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F i n a l R e p o r t

Analysis of Whole Effluent Toxicity (WET) Data from the Tres Rios Demonstration

Constructed Wetlands for the Period from January 1996 – August 2002

Prepared for

City of Phoenix August 2004

F i n a l R e p o r t

Analysis of Whole Effluent Toxicity (WET) Data from the Tres Rios Demonstration

Constructed Wetlands for the Period from January 1996 – August 2002

Prepared for

City of Phoenix

August 2004

WETLAND SOLUTIONS, INC.

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Table of Contents

Table of Contents ........................................................................................................................ ii Executive Summary......................................................................................................................1 Introduction...................................................................................................................................1 Whole Effluent Toxicity Testing ...............................................................................................2 Effects of Wetlands on Whole Effluent Toxicity ....................................................................5 Methods and Materials..............................................................................................................12

Study Site .........................................................................................................................12 Sample Collection and Handling .................................................................................20 WET Test Methods and Endpoints ..............................................................................20

Results ..........................................................................................................................................21 Overview .........................................................................................................................21 Time Series Data .............................................................................................................26 Effluent Concentration Effects......................................................................................28 Toxic Events ....................................................................................................................34 Possible Water Quality Effects......................................................................................37

Summary and Recommendations ...........................................................................................42 Acknowledgements....................................................................................................................44 References ....................................................................................................................................45 Appendices A Detailed Biomonitoring Data from the Tres Rios Constructed Demonstration

Wetlands, Phoenix, Arizona Exhibit 1 Summary of WET Reduction Data from Treatment Wetlands ..................................6 2 Summary of whole effluent toxicity results from wetland treatment

technologies being tested by the SFWMD for Everglades (US) protection............10 3 Tres Rios Demonstration Constructed Wetland Project - Cobble,

Hayfield, and Research Cells Wetland Sites...............................................................13 4 Tres Rios Demonstration Constructed Wetland Project - Hayfield Site .................14 5 Tres Rios Demonstration Constructed Wetland Project - Cobble Site ....................15 6 Tres Rios Demonstration Constructed Wetland Project - Research Cells ..............16 7 Summary of Hydraulic Loading (cm/d) in the Tres Rios Demonstration

Wetlands, AZ ..................................................................................................................17 8 Summary of Inlet Water Quality to the Tres Rios Demonstration

Wetlands, AZ - August 1995 - December 2002...........................................................18


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Exhibit 9 Summary of End Points for Survival and Reproduction Tests of

Ceriodaphnia dubia in the Hayfield Cells at the Tres Rios Demonstration Wetlands, AZ - January 1996 - August 2002...............................................................22

10 Summary of End Points for Survival and Reproduction Tests of Ceriodaphnia dubia in the Cobble Cells at the Tres Rios Demonstration Wetlands, AZ - January 1996 - May 2002....................................................................23

11 Summary of Average End Points for Ceriodaphnia dubia 7-day Survival and Reproduction Test in the Tres Rios Demonstration Wetlands, AZ - January 1996 - August 2002...........................................................................................24

12 Average Survival and Reproduction of Ceriodaphnia dubia by Percent Dilution in the Tres Rios Demonstration Wetlands, AZ - January 1996 - August 2002.....................................................................................................................25

13 Reproduction (# young/female) and Percent Mortality differences for Ceridaphnia dubia between Test and Control samples as a function of dilution in the 91st Avenue WWTP Final Effluent to the Tres Rios Demonstration Wetlands, AZ.......................................................................................27

14 Reproduction (# young/female) differences for Ceridaphnia dubia between Test and Control samples as a function of dilution in the Cobble Site Tres Rios Demonstration Wetlands, AZ ..............................................................................29

15 Percent Mortality differences for Ceridaphnia dubia between Test and Control samples as a function of dilution in the Cobble Site Tres Rios Demonstration Wetlands, AZ.......................................................................................30

16 Reproduction (# young/female) differences for Ceridaphnia dubia between Test and Control samples as a function of dilution in the Hayfield Site Tres Rios Demonstration Wetlands, AZ......................................................................31

17 Percent Mortality differences for Ceridaphnia dubia between Test and Control samples as a function of dilution in the Hayfield Site Tres Rios Demonstration Wetlands, AZ.......................................................................................32

18 Cobble Site Ceriodaphnia dubia Survival and Reproduction Biomonitoring Summary - Average (± SE) 1996-2002..........................................................................33

19 Hayfield Site Ceriodaphnia dubia Survival and Reproduction Biomonitoring Summary - Average (± SE) 1996-2002 ...............................................35

20 Tres Rios Cobble Site Ceriodaphnia dubia 7-day Survival and Reproduction Chronic Toxicity Test (± SE) - October 97 ..................................................................36

21 Tres Rios Hayfield Site Ceriodaphnia dubia 7-day Survival and Reproduction Chronic Toxicity Test (± SE) - September 97 ....................................38

22 Tres Rios Hayfield Site Ceriodaphnia dubia 7-day Survival and Reproduction Chronic Toxicity Test (± SE) - October 97 ..........................................39

23 Summary of Monthly Average Electrical Conductivity and Total Ammonia Nitrogen at the Tres Rios Demonstration Constructed Wetlands, January 1996 - August 2002........................................................................41


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Executive Summary

The Clean Water Act (CWA) prohibits the discharge of toxic substances in toxic amounts to Waters of the U.S. Whole effluent biomonitoring tests have been developed to provide a relatively rapid measure of the toxicity of effluents regulated under the National Pollutant Discharge Elimination System (NPDES) permitting program of the CWA. Like many municipal wastewater treatment plants (WWTPs) across the U.S., the City of Phoenix 91st Avenue WWTP conducts effluent bioassays as part of their NPDES permit requirements. Also, like many WWTPs across the U.S., these tests sporadically detect varying levels of toxicity. Due to the infrequent occurrence of test failures, detection and elimination of toxicants in the WWTP effluent is difficult and expensive.

The City of Phoenix is embarking on incorporation of constructed wetlands into their overall effluent management strategy. These proposed wetlands will have multiple purposes including restoration of habitat within and adjacent to the Salt River, public recreational use (e.g., nature study), and water quality polishing before eventual discharge into the Salt River channel. A constructed wetland demonstration project has been underway since August 1995 at the City’s 91st Avenue WWTP to investigate and document the effects of various designs and loading scenarios on wetland treatment and habitat performance. Two of the issues being investigated are the effects of the City’s treated effluent on the wetlands and in turn the effects of the wetlands on the effluent quality. This report focuses on the effects of the constructed wetlands on effluent toxicity.

A review of published and unpublished biomonitoring results from treatment wetlands indicates that these systems typically reduce levels of acute and chronic toxicity to plants, invertebrates, and vertebrates in standard whole effluent toxicity (WET) tests. Toxicity reduction in wetlands appears to be related to hydraulic loading rates and design features that promote effective flow distribution and long hydraulic residence times, the same factors that contribute to effective wetland performance for pollutant reductions. In a very few cases limited to arid climates, WET has been found to increase in wetlands, possibly indicating an effect of salt concentration in those systems. Simultaneous inflow/outflow toxicity results for wetlands must also be interpreted in light of the transient nature of some toxic events at the inflow and the residence time within the wetland, leading to the possibility of comparison of WET in different masses of water.

On a routine basis the acute and chronic toxicity of both the 91st Avenue WWTP final effluent and the wetland outflows is nearly non-existent or difficult to detect. The intermittent nature of toxicity in these discharges makes identification of responsible toxicants difficult. The purpose of this report is to assess the effects of constructed treatment wetlands on toxicity, not to specifically identify toxicity-causing pollutants in the effluent. From this review it can be concluded that the wetlands typically reduced low levels of toxicity when they occurred in the WWTP effluent and did not add to toxicity in any samples tested. High levels of toxicity, when present in the WWTP effluent, passed through the wetlands with minor alteration, probably due to high


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loading rates and resulting short hydraulic residence times. Low toxicity levels were attenuated within the loading regimes experienced in the wetlands. It can be concluded from this research that use of full-scale constructed wetlands for final effluent polishing of the 91st Avenue WWTP final effluent will reduce the frequency and magnitude of WET and provide enhancement of water quality in the ultimate receiving waters in the Salt River.

Since WET may be related to a vast array of chemical constituents in these complex effluents, identification of specific causative agents and quantification of their removal in wetlands has typically not been accomplished. Also, because of the significant cost of most WET tests, they are typically not conducted at a frequency and with sufficient replication that allows careful quantitative data analysis. Additional testing at a variety of sites under carefully controlled conditions is necessary to better understand and predict the effectiveness of treatment wetlands to consistently reduce WET in municipal and industrial effluents. Research-oriented biomonitoring and ecological risk assessments could be conducted at the Tres Rios Demonstration Wetlands. The Research Cells could provide a replicated platform for this type of research. The cells could be re-started with a range of hydraulic loading rates and the existing internal deep zone configurations. WET monitoring could also be conducted along the flow path in the full-scale treatment wetlands to demonstrate the benefits for toxicity reduction.

For future monitoring efforts a sensitive indicator of toxicity such as Microtox™ could be utilized to provide rapid detection of any toxic events in the 91st Avenue WWTP effluent. Wetland inflow and outflow toxicity would then be monitored throughout the toxic event. Fathead minnow and algal tests could be used to augment the existing Ceriodaphnia bioassays. Screening rather than definitive tests can be used to reduce expenses associated with this program. Tests should probably be conducted by the City’s own laboratory because of their apparent ability to provide more sensitive detection levels. Statistical comparisons of the raw test data including neonates produced, weight gain of fish, and algal cell volumes or weight should be used to detect effects of the various wetland designs on toxicity.


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Introduction

Constructed wetlands are being used worldwide to effectively improve the quality of municipal effluents (IWA 2000; Kadlec and Knight, 1996). While greatest interest in treatment wetlands has been focused on their performance for the major contaminants such as biochemical oxygen demand (BOD), total suspended solids (TSS), and nutrients, there is a growing interest in their ability to improve the overall health of downstream waters by reducing concentrations of a broad spectrum of contaminants that may occur at acute or sub-lethal concentrations in treated municipal effluents (USEPA, 1999; API 1998; Knight et al., 1994). These contaminants may include elevated levels of unionized ammonia nitrogen, dissolved ions and salts, a variety of trace metals, and complex trace organic compounds that independently or synergistically exert toxic effects on biota.

While the concentration of individual pollutants may be monitored in some instances, biomonitoring is required to assess the overall net potential for acute and chronic toxicity to receiving water biota. The Clean Water Act of 1972 specifically prohibits the discharge of toxic substances in toxic amounts to waters of the U.S. For this reason, whole effluent toxicity (WET) monitoring is included in the National Pollutant Discharge Elimination System (NPDES) permits for nearly all municipal treatment plants. WET tests are the standardized procedure to detect levels of acute and chronic toxicity in municipal and industrial effluents.

The City of Phoenix and other regional partners have been evaluating the use of constructed wetlands for final effluent polishing and habitat enhancement since the early 1990’s. The Tres Rios Wetland Project, named for its location near the confluence of three major rivers – the Salt, Gila, and Aqua Fria, is intended to reuse treated municipal effluent from the Phoenix area to restore lost and degraded wildlife habitat along the Salt River. Highly treated effluent from the City’s 91st Avenue Wastewater Treatment Plant (WWTP) is the source of the water for this habitat restoration project. Existing excess effluent is discharged into the channel of the Salt River. As an alternative to the existing point discharge, an 800-acre constructed wetland project is envisioned to create a large habitat area adjacent to the Salt River and to provide a diffuse discharge into the existing river floodplain (USACOE, 2000).

One of the additional goals of the proposed Tres Rios Constructed Wetland Project is the reduction of sporadic and poorly defined occurrences of toxicity in the effluent from the City’s 91st Avenue WWTP before this water is released to waters of the state. A demonstration-scale wetland project was constructed in 1994-1995 to better define the potential role of constructed wetlands for improvement of the water quality from the 91st Avenue WWTP. Monitoring of the demonstration wetlands has been underway since July 1995. Preliminary WET testing on the wetlands effluent was conducted in November 1995 and routine testing began in 1996. The purpose of this testing is to document what changes in WET occur with passage of the treated effluent through the constructed wetlands. The WET testing program and results are described below.


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Whole Effluent Toxicity Testing

It has long been recognized that all substances can be poisonous, depending upon the dose. Environmental protection, including the health of people and ecosystems, is dependent upon the knowledge of what concentrations or dosages of substances are safe and what levels do not inflict harm. For many years, scientific study of this issue focused on dose-response studies of individual chemicals. A massive literature is available documenting the toxicology of chemicals to humans, and a considerable but much smaller knowledge base exists concerning the toxicity of individual chemicals to ecosystems and their component parts. Until relatively recently, the effects of complex mixtures of chemical pollutants on the environment were poorly known.

Passage of the 1972 amendments to the Clean Water Act (Federal Water Pollution Control Act) instituted the national mandate to control the discharge of “toxic substances in toxic amounts” to the Nation’s waterways. Methods to implement this important legislation were under development on many fronts. Numerous mesocosm, pond, and artificial stream studies were initiated to assess the effects of individual pollutants on ambient waters and their ecosystems. Laboratory studies with mammal surrogates continued to be the main method for assessing toxicity of chemicals to humans. However, a method was sorely needed for assessing the effects of complex effluents on receiving waters. Most municipal and many industrial wastewater effluents contain from dozens to hundreds of chemicals, precluding the itemization of every possible toxicant. A method to assess both the acutely lethal and the sub-lethal or chronic effects of whole effluents was developed by the U.S. EPA and proposed for use in 1984 (Mount and Norberg, 1984).

The short-term chronic toxicity tests that were eventually developed by the U.S. EPA have found worldwide use as a relatively inexpensive and relevant approach to assessing WET (USEPA, 1994). Fresh water chronic toxicity tests currently encompass three principal organisms:

• An invertebrate, the water flea (Ceriodaphnia dubia)

• A vertebrate, the fathead minnow (Pimephales promelus)

• A microscopic plant, the green alga (Selenastrum capricornutum)

Testing methods have been refined for each of these three organisms over the years. Current test methods typically require a 7-day (96 hours for the algal test) testing period with renewals of the testing solution using the tested effluent three times during that 7-day period. For the water flea the test encompasses three reproductive cycles where female water fleas give birth to three broods of young (neonates). The adult water fleas are typically fed three times per day during the testing period. Acute toxicity is assessed over this testing period by recording mortality of the mature female water fleas. Chronic toxicity is assessed by recording the number of young produced per female over the 7-day testing period. Tests may be conducted using just control water and 100 percent effluent, or with one or more diluted effluent concentrations.


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Short-term chronic fathead minnow testing utilizes larval fish, less than 48-hours old at the time of test initiation. These immature fish are also tested over a 7-day growth period with test water renewals and are fed each day. Acute toxicity is assessed through observed mortality of these young fish. Chronic toxicity is assessed by measurement of the final dry weight of the tiny fish at the end of the test period. Laboratory controls are utilized and one or more effluent concentrations are tested to assess the lethal and sub-lethal effects of the effluent on the fish.

The green algae are typically tested over a 96-hour growth period. The algae are grown in flasks under artificial light and on mechanical shakers without any renewals of the test solution. A nutrient broth is added to control and tested waters to provide sustenance for the test organisms. After 96 hours, the total algal cell density, cell volume, or dry weight is determined by microscopic count and compared to the controls.

Chronic toxicity tests may be run for a single effluent concentration (screening test) or for a range of effluent concentrations (definitive test). Screening tests are less expensive and therefore more common and look for any signs of toxicity by the effluent, acute or chronic, to the test organisms. Definitive tests are used to estimate a number of possible metrics useful for assessment of the absolute toxicity of the effluent. For example, the lethal concentration to 50 percent of the organisms (LC50) within a specified time interval (for example 24-hr LC50) is often calculated from definitive test results. Concentrations that cause inhibition to 25 or 50 percent of the test organisms may also be reported (IC25 and IC50). The inverse of the IC25 is reported as the “toxicity units” with a value of 1 indicating no toxicity and values over 1 indicating toxicity to the test organism. Other common metrics determined through statistical analysis of test results include the lowest-observable effect concentration (LOEC) and the no-observable effect concentration (NOEC). If screening tests detect acute or chronic toxicity, most permits trigger the need to run definitive tests to better define the level of effect on the test organisms.

In addition to the standard tests described above, there are many variations in testing protocols that have been used over the past two decades. Also, many states have specific protocols that vary somewhat from the federal WET testing guidelines. Many other vertebrate and invertebrate species have been used for testing. Two of the more common alternate species that have been used are rainbow trout (Salmo gairdneri) in regions of the country with natural salmonid fish populations and an alternate water flea, Daphnia magna. Some states replace fathead minnows with indigenous fish species such as the bluegill (Lepomis macrochirus) or the bannerfin shiner (Cyprinella leedsi). Effluents discharged to salt waters have an entirely different set of invertebrate, vertebrate, and plant test organisms. A luminescent bacterium test (Microtox™) has also been developed to provide a protocol for very rapid and relatively less expensive toxicity assessments. In addition to the test organisms used, WET testing protocols differ with respect to their duration, number of water renewals, dilution water requirements, numbers of replicates, feeding intensity, water pretreatment, etc.

Many concerns about the technical adequacy and environmental relevance of WET tests have been raised. Some of the key concerns include the following (Chapman, 2000; La Point and Waller, 2000):

• Substantial inter- and intra-laboratory variability exists in WET results


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• The specific organisms selected for the tests and their age at the time of testing have varying sensitivities to different types of toxicants and may be more or less sensitive than the actual organisms in the receiving water environment

• Permit-mandated testing frequently only requires one or two of the test methods to be routinely performed, potentially missing an effect on a more sensitive phylogenetic group

• The results of single-species assays have limited relevance to multi-species assemblages typical of most receiving waters

• Chemical and physical conditions in the receiving water might ameliorate or enhance the effects of the effluent in ways not duplicated in the laboratory test environment

In spite of limitations inherent in any analytical or biological test, the use of WET methods continues to be the primary approach to assessing the prohibition against the release of toxic substances in effluents to U.S. waters. Apparently this testing program is working since it is reported that about 25 percent of NPDES point-source discharges were acutely toxic in 1986 and that the number of permitted facilities that are acutely and chronically toxic is currently less than 10 percent (Ausley, 2000).


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Effects of Wetlands on Whole Effluent Toxicity

The bulk of research incorporating WET testing of municipal and industrial effluents focuses on effects of various conventional treatment technologies on WET or on the effects of the measured WET on receiving waters such as streams, lakes, and estuaries. Of interest to this report are those studies where researchers have reported the effects of treatment wetlands on modifying the WET of effluents. While there are numerous studies on the fate of individual toxicants in wetlands, this report directs attention to those studies that measure the overall change in toxicant effect by wetlands by use of WET testing methods.

The relevance of WET results to assessing or predicting the effects of effluents on wetland biota is also of interest but not discussed further here. The relevancy of acute and chronic WET tests to assessing effects on wetland biota has not been examined. Due to the relative simplicity of these test conditions in comparison to the complexity of interactions within a typical wetland ecosystem, it is not considered likely that these tests will correlate well with impacts measured using standard wetland bioassessment techniques. Wetland environments tend to be harsh compared to other aquatic ecosystems because of naturally low levels of dissolved oxygen, high organic matter, and fluctuating water levels. These wetland environments tend to be colonized by organisms (plants and animals) adapted to life in relatively harsh environments and therefore they may not be as sensitive to the effects of some pollutants. From this standpoint, using sensitive WET test organisms such as embryo-larval fathead minnows or water fleas to determine the potential for effluent toxicity within a treatment or receiving wetland may be overly conservative. Wetlands as receptors for releases of toxic substances is a field of growing interest (USEPA, 1999); however, considerable work needs to be conducted before there is a general understanding of the susceptibility to these environments to anthropogenic toxicants.

