboratory experiments.	This finding somewhat weakens the case for the candidate cause, but is not strongly weakening because there may be differences organisms or conditions between the case and the laboratory.	-
The observed relationship between exposure and effects in the case does not even qualitatively agree with stressor-response relationships in controlled laboratory experiments, or the quantitative differences are very large.	This finding strongly weakens the case for the candidate cause, but is not convincing because there may be substantial and consistent differences in organisms or conditions between the case and the laboratory.	--

Table 28. Stressor-response from laboratory studies scores for metals.

Candidate Cause	Result	Score
Metals	Metals concentrations did not exceed PEL	-

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Worksheet 11. Mechanistically Plausible Cause

Does a plausible mechanism exist for increased metals to cause the observed effects?

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Analysis

Table 29. Plausibility of different effects.

Specific Effect	Mechanism	References
Increased relative weight	No known mechanism
Increased DELT	Metals cause fin erosion and lesions; Pb, Zn, and Cu cause deformities	1,2,3
Decreased percent mayflies	Metals cause lethal and sublethal effects on invertebrates	4,5

Discussion

Decreased percent mayflies and increased DELT can be linked to metals contamination. No plausible mechanism is known by which increased metals leads to increased relative weight.

Table 30. Evidence scoring table for mechanistically plausible cause.

Finding	Interpretation	Score
A plausible mechanism exists.	This finding somewhat supports the case for the candidate cause, but is not strongly supportive because levels of the agent may not be sufficient to cause the observed effect.	+
No mechanism is known.	This finding neither supports nor weakens the cause for the candidate cause.	0
The candidate cause is mechanistically implausible.	This finding strongly weakens the case for the candidate cause, but is not convincing because the mechanism could be unknown.	--

Table 31. Mechanistically plausible cause scores for metals.

Candidate Cause	Specific Effects	Result	Score
Metals	Increased relative weight	Implausible: no known mechanisms	--
	Increased DELT	Plausible	+
	Decreased percent mayflies	Plausible	+

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Worksheet 12. Summary of Scores from Elsewhere

Increased % DELT | Increased Relative Weight | Decreased % Mayflies and Increased % Tolerant Macroinvertebrates

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Increased % DELT

Table 32. Scores for increased % DELT, for evidence that uses data from elsewhere.

Types of Evidence	Algae	Ammonia	Dissolved Oxygen	Metals	PAHs	Riffles/Pools	Sediment
Mechanistically Plausible Cause	0	0	+	+		--	--
Stressor-Response Relationships from Other Field Studies	-	NE	NE	NE		NE	NE
Stressor-Response Relationships from Laboratory Studies	NE	NE	NE	NE		NE	NE
Stressor-Response Relationships from Ecological Simulation Models	NE	NE	NE	NE		NE	NE
Manipulation of Exposure at Other Sites	NE	NE	NE	NE		NE	NE
Analogous Stressors	NA	NA	NA	NA		NA	NA

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Increased Relative Weight

Table 33. Scores for increased relative weight, for evidence that uses data from elsewhere.

Types of Evidence	Algae	Ammonia	Dissolved Oxygen	Metals	PAHs	Riffles/Pools	Sediment
Mechanistically Plausible Cause	+	--	--	--		+	--
Stressor-Response Relationships from Other Field Studies	NE	NE	NE	NE		NE	NE
Stressor-Response Relationships from Laboratory Studies	NE	NE	NE	NE		NE	NE
Stressor-Response Relationships from Ecological Simulation Models	NE	NE	NE	NE		NE	NE
Manipulation of Exposure at Other Sites	NE	NE	NE	NE		NE	NE
Analogous Stressors	NA	NA	NA	NA		NA	NA

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Decreased % Mayflies and Increased % Tolerant Macroinvertebrates

Table 34. Scores for decreased % mayflies and increased % tolerant macroinvertebrates, for evidence that uses data from elsewhere.

Types of Evidence	Algae	Ammonia	Dissolved Oxygen	Metals	PAHs	Riffles/Pools	Sediment
Mechanistically Plausible Cause	+	+	+	+		+	+
Stressor-Response Relationships from Other Field Studies	-	NE	NE	NE		NE	NE
Stressor-Response Relationships from Laboratory Studies	NE	-	+	-		NE	NE
Stressor-Response Relationships from Ecological Simulation Models	NE	NE	NE	NE		NE	NE
Manipulation of Exposure at Other Sites	NE	NE	NE	NE		NE	NE
Analogous Stressors	NA	NA	NA	NA		NA	NA

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Worksheet 13. Evaluating Multiple Lines of Evidence

Increased % DELT | Increased Relative Weight | Decreased % Mayflies and Increased % Tolerant Macroinvertebrates

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Increased % DELT

Table 35. Scores for evaluating multiple lines of evidence for increased % DELT.