API (1998) provides one of the few published summaries of the effects of wetlands on WET. This study was sponsored and reviewed by the American Petroleum Institute’s (API’s) Biomonitoring Task Force and focuses on the use of wetlands for treatment of petroleum-bearing effluents. Additional review and summarization of the effects of treatment wetlands on WET is provided in USEPA (1999) and summarized in the North American Treatment Wetland Database (NADB) v. 2.0. Exhibit 1 provides a summary of the key published results on the effects of wetlands on whole effluent toxicity measurements.

McAllister published the results of investigations at 6 constructed treatment wetlands in the United States conducted during 1992 (McAllister, 1992, 1993a, 1993b). Standardized tests to assess the ecological structure and function of each of these six sites were conducted. These tests included 7-day renewal, screening tests using Ceriodaphnia dubia. Results of these tests are summarized in Exhibit 1 for two arid wetlands (Show Low,


EXHIBIT 1Summary of WET Reduction Data From Treatment Wetlands

Toxicity Measurement

Description In OutImprovement

(%) ReferenceMicrotox™ EC50 (luminescence as a percentage of control) for wastewaters from an oil terminal 1.8 88 98

Farmer, 1996 (unpublished)

Ceriodaphnia dubia LC50 (static-renewal, 7-day tests) in laboratory-scale wetlands for Zn-amended water to simulate wastewater at the St. Charles, Louisian (US) oil refinery (average inflow 1.70 mg/L Zn) 0.155 mg/L 0.189 mg/L 22 Hawkins et al. , 1995Ceriodaphnia dubia survival (%) (static-renewal, 7-day tests) in laboratory-scale wetlands for Zn-amended water to simulate wastewater at the St. Charles, Louisian(US) oil refinery

Hawkins et al. , 1995 (estimated from graph)

Control (no zinc) 100 93 -7 1.70 mg/L zinc 0 23 >100 0.85 mg/L zinc 0 38 >100 0.43 mg/L zinc 0 88 >100 0.22 mg/L zinc 10 98 90 0.11 mg/L zinc 88 100 12Daphnia magna survival (%) in pilot-scale wetlands used to treat wastewaters fromoil sands processing facility in Alberta (Canada)

Bishay et al. , 1995 (estimated from graph)

Ditch (reference water consisting of surface runoff and groundwater seepage) 75 63 -16 Dike (seepage from saturated tailings) 61 80 31 Pond (tailings pond top water with highest expected contaminant levels) 0 63 >100

Daphnia magna reproduction (neonates per female) in pilot-scale wetlands used totreat wastewaters from oil sands processing facility in Alberta (Canada)

Bishay et al. , 1995 (estimated from graph)

Ditch (reference water consisting of surface runoff and groundwater seepage) 113 82 -32 Dike (seepage from saturated tailings) 115 138 20 Pond (tailings pond top water with highest expected contaminant levels) 0 123 >100

Microtox™ EC50 (as a percent of control luminescence) in pilot-scale wetlands used to treat wastewaters from oil sands processing facility in Alberta (Canada) Bishay et al. , 1995 Ditch (reference water consisting of surface runoff and groundwater seepage) >100% >100% 0 Dike (seepage from saturated tailings) 64% 75% 18 Pond (tailings pond top water with highest expected contaminant levels) 38% 85% 126Ceriodaphnia dubia reproduction (neonates per female) in constructed wetlands receiving municipal effluent at Collins, Mississippi (US) 0 31.1 >100 McAllister, 1992Ceriodaphnia dubia survival (%) in constructed wetlands receiving municipal effluent at Collins, Mississippi (US) 0 100 >100 McAllister, 1992Ceriodaphnia dubia reproduction (neonates per female) in constructed wetlands receiving municipal effluent at West Jackson Co., Mississippi (US) 22.7 24.3 7 McAllister, 1992Ceriodaphnia dubia survival (%) in constructed wetlands receiving municipal effluent at West Jackson Co., Mississippi (US) 90 100 11 McAllister, 1992

Rainbow trout survival (%) in a constructed wetland at Arcata, California (US) 94 96 2 USEPA, 1999Ceriodaphnia dubia reproduction (neonates per female) in constructed wetlands receiving municipal effluent at Incline City, Nevada (US) 28.1 5.3 -81 McAllister, 1993aCeriodaphnia dubia survival (%) in constructed wetlands receiving municipal effluent at Incline Village, Nevada (US) 100 70 -30 McAllister, 1993aCeriodaphnia dubia reproduction (neonates per female) in constructed wetlands receiving municipal effluent at Orlando, Florida (US) 26.4 25.6 -3 McAllister, 1993bCeriodaphnia dubia survival (%) in constructed wetlands receiving municipal effluent at Orlando, Florida (US) 100 100 0 McAllister, 1993bCeriodaphnia dubia reproduction (neonates per female) in constructed wetlands receiving municipal effluent at Lakeland, Florida (US) 19.9 25.7 29 McAllister, 1993bCeriodaphnia dubia survival (%) in constructed wetlands receiving municipal effluent at Lakeland, Florida (US) 80 90 12.5 McAllister, 1993bCeriodaphnia dubia reproduction (neonates per female) in constructed wetlands receiving municipal effluent at Show Low, Arizona (US) 30.6 24.5 -20 McAllister, 1993aCeriodaphnia dubia survival (%) in constructed wetlands receiving municipal effluent at Show Low, Arizona (US) 100 100 0 McAllister, 1993a

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Arizona and Incline Village, Nevada), two Gulf of Mexico coastal plain wetlands (Collins and West Jackson County, Mississippi), and two wetlands in peninsular Florida (Lakeland and Orlando’s Iron Bridge).

The Collins wetland had significant acute and chronic toxicity at the wetland inflow, probably due to high unionized ammonia concentrations. Both acute and chronic toxicity was absent in the wetland outflow samples. West Jackson County had very slight acute and chronic toxicity to the water flea at the wetland inflow but no toxicity indicated at the outflow. Both of these wetlands provide substantial reductions in concentrations of total ammonium nitrogen as well as biochemical oxygen demand (BOD) and total suspended solids (TSS).

Both survival and reproduction of the water fleas were also increased with passage through the Lakeland constructed wetland. No chronic toxicity was detected at either the inflow or the outflow of the Iron Bridge constructed wetland.

While no toxicity was detected at the inflow of the Incline Village wetland in Nevada, the outflow (collected from the terminal cell in this total evaporative wetland) was acutely and chronically toxic to the water flea. A similar reduction in reproduction was observed with passage through the Show Low constructed wetland, another zero discharge system. It appears that toxicity was increased in these wetlands as a function of their evaporative concentration of salts and other conservative constituents. Visual and biological observations at both of these arid wetlands indicate that their biotic communities undergo similar changes from the inlet to the final evaporative cells, with shifts to salt-tolerant plant and animal species.

Acute toxicity test results were reported for four treatment wetlands in the NADB v. 2.0 (USEPA, 1999). These data include acute static results for the pilot wetland cells at Santa Rosa, California (US), the Minot, North Dakota (US) constructed wetland, and the American Crystal Sugar Company constructed wetland in Hillsboro, North Dakota (US). Acute renewal test data were also available for the Hillsboro wetland and for the Arcata, California (US) constructed treatment wetland.

Data from five 96-hour acute static tests using rainbow trout are reported for the Santa Rosa wetland. This system receives nitrified reuse-quality municipal wastewater. An average of 9 percent mortality was observed in these samples collected at three points in the wetland system. No influent data were available from this site for comparison.

Acute biomonitoring data were available for water fleas (48-hour static) and fathead minnows (96-hour static) at the Minot constructed wetland outlet. No significant mortality to either organism was recorded for test dilutions up to 87.5 percent effluent.

Similar acute WET data are reported for the Hillsboro food processing wastewater treatment wetland. These data indicate that the undiluted wetland outflow was slightly toxic to fathead minnows (average of 15 percent mortality at 96 hours) and to water fleas (average of 16 percent mortality at 48 hours). No wetland influent data are available from this site for comparison.

WET to rainbow trout was tested at the Arcata wetland in 1981 and 1982. These data indicated little change in survival between the wetland inlet and outlet (94 to 96 percent).


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Champion International tested 6 pilot wetlands for effluent polishing at their Cantonment, Florida, bleached kraft paper mill (Knight et al., 1994). Each cell had a different design configuration and pairs of cells received a range of hydraulic loads. Whole water toxicity was measured with water fleas and fathead minnows using 7-day, definitive renewal tests quarterly during the first year of pilot wetland operation. No acute toxicity to either organism was reported. Influent samples were chronically toxic to the water flea in all cases and this chronic toxicity was reduced by passage through the wetland cells in 10 of 11 test comparisons. Chronic toxicity measured with the water flea was reduced from a chronic value of less than 12.5 to 65 percent influent, to a chronic value between 17.8 and >100 percent in the outflow. Inflow samples were chronically toxic to fathead minnows in two of three samples tested. No chronic toxicity to fathead minnows was observed in any of the wetland outflow samples tested. It was concluded that reduction of chronic toxicity to the water fleas and fathead minnows was strongly related to hydraulic loading rate and resulting reductions in concentrations of unionized ammonia and a variety of unidentified chlorinated organics.

In studies of pilot-scale subsurface flow and surface flow treatment wetlands at a U.S. refinery (API, 1998), reductions in chronic toxicity to fathead minnows were found to be positively correlated with hydraulic retention time (HRT). More than 50 percent of the toxicity was removed using a 12-hour HRT, with increasing but smaller incremental reductions using 24-, 36-, and 48-hour HRTs. Nearly all toxicity to fathead minnows was eliminated at the 48-hour HRT.

At a non-U.S. oil terminal, Microtox™ (bacteria luminescence) tests using zero dilution indicated a reduction of EC50 values by 98 percent with passage through a treatment wetland (Farmer, 1996).

A full-scale surface flow constructed wetland at the Chevron refinery at Richmond, California (US), has used rainbow trout to assess effluent toxicity. These tests have consistently shown no mortality (Duda, 1992).

Short-term (7-day) chronic WET tests using water fleas were conducted on wetland mesocosms by using zinc-amended simulated wastewater from an oil refinery in St. Charles, Louisiana (US) by Hawkins et al., 1995. Results indicated that zinc (Zn) removal by the wetland (80 percent) reduced toxicity measured at the inflow and outflow of the wetland. Static renewal 7-day tests with Ceriodaphnia dubia indicated that at inflow Zn concentrations of 1.7, 0.85, and 0.43 mg/L, survival was zero (estimated LC50 of 0.16 mg/L, while survival in the wetland outflow from these test units was approximately 23, 38, and 88 percent respectively, increasing the apparent 7-day LC50 for Zn to 0.19 mg/L. At an inflow Zn concentration of 0.22 mg/L, the survival rate was about 10 percent while the survival measured in the wetland outflow was 98 percent. When the influent Zn concentration was 0.11 mg/L the wetland mesocosm inflow and outflow survival rates were 88 and 100 percent, respectively.

A pilot-scale wetland has been used to treat wastewater from an oil sand processing facility at Fort MacMurray, in Alberta, Canada. Studies indicate that the treatment wetlands have reduced toxicity to the cladoceran Daphnia magna, as well as in Microtox™ tests (Bishay et al., 1995).


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A brackish marsh wetland treatment system for urban runoff was monitored near San Francisco Bay, California (US) by using 7-day water flea tests from 1991 through 1994 (WCC, 1995). Reductions in WET were observed at various points within the stormwater treatment wetland. The study demonstrated that toxicity was reduced by elimination of short-circuiting within the wetland by use of log-baffles.

The South Florida Water Management District (SFWMD) located in West Palm Beach, Florida (US) tested a variety of natural and conventional technologies to achieve very low total phosphorus (TP) discharge standards (<10 ppb) in agricultural and urban stormwaters discharged to the Everglades (SFWMD, 2001). Several of the natural and hybrid technologies included wetland components. Bioassay tests were required as part of these technology evaluations to assess the “marsh readiness” of the treated water. The “managed wetland” technology combined a chemical treatment system using iron (Fe) or aluminum (Al) salts with a cattail wetland marsh to achieve low outflow TP concentrations. Three different biomonitoring test organisms were used in December 2000 (CH2M HILL, 2001) to assess the marsh readiness of the water leaving control and treatment wetlands at two different TP inlet loads (high = 51 ug TP/L and low = 21 ug TP/L). Results of these tests are summarized in Exhibit 2.

Water flea short-term chronic tests found no effects of the outflow from any of the treatment or control wetlands on survival, but the high TP control wetland and two of the wetlands receiving Al treated effluents had slightly reduced reproduction rates. Survival of the test vertebrate, the bannerfin shiner (Cyprinella leedsi), was significantly reduced for all of the wetland cells receiving chemical pretreatments and slightly reduced for the low TP inlet control wetland cell. Growth of the fish was also significantly reduced in the high TP Al and Fe treatments and in both the control and Fe treatment low TP wetland outflows. Algal growth as measured by the 96-hour non-renewal Selenastrum capricornutum test was higher in all of the wetland outflows compared to the laboratory control. There was negligible algal growth potential in wetland outflow samples from both the high and low TP research sites.

A second system tested for Everglades TP removal combined a submerged aquatic vegetation (SAV) dominated wetland with a permeable limerock (LR) berm. Exhibit 2 also summarizes results from biomonitoring tests conducted on inflows and outflows to the SAV and SAV+LR treatment systems at the two research sites (high and low inlet TP). Ceriodaphnia survival and reproduction were significantly lowered in the LR effluent compared to the inlet at the North research site (high inlet TP). No chemical factor could be discovered that explained this surprising effect. Survival and growth of the bannerfin shiner was not markedly affected in any of the treatments. All of the samples promoted algal growth compared to controls in the 96-hour Selenastrum test. As was observed for the managed wetland technology tests, there was basically no measurable response with this low TP water during the 14-day algal growth potential test.

In summary, existing studies indicate that treatment wetlands generally reduce levels of acute and chronic toxicity if present in the inflow. The magnitude of toxicity reduction appears to be dependent upon wetland design and operational conditions, especially those that affect contact between the wastewater and the wetland plants and soils. As with the majority of the pollutant treatment processes acting in wetlands, the effects of


EXHIBIT 2Summary of whole effluent toxicity results from wetland treatment technologies being tested by the SFWMD for Everglades (US) protectiona.

Ceriodaphnia dubia Cyprinella leedsiSelenastrum

capricornutum

DescriptionSurvival

(%)

Reproduction (total

neonates)Survival

(%)Growth

(mg/fish)96-Hr Growth

(cells/mL x 105)

Algal Growth

Potential (mg dry wt./L) Reference

Managed Wetland Treatment Technology CH2M HILL, 2001

North Test Cell 3 (high inlet TP control) 100 (100) 296 (338)* 80.0 (90.0) 0.19 (0.23) 12.3 (5.53) 1 (103)

North Test Cell 2 (high inlet TP, Fe coagulent) 90 (100) 236 (267) 32.5 (87.5)* 0.08 (0.21)* 15.0 (5.53) 0 (103)

North Test Cell 4 (high inlet TP, Al coagulent) 100 (100) 301 (338)* 17.5 (90.0)* 0.02 (0.23)* 12.4 (5.53) 1 (103)

South Test Cell 6 (low inlet TP, control) 100 (100) 332 (338) 55.0 (90.0)* 0.11 (0.23)* 8.60 (5.53) 0 (103)

South Test Cell 7 (low inlet TP, Al coagulent) 100 (100) 270 (338)* 35.0 (90.0)* 0.07 (0.23)* 13.0 (5.53) 0 (103)

Submerged Aquatic Vegetation/Limerock (SAV/LR) Technology96-Hr Growth (mg dry wt./L) FDEP, 1999

North ENR Technology Site Influent 100 (100) 165 (161) 92.5 (97.5) 0.32 (0.37) 7.29 (4.29) 1.67

North ENR Technology Site SAV Effluent 100 (100) 148 (173) 87.5 (100)* 0.39 (0.36) 7.92 (4.29) 0.114

North ENR Technology Site SAV+LR Effluent 60 (100)* 74 (190)* 100 (95) 0.36 (0.35) 6.37 (4.29) 0.334

South ENR Technology Site Influent 90 (100) 175 (153) 92.5 (100) 0.47 (0.49) 5.12 (2.43) 0.355

South ENR Technology Site SAV Effluent 80 (100) 172 (184) 97.5 (97.5) 0.55 (0.50) 4.61 (2.43) 0.104

South ENR Technology Site SAV+LR Effluent 100 (100) 182 (200) 95 (97.5) 0.56 (0.50) 7.45 (2.43) 0.093aLaboratory control values for each respective test are given in parentheses. * Indicates significant effect compared to the control. Methods follow EPA/600/4-91/002 for static, renewal, screening with substitution of C. leedsi for P. pimephales

10


11

hydraulic loading generally outweigh hydraulic residence time, indicating that increasing water depth may not be as effective as increasing area to reduce WET in treatment wetlands. Some of the reported exceptions to the general reduction of WET by wetlands are located in the arid western U.S. Such systems are exposed to high temperatures and low rainfall which can concentrate dissolved ions (salts) and in several cases have been found to have increasing toxicity between the wetland inflow and final cells. While this observed effect could be the result of salt buildup within these wetlands, it could also be in response to increasing concentrations of other conservative elements and compounds that are toxic at lower amounts. Correlation between water quality, biomonitoring, and in situ biological data from these arid wetlands would be useful to further clarify the important processes affecting WET in these systems. While not a total evaporative wetland concept, the Tres Rios Demonstration Constructed Wetland Project data offer an opportunity to better understand the effects of treatment wetlands on WET.


12

Methods and Materials

Study Site The Tres Rios Demonstration Constructed Wetland Project consists of three discrete constructed wetland sites, totaling about 6 ha of wetted area (Exhibit 3). These demonstration wetland units are adjacent to the City of Phoenix’s 91st Avenue WWTP west of Phoenix, on the north side of the Salt River floodway, and include:

• Hayfield Riparian Wetlands (Exhibit 4) – two cells, each 1.3 ha built on silty soils on the first terrace above the Salt River floodway

• Cobble Site (Exhibit 5) – two wetland cells with areas of 1.1 and 1.2 ha each built on mixed cobble (water-polished rock) within the floodway of the Salt River

• Research site (Exhibit 6) – 12 wetland test cells, each 0.1 ha, with various numbers of internal transverse deep zones

WET testing has only been conducted on the Hayfield and Cobble Site wetlands.

The Tres Rios wetlands are surface flow systems and are planted with emergent marsh plant species including primarily giant bulrush (Schoenoplectus = Scirpus tabernaemontani) and three-square bulrush (S. americanus). Each of the four wetland cells tested has an inlet deep zone for initial distribution of the WWTP effluent, from one to 5 internal deep zones for re-distribution of flows across the width of the wetland cells, and a final, outlet deep zone for final collection. Treated wastewater for this research is derived from a combination of treatment trains at the 91st Avenue WWTP, Plant 3A, with effluent diverted to the wetland facilities from the chlorine contact channel. Plant 3 produces advanced effluent with nitrification/denitrification, but carries a free chlorine residual into the front end of the wetland cells (typically 2.5 to 3.5 mg/L). Internal sampling within the wetlands indicates that this free chlorine is quickly dissipated within all of the wetland cells, prior to the first deep water zone where measurements can be conducted.

The demonstration constructed wetland cells received initial flows in the summer of 1995 and have been operational since that time. Several shutdowns have occurred at individual cells as documented in Exhibit 7 that lists the average monthly hydraulic loads to each cell through the period of this study. The two Cobble Cells were reconfigured by inclusion of additional longitudinal deep zones and islands in early 1998. A summary of the WWTP effluent (wetland influent) data for the period-of-record covered in this report is provided in Exhibit 8. It should be noted that this effluent typically has low concentrations of biochemical oxygen demand (BOD5), total suspended solids (TSS), and ammonium nitrogen.


EXHIBIT 3Tres Rios Demonstration Constructed Wetland Project - Cobble, Hayfield, and Research Cells Wetland Sites

Cobble Site

Hayfield SiteResearchCells

91st AveWWTP

Salt River

C1C2

H1

H2

91st Ave

13


EXHIBIT 4Tres Rios Demonstration Constructed Wetland Project - Hayfield Site

Inlet DZ

Inlet DZDZ 1

DZ 2

Outlet DZ

DZ 1

DZ 2

Outlet DZ

DZ 3

DZ 4

DZ 5

H 2

H 1

Station H2Effluent

Station H1Effluent

Station HSInlet Splitter

flow

flow

14


EXHIBIT 5Tres Rios Demonstration Constructed Wetland Project - Cobble Site

Station CSInlet Splitter

InletDZ

DZ 1C 1

Station C1Effluent

flow

C 2DZ 1

DZ 2

DZ 2

DZ 3

DZ 3

OutletDZ

flow

InletDZ

OutletDZ

Station C2Effluent

islands

Station CSInlet Splitter

InletDZ

C 1Station C1

Effluent

flow

C 2

OutletDZ

flow InletDZ

OutletDZ

Station C2Effluent

islands

islands

Post-Reconfiguration (After July 1998)

Pre-Reconfiguration (Prior to July 1998)

15


EXHIBIT 6Tres Rios Demonstration Constructed Wetland Project - Research Cells

R1R2

R3R4

R5R6

R7R8

R9R10

R11R12

R7 Eff

R8 Eff

R9 Eff

R10 Eff

R12 Eff

R11 Eff

R1 EffR2 Eff

R3 EffR4 Eff

R5 EffR6 Eff

Inlet DZInlet DZ

Inlet DZInlet DZ

Inlet DZ

Inlet DZ

Inlet DZ

Inlet DZInlet DZ

Inlet DZInlet DZ

Inlet DZ

Outlet DZOutlet DZ

Outlet DZOutlet DZ

Outlet DZOutlet DZ

Outlet DZ

Outlet DZOutlet DZ

Outlet DZOutlet DZ

Outlet DZ

A

B

A

B

A

B

A

B

A

B

A

B

B

A

B

A

B

A

B

A

B

A

B

A

flow

flow

flow

flow

flow

flowflow

flow

flow

flowflow

flow

A, B = Internal Sampling Points

Splitter Inflow

16


EXHIBIT 7Summary of Hydraulic Loading (cm/d) in the Tres Rios Demonstration Wetlands, AZ

0

20

40

60

80

100

120

140

Aug-95

Nov-95

Feb-96

May-96

Aug-96

Nov-96

Feb-97

May-97

Aug-97

Nov-97

Feb-98

May-98

Aug-98

Nov-98

Feb-99

May-99

Aug-99

Nov-99

Feb-00

May-00

Aug-00

Nov-00

Feb-01

May-01

Aug-01

Nov-01

Feb-02

May-02

Aug-02

Nov-02

HLR

(cm

/d)

H1 H2 C1 C2

Average 11.9 13.9 37.7 19.5Median 14.2 15.1 33.2 17.0

Maximum 20.8 38.7 121 55.6Minimum 0.00 0.00 0.00 0.00Std Dev 5.32 6.92 25.7 12.6Count 82 82 86 86

Statistics

17


EXHIBIT 8Summary of Inlet Water Quality to the Tres Rios Demonstration Wetlands, AZAugust 1995 - December 2002

DateAlkalinity(mg/L)

TSS(mg/L)

TDS(mg/L)

Cl(mg/L)

TKN(mg/L)

NO2-N(mg/L)

NO3-N(mg/L)

NOx-N(mg/L)

PO4-P(mg/L)

Diss. P(mg/L)

COD(mg/L)

cBOD(mg/L)

NH3-N(mg/l)

TOC(mg/L)

TN(mg/L)

Org. N(mg/L)

Sulfate(mg/L)

UOD(mg/L)

Temp(C)

pH(units)

Cond.(µmhos/cm)

DO(mg/L)

Aug-95 182 3.7 858 251 3.05 0.05 1.59 1.64 3.58 4.56 32.1 2.5 2.86 7.0 4.7 0.43 --- 16.8 31.06 7.03 --- 3.69Sep-95 184 1.9 783 248 2.67 --- --- 3.09 --- 3.13 38.7 1.9 1.90 6.5 5.8 0.78 --- 11.5 30.17 6.83 --- 5.14Oct-95 175 3.3 764 245 2.98 0.05 1.02 1.18 3.79 4.15 22.0 3.1 1.83 7.6 4.0 1.15 --- 13.0 28.79 6.98 1398 7.10Nov-95 176 3.7 820 217 3.75 0.25 2.64 2.89 3.50 3.50 23.0 3.7 3.17 8.0 6.6 0.58 --- 20.0 26.93 6.95 1363 6.08Dec-95 153 3.6 813 212 4.25 0.25 3.80 4.04 4.10 3.60 133.6 4.5 2.37 8.6 8.3 1.88 --- 17.6 24.19 7.05 1411 5.10Jan-96 177 4.2 849 235 3.67 0.06 3.81 3.83 4.11 4.20 38.7 3.8 2.29 9.0 7.5 1.38 --- 16.2 22.22 7.11 1454 3.67Feb-96 177 4.4 868 250 4.51 0.23 1.85 1.87 3.36 3.66 53.8 5.6 2.57 10.9 6.4 1.94 --- 20.2 23.03 7.04 1483 3.42Mar-96 178 8.4 842 244 4.16 0.15 2.77 2.89 4.84 5.15 54.1 5.0 2.22 10.1 7.1 1.94 --- 17.7 23.78 6.98 1471 3.38Apr-96 185 2.8 856 238 3.55 1.12 2.98 3.87 4.18 4.84 48.1 3.2 2.54 9.3 7.4 1.01 --- 16.5 25.48 7.02 1465 3.61May-96 170 2.4 891 265 3.10 0.48 3.57 3.89 3.34 3.78 42.1 2.3 1.83 8.1 7.0 1.27 --- 11.8 27.98 6.91 1529 3.24Jun-96 173 4.4 883 289 3.33 0.05 2.85 2.85 10.59 3.81 46.9 3.4 1.72 9.6 6.2 1.61 --- 12.9 30.22 6.95 1505 3.01Jul-96 186 2.1 899 302 3.57 0.05 1.34 1.36 3.01 3.41 48.1 3.3 1.76 9.5 4.9 1.81 --- 13.0 31.76 6.87 1530 2.58Aug-96 168 4.0 895 286 3.98 0.05 2.90 1.99 2.94 3.95 39.9 2.8 2.27 9.9 4.5 1.71 --- 14.5 32.44 6.89 1530 2.75Sep-96 164 3.0 906 269 3.00 0.05 0.88 0.93 3.26 3.40 35.6 4.6 1.70 10.3 3.9 1.30 --- 14.7 31.86 6.80 1509 3.11Oct-96 193 2.2 863 253 3.64 0.05 3.62 3.67 3.60 4.23 65.6 2.4 1.91 10.3 7.6 1.73 --- 12.3 29.78 6.88 1548 3.36Nov-96 183 2.3 868 202 3.70 0.06 2.94 2.96 3.11 3.42 51.8 3.8 1.65 9.2 6.7 2.05 --- 13.2 26.42 7.06 1476 3.32Dec-96 163 3.5 896 248 3.15 0.30 5.16 5.46 4.29 4.96 35.1 3.7 1.17 9.7 8.6 1.98 --- 10.9 23.68 7.05 1496 3.56Jan-97 166 3.7 898 219 2.96 0.06 4.63 4.68 --- 3.90 37.6 3.6 1.17 10.2 7.6 1.79 210 10.7 22.24 7.01 1547 3.49Feb-97 172 2.0 894 191 2.96 0.09 5.11 5.16 3.79 4.47 52.4 3.1 1.15 9.8 8.1 1.81 224 9.9 22.25 7.05 1468 3.58Mar-97 182 2.2 935 --- 3.47 --- --- 1.13 --- 3.98 38.4 2.6 1.54 9.4 4.6 1.94 --- 10.9 24.20 7.03 1518 3.39Apr-97 167 3.7 859 233 1.29 0.30 3.90 4.20 3.68 5.25 32.5 3.4 1.34 9.7 5.5 -0.05 189 11.2 25.16 7.04 1473 2.74May-97 162 3.6 954 266 2.55 --- --- 2.18 --- 3.92 38.9 2.9 1.81 8.8 4.7 0.75 156 12.6 28.50 6.92 1581 2.77Jun-97 158 2.5 982 306 1.74 0.07 2.68 2.72 1.41 1.55 26.9 1.0 0.64 7.9 4.0 1.04 162 4.4 29.88 6.96 1698 3.61Jul-97 162 1.3 1017 346 2.58 0.21 2.91 2.95 1.72 2.04 30.7 2.0 1.41 7.4 5.5 1.17 154 9.4 30.52 6.85 1830 3.09Aug-97 154 3.0 1043 339 2.57 0.07 2.79 2.83 2.56 3.68 18.9 2.4 1.27 7.4 5.4 1.30 151 9.4 31.77 6.83 1824 2.38Sep-97 187 3.0 956 319 2.38 0.05 0.84 0.89 0.70 0.96 25.6 1.4 1.10 6.8 3.3 1.28 134 7.1 31.49 6.93 1693 2.43Oct-97 173 2.6 886 290 3.04 0.03 0.90 0.92 3.70 3.40 26.4 2.0 1.24 6.7 4.0 1.80 170 8.7 29.42 6.87 1667 2.85Nov-97 188 2.6 797 205 2.88 0.05 1.00 1.05 1.72 2.32 31.8 3.2 1.40 7.2 3.9 1.48 175 11.2 26.03 7.17 1422 3.22Dec-97 168 2.6 872 220 3.96 0.32 6.00 6.32 3.48 3.60 28.6 4.0 2.06 7.7 10.3 1.90 192 15.4 22.48 7.02 1362 2.84Jan-98 172 1.0 866 209 3.46 0.20 2.70 2.90 2.80 3.60 42.8 2.0 1.76 9.0 6.4 1.70 187 11.0 22.03 7.02 1403 2.89Feb-98 --- --- --- 164 2.62 0.07 1.66 1.73 2.80 3.76 --- --- 1.28 --- 4.4 1.34 161 --- 21.83 7.04 1388 2.80Mar-98 --- --- --- 177 1.90 0.10 1.10 1.20 3.00 3.50 --- --- 0.93 9.9 3.1 0.97 145 --- 22.64 7.07 1326 2.48Apr-98 --- --- --- 182 3.70 0.10 2.60 2.70 2.00 2.70 --- --- 1.80 9.0 6.4 1.90 120 --- 23.34 7.01 1241 2.31May-98 --- --- --- 230 2.30 0.10 2.20 2.30 3.00 3.50 --- --- 1.70 9.0 4.6 0.60 122 --- 26.91 7.01 1299 2.49Jun-98 --- --- --- 210 7.70 0.20 0.90 1.10 1.60 --- --- --- 4.90 10.2 8.8 2.80 112 --- 28.75 7.02 1284 2.34Jul-98 163 --- --- 342 2.70 0.05 2.00 2.05 2.00 2.40 --- --- 1.50 9.1 4.8 1.20 143 --- 31.40 7.13 1583 2.57Aug-98 162 3.0 1070 330 3.80 0.05 1.10 1.15 --- 2.30 116.0 7.0 0.79 7.2 5.0 3.01 148 14.1 32.72 6.97 1740 2.71Sep-98 --- --- --- 318 4.18 0.05 2.65 2.70 1.53 3.25 --- --- 1.70 7.6 6.9 2.48 142 --- 32.63 6.83 1704 2.71Oct-98 172 3.0 748 207 4.20 0.20 1.78 1.98 1.20 1.00 21.0 2.0 2.23 8.0 6.2 1.98 152 13.2 30.56 6.63 1399 2.59Nov-98 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- 28.18 7.13 1294 2.40Dec-98 168 4.0 742 197 3.10 0.05 2.70 2.75 3.30 4.80 5.0 3.0 1.20 7.9 5.9 1.90 150 10.0 25.73 6.65 1324 2.57Jan-99 176 2.0 856 240 4.20 0.26 4.06 4.32 3.20 3.00 36.8 1.6 1.68 10.2 8.5 2.52 128 10.1 23.50 6.75 1361 3.16Feb-99 201 3.0 756 166 4.50 0.20 3.70 3.90 3.45 4.00 31.0 4.0 1.60 --- 8.4 2.90 126 13.3 23.88 7.21 1240 3.03Mar-99 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- 26.40 7.01 1498 3.66Apr-99 168 6.3 925 250 4.33 0.27 1.53 1.80 1.70 --- 28.3 3.3 2.37 10.1 6.1 1.97 196 15.8 25.90 6.89 1476 3.28May-99 163 12.4 931 257 3.70 0.23 2.94 3.17 2.38 --- 33.2 3.0 2.08 --- 6.9 1.62 171 14.0 25.78 7.00 1440 2.34Jun-99 165 2.8 1020 309 2.44 0.25 4.88 5.13 1.92 3.40 38.2 1.0 1.43 8.0 7.6 1.01 143 8.0 31.36 6.92 1701 3.40Jul-99 169 2.6 995 300 2.08 0.05 3.16 3.21 --- 3.00 32.4 1.0 1.08 7.9 5.3 1.00 143 6.5 32.50 6.89 1610 2.94Aug-99 177 1.6 998 288 2.48 0.05 2.52 2.57 --- 2.00 38.4 2.4 1.76 9.0 5.1 0.72 149 11.6 31.81 6.88 1649 2.50Sep-99 179 4.8 982 307 2.16 0.05 2.82 2.87 --- 4.00 20.8 1.0 1.56 7.8 5.0 0.60 147 8.6 31.03 6.84 1675 2.90Oct-99 171 4.4 917 269 2.96 0.07 2.64 2.71 --- 3.00 26.2 1.4 1.12 7.4 5.7 1.84 143 7.2 29.29 7.03 1601 3.25Nov-99 128 3.0 809 220 0.86 0.05 3.00 3.05 --- 2.00 32.2 2.2 0.10 7.5 3.9 0.74 139 3.8 27.38 6.82 1379 2.21Dec-99 182 4.4 848 209 2.68 0.20 4.04 4.24 --- 1.00 39.4 3.0 1.96 8.8 6.9 0.72 146 13.5 24.98 6.92 1349 2.43Jan-00 166 26.6 856 214 1.18 0.05 4.68 4.73 --- 3.60 32.6 1.0 0.30 8.8 5.9 0.86 165 2.9 24.12 6.78 1356 2.69Feb-00 170 3.0 847 197 2.60 0.05 3.25 3.30 --- 3.05 44.5 1.0 1.30 9.7 5.9 1.30 200 7.4 24.69 6.90 1420 3.45Mar-00 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- 25.26 6.70 1499 4.02Apr-00 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- 27.14 6.84 1537 4.42May-00 172 2.7 997 319 1.90 0.25 1.52 1.83 --- 2.30 32.5 1.8 1.30 8.4 3.7 0.30 183 8.6 29.45 6.83 1594 3.33Jun-00 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- 31.74 7.04 1716 2.78Jul-00 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- 32.18 6.94 1792 3.34Aug-00 194 3.0 1150 362 1.40 0.10 1.70 1.80 --- 1.50 20.0 2.0 0.88 6.9 3.2 0.52 189 7.0 31.69 6.88 1795 2.91Sep-00 177 4.0 1040 232 2.60 0.10 2.20 2.30 1.90 2.20 35.0 1.0 1.30 7.9 4.9 1.30 216 7.4 32.90 6.94 1672 3.41Oct-00 190 2.0 970 280 2.20 0.20 2.40 2.60 --- 1.80 10.0 1.0 1.50 7.1 4.8 0.70 234 8.4 31.57 7.27 1794 1.86Nov-00 192 3.0 943 260 3.45 0.18 4.85 5.05 --- 3.05 49.0 2.0 1.88 7.6 8.5 1.45 183 11.6 25.00 7.07 1967 1.98Dec-00 188 2.5 899 250 1.80 0.10 4.30 4.40 --- 1.15 30.0 1.5 0.53 7.6 6.2 1.27 199 4.7 23.31 7.23 1638 2.45Jan-01 193 8.5 875 239 4.45 0.80 3.95 4.75 0.95 --- 46.0 1.0 2.80 8.5 9.2 1.65 181 14.3 23.41 6.74 1704 4.45Feb-01 205 2.0 910 238 8.70 1.00 3.60 4.60 1.15 --- 27.5 1.5 6.45 8.1 13.3 2.25 192 31.7 24.40 6.90 1713 3.88Mar-01 174 2.5 879 226 3.10 0.05 3.35 3.35 2.15 --- 36.5 --- 1.20 9.0 6.5 1.90 198 --- 24.23 6.88 1701 3.16Apr-01 --- 1.5 1285 225 2.25 0.20 3.45 3.65 --- --- 5.0 --- 1.55 8.6 5.9 0.70 193 --- --- 6.92 1684 3.67May-01 --- 6.0 1090 337 2.00 0.20 3.40 3.60 --- --- 21.0 1.0 1.25 7.9 5.6 0.75 190 7.2 --- 6.86 1923 2.23Jun-01 --- 1.5 1230 395 1.35 0.10 4.30 4.40 --- --- 40.5 1.0 0.84 4.8 5.8 0.52 209 5.3 30.40 7.04 2080 3.60Jul-01 --- 2.5 1140 391 2.25 0.40 3.05 3.45 --- --- 29.5 1.5 1.01 6.2 5.7 1.24 205 6.9 --- 6.90 3000 5.40Aug-01 208 1.5 1255 400 0.90 0.30 7.05 7.35 --- --- 19.0 1.0 0.59 5.5 8.3 0.32 195 4.2 --- --- --- ---Sep-01 195 1.5 1040 363 1.20 0.15 3.30 3.45 --- --- 21.5 1.0 0.49 6.8 4.7 0.72 162 3.7 31.59 7.00 1812 5.13Oct-01 --- 0.8 1145 393 0.75 0.10 3.70 3.80 --- --- 24.0 1.0 0.57 5.0 4.5 0.18 190 4.1 32.02 7.20 1437 5.89Nov-01 196 2.0 776 188 3.10 0.15 2.30 2.45 --- --- 29.5 1.0 0.96 7.0 5.6 2.14 149 5.9 25.65 7.09 1511 3.76Dec-01 --- 1.3 805 194 3.10 0.10 4.00 4.10 --- --- 23.5 1.0 1.65 6.7 7.2 1.45 163 9.0 --- --- --- ---Jan-02 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- 22.58 6.99 1736 3.18Feb-02 --- 2.0 735 287 3.85 0.40 3.50 3.90 --- --- 34.0 1.5 2.15 7.8 7.8 1.70 194 12.1 22.89 7.13 1664 3.28Mar-02 181 2.0 947 256 1.95 0.13 3.60 3.73 --- --- 44.0 1.0 3.44 8.1 5.7 0.48 221 17.2 25.72 7.04 1577 3.08Apr-02 162 2.0 1010 312 2.50 0.05 9.20 9.25 --- --- 23.0 2.0 0.96 7.1 11.8 1.58 192 7.4 28.12 6.94 1473 3.54