Types of Evidence	Algae	Ammonia	Dissolved Oxygen	Metals	PAHs	Riffles/Pools	Sediment
Consistency of Evidence	--	--	+	+		--	--
Explanation of the Evidence

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Increased Relative Weight

Table 36. Scores for evaluating multiple lines of evidence for increased relative weight.

Types of Evidence	Algae	Ammonia	Dissolved Oxygen	Metals	PAHs	Riffles/Pools	Sediment
Consistency of Evidence	--	---	--	--		+	--
Explanation of the Evidence

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Decreased % Mayflies and Increased % Tolerant Macroinvertebrates

Table 37. Scores for evaluating multiple lines of evidence for decreased % mayflies and increased % tolerant macroinvertebrates.

Types of Evidence	Algae	Ammonia	Dissolved Oxygen	Metals	PAHs	Riffles/Pools	Sediment
Consistency of Evidence	--	--	+	--		+	+
Explanation of the Evidence

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Worksheet 14. Identify Probable Cause

Table 38. Probable cause(s) for increased relative weight.

	Probable Cause
	Degraded riffles/pools
Evidence for:	Stressor co-occurs with effect
	Cause is mechanistically plausible

Table 39. Probable cause(s) for increased % DELT.

	Probable Causes
	Decreased DO	Increased Metals
	Stressor co-occurs with effect	Stressor co-occurs with effect
Evidence for:	Cause is mechanistically plausible	Cause is mechanistically plausible
	Causal pathway exists

Table 40. Probable cause(s) for decreased % mayflies and increased % tolerant macroinvertebrates.

	Probable Causes
	Increased sediment	Degraded riffle/pools	Decreased DO
Evidence for:	Stressor co-occurs with effect	Stressor co-occurs with effect	Stressor co-occurs with effect
	Cause is mechanistically plausible	Cause is mechanistically plausible	Cause is mechanistically plausible
			Causal pathway established
			Effect consistent with field stressor-response relationship
			DO is less than minimum value from criterion

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Final Conclusion

Different candidate causes seem to be responsible for each of the biological effects that were considered in this analysis. However, closer examination of the candidate causes suggests a common thread. First, it is likely that the three causes associated with decreased mayflies (increased sediment, degraded pools and riffles and decreased DO) all derive from the same human activity, stream channelization. When the streamcourse is channelized, fine sediment can increase and riffle habitats can be degraded. Furthermore, the deepening of the channel and lost riffles can reduce dissolved oxygen. The deepened channel is also identified as a likely cause for the increased fish weight. Finally, reduced DO from the deepened channel may also be the cause of the increased proportion of DELT. Therefore, we identify the channelization of the stream as the most likely source of the observed impairment at this site.

Discussion

Increased relative weight: Only one candidate cause was found to be probable from the strength of evidence analysis. The available evidence suggested that the change in channel structure, and the resulting deepening of the stream was the most likely factor causing the increase in fish weight. Most other candidate causes were unlikely because no plausible mechanisms existed by which they could increase fish weights (sediment, dissolved oxygen, ammonia, metals). The argument for increased algae was weakened by a negative relationship between increased nutrients (used as a surrogate for algae) and relative fish weight. That is, the largest fish weight was observed at the site with one of the lowest nutrient concentrations. Although only one cause was identified, the evidence in support of this cause is weak, and further data collection is suggested. In particular, the effects of different sampling methodologies between the upstream site and the site of interest should be investigated. Increased relative weight is not a metric in either IBI or ICI, and so this effect is difficult to relate back to the original definition of the impairment.

Increased proportion of DELT anomalies: The cause of the change in DELT was either decreased DO or increased metal concentrations, but the available evidence does not distinguish between these two possibilities. The available lines of evidence for these two candidate causes were virtually identical. Decreased DO included the extra line of evidence that a causal pathway existed. Increased metals lacks this line of evidence only because the causal pathway is simple: increased metals can directly cause increased DELT. Other candidate causes were unlikely for a variety of reasons: Sediment and riffle/pool quality were unlikely causes because they lacked plausible mechanisms. Increased ammonia concentrations were not found at the site, and therefore an unlikely cause. Nutrient concentrations (used as a surrogate for increased autotrophs) were lower than the levels associated with increased DELT anomalies from other field studies in Ohio. Collecting additional evidence would be useful, particularly data that establishes relationships between DO or metal concentrations and the proportion of DELT anomalies. Information on the specific types of anomolies found would also be useful.

Decreased proportion of mayflies and increased proportion of tolerant macroinvertebrate taxa: Three candidate causes possibly explain the changes observed in the macroinvertebrate assemblage. Stressor-response relationships between sediment levels, habitat quality, and the observed biological responses were difficult to establish using the available data. However, changes in substrate composition and the quality of riffle habitat are known to have profound effects on macroinvertebrate assemblages. Several lines of evidence suggest that decreased DO is the cause of the observed changes. Collecting additional evidence linking sediment levels and habitat quality with the macroinvertebrate assemblage response would strengthen the argument for these causes.

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