18


EXHIBIT 8Summary of Inlet Water Quality to the Tres Rios Demonstration Wetlands, AZAugust 1995 - December 2002

DateAlkalinity(mg/L)

TSS(mg/L)

TDS(mg/L)

Cl(mg/L)

TKN(mg/L)

NO2-N(mg/L)

NO3-N(mg/L)

NOx-N(mg/L)

PO4-P(mg/L)

Diss. P(mg/L)

COD(mg/L)

cBOD(mg/L)

NH3-N(mg/l)

TOC(mg/L)

TN(mg/L)

Org. N(mg/L)

Sulfate(mg/L)

UOD(mg/L)

Temp(C)

pH(units)

Cond.(µmhos/cm)

DO(mg/L)

May-02 156 3.0 1090 337 4.80 0.05 4.10 4.15 --- --- 34.0 1.0 1.34 7.7 9.0 3.10 178 7.6 30.18 6.97 1776 3.25Jun-02 --- 2.0 1070 394 0.90 0.05 2.20 2.25 --- --- 42.0 1.0 0.10 7.3 3.2 0.80 170 2.0 28.95 6.56 --- 3.96Jul-02 --- 2.0 1080 358 1.20 0.05 2.70 2.75 --- --- 15.0 1.0 0.32 6.6 4.0 0.88 191 3.0 29.81 6.57 --- 3.32Aug-02 166 20.0 1030 356 2.10 0.05 3.10 3.15 --- --- 14.0 1.0 1.51 6.6 5.3 0.90 175 8.4 30.51 6.00 1513 3.61Sep-02 --- 2.0 1010 295 2.60 0.05 4.50 4.55 --- --- 5.0 1.0 --- 8.2 7.2 --- 221 --- 29.57 6.94 987 3.23Oct-02 --- 2.0 952 263 1.70 0.10 7.90 8.00 --- --- 27.0 1.0 0.82 68.7 9.7 0.88 216 5.2 27.93 6.74 746 3.60Nov-02 --- 3.0 900 233 2.60 0.10 4.20 4.30 --- --- 27.0 2.0 1.50 6.7 6.9 1.10 200 9.9 25.43 6.88 --- 2.90Dec-02 --- 7.0 920 219 2.40 0.10 3.40 3.50 --- --- 20.0 2.0 1.50 7.4 5.9 0.90 210 9.9 22.99 6.84 1542 2.71

Average 175 3.7 933 266 2.94 0.17 3.16 3.26 2.96 3.24 34.7 2.3 1.61 8.9 6.2 1.36 173 10.7 27.5 6.93 1556 3.29Median 173 2.8 900 251 2.92 0.10 2.98 3.07 3.01 3.46 32.5 2.0 1.50 8.0 5.9 1.30 175 10.4 27.7 6.94 1524 3.22

Maximum 208 27 1285 400 8.70 1.12 9.20 9.25 10.6 5.25 134 7.0 6.45 69 13.3 3.10 234 31.7 32.9 7.27 3000 7.10Minimum 128 0.8 735 164 0.75 0.03 0.84 0.89 0.70 0.96 5.0 1.0 0.10 4.8 3.1 -0.05 112 2.0 21.8 6.00 746 1.86StdDev 14 3.8 119 60 1.29 0.19 1.51 1.55 1.56 1.08 19.2 1.3 0.93 6.9 1.9 0.67 29 4.9 3.45 0.17 259 0.90Count 63 75 75 81 82 79 79 82 44 56 75 73 81 79 82 81 64 72 84 87 82 87

19


20

Sample Collection and Handling Biomonitoring samples were typically collected during the first week of each month. Four-liter samples were obtained at the combined inlets to each site (Hayfield and Cobble). These samples are representative of chlorinated effluent from Plant 3A. At roughly the same time, 4-liter samples were also collected from the outlet discharge structures of the four wetlands (H1, H2, C1, and C2). This report summarizes biomonitoring data at the Tres Rios Demonstration Constructed Wetland Project site for the period: January 1996 through August 2002.

WET Test Methods and Endpoints Samples were analyzed either in the City of Phoenix Water Services Laboratory, by Aquatic Consulting and Testing, Inc. located in Tempe, Arizona, or by ENSR in Fort Collins, Colorado. Test methods followed EPA (1994) for the static renewal, definitive, 7-day Ceriodaphnia dubia survival and reproduction test. End points identified in these tests are defined as follows:

• LC50 or EC50 – effluent concentration as a percent of the undiluted sample that adversely affects 50% of the test organisms

• No Observable Effect Concentration (NOEC) – the highest concentration as a percent of the undiluted sample which caused no statistically significant adverse effect on survival or reproduction

• Lowest Observable Effect Concentration (LOEC) – the lowest concentration as a percent of the undiluted sample that caused a statistically significant adverse effect on survival or growth

• Inhibition Concentration (IC25 or IC50) - an estimate of the concentration as a percent of the undiluted sample that causes 25 or 50 percent reduction in reproduction compared to the laboratory control


21

Results

Overview Detailed results for Ceriodaphnia dubia reproduction and mortality for all of the tests conducted with the Tres Rios Demonstration Wetland influent and effluent are provided in Appendices A-1 and A-2. This section provides a general overview of those results prior to a more detailed analysis that follows.

Exhibits 9 and 10 present summaries of the test endpoints (measured as percent of sample concentration) for the Hayfield and Cobble Site wetlands, respectively. Exhibit 11 presents a summary (averages) of these endpoint data for the wetlands. Very little toxicity has been detected in the wetland influent or at the outlet of either wetland site. Of the 40 tests recorded for the influent (91st Avenue WWTP effluent), only 7 have shown any indication of acute toxicity and 5 have shown acute and chronic toxicity. For the Hayfield Wetland Cells, H1 outflow has shown no evidence of acute or chronic toxicity in any of the 12 tests reported while the H2 effluent has been acutely and chronically toxic in one out of 9 tests. For the Cobble Site Wetland Cells, C1 effluent has shown no acute or chronic toxicity to the water flea in 10 tests over the 7 year period of record, while C2 effluent was acutely and chronically toxic in 1 of 12 tests conducted. Details of these test results must be examined to develop an understanding of their patterns and significance.

The averages in Exhibit 11 seem to indicate that the wetland effluent is slightly more toxic than the influent (WWTP effluent). These differences are an artifact of averaging a single high toxicity value for each wetland with a number of no-effect values. Additional data evaluation is provided below to better illustrate the subtleties of these results.

Exhibit 12 provides a summary of all of the biomonitoring data for both the Hayfield and Cobble sites, averaged by effluent dilution. Water flea reproduction (average neonates per female) and percent mortality data are provided and results from the two wetland cells at each site are lumped together. Controls typically averaged between 18.4 and 28.3 neonates per female. The influent data averaged between 19.1 and 30.2 neonates per female, and were typically higher than the controls, especially at the low dilutions. This is an indication that the relatively high-quality WWTP effluent was actually enhancing nutrition of the water fleas. The test reproduction results were more likely to be less than the control reproduction at high effluent percentages, indicating a subtle stress with the higher strength effluent. This “subsidy-stress” relationship between effluent concentration and Ceriodaphnia reproduction is a relatively common occurrence in biomonitoring tests and is referred to as hormesis (Chapman, 1998; 2000). Hormesis has been described over a wide variety of taxa and biological endpoints, and can be considered to be a general principle of ecological systems (Odum et al. 1979; Knight 1983).


EXHIBIT 9Summary of End Points for Survival and Reproduction Tests of Ceriodaphnia dubia in the Hayfield Cells at the Tres Rios Demonstration Wetlands, AZJanuary 1996 - August 2002

SITE_NAME TIMEPERIOD LAB LC50 LOECr LOECs NOECr NOECs IC25 IC50 TU LC50 LOECr LOECs NOECr NOECs IC25 IC50 TU LC50 LOECr LOECs NOECr NOECs IC25 IC50 TUTres Rios, AZ Feb-96 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Apr-96 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Jun-96 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Jun-96 PHX 88 100 100 75 75 98 >100 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Aug-96 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Oct-96 ACT 69.8 75 75 50 50 56.7 64.8 1.8 16.1 25 25 <25 <25 6.9 13.7 14.5Tres Rios, AZ Nov-96 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Jan-97 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Mar-97 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ May-97 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Jul-97 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Sep-97 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Sep-97 PHX 94 100 100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Oct-97 PHX 33 25 100 <25 90 63 >100 1.6 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Oct-97 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Apr-98 PHX >100 >100 90 100 75 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Dec-98 ENSR >100 >100 >100 100 100 >100 < 1.0Tres Rios, AZ Dec-00 PHX >100 >100 100 >100 < 1.0 >100 >100 100 >100 >100 < 1.0Tres Rios, AZ Sep-01 PHX >100 >100 >100 100 100 >100 >100 <1.0 >100 >100 >100 100 100 >100 >100 <1.0Tres Rios, AZ Mar-02 PHX >100 >100 >100 <1.0 >100 >100 >100 100 100 >100 >100 <1.0Tres Rios, AZ Aug-02 PHX >100 >100 >100 100 100 >100 >100 <1.0 >100 >100 >100 100 100 >100 >100 <1.0

H1 Eff H2 EffHS Inlet

22


EXHIBIT 10Summary of End Points for Survival and Reproduction Tests of Ceriodaphnia dubia in the Cobble Cells at the Tres Rios Demonstration Wetlands, AZJanuary 1996 - May 2002

SITE_NAME TIMEPERIOD LAB LC50 LOECr LOECs NOECr NOECs IC25 IC50 TU LC50 LOECr LOECs NOECr NOECs IC25 IC50 TU LC50 LOECr LOECs NOECr NOECs IC25 IC50 TUTres Rios, AZ Jan-96 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 100Tres Rios, AZ Mar-96 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 <1.0Tres Rios, AZ May-96 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Jul-96 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Sep-96 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 <1.0Tres Rios, AZ Oct-96 ACT 32.4 25 50 <25 25 15.9 30.3 6.3Tres Rios, AZ Dec-96 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Feb-97 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 <1.0Tres Rios, AZ Apr-97 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Jun-97 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ Aug-97 ACT >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 <1.0Tres Rios, AZ Oct-97 PHX >100 >100 >100 100 100 >100 >100 <1.0Tres Rios, AZ Jan-98 PHX >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 < 1.0Tres Rios, AZ May-99 PHX >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 <1.0Tres Rios, AZ Sep-99 PHX >100 >100 >100 100 100 >100 >100 < 1.0 >100 >100 >100 100 100 >100 >100 <1.0Tres Rios, AZ Dec-99 PHX 82 100 100 50 50 64 80 1.6 >100 >100 >100 50 100 >100 >100 < 1.0Tres Rios, AZ Mar-00 PHX 61.2 71.8 >100 1.4 >100 >100 <1.0Tres Rios, AZ Sep-00 PHX 95.3 >100 < 1.0 >100 >100 < 1.0Tres Rios, AZ Mar-01 PHX >100 >100 >100 100 100 >100 >100 <1.0 >100 >100 >100 100 100 >100 >100 <1.0Tres Rios, AZ Nov-01 PHX >100 >100 >100 100 100 >100 >100 <1.0 >100 >100 >100 100 100 >100 >100 <1.0Tres Rios, AZ May-02 PHX >100 >100 >100 100 100 >100 >100 <1.0 >100 >100 >100 100 100 >100 >100 <1.0

CS Inlet C1 Eff C2 Eff

23


EXHIBIT 11Summary of Average End Points for Ceriodaphnia dubia 7-day Survival and Reproduction Test in the Tres RiosDemonstration Wetlands, AZ - January 1996 - August 2002

End Points Influent Effluent Influent EffluentSurvivalLC50 96.6 96.9 94.2 96.0LOEC 100.0 97.4 98.1 96.3NOEC 96.9 96.1 93.3 96.3

ReproductionLOEC 100.0 96.3 94.7 96.4NOEC 96.9 93.8 91.6 96.4IC25 96.4 96.0 95.9 95.6IC50 98.8 96.3 98.0 95.7TUC 1.1 1.3 1.1 1.6

Note(s):

Results expressed as percent of undiluted sample

Lowest Observed Effect Concentration (LOEC) - the lowest concentration of effluent to which organisms were exposed which caused a statistically significant adverse effect.No Observed Effect Concentration (NOEC) - the highest concentration of effluent to which organisms were exposed which caused a statistically significant adverse effect.

TUC - (Toxic Units) - the inverse of the IC25. A TUC of greater than 1.0 is considered a toxic response.

Cobble Hayfield

Ceriodaphnia dubia chronic static renewal 7-Day survival and reproduction test

LC50 - represents a point estimate of the effluent concentration that would adversely affect 50 percent of the test organisms.

IC - (Inhibition concentration) - point estimate of effluent concentration that causes a given percent reduction in the reproduction or growth of test organism.

24


EXHIBIT 12Average Survival and Reproduction of Ceriodaphnia dubia by Percent Dilution in the Tres Rios Demonstration Wetlands, AZJanuary 1996 - August 2002

System Dilution Effluent Inf/Eff% % Control Test Control Test % Change

Cobble 100.00 0 22.8 --- 21.5 --- ---93.75 6.25 28.3 30.2 27.3 26.6 -12.087.50 12.50 28.3 29.7 26.9 28.7 -3.475.00 25.00 22.8 24.6 21.5 23.3 -5.350.00 50.00 22.8 24.4 21.5 23.2 -4.925.00 75.00 21.8 22.1 20.6 22.5 1.810.00 90.00 18.8 19.3 18.4 19.9 3.00.00 100.00 22.8 21.9 21.5 23.0 5.0

Hayfield 100.00 0 21.4 --- 20.4 --- ---93.75 6.25 24.0 28.2 --- --- ---87.50 12.50 23.9 21.1 22.4 27.9 32.275.00 25.00 21.4 21.4 20.4 22.4 4.550.00 50.00 21.4 22.5 20.4 23.4 4.325.00 75.00 21.2 21.3 20.4 23.8 11.810.00 90.00 20.4 19.1 19.8 22.3 17.20.00 100.00 21.4 20.0 20.4 24.0 20.3

System Dilution Effluent Inf/Eff% % Control Test Control Test % Change

Cobble 100.00 0 1.1 --- 2.3 --- ---93.75 6.25 0.0 0.0 0.0 3.3 ---87.50 12.5 1.3 1.3 3.8 0.0 -100.075.00 25 1.1 0.5 2.3 1.4 ---50.00 50 1.1 7.4 2.3 6.4 -13.725.00 75 1.3 15.0 2.6 6.3 -57.910.00 90 0.9 12.7 1.4 9.3 -27.00.00 100 1.1 20.0 2.3 8.2 -59.1

Hayfield 100.00 0 0.0 --- 0.5 --- ---93.75 6.25 0.0 0.0 --- --- ---87.50 12.5 0.0 1.7 0.0 0.0 ---75.00 25 0.0 4.0 0.5 4.8 19.050.00 50 0.0 6.0 0.5 5.2 -12.725.00 75 0.0 12.1 0.5 4.8 -60.710.00 90 0.0 16.4 0.6 6.9 -58.10.00 100 0.0 20.5 0.5 6.2 -69.8

Reproduction (# young/female)

Mortality (%)Influent

Influent Effluent

Effluent

25


26

A slight hormetic effect is also apparent for the wetland outflow water at both the Hayfield and Cobble sites (Exhibit 12). This effect is extended along the dilution series for the Hayfield site with water flea reproduction in every dilution higher than the control.

Exhibit 12 presents the calculated percent change (increase) in the number of neonates per female for the wetland influent and effluent. This percent change typically increases at increasing effluent dilutions, indicating that some of the stimulatory effects of the high dilution influent samples are lost with passage through the wetland, while the toxic effects of the influent at the low dilution end of the spectrum were reduced by passage through the wetlands.

Exhibit 12 also illustrates the overall results for Ceriodaphnia mortality for both wetland sites with the two cells lumped at each site. Acute effects of the WWTP effluent (wetland influent) are clear in this table. Mortality increases in a generally consistent pattern for the wetland influent versus control at each of the two wetlands. Average mortality at the wetland outflows was reduced compared to the influent only at the lower dilutions.

These general data summaries indicate that the Tres Rios Demonstration wetlands alter the biologically stimulatory and toxic effects of the WWTP effluent in subtle ways. Diluted WWTP effluent is likely to be beneficial to some invertebrate populations in the wetlands. Higher concentrations are periodically toxic, both acutely and chronically. With passage through the wetlands, the nutritional aspects of relatively low influent pollutant concentrations are diminished as are the toxic effects. It can be cautiously concluded that overall, the wetland outflow provides a safe and slightly stimulatory source of water for invertebrates and other organisms with similar requirements. These subtle results are examined in more detail in the subsequent data analyses presented below.

Time Series Data Observed toxicity at the Tres Rios 91st Avenue WWTP and in the wetland effluent was sporadic. Exhibits 13 through 17 illustrate the time series patterns for Ceriodaphnia reproduction and mortality data for the Demonstration Wetlands.

Toxicity tests at the Cobble and Hayfield wetlands were generally run on different days. On each occasion the inlet water was tested as well as the outlet water from the two parallel wetland cells at each site. To best examine the chronic toxicity time series data for the wetland inlet water (91st Avenue WWTP effluent), the two inlet data sets have been combined in Exhibit 13. Chronic (7-day) reproduction and mortality data for Ceriodaphnia are summarized by reporting the difference between the test sample result and the control sample result for the entire range of test sample dilutions (presented as % effluent). For the reproduction graph in Exhibit 13 a positive number indicates an enhancement in neonate production. A negative response, especially one that increases with % effluent, indicates a chronically toxic response. Water flea reproduction was generally enhanced by the WWTP effluent. However, a number of apparently toxic events can be observed in this data set over the period-of-record. The most significant events occurred in February and October 1996, October 1997, December 1999, and March 2000. These observations are reinforced by the lower graph in Exhibit 13 for percent


EXHIBIT 13Reproduction (# young/female) and Percent Mortality differences for Ceridaphnia dubia between Test and Control samples as a function of dilution in the 91st Avenue WWTP Final Effluent to the Tres Rios Demonstration Wetlands, AZ

-30

-20

-10

0

10

20

30Ja

n-96

Apr

-96

Jul-9

6

Oct

-96

Jan-

97

Apr

-97

Jul-9

7

Oct

-97

Jan-

98

Apr

-98

Jul-9

8

Oct

-98

Jan-

99

Apr

-99

Jul-9

9

Oct

-99

Jan-

00

Apr

-00

Jul-0

0

Oct

-00

Jan-

01

Apr

-01

Jul-0

1

Oct

-01

Jan-

02

Apr

-02

Jul-0

2

Rep

rodu

ctio

n (#

/fem

ale)

diff

eren

ce(T

est -

Con

trol

)

6.25 % Eff 12.5 % Eff 25 % Eff 50 % Eff 75 % Eff 90 % Eff 100 % Eff

% Eff Average6.25 2.4512.5 -0.3825 0.9250 1.3675 0.2890 -0.60100 -1.06

Reproduction

-20

0

20

40

60

80

100

120

Jan-

96

Apr

-96

Jul-9

6

Oct

-96

Jan-

97

Apr

-97

Jul-9

7

Oct

-97

Jan-

98

Apr

-98

Jul-9

8

Oct

-98

Jan-

99

Apr

-99

Jul-9

9

Oct

-99

Jan-

00

Apr

-00

Jul-0

0

Oct

-00

Jan-

01

Apr

-01

Jul-0

1

Oct

-01

Jan-

02

Apr

-02

Jul-0

2

Perc

ent M

orta

lity

diffe

renc

e(T

est -

Con

trol

)


Percent Mortality % Eff Average6.25 0.0012.5 0.7125 1.1150 5.5675 12.1990 13.48100 17.50

27


28

mortality. A positive response on this graph indicates chronic toxicity to the water fleas. Increased mortality compared to the controls was greatest on the same dates as listed above for reproduction.

Exhibits 14 and 15 illustrate Ceriodaphnia reproduction and mortality time series data for the Cobble Site wetlands. Time series plots for the C1 outflow indicate that the wetland outflow was typically stimulatory for Ceriodaphnia reproduction; however three events of reduced reproduction compared to the controls were observed, the first in January 1996, the second in January 2000, and the third in May 2002. C2 outflow data also indicate a fairly consistent stimulatory effect on reproduction. The only clear chronic effect in the C2 outflow was observed in October 1996 with a smaller event in May 1999. C2 effluent was generally healthier for water fleas than was effluent from C1. Exhibit 15 illustrates the time series of observed mortality of the various tested effluent dilutions for the Cobble Site wetland cell inlet and outlets. Consistent increased mortality compared to the controls for the WWTP effluent was observed in October 1997, January 1998, December 1999, March and September 2000, and November 2001. The mortality observed at the Cobble Site wetland inlet was substantially or completely reduced at the wetland outlets throughout most of the study period except in C2 in October 1996.

Data plotted in Exhibits 16 and 17 illustrate reproduction and mortality data for the Hayfield Site wetlands. Data from wetland station H1 outflow were consistently stimulatory to the water flea reproduction. H2 outflow was severely toxic in October 1996 at the same time the inflow was toxic. Mortality results for H2 in October 1996 confirmed the low reproduction results for that set of samples (Exhibit 17).

Effluent Concentration Effects WET dilution series data for the Cobble Site wetland inflow and each of the two wetland outflows are summarized in Exhibit 18. These data points include plus/minus one standard error to allow a visual assessment of significance of the slight differences observed between the wetland inflow and outflow samples. The reproduction data illustrate a typical hormesis curve of apparent stimulation at a low effluent concentration with less reproduction at the higher effluent concentrations. The stimulation of reproduction at an effluent concentration in the range of 10 percent appears to be significant compared to the control data (0 percent effluent), but the higher effluent concentration reproduction results are not significantly different from the control results.

The trend of increasing water flea mortality with increasing effluent concentration is clear for the Cobble Site influent and for the C2 wetland outflow but the high variability in these results masks significant differences. It is also clear from this graph that the C1 outflow samples did not create measurable mortality compared to the control (0 percent effluent).


EXHIBIT 14Reproduction (# young/female) differences for Ceridaphnia dubia between Test and Control samples as a function of dilution in thCobble Site Tres Rios Demonstration Wetlands, AZ

-30

-20

-10

0

10

20

30

Jan-9

6

Apr-96

Jul-9

6

Oct-96

Jan-9

7

Apr-97

Jul-9

7

Oct-97

Jan-9

8

Apr-98

Jul-9

8

Oct-98

Jan-9

9

Apr-99

Jul-9

9

Oct-99

Jan-0

0

Apr-00

Jul-0

0

Oct-00

Jan-0

1

Apr-01

Jul-0

1

Oct-01

Jan-0

2

Apr-02

Rep

rodu

ctio

n (#

/fem

ale)

diff

eren

ce(T

est -

Con

trol

)


C1 Effluent % Eff Average6.25 -1.2012.5 -0.8325 -0.3050 0.2775 0.9390 1.73100 0.74

-30

-20

-10

0

10

20

30

Jan-9

6

Apr-96

Jul-9

6

Oct-96

Jan-9

7

Apr-97

Jul-9

7

Oct-97

Jan-9

8

Apr-98

Jul-9

8

Oct-98

Jan-9

9

Apr-99

Jul-9

9

Oct-99

Jan-0

0

Apr-00

Jul-0

0

Oct-00

Jan-0

1

Apr-01

Jul-0

1

Oct-01

Jan-0

2

Apr-02

Rep

rodu

ctio

n (#

/fem

ale)

diff

eren

ce(T

est -

Con

trol

)


C2 Effluent % Eff Average6.25 -0.5512.5 3.4225 3.5050 3.0075 2.8890 1.19100 2.10

29


EXHIBIT 15Percent Mortality differences for Ceridaphnia dubia between Test and Control samples as a function of dilution in theCobble Site Tres Rios Demonstration Wetlands, AZ

-20

0

20

40

60

80

100

120

Jan-9

6

Apr-96

Jul-9

6

Oct-96

Jan-9

7

Apr-97

Jul-9

7

Oct-97

Jan-9

8

Apr-98

Jul-9

8

Oct-98

Jan-9

9

Apr-99

Jul-9

9

Oct-99

Jan-0

0

Apr-00

Jul-0

0

Oct-00

Jan-0

1

Apr-01

Jul-0

1

Oct-01

Jan-0

2

Apr-02

Perc

ent M

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diffe

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e(T

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)


C1 Effluent % Eff Average6.25 0.0012.5 0.0025 1.0050 2.0075 2.2290 2.86100 1.00

-20

0

20

40

60

80

100

120

Jan-9

6

Apr-96

Jul-9

6

Oct-96

Jan-9

7

Apr-97

Jul-9

7

Oct-97

Jan-9

8

Apr-98

Jul-9

8

Oct-98

Jan-9

9

Apr-99

Jul-9

9

Oct-99

Jan-0

0

Apr-00

Jul-0

0

Oct-00

Jan-0

1

Apr-01

Jul-0

1

Oct-01

Jan-0

2

Apr-02

Perc

ent M

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diffe

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e(T

est -

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)


C2 Effluent % Eff Average6.25 5.0012.5 -6.0025 -2.5050 5.8375 5.0090 12.86100 10.00

30


EXHIBIT 16Reproduction (# young/female) differences for Ceridaphnia dubia between Test and Control samples as a function of dilution in theHayfield Site Tres Rios Demonstration Wetlands, AZ

-20

-15

-10

-5

0

5

10

15

Jan-9

6

Apr-96

Jul-9

6

Oct-96

Jan-9

7

Apr-97

Jul-9

7

Oct-97

Jan-9

8

Apr-98

Jul-9

8

Oct-98

Jan-9

9

Apr-99

Jul-9

9

Oct-99

Jan-0

0

Apr-00

Jul-0

0

Oct-00

Jan-0

1

Apr-01

Jul-0

1

Oct-01

Jan-0

2

Apr-02

Jul-0

2

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rodu

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diff

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ce(T

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trol

)


H1 Effluent

% Eff Average6.25 ---12.5 5.3325 2.9650 3.9975 3.7690 3.82100 4.41

-20

-15

-10

-5

0

5

10

15

Jan-9

6

Apr-96

Jul-9

6

Oct-96

Jan-9

7

Apr-97

Jul-9

7

Oct-97

Jan-9

8

Apr-98

Jul-9

8

Oct-98

Jan-9

9

Apr-99

Jul-9

9

Oct-99

Jan-0

0

Apr-00

Jul-0

0

Oct-00

Jan-0

1

Apr-01

Jul-0

1

Oct-01

Jan-0

2

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Jul-0

2

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rodu

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/fem

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diff

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ce(T

est -

Con

trol

)


H2 Effluent

% Eff Average6.25 ---12.5 5.7025 -0.1350 0.8875 1.4790 -0.70100 1.04

31


EXHIBIT 17Percent Mortality differences for Ceridaphnia dubia between Test and Control samples as a function of dilution in theHayfield Site Tres Rios Demonstration Wetlands, AZ

0

20

40

60

80

100

120

Jan-9

6

Apr-96

Jul-9

6

Oct-96

Jan-9

7

Apr-97

Jul-9

7

Oct-97

Jan-9

8

Apr-98

Jul-9

8

Oct-98

Jan-9

9

Apr-99

Jul-9

9

Oct-99

Jan-0

0

Apr-00

Jul-0

0

Oct-00

Jan-0

1

Apr-01

Jul-0

1

Oct-01

Jan-0

2

Apr-02

Jul-0

2

Perc

ent M

orta

lity

diffe

renc

e(T

est -

Con

trol

)


H1 Effluent % Eff Average6.25 ---12.5 0.0025 1.8250 0.0075 0.0090 1.25100 1.82

0

20

40

60

80

100

120

Jan-9

6

Apr-96

Jul-9

6

Oct-96

Jan-9

7

Apr-97

Jul-9

7

Oct-97

Jan-9

8

Apr-98

Jul-9

8

Oct-98

Jan-9

9

Apr-99

Jul-9

9

Oct-99

Jan-0

0

Apr-00

Jul-0

0

Oct-00

Jan-0

1

Apr-01

Jul-0

1

Oct-01

Jan-0

2

Apr-02

Jul-0

2

Perc

ent M

orta

lity

diffe

renc

e(T

est -

Con

trol

)


H2 Effluent % Eff Average6.25 ---12.5 0.0025 10.0050 14.2975 12.8690 15.00100 13.57

32


EXHIBIT 18Cobble Site Ceriodaphnia dubia Survival and Reproduction Biomonitoring Summary - Average (± SE) 1996-2002

10

15

20

25

30

35

0 10 20 30 40 50 60 70 80 90 100% Effluent

Rep

rodu

ctio

n (#

you

ng/fe

mal

e)

CS InletC1 EffC2 EFF

Reproduction

0

5

10

15

20

25

30

35

0 10 20 30 40 50 60 70 80 90 100% Effluent

Mor

talit

y (%

)

CS InletC1 EffC2 EFFMortality

33


34

Exhibit 19 illustrates the same data analysis for the Hayfield Site wetlands. The hormesis effect on Ceriodaphnia reproduction is also evident for the Hayfield inflow and outflows and the H1 outflow had consistently higher reproduction than the control regardless of sample dilution. Mortality results for the inlet and the H2 outlet also illustrate a relatively consistent pattern with respect to sample concentration, except for data from H1 where there was no observable effect.

Toxic Events Detailed data from a few of the acutely and chronically toxic events are described in this section to provide insight into the nature of these responses.

Measured toxicity was greater in the wetland outflow compared to the inflow during one event in October 1996 (Exhibit 9). The Hayfield inflow sample had a measured LC50 of 70 percent effluent, an NOEC of 50 percent, and 1.8 toxic units. The outflows from both H2 and C2 were more toxic than the inflows, with LC50s of 16 and 32 percent, NOECs of <25 percent, and 14.5 and 6.3 toxic units, respectively. Examination of detailed test results indicates that mortality was occurring within the first two days of the test for each of three sampling locations. There was nothing detected in the routine test water chemistry that provides an explanation for the increasing toxicity through the wetland. However, the mosquito adulticide malathion had been sprayed at both wetland sites and adjacent to the WWTP effluent channel on September 25 and October 4, 1996 and was probably the causative agent for this observed toxicity. It appears that the most likely explanation for the observed higher outflow than inflow toxicity is the different timing and location of sampling. Wetland inflow samples (collected at the Hayfield wetland splitter inlet box) may have received less of the malathion spray than the wetland outflow samples.

Exhibit 20 illustrates data for tests at the Cobble Site C2 wetland in October 1997. The controls failed for the influent sample test due to low reproduction (there was no mortality in the control populations). A slight hormesis effect on reproduction was observed when the number of neonates per original females was plotted. This reproduction occurred prior to 100 percent mortality of the female water fleas after the 4th day of the test. This mortality only occurred at the 50 percent effluent and above concentrations, confirming that it was sample toxicity and not a laboratory anomaly. C2 outflow samples collected during this same period exhibited a strong stimulatory response with increasing sample concentration. Neonate production was over 3 times as high in the 100 percent sample concentration as it was in the controls. One possible explanation of this difference in results is that the toxic agent in the wetland influent sample was not present until the renewal of the test water on Day 5. The wetland outflow test being conducted at the same time may not have been exposed to this hypothetical toxicant because the moving front sampled at the wetland inlet may not have reached the wetland outlet at the time of sampling due to the lag time for the water mass to move between the inflow and outflow wetland stations (measured hydraulic residence time of about 2 to 3 days based on Kadlec, 2001). A second possible explanation is that the wetland cell C2 was very effective at removing toxicity in the wetland influent during this testing period.


EXHIBIT 19Hayfield Site Ceriodaphnia dubia Survival and Reproduction Biomonitoring Summary - Average (± SE) 1996-2002

15

17

19

21

23

25

27

29

31

0 10 20 30 40 50 60 70 80 90 100% Effluent

Rep

rodu

ctio

n (#

you

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mal

e)

HS InletH1 EffH2 EFF

Reproduction

0

5

10

15

20

25

30

35

0 10 20 30 40 50 60 70 80 90 100% Effluent

Mor

talit

y (%

)

HS InletH1 EffH2 EFFMortality

35


EXHIBIT 20Tres Rios Cobble Site Ceriodaphnia dubia 7-day Survival and Reproduction Chronic Toxicity Test (± SE) - October 97

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0 25 50 75 90 100

% Effluent

Rep

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n (#

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e)

CS Inlet (PHX)C2 Eff (PHX)

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70

80

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100

0 25 50 75 90 100

% Effluent

Perc

ent M

orta

lity

CS Inlet (PHX)C2 Eff (PHX)

CS Inlet - control failure < 3 broods within 7 days

36


37

Exhibits 21 and 22 illustrate biomonitoring data for the Hayfield Site in September and October 1997. Plus/minus one standard error bars for Ceriodaphnia reproduction are provided on these graphs to assist with interpretation of statistical significance. Detailed data are provided in Appendix A-3.

On both of these dates two different biomonitoring labs conducted identical tests. In general, the differences between the labs were greater than the differences observed between sample concentrations. In September 1997 the City of Phoenix Water Services Laboratory detected both chronic and acute toxicity in the wetland inlet samples. Reproduction results did not display a predictable curve of subsidy and stress over the gradient of sample concentrations tested, but the mortality data did show a predictable increasing mortality with concentration. The H2 wetland outflow samples tested at the same time were clearly stimulatory compared to the control. Data reported by ACT for the same samples showed no detrimental or stimulatory effect of the inlet sample concentration on Ceriodaphnia reproduction and a slight stimulatory effect on the H2 reproduction at increasing sample concentrations. No mortality was observed in either of the definitive tests run by ACT.

A similar comparison is shown for October 1997 for the Hayfield Site inlet and the H1 outlet samples (Exhibit 21). Once again the City of Phoenix Water Services Laboratory showed both chronic and acute toxicity for the WWTP effluent while ACT did not, while there were only stimulatory effects shown for the H1 outlet sample. From these results it appears that the City of Phoenix Water Services Laboratory has greater sensitivity at detecting both acute and chronic toxicity to the water fleas than ACT. This finding may indicate that the overall scarcity of toxicity recorded by ACT (who conducted the majority of the tests) does not reflect the result that would have been obtained if the City’s lab had conducted those same tests. Laboratory variability is a common occurrence in these complicated tests (Warren-Hicks, et al. 2000). Split sampling between multiple labs with complete audits of test results is necessary to determine if the City’s lab is more capable of detecting real acute and chronic effects or is more likely to record a false positive result.

Possible Water Quality Effects Previous toxicity identification programs at the City of Phoenix 91st Avenue WWTP have identified diazinon as a contributor to whole effluent toxicity (Karen Barten [ENSR] letter to Robert Hollander [City of Phoenix] dated January 6, 1995). The report prepared by Wass (1997) indicated that diazinon concentration is reduced in the wetland influent compared to the WWTP effluent by about 50 percent from an average wetland inflow of 0.215 ug/L to an average outflow concentration of 0.104 ug/L and concluded that toxicity associated with diazinon may be reduced by passage of the effluent through the wetland cells.


EXHIBIT 21Tres Rios Hayfield Site Ceriodaphnia dubia 7-day Survival and Reproduction Chronic Toxicity Test (± SE) - September 97

0

5

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0 25 50 75 90 100

% Effluent

Rep

rodu

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n (#

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e)

HS Inlet (ACT)H2 Eff (ACT)HS Inlet (PHX)H2 Eff (PHX)

0

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30

40

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60

70

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0 25 50 75 90 100

% Effluent

Perc

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38


EXHIBIT 22Tres Rios Hayfield Site Ceriodaphnia dubia 7-day Survival and Reproduction Chronic Toxicity Test (± SE) - October 97

0

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0 25 50 75 90 100

% Effluent

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% Effluent

Perc

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39


40

Other chemical constituents that may be associated with the observed toxicity are salts (as estimated by electrical conductivity), un-ionized ammonia, and trace concentrations of metals or organics. Due to the arid location of the Tres Rios wetlands, salt concentrations could reasonably be expected to increase under some conditions. On the other hand, concentrations of un-ionized ammonia, trace metals, and organics would be expected to decrease due to passage through the wetland cells. Detailed data for ammonia nitrogen and electrical conductivity were available for a comparison to WET results. Data for trace metals and organics are not sufficient to correlate to WET results.

Conductivity and total ammonium nitrogen (roughly proportional to un-ionized ammonia based on temperature and pH) data were reported by the biomonitoring testing laboratories and are summarized in Exhibit 23. The conductivity of the WWTP effluent averaged about 1,538 umhos/cm over the period of record. Water quality data collected during the biomonitoring events indicated that there was relatively little increase in salt concentrations between the inlet and outlets of the wetland cells (typically less than 10 percent). Total ammonium nitrogen concentrations were never very high in the wetland inflow (average about 1.54 mg/L) and were reduced through the wetland cells to averages typically between 0.3 and 0.6 mg/L. A few samples had outflow ammonium nitrogen concentrations higher than 3 mg/L but there was no measurable toxicity recorded on those dates.

Based on this review of limited water quality data collected at the time of WET testing from the wetland cells it appears that slight reductions in concentrations of total ammonium nitrogen, diazinon, or other un-recorded trace contaminants may have contributed to the relatively subtle decline in toxicity effects observed through the Tres Rios Demonstration Constructed Wetlands. This decline in toxicity occurred in spite of a slight observed rise in electrical conductivity (dissolved salts).


EXHIBIT 23

DateEC

(uS/cm)NH3-N (mg/L)

EC (uS/cm)

NH3-N (mg/L)

EC (uS/cm)

NH3-N (mg/L)

EC (uS/cm)

NH3-N (mg/L)

EC (uS/cm)

NH3-N (mg/L)

EC (uS/cm)

NH3-N (mg/L)

Jan-96 1270 0.80Feb-96 1396 0.24 1379 0.05Mar-96 1466 2.46 1513 0.05Apr-96 1461 2.73 1497 0.40May-96 1499 1.89 1499 0.05Jun-96 1459 1.31 1532 0.15Jul-96 1340 1.03 1367 0.05Aug-96 1343 1.23 1397 0.05Sep-96 1327 0.93 1420 0.05Oct-96 1343 0.05 1377 1.20 1380 0.05Nov-96 1280 1.77 1300 0.05Dec-96 890 1.33 887 0.05Jan-97 1517 2.07 1513 0.05Feb-97 1403 1.03 1407 0.05Mar-97 1397 1.60 1407 0.05Apr-97 1510 0.93 1557 0.05May-97 1590 1.77 1540 0.05Jun-97 1647 1.43 1667 0.05Jul-97 1847 1.97 1893 0.05Aug-97 1737 0.60 1990 0.47Sep-97 1669 0.97 1722 0.14Oct-97 1529 1.27 1694 0.13 1483 1.07 1552 0.08Jan-98 1409 1.91 1441 0.32Apr-98 1168 1.13 1212 1.10Dec-98 1358 1.10May-99 1548 1.88 1645 0.22Sep-99 1588 0.97 1583 0.91Oct-99 1365 2.11 1388 3.56Mar-00 1525 1.25 1512 0.48Sep-00 1609 1.54 1613 0.80Dec-00 1510 1.31 1531 0.72Mar-01 1473 0.18 1453 0.16Sep-01 2041 0.41 2053 0.29Nov-01 1240 0.75 1241 0.37Mar-02 1620 4.00 1641 2.13Apr-02 1760 1.00 1680 0.34May-02 1714 1.20 1734 0.18Aug-02 1783 1.60 1843 0.50

Average 1479 1.29 1464 0.57 1527 0.27 1524 1.60 1553 0.32 1530 0.37

Summary of Monthly Average Electrical Conductivity and Total Ammonia Nitrogen at the Tres Rios Demonstration Constructed Wetlands, January 1996 - August 2002

H1 Effluent H2 EffluentCS Inlet C1 Effluent C2 Effluent HS Inlet

41


42

Summary and Recommendations

On a routine basis the acute and chronic toxicity of both the 91st Avenue WWTP final effluent and the wetland outflows is nearly non-existent or difficult to detect. The intermittent nature of toxicity in these discharges makes identification of responsible toxicants difficult. The purpose of this report is to assess the effects of constructed treatment wetlands on toxicity, not to specifically identify toxicity-causing pollutants in the effluent. From this review it can be concluded that the wetlands typically reduced low levels of toxicity when they occurred in the WWTP effluent and did not add to toxicity in any samples tested. High levels of toxicity, when present in the WWTP effluent, passed through the wetlands with minor alteration, probably due to high loading rates and resulting short hydraulic residence times. Low toxicity levels were attenuated within the loading regimes experienced in the wetlands.

It can be concluded from this research that use of full-scale constructed wetlands for final effluent polishing of the 91st Avenue WWTP final effluent will reduce the frequency and magnitude of WET and provide enhancement of water quality in the ultimate receiving waters in the Salt River. Additional detailed quantification of those benefits could be accomplished by research-oriented biomonitoring and ecological risk assessments at the Tres Rios Demonstration Wetlands. The Research Cells could provide a replicated platform for this type of research. The cells could be re-started with a range of hydraulic loading rates and the existing internal deep zone configurations. WET monitoring should also be conducted along the flow path in the full-scale treatment wetlands.

For future monitoring efforts a sensitive indicator of toxicity such as Microtox™ could be utilized to provide rapid detection of any toxic events in the 91st Avenue WWTP effluent. Wetland inflow and outflow toxicity would then be monitored throughout the toxic event. Fathead minnow and algal tests could be used to augment the existing Ceriodaphnia bioassays. Screening rather than definitive tests can be used to reduce expenses associated with this proposed WET testing program. Tests should probably be conducted by the City’s own laboratory because of their apparent ability to provide more sensitive detection levels. Statistical comparisons of the raw test data including neonates produced, weight gain of fish, and algal cell volumes or weight should be used to detect effects of the various wetland designs on toxicity. Detailed analysis of wetland influent and effluent quality for major and minor constituents should be conducted at the time of toxicity testing.

Important research issues related to WET and treatment wetlands in general include the following:

• A demonstration of the relevance of the response of the existing standard test organisms as an indicator of actual response in a receiving water, whether it is a wetland, stream, or lake

• The nature of differing sensitivities between standardized WET test organisms


43

• Inter- and intra-laboratory variability when conducting standardized tests

• Analysis and reporting of the detailed WET laboratory test data to better reflect the full response of the test organisms to the effluents tested

Portions of the issues listed above are addressed in the data summarized for this review of nearly seven years of biomonitoring at the City of Phoenix Tres Rios Demonstration Constructed Wetlands. Additional focused research is needed to fully address each of these issues to enlighten designers and operators of the proposed full-scale Tres Rios Constructed Wetlands.


44

Acknowledgements

Data from the Tres Rios Demonstration Constructed Wetlands were collected and provided by staff of the City of Phoenix, Water Services Department. Alice Brawley-Chesworth and Aimee Conroy were the project managers for this data review and analysis project. Roland Wass, currently with Wass Gerke & Associates was the City’s project manager during most of the period covered by this report. Able assistance with sample collection was provided by Wes Camfield and Ron Elkins. Ron Clarke with Wetland Solutions, Inc. assisted with data analysis and report preparation.


45

References

American Petroleum Institute (API). 1998. The Use of Treatment Wetlands for Petroleum Industry Effluents. Prepared by Robert L. Knight, Robert H. Kadlec, and Harry M. Ohlendorf under contract to CH2M HILL. Health and Environmental Sciences Department. Publication No. 4672.

Ausley, L.W. 2000. Reflection on whole effluent toxicity: the Pellston workshops. Environmental Toxicology and Chemistry 19(1): 1-2.

Bishay, F.S., J.R. Gulley, S.H. Hamilton, and P.G. Nix. 1995. Constructed Wetlands as a Treatment System for Wastewater from an Oil Sands Mining and Extraction Operation. Poster presentation at SETAC World Congress, Vancouver, B.C., Canada, November 5-9.

Chapman, P.M. 1998. New and emerging issues in ecotoxicology – the shape of testing to come? Australian Journal of Ecotoxicology 4: 1-7.

Chapman, P.M. 2000. Whole effluent toxicity testing – usefulness, level of protection, and risk assessment. Environmental Toxicology and Chemistry 19(1): 3-13.

Duda, P.J. 1992. Chevron’s Richmond Refinery Water Enhancement Wetland. Report to the Regional Water Quality Control Board, Oakland, CA.

Farmer, J.M. 1996. Personal communication.

Hawkins, W.B., A. Dunn, and J.H. Rodgers, Jr. 1995. Evaluation of Pilot-Scale Constructed Wetlands for Tertiary Treatment of Refinery Effluent. Presented at the Second SETAC World Congress, Vancouver, B.C., Canada, November 5-9.

International Water Association (IWA). 2000. Constructed Wetlands for Pollution Control. Processes, Performance, Design, and Operation. Scientific and Technical Report No. 8. 156 pp.

Kadlec, R.H. 2001. Water movement in surface flow wetlands in hot climates. Draft Technical Memorandum prepared for City of Phoenix Department of Water Services. 30 pp.

Kadlec, R.H. and R.L. Knight. 1996. Treatment Wetlands. Lewis Publishers/CRC Press, Boca Raton, FL 893 pp.

Knight, R.L., J. Hilleke, and S. Grayson. 1994. Design and performance of the Champion pilot constructed wetland treatment system. TAPPI Journal 77(5): 240-245.

Knight, R.L. 1983. Energy basis of ecosystem control at Silver Springs, Florida. Chapter 8 (pp. 161-179) in: T.D. Fontaine and S.M. Bartell (eds.) Dynamics of Lotic Ecosystems. Ann Arbor Science, Ann Arbor, MI.

LaPoint, T.W. and W.T. Waller. 2000. Field assessments in conjunction with whole effluent toxicity testing. Environmental Toxicology and Chemistry 19(1): 14-24.


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McAllister, L.S. 1992. Habitat Quality Assessment of Two Wetland Treatment Systems in Mississippi – A Pilot Study. U.S. Environmental Protection Agency Environmental Research Laboratory, Corvallis, OR, EPA/600/R-92/229.

McAllister, L.S. 1993a. Habitat Quality Assessment of Two Wetland Treatment Systems in the Arid West – A Pilot Study. U.S. Environmental Protection Agency Environmental Research Laboratory, Corvallis, OR, EPA/600/R-93/117.

McAllister, L.S. 1993b. Habitat Quality Assessment of Two Wetland Treatment Systems in Florida – A Pilot Study. U.S. Environmental Protection Agency Environmental Research Laboratory, Corvallis, OR, EPA/600/R-93/222.

Mount, D.I. and T.J. Norberg. 1984. A seven-day life-cycle cladoceran toxicity test. Environmental Toxicology and Chemistry 3:433.

Odum, E.P., J.T. Finn, and E.H. Franz. 1979. Perturbation theory and the subsidy-stress gradient. Bioscience 29: 349-352.

U.S. Army Corps of Engineers (USACOE). 2000.Tres Rios, Arizona Feasibility Study. Feasibility Report and Final Environmental Impact Statement. Los Angeles District, South Pacific Division. Aoril, 2000.

U.S. Environmental Protection Agency (USEPA). 1994. Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Water to Freshwater Organisms. Third Edition. EPA/600/4-91/002. Environmental Monitoring and Support Laboratory, Cincinnati, Ohio. 341 pp.

U.S. Environmental Protection Agency (USEPA). 1999. Treatment Wetland Habitat and Wildlife Use Assessment. Prepared by CH2M HILL for the USEPA, U.S. Department of the Interior, and the City of Phoenix. EPA 832-S-99-001. Executive Summary (31 pp) and CD-ROM.

Warren-Hicks, W.J., B.R. Parkhurst, D.R.J. Moore, R.S. Teed, R.B. Baird, R. Berger, D.L. Denton, J.J. Pletl. 2000. Assessment of whole effluent toxicity test variability: partitioning sources of variability. Environmental Toxicology and Chemistry 19(1): 94-104.

Woodward-Clyde Consultants (WCC). 1995. Demonstration Urban Stormwater Treatment (DUST) Marsh Special Study RY 93-94. Alameda County Urban Runoff Clean Water Program. January 1995.


Appendix A Detailed Biomonitoring Data from the Tres Rios Constructed

Demonstration Wetlands, Phoenix, Arizona


Appendix A-1Summary of Reproduction (# young/female) of Ceriodaphnia dubia in the Tres Rios Demonstration Wetlands, AZJanuary 1996 - August 2002

SITE_NAME TIMEPERIOD LAB DILUTION %_EFF CS Inlet C1 Eff C2 Eff HS Inlet H1 Eff H2 EffTres Rios, AZ Jan-96 ACT 0 100 13.9 13.5Tres Rios, AZ Jan-96 ACT 10 90 13.2 11.0Tres Rios, AZ Jan-96 ACT 25 75 13.8 15.3Tres Rios, AZ Jan-96 ACT 50 50 14.2 16.6Tres Rios, AZ Jan-96 ACT 75 25 14.8 15.4Tres Rios, AZ Jan-96 ACT 100 0 15.7 15.5Tres Rios, AZ Feb-96 ACT 0 100 0.0 17.5 18.4Tres Rios, AZ Feb-96 ACT 10 90 0.0 17.0 17.6Tres Rios, AZ Feb-96 ACT 25 75 14.9 17.2 19.9Tres Rios, AZ Feb-96 ACT 50 50 18.4 18.8 20.0Tres Rios, AZ Feb-96 ACT 75 25 18.4 17.4 18.1Tres Rios, AZ Feb-96 ACT 100 0 18.4 16.9 18.1Tres Rios, AZ Mar-96 ACT 0 100 16.0 19.8Tres Rios, AZ Mar-96 ACT 10 90 17.9 18.8Tres Rios, AZ Mar-96 ACT 25 75 16.4 18.4Tres Rios, AZ Mar-96 ACT 50 50 16.5 17.5Tres Rios, AZ Mar-96 ACT 75 25 16.5 18.2Tres Rios, AZ Mar-96 ACT 100 0 16.0 16.7Tres Rios, AZ Apr-96 ACT 0 100 19.1 22.6Tres Rios, AZ Apr-96 ACT 10 90 18.3 22.5Tres Rios, AZ Apr-96 ACT 25 75 18.3 21.6Tres Rios, AZ Apr-96 ACT 50 50 19.0 19.7Tres Rios, AZ Apr-96 ACT 75 25 18.2 19.8Tres Rios, AZ Apr-96 ACT 100 0 17.1 18.7Tres Rios, AZ May-96 ACT 0 100 16.5 17.2Tres Rios, AZ May-96 ACT 10 90 21.8 19.2Tres Rios, AZ May-96 ACT 25 75 22.5 18.7Tres Rios, AZ May-96 ACT 50 50 25.0 20.0Tres Rios, AZ May-96 ACT 75 25 22.3 19.2Tres Rios, AZ May-96 ACT 100 0 17.9 18.2Tres Rios, AZ Jun-96 ACT 0 100 20.4 20.2Tres Rios, AZ Jun-96 ACT 10 90 20.6 20.2Tres Rios, AZ Jun-96 ACT 25 75 19.6 21.1Tres Rios, AZ Jun-96 ACT 50 50 21.1 18.4Tres Rios, AZ Jun-96 ACT 75 25 18.8 19.9Tres Rios, AZ Jun-96 ACT 100 0 17.2 18.9Tres Rios, AZ Jun-96 PHX 0 100 17.3 36.2Tres Rios, AZ Jun-96 PHX 25 75 25.6 37.2Tres Rios, AZ Jun-96 PHX 50 50 26.5 33.1Tres Rios, AZ Jun-96 PHX 75 25 23.6 29.0Tres Rios, AZ Jun-96 PHX 87.5 12.5 20.4 28.8Tres Rios, AZ Jun-96 PHX 100 0 22.8 24.1Tres Rios, AZ Jul-96 ACT 0 100 22.6 22.0Tres Rios, AZ Jul-96 ACT 10 90 21.7 22.0Tres Rios, AZ Jul-96 ACT 25 75 22.5 21.7Tres Rios, AZ Jul-96 ACT 50 50 21.4 21.8Tres Rios, AZ Jul-96 ACT 75 25 22.1 21.4Tres Rios, AZ Jul-96 ACT 100 0 22.4 20.4Tres Rios, AZ Aug-96 ACT 0 100 24.9 24.9Tres Rios, AZ Aug-96 ACT 10 90 24.7 27.3Tres Rios, AZ Aug-96 ACT 25 75 24.0 23.8Tres Rios, AZ Aug-96 ACT 50 50 21.7 24.2Tres Rios, AZ Aug-96 ACT 75 25 26.2 22.1Tres Rios, AZ Aug-96 ACT 100 0 25.4 22.5

A-1



SITE_NAME TIMEPERIOD LAB DILUTION %_EFF CS Inlet C1 Eff C2 Eff HS Inlet H1 Eff H2 EffTres Rios, AZ Sep-96 ACT 0 100 20.4 17.4Tres Rios, AZ Sep-96 ACT 10 90 19.5 17.6Tres Rios, AZ Sep-96 ACT 25 75 20.2 19.4Tres Rios, AZ Sep-96 ACT 50 50 19.6 18.3Tres Rios, AZ Sep-96 ACT 75 25 18.7 18.8Tres Rios, AZ Sep-96 ACT 100 0 18.5 17.6Tres Rios, AZ Oct-96 ACT 0 100 1.9 2.0 0.4Tres Rios, AZ Oct-96 ACT 10 90 1.8 1.9 1.2Tres Rios, AZ Oct-96 ACT 25 75 1.6 3.4 1.2Tres Rios, AZ Oct-96 ACT 50 50 1.6 17.7 0.9Tres Rios, AZ Oct-96 ACT 75 25 10.7 16.9 1.4Tres Rios, AZ Oct-96 ACT 100 0 17.6 18.1 15.7Tres Rios, AZ Nov-96 ACT 0 100 24.4 23.6Tres Rios, AZ Nov-96 ACT 10 90 23.8 25.9Tres Rios, AZ Nov-96 ACT 25 75 25.9 24.1Tres Rios, AZ Nov-96 ACT 50 50 24.5 24.0Tres Rios, AZ Nov-96 ACT 75 25 22.7 22.9Tres Rios, AZ Nov-96 ACT 100 0 21.6 19.5Tres Rios, AZ Dec-96 ACT 0 100 23.0 22.6Tres Rios, AZ Dec-96 ACT 10 90 24.7 25.7Tres Rios, AZ Dec-96 ACT 25 75 22.0 23.0Tres Rios, AZ Dec-96 ACT 50 50 21.8 22.2Tres Rios, AZ Dec-96 ACT 75 25 22.1 21.2Tres Rios, AZ Dec-96 ACT 100 0 20.4 22.2Tres Rios, AZ Jan-97 ACT 0 100 20.4 21.0Tres Rios, AZ Jan-97 ACT 10 90 19.6 22.0Tres Rios, AZ Jan-97 ACT 25 75 20.8 21.3Tres Rios, AZ Jan-97 ACT 50 50 20.2 23.1Tres Rios, AZ Jan-97 ACT 75 25 19.4 19.7Tres Rios, AZ Jan-97 ACT 100 0 20.1 19.8Tres Rios, AZ Feb-97 ACT 0 100 22.9 21.6Tres Rios, AZ Feb-97 ACT 10 90 21.3 22.7Tres Rios, AZ Feb-97 ACT 25 75 23.4 24.0Tres Rios, AZ Feb-97 ACT 50 50 22.3 24.6Tres Rios, AZ Feb-97 ACT 75 25 23.3 23.7Tres Rios, AZ Feb-97 ACT 100 0 21.2 17.6Tres Rios, AZ Mar-97 ACT 0 100 19.4 17.3Tres Rios, AZ Mar-97 ACT 10 90 23.5 19.7Tres Rios, AZ Mar-97 ACT 25 75 21.1 20.2Tres Rios, AZ Mar-97 ACT 50 50 21.3 21.2Tres Rios, AZ Mar-97 ACT 75 25 20.2 21.9Tres Rios, AZ Mar-97 ACT 100 0 17.8 17.3Tres Rios, AZ Apr-97 ACT 0 100 21.4 21.9Tres Rios, AZ Apr-97 ACT 10 90 20.7 20.8Tres Rios, AZ Apr-97 ACT 25 75 19.9 22.7Tres Rios, AZ Apr-97 ACT 50 50 19.7 24.4Tres Rios, AZ Apr-97 ACT 75 25 20.0 21.9Tres Rios, AZ Apr-97 ACT 100 0 19.4 21.0Tres Rios, AZ May-97 ACT 0 100 18.6 18.6Tres Rios, AZ May-97 ACT 10 90 22.0 19.4Tres Rios, AZ May-97 ACT 25 75 19.0 21.0Tres Rios, AZ May-97 ACT 50 50 21.1 20.3Tres Rios, AZ May-97 ACT 75 25 18.0 18.5Tres Rios, AZ May-97 ACT 100 0 16.3 16.8

A-2



SITE_NAME TIMEPERIOD LAB DILUTION %_EFF CS Inlet C1 Eff C2 Eff HS Inlet H1 Eff H2 EffTres Rios, AZ Jun-97 ACT 0 100 20.4 20.0Tres Rios, AZ Jun-97 ACT 10 90 14.8 21.2Tres Rios, AZ Jun-97 ACT 25 75 17.7 20.4Tres Rios, AZ Jun-97 ACT 50 50 17.2 18.1Tres Rios, AZ Jun-97 ACT 75 25 18.0 18.7Tres Rios, AZ Jun-97 ACT 100 0 16.6 16.0Tres Rios, AZ Jul-97 ACT 0 100 21.2 21.5Tres Rios, AZ Jul-97 ACT 10 90 21.8 21.3Tres Rios, AZ Jul-97 ACT 25 75 22.1 19.2Tres Rios, AZ Jul-97 ACT 50 50 22.0 22.8Tres Rios, AZ Jul-97 ACT 75 25 20.8 21.6Tres Rios, AZ Jul-97 ACT 100 0 18.3 18.8Tres Rios, AZ Aug-97 ACT 0 100 21.8 21.2Tres Rios, AZ Aug-97 ACT 10 90 23.0 22.4Tres Rios, AZ Aug-97 ACT 25 75 21.8 20.9Tres Rios, AZ Aug-97 ACT 50 50 23.4 23.6Tres Rios, AZ Aug-97 ACT 75 25 22.5 21.0Tres Rios, AZ Aug-97 ACT 100 0 17.8 20.0Tres Rios, AZ Sep-97 ACT 0 100 21.5 21.9Tres Rios, AZ Sep-97 ACT 10 90 22.3 22.0Tres Rios, AZ Sep-97 ACT 25 75 22.4 22.5Tres Rios, AZ Sep-97 ACT 50 50 22.4 22.5Tres Rios, AZ Sep-97 ACT 75 25 23.0 19.9Tres Rios, AZ Sep-97 ACT 100 0 21.2 17.4Tres Rios, AZ Sep-97 PHX 0 100 16.0 26.9Tres Rios, AZ Sep-97 PHX 10 90 24.9 26.7Tres Rios, AZ Sep-97 PHX 25 75 20.7 25.3Tres Rios, AZ Sep-97 PHX 50 50 17.2 23.4Tres Rios, AZ Sep-97 PHX 75 25 16.4 21.9Tres Rios, AZ Sep-97 PHX 100 0 21.1 21.9Tres Rios, AZ Oct-97 ACT 0 100 23.9 23.8Tres Rios, AZ Oct-97 ACT 10 90 21.4 25.0Tres Rios, AZ Oct-97 ACT 25 75 19.5 23.1Tres Rios, AZ Oct-97 ACT 50 50 25.8 24.3Tres Rios, AZ Oct-97 ACT 75 25 25.1 23.8Tres Rios, AZ Oct-97 ACT 100 0 21.9 19.4Tres Rios, AZ Oct-97 PHX 0 100 5.3 44.3 11.6 36.2Tres Rios, AZ Oct-97 PHX 10 90 4.8 36.3 17.1 37.2Tres Rios, AZ Oct-97 PHX 25 75 5.9 35.5 11.3 33.1Tres Rios, AZ Oct-97 PHX 50 50 7.5 36.7 19.1 29.0Tres Rios, AZ Oct-97 PHX 75 25 24.8 34.1 18.7 28.8Tres Rios, AZ Oct-97 PHX 100 0 14.8 17.3 21.9 24.1Tres Rios, AZ Jan-98 PHX 0 100 23.5 29.4Tres Rios, AZ Jan-98 PHX 10 90 21.9 25.2Tres Rios, AZ Jan-98 PHX 25 75 22.7 24.4Tres Rios, AZ Jan-98 PHX 50 50 19.6 24.2Tres Rios, AZ Jan-98 PHX 75 25 22.0 20.2Tres Rios, AZ Jan-98 PHX 100 0 22.1 21.7Tres Rios, AZ Apr-98 PHX 0 100 24.1 34.7Tres Rios, AZ Apr-98 PHX 10 90 24.0 32.5Tres Rios, AZ Apr-98 PHX 25 75 29.4 32.8Tres Rios, AZ Apr-98 PHX 50 50 27.7 31.9Tres Rios, AZ Apr-98 PHX 75 25 27.7 30.5Tres Rios, AZ Apr-98 PHX 100 0 29.1 31.3

A-3



SITE_NAME TIMEPERIOD LAB DILUTION %_EFF CS Inlet C1 Eff C2 Eff HS Inlet H1 Eff H2 EffTres Rios, AZ Dec-98 ENSR 0 100 28.8Tres Rios, AZ Dec-98 ENSR 50 50 27.8Tres Rios, AZ Dec-98 ENSR 75 25 21.0Tres Rios, AZ Dec-98 ENSR 87.5 12.5 23.0Tres Rios, AZ Dec-98 ENSR 93.75 6.25 28.2Tres Rios, AZ Dec-98 ENSR 100 0 24.0Tres Rios, AZ May-99 PHX 0 100 27.8 16.5Tres Rios, AZ May-99 PHX 50 50 29.4 19.7Tres Rios, AZ May-99 PHX 75 25 24.3 22.4Tres Rios, AZ May-99 PHX 87.5 12.5 22.8 20.7Tres Rios, AZ May-99 PHX 93.75 6.25 24.4 18.6Tres Rios, AZ May-99 PHX 100 0 21.0 21.1Tres Rios, AZ Sep-99 PHX 0 100 37.9 31.1Tres Rios, AZ Sep-99 PHX 50 50 33.5 32.6Tres Rios, AZ Sep-99 PHX 75 25 33.4 34.5Tres Rios, AZ Sep-99 PHX 87.5 12.5 33.4 31.4Tres Rios, AZ Sep-99 PHX 93.75 6.25 32.0 30.2Tres Rios, AZ Sep-99 PHX 100 0 31.4 28.8Tres Rios, AZ Dec-99 PHX 0 100 6.0 27.4Tres Rios, AZ Dec-99 PHX 50 50 32.6 27.1Tres Rios, AZ Dec-99 PHX 75 25 33.5 28.9Tres Rios, AZ Dec-99 PHX 87.5 12.5 34.6 28.7Tres Rios, AZ Dec-99 PHX 93.75 6.25 34.2 30.9Tres Rios, AZ Dec-99 PHX 100 0 32.6 32.1Tres Rios, AZ Mar-00 PHX 0 100 16.0 30.4Tres Rios, AZ Mar-00 PHX 25 75 19.8 29.9Tres Rios, AZ Mar-00 PHX 50 50 30.4 30.7Tres Rios, AZ Mar-00 PHX 75 25 26.8 29.7Tres Rios, AZ Mar-00 PHX 87.5 12.5 30.5 29.2Tres Rios, AZ Mar-00 PHX 100 0 30.4 21.4Tres Rios, AZ Sep-00 PHX 0 100 30.1 31.8Tres Rios, AZ Sep-00 PHX 25 75 29.0 31.6Tres Rios, AZ Sep-00 PHX 50 50 31.1 30.9Tres Rios, AZ Sep-00 PHX 75 25 30.3 29.5Tres Rios, AZ Sep-00 PHX 87.5 12.5 29.8 31.7Tres Rios, AZ Sep-00 PHX 100 0 24.0 27.3Tres Rios, AZ Dec-00 PHX 0 100 35.3 33.5Tres Rios, AZ Dec-00 PHX 25 75 35.0 33.3Tres Rios, AZ Dec-00 PHX 50 50 33.8 33.3Tres Rios, AZ Dec-00 PHX 75 25 32.4 31.3Tres Rios, AZ Dec-00 PHX 87.5 12.5 27.0 28.2Tres Rios, AZ Dec-00 PHX 100 0 30.7 29.1Tres Rios, AZ Mar-01 PHX 0 100 31.6 36.9Tres Rios, AZ Mar-01 PHX 25 75 34.6 38.9Tres Rios, AZ Mar-01 PHX 50 50 34.8 38.4Tres Rios, AZ Mar-01 PHX 75 25 31.6 38.3Tres Rios, AZ Mar-01 PHX 87.5 12.5 35.4 37.8Tres Rios, AZ Mar-01 PHX 100 0 32.6 34.5Tres Rios, AZ Sep-01 PHX 0 100 26.0 27.0Tres Rios, AZ Sep-01 PHX 25 75 28.3 26.6Tres Rios, AZ Sep-01 PHX 50 50 23.7 27.3Tres Rios, AZ Sep-01 PHX 75 25 23.5 26.2Tres Rios, AZ Sep-01 PHX 87.5 12.5 21.5 25.6Tres Rios, AZ Sep-01 PHX 100 0 22.5 20.8

A-4



SITE_NAME TIMEPERIOD LAB DILUTION %_EFF CS Inlet C1 Eff C2 Eff HS Inlet H1 Eff H2 EffTres Rios, AZ Nov-01 PHX 0 100 17.0 15.9Tres Rios, AZ Nov-01 PHX 25 75 21.0 20.3Tres Rios, AZ Nov-01 PHX 50 50 21.7 21.0Tres Rios, AZ Nov-01 PHX 75 25 19.9 20.5Tres Rios, AZ Nov-01 PHX 87.5 12.5 18.5 21.0Tres Rios, AZ Nov-01 PHX 100 0 19.6 17.2Tres Rios, AZ Mar-02 PHX 0 100 21.6 26.7Tres Rios, AZ Mar-02 PHX 25 75 22.0 24.3Tres Rios, AZ Mar-02 PHX 50 50 20.9 23.6Tres Rios, AZ Mar-02 PHX 75 25 17.9 26.5Tres Rios, AZ Mar-02 PHX 87.5 12.5 16.7 25.6Tres Rios, AZ Mar-02 PHX 100 0 20.5 18.9Tres Rios, AZ May-02 PHX 0 100 35.3 28.5Tres Rios, AZ May-02 PHX 25 75 34.5 27.3Tres Rios, AZ May-02 PHX 50 50 36.9 27.1Tres Rios, AZ May-02 PHX 75 25 34.6 28.5Tres Rios, AZ May-02 PHX 87.5 12.5 32.8 29.2Tres Rios, AZ May-02 PHX 100 0 34.9 32.7Tres Rios, AZ Aug-02 PHX 0 100 23.3 31.9Tres Rios, AZ Aug-02 PHX 25 75 22.1 30.4Tres Rios, AZ Aug-02 PHX 50 50 19.5 30.0Tres Rios, AZ Aug-02 PHX 75 25 20.5 28.5Tres Rios, AZ Aug-02 PHX 87.5 12.5 18.1 31.4Tres Rios, AZ Aug-02 PHX 100 0 22.8 19.3

Average 0 100 21.9 23.5 22.5 20.0 26.2 21.110 90 19.3 21.0 18.7 19.1 25.4 18.425 75 22.1 22.6 22.4 21.3 25.5 21.550 50 24.4 23.0 23.4 22.4 25.5 20.675 25 24.6 22.4 23.9 21.4 24.6 19.4

87.5 12.5 29.7 29.9 28.0 21.1 28.4 27.293.75 6.25 30.2 30.9 24.4 28.2100 0 22.8 22.7 20.4 21.4 21.5 19.1

Minimum 0 100 5.3 13.9 1.9 0.0 17.3 0.410 90 4.8 13.2 1.8 0.0 17.0 1.225 75 5.9 13.8 1.6 3.4 17.2 1.250 50 7.5 14.2 1.6 17.2 18.8 0.975 25 16.5 14.8 10.7 16.4 17.4 1.4

87.5 12.5 18.5 28.7 20.7 16.7 25.6 25.693.75 6.25 24.4 30.9 18.6 28.2100 0 14.8 15.7 15.5 16.3 16.9 15.7

Maximum 0 100 37.9 31.8 44.3 35.3 36.2 36.210 90 24.7 25.7 36.3 24.9 37.2 26.725 75 34.6 31.6 38.9 35.0 33.3 37.250 50 36.9 30.9 38.4 33.8 33.3 33.175 25 34.6 29.5 38.3 32.4 31.3 29.0

87.5 12.5 35.4 31.7 37.8 27.0 31.4 28.893.75 6.25 34.2 30.9 30.2 28.2100 0 34.9 32.7 34.5 30.7 31.3 24.1

A-5



SITE_NAME TIMEPERIOD LAB DILUTION %_EFF CS Inlet C1 Eff C2 Eff HS Inlet H1 Eff H2 EffStdDev 0 100 8.5 5.7 11.4 7.9 6.5 9.6

10 90 5.5 4.2 10.7 7.7 6.3 8.125 75 6.8 5.1 10.6 6.6 5.7 9.350 50 7.5 4.9 10.2 4.1 4.7 8.575 25 5.6 4.9 8.4 4.0 4.4 7.7

87.5 12.5 6.0 1.6 7.3 3.7 2.9 2.393.75 6.25 5.1 8.2100 0 6.3 6.1 5.7 3.8 4.6 2.6

Count 0 100 19 10 12 21 12 910 90 11 7 7 15 9 725 75 16 9 10 20 12 950 50 19 10 12 21 12 975 25 19 10 12 21 12 9

87.5 12.5 8 3 5 6 3 293.75 6.25 3 1 2 1100 0 19 10 12 21 12 9

A-6


Appendix A-2Summary of Mortality (%) of Ceriodaphnia dupia in the Tres Rios Demonstration Wetlands, AZJanuary 1996 - August 2002

SITE_NAME TIMEPERIOD LAB DILUTION %_EFF CS Inlet C1 Eff C2 Eff HS Inlet H1 Eff H2 EffTres Rios, AZ Jan-96 ACT 0 100 10 10Tres Rios, AZ Jan-96 ACT 10 90 10 10Tres Rios, AZ Jan-96 ACT 25 75 0 0Tres Rios, AZ Jan-96 ACT 50 50 0 0Tres Rios, AZ Jan-96 ACT 75 25 0 0Tres Rios, AZ Jan-96 ACT 100 0 0 0Tres Rios, AZ Feb-96 ACT 0 100 0 0Tres Rios, AZ Feb-96 ACT 10 90 0 0Tres Rios, AZ Feb-96 ACT 25 75 0 0Tres Rios, AZ Feb-96 ACT 50 50 0 0Tres Rios, AZ Feb-96 ACT 75 25 0 0Tres Rios, AZ Feb-96 ACT 100 0 0 0Tres Rios, AZ Mar-96 ACT 0 100 0 0Tres Rios, AZ Mar-96 ACT 10 90 0 0Tres Rios, AZ Mar-96 ACT 25 75 0 0Tres Rios, AZ Mar-96 ACT 50 50 0 0Tres Rios, AZ Mar-96 ACT 75 25 0 0Tres Rios, AZ Mar-96 ACT 100 0 0 0Tres Rios, AZ Apr-96 ACT 0 100 0 0Tres Rios, AZ Apr-96 ACT 10 90 10 10Tres Rios, AZ Apr-96 ACT 25 75 0 0Tres Rios, AZ Apr-96 ACT 50 50 0 0Tres Rios, AZ Apr-96 ACT 75 25 0 0Tres Rios, AZ Apr-96 ACT 100 0 0 0Tres Rios, AZ May-96 ACT 0 100 0 0Tres Rios, AZ May-96 ACT 10 90 0 0Tres Rios, AZ May-96 ACT 25 75 0 0Tres Rios, AZ May-96 ACT 50 50 0 0Tres Rios, AZ May-96 ACT 75 25 0 0Tres Rios, AZ May-96 ACT 100 0 0 0Tres Rios, AZ Jun-96 ACT 0 100 0 10Tres Rios, AZ Jun-96 ACT 10 90 0 0Tres Rios, AZ Jun-96 ACT 25 75 10 0Tres Rios, AZ Jun-96 ACT 50 50 0 0Tres Rios, AZ Jun-96 ACT 75 25 0 0Tres Rios, AZ Jun-96 ACT 100 0 0 0Tres Rios, AZ Jun-96 PHX 0 100 90 0Tres Rios, AZ Jun-96 PHX 25 75 0 0Tres Rios, AZ Jun-96 PHX 50 50 0 0Tres Rios, AZ Jun-96 PHX 75 25 0 0Tres Rios, AZ Jun-96 PHX 87.5 12.5 0 0Tres Rios, AZ Jun-96 PHX 100 0 0 0Tres Rios, AZ Jul-96 ACT 0 100 0 0Tres Rios, AZ Jul-96 ACT 10 90 0 0Tres Rios, AZ Jul-96 ACT 25 75 0 0Tres Rios, AZ Jul-96 ACT 50 50 0 0Tres Rios, AZ Jul-96 ACT 75 25 0 0Tres Rios, AZ Jul-96 ACT 100 0 0 0Tres Rios, AZ Aug-96 ACT 0 100 0 0Tres Rios, AZ Aug-96 ACT 10 90 0 0Tres Rios, AZ Aug-96 ACT 25 75 10 0Tres Rios, AZ Aug-96 ACT 50 50 10 0Tres Rios, AZ Aug-96 ACT 75 25 0 10Tres Rios, AZ Aug-96 ACT 100 0 0 0

A-7



SITE_NAME TIMEPERIOD LAB DILUTION %_EFF CS Inlet C1 Eff C2 Eff HS Inlet H1 Eff H2 EffTres Rios, AZ Sep-96 ACT 0 100 0 0Tres Rios, AZ Sep-96 ACT 10 90 0 0Tres Rios, AZ Sep-96 ACT 25 75 0 0Tres Rios, AZ Sep-96 ACT 50 50 0 0Tres Rios, AZ Sep-96 ACT 75 25 0 0Tres Rios, AZ Sep-96 ACT 100 0 0 0Tres Rios, AZ Oct-96 ACT 0 100 100 100 100Tres Rios, AZ Oct-96 ACT 10 90 100 70 100Tres Rios, AZ Oct-96 ACT 25 75 100 70 100Tres Rios, AZ Oct-96 ACT 50 50 100 0 100Tres Rios, AZ Oct-96 ACT 75 25 20 0 80Tres Rios, AZ Oct-96 ACT 100 0 0 0 10Tres Rios, AZ Nov-96 ACT 0 100 0 0Tres Rios, AZ Nov-96 ACT 10 90 0 0Tres Rios, AZ Nov-96 ACT 25 75 0 0Tres Rios, AZ Nov-96 ACT 50 50 0 0Tres Rios, AZ Nov-96 ACT 75 25 0 0Tres Rios, AZ Nov-96 ACT 100 0 0 0Tres Rios, AZ Dec-96 ACT 0 100 0 0Tres Rios, AZ Dec-96 ACT 10 90 0 0Tres Rios, AZ Dec-96 ACT 25 75 0 0Tres Rios, AZ Dec-96 ACT 50 50 0 0Tres Rios, AZ Dec-96 ACT 75 25 0 0Tres Rios, AZ Dec-96 ACT 100 0 0 0Tres Rios, AZ Jan-97 ACT 0 100 0 0Tres Rios, AZ Jan-97 ACT 10 90 0 0Tres Rios, AZ Jan-97 ACT 25 75 0 0Tres Rios, AZ Jan-97 ACT 50 50 0 0Tres Rios, AZ Jan-97 ACT 75 25 0 0Tres Rios, AZ Jan-97 ACT 100 0 0 0Tres Rios, AZ Feb-97 ACT 0 100 0 0Tres Rios, AZ Feb-97 ACT 10 90 10 0Tres Rios, AZ Feb-97 ACT 25 75 0 0Tres Rios, AZ Feb-97 ACT 50 50 0 0Tres Rios, AZ Feb-97 ACT 75 25 0 0Tres Rios, AZ Feb-97 ACT 100 0 0 0Tres Rios, AZ Mar-97 ACT 0 100 0 10Tres Rios, AZ Mar-97 ACT 10 90 0 0Tres Rios, AZ Mar-97 ACT 25 75 0 0Tres Rios, AZ Mar-97 ACT 50 50 0 0Tres Rios, AZ Mar-97 ACT 75 25 0 0Tres Rios, AZ Mar-97 ACT 100 0 0 0Tres Rios, AZ Apr-97 ACT 0 100 0 0Tres Rios, AZ Apr-97 ACT 10 90 0 0Tres Rios, AZ Apr-97 ACT 25 75 0 0Tres Rios, AZ Apr-97 ACT 50 50 0 0Tres Rios, AZ Apr-97 ACT 75 25 0 0Tres Rios, AZ Apr-97 ACT 100 0 0 0Tres Rios, AZ May-97 ACT 0 100 0 0Tres Rios, AZ May-97 ACT 10 90 0 0Tres Rios, AZ May-97 ACT 25 75 0 0Tres Rios, AZ May-97 ACT 50 50 0 0Tres Rios, AZ May-97 ACT 75 25 0 0Tres Rios, AZ May-97 ACT 100 0 0 0

A-8



SITE_NAME TIMEPERIOD LAB DILUTION %_EFF CS Inlet C1 Eff C2 Eff HS Inlet H1 Eff H2 EffTres Rios, AZ Jun-97 ACT 0 100 0 0Tres Rios, AZ Jun-97 ACT 10 90 10 0Tres Rios, AZ Jun-97 ACT 25 75 10 0Tres Rios, AZ Jun-97 ACT 50 50 10 10Tres Rios, AZ Jun-97 ACT 75 25 0 0Tres Rios, AZ Jun-97 ACT 100 0 0 0Tres Rios, AZ Jul-97 ACT 0 100 0 0Tres Rios, AZ Jul-97 ACT 10 90 0 0Tres Rios, AZ Jul-97 ACT 25 75 0 0Tres Rios, AZ Jul-97 ACT 50 50 0 0Tres Rios, AZ Jul-97 ACT 75 25 0 0Tres Rios, AZ Jul-97 ACT 100 0 0 0Tres Rios, AZ Aug-97 ACT 0 100 0 0Tres Rios, AZ Aug-97 ACT 10 90 0 0Tres Rios, AZ Aug-97 ACT 25 75 0 0Tres Rios, AZ Aug-97 ACT 50 50 0 0Tres Rios, AZ Aug-97 ACT 75 25 0 0Tres Rios, AZ Aug-97 ACT 100 0 0 0Tres Rios, AZ Sep-97 ACT 0 100 0 0Tres Rios, AZ Sep-97 ACT 10 90 0 0Tres Rios, AZ Sep-97 ACT 25 75 0 0Tres Rios, AZ Sep-97 ACT 50 50 0 0Tres Rios, AZ Sep-97 ACT 75 25 0 0Tres Rios, AZ Sep-97 ACT 100 0 0 0Tres Rios, AZ Sep-97 PHX 0 100 90 0Tres Rios, AZ Sep-97 PHX 10 90 20 0Tres Rios, AZ Sep-97 PHX 25 75 20 0Tres Rios, AZ Sep-97 PHX 50 50 20 0Tres Rios, AZ Sep-97 PHX 75 25 20 0Tres Rios, AZ Sep-97 PHX 100 0 0 0Tres Rios, AZ Oct-97 ACT 0 100 0 0Tres Rios, AZ Oct-97 ACT 10 90 0 0Tres Rios, AZ Oct-97 ACT 25 75 0 0Tres Rios, AZ Oct-97 ACT 50 50 0 0Tres Rios, AZ Oct-97 ACT 75 25 0 0Tres Rios, AZ Oct-97 ACT 100 0 0 0Tres Rios, AZ Oct-97 PHX 0 100 100 10 100 0Tres Rios, AZ Oct-97 PHX 10 90 100 0 80 0Tres Rios, AZ Oct-97 PHX 25 75 100 0 90 0Tres Rios, AZ Oct-97 PHX 50 50 100 0 60 0Tres Rios, AZ Oct-97 PHX 75 25 0 0 40 0Tres Rios, AZ Oct-97 PHX 100 0 0 0 0 0Tres Rios, AZ Jan-98 PHX 0 100 20 0Tres Rios, AZ Jan-98 PHX 10 90 20 10Tres Rios, AZ Jan-98 PHX 25 75 30 20Tres Rios, AZ Jan-98 PHX 50 50 30 0Tres Rios, AZ Jan-98 PHX 75 25 0 10Tres Rios, AZ Jan-98 PHX 100 0 0 0Tres Rios, AZ Apr-98 PHX 0 100 30 0Tres Rios, AZ Apr-98 PHX 10 90 50 0Tres Rios, AZ Apr-98 PHX 25 75 20 0Tres Rios, AZ Apr-98 PHX 50 50 10 0Tres Rios, AZ Apr-98 PHX 75 25 0 0Tres Rios, AZ Apr-98 PHX 100 0 0 0

A-9



SITE_NAME TIMEPERIOD LAB DILUTION %_EFF CS Inlet C1 Eff C2 Eff HS Inlet H1 Eff H2 EffTres Rios, AZ Dec-98 ENSR 0 100 0Tres Rios, AZ Dec-98 ENSR 50 50 0Tres Rios, AZ Dec-98 ENSR 75 25 10Tres Rios, AZ Dec-98 ENSR 87.5 12.5 0Tres Rios, AZ Dec-98 ENSR 93.75 6.25 0Tres Rios, AZ Dec-98 ENSR 100 0 0Tres Rios, AZ May-99 PHX 0 100 0 20Tres Rios, AZ May-99 PHX 50 50 0 10Tres Rios, AZ May-99 PHX 75 25 0 0Tres Rios, AZ May-99 PHX 87.5 12.5 10 0Tres Rios, AZ May-99 PHX 93.75 6.25 0 10Tres Rios, AZ May-99 PHX 100 0 0 0Tres Rios, AZ Sep-99 PHX 0 100 0 0Tres Rios, AZ Sep-99 PHX 50 50 0 10Tres Rios, AZ Sep-99 PHX 75 25 0 0Tres Rios, AZ Sep-99 PHX 87.5 12.5 0 0Tres Rios, AZ Sep-99 PHX 93.75 6.25 0 0Tres Rios, AZ Sep-99 PHX 100 0 0 0Tres Rios, AZ Dec-99 PHX 0 100 70 0Tres Rios, AZ Dec-99 PHX 50 50 0 0Tres Rios, AZ Dec-99 PHX 75 25 0 0Tres Rios, AZ Dec-99 PHX 87.5 12.5 0 0Tres Rios, AZ Dec-99 PHX 93.75 6.25 0 0Tres Rios, AZ Dec-99 PHX 100 0 0 0Tres Rios, AZ Mar-00 PHX 0 100 100 0Tres Rios, AZ Mar-00 PHX 25 75 100 0Tres Rios, AZ Mar-00 PHX 50 50 0 0Tres Rios, AZ Mar-00 PHX 75 25 0 0Tres Rios, AZ Mar-00 PHX 87.5 12.5 0 0Tres Rios, AZ Mar-00 PHX 100 0 0 20Tres Rios, AZ Sep-00 PHX 0 100 70 0Tres Rios, AZ Sep-00 PHX 25 75 0 0Tres Rios, AZ Sep-00 PHX 50 50 0 0Tres Rios, AZ Sep-00 PHX 75 25 0 0Tres Rios, AZ Sep-00 PHX 87.5 12.5 0 0Tres Rios, AZ Sep-00 PHX 100 0 0 0Tres Rios, AZ Dec-00 PHX 0 100 0 0Tres Rios, AZ Dec-00 PHX 25 75 10 0Tres Rios, AZ Dec-00 PHX 50 50 10 0Tres Rios, AZ Dec-00 PHX 75 25 0 0Tres Rios, AZ Dec-00 PHX 87.5 12.5 10 0Tres Rios, AZ Dec-00 PHX 100 0 0 0Tres Rios, AZ Mar-01 PHX 100 0 0 0Tres Rios, AZ Mar-01 PHX 87.5 12.5 0 0Tres Rios, AZ Mar-01 PHX 75 25 10 0Tres Rios, AZ Mar-01 PHX 50 50 0 0Tres Rios, AZ Mar-01 PHX 25 75 0 0Tres Rios, AZ Mar-01 PHX 0 100 0 10Tres Rios, AZ Sep-01 PHX 100 0 0 0Tres Rios, AZ Sep-01 PHX 87.5 12.5 0 0Tres Rios, AZ Sep-01 PHX 75 25 0 0Tres Rios, AZ Sep-01 PHX 50 50 10 0Tres Rios, AZ Sep-01 PHX 25 75 0 0Tres Rios, AZ Sep-01 PHX 0 100 0 10

A-10



SITE_NAME TIMEPERIOD LAB DILUTION %_EFF CS Inlet C1 Eff C2 Eff HS Inlet H1 Eff H2 EffTres Rios, AZ Nov-01 PHX 100 0 0 10Tres Rios, AZ Nov-01 PHX 87.5 12.5 0 0Tres Rios, AZ Nov-01 PHX 75 25 0 0Tres Rios, AZ Nov-01 PHX 50 50 0 0Tres Rios, AZ Nov-01 PHX 25 75 0 0Tres Rios, AZ Nov-01 PHX 0 100 20 20Tres Rios, AZ Mar-02 PHX 100 0 0 0Tres Rios, AZ Mar-02 PHX 87.5 12.5 0 0Tres Rios, AZ Mar-02 PHX 75 25 10 0Tres Rios, AZ Mar-02 PHX 50 50 0 10Tres Rios, AZ Mar-02 PHX 25 75 0 0Tres Rios, AZ Mar-02 PHX 0 100 0 0Tres Rios, AZ May-02 PHX 100 0 10 0Tres Rios, AZ May-02 PHX 87.5 12.5 0 0Tres Rios, AZ May-02 PHX 75 25 0 0Tres Rios, AZ May-02 PHX 50 50 0 10Tres Rios, AZ May-02 PHX 25 75 0 0Tres Rios, AZ May-02 PHX 0 100 0 0Tres Rios, AZ Aug-02 PHX 100 0 0 0Tres Rios, AZ Aug-02 PHX 87.5 12.5 0 0Tres Rios, AZ Aug-02 PHX 75 25 0 10Tres Rios, AZ Aug-02 PHX 50 50 0 0Tres Rios, AZ Aug-02 PHX 25 75 0 0Tres Rios, AZ Aug-02 PHX 0 100 0 0

Average 0 100 20.0 1.0 14.2 20.5 1.7 12.210 90 12.7 2.9 15.7 16.4 1.1 14.325 75 15.0 2.2 10.0 12.1 0.0 11.150 50 7.4 2.0 10.0 6.0 0.0 12.275 25 0.5 1.0 1.7 4.0 1.7 8.9

87.5 12.5 1.3 0.0 0.0 1.7 0.0 0.093.75 6.25 0.0 0.0 5.0 0.0100 0 1.1 0.0 4.2 0.0 0.0 1.1

Minimum 0 100 0 0 0 0 0 010 90 0 0 0 0 0 025 75 0 0 0 0 0 050 50 0 0 0 0 0 075 25 0 0 0 0 0 0

87.5 12.5 0 0 0 0 0 093.75 6.25 0 0 0 0100 0 0 0 0 0 0 0

Maximum 0 100 100 10 100 100 10 10010 90 100 10 100 80 10 10025 75 100 20 100 90 0 10050 50 100 10 100 60 0 10075 25 10 10 20 40 10 80

87.5 12.5 10 0 0 10 0 093.75 6.25 0 0 10 0100 0 10 0 20 0 0 10

A-11



SITE_NAME TIMEPERIOD LAB DILUTION %_EFF CS Inlet C1 Eff C2 Eff HS Inlet H1 Eff H2 EffStdDev 0 100 35.8 3.2 28.1 38.9 3.9 33.1

10 90 29.7 4.9 37.4 28.5 3.3 37.825 75 34.1 6.7 31.6 25.1 0.0 33.350 50 23.5 4.2 28.6 13.9 0.0 33.175 25 2.3 3.2 5.8 10.0 3.9 26.7

87.5 12.5 3.5 0.0 0.0 4.1 0.0 0.093.75 6.25 0.0 7.1100 0 3.2 0.0 6.7 0.0 0.0 3.3

Count 0 100 19 10 12 20 12 910 90 11 7 7 14 9 725 75 16 9 10 19 12 950 50 19 10 12 20 12 975 25 19 10 12 20 12 9

87.5 12.5 8 3 5 6 3 293.75 6.25 3 1 2 1100 0 19 10 12 20 12 9

A-12


Appendix A-3Tres Rios Ceriodaphnia dubia 7-day Chronic Toxicity Testing

STATION TIMEPERIOD LAB %_EFF REP_1 REP_2 REP_3 REP_4 REP_5 REP_6 REP_7 REP_8 REP_9 REP_10 AVG STDEV N SEC2 EFF Oct-97 PHX 0 11 35 male 24 26 28 16 6 6 22 19.3 10.19 9 3.40C2 EFF Oct-97 PHX 25 37 40 male 39 37 38 38 38 37 37 37.9 1.05 9 0.35C2 EFF Oct-97 PHX 50 43 42 male 47 31 40 37 45 40 42 40.8 4.68 9 1.56C2 EFF Oct-97 PHX 75 48 46 male 45 43 38 45 male 43 47 44.4 3.11 8 1.10C2 EFF Oct-97 PHX 90 47 47 male 39 44 49 46 male 43 48 45.4 3.25 8 1.15C2 EFF Oct-97 PHX 100 50 62 male 41 50 48 44 53 49 46 49.2 5.97 9 1.99CS Inlet Oct-97 PHX 0 25 25 5 6 25 0 9 13 14 26 14.8 9.82 10 3.10CS Inlet Oct-97 PHX 25 27 25 24 26 25 25 24 23 24 25 24.8 1.14 10 0.36CS Inlet Oct-97 PHX 50 5 6 9 6 15 6 5 14 4 5 7.5 3.92 10 1.24CS Inlet Oct-97 PHX 75 5 5 6 6 6 6 5 8 6 6 5.9 0.88 10 0.28CS Inlet Oct-97 PHX 90 4 5 4 4 5 6 6 5 4 5 4.8 0.79 10 0.25CS Inlet Oct-97 PHX 100 5 6 6 4 5 6 7 4 5 5 5.3 0.95 10 0.30H1 EFF Oct-97 ACT 0 20 19 15 18 22 16 21 17 24 22 19.4 2.91 10 0.92H1 EFF Oct-97 ACT 25 25 19 24 26 18 23 26 23 31 23 23.8 3.68 10 1.16H1 EFF Oct-97 ACT 50 24 26 26 25 25 21 24 27 21 24 24.3 2.00 10 0.63H1 EFF Oct-97 ACT 75 29 28 26 26 22 14 25 22 21 18 23.1 4.65 10 1.47H1 EFF Oct-97 ACT 90 26 26 22 27 34 31 30 19 18 21 25.4 5.34 10 1.69H1 EFF Oct-97 ACT 100 24 18 27 27 26 20 23 24 26 23 23.8 2.97 10 0.94H1 EFF Oct-97 PHX 0 5 29 26 26 26 28 10 29 19 27 22.5 8.48 10 2.68H1 EFF Oct-97 PHX 25 30 25 25 25 17 17 0 23 23 26 21.1 8.40 10 2.66H1 EFF Oct-97 PHX 50 27 25 25 28 21 25 25 25 24 21 24.6 2.22 10 0.70H1 EFF Oct-97 PHX 75 27 29 28 22 17 31 24 29 27 29 26.3 4.19 10 1.33H1 EFF Oct-97 PHX 90 29 30 31 24 28 18 28 28 3 31 25.0 8.65 10 2.74H1 EFF Oct-97 PHX 100 33 27 28 29 25 28 19 30 25 32 27.6 4.01 10 1.27H2 EFF Sep-97 ACT 0 13 14 25 20 18 14 18 17 20 15 17.4 3.66 10 1.16H2 EFF Sep-97 ACT 25 21 19 14 17 26 26 22 17 20 17 19.9 3.96 10 1.25H2 EFF Sep-97 ACT 50 27 23 23 23 23 22 17 20 26 21 22.5 2.84 10 0.90H2 EFF Sep-97 ACT 75 26 24 26 17 19 11 27 24 26 25 22.5 5.19 10 1.64H2 EFF Sep-97 ACT 90 20 23 20 23 17 21 23 26 25 22 22.0 2.62 10 0.83H2 EFF Sep-97 ACT 100 22 23 19 21 22 20 18 22 27 25 21.9 2.69 10 0.85H2 EFF Sep-97 PHX 0 20 19 21 22 25 21 24 25 17 25 21.9 2.81 10 0.89H2 EFF Sep-97 PHX 25 20 24 24 22 21 22 22 21 22 21 21.9 1.29 10 0.41H2 EFF Sep-97 PHX 50 20 23 26 23 26 23 23 24 23 23 23.4 1.71 10 0.54H2 EFF Sep-97 PHX 75 22 25 25 22 26 25 25 28 28 27 25.3 2.11 10 0.67H2 EFF Sep-97 PHX 90 25 29 27 28 24 27 29 26 25 27 26.7 1.70 10 0.54H2 EFF Sep-97 PHX 100 27 25 27 27 29 23 26 27 25 33 26.9 2.69 10 0.85HS Inlet Sep-97 ACT 0 24 19 19 21 23 27 20 19 19 21 21.2 2.70 10 0.85HS Inlet Sep-97 ACT 25 24 25 24 26 20 19 25 17 26 24 23.0 3.16 10 1.00HS Inlet Sep-97 ACT 50 25 23 22 24 25 20 24 21 16 24 22.4 2.80 10 0.88HS Inlet Sep-97 ACT 75 24 18 22 25 25 24 24 17 27 18 22.4 3.50 10 1.11HS Inlet Sep-97 ACT 90 26 18 19 28 27 18 21 24 23 19 22.3 3.83 10 1.21HS Inlet Sep-97 ACT 100 22 28 18 22 21 19 11 22 28 24 21.5 4.95 10 1.57HS Inlet Sep-97 PHX 0 26 23 23 18 22 21 14 22 21 21 21.1 3.21 10 1.02HS Inlet Sep-97 PHX 25 22 22 22 0 0 17 21 18 27 15 16.4 9.25 10 2.93HS Inlet Sep-97 PHX 50 27 3 21 0 21 26 22 17 26 9 17.2 9.82 10 3.10HS Inlet Sep-97 PHX 75 30 20 29 26 0 23 0 25 29 25 20.7 11.31 10 3.58HS Inlet Sep-97 PHX 90 25 27 28 27 26 27 21 28 31 9 24.9 6.14 10 1.94HS Inlet Sep-97 PHX 100 14 16 20 15 15 15 21 14 10 20 16.0 3.40 10 1.07HS Inlet Oct-97 ACT 0 18 19 19 16 24 23 18 15 23 20 19.5 3.03 10 0.96HS Inlet Oct-97 ACT 25 21 17 28 27 17 22 16 23 22 21 21.4 4.03 10 1.28HS Inlet Oct-97 ACT 50 30 17 32 22 22 20 24 28 16 28 23.9 5.47 10 1.73HS Inlet Oct-97 ACT 75 24 25 27 20 17 22 22 24 18 20 21.9 3.18 10 1.00HS Inlet Oct-97 ACT 90 31 28 28 14 29 27 31 15 23 25 25.1 6.10 10 1.93HS Inlet Oct-97 ACT 100 27 25 26 30 30 23 27 26 20 24 25.8 3.05 10 0.96HS Inlet Oct-97 PHX 0 17 25 25 6 23 29 22 30 21 21 21.9 6.79 10 2.15HS Inlet Oct-97 PHX 25 6 9 25 26 27 24 14 8 24 24 18.7 8.42 10 2.66HS Inlet Oct-97 PHX 50 6 31 24 26 9 6 16 29 18 26 19.1 9.52 10 3.01HS Inlet Oct-97 PHX 75 6 6 6 8 13 8 5 33 12 16 11.3 8.45 10 2.67HS Inlet Oct-97 PHX 90 12 17 18 26 13 19 16 36 6 8 17.1 8.76 10 2.77HS Inlet Oct-97 PHX 100 18 7 8 5 6 21 11 16 4 20 11.6 6.55 10 2.07

Note: CS Inlet October 1997 - control failure < 3 broods within 7 days

A-13

analysis of whole effluent toxicity (wet) data from the ... · data from the tres rios...

